Charles R. Vanderburg

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder. Ten percent of cases are inherited; most involve unidentified genes. We report here 13 mutations in the fused in sarcoma/translated in liposarcoma (FUS/TLS) gene on chromosome 16 that were specific for familial ALS. The FUS/TLS protein binds to RNA, functions in diverse processes, and is normally located predominantly in the nucleus. In contrast, the mutant forms of FUS/TLS accumulated in the cytoplasm of neurons, a pathology that is similar to that of the gene TAR DNA-binding protein 43 (TDP43), whose mutations also cause ALS. Neuronal cytoplasmic protein aggregation and defective RNA metabolism thus appear to be common pathogenic mechanisms involved in ALS and possibly in other neurodegenerative disorders.

Gene expression at fine scale Mapping gene expression at the single-cell level within tissues remains a technical challenge. Rodriques et al. developed a method called Slide-seq, whereby RNA was spatially resolved from tissue sections by transfer onto a surface covered with DNA-barcoded beads. Applying Slide-seq to regions of a mouse brain revealed spatial gene expression patterns in the Purkinje layer of the cerebellum and axes of variation across Purkinje cell compartments. The authors used this method to dissect the temporal evolution of cell type–specific responses in a mouse model of traumatic brain injury. Science , this issue p. 1463

Article22 February 2018Open Access Transparent process Tau protein liquid–liquid phase separation can initiate tau aggregation Susanne Wegmann Corresponding Author Susanne Wegmann [email protected] orcid.org/0000-0002-5388-2479 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Bahareh Eftekharzadeh Bahareh Eftekharzadeh Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Katharina Tepper Katharina Tepper German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Katarzyna M Zoltowska Katarzyna M Zoltowska Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Rachel E Bennett Rachel E Bennett Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Simon Dujardin Simon Dujardin Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Pawel R Laskowski Pawel R Laskowski orcid.org/0000-0002-8118-9030 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Danny MacKenzie Danny MacKenzie Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Tarun Kamath Tarun Kamath Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Caitlin Commins Caitlin Commins Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Charles Vanderburg Charles Vanderburg Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Allyson D Roe Allyson D Roe Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Zhanyun Fan Zhanyun Fan Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Amandine M Molliex Amandine M Molliex Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Amayra Hernandez-Vega Amayra Hernandez-Vega Max-Planck Institute for Molecular Cell Biology & Genetics, Dresden, Germany Search for more papers by this author Daniel Muller Daniel Muller orcid.org/0000-0003-3075-0665 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Anthony A Hyman Anthony A Hyman Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Eckhard Mandelkow Eckhard Mandelkow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck Institute for Metabolism Research, Hamburg Outstation c/o DESY, Hamburg, Germany CAESAR Research Center, Bonn, Germany Search for more papers by this author J Paul Taylor J Paul Taylor Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Bradley T Hyman Corresponding Author Bradley T Hyman [email protected] orcid.org/0000-0002-7959-9401 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Susanne Wegmann Corresponding Author Susanne Wegmann [email protected] orcid.org/0000-0002-5388-2479 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Bahareh Eftekharzadeh Bahareh Eftekharzadeh Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Katharina Tepper Katharina Tepper German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Katarzyna M Zoltowska Katarzyna M Zoltowska Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Rachel E Bennett Rachel E Bennett Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Simon Dujardin Simon Dujardin Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Pawel R Laskowski Pawel R Laskowski orcid.org/0000-0002-8118-9030 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Danny MacKenzie Danny MacKenzie Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Tarun Kamath Tarun Kamath Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Caitlin Commins Caitlin Commins Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Charles Vanderburg Charles Vanderburg Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Allyson D Roe Allyson D Roe Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Zhanyun Fan Zhanyun Fan Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Amandine M Molliex Amandine M Molliex Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Amayra Hernandez-Vega Amayra Hernandez-Vega Max-Planck Institute for Molecular Cell Biology & Genetics, Dresden, Germany Search for more papers by this author Daniel Muller Daniel Muller orcid.org/0000-0003-3075-0665 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Anthony A Hyman Anthony A Hyman Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Eckhard Mandelkow Eckhard Mandelkow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck Institute for Metabolism Research, Hamburg Outstation c/o DESY, Hamburg, Germany CAESAR Research Center, Bonn, Germany Search for more papers by this author J Paul Taylor J Paul Taylor Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Bradley T Hyman Corresponding Author Bradley T Hyman [email protected] orcid.org/0000-0002-7959-9401 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Author Information Susanne Wegmann *,1,‡, Bahareh Eftekharzadeh1,‡, Katharina Tepper2,‡, Katarzyna M Zoltowska1, Rachel E Bennett1, Simon Dujardin1, Pawel R Laskowski3, Danny MacKenzie1, Tarun Kamath1, Caitlin Commins1, Charles Vanderburg1, Allyson D Roe1, Zhanyun Fan1, Amandine M Molliex4, Amayra Hernandez-Vega5, Daniel Muller3, Anthony A Hyman3, Eckhard Mandelkow2,6,7, J Paul Taylor4,8 and Bradley T Hyman *,1 1Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA 2German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 3Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland 4Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA 5Max-Planck Institute for Molecular Cell Biology & Genetics, Dresden, Germany 6Max-Planck Institute for Metabolism Research, Hamburg Outstation c/o DESY, Hamburg, Germany 7CAESAR Research Center, Bonn, Germany 8Howard Hughes Medical Institute, Chevy Chase, MD, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 617 230 7184; E-mail: [email protected] *Corresponding author. Tel: +1 617 726 3987; E-mail: [email protected] The EMBO Journal (2018)37:e98049https://doi.org/10.15252/embj.201798049 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase-separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho-tau isolated from human Alzheimer brain. Droplet-like tau can also be observed in neurons and other cells. We found that tau droplets become gel-like in minutes, and over days start to spontaneously form thioflavin-S-positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases. Synopsis The microtubule binding protein tau can undergo liquid-liquid phase separation under physiological conditions. Phosphorylated, FTD-mutant, and Alzheimer's disease brain tau is capable of forming droplets that can initiate the formation of aberrant, aggregated tau “seeds”. LLPS may play a role in different tauopathies. Full-length human tau can undergo liquid-liquid phase separation in neurons. In vitro studies show importance of phosphorylation and Frontotemporal Dementia mutations for tau LLPS. AD brain tau is competent of forming droplets. Tau droplets can transition into aggregates. Tau LLPS is a potential mechanism for aggregation initiation in tauopathies. Introduction Tau protein is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD) and of various other forms of intracellular inclusions in frontotemporal dementias (FTDs). Tau is classically described as a soluble neuron-specific microtubule binding (MTB) protein; however, the connection between tau (mis)function and neurodegeneration is uncertain. It is clear, for example, that post-translational modifications (PTMs) and tau mutations predisposing to aggregation are both associated with neurodegeneration. Recently, soluble hyperphosphorylated high molecular weight tau was identified as a bioactive form, which can be released and taken up by neurons and initiate templated misfolding of cytoplasmic tau in neurons (Takeda et al, 2015). This soluble hyperphosphorylated tau is, however, clearly distinct from aggregated fibrillary tau in neurofibrillary tangles, despite both being implicated in tau toxicity. Tau is an exceptionally soluble protein, and the molecular mechanisms that link soluble tau to aggregated tau are unknown. We now report that tau—similar to several other neurodegenerative disease-associated proteins such as the prion-domain harboring RNA binding proteins FUS, TDP43, hnRNPA1 (King et al, 2012)—can undergo liquid–liquid phase separation (LLPS), and we suggest that this observation may provide a biological mechanism for tau aggregation in neurodegenerative diseases. The longest isoform of tau in the human CNS contains a MTB region that contains four pseudo-repeats (R1–R4) plus flanking proline-rich regions (P1, P2, and P3; Gustke et al, 1994), a shorter (≈40 aa) C-terminal tail, and a long (≈250 aa) flexible N-terminal half of tau, which projects from the surface of microtubules in the MT-bound state (Goode et al, 1997), and forms a polyelectrolyte brush around fibrillary aggregates of tau (Sillen et al, 2005; Wegmann et al, 2013). The lack of a fixed tertiary protein structure classifies tau as an intrinsically disordered protein (IDP). Proteins that contain intrinsically disordered regions often have multiple biological functions (Wright & Dyson, 2014), and some of them aggregate in protein aggregation diseases, such as huntingtin protein in Huntington's disease, α-synuclein in Parkinson's disease, TDP43 and FUS in ALS, and tau in Alzheimer's disease and tauopathies (Uversky et al, 2008). Recent studies revealed that the RNA binding and stress granule-associated proteins FUS (Patel et al, 2015), hnRNPA1 (Molliex et al, 2015), and TDP43 (Conicella et al, 2016) have the ability to reversibly form intracellular membrane-less organelles. These reversible droplets represent physiologically active protein or protein-nucleic acid “bioreactors” (Hyman et al, 2014), which form through a process known as LLPS (Brangwynne et al, 2015). Such transient membrane-less organelles have multiple cellular functions, such as p-granule formation to establish intracellular gradients of RNA transcription (Brangwynne et al, 2009), the enrichment of RNA binding proteins in stress granules (Lin et al, 2015; Molliex et al, 2015), concentrating transcription factors in nucleoli (Berry et al, 2015), and the initiation of microtubule spindle formation (Jiang et al, 2015). However, the functional phase separation of nuclear proteins was shown to be disrupted by C9orf72 GR/PR dipeptide repeats (Lee et al, 2016) and is related to protein aggregation in neurodegeneration (Schmidt & Rohatgi, 2016). In most cases, the phase transition of these proteins is driven by so-called low complexity domains (LCDs) in their sequence, a term of somewhat inconclusive nomenclature that is often used to describe protein domains of low amino acid variance leading to inhomogeneous charge distribution or polarity distribution along the peptide backbone (Nott et al, 2015). For example, FUS, TDP-43, and hnRNPA1 contain “prion-like” LCDs that drive their phase separation. However, in the case of tau, no typical low complexity domain (LCD) exists in the protein sequence, but the intrinsic disorder and the inhomogeneous charge distribution of full-length tau (Lee et al, 1988) led us to postulate that tau may undergo a similar phase separation. In fact, recent reports using recombinant tau constructs support the argument that tau, despite having no defined LCDs, can undergo LLPS facilitated by crowding agents or RNA in vitro (Ambadipudi et al, 2017; Hernandez-Vega et al, 2017; Zhang et al, 2017). We now show that full-length human tau protein can efficiently undergo LLPS to form condensed liquid tau droplets in cell physiological conditions. We observed the formation of tau droplets not only ex vivo for post-translationally modified recombinant tau, but also in neurons in vitro and with strong evidence even in vivo, using high molecular weight hyperphosphorylated tau isolated from human Alzheimer brain. Importantly, tau LLPS is enabled and regulated by physiological and pathological-like tau phosphorylation, by disease-associated mutations, and can be induced in in vitro aggregation conditions. Similar to FUS and hnRNPA1 proteins (Molliex et al, 2015; Murakami et al, 2015; Patel et al, 2015), droplets of pathological tau “mature” through a viscous gel phase into protein aggregates; the tau aggregates become thioflavin-S positive, suggesting the emergence of the beta-pleated sheet conformation present in tau aggregates in vivo. It appears that the phase separation of tau at physiological protein concentrations may be catalyzed by an interplay of electrostatic interactions in the unstructured N-terminal half of tau, combined with hydrophobic interactions of the C-terminal MTB domain, which can stabilize tau droplets possibly through β-sheet structures. These findings complement and provide additional biological relevance to the recent reports showing in vitro tau LLPS of repeat domain constructs at rather high concentrations (Ambadipudi et al, 2017) and in the presence of aggregation triggering polyanionic RNA (Zhang et al, 2017). Together, our data strongly support the hypothesis that intracellular phase separation of tau leads to subcellular foci of high local concentration of tau, which may be important for physiological roles of tau, and—in the setting of aberrant phosphorylation or disease-relevant pro-aggregation mutations—lead to tau aggregation. By analogy to hnRNPA1 (Molliex et al, 2015)-, TDP-43 (Conicella et al, 2016)-, FUS (Murakami et al, 2015)-, and C9orf72-derived dipeptide repeats (Lee et al, 2016), tau LLPS could act to initiate tau aggregation in AD and FTD. We suggest that protein LLPS may be a biophysical mechanism underlying multiple protein aggregation and neurodegenerative diseases. Results Intrinsically disordered tau protein can accumulate in droplet-like assemblies in neurons Several disordered proteins with LCDs have been shown to undergo LLPS leading to liquid droplet formation in aqueous solution upon increasing the molecular crowding in the solution (Mitrea et al, 2016). Cells use LLPS to reversibly assemble membrane-less organelles such as stress granules, p-granules, or nucleoli (reviewed in Mitrea et al, 2016; Hyman et al, 2014). In a number of LLPS proteins, including Ddx4 (Nott et al, 2014), domains of low amino acid complexity introduce an unequal charge distribution along the protein backbone resulting in multivalency of these polypeptides with alternating patches of high positive or negative charge densities (Toretsky & Wright, 2014). The phase separation of these proteins is often driven by electrostatic interactions within the LCDs, or between them. Tau is intrinsically disordered (Fig 1A, Appendix Fig S1), and the distribution of charges in the longest human isoform of tau (2N4R, 441 aa) at physiological pH 7.4 shows per se a similar multivalent pattern (Wegmann et al, 2013): The N-terminus (≈aa 1–120) has a negative net charge, the middle domain (≈aa 121–250) has a large excess of positive charges, the repeat domain (TauRD, ≈aa 251–390) has a moderate excess of positive charges, and the C-terminus (≈aa 391–441) is negatively charged (Fig 1B). Figure 1. Droplet-like condensation of intrinsically disordered human tau protein in neurons Protein sequence and disorder prediction (PONDR) of the longest human tau isoform [2N4R, 441 amino acids (aa)]. The highly disordered N-terminal half (projection domain) contains two N-terminal repeats (N1, N2) and two proline-rich regions (P1, P2). The repeat domain (TauRD) consists of four pseudo-repeats (R1–R4), can transiently adopt short stretches of β-strand structure in R2 and R3, facilitates microtubule binding together with P2 + P3, and mediates the aggregation of tau. Single amino acid and average (sliding window of 6 aa) charge distribution along the tau441 sequence (at pH 7.4) reveal the following charged domains in tau441: the negatively charged N-terminal end (≈aa 1–120), a strongly positively charged middle domain (aa 121–250), the positively charged TauRD (aa 251–400), and a negatively charged C-terminal end (aa 401–441). Intracellular droplet-like accumulations of GFP-tau441 (white arrows) in primary cortical mouse neurons. Expression of GFP alone does not lead to droplets. Droplet dynamics in neurons is shown in Movie EV1. FRAP reveals incomplete recovery of droplet-like tau in neuronal cell bodies indicating an immobile fraction of GFP-tau441 molecules of ≈30%. Neurons growing in microfluidic chambers extend their axons in microgrooves (Takeda et al, 2015). Time-lapse imaging (#1–5, time interval ≈3 s) shows the movement of droplet-like GFP-tau441 (white arrows) in axons over time. Immunofluorescent labeling of neurons expressing GFP-tau441 shows that tau accumulations (white arrows) do not co-localize with lysosomes (LAMP1). In murine neuroblastoma cells (N2a) expressing GFP-tau441, droplet-like tau occurs in cells having a critical GFP-tau441 concentration (white star). In cells with low expression level (white square), GFP-tau binds to microtubule bundles; in cells with medium GFP-tau441 (white circle), excess GFP-tau441 fills the cell bodies. The graph shows the GFP fluorescence intensity in cell bodies with (pink) and without (gray) droplets. Cross-sectional profiles of cells with droplets suggest a similar tau concentration of GFP-tau on microtubules (white lines, black traces) and in droplets (pink lines and traces). Data information: In (D), data are presented as mean ± s.e.m., n = 9 droplets; data have been fitted with a one-phase exponential fit, r2 = 0.08215. In (G), average fluorescence intensity in cell bodies is plotted as mean ± s.e.m., n = 45 for “− droplets”, n = 17 for “+ droplets”. Download figure Download PowerPoint Based on this knowledge about the disorder and inhomogeneous charge distribution of tau, we postulated that human tau may also be able to undergo LLPS in neurons. To test this hypothesis, we expressed GFP-tagged full-length tau (GFP-tau441) in primary cortical mouse neurons. The expression of GFP-tau441 led to the formation of mobile intracellular droplet-like tau accumulations in the cytosol (Fig 1C, Movies EV1–EV3), and fluorescence recovery after photobleaching (FRAP) of intraneuronal GFP-tau441 droplets revealed a fast recovery rate with an immobile tau molecule fraction of ≈30% (Figs 1D and EV1A). Some droplet-like tau accumulations in axons of neurons grown in microfluidic chambers moved retro- as well as anterograde (Fig 1E, Movie EV4). Interestingly, GFP-tau441 droplets (Movies EV1–EV3) appeared less mobile compared to droplets formed by the N-terminal projection domain of tau441 (aa 1–256), GFP-tau256 (Movies EV5,EV6, EV7), maybe because of the ability of full-length GFP-tau441 but not GFP-tau256 to bind microtubules via the repeat domain. Click here to expand this figure. Figure EV1. Tau phosphorylated in cells can undergo LLPS Example FRAP images of GFP-tau441 droplet (green circle and arrowhead) in primary neurons. Recovery after photobleaching event appears rapid but incomplete. FRAP of tau aggregates in HEK TauRDP301S-CFP/YFP cells shows no recovery after photobleaching of the aggregates that have been initiated with brain lysate (5 μg/μl total protein) from human tauP301L transgenic mice (rTg4510 line). Insets show example images taken before bleaching and at t = 0 and 300 s. FRAP parameters were the same as in (A, C). Graph represents mean ± s.e.m. of n = 3 aggregates. FRAP of GFP-PolyQ aggregates that formed in primary cortical mouse neurons after overexpression for 24 h. PolyQ aggregates show no recovery after photobleaching. Insets show example images taken before bleaching and at t = 0 and 300 s. FRAP parameters were the same as in (A, B). Graph represents mean ± s.e.m. of n = 4 aggregates. Example FRAP experiment for GFP-tau441 expressed in primary cortical neurons. Cytosolic soluble GFP-tau441 shows rapid and complete recovery, whereas microtubule-bound GFP-tau441 in a neighboring cell recovers slightly slower. FRAP parameters were the same as in (A– C). Expression of AAV8 Dendra2-tau441 in the cortex of wild-type mice allows the visualization of droplet-like tau in cortical neuronal cell bodies (1–3) and processes (4–6) in vivo by two-photon microscopy. GFP expressing control neurons show a homogenous GFP distribution instead. Cell lysates from murine N2a cells and primary cortical mouse neurons (DIV7) expressing GFP-tau256 or GFP-tau441 were analyzed by Western blot for the content of human tau (Tau13) and phospho-tau using antibodies PHF-1 or a mix of p-Tau antibodies. Most abundant phosphorylation sites previously found in p-tau441 and deP-tau441 (*) by mass spectrometry (Mair et al, 2016). Most of these phospho-sites, for which specific antibodies were available, could be verified (red; blue = not detected) in the p-tau441 preparation used in this manuscript. Download figure Download PowerPoint We did not observe the fusion or fission of GFP-tau441 droplets in neurons, which can be seen as a typical behavior of liquid droplets. However, the observed FRAP recovery of the GFP-tau droplets excludes the possibility that the observed spherical droplets resemble large tau aggregates previously reported for mutant tau expressed in neurons (Hoover et al, 2010). Tau aggregates induced in HEK TauRDP301S-CFP/YFP cells, or GFP-polyQ aggregates in primary neurons, did not show recovery after photobleaching (Fig EV1B and C), whereas soluble GFP-tau in the cytosol and bound on microtubules showed fast recovery (Fig EV1D). Moreover, the expression of wild-type tau does not lead to intracellular tau aggregation, neither in vitro (Lim et al, 2014) nor in vivo. Furthermore, GFP-tau441 droplets did not co-localize with membrane-bound organelles like lysosomes (Fig 1F), endosomes (Appendix Fig S2A), or the endoplasmic reticulum (ER, Appendix Fig S2B). Interestingly, when tau441 N-terminally fused to the fluorescent protein Dendra2, Dendra2-tau441, was expressed in the cortex of living wild-type mice upon stereotactical injection of AAV Dendra2-tau441 into the somatosensory cortex, two-photon imaging through a cranial window revealed a heterogeneous distribution of unconverted green Dendra2-tau441 with spherical droplet-shaped accumulations in the cell bodies of neurons in cortex layer 2/3 (Fig EV1E, neuron #1–3). Some of these accumulations also occurred along neuritic projections (Fig EV1E, #4–6). In contrast, the distribution of GFP in neurons of the control AAV GFP-injected hemisphere was homogeneous in cell bodies and projections of transduced neurons. These data indicated that tau LLPS may also occur in the living brain. Notably, in N2a cells, GFP-tau droplets could mostly be observed in cells with sufficiently high GFP-tau expression levels (Fig 1G), whereas in primary neurons, droplets could also occur in neurons with rather low or medium GFP-tau441 expression levels. This indicated that tau LLPS in neurons may be regulated by additional factors. In neurons, tau is phosphorylated at multiple sites (Fig EV1F; Iqbal et al, 2005). Many of tau's physiological and pathological phosphorylation sites are located in the positively charged middle domain of the N-terminal half and in the repeat domain (Johnson & Stoothoff, 2004), where phosphorylation causes a local increase or a change in domain charge from positive to neutral or negative, and hence can change intra- and intermolecular interactions of tau. However, most previous in vitro studies on tau aggregation utilized recombinant non-phosphorylated tau from Escherichia coli, and in these studies, tau LLPS has not been observed. Phosphorylated human full-length tau undergoes LLPS in vitro We decided to describe the conditions for tau LLPS in more detail and produced recombinant full-length human tau (p-tau441, aa 1–441; Fig 2A) in SF9 insect cells, which are able to introduce PTMs including phosphorylation into recombinant tau (Tepper et al, 2014). Previously, the phosphorylation sites found in p-tau441 by mass spectrometry were reported to be similar to the phosphorylation of tau extracted from AD brains (Mair et al, 2016; Fig EV1G), with phosphorylation in the repeat domain (R1–R4), in the proline-rich region (P1 + P2), and some in the N-terminal insets (N1 and N2) of tau441 (Fig 2A). Here, we used Sf9 p-tau441 protein from the same source that was expressed and purified under identical conditions, and verified most of the reported phosphorylation sites in p-tau441 by Western blot analysis (Fig EV1G). Figure 2. Liquid droplet characteristics of p-tau441 Qualitative distribution of phosphorylation sites in p-tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al, 2016)]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p-tau441 in presence of molecular crowding (12.5% w/v Ficoll-400). No phase separation is observed without crowding agent or in the absence of p-tau441 protein. Liquid droplets formed by p-tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl-acetate and visualized by transmission electron microscopy (TEM). p-tau441 droplets are decorated with gold particles after immunogold labeling using anti-tau antibody K9JA. Shortly after formation (15 min), p-tau441 droplets stop to coalesce and often occur as doublets or triplets. With time (60 min), droplets grow in size but remain colloidal. Droplet fusion is shown in Movie EV1. p-tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p-tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p-tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium

Aggregation of alpha-synuclein (αsyn) and resulting cytotoxicity is a hallmark of sporadic and familial Parkinson's disease (PD) as well as dementia with Lewy bodies, with recent evidence implicating oligomeric and pre-fibrillar forms of αsyn as the pathogenic species. Recent in vitro studies support the idea of transcellular spread of extracellular, secreted αsyn across membranes. The aim of this study is to characterize the transcellular spread of αsyn oligomers and determine their extracellular location.Using a novel protein fragment complementation assay where αsyn is fused to non-bioluminescent amino-or carboxy-terminus fragments of humanized Gaussia Luciferase we demonstrate here that αsyn oligomers can be found in at least two extracellular fractions: either associated with exosomes or free. Exosome-associated αsyn oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free αsyn oligomers. Specifically, we determine that αsyn oligomers are present on both the outside as well as inside of exosomes. Notably, the pathway of secretion of αsyn oligomers is strongly influenced by autophagic activity.Our data suggest that αsyn may be secreted via different secretory pathways. We hypothesize that exosome-mediated release of αsyn oligomers is a mechanism whereby cells clear toxic αsyn oligomers when autophagic mechanisms fail to be sufficient. Preventing the early events in αsyn exosomal release and uptake by inducing autophagy may be a novel approach to halt disease spreading in PD and other synucleinopathies.

Objective To examine region‐ and substrate‐specific autoradiographic and in vitro binding patterns of positron emission tomography tracer [F‐18]‐AV‐1451 (previously known as T807), tailored to allow in vivo detection of paired helical filament‐tau–containing lesions, and to determine whether there is off‐target binding to other amyloid/non‐amyloid proteins. Methods We applied [F‐18]‐AV‐1451 phosphor screen autoradiography, [F‐18]‐AV‐1451 nuclear emulsion autoradiography, and [H‐3]‐AV‐1451 in vitro binding assays to the study of postmortem samples from patients with a definite pathological diagnosis of Alzheimer disease, frontotemporal lobar degeneration–tau, frontotemporal lobar degeneration–transactive response DNA binding protein 43 (TDP‐43), progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy, cerebral amyloid angiopathy and elderly controls free of pathology. Results Our data suggest that [F‐18]‐AV‐1451 strongly binds to tau lesions primarily made of paired helical filaments in Alzheimer brains (eg, intraneuronal and extraneuronal tangles and dystrophic neurites), but does not seem to bind to a significant extent to neuronal and glial inclusions mainly composed of straight tau filaments in non‐Alzheimer tauopathy brains or to lesions containing β‐amyloid, α‐synuclein, or TDP‐43. [F‐18]‐AV‐1451 off‐target binding to neuromelanin‐ and melanin‐containing cells and, to a lesser extent, to brain hemorrhagic lesions was identified. Interpretation Our data suggest that [F‐18]‐AV‐1451 holds promise as a surrogate marker for the detection of brain tau pathology in the form of tangles and paired helical filament‐tau–containing neurites in Alzheimer brains but also point to its relatively lower affinity for lesions primarily made of straight tau filaments in non‐Alzheimer tauopathy cases and to the existence of some [F‐18]‐AV‐1451 off‐target binding. These findings provide important insights for interpreting in vivo patterns of [F‐18]‐AV‐1451 retention. Ann Neurol 2015 Ann Neurol 2015;78:Ann Neurol 2015;78:679–696

Abstract Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization 1–5 . First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas.

Alzheimer's disease (AD) is a multifactorial and fatal neurodegenerative disorder for which the mechanisms leading to profound neuronal loss are incompletely recognized. MicroRNAs (miRNAs) are recently discovered small regulatory RNA molecules that repress gene expression and are increasingly acknowledged as prime regulators involved in human brain pathologies. Here we identified two homologous miRNAs, miR-132 and miR-212, downregulated in temporal cortical areas and CA1 hippocampal neurons of human AD brains. Sequence-specific inhibition of miR-132 and miR-212 induces apoptosis in cultured primary neurons, whereas their overexpression is neuroprotective against oxidative stress. Using primary neurons and PC12 cells, we demonstrate that miR-132/212 controls cell survival by direct regulation of PTEN, FOXO3a and P300, which are all key elements of AKT signaling pathway. Silencing of these three target genes by RNAi abrogates apoptosis caused by the miR-132/212 inhibition. We further demonstrate that mRNA and protein levels of PTEN, FOXO3a, P300 and most of the direct pro-apoptotic transcriptional targets of FOXO3a are significantly elevated in human AD brains. These results indicate that the miR-132/miR-212/PTEN/FOXO3a signaling pathway contributes to AD neurodegeneration.

Exosomes are cellular secretory vesicles containing microRNAs (miRNAs). Once secreted, exosomes are able to attach to recipient cells and release miRNAs potentially modulating the function of the recipient cell. We hypothesized that exosomal miRNA expression in brains of patients diagnosed with schizophrenia (SZ) and bipolar disorder (BD) might differ from controls, reflecting either disease-specific or common aberrations in SZ and BD patients. The sources of the analyzed samples included McLean 66 Cohort Collection (Harvard Brain Tissue Resource Center), BrainNet Europe II (BNE, a consortium of 18 brain banks across Europe) and Boston Medical Center (BMC). Exosomal miRNAs from frozen postmortem prefrontal cortices with well-preserved RNA were isolated and submitted to profiling by Luminex FLEXMAP 3D microfluidic device. Multiple statistical analyses of microarray data suggested that certain exosomal miRNAs were differentially expressed in SZ and BD subjects in comparison to controls. RT-PCR validation confirmed that two miRNAs, miR-497 in SZ samples and miR-29c in BD samples, have significantly increased expression when compared to control samples. These results warrant future studies to evaluate the potential of exosome-derived miRNAs to serve as biomarkers of SZ and BD.

The loss of dopamine (DA) neurons within the substantia nigra pars compacta (SNpc) is a defining pathological hallmark of Parkinson's disease (PD). Nevertheless, the molecular features associated with DA neuron vulnerability have not yet been fully identified. Here, we developed a protocol to enrich and transcriptionally profile DA neurons from patients with PD and matched controls, sampling a total of 387,483 nuclei, including 22,048 DA neuron profiles. We identified ten populations and spatially localized each within the SNpc using Slide-seq. A single subtype, marked by the expression of the gene AGTR1 and spatially confined to the ventral tier of SNpc, was highly susceptible to loss in PD and showed the strongest upregulation of targets of TP53 and NR2F2, nominating molecular processes associated with degeneration. This same vulnerable population was specifically enriched for the heritable risk associated with PD, highlighting the importance of cell-intrinsic processes in determining the differential vulnerability of DA neurons to PD-associated degeneration.

Objective Recent studies have shown that positron emission tomography (PET) tracer AV‐1451 exhibits high binding affinity for paired helical filament (PHF)‐tau pathology in Alzheimer's brains. However, the ability of this ligand to bind to tau lesions in other tauopathies remains controversial. Our goal was to examine the correlation of in vivo and postmortem AV‐1451 binding patterns in three autopsy‐confirmed non‐Alzheimer tauopathy cases. Methods We quantified in vivo retention of [F‐18]‐AV‐1451 and performed autoradiography, [H‐3]‐AV‐1451 binding assays, and quantitative tau measurements in postmortem brain samples from two progressive supranuclear palsy (PSP) cases and a MAPT P301L mutation carrier. They all underwent [F‐18]‐AV‐1451 PET imaging before death. Results The three subjects exhibited [F‐18]‐AV‐1451 in vivo retention predominantly in basal ganglia and midbrain. Neuropathological examination confirmed the PSP diagnosis in the first two subjects; the MAPT P301L mutation carrier had an atypical tauopathy characterized by grain‐like tau‐containing neurites in gray and white matter with heaviest burden in basal ganglia. In all three cases, autoradiography failed to show detectable [F‐18]‐AV‐1451 binding in multiple brain regions examined, with the exception of entorhinal cortex (reflecting incidental age‐related neurofibrillary tangles) and neuromelanin‐containing neurons in the substantia nigra (off‐target binding). The lack of a consistent significant correlation between in vivo [F‐18]‐AV‐1541 retention and postmortem in vitro binding and tau measures in these cases suggests that this ligand has low affinity for tau lesions primarily made of straight tau filaments. Interpretation AV‐1451 may have limited utility for in vivo selective and reliable detection of tau aggregates in these non‐Alzheimer tauopathies. ANN NEUROL 2017;81:117–128

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types, and their positions within individual anatomical structures, remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA-seq with Slide-seq-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain, and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signaling, elucidated region-specific specializations in activity-regulated gene expression, and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource (BrainCellData.org) should find diverse applications across neuroscience, including the construction of new genetic tools, and the prioritization of specific cell types and circuits in the study of brain diseases.

<h2>Summary</h2> Cellular perturbations underlying Alzheimer's disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes—which we refer to as the early cortical amyloid response—were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with β-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in 'Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia'. Neuron, 21, 1315-1325, 1998; Sheen et al. in 'Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex'. Nat. Genet., 36, 69-76, 2004). Recent studies have shown that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in 'MEKK4 signaling regulates filamin expression and neuronal migration'. Neuron, 52, 789-801, 2006). Here we show that neurons in post-mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attachment protein alpha (alpha-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to periventricular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafficking and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafficking of transport vesicles. Our current findings suggest that PH formation arises from a final common pathway involving disruption of vesicle trafficking, leading to impaired cell adhesion and loss of neuroependymal integrity.

Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5′-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2′-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation. Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5′-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2′-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.

The most common monogenic cause of small-vessel disease leading to ischemic stroke and vascular dementia is the neurodegenerative syndrome cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is associated with mutations in the Notch 3 receptor. CADASIL pathology is characterized by vascular smooth muscle cell degeneration and accumulation of diagnostic granular osmiophilic material (GOM) in vessels. The functional nature of the Notch 3 mutations causing CADASIL and their mechanistic connection to small-vessel disease and GOM accumulation remain enigmatic. To gain insight into how Notch 3 function is linked to CADASIL pathophysiology, we studied two phenotypically distinct mutations, C455R and R1031C, respectively associated with early and late onset of stroke, by using hemodynamic analyses in transgenic mouse models, receptor activity assays in cell culture, and proteomic examination of postmortem human tissue. We demonstrate that the C455R and R1031C mutations define different hypomorphic activity states of Notch 3, a property linked to ischemic stroke susceptibility in mouse models we generated. Importantly, these mice develop osmiophilic deposits and other age-dependent phenotypes that parallel remarkably the human condition. Proteomic analysis of human brain vessels, carrying the same CADASIL mutations, identified clusterin and collagen 18 α1/endostatin as GOM components. Our findings link loss of Notch signaling with ischemic cerebral small-vessel disease, a prevalent human condition. We determine that CADASIL pathophysiology is associated with hypomorphic Notch 3 function in vascular smooth muscle cells and implicate the accumulation of clusterin and collagen 18 α1/endostatin in brain vessel pathology.

[F-18]-AV-1451 is a novel positron emission tomography (PET) tracer with high affinity to neurofibrillary tau pathology in Alzheimer’s disease (AD). PET studies have shown increased tracer retention in patients clinically diagnosed with dementia of AD type and mild cognitive impairment in regions that are known to contain tau lesions. In vivo uptake has also consistently been observed in midbrain, basal ganglia and choroid plexus in elderly individuals regardless of their clinical diagnosis, including clinically normal whose brains are not expected to harbor tau pathology in those areas. We and others have shown that [F-18]-AV-1451 exhibits off-target binding to neuromelanin, melanin and blood products on postmortem material; and this is important for the correct interpretation of PET images. In the present study, we further investigated [F-18]-AV-1451 off-target binding in the first autopsy-confirmed Parkinson’s disease (PD) subject who underwent antemortem PET imaging. The PET scan showed elevated [F-18]-AV-1451 retention predominantly in inferior temporal cortex, basal ganglia, midbrain and choroid plexus. Neuropathologic examination confirmed the PD diagnosis. Phosphor screen and high resolution autoradiography failed to show detectable [F-18]-AV-1451 binding in multiple brain regions examined with the exception of neuromelanin-containing neurons in the substantia nigra, leptomeningeal melanocytes adjacent to ventricles and midbrain, and microhemorrhages in the occipital cortex (all reflecting off-target binding), in addition to incidental age-related neurofibrillary tangles in the entorhinal cortex. Additional legacy postmortem brain samples containing basal ganglia, choroid plexus, and parenchymal hemorrhages from 20 subjects with various neuropathologic diagnoses were also included in the autoradiography experiments to better understand what [F-18]-AV-1451 in vivo positivity in those regions means. No detectable [F-18]-AV-1451 autoradiographic binding was present in the basal ganglia of the PD case or any of the other subjects. Off-target binding in postmortem choroid plexus samples was only observed in subjects harboring leptomeningeal melanocytes within the choroidal stroma. Off-target binding to parenchymal hemorrhages was noticed in postmortem material from subjects with cerebral amyloid angiopathy. The imaging-postmortem correlation analysis in this PD case reinforces the notion that [F-18]-AV-1451 has strong affinity for neurofibrillary tau pathology but also exhibits off-target binding to neuromelanin, melanin and blood components. The robust off-target in vivo retention in basal ganglia and choroid plexus, in the absence of tau deposits, meningeal melanocytes or any other identifiable binding substrate by autoradiography in the PD case reported here, also suggests that the PET signal in those regions may be influenced, at least in part, by biological or technical factors that occur in vivo and are not captured by autoradiography.

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( www.BrainCellData.org ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed. Missing from these measurements, however, is the ability to routinely and easily spatially localise these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are 'tagged' with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 micron spatial resolution, and delivered whole-transcriptome data that was indistinguishable in quality from ordinary snRNA-seq. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil, and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualised receptor-ligand interactions driving B-cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to virtually any single-cell measurement technology. As proof of principle, we performed multiomic measurements of open chromatin, RNA, and T-cell receptor sequences in the same cells from metastatic melanoma. We identified spatially distinct tumour subpopulations to be differentially infiltrated by an expanded T-cell clone and undergoing cell state transition driven by spatially clustered accessible transcription factor motifs. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

Epithelial-mesenchymal transformation plays an important role in the disappearance of the midline line epithelial seam in rodent palate, leading to confluence of the palate. The aim of this study was to test the potential of the naturally cleft chicken palate to become confluent under the influence of growth factors, such as TGFbeta3, which are known to promote epithelial-mesenchymal transformation. After labeling medial edge epithelia with carboxyfluorescein, palatal shelves (E8-9) with or without beak were dissected and cultured on agar gels. TGFbeta1, TGFbeta2 or TGFbeta3 was added to the chemically defined medium. By 24 hours in culture, medial edge epithelia form adherent midline seams in all paired groups without intact beaks. After 72 hours, seams in the TGFbeta3 groups disappear and palates become confluent due to epithelial-mesenchymal transformation, while seams remain mainly epithelial in control, TGFbeta1 and TGFbeta2 groups. Epithelium-derived mesenchymal cells are identified by carboxyfluorescein fluorescence with confocal microscopy and by membrane-bound carboxyfluorescein isolation bodies with electron microscopy. Labeled fibroblasts completely replace the labeled epithelia of origin in TGFbeta3-treated palates without beaks. Single palates are unable to undergo transformation, and paired palatal shelves with intact beaks do not adhere or undergo transformation, even when treated with TGFbeta3. Thus, physical contact of medial edge epithelia and formation of the midline seam are necessary for epithelial-mesenchymal transformation to be triggered. We conclude that there may be no fundamental difference in developmental potential of the medial edge epithelium for transformation to mesenchyme among reptiles, birds and mammals. The bird differs from other amniotes in having developed a beak and associated craniofacial structures that seemingly keep palatal processes separated in vivo. Even control medial edge epithelia partly transform to mesenchyme if placed in close contact. However, exogenous TGFbeta3 is required to achieve complete confluence of the chicken palate.

With the growing aging population in Western countries, Alzheimer’s disease (AD) has become a major public health concern. No preventive measure and effective treatment for this burdensome disease is currently available. Genetic, biochemical, and neuropathological data strongly suggest that Aβ amyloidosis, which originates from the amyloidogenic processing of a metalloprotein-amyloid precursor protein (APP), is the key event in AD pathology. However, neurochemical factors that impact upon the age-dependent cerebral Aβ amyloidogenesis are not well recognized. Growing data indicate that cerebral dysregulation of biometals, environmental metal exposure, and oxidative stress contribute to AD pathology. Herein we provided further evidence that both metals (such as Cu) and H2O2 promote formation of neurotoxic Aβ oligomers. Moreover, we first demonstrated that laser capture microdissection coupled with X-ray fluorescence microscopy can be applied to determine elemental profiles (S, Fe, Cu, and Zn) in Aβ amyloid plaques. Clearly the fundamental biochemical mechanisms linking brain biometal metabolism, environmental metal exposure, and AD pathophysiology warrant further investigation. Nevertheless, the study of APP and Aβ metallobiology may identify potential targets for therapeutic intervention and/or provide diagnostic methods for AD.

Levels of brain-derived neurotrophic factor (BDNF) are reduced in specific brain regions in Alzheimer's disease (AD) and BDNF gene polymorphisms have been suggested to influence AD risk, hippocampal function, and memory. We investigated whether the polymorphisms at the BDNF 196 and 270 loci were associated with AD in a clinical and neuropathological cohort of 116 AD cases and 77 control subjects. To determine how BDNF protein levels relate to BDNF polymorphisms and AD pathology, we also measured BDNF in temporal association cortex, frontal association cortex, and cerebellum in 57 of the AD and 21 control cases. BDNF protein levels in temporal neocortex of the AD brains were reduced by 33% compared to control brains, whereas levels were unchanged in frontal and cerebellar cortex. The BDNF genotypes were not significantly associated with a diagnosis of AD, although the BDNF 270 C allele was slightly overrepresented among carriers of the APOEepsilon4 allele. Moreover, BDNF protein levels did not differ between the various BDNF genotypes and alleles. Neuropathologically, the loss of BDNF in AD showed a weak correlation with accumulation of neuritic amyloid plaques and loss of the neuronal/synaptic marker synaptophysin. The results suggest that the investigated BDNF polymorphisms are neither robust genetic risk factors nor determinants of BDNF protein levels in AD.

Independent component analysis (ICA) methods have received growing attention as effective data-mining tools for microarray gene expression data. As a technique of higher-order statistical analysis, ICA is capable of extracting biologically relevant gene expression features from microarray data. Herein we have reviewed the latest applications and the extended algorithms of ICA in gene clustering, classification, and identification. The theoretical frameworks of ICA have been described to further illustrate its feature extraction function in microarray data analysis.

Abstract Although the amyloid β-protein (Aβ) is believed to play an initiating role in Alzheimer’s disease (AD), the molecular characteristics of the key pathogenic Aβ forms are not well understood. As a result, it has proved difficult to identify optimal agents that target disease-relevant forms of Aβ. Here, we combined the use of Aβ-rich aqueous extracts of brain samples from AD patients as a source of human Aβ and live-cell imaging of iPSC-derived human neurons to develop a bioassay capable of quantifying the relative protective effects of multiple anti-Aβ antibodies. We report the characterization of 1C22, an aggregate-preferring murine anti-Aβ antibody, which better protects against forms of Aβ oligomers that are toxic to neurites than do the murine precursors of the clinical immunotherapeutics, bapineuzumab and solanezumab. These results suggest further examination of 1C22 is warranted, and that this bioassay maybe useful as a primary screen to identify yet more potent anti-Aβ therapeutics.

The cerebellum is a well-studied brain structure with diverse roles in motor learning, coordination, cognition, and autonomic regulation. Nonetheless, a complete inventory of cerebellar cell types is presently lacking. We used high-throughput transcriptional profiling to molecularly define cell types across individual lobules of the adult mouse cerebellum. Purkinje and granule neurons showed considerable regional specialization, with the greatest diversity occurring in the posterior lobules. For multiple types of cerebellar interneurons, the molecular variation within each type was more continuous, rather than discrete. For the unipolar brush cells (UBCs)—an interneuron population previously subdivided into two discrete populations—the continuous variation in gene expression was associated with a graded continuum of electrophysiological properties. Most surprisingly, we found that molecular layer interneurons (MLIs) were composed of two molecularly and functionally distinct types. Both show a continuum of morphological variation through the thickness of the molecular layer, but electrophysiological recordings revealed marked differences between the two types in spontaneous firing, excitability, and electrical coupling. Together, these findings provide the first comprehensive cellular atlas of the cerebellar cortex, and outline a methodological and conceptual framework for the integration of molecular, morphological, and physiological ontologies for defining brain cell types.

Abstract Single cell transcriptomics has transformed the characterization of brain cell identity by providing quantitative molecular signatures for large, unbiased samples of brain cell populations. With the proliferation of taxonomies based on individual datasets, a major challenge is to integrate and validate results toward defining biologically meaningful cell types. We used a battery of single-cell transcriptome and epigenome measurements generated by the BRAIN Initiative Cell Census Network (BICCN) to comprehensively assess the molecular signatures of cell types in the mouse primary motor cortex (MOp). We further developed computational and statistical methods to integrate these multimodal data and quantitatively validate the reproducibility of the cell types. The reference atlas, based on more than 600,000 high quality single-cell or -nucleus samples assayed by six molecular modalities, is a comprehensive molecular account of the diverse neuronal and non-neuronal cell types in MOp. Collectively, our study indicates that the mouse primary motor cortex contains over 55 neuronal cell types that are highly replicable across analysis methods, sequencing technologies, and modalities. We find many concordant multimodal markers for each cell type, as well as thousands of genes and gene regulatory elements with discrepant transcriptomic and epigenomic signatures. These data highlight the complex molecular regulation of brain cell types and will directly enable design of reagents to target specific MOp cell types for functional analysis.

ABSTRACT The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella -induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella , 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1α (IL-1α), IL-1β, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor β1 mRNA and prostaglandin E 2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.

Objective Individuals with Parkinson disease are more likely to develop melanoma, and melanoma patients are reciprocally at higher risk of developing Parkinson disease. Melanoma is strongly tied to red hair/fair skin, a phenotype of loss‐of‐function polymorphisms in the MC1R (melanocortin 1 receptor) gene. Loss‐of‐function variants of MC1R have also been linked to increased risk of Parkinson disease. The present study is to investigate the role of MC1R in dopaminergic neurons in vivo. Methods Genetic and pharmacological approaches were employed to manipulate MC1R, and nigrostriatal dopaminergic integrity was determined by comprehensive behavioral, neurochemical, and neuropathological measures. Results MC1R e/e mice, which carry an inactivating mutation of MC1R and mimic the human redhead phenotype, have compromised nigrostriatal dopaminergic neuronal integrity, and they are more susceptible to dopaminergic neuron toxins 6‐hydroxydopamine and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Furthermore, a selective MC1R agonist protects against MPTP‐induced dopaminergic neurotoxicity. Interpretation Our findings reveal a protective role of MC1R in the nigrostriatal dopaminergic system, and they provide a rationale for MC1R as a potential therapeutic target for Parkinson disease. Together with its established role in melanoma, MC1R may represent a common pathogenic pathway for melanoma and Parkinson disease. Ann Neurol 2017;81:395–406

Charting a biological atlas of an organ, such as the brain, requires us to spatially-resolve whole transcriptomes of single cells, and to relate such cellular features to the histological and anatomical scales. Single-cell and single-nucleus RNA-Seq (sc/snRNA-seq) can map cells comprehensively 5,6 , but relating those to their histological and anatomical positions in the context of an organ’s common coordinate framework remains a major challenge and barrier to the construction of a cell atlas 7–10 . Conversely, Spatial Transcriptomics allows for in-situ measurements 11–13 at the histological level, but at lower spatial resolution and with limited sensitivity. Targeted in situ technologies 1–3 solve both issues, but are limited in gene throughput which impedes profiling of the entire transcriptome. Finally, as samples are collected for profiling, their registration to anatomical atlases often require human supervision, which is a major obstacle to build pipelines at scale. Here, we demonstrate spatial mapping of cells, histology, and anatomy in the somatomotor area and the visual area of the healthy adult mouse brain. We devise Tangram, a method that aligns snRNA-seq data to various forms of spatial data collected from the same brain region, including MERFISH 1 , STARmap 2 , smFISH 3 , and Spatial Transcriptomics 4 (Visium), as well as histological images and public atlases. Tangram can map any type of sc/snRNA-seq data, including multi-modal data such as SHARE-seq data 5 , which we used to reveal spatial patterns of chromatin accessibility. We equipped Tangram with a deep learning computer vision pipeline, which allows for automatic identification of anatomical annotations on histological images of mouse brain. By doing so, Tangram reconstructs a genome-wide, anatomically-integrated, spatial map of the visual and somatomotor area with ∼30,000 genes at single-cell resolution, revealing spatial gene expression and chromatin accessibility patterning beyond current limitation of in-situ technologies.

Abstract Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed 1–6 . However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 μm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor–ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

Bipolar disorder (BD) is a common, recurring psychiatric illness with unknown pathogenesis. Recent studies suggest that microRNA (miRNA) levels in brains of BD patients are significantly altered, and these changes may offer insight into BD pathology or etiology. Previously, we observed significant alterations of miR‐29c levels in extracellular vesicles (EVs) extracted from prefrontal cortex (Brodmann area 9, BA9) of BD patients. In this study, we show that EVs extracted from the anterior cingulate cortex (BA24), a crucial area for modulating emotional expression and affect, have increased levels of miR‐149 in BD patients compared to controls. Because miR‐149 has been shown to inhibit glial proliferation, increased miR‐149 expression in BA24‐derived EVs is consistent with the previously reported reduced glial cell numbers in BA24 of patients diagnosed with either familial BD or familial major depressive disorder. qPCR analysis of laser‐microdissected neuronal and glial cells from BA24 cortical samples of BD patients verified that the glial, but not neuronal, population exhibits significantly increased miR‐149 expression. Finally, we report altered expression of both miR‐149 and miR‐29c in EVs extracted from brains of Flinders Sensitive Line rats, a well‐validated animal model exhibiting depressive‐like behaviors and glial (astrocytic) dysfunction. These findings warrant future investigations into the potential of using EV miRNA signatures as biomarkers to further enhance the biological definition of BD. © 2017 Wiley Periodicals, Inc.

Cellular perturbations underlying Alzheimer's disease are primarily studied in human postmortem samples and model organisms. Here we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of Alzheimer's disease pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the Early Cortical Amyloid Response-were prominent in neurons, wherein we identified a transient state of hyperactivity preceding loss of excitatory neurons, which correlated with the selective loss of layer 1 inhibitory neurons. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathological burden increased. Lastly, both oligodendrocytes and pyramidal neurons upregulated genes associated with amyloid beta production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

Living bone is a complex, three-dimensional composite material consisting of numerous cell types spatially organized within a mineralized extracellular matrix. To date, mechanistic investigation of the complex cellular level cross-talk between the major bone-forming cells involved in the response of bone to mechanical and biochemical stimuli has been hindered by the lack of a suitable in vitro model that captures the "coupled" nature of this response. Using a novel rotational co-culture approach, we have generated large (>4mm diameter), three-dimensional mineralized tissue constructs from a mixture of normal human primary osteoblast and osteoclast precursor cells without the need for any exogenous osteoconductive scaffolding material that might interfere with such cell-cell interactions. Mature, differentiated bone constructs consist of an outer region inhabited by osteoclasts and osteoblasts and a central region containing osteocytes encased in a self-assembled, porous mineralized extracellular matrix. Bone constructs exhibit morphological, mineral and biochemical features similar to remodeling human trabecular bone, including the expression of mRNA for SOST, BGLAP, ACP5, BMP-2, BMP-4 and BMP-7 within the construct and the secretion of BMP-2 protein into the medium. This "coupled" model of bone formation will allow the future investigation of various stimuli on the process of normal bone formation/remodeling as it relates to the cellular function of osteoblasts, osteoclasts and osteocytes in the generation of human mineralized tissue.

Summary Defining cell types requires integrating diverse measurements from multiple experiments and biological contexts. Recent technological developments in single-cell analysis have enabled high-throughput profiling of gene expression, epigenetic regulation, and spatial relationships amongst cells in complex tissues, but computational approaches that deliver a sensitive and specific joint analysis of these datasets are lacking. We developed LIGER, an algorithm that delineates shared and dataset-specific features of cell identity, allowing flexible modeling of highly heterogeneous single-cell datasets. We demonstrated its broad utility by applying it to four diverse and challenging analyses of human and mouse brain cells. First, we defined both cell-type-specific and sexually dimorphic gene expression in the mouse bed nucleus of the stria terminalis, an anatomically complex brain region that plays important roles in sex-specific behaviors. Second, we analyzed gene expression in the substantia nigra of seven postmortem human subjects, comparing cell states in specific donors, and relating cell types to those in the mouse. Third, we jointly leveraged in situ gene expression and scRNA-seq data to spatially locate fine subtypes of cells present in the mouse frontal cortex. Finally, we integrated mouse cortical scRNA-seq profiles with single-cell DNA methylation signatures, revealing mechanisms of cell-type-specific gene regulation. Integrative analyses using the LIGER algorithm promise to accelerate single-cell investigations of cell-type definition, gene regulation, and disease states.

Important and precisely regulated transitions in tissue phenotype from epithelium to mesenchyme and from mesenchyme to epithelium occur in the developing embryo. The gene for E-cadherin has been shown to cause fibroblastic cell lines to become epithelioid in culture. We asked whether or not the activities of the E-cadherin gene could cause a definitive embryonic mesenchyme to transdifferentiate into an epithelial phenotype. Primary corneal fibroblasts from 6- to 7-day-old chick embryos were contransfected by impact loading with plasmids containing E-cadherin and Neo genes and selected in G418. The fibroblasts expressing E-cadherin aggregate, localize E-cadherin to lateral surfaces, and form stratified epithelia that develop zonulae occludentes and adherentes, connexin 43, cytokeratin, desmoplakin, and desmosomes. Vimentin intermediate filaments persist and no basement membranes appear, even though the cells synthesize laminin and type IV collagen. Our study is the first to demonstrate the ability of E-cadherin to induce fibroblasts to form desmosomes and stratified epithelia. The primary embryonic fibroblasts apparently have more developmental potential to transdifferentiate into epithelia than do the fibroblastic cell lines previously studied. We conclude that E-cadherin is likely to play an important role in transformation of mesenchyme to epithelium in the embryo.

Brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB) may influence brain reserve, the ability of the brain to tolerate pathological changes without significant decline in function. Here, we explore whether a specifically vulnerable population of human neurons shows a compensatory response to the neuropathological changes of Alzheimer disease (AD) and whether that response depends on an upregulation of the BDNF pathway. We observed increased neuronal TrkB expression associated with early-stage AD pathology (Braak and Braak stages I-II) in hippocampal CA1 region samples from cognitively intact Framingham Heart Study subjects (n = 5) when compared with cognitively intact individuals with no neurofibrillary tangles (n = 4). Because BDNF/TrkB signaling affects memory formation and retention through modification of the actin cytoskeleton, we examined the expression of actin capping protein β2 (Capzb2), a marker of actin cytoskeleton reorganization. Capzb2 expression was also significantly increased in CA1 hippocampal neurons of cognitively intact subjects with early-stage AD pathology. Our data suggest that increased expression of TrkB and Capzb2 accompanies adequate brain reserve in the initial stages of AD pathology. In subsequent stages of AD, the higher levels of TrkB and Capzb2 expression achieved may not be sufficient to prevent cognitive decline.

Extensive epidemiological data have demonstrated an exponential rise in the incidence of non-Hodgkin lymphoma (NHL) that is associated with increasing age. The molecular etiology of this remains largely unknown, which impacts the effectiveness of treatment for patients. We proposed that age-dependent circulating microRNA (miRNA) signatures in the host influence diffuse large B cell lymphoma (DLBCL) development. Our objective was to examine tumor development in an age-based DLBCL system using an inventive systems biology approach. We harnessed a novel murine model of spontaneous DLBCL initiation (Smurf2-deficient) at two age groups: 3 and 15 months old. All Smurf2-deficient mice develop visible DLBCL tumor starting at 15 months of age. Total miRNA was isolated from serum, bone marrow and spleen and were collected for all age groups for Smurf2-deficient mice and age-matched wild-type C57BL/6 mice. Using systems biology techniques, we identified a list of 10 circulating miRNAs being regulated in both the spleen and bone marrow that were present in DLBCL forming mice starting at 3 months of age that were not present in the control mice. Furthermore, this miRNA signature was found to occur circulating in the blood and it strongly impacted JUN and MYC oncogenic signaling. In addition, quantification of the miRNA signature was performed via Droplet Digital PCR technology. It was discovered that a key miRNA signature circulates throughout a host prior to the formation of a tumor starting at 3 months old, which becomes further modulated by age and yielded calculation of a 'carcinogenic risk score'. This novel age-based circulating miRNA signature may potentially be leveraged as a DLBCL risk profile at a young age to predict future lymphoma development or disease progression as well as for potential innovative miRNA-based targeted therapeutic strategies in lymphoma.

Potential molecular alterations based on age and sex are not well defined in diffuse large B-cell lymphoma (DLBCL). We examined global transcriptome DLBCL data from The Cancer Genome Atlas (TCGA) via a systems biology approach to determine the molecular differences associated with age and sex. Collectively, sex and age revealed striking transcriptional differences with older age associated with decreased metabolism and telomere functions and female sex was associated with decreased interferon signaling, transcription, cell cycle, and PD-1 signaling. We discovered that the key genes for most groups strongly regulated immune function activity. Furthermore, older females were predicted to have less DLBCL progression versus older males and young females. Finally, analyses in systems biology revealed that JUN and CYCS signaling were the most critical factors associated with tumor progression in older and male patients. We identified important molecular perturbations in DLBCL that were strongly associated with age and sex and were predicted to strongly influence tumor progression.

Abstract Synaptic dysfunction and loss are core pathological features in Alzheimer disease ( AD ). In the vicinity of amyloid‐β plaques in animal models, synaptic toxicity occurs and is associated with chronic activation of the phosphatase calcineurin ( CN ). Indeed, pharmacological inhibition of CN blocks amyloid‐β synaptotoxicity. We therefore hypothesized that CN ‐mediated transcriptional changes may contribute to AD neuropathology and tested this by examining the impact of CN over‐expression on neuronal gene expression in vivo . We found dramatic transcriptional down‐regulation, especially of synaptic mRNA s, in neurons chronically exposed to CN activation. Importantly, the transcriptional profile parallels the changes in human AD tissue. Bioinformatics analyses suggest that both nuclear factor of activated T cells and numerous micro RNA s may all be impacted by CN , and parallel findings are observed in AD . These data and analyses support the hypothesis that at least part of the synaptic failure characterizing AD may result from aberrant CN activation leading to down‐regulation of synaptic genes, potentially via activation of specific transcription factors and expression of repressive micro RNA s. Open science badges This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/ . image Read the Editorial Highlight for this article on page 8 .

Pancreatic ductal adenocarcinoma (PDAC) lethality is multifactorial; although studies have identified transcriptional and genetic subsets of tumors with different prognostic significance, there is limited understanding of features associated with the minority of patients who have durable remission after surgical resection. In this study, we performed laser capture microdissection (LCM) of PDAC samples to define their cancer- and stroma-specific molecular subtypes and identify a prognostic gene expression signature for short-term and long-term survival.LCM and RNA sequencing (RNA-seq) analysis of cancer and adjacent stroma of 19 treatment-naïve PDAC tumors was performed. Gene expression signatures were tested for their robustness in a large independent validation set. An RNA-ISH assay with pooled probes for genes associated with disease-free survival (DFS) was developed to probe 111 PDAC tumor samples.Gene expression profiling identified four subtypes of cancer cells (C1-C4) and three subtypes of cancer-adjacent stroma (S1-S3). These stroma-specific subtypes were associated with DFS (P = 5.55E-07), with S1 associated with better prognoses when paired with C1 and C2. Thirteen genes were found to be predominantly expressed in cancer cells and corresponded with DFS in a validation using existing RNA-seq datasets. A second validation on an independent cohort of patients using RNA-ISH probes to six of these prognostic genes demonstrated significant association with overall survival (median 17 vs. 25 months; P < 0.02).Our results identified specific signatures from the epithelial and the stroma components of PDAC, which add clarity to the nature of PDAC molecular subtypes and may help predict survival.

Clustering is the grouping of similar objects into a class. Local clustering feature refers to the phenomenon whereby one group of data is separated from another, and the data from these different groups are clustered locally. A compact class is defined as one cluster in which all similar elements cluster tightly within the cluster. Herein, the essence of the local clustering feature, revealed by mathematical manipulation, results in a novel clustering algorithm termed as the special local clustering (SLC) algorithm that was used to process gene microarray data related to Alzheimer's disease (AD). SLC algorithm was able to group together genes with similar expression patterns and identify significantly varied gene expression values as isolated points. If a gene belongs to a compact class in control data and appears as an isolated point in incipient, moderate and/or severe AD gene microarray data, this gene is possibly associated with AD. Application of a clustering algorithm in disease-associated gene identification such as in AD is rarely reported.

During the progression of Alzheimer's disease (AD), hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB) III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF) receptor tyrosine kinase B (TrkB), mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.

Metallosis is the accumulation of metallic debris in soft tissues resulting from wear following total joint replacement. A dog was evaluated for lameness 4 years after total hip arthroplasty using a titanium alloy and cobalt chromium total hip system. Radiographs revealed severe acetabular component wear, implant-bone interface deterioration, and peri-acetabular osteolysis. During surgical revision, black periarticular tissue surrounded the implants. Histologically, there was fibrosis and granulomatous inflammation with abundant, intra- and extracellular, black, granular material and smaller amounts of clear punctate to acicular material. Laser capture microdissection followed by x-ray fluorescence microscopy indicated the material contained large amounts of titanium with smaller amounts of vanadium, cobalt, and chromium, confirming the diagnosis of metallosis. The clear material was birefringent under cross-polarized light, stained positive with Oil-Red-O, and thus was consistent with polyethylene. Metallosis exhibits characteristic gross and histologic lesions and is a differential diagnosis for aseptic loosening of hip implants.

Summary MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 positive patients, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we have developed a novel miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters and may potentially inhibit a COVID-19 disease state in humans.

Abstract Midbrain dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) project widely throughout the central nervous system, playing critical roles in voluntary movements, reward processing, and working memory. Many of these neurons are highly sensitive to neurodegeneration in Parkinson’s Disease (PD), and their loss correlates strongly with the pathognomonic symptoms. To characterize these populations molecularly, we developed a protocol to enrich and transcriptionally profile DA neuron nuclei from postmortem human SNpc of both PD patients and matched controls. We identified a total of ten distinct populations, including one that was primate-specific. A single subtype, marked by the gene AGTR1 , was highly susceptible to degeneration, and was enriched for expression of genes associated with PD in genetic studies, suggesting many risk loci act within this subtype to influence its neurodegeneration. The AGTR1 subtype also showed the strongest upregulation of TP53 and its downstream targets, nominating a potential pathway of degeneration in vivo . The transcriptional characterization of differentially disease-vulnerable DA neurons in the SNpc will inform the development of laboratory models, enable the nomination of novel disease biomarkers, and guide further studies of pathogenic disease mechanisms.

Chordoma is a rare malignant tumor demonstrating notochordal differentiation. It is dependent on brachyury (TBXT), a hallmark notochordal gene and transcription factor, and shares histologic features and the same anatomic location as the notochord. This study involved a molecular comparison of chordoma and notochord to identify dysregulated cellular pathways. The lack of a molecular reference from appropriate control tissue limits our understanding of chordoma and its relationship to notochord. Therefore, an unbiased comparison of chordoma, human notochord, and an atlas of normal and cancerous tissue was conducted using gene expression profiling to clarify the chordoma/notochord relationship and potentially identify novel drug targets. The study found striking consistency in gene expression profiles between chordoma and notochord, supporting the hypothesis that chordoma develops from notochordal remnants. A 12-gene diagnostic chordoma signature was identified and the TBXT/transforming growth factor beta (TGF-β)/SOX6/SOX9 pathway was hyperactivated in the tumor, suggesting that pathways associated with chondrogenesis were a central driver of chordoma development. Experimental validation in chordoma cells confirmed these findings and emphasized the dependence of chordoma proliferation and survival on TGF-β. The computational and experimental evidence provided the first molecular connection between notochord and chordoma and identified core members of a chordoma regulatory pathway involving TBXT. This pathway provides new therapeutic targets for this unique malignant neoplasm and highlights TGF-β as a prime druggable candidate. Chordoma is a rare malignant tumor demonstrating notochordal differentiation. It is dependent on brachyury (TBXT), a hallmark notochordal gene and transcription factor, and shares histologic features and the same anatomic location as the notochord. This study involved a molecular comparison of chordoma and notochord to identify dysregulated cellular pathways. The lack of a molecular reference from appropriate control tissue limits our understanding of chordoma and its relationship to notochord. Therefore, an unbiased comparison of chordoma, human notochord, and an atlas of normal and cancerous tissue was conducted using gene expression profiling to clarify the chordoma/notochord relationship and potentially identify novel drug targets. The study found striking consistency in gene expression profiles between chordoma and notochord, supporting the hypothesis that chordoma develops from notochordal remnants. A 12-gene diagnostic chordoma signature was identified and the TBXT/transforming growth factor beta (TGF-β)/SOX6/SOX9 pathway was hyperactivated in the tumor, suggesting that pathways associated with chondrogenesis were a central driver of chordoma development. Experimental validation in chordoma cells confirmed these findings and emphasized the dependence of chordoma proliferation and survival on TGF-β. The computational and experimental evidence provided the first molecular connection between notochord and chordoma and identified core members of a chordoma regulatory pathway involving TBXT. This pathway provides new therapeutic targets for this unique malignant neoplasm and highlights TGF-β as a prime druggable candidate. Chordoma is an indolent malignant tumor of the axial skeleton. It is uncommon and accounts for <4% of all primary bone tumors.1Scott D. Pedlow F. Hecht A. Hornicek F. Chapter 11: Tumors: primary benign and malignant extradural spine tumors.in: The Adult and Pediatric Spine. ed 3. Lippincott Williams & Wilkins, Philadelphia, PA2004: 191-246Google Scholar The yearly incidence of chondroma is 0.5 cases per million in the United States.1Scott D. Pedlow F. Hecht A. Hornicek F. Chapter 11: Tumors: primary benign and malignant extradural spine tumors.in: The Adult and Pediatric Spine. ed 3. Lippincott Williams & Wilkins, Philadelphia, PA2004: 191-246Google Scholar It usually arises in adults aged >50 years, although occasional pediatric cases are observed.1Scott D. Pedlow F. Hecht A. Hornicek F. Chapter 11: Tumors: primary benign and malignant extradural spine tumors.in: The Adult and Pediatric Spine. ed 3. Lippincott Williams & Wilkins, Philadelphia, PA2004: 191-246Google Scholar Chordoma typically develops in the axial skeleton, and approximately 50% of cases arise in the sacrum.1Scott D. Pedlow F. Hecht A. Hornicek F. Chapter 11: Tumors: primary benign and malignant extradural spine tumors.in: The Adult and Pediatric Spine. ed 3. Lippincott Williams & Wilkins, Philadelphia, PA2004: 191-246Google Scholar The 5-year survival rate of patients with chordoma is 67%, and the median survival time is 6.3 years.2McMaster M.L. Goldstein A.M. Bromley C.M. Ishibe N. Parry D.M. Chordoma: incidence and survival patterns in the United States, 1973-1995.Cancer Causes Control. 2001; 12: 1-11Crossref PubMed Scopus (744) Google Scholar Surgery is the primary treatment option, with radiotherapy used as an adjuvant.3DeLaney T.F. Liebsch N.J. Pedlow F.X. Adams J. Dean S. Yeap B.Y. McManus P. Rosenberg A.E. Nielsen G.P. Harmon D.C. Spiro I.J. Raskin K.A. Suit H.D. Yoon S.S. Hornicek F.J. Phase II study of high-dose photon/proton radiotherapy in the management of spine sarcomas.Int J Radiat Oncol Biol Phys. 2009; 74: 732-739Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar Clinical trials using cytotoxic chemotherapy have shown little benefit for the treatment of chordoma; however, initial tests of targeted therapies have shown some promising results in small cohorts of patients.4Hofheinz R.D. Kubicka S. Wollert J. Arnold D. Hochhaus A. Gefitinib in combination with 5-fluorouracil (5-FU)/folinic acid and irinotecan in patients with 5-FU/oxaliplatin-refractory colorectal cancer: a phase I/II study of the Arbeitsgemeinschaft fur Internistische Onkologie (AIO).Onkologie. 2006; 29: 563-567Crossref PubMed Scopus (16) Google Scholar, 5Stacchiotti S. Longhi A. Ferraresi V. Grignani G. Comandone A. Stupp R. Bertuzzi A. Tamborini E. Pilotti S. Messina A. Spreafico C. Gronchi A. Amore P. Vinaccia V. Casali P.G. Phase II study of imatinib in advanced chordoma.J Clin Oncol. 2012; 30: 914-920Crossref PubMed Scopus (200) Google Scholar, 6Meng T. Jin J. Jiang C. Huang R. Yin H. Song D. Cheng L. Molecular targeted therapy in the treatment of chordoma: a systematic review.Front Oncol. 2019; 9: 30Crossref PubMed Scopus (62) Google Scholar The molecular mechanisms underlying chordoma are poorly understood; therefore, clinical trials based on genetic mechanisms are limited. The current study attempted to expand our understanding of genetically driven cellular pathways to provide new therapeutic targets to explore. Current hypotheses about chordoma development rely mostly on histologic and immunohistochemical studies that show similarities between chordoma and embryonic human notochord. During higher vertebrate development, most fetal notochordal cells regress during embryogenesis; however, remnants within the adult vertebral disk and bone can occur. Benign notochordal cell tumors are relatively common lesions in the adult vertebral bodies, and their etiology is unclear.7Picci P. Vanel D. Alberghini M. Mirra J.M. Errani C. Staals E.L. Mercuri M. Giant notochordal rests misdiagnosed and treated as chordomas: a retrospective clinical, radiological and histologic study of four cases.JCO. 2008; 26: 21503Crossref Google Scholar,8Yamaguchi T. Iwata J. Sugihara S. McCarthy E.F. Karita M. Murakami H. Kawahara N. Tsuchiya H. Tomita K. Distinguishing benign notochordal cell tumors from vertebral chordoma.Skeletal Radiol. 2008; 37: 291-299Crossref PubMed Scopus (87) Google Scholar In some cases of chordoma, benign notochordal cell tumors have been found within the affected vertebral body,9Arain A. Hornicek F.J. Schwab J.H. Chebib I. Damron T.A. Chordoma arising from benign multifocal notochordal tumors.Skeletal Radiol. 2017; 46: 1745-1752Crossref PubMed Scopus (16) Google Scholar suggesting that benign notochordal cell tumors may be a precursor lesion. Limited molecular examinations support the connection between chordoma and embryonic notochord vestiges. The transcription factor brachyury (TBXT), encoded by the gene TBXT (previously named T), has been previously identified as being important in the development of the normal notochord10Kispert A. Koschorz B. Herrmann B.G. The T protein encoded by Brachyury is a tissue-specific transcription factor.EMBO J. 1995; 14: 4763-4772Crossref PubMed Scopus (259) Google Scholar and appears essential for chordoma survival.11Presneau N. Shalaby A. Ye H. Pillay N. Halai D. Idowu B. Tirabosco R. Whitwell D. Jacques T.S. Kindblom L.-G. Brüderlein S. Möller P. Leithner A. Liegl B. Amary F.M. Athanasou N.N. Hogendoorn P.C. Mertens F. Szuhai K. Flanagan A.M. Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study.J Pathol. 2010; 223: 327-335Crossref PubMed Scopus (155) Google Scholar The TBXT gene has been found mutated in some chordomas, and copy number variants have been identified in familial cases.12Yang X.R. Ng D. Alcorta D.A. Liebsch N.J. Sheridan E. Li S. Goldstein A.M. Parry D.M. Kelley M.J. T (brachyury) gene duplication confers major susceptibility to familial chordoma.Nat Genet. 2009; 41: 1176-1178Crossref PubMed Scopus (228) Google Scholar,13Pillay N. Plagnol V. Tarpey P.S. Lobo S.B. Presneau N. Szuhai K. Halai D. Berisha F. Cannon S.R. Mead S. Kasperaviciute D. Palmen J. Talmud P.J. Kindblom L.G. Amary M.F. Tirabosco R. Flanagan A.M. A common single-nucleotide variant in T is strongly associated with chordoma.Nat Genet. 2012; 44: 1185-1187Crossref PubMed Scopus (97) Google Scholar Chordoma cell lines have been shown to depend on TBXT, where its decreased expression leads to cell cycle arrest.11Presneau N. Shalaby A. Ye H. Pillay N. Halai D. Idowu B. Tirabosco R. Whitwell D. Jacques T.S. Kindblom L.-G. Brüderlein S. Möller P. Leithner A. Liegl B. Amary F.M. Athanasou N.N. Hogendoorn P.C. Mertens F. Szuhai K. Flanagan A.M. Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study.J Pathol. 2010; 223: 327-335Crossref PubMed Scopus (155) Google Scholar However, the mechanism that brachyury plays in the context of the disease requires further investigation. TBXT is required for most chordoma tumors to survive, yet it does not have a role in most normal adult tissues, making it an ideal drug target. However, brachyury is a transcription factor, a challenging category of molecules.14Chen A. Koehler A.N. Transcription factor inhibition: lessons learned and emerging targets.Trends Mol Med. 2020; 26: 508-518Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Currently, there are no US Food and Drug Administration–approved drugs targeting a transcription factor, but it is an active area of research.15Henley M.J. Koehler A.N. Advances in targeting “undruggable” transcription factors with small molecules.Nat Rev Drug Discov. 2021; 20: 669-688Crossref PubMed Scopus (94) Google Scholar A better understanding of the molecular mechanisms active in chordoma pathogenesis is necessary to provide additional targets for therapeutic development. Standard molecular profiling approaches require a control sample for comparison against a disease state, and they generally highlight differences but not similarities between the samples. Chordoma is problematic as its normal tissue counterpart is the notochord, which is only present in the human during the early stages of embryogenesis. To overcome these challenges, gene expression profiling of chordoma and human notochord was conducted and compared with many non-chordoma tissues to identify the best control tissue and find key pathways associated with tumor pathogenesis. We identified a chordoma diagnostic gene signature, overactive pathways within chordoma cells, and a core gene interaction network required for chordoma survival and proliferation. Finally, shRNA knockdowns and chemical inhibitor experiments support the relevance of the gene network and highlight the importance of transforming growth factor beta (TGF-β) in chordoma cell survival. We conclude that the pathways related to chondrogenesis are a vital driver of chordoma progression and a promising candidate for therapeutic disruption. Our findings indicate that TGF-β is central to chordoma and is an important druggable target. This study used discarded tumor tissues from patients with chordoma as well as notochord cells from discarded human embryonic tissues. The Institutional Review Board at Massachusetts General Hospital (Boston, MA) approved the chordoma study protocol (2009A052093) and the notochord study protocol (2007P-002239). As both protocols used discarded material, the Institutional Review Board waived the need for written informed consent. Cell lines U-CH1, U-CH2, U-CH12, HEK293, and 293T were purchased from ATCC (Manassas, VA), and CH22 cells were provided by F.J.H. Cells were cultured in a biosafety level 2 environment according to ATCC guidelines. Fresh chordoma specimens from five patients were obtained immediately on surgical resection. The specimens were low in cellularity with abundant extracellular matrix, presenting difficulties in capturing high-quality and adequate amounts of RNA. Tumor samples were cut into 0.5- to 1-cm–diameter pieces. Each sample was either stored in 10 mL of RNAlater (Qiagen, Germantown, MD) or frozen immediately in liquid nitrogen. Homogenization and RNA extraction were performed in the same step. RNA was extracted by placing each tissue sample in 4 mL of Trizol along with 25% (by volume) silicon carbide beads (1 mm; BioSpec, Bartlesville, OK). The tube was placed in a bead beater (BioSpec Mini-Beadbeater-96) and shaken vigorously for 1 to 5 minutes in 30-second intervals. The duration of the homogenization was determined by examining the solution for the disappearance of large masses of tissue. It was not uncommon for tumor samples to contain bone fragments embedded within the tumor mass. In such cases, the tissue was homogenized until only the bone fragments remained intact. The Trizol/tissue homogenate was then transferred to a fresh tube, to which 5 mL of Trizol was added. The tubes were centrifuged for 5 minutes at 12,000 × g to pellet the debris and silicon carbide dust. The homogenate was then transferred to a fresh tube, and 1.6 mL of chloroform was added to the extracted aqueous phase. To precipitate the RNA, 4 mL of isopropanol was immediately added. The pellet was washed in 80% ethanol, dried, and dissolved in 100 μL of water. In cases in which there was still significant visible insoluble material (protein), additional RNA isolation with Trizol was performed on the purified RNA. The RNA solution was then further purified using an RNeasy silica column (Qiagen), according to the manufacturer's protocol. The RNA quality was assessed with a Bioanalyzer RNA Pico kit (Agilent, Santa Clara, CA), and mRNA was purified from samples that passed the quality control step. Human embryonic notochord specimens were obtained from discarded tissues. To obtain sufficient high-quality mRNA, it was necessary to laser microdissect the cellular vestige of the notochord from the fetal spine. Fetal spinal columns were grossly dissected and frozen to −20°C. The specimens were then mounted on a cryostat chuck using M-1 embedding matrix (ThermoFisher, Waltham, MA) and sectioned every 25 μm until the central region of the spine was approximated. At this point, the cryomicrotomy blades were changed to a new blade, and 12-μm thin cryosections were mounted on microscopy slides (Gold Seal Rite-On Micro Slides; VWR, Radnor, PA) and immediately processed for laser capture microdissection. For the cytoarchitectural visualization of the notochord cells, each tissue section was fixed in 70% ethanol for 30 seconds. The sections were then rinsed with RNase-free distilled water and incubated in HistoGene staining solution (Arcturus; MDS Analytical Technologies, Sunnyvale, CA) for 1 minute, followed by dehydration in increasingly concentrated ethanol (75% to 100%) into xylene and subsequent laser capture microdissection. All incubations and washes were performed at room temperature. Cells representative of the degenerating notochord were clearly visible residing in central lacunae of the nucleus pulposus, and approximately 2000 of these notochord cells from each fetus were captured onto separate polyethylene collecting caps (Macro Cap, Arcturus; MDS Analytical Technologies). RNA isolation was performed using the PicoPure RNA isolation system (Arcturus; MDS Analytical Technologies). Plastic laser capture microdissection collecting caps were incubated at 42°C for 30 minutes in 20 μL of the extraction buffer containing guanidinium thiocyanate, centrifuged briefly to collect the extracted solution, and then frozen at −80°C. Genomic DNA was removed via RNase-free DNase (Qiagen) digestion on the columns. Total cellular RNA from each column was eluted in a two-step process with 6 μL per step for a total of 12 μL of elution buffer. Isolated RNA was then stored at −80°C until further analysis. The quality of the RNA preparations at various stages was measured using an RNA 6000 Pico chip (Agilent). Given the generally low RNA yield from the chordoma samples, the mRNA was amplified to produce cDNA in sufficient quantity for both microarray and RNA sequencing (RNA-Seq). Single-primer isothermal amplification (Nugen, San Carlos, CA) was used to linearly amplify the purified RNA. For the initial amplification, an Ovation Pico WTA kit (Nugen) was used. Samples destined for microarray were further processed with the Encore Biotin Module (Tecan, Männedorf, Switzerland). RNA-Seq samples were also processed with the WT-Ovation Exon Module (Nugen) to synthesize the cDNA second strand. High-throughput sequencing libraries for the GAII Illumina platform were constructed using an Illumina kit (Illumina, San Diego, CA). An RNA Amplification System V2 (Ovation) was also used, which includes a complete solution and was not available at the start of the project. In all cases, the manufacturer's directions were followed. Primary normal tissue and cell type expression files were obtained from Gene Expression Omnibus16Barrett T. Suzek T.O. Troup D.B. Wilhite S.E. Ngau W.C. Ledoux P. Rudnev D. Lash A.E. Fujibuchi W. Edgar R. NCBI GEO: mining millions of expression profiles--database and tools.Nucleic Acids Res. 2005; 33: D562-D566Crossref PubMed Scopus (878) Google Scholar and ArrayExpress.17Parkinson H. Kapushesky M. Shojatalab M. Abeygunawardena N. Coulson R. Farne A. Holloway E. Kolesnykov N. Lilja P. Lukk M. Mani R. Rayner T. Sharma A. William E. Sarkans U. Brazma A. ArrayExpress--a public database of microarray experiments and gene expression profiles.Nucleic Acids Res. 2007; 35: D747-D750Crossref PubMed Scopus (514) Google Scholar All samples obtained for analysis were profiled on the Affymetrix U133plus2 platform (ThermoFisher). The authors used version 14 of the custom transcript definition files provided by Brainarray.18Dai M. Wang P. Boyd A.D. Kostov G. Athey B. Jones E.G. Bunney W.E. Myers R.M. Speed T.P. Akil H. Watson S.J. Meng F. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data.Nucleic Acids Res. 2005; 33: e175Crossref PubMed Scopus (1415) Google Scholar These files redefine Affymetrix probes by remapping individual probes to the human genome and adjusting them to the most up-to-date annotation. The data files were then normalized using the GCRMA module of the Bioconductor software library version 2.22.0 (https://www.bioconductor.org/packages/release/bioc/html/gcrma.html), and present/absent calls were calculated for each probe using the MAS5 module.19Gautier L. Cope L. Bolstad B.M. Irizarry R.A. affy—analysis of Affymetrix GeneChip data at the probe level.Bioinformatics. 2004; 20: 307-315Crossref PubMed Scopus (3995) Google Scholar All probes with no present calls were removed and, from the remaining probes, at least one sample was required to have an expression value larger than log2(100). Tumor expression profiles were obtained from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse2109; accession number GSE2109), and cell line profiles were obtained from the Cancer Cell Line Encyclopedia.20Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. et al.The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (5132) Google Scholar The gene enrichment algorithm has previously been described21Benita Y. Cao Z. Giallourakis C. Li C. Gardet A. Xavier R.J. Gene enrichment profiles reveal T-cell development, differentiation, and lineage-specific transcription factors including ZBTB25 as a novel NF-AT repressor.Blood. 2010; 115: 5376-5384Crossref PubMed Scopus (104) Google Scholar (source code can be found at (http://xavierlab2.mgh.harvard.edu/chordoma/code.html, last accessed November 7, 2022). Briefly, all normal primary cell types and tissues contained replicates, and each such group was compared with each of the other normal tissues using the Limma module of Bioconductor version 3.6.9.22Smyth G.K. Michaud J. Scott H.S. Use of within-array replicate spots for assessing differential expression in microarray experiments.Bioinformatics. 2005; 21: 2067-2075Crossref PubMed Scopus (1080) Google Scholar Limma uses linear models and Bayes methods to assess differential expression. For each group, a linear model coefficient was obtained, which is a measure of differences between two cell types. The enrichment score for each probe was defined as the sum of all statistically significant (Bonferroni-adjusted P < 0.05) coefficients. In this study, the authors extended this method further by calculating the gene enrichment in cell lines and tumors. To avoid a bias due to the large number of tumors and cell lines, each individual sample was compared with all normal cell types. Thus, the enrichment value in each tumor or cell line sample reflects enrichment with respect to a body atlas of all normal cell types. To assemble BA0, the authors identified and downloaded 1499 publicly available Affymetrix gene expression microarray data files representing 127 normal human cell types and tissues. Next, the authors filtered and normalized the chordoma and notochord gene expression microarrays together with those of the assembled body atlas as a single data set (BA0). To identify enriched genes, the authors compared chordoma samples pairwise with each primary cell type using a linear model. This comparison yielded 126 linear models, each with a coefficient and an associated P value for each gene. The coefficient is a measure of differences between two samples, with larger coefficient values associated with larger differences. In each comparison, the enrichment score for each gene was defined as the sum of all individual coefficients with an adjusted P < 0.05. Thus, a gene that was highly expressed in one cell type would produce a high enrichment score, as a result of its 126 large and statistically significant coefficient values. This score is comparable between genes within a sample, enabling ranking to identify the specific genes that define chordoma or notochord. The authors obtained 2158 tumor profiles [Expression Project for Oncology (ExpO) data set] and 810 cell lines from the public domain and normalized and processed all samples (including BA0) as a single data set containing 4475 microarrays (BA1). The authors derived enrichment scores for each tumor and cell line by comparing it with all normal cell types, as described above, one sample at a time. The authors used two parameters to identify genes that differentiate chordoma from other tumors. First, the authors identified genes enriched within each chordoma sample with a z-score ≥5; this stringent cutoff was used to increase specificity. Next, using the normalized gene expression data, the authors calculated a z-score for each gene across all the tumor samples (expression z-score). The authors deemed genes with a chordoma enrichment z-score ≥5, as well as a chordoma expression z-score ≥5, to be both specific and highly expressed in chordoma. Next-generation sequencing data were first mapped to ribosomal RNA (12s, 16s, 18s, 28s, and 5.8s) using Bowtie version 0.12.7.23Langmead B. Trapnell C. Pop M. Salzberg S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biol. 2009; 10: R25Crossref PubMed Scopus (15598) Google Scholar All matching reads were discarded. The human genome sequence version HG19 and associated gene models (knownGenes) were obtained from the University of California, Santa Cruz, genome browser.24Karolchik D. Kuhn R.M. Baertsch R. Barber G.P. Clawson H. Diekhans M. Giardine B. Harte R.A. Hinrichs A.S. Hsu F. Kober K.M. Miller W. Pedersen J.S. Pohl A. Raney B.J. Rhead B. Rosenbloom K.R. Smith K.E. Stanke M. Thakkapallayil A. Trumbower H. Wang T. Zweig A.S. Haussler D. Kent W.J. The UCSC genome browser database: 2008 update.Nucleic Acids Res. 2008; 36: D773-D779Crossref PubMed Scopus (429) Google Scholar Short reads were mapped to the genome using TopHat version 1.4.1.25Trapnell C. Pachter L. Salzberg S.L. TopHat: discovering splice junctions with RNA-Seq.Bioinformatics. 2009; 25: 1105-1111Crossref PubMed Scopus (9225) Google Scholar Only reads mapping at least partially to defined exons were retained and were aggregated for a single gene locus. The number of reads per sample was normalized to reads per million; and for each gene, the expression value was calculated as the normalized number of reads/observed gene length. The observed length of a gene was defined as the number of bases detected by sequencing. This adjustment was necessary because the Nugen kit applied to the samples was based on nonrandom probes and resulted in inconsistent coverage of genes with typical 3′-end enrichment (Supplemental Figure S1 provides a representative chordoma/notochord gene coverage plot). rRNA-depleted data, as well as count-based expression profiles generated using STAR version 2.7.10a,26Dobin A. Davis C.A. Schlesinger F. Drenkow J. Zaleski C. Jha S. Batut P. Chaisson M. Gingeras T.R. STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2013; 29: 15-21Crossref PubMed Scopus (21993) Google Scholar have been uploaded to Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE205458; accession number GSE205458). Pathway data were obtained from the following data sources: MetaBase from GeneGo version 6.3 (Clarivate, Chandler, AZ), Ingenuity Pathway Analysis from Ingenuity Systems (Redwood City, CA), Reactome Pathway,27Vastrik I. D'Eustachio P. Schmidt E. Joshi-Tope G. Gopinath G. Croft D. de Bono B. Gillespie M. Jassal B. Lewis S. Matthews L. Wu G. Birney E. Stein L. Reactome: a knowledge base of biologic pathways and processes.Genome Biol. 2007; 8: R39Crossref PubMed Scopus (491) Google Scholar and Gene Ontology.28Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. Harris M.A. Hill D.P. Issel-Tarver L. Kasarskis A. Lewis S. Matese J.C. Richardson J.E. Ringwald M. Rubin G.M. Sherlock G. The Gene Ontology Consortium: Gene ontology: tool for the unification of biology..Nat Genet. 2000; 25: 25-29Crossref PubMed Scopus (28424) Google Scholar To identify pathways and processes that were enriched in a gene list, a hypergeometric-based enrichment analysis was implemented. The hypergeometric P value was calculated using the R program version 3.5.0 (https://www.R-project.org, last accessed November 7, 2022) with the following command: phyper(x − 1, m, n-m, k and lower.tail = FALSE), where x is the number of genes from the gene list that are members of the pathway, m is the number of genes in the pathway, n is the total number of unique genes in all pathways, and k is the number of genes from the list that were present in at least one pathway. The resulting P value is indicative of the likelihood of enrichment for a specific pathway by chance given the size of the gene list. This approach typically results in multiple significant pathways because of redundancy. To control for redundancy, this approach was employed iteratively. In each iteration, the most significantly enriched pathway and associated genes from the list were set aside, shortening the gene list. The process was repeated until no pathway was significant (P < 0.05). Protein interaction data were obtained from National Center for Biotechnology Information GeneRIF, MetaBase from GeneGo version 6.3, Ingenuity Pathway Analysis from Ingenuity Systems, and NetPro from Molecular Connections (Bangalore, India). Nonhuman protein interactions were mapped to human homologs using National Center for Biotechnology Information HomoloGene.29Wheeler D.L. Barrett T. Benson D.A. Bryant S.H. Canese K. Church D.M. DiCuccio M. Edgar R. Federhen S. Helmberg W. Kenton D.L. Khovayko O. Lipman D.J. Madden T.L. Maglott D.R. Ostell J. JU Pontius Pruitt K.D. Schuler G.D. Schriml L.M. Sequeira E. Sherry S.T. Sirotkin K. Starchenko G. Suzek T.O. Tatusov R. Tatusova T.A. Wagner L. Yaschenko E. Database resources of the National Center for Biotechnology Information.Nucleic Acids Res. 2005; 33: D39-D45Crossref PubMed Scopus (379) Google Scholar Data were integrated on the basis of PubMed identifier references supporting each interaction. PubMed identifiers associated with >10 interactions were discarded, and from the remaining interactions, at least two PubMed identifiers were required for the interaction to be retained. Pathway-associated interactions

Computational genomics of Alzheimer disease (AD), the most common form of senile dementia, is a nascent field in AD research. The field includes AD gene clustering by computing gene order which generates higher quality gene clustering patterns than most other clustering methods. However, there are few available gene order computing methods such as Genetic Algorithm (GA) and Ant Colony Optimization (ACO). Further, their performance in gene order computation using AD microarray data is not known. We thus set forth to evaluate the performances of current gene order computing methods with different distance formulas, and to identify additional features associated with gene order computation.Using different distance formulas- Pearson distance and Euclidean distance, the squared Euclidean distance, and other conditions, gene orders were calculated by ACO and GA (including standard GA and improved GA) methods, respectively. The qualities of the gene orders were compared, and new features from the calculated gene orders were identified.Compared to the GA methods tested in this study, ACO fits the AD microarray data the best when calculating gene order. In addition, the following features were revealed: different distance formulas generated a different quality of gene order, and the commonly used Pearson distance was not the best distance formula when used with both GA and ACO methods for AD microarray data.Compared with Pearson distance and Euclidean distance, the squared Euclidean distance generated the best quality gene order computed by GA and ACO methods.

Abstract In addition to its well-known contributions to motor function, the cerebellum is involved in emotional regulation, anxiety, and affect 1-4 . We found that suppressing the firing of cerebellar Purkinje cells (PCs) rapidly excites forebrain areas that could contribute to such functions (including the amygdala, basal forebrain, and septum), but that the classic cerebellar outputs, the deep cerebellar nuclei (DCN), do not project to these forebrain regions. Here we show that PCs directly inhibit parabrachial nuclei (PBN) neurons that project to and influence numerous forebrain regions in a manner distinct from the DCN pathway. We also found that the PBN and DCN output pathways differentially influence behavior: suppressing the PC to PBN pathway is aversive and does not affect the speed of movement, whereas suppressing the PC to DCN pathway is not aversive and reduces speed. Molecular profiling shows that PCs inhibit numerous types of PBN neurons that control diverse nonmotor behaviors 5-9 . Therefore, the PBN pathway allows the cerebellum to regulate activity in many forebrain regions and may be an important substrate for nonmotor disorders related to cerebellar dysfunction.

Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction in the ubiquitin–proteasome system (UPS) specifically has been implicated in several important neurodegenerative diseases including Alzheimer’s Disease, Parkinson’s Disease, and amyotrophic lateral sclerosis. Research in this area has been limited by the fact that many inhibitors of the UPS given systemically do not cross the blood–brain barrier (BBB) in appreciable levels. This limits the ability to easily test in vivo specific hypotheses generated in reduced systems, like brain slice or dissociated cell culture, about whether the UPS may degrade a particular protein of interest. Although several techniques including intracerebral application via direct syringe injection, catheter-pump systems and drug-eluting beads are available to introduce BBB-impermeant drugs into brain they each have certain limitations and new approaches could provide further insights into this problem. In order to test the role of the UPS in protein degradation in vivo we have developed a strategy to treat mouse cortex with the UPS inhibitor clasto-lactacystin beta-lactone (CLBL) via a “cranial window” and recover the treated tissue for immunoblot analysis. This approach can be used in several different cranial window configurations including single window and double hemi-window arrangements that are tailored for different applications. We have also developed two different strategies for recovering treated cortical tissue including a vibratome/laser capture microscopy (LCM)-based and a vibratome only-based approach, each with its own specific advantages. We have documented UPS inhibition >600 μm deep into the cortex with this strategy. This set of techniques in the living mammalian brain is complementary to previously developed approaches and extends the repertoire of tools that can be used to the study protein degradation pathways relevant to neurodegenerative disease.

&lt;p&gt;Supplementary Tables S1-S15&lt;/p&gt;

&lt;p&gt;Figures S1-S6&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Pancreatic ductal adenocarcinoma (PDAC) lethality is multifactorial; although studies have identified transcriptional and genetic subsets of tumors with different prognostic significance, there is limited understanding of features associated with the minority of patients who have durable remission after surgical resection. In this study, we performed laser capture microdissection (LCM) of PDAC samples to define their cancer- and stroma-specific molecular subtypes and identify a prognostic gene expression signature for short-term and long-term survival.&lt;/p&gt;Experimental Design:&lt;p&gt;LCM and RNA sequencing (RNA-seq) analysis of cancer and adjacent stroma of 19 treatment-naïve PDAC tumors was performed. Gene expression signatures were tested for their robustness in a large independent validation set. An RNA-ISH assay with pooled probes for genes associated with disease-free survival (DFS) was developed to probe 111 PDAC tumor samples.&lt;/p&gt;Results:&lt;p&gt;Gene expression profiling identified four subtypes of cancer cells (C1–C4) and three subtypes of cancer-adjacent stroma (S1–S3). These stroma-specific subtypes were associated with DFS (&lt;i&gt;P&lt;/i&gt; = 5.55E-07), with S1 associated with better prognoses when paired with C1 and C2. Thirteen genes were found to be predominantly expressed in cancer cells and corresponded with DFS in a validation using existing RNA-seq datasets. A second validation on an independent cohort of patients using RNA-ISH probes to six of these prognostic genes demonstrated significant association with overall survival (median 17 vs. 25 months; &lt;i&gt;P&lt;/i&gt; &lt; 0.02).&lt;/p&gt;Conclusions:&lt;p&gt;Our results identified specific signatures from the epithelial and the stroma components of PDAC, which add clarity to the nature of PDAC molecular subtypes and may help predict survival.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Pancreatic ductal adenocarcinoma (PDAC) lethality is multifactorial; although studies have identified transcriptional and genetic subsets of tumors with different prognostic significance, there is limited understanding of features associated with the minority of patients who have durable remission after surgical resection. In this study, we performed laser capture microdissection (LCM) of PDAC samples to define their cancer- and stroma-specific molecular subtypes and identify a prognostic gene expression signature for short-term and long-term survival.&lt;/p&gt;Experimental Design:&lt;p&gt;LCM and RNA sequencing (RNA-seq) analysis of cancer and adjacent stroma of 19 treatment-naïve PDAC tumors was performed. Gene expression signatures were tested for their robustness in a large independent validation set. An RNA-ISH assay with pooled probes for genes associated with disease-free survival (DFS) was developed to probe 111 PDAC tumor samples.&lt;/p&gt;Results:&lt;p&gt;Gene expression profiling identified four subtypes of cancer cells (C1–C4) and three subtypes of cancer-adjacent stroma (S1–S3). These stroma-specific subtypes were associated with DFS (&lt;i&gt;P&lt;/i&gt; = 5.55E-07), with S1 associated with better prognoses when paired with C1 and C2. Thirteen genes were found to be predominantly expressed in cancer cells and corresponded with DFS in a validation using existing RNA-seq datasets. A second validation on an independent cohort of patients using RNA-ISH probes to six of these prognostic genes demonstrated significant association with overall survival (median 17 vs. 25 months; &lt;i&gt;P&lt;/i&gt; &lt; 0.02).&lt;/p&gt;Conclusions:&lt;p&gt;Our results identified specific signatures from the epithelial and the stroma components of PDAC, which add clarity to the nature of PDAC molecular subtypes and may help predict survival.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Detailed methods for sample preparation and RNA seqeucning&lt;/p&gt;

&lt;p&gt;Supplementary Tables S1-S15&lt;/p&gt;

&lt;p&gt;Figures S1-S6&lt;/p&gt;

&lt;p&gt;Detailed methods for sample preparation and RNA seqeucning&lt;/p&gt;

The silencing of actin capping protein β2, Capzb2, by RNAi in developing cultured neurons results in short, dystrophic neurites reminiscent of cytoskeletal changes seen in diverse neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). Actin and tubulin are two major cytoskeletal proteins indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that Capzb2 binds tubulin and, in the presence of microtubule- associated protein tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons results in short neurites with abnormal growth cones. Decreased neurite length is found in both AD and HD. In the first step towards uncovering the possible role of Capzb2 in these diseases, we studied Capzb2 protein expression in the postmortem brains of AD and HD patients. To determine whether disease-specific changes in Capzb2 protein accompany the progression of neurodegeneration, we performed Western Blot analysis of prefrontal cortices (PFC) and hippocampi (HPC) in AD patients and of PFC and heads of caudate nuclei (HCN) in HD patients. Our results show disease- and area-specific dynamics in the levels of Capzb2 protein expression in the progressive stages of AD and HD.

Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). This protocol is based strongly on a similar extraction protocol from the McCarroll lab.

Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). This protocol is based strongly on a similar extraction protocol from the McCarroll lab.

This chapter describes impact-mediated cytoplasmic loading of macromolecules into adherent cells. The advent of modern molecular biology, including the development of gene array technologies, has resulted in an explosion of information concerning the specific genes activated during normal cellular development, as well as those associated with a variety of pathological conditions. The critical importance of post-translational modification to protein function has been recognized with regard to a number of proteins involved in a variety of important disease states. Aberrant sialylation of cell surface antigens has been detected in a number of different tumor cell types and has been linked to the acquisition of a neoplastic phenotype, whereas improper sialylation of sodium channels in cardiac tissue has been linked to heart failure. One approach to overcome the limitations of genetic analysis is to insert a protein of interest into a cell and directly observe the effects on cell function. Prior to use on living cells, it is important to remove any preservative from the immunoglobulin solution, as exposure of living cells to such contaminants can result in rapid cell death.