Susan L. Cotman

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders that affect children and adults and are grouped together by similar clinical features and the accumulation of autofluorescent storage material. More than a dozen genes containing over 430 mutations underlying human NCLs have been identified. These genes encode lysosomal enzymes (CLN1, CLN2, CLN10, CLN13), a soluble lysosomal protein (CLN5), a protein in the secretory pathway (CLN11), two cytoplasmic proteins that also peripherally associate with membranes (CLN4, CLN14), and many transmembrane proteins with different subcellular locations (CLN3, CLN6, CLN7, CLN8, CLN12). For most NCLs, the function of the causative gene has not been fully defined. Most of the mutations in these genes are associated with a typical disease phenotype, but some result in variable disease onset, severity, and progression, including distinct clinical phenotypes. There remain disease subgroups with unknown molecular genetic backgrounds. This article is part of a Special Issue entitled: "Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease)."

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.

Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by <i>CLN3</i>. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in <i>Cln3</i>^Δex7/8 knock-in mice and Cb<i>Cln3</i>^Δex7/8 cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process.

The <i>CLN6</i> gene that causes variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a recessively inherited neurodegenerative disease that features blindness, seizures, and cognitive decline, maps to 15q21-23. We have used multiallele markers spanning this ∼4-Mb candidate interval to reveal a core haplotype, shared in Costa Rican families with vLINCL but not in a Venezuelan kindred, that highlighted a region likely to contain the <i>CLN6</i> defect. Systematic comparison of genes from the minimal region uncovered a novel candidate, FLJ20561, that exhibited DNA sequence changes specific to the different disease chromosomes: a G→T transversion in exon 3, introducing a stop codon on the Costa Rican haplotype, and a codon deletion in exon 5, eliminating a conserved tyrosine residue on the Venezuelan chromosome. Furthermore, sequencing of the murine homologue in the <i>nclf</i> mouse, which manifests recessive NCL-like disease, disclosed a third lesion—an extra base pair in exon 4, producing a frameshift truncation on the <i>nclf</i> chromosome. Thus, the novel ∼36-kD <i>CLN6</i>-gene product augments an intriguing set of unrelated membrane-spanning proteins, whose deficiency causes NCL in mouse and man.

Juvenile-onset neuronal ceroid lipofuscinosis (JNCL; Batten disease) features hallmark membrane deposits and loss of central nervous system (CNS) neurons. Most cases of the disease are due to recessive inheritance of an approximately 1 kb deletion in the CLN3 gene, encoding battenin. To investigate the common JNCL mutation, we have introduced an identical genomic DNA deletion into the murine CLN3 homologue (Cln3) to create Cln3( Deltaex7/8) knock-in mice. The Cln3( Deltaex7/8) allele produced alternatively spliced mRNAs, including a variant predicting non-truncated protein, as well as mutant battenin that was detected in the cytoplasm of cells in the periphery and CNS. Moreover, Cln3( Deltaex7/8) homozygotes exhibited accrual of JNCL-like membrane deposits from before birth, in proportion to battenin levels, which were high in liver and select neuronal populations. However, liver enzymes and CNS development were normal. Instead, Cln3( Deltaex7/8) mice displayed recessively inherited degenerative changes in retina, cerebral cortex and cerebellum, as well as neurological deficits and premature death. Thus, the harmful impact of the common JNCL mutation on the CNS was not well correlated with membrane deposition per se, suggesting instead a specific battenin activity that is essential for the survival of CNS neurons.

Neuronal ceroid lipofuscinosis (NCL) comprises ∼13 genetically distinct lysosomal disorders primarily affecting the central nervous system. Here we report successful reprograming of patient fibroblasts into induced pluripotent stem cells (iPSCs) for the two most common NCL subtypes: classic late-infantile NCL, caused by TPP1(CLN2) mutation, and juvenile NCL, caused by CLN3 mutation. CLN2/TPP1- and CLN3-iPSCs displayed overlapping but distinct biochemical and morphological abnormalities within the endosomal-lysosomal system. In neuronal derivatives, further abnormalities were observed in mitochondria, Golgi and endoplasmic reticulum. While lysosomal storage was undetectable in iPSCs, progressive disease subtype-specific storage material was evident upon neural differentiation and was rescued by reintroducing the non-mutated NCL proteins. In proof-of-concept studies, we further documented differential effects of potential small molecule TPP1 activity inducers. Fenofibrate and gemfibrozil, previously reported to induce TPP1 activity in control cells, failed to increase TPP1 activity in patient iPSC-derived neural progenitor cells. Conversely, nonsense suppression by PTC124 resulted in both an increase of TPP1 activity and attenuation of neuropathology in patient iPSC-derived neural progenitor cells. This study therefore documents the high value of this powerful new set of tools for improved drug screening and for investigating early mechanisms driving NCL pathogenesis.

Neuronal ceroid lipofuscinosis (NCL) is a genetically heterogeneous group of lysosomal diseases that collectively compose the most common Mendelian form of childhood-onset neurodegeneration. It is estimated that ∼8% of individuals diagnosed with NCL by conservative clinical and histopathologic criteria have been ruled out for mutations in the nine known NCL-associated genes, suggesting that additional genes remain unidentified. To further understand the genetic underpinnings of the NCLs, we performed whole-exome sequencing on DNA samples from a Mexican family affected by a molecularly undefined form of NCL characterized by infantile-onset progressive myoclonic epilepsy (PME), vision loss, cognitive and motor regression, premature death, and prominent NCL-type storage material. Using a recessive model to filter the identified variants, we found a single homozygous variant, c.550C>T in KCTD7, that causes a p.Arg184Cys missense change in potassium channel tetramerization domain-containing protein 7 (KCTD7) in the affected individuals. The mutation was predicted to be deleterious and was absent in over 6,000 controls. The identified variant altered the localization pattern of KCTD7 and abrogated interaction with cullin-3, a ubiquitin-ligase component and known KCTD7 interactor. Intriguingly, murine cerebellar cells derived from a juvenile NCL model (CLN3) showed enrichment of endogenous KCTD7. Whereas KCTD7 mutations have previously been linked to PME without lysosomal storage, this study clearly demonstrates that KCTD7 mutations also cause a rare, infantile-onset NCL subtype designated as CLN14.

To date, microglia subsets in the healthy CNS have not been identified. Utilizing autofluorescence (AF) as a discriminating parameter, we identified two novel microglia subsets in both mice and non-human primates, termed autofluorescence-positive (AF+) and negative (AF−). While their proportion remained constant throughout most adult life, the AF signal linearly and specifically increased in AF+ microglia with age and correlated with a commensurate increase in size and complexity of lysosomal storage bodies, as detected by transmission electron microscopy and LAMP1 levels. Post-depletion repopulation kinetics revealed AF− cells as likely precursors of AF+ microglia. At the molecular level, the proteome of AF+ microglia showed overrepresentation of endolysosomal, autophagic, catabolic, and mTOR-related proteins. Mimicking the effect of advanced aging, genetic disruption of lysosomal function accelerated the accumulation of storage bodies in AF+ cells and led to impaired microglia physiology and cell death, suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

Agrin is an extracellular matrix heparan sulfate proteoglycan (HSPG) well known for its role in modulation of the neuromuscular junction during development. Although agrin is one of the major HSPGs of the brain, its function there remains elusive. Here we provide evidence suggesting a possible function for agrin in Alzheimer's disease brain. Agrin protein binds the amyloidogenic peptide Abeta (1-40) in its fibrillar state via a mechanism that involves the heparan sulfate glycosaminoglycan chains of agrin. Furthermore, agrin is able to accelerate Abeta fibril formation and protect Abeta (1-40) from proteolysis, in vitro. Supporting a biological significance for these in vitro data, immunocytochemical studies demonstrate agrin's presence within senile plaques and cerebrovascular amyloid deposits, and agrin immunostained capillaries exhibit pathological alterations in AD brain. These data therefore suggest that agrin may be an important factor in the progression of Abeta peptide aggregation and/or its persistence in Alzheimer's disease brain.

JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a approximately 1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Deltaex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology.To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Deltaex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced.These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is the result of mutations in the Cln3 gene. The Cln3 knock-in mouse (Cln3Deltaex7/8) reproduces the most common Cln3 mutation and we have now characterized the CNS of these mice at 12 months of age. With the exception of the thalamus, Cln3Deltaex7/8 homozygotes displayed no significant regional atrophy, but a range of changes in individual laminar thickness that resulted in variable cortical thinning across subfields. Stereological analysis revealed a pronounced loss of neurons within individual laminae of somatosensory cortex of affected mice and the novel finding of a loss of sensory relay thalamic neurons. These affected mice also exhibited profound astrocytic reactions that were most pronounced in the neocortex and thalamus, but diminished in other brain regions. These data provide the first direct evidence for neurodegenerative and reactive changes in the thalamocortical system in JNCL and emphasize the localized nature of these events.

Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, which generates considerable cellular debris, is synapse elimination, in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning.

Mucolipidosis type IV is a neurodegenerative lysosomal disease clinically characterized by psychomotor retardation, visual impairment, and achlorhydria. In this study we report the development of a neuronal cell model generated from cerebrum of Mcoln1−/− embryos. Prior functional characterization of MLIV cells has been limited to fibroblast cultures gleaned from patients. The current availability of the mucolipin-1 knockout mouse model Mcoln1−/− allows the study of mucolipin-1-defective neurons, which is important since the disease is characterized by severe neurological impairment. Electron microscopy studies reveal significant membranous intracytoplasmic storage bodies, which correlate with the storage morphology observed in cerebral cortex of Mcoln1−/− P7 pups and E17 embryos. The Mcoln1−/− neuronal cultures show an increase in size of LysoTracker and Lamp1 positive vesicles. Using this neuronal model system, we show that macroautophagy is defective in mucolipin-1-deficient neurons and that LC3-II levels are significantly elevated. Treatment with rapamycin plus protease inhibitors did not increase levels of LC3-II in Mcoln1−/− neuronal cultures, indicating that the lack of mucolipin-1 affects LC3-II clearance. P62/SQSTM1 and ubiquitin levels were also increased in Mcoln1−/− neuronal cultures, suggesting an accumulation of protein aggregates and a defect in macroautophagy which could help explain the neurodegeneration observed in MLIV. This study describes, for the first time, a defect in macroautophagy in mucolipin-1-deficient neurons, which corroborates recent findings in MLIV fibroblasts and provides new insight into the neuronal pathogenesis of this disease.

AbstractLoss-of-function mutations in CLN3 are responsible for juvenile-onset neuronal ceroid lipofuscinosis (JNCL), or Batten disease, which is an incurable lysosomal disease that manifests with vision loss, followed by seizures and progressive neurodegeneration, robbing children of motor skills, speech and cognition, and eventually leading to death in the second or third decade of life. Emerging clinical evidence points to JNCL pathology outside of the CNS, including the cardiovascular system. The CLN3 gene encodes an unusual transmembrane protein, CLN3 or battenin, whose elusive function has been the subject of intense study for more than 10 years. Owing to the detailed characterization of a large number of disease models, our knowledge of CLN3 protein function is finally coming into focus. This review will describe the most current understanding of CLN3 structure, function and dysfunction in JNCL.Key Words: autophagyBatten diseaseCLN3endosomeGolgijuvenile neuronal ceroid lipofuscinosislysosomepalmitoylation

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca2+-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca2+ chelation. Interrogation of intracellular Ca2+ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca2+ pools and in store-operated Ca2+ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca2+ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening. Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca2+-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca2+ chelation. Interrogation of intracellular Ca2+ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca2+ pools and in store-operated Ca2+ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca2+ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.

Neuronal ceroid lipofuscinosis (NCL), first clinically described in 1826 and pathologically defined in the 1960s, refers to a group of disorders mostly diagnosed in the childhood years that involve the accumulation of lysosomal storage material with characteristic ultrastructure and prominent neurodegenerative features including vision loss, seizures, motor and cognitive function deterioration, and often times, psychiatric disturbances. All NCL disorders evidence early morbidity and treatment options are limited to symptomatic and palliative care. While distinct genetic forms of NCL have long been recognized, recent genetic advances are considerably widening the NCL genotypic and phenotypic spectrum, highlighting significant overlap with other neurodegenerative diseases. This review will discuss these recent advances and the expanded potential for increased awareness and new research that will ultimately lead to effective treatments for NCL and related disorders.

Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The <i>CLN3</i> gene encodes a lysosomal membrane protein of unknown function, and <i>CLN3</i> mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.

The neuronal ceroid lipofuscinoses (NCL) are a group of disorders defined by shared clinical and pathological features, including seizures and progressive decline in vision, neurocognition, and motor functioning, as well as accumulation of autofluorescent lysosomal storage material, or ‘ceroid lipofuscin’. Research has revealed thirteen distinct genetic subtypes. Precisely how the gene mutations lead to the clinical phenotype is still incompletely understood, but recent research progress is starting to shed light on disease mechanisms, in both gene-specific and shared pathways. As the application of new sequencing technologies to genetic disease diagnosis has grown, so too has the spectrum of clinical phenotypes caused by mutations in the NCL genes. Most genes causing NCL have probably been identified, underscoring the need for a shift towards applying genomics approaches to achieve a deeper understanding of the molecular basis of the NCLs and related disorders. Here, we summarize the current understanding of the thirteen identified NCL genes and the proteins they encode, touching upon the spectrum of clinical manifestations linked to each of the genes, and we highlight recent progress leading to a broader understanding of key pathways involved in NCL disease pathogenesis and commonalities with other neurodegenerative diseases.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.

Agrin is a major brain heparan sulfate proteoglycan which is expressed in nearly all basal laminae and in early axonal pathways of the developing central nervous system. To further understand agrin's function during nervous system development, we have examined agrin's ability to interact with several heparin-binding extracellular matrix proteins. Our data show that agrin binds FGF-2 and thrombospondin by a heparan sulfate-dependent mechanism, merosin and laminin by both heparan sulfate-dependent and -independent mechanisms, and tenascin solely via agrin's protein core. Furthermore, agrin's heparan sulfate side chains encode a specificity in interactions with heparin-binding molecules since fibronectin and the cell adhesion molecule L1 do not bind agrin. Surface plasmon resonance studies (BIAcore) reveal a high affinity for agrin's interaction with FGF-2 and merosin (2.5 and 1.8 nM, respectively). Demonstrating a biological significance for these interactions, FGF-2, laminin, and tenascin copurify with immunopurified agrin and immunohistochemistry reveals a partial codistribution of agrin and its ECM ligands in the chick developing visual system. These studies and our previous studies, showing that merosin and NCAM also colocalize with agrin, provide evidence that agrin plays a crucial role in the function of the extracellular matrix and suggest a role for agrin in axon pathway development.

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6nclf/nclf cerebellar cells and compared them to wild-type and CbCln3Δex7/8/Δex7/8 cerebellar cells. CbCln6nclf/nclf cells and CbCln3Δex7/8/Δex7/8 cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6nclf/nclf and CbCln3Δex7/8/Δex7/8 cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3Δex7/8 and Cln6nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.

The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a family of neurodegenerative diseases that affect all age groups and ethnicities around the globe. At least a dozen NCL subtypes have been identified that are each linked to a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene. Mutations in CLN genes cause the accumulation of autofluorescent lipoprotein aggregates, called ceroid lipofuscin, in neurons and other cell types outside the central nervous system. The mechanisms regulating the accumulation of this material are not entirely known. The CLN genes encode cytosolic, lysosomal, and integral membrane proteins that are associated with a variety of cellular processes, and accumulated evidence suggests they participate in shared or convergent biological pathways. Research across a variety of non-mammalian and mammalian model systems clearly supports an effect of CLN gene mutations on autophagy, suggesting that autophagy plays an essential role in the development and progression of the NCLs. In this review, we summarize research linking the autophagy pathway to the NCLs to guide future work that further elucidates the contribution of altered autophagy to NCL pathology.

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin-binding growth factor basic fibroblast growth factor (FGF-2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF-2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF-2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF-2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c-fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF-2, and augments and sustains FGF-2 mediated increases in c-fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF-2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF-2.

Mutations in the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis, a pediatric neurodegenerative disorder characterized by visual loss, epilepsy and psychomotor deterioration. Although most CLN3 patients carry the same 1-kb deletion in the CLN3 gene, their disease phenotype can be variable. The aims of this study were to (i) study the clinical phenotype in CLN3 patients with identical genotype, (ii) identify genes that are dysregulated in CLN3 disease regardless of the clinical course that could be useful as biomarkers, and (iii) find modifier genes that affect the progression rate of the disease. A total of 25 CLN3 patients homozygous for the 1-kb deletion were classified into groups with rapid, average or slow disease progression using an established clinical scoring system. Genome-wide expression profiling was performed in eight CLN3 patients with different disease progression and matched controls. The study showed high phenotype variability in CLN3 patients. Five genes were dysregulated in all CLN3 patients and present candidate biomarkers of the disease. Of those, dual specificity phosphatase 2 (DUSP2) was also validated in acutely CLN3-depleted cell models and in CbCln3Δex7/8 cerebellar precursor cells. A total of 13 genes were upregulated in patients with rapid disease progression and downregulated in patients with slow disease progression; one gene showed dysregulation in the opposite way. Among these potential modifier genes, guanine nucleotide exchange factor 1 for small GTPases of the Ras family (RAPGEF1) and transcription factor Spi-B (SPIB) were validated in an acutely CLN3-depleted cell model. These findings indicate that differential perturbations of distinct signaling pathways might alter disease progression and provide insight into the molecular alterations underlying neuronal dysfunction in CLN3 disease and neurodegeneration in general.

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3− cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3− cells was precocious and cln3− slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3− cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3− cells, strongly supports the use of this new model for JNCL research.

XFM approach detects subcellular zinc and calcium mishandling in a fatal neurodegenerative disease, that is corrected by delivery of bioavailable zinc.

Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3(Δex7/8) knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3(Δex7/8) mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3(Δex7/8) mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and other diseases in this category.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a neurodegenerative disorder caused by mutation in CLN3. Defective autophagy and concomitant accumulation of autofluorescence enriched with mitochondrial ATP synthase subunit c were previously discovered in Cln3 mutant knock-in mice. In this study, we show that treatment with lithium reduces numbers of LC3-positive autophagosomes and accumulation of LC3-II in Cln3 mutant knock-in cerebellar cells (CbCln3(Δex7/8/Δex7/8) ). Lithium, an inhibitor of GSK3 and IMPase, reduces the accumulation of mitochondrial ATP synthase subunit c and autofluorescence in CbCln3(Δex7/8/Δex7/8) cells, and mitigates the abnormal subcellular distribution of acidic vesicles in the cells. L690,330, an IMPase inhibitor, is as effective as lithium in restoring autophagy in CbCln3(Δex7/8/Δex7/8) cells. Moreover, lithium or down-regulation of IMPase expression protects CbCln3(Δex7/8/Δex7/8) cells from cell death induced by amino acid deprivation. These results suggest that lithium overcomes the autophagic defect in CbCln3(Δex7/8/Δex7/8) cerebellar cells probably through IMPase, thereby reducing their vulnerability to cell death.

Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, refers to a group of severe neurodegenerative disorders that primarily affect children. The most common subtype of the disease is caused by loss-of-function mutations in CLN3, which is conserved across model species from yeast to human. The precise function of the CLN3 protein is not known, which has made targeted therapy development challenging. In the social amoeba Dictyostelium discoideum, loss of Cln3 causes aberrant mid-to-late stage multicellular development. In this study, we show that Cln3-deficiency causes aberrant adhesion and aggregation during the early stages of Dictyostelium development. cln3- cells form ∼30% more multicellular aggregates that are comparatively smaller than those formed by wild-type cells. Loss of Cln3 delays aggregation, but has no significant effect on cell speed or cAMP-mediated chemotaxis. The aberrant aggregation of cln3- cells cannot be corrected by manually pulsing cells with cAMP. Moreover, there are no significant differences between wild-type and cln3- cells in the expression of genes linked to cAMP chemotaxis (e.g., adenylyl cyclase, acaA; the cAMP receptor, carA; cAMP phosphodiesterase, pdsA; g-protein α 9 subunit, gpaI). However, during this time in development, cln3- cells show reduced cell-substrate and cell-cell adhesion, which correlate with changes in the levels of the cell adhesion proteins CadA and CsaA. Specifically, loss of Cln3 decreases the intracellular level of CsaA and increases the amount of soluble CadA in conditioned media. Together, these results suggest that the aberrant aggregation of cln3- cells is due to reduced adhesion during the early stages of development. Revealing the molecular basis underlying this phenotype may provide fresh new insight into CLN3 function.

To critically re-evaluate cases diagnosed as adult neuronal ceroid lipofuscinosis (ANCL) in order to aid clinicopathologic diagnosis as a route to further gene discovery.Through establishment of an international consortium we pooled 47 unsolved cases regarded by referring centers as ANCL. Clinical and neuropathologic experts within the Consortium established diagnostic criteria for ANCL based on the literature to assess each case. A panel of 3 neuropathologists independently reviewed source pathologic data. Cases were given a final clinicopathologic classification of definite ANCL, probable ANCL, possible ANCL, or not ANCL.Of the 47 cases, only 16 fulfilled the Consortium's criteria of ANCL (5 definite, 2 probable, 9 possible). Definitive alternate diagnoses were made in 10, including Huntington disease, early-onset Alzheimer disease, Niemann-Pick disease, neuroserpinopathy, prion disease, and neurodegeneration with brain iron accumulation. Six cases had features suggesting an alternate diagnosis, but no specific condition was identified; in 15, the data were inadequate for classification. Misinterpretation of normal lipofuscin as abnormal storage material was the commonest cause of misdiagnosis.Diagnosis of ANCL remains challenging; expert pathologic analysis and recent molecular genetic advances revealed misdiagnoses in >1/3 of cases. We now have a refined group of cases that will facilitate identification of new causative genes.

The CLN3 gene was identified over two decades ago, but the primary function of the CLN3 protein remains unknown. Recessive inheritance of loss of function mutations in CLN3 are responsible for juvenile neuronal ceroid lipofuscinosis (Batten disease, or CLN3 disease), a fatal childhood onset neurodegenerative disease causing vision loss, seizures, progressive dementia, motor function loss and premature death. CLN3 is a multipass transmembrane protein that primarily localizes to endosomes and lysosomes. Defects in endocytosis, autophagy, and lysosomal function are common findings in CLN3-deficiency model systems. However, the molecular mechanisms underlying these defects have not yet been fully elucidated. In this mini-review, we will summarize the current understanding of the CLN3 protein interaction network and discuss how this knowledge is starting to delineate the molecular pathogenesis of CLN3 disease. Accumulating evidence strongly points towards CLN3 playing a role in regulation of the cytoskeleton and cytoskeletal associated proteins to tether cellular membranes, regulation of membrane complexes such as channels/transporters, and modulating the function of small GTPases to effectively mediate vesicular movement and membrane dynamics.

The neuronal ceroidlipofuscinoses (NCL) are a group of neurodegenerative disorders and are the most common lysosomal storage diseases of infancy and childhood. Juvenile NCL is caused by CLN3 mutation, producing retinal degeneration, uncontrollable seizures, cognitive and motor decline, and early death before the age of 30 years. To study the pathogenetic mechanisms of the disease, Cln3 knock-in mice (Cln3(Deltaex7/8)) have been generated, which reproduce the 1.02-kb deletion in the CLN3 gene observed in more than 85% of juvenile NCL patients. To characterize the impact of the common Cln3 mutation on development of autofluorescent storage material, gliosis, glucose metabolism, oxidative stress, and transmitter receptors during postnatal brain maturation, brain tissue of Cln3(Deltaex7/8) mice at the ages of 3, 4, 5, 6, 9, and 19 months was subjected to immunocytochemistry to label gliotic markers and nitric oxide synthases; photometric assays to assess enzyme activities of glycolysis and antioxidative defense systems; and level of reactive nitrogen species as well as quantitative receptor autoradiography to detect select cholinergic, glutamatergic, and GABAergic receptor subtypes. The developmental increase in cerebral cortical autofluorescent lipofuscin-like deposition is accompanied by a significant astro- and microgliosis, increased activities of lactate dehydrogenase and phosphofructokinase, decreased level of glutathione peroxidase, enhanced amount of reactive nitrogen species, and lowered binding levels of N-methyl-D-aspartate- and M1-muscarinic acetylcholine receptors in select brain regions but hardly in GABA(A) receptor sites compared with wild-type mice. Detailed elucidation of the sequence of pathological events during postnatal development highlights new potential strategies for symptomatic treatment of the disease.

The neuronal ceroid lipofuscinoses (NCLs, or Batten disease) comprise the most common Mendelian form of childhood-onset neurodegeneration, but the functions of the known underlying gene products remain poorly understood. The clinical heterogeneity of these disorders may shed light on genetic interactors that modify disease onset and progression.We describe a proband with congenital hypotonia and an atypical form of infantile-onset, biopsy-proven NCL. Pathologic and molecular work-up of this patient identified CLN5 mutations as well as a mutation-previously described as incompletely penetrant or a variant of unknown significance-in POLG1, a nuclear gene essential for maintenance of mitochondrial DNA (mtDNA) copy number. The congenital presentation of this patient is far earlier than that described for either CLN5 patients or affected carriers of the POLG1 variant (c.1550 G > T, p.Gly517Val). Assessment of relative mtDNA copy number and mitochondrial membrane potential in the proband and control subjects suggested a pathogenic effect of the POLG1 change as well as a possible functional interaction with CLN5 mutations.These findings suggest that an incompletely penetrant variant in POLG1 may modify the clinical phenotype in a case of CLN5 and are consistent with emerging evidence of interactions between NCL-related genes and mitochondrial physiology.

The CAG repeat expansion that elongates the polyglutamine tract in huntingtin is the root genetic cause of Huntington's disease (HD), a debilitating neurodegenerative disorder. This seemingly slight change to the primary amino acid sequence alters the physical structure of the mutant protein and alters its activity. We have identified a set of G-quadruplex-forming DNA aptamers (MS1, MS2, MS3, MS4) that bind mutant huntingtin proximal to lysines K2932/K2934 in the C-terminal CTD-II domain. Aptamer binding to mutant huntingtin abrogated the enhanced polycomb repressive complex 2 (PRC2) stimulatory activity conferred by the expanded polyglutamine tract. In HD, but not normal, neuronal progenitor cells (NPCs), MS3 aptamer co-localized with endogenous mutant huntingtin and was associated with significantly decreased PRC2 activity. Furthermore, MS3 transfection protected HD NPCs against starvation-dependent stress with increased ATP. Therefore, DNA aptamers can preferentially target mutant huntingtin and modulate a gain of function endowed by the elongated polyglutamine segment. These mutant huntingtin binding aptamers provide novel molecular tools for delineating the effects of the HD mutation and encourage mutant huntingtin structure-based approaches to therapeutic development.

Background: Microglia are the primary brain cell type regulating neuroinflammation and they are important for healthy aging. Genes regulating microglial function are associated with an increased risk of neurodegenerative disease. Loss-of-function mutations in CLN3, which encodes an endolysosomal membrane protein, lead to the most common childhood-onset form of neurodegeneration, featuring early-stage neuroinflammation that long precedes neuronal cell loss. How loss of CLN3 function leads to this early neuroinflammation is not yet understood. Methods: Here, we have comprehensively studied microglia from Cln3∆ex7/8 mice, a genetically accurate CLN3 disease model. Microglia were isolated from young and old Cln3∆ex7/8 mice for downstream molecular and functional studies. Results: We show that loss of CLN3 function in microglia leads to classic age-dependent CLN3-disease lysosomal storage as well as an altered morphology of the lysosome, mitochonodria and Golgi compartments. Consistent with these morphological alterations, we also discovered pathological proteomic signatures implicating defects in lysosomal function and lipid metabolism processes at an early disease stage. CLN3-deficient microglia were unable to efficiently turnover myelin and metabolize its associated lipids, showing severe defects in lipid droplet formation and significant accumulation of cholesterol, phenotypes that were corrected by treatment with autophagy inducers and cholesterol lowering drugs. Finally, we observed reduced myelination in aging homozygous Cln3∆ex7/8 mice suggesting altered myelin turnover by microglia impacts myelination in the CLN3-deficient brain. Conclusion: Our results implicate a cell autonomous defect in CLN3-deficient microglia that impacts the ability of these cells to support neuronal cell health. These results strongly suggest microglial targeted therapies should be considered for CLN3 disease.

Alterations in the autophagosomal–lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal–lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis.

Clinicians, basic researchers, representatives from pharma and families from around the world met in Cordoba, Argentina in October, 2014 to discuss recent research progress at the 14th International Congress on Neuronal Ceroid Lipofuscinoses (NCLs; Batten disease), a group of clinically overlapping fatal, inherited lysosomal disorders with primarily neurodegenerative symptoms. This brief review article will provide perspectives on the anticipated future directions of NCL basic and clinical research as we move towards improved diagnosis, care and treatment of NCL patients. This article is part of a Special Issue entitled: Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease).

A novel cDNA clone, CR16, was isolated from a rat hippocampal cDNA library and characterized for responses to corticosteroids and regional expression. The 4-kb RNA was increased 3-fold by treatment of adrenalectomized (ADX) rats with corticosterone (CORT). Overlapping cDNA totaling 4,374 nt were used to define an open reading frame of 1,356 nt beginning 191 nt from the 5’-end and encoding a 45-kD protein containing 32% proline. CR16 has no obvious homologies to GenBank or protein databases. CR16 RNA was detected by in situ hybridization in neuron-rich layers of the hippocampal formation, layers II, III and VI of the cerebral cortex, thalamus, ventromedial nucleus of the hypothalamus, bed nucleus of the stria terminalis, lateral septal nucleus, nucleus accumbens, olfactory bulb, inferior colliculus, pons and inferior olive. The CR16 RNA has low prevalence in the hippocampus and cortex (<10 pg/µg total RNA) and is elevated 3-fold in both structures in a dose-dependent manner by CORT in ADX rats. Treatment of ADX rats with aldosterone (ALDO), CORT, or RU28362 increased CR16 RNA to similar levels in the hippocampus while ALDO had minimal effects on the level of CR16 RNA relative to CORT or RU28362 in the cortex. Neither shaking stress (2 h) nor 2 h CORT significantly elevated CR16 RNA in the hippocampus, suggesting that its response to elevated CORT is not rapid. ADX lowered CR16 RNA levels by 50% relative to intact rats while low-level CORT replacement (≧4 ng/ml serum CORT) significantly elevated CR16 RNA 2-fold in ADX rats. These results are consistent with both the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) regulating the CR16 gene. This gene will be useful in dissecting the role of MR and GR in CNS neurons.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract To date, microglia subsets in the healthy CNS have not been identified. Utilizing autofluorescence (AF) as a discriminating parameter, we identified two novel microglia subsets in both mice and non-human primates, termed autofluorescence-positive (AF+) and negative (AF−). While their proportion remained constant throughout most adult life, the AF signal linearly and specifically increased in AF+ microglia with age and correlated with a commensurate increase in size and complexity of lysosomal storage bodies, as detected by transmission electron microscopy and LAMP1 levels. Post-depletion repopulation kinetics revealed AF− cells as likely precursors of AF+ microglia. At the molecular level, the proteome of AF+ microglia showed overrepresentation of endolysosomal, autophagic, catabolic, and mTOR-related proteins. Mimicking the effect of advanced aging, genetic disruption of lysosomal function accelerated the accumulation of storage bodies in AF+ cells and led to impaired microglia physiology and cell death, suggestive of a mechanistic convergence between aging and lysosomal storage disorders. eLife digest Microglia are a unique type of immune cell found in the brain and spinal cord. Their job is to support neurons, defend against invading microbes, clear debris and remove dying neurons by engulfing them. Despite these diverse roles, scientists have long believed that there is only a single type of microglial cell, which adapts to perform whatever task is required. But more recent evidence suggests that this is not the whole story. Burns et al. now show that we can distinguish two subtypes of microglia based on a property called autofluorescence. This is the tendency of cells and tissues to emit light of one color after they have absorbed light of another. Burns et al. show that about 70% of microglia in healthy mouse and monkey brains display autofluorescence. However, about 30% of microglia show no autofluorescence at all. This suggests that there are two subtypes of microglia: autofluorescence-positive and autofluorescence-negative. But does this difference have any implications for how the microglia behave? Autofluorescence occurs because specific substances inside the cells absorb light. In the case of microglia, electron microscopy revealed that autofluorescence was caused by structures within the cell called lysosomal storage bodies accumulating certain materials. The stored material included fat molecules, cholesterol crystals and other substances that are typical of disorders that affect these compartments. Burns et al. show that autofluorescent microglia contain larger amounts of proteins involved in storing and digesting waste materials than their non-autofluorescent counterparts. Moreover, as the brain ages, lysosomal storage material builds up inside autofluorescent microglia, which increase their autofluorescence as a result. Unfortunately, this accumulation of cellular debris also makes it harder for the microglia to perform their tasks. Increasing evidence suggests that the accumulation of waste materials inside the brain contributes to diseases of aging. Future work should examine how autofluorescent microglia behave in animal models of neurodegenerative diseases. If these cells do help protect the brain from the effects of aging, targeting them could be a new strategy for treating aging-related diseases. Introduction Microglia are a unique population of tissue resident macrophages residing in the central nervous system (CNS) accounting for 10% to 15% of all cells within the CNS. While displaying some canonical macrophage activities such as the phagocytosis of debris and apoptotic bodies (Chan et al., 2001; Janda et al., 2018), microglia are also endowed with functions specific to the CNS microenvironment (Clayton et al., 2017; Li and Barres, 2018; Ransohoff, 2016; Ransohoff and Khoury, 2016), such as synaptic remodeling (Paolicelli et al., 2011; Stephan et al., 2012; Stevens et al., 2007; Weinhard et al., 2018), neuronal support (Parkhurst et al., 2013; Ueno et al., 2013), and oligodendrogenesis (Hagemeyer et al., 2017; Wlodarczyk et al., 2017). However, despite this diversity of functions, no durable subsets have been identified in the healthy adult brain at steady-state. The disease-associated microglia (DAM) and the closely-related microglia subset expressing a neurodegenerative phenotype (MGnD) were reported to arise in response to the accumulation of β-amyloid plaques in Alzheimer’s Disease (AD) transgenic mouse models (Kamphuis et al., 2016; Keren-Shaul et al., 2017; Krasemann et al., 2017; Mrdjen et al., 2018), as well as in other models of neurodegeneration and aging (Chiu et al., 2013; Holtman et al., 2015; Spiller et al., 2018; Wlodarczyk et al., 2014). Mirroring the gene signature and function of DAM and MGnD subsets in disease conditions, the proliferative-region-associated microglia (PAM) subset was associated with the phagocytosis of newly formed and dying oligodendrocytes during normal post-natal development (Felsky et al., 2019; Hagemeyer et al., 2017; Li et al., 2019). Dark microglia, a subset identified by their condensed, electron-dense cytoplasm visible by transmission electron microscopy, are frequently observed during pathologic states and believed to actively associate with neuronal synapses (Bisht et al., 2016). Beyond conditions of neuronal or oligodendrocyte cell death, microglia heterogeneity was also observed within the adult steady-state CNS. In particular, regional variations in cellular density (Lawson et al., 1990), but also in microglia gene expression profiles were reported, pointing to region-specific microglia functions and possibly subsets in healthy brain (Ayata et al., 2018; Grabert et al., 2016). While these emerging transcriptomic findings suggest that steady-state microglia subsets are likely present, they remain to be identified and characterized. By using cellular autofluorescence (AF) as a novel photophysical parameter to explore microglia heterogeneity in unperturbed conditions, we report that steady-state microglia exist in two discrete states at a regulated ratio throughout the entire lifespan of rodent and non-human primate species. Leveraging this physical property, we devised a novel probe-free method to isolate and characterize these subsets and established that AF+ and AF− microglia differed in their ultrastructural features, homeostatic dynamics, proteomic content and physiological properties. Results Cellular autofluorescence identifies two discrete microglia subsets Based on the observation that certain peripheral myeloid populations are highly autofluorescent, we initially included an empty fluorescence channel in our microglia flow cytometry analyses. Doing so revealed an unexpected bimodal distribution of autofluorescence (AF) intensity in CD45dimCD11B+ microglia isolated from 6-month-old naïve C57BL/6 mice (Figure 1A and Figure 1—figure supplement 1A). This bimodal distribution identified two subsets of microglia: an AF-positive (AF+) subset showing a strong AF signal, and an AF-negative (AF−) subset which displayed no or minimal levels of AF. The two subsets appeared at a highly reproducible ratio of 1:2.5 (AF−:AF+), with an average frequency of AF+ microglia of 71 ± 1.2% (n = 13; CV = 1.6%) (Figure 1—figure supplement 1B). To further characterize the spectral properties of the AF signal, we included additional empty fluorescence channels. The AF signal showed maximal intensity in the 660–735 nm emission range upon excitation with a 488 nm (Blue) laser but was also detected across multiple combinations of laser excitation wavelengths and emission filter ranges (Figure 1B). Extraction of single cell-level data from flow cytometry analyses revealed that the majority of microglia that were either positive or negative for AF in the Blue-710 nm AF channel were also positive or negative across most other AF channels tested, respectively (Figure 1C). In a few channels such as Red-780 nm for instance, 10% to 20% of the cells that were positive in Blue-710 nm appeared low or negative, which likely resulted from a differential sensitivity of the channels used to detect AF as shown in Figure 1B by the differential brightness of the AF+ population in those two channels. Both AF microglia subsets were detected across different regions of the brain and while there were minor differences in the frequency of AF+ microglia between regions, AF+ cells displayed similar levels of AF signal in cerebellum, cortex and hippocampus (Figure 1—figure supplement 1C,D). Both AF+ and AF− subsets of microglia were positive for microglia homeostatic markers, including CX3CR1, P2RY12 and TMEM119 (Figure 1D and Figure 1—figure supplement 1E). Finally, there were no gender differences observed in the frequency of AF+ microglia nor the intensity of AF (Figure 1—figure supplement 1F,G). This observation was conserved across species as microglia isolated from the brains of 3- to 4-year-old Cynomolgus monkeys showed a very consistent bimodal pattern of AF in the Blue-710 nm channel with 80 ± 3.6% of microglia that were AF+ on average (Figure 1E,F). Altogether, these analyses identified cellular AF as a novel photophysical property for discriminating two discrete subsets of microglia present at steady-state and conserved between rodents and non-human primates. Figure 1 with 1 supplement see all Download asset Open asset Cellular autofluorescence identifies two discrete and novel microglia subsets. (A) Representative flow cytometry scatterplot showing CD45 and CD11B surface levels in a brain single cell suspension and histogram of autofluorescence (AF) signal in DAPI− CD45dim CD11B+ microglia detected with a blue laser and 710 nm detector. (B) Representative flow cytometry histograms of AF intensity in the entire microglia population for multiple excitation lasers and emission filters. (C) Heatmap of AF signal detected across multiple cytometer channels (columns) in 500 single microglial cells (rows) and hierarchically clustered by Manhattan distance. (D) Representative flow cytometry histograms of TMEM119, CX3CR1 and P2RY12 surface levels in AF+ and AF− microglia subsets. (E) Representative flow cytometry scatterplots used to identify microglia in brain single cell suspensions from Cynomolgus macaques and representative histogram of AF levels detected in microglia. (F) Quantitation of results presented in (E). (G) Representative scatterplots of AF levels detected in CD45dimCD11B+ microglia and corresponding microscopy images of selected AF+ and AF− cells (highlighted in orange and numbered in the scatterplots and images) analyzed using imaging flow cytometry. Scale bar = 7 μm. (A, B) Representative of n = 12 animals from at least four independent experiments. (D) n = 6 animals from at least two independent experiments. (E, F) n = 5 animals from two independent experiments. B = blue laser, R = red laser, YG = yellow green; AF = autofluorescence; BF = bright field; SSC = side scatter. See also Figure 1—figure supplement 1. The autofluorescence signal in AF+ microglia originates from intracellular organelles To define the subcellular origin of the AF signal in microglia, we utilized imaging cytometry. Microscopy images of AF+ cells identified by the fluorescence intensity detected in two empty channels (Figure 1G) revealed that the AF signal was not diffusely distributed throughout the cellular volume but was punctate and localized within intracellular organelles. The subcellular AF compartments were observed in all tested AF channels and systematically colocalized (Figure 1—figure supplement 1H and I). AF− cells did not display detectable AF subcellular compartments in any of the tested channels, altogether establishing that these two subsets of microglia, identified solely by their AF profiles, differed by the presence of highly autofluorescent intracellular organelles that were restricted to the AF+ microglia subset. AF signal intensity increases linearly with aging, but solely within AF+ microglia In contrast to the punctate AF signal detected in 3-month-old animals, imaging flow cytometry applied to naïve mice from a range of ages revealed that AF subcellular structures became largely confluent in 10-month-old animals and occupied a larger fraction of the cytosol (from 11 μm2 to 17 μm2 on average in 3- and 10- month-old animals, respectively) (Figure 2A,B). Consistent with these results, flow cytometry analyses established a nearly linear increase of AF signal intensity in AF+ microglia with aging (Figure 2C,D), resulting in a cumulative 3-fold increase of AF signal between 3- and 12-month-old mice across multiple fluorescence channels (Figure 2D and Figure 2—figure supplement 1A). While a clear AF+ population was not detectable in mice aged 15 days post-natal or younger, a bimodal distribution appeared as early as 30 days post-natal and AF increased linearly with age from that point on (Figure 2—figure supplement 1B,C). In contrast, age-dependent increases in cellular AF were not detected in peripheral CD11B+ cells residing in the spleen (Figure 2—figure supplement 1D). Despite the large increase in the AF signal observed in AF+ cells, their frequency remained largely unchanged during aging, decreasing only slightly from 75% to 72% from 3 to 12 months of age (Figure 2E). Finally, neither the proportion of AF− cells (Figure 2E) nor the area of intracellular AF signal detected in AF− microglia (Figure 2A,B) were altered by aging, revealing a selective impact of aging on the AF+ microglia subset. Figure 2 with 1 supplement see all Download asset Open asset Natural aging increases microglia AF signal intensity solely within AF+ cells. (A) Representative imaging flow cytometry images and (B) corresponding quantitation of average AF area per cell in AF+ and AF− CD45dimCD11B+ microglia isolated from 3- and 10-month old mice. Significance established with 2-way ANOVA followed by Sidak’s post-hoc correction, n = 4000–8000 cells. (C) Representative flow cytometry histogram and (D) corresponding quantitation of AF intensity in AF+ CD45dim CD11B+ microglia isolated from mice at the indicated ages and presented as normalized values to AF levels observed in 3 month old animals. Significance established with 1-way ANOVA followed by Dunnett’s post-hoc. The line depicts the linear regression (R2 = 0.95). (E) Percent AF+ microglia at indicated ages. Significance established with 1-way ANOVA followed by Dunnett’s post-hoc. All data are presented as mean ± SD. For panels C-E, n ≥ 6 animals per age from at least two independent experiments. BF = bright field, AF = autofluorescence, SSC = side scatter, ***p<0.001. See also Figure 2—figure supplement 1. AF+ microglia selectively accumulate intracellular storage bodies with age We next isolated AF+ and AF− microglia from 3- and 18-month-old mice using fluorescence-activated cell sorting (FACS) and performed transmission electron microscopy (TEM) analyses. At 3 months of age, the intracellular organization of AF microglia subsets differed in that AF+ cells almost systematically contained large storage bodies filled with osmophilic electron-dense deposits (Figure 3A). These electron-dense storage bodies were observed in 80% of AF+ microglia whereas only 46% of AF− microglia contained electron-dense organelles (Figure 3A,C). When observed in AF− cells, storage bodies were devoid of complex storage material and were more regularly shaped than those observed in AF+ cells (Figure 3A). In aged mice, the storage bodies within AF+ microglia changed dramatically in both size and complexity (Figure 3A). Between 3 and 18 months of age, the percentage of visible cytoplasm occupied by storage bodies increased from 9% to 23% on average and approximately 30% of AF+ cells from aged mice contained AF material that occupied more than a third of the cytoplasm (Figure 3D). The ultrastructural composition of the storage bodies in AF+ cells varied with frequent curvilinear (black arrowheads) and fingerprint-like profiles (red arrowheads) (Figure 3B). White, linear, rod-like material with fine-tipped ends (white arrowheads) were observed within membrane-bound lipoid bodies and closely resembled cholesterol crystal deposits (Figure 3B). Most prominent, however, was the proportion and volume of lipid droplets (asterisks) (Figure 3B). In contrast, both the frequency of cells containing storage bodies and the proportion of the cytosol occupied by storage bodies remained unchanged in the AF− microglia subset (Figure 3A,C,D). Figure 3 Download asset Open asset AF+ microglia selectively accumulate intracellular storage bodies with age. (A) Representative TEM images of sorted microglia subsets from 3- to 18-month-old mice. Red arrowheads point to storage bodies. Scale bars = 2 µm. Insets depict higher magnification images of selected AF+ microglia (dashed square lines). Inset scale bars = 500 nm. (B) Representative TEM images of storage bodies observed within aged AF+ microglia. Black arrowheads, curvilinear storage material; white arrowheads, crystal bodies; red arrows, fingerprint-like storage material; asterisks, lipid storage content. Scale bars = 500 nm. (C) Frequency of cells containing storage bodies in microglia AF subsets sorted and pooled from n = 10 mice at the indicated ages. Significance established with Fischer’s exact test. n = 48–59 cells/group. (D) Average percent of cytosolic area occupied by storage material in cells containing at least one storage body. Significance established with 2-way ANOVA followed by Tukey’s post-hoc. n = 11–21 cells/group. (E) Representative flow cytometry histograms and (F) corresponding quantitation of LAMP1 and CD68 staining in AF− and AF+ subsets at indicated ages. Relative levels of LAMP1 and CD68 calculated as net geometric mean fluorescence intensity after subtraction of background AF signal and normalization to AF intensity detected in the AF− subset at 3 months of age. Significance established with 2-way repeated-measures ANOVA followed by Tukey’s post-hoc. n ≥ 6 animals per age from at least two independent experiments. All data presented as mean ± SD. ns = not significant, **p<0.01 ***p<0.001. In addition to these ultrastructural differences, AF+ cells expressed higher levels of LAMP1 and CD68 compared to AF− cells (Figure 3E,F), indicating an enlargement of endolysosomal storage compartments in AF+ cells. Furthermore, a gradual age-dependent increase in LAMP1 and CD68 protein levels was observed in the AF+ subset (Figure 3E,F) whereas AF− microglia did not show changes. Altogether these observations indicated that AF+ microglia differed from AF− cells by their unique accumulation of endolysosomal storage compartments with aging. Microglia AF subsets exhibit differential population dynamics upon depletion and replenishment of the microglia niche To explore the cellular dynamics and ontogenic relationships between these two novel subsets of microglia, we depleted microglia in 14-month-old mice with the CSF1R-small molecule antagonist BLZ945 (Krauser et al., 2015; Pyonteck et al., 2013). Twenty-four hours following the treatment period, depletion was nearly complete, as assessed by the remaining frequency and absolute numbers of microglia (Figure 4A,B and data not shown). Consistent with previous reports (Huang et al., 2018; O’Neil et al., 2018), microglia rapidly repopulated the CNS at 7 and 14 days post-treatment, recovering on average to 25% (at 7 days) and 87% (at 14 days) of steady-state microglia numbers (Figure 4A and data not shown). However, repopulation by AF+ and AF− subsets showed distinct kinetics. While AF− microglia cell numbers reached 67% of steady-state levels by day 7, AF+ cells only repopulated to 4% of steady-state levels by that time (Figure 4B). At 14 days repopulating AF+ cell numbers only reached 14% of steady-state AF+ values in vehicle-treated mice while the repopulating AF− subset surpassed steady-state levels by approximately 2-fold (Figure 4B) before normalizing to steady-state levels at day 70. Altogether these results established that the AF− subset was the first subset to repopulate the depleted brain while the repopulation by AF+ cells was delayed, suggesting a possible conversion from AF− to AF+ state during repopulation. Figure 4 with 1 supplement see all Download asset Open asset Microglia AF subsets exhibit differential population dynamics upon depletion and replenishment of the microglia niche. (A) Representative flow cytometry scatterplots showing CD45 and CD11B surface levels in brain single cell suspensions from mice at indicated timepoints post treatment with vehicle or BLZ945 and (B) corresponding quantitation of absolute cell numbers of AF+ and AF− microglia post-treatment. Significance established with 1-way ANOVA followed by Dunnett’s post-hoc test. (C) Representative flow cytometry histograms and (D) corresponding quantitation of AF intensity in the entire microglia population detected across multiple combinations of excitation lasers and emission filters and normalized to levels observed in vehicle-treated mice. In the scatter plot, the line represents the linear regression (R2 = 0.99). Significance established with 1-way ANOVA followed by Dunnett’s post-hoc test for each AF channel. (E) Representative flow cytometry scatterplots depicting KI-67 levels and AF intensity in microglia isolated at indicated time points post-treatment and (F) corresponding quantitation of the percent microglia positive for KI-67. Significance established with Welch’s ANOVA followed by Dunnett’s T3 post-hoc. n ≥ 8 animals per genotype group from at least two independent experiments. All data represented as mean ± SD. d = day; h = hour; B = blue laser; YG = yellow green laser; AF = autofluorescence; ns = not significant, ***p<0.001. See also Figure 4—figure supplement 1. Supporting this hypothesis, the AF intensity of the repopulated microglia slowly increased from barely detectable levels at day seven to slightly increased levels at day 14, ultimately returning to a bimodal distribution by day 70 (Figure 4C,D). Even at the latter timepoint, only a small fraction of this newly-formed AF+ subset (19% on average) displayed AF intensity levels comparable to those seen in vehicle-treated mice (Figure 4C,D and Figure 4—figure supplement 1A,B) while most repopulating AF+ cells displayed 35% weaker AF signal intensity on average. This gradual accumulation of AF material over time in repopulating microglia supported the possibility that AF+ microglia were derived from the conversion of AF− cells during replenishment. Further validating this conclusion, 56% of microglia displayed positivity for the proliferation-associated marker KI-67 at day seven before returning to steady-state values by day 14 (Figure 4E,F). Because AF+ microglia were virtually absent during this early repopulation phase (Figure 4B), these proliferation kinetics implied that the AF− subset of microglia was the predominant subset responsible for the repopulation of the microglia compartment following depletion and that AF+ microglia were derived from the conversion over time of a subset of AF− microglia. Proteomic analysis of isolated AF+ and AF− microglia subsets reveals molecular differences in endolysosomal, autophagic and metabolic pathways To determine whether microglia AF subsets were distinct at the molecular level, FACS-isolated AF+ and AF− microglia were analyzed by nano liquid chromatography mass spectrometry (nLC-MS). A total of 4231 proteins were detected by label-free LC-MS/MS and after filtering for proteins quantified in at least 50% of samples in either subset, 3492 remained for analysis. 351 proteins showed significant differences in abundance between AF+ and AF− microglia (Benjamini-Hochberg adj. p value < 0.01, fold-change > |1.3|), with 254 and 97 upregulated and downregulated differentially expressed proteins (DEPs), respectively (Figure 5A and Figure 5—source data 1). When ranked by significance, 32 of the top 50 DEPs upregulated in AF+ microglia were associated with endolysosomal biology, among which was LAMP1, validating prior flow cytometry results (Figure 3E,F). Furthermore, a large number of lysosomal enzymes were upregulated in AF+ microglia including cathepsins (CTSA, CTSB, CTSD, CTSF, CTSL, CTSZ) as well as several enzymes involved in lysosomal degradation such as amidases (ASAH1, GBA, NAAA), thioesterases (PPT1, PSAP, NAGA), proteases (TPP1, LGMN) and glycosyl hydrolases (HEXA, HEXB, GLB1). Many other DEPs involved in the biology, trafficking, or fusion of endosomes with lysosomes and phagosomes (ATP6V0D1, SCARB2, GRN, ARL8B, TOM1, STX7, TMEM55B) were also upregulated in AF+ microglia. Underscoring the importance of these proteins towards maintaining proper CNS homeostasis, genetic perturbations in 15 of the top 50 DEPs were associated with severe CNS-related storage disorders, such as Neuronal Ceroid Lipofucinosis and Niemann-Pick. Lastly, DEPs that are typically associated with neurons (CBLN1, CBLN4, SNAP25), oligodendrocytes (MOBP, SYNE2) and astrocytes (GJA1) were detected at significantly higher levels in AF+ microglia, suggesting functional differences in either the phagocytic capacity of this subset or its ability to fully degrade ingested material. Figure 5 Download asset Open asset Proteomic analysis of isolated AF+ and AF− microglia subsets reveals molecular differences in endolysosomal, autophagic and metabolic pathways. (A) Volcano plot comparing protein abundance in AF+ and AF− microglia (x-axis: Log2 abundance difference, y-axis: negative log Benjamini-Hochberg adjusted p value from two-tailed Student’s t-test) with most-differentially expressed proteins annotated. Red lines: significance cutoffs (adjusted p value < 0.01, |Fold change| > 1.3). (B) Scatter plot displaying adjusted p value and GO term fold-enrichment results among differentially expressed proteins (DEPs) upregulated in AF+, with selected pathways highlighted. Dot color indicates number of proteins within GO pathway detected in the dataset (see legend). (C) Volcano plots comparing protein levels in AF+ and AF− microglia with indicated GO term signatures overlaid in red. p value, χ2 statistical test for bias in distribution of GO term pathway members. (D) Top canonical pathways and (E) upstream regulators identified by IPA as differentially regulated between AF subsets. (D) Bars and dots respectively indicate p values and the percent of pathway proteins detected. (D, E) Predicted activation or inhibition of pathway or transcriptional regulator is indicated by positive and negative Z-scores, respectively, see legend. Analysis based on 351 DEPs identified using adjusted p value < 0.01 and fold change > |1.3|. See also Figure 5—source data 1, 2 and 3. Figure 5—source data 1 AF+ versus AF- subset associated proteins. Sheet 1: Description of the column headers. Sheet 2: Proteins which were differentially regulated (adj. p value < 0.05) between subsets. https://cdn.elifesciences.org/articles/57495/elife-57495-fig5-data1-v2.xlsx Download elife-57495-fig5-data1-v2.xlsx Figure 5—source data 2 Panther overrepresentation test of AF+ and AF- associated proteins. Sheet 1: Description of the column headers. Sheet 2: Pathway overrepresentation testing for proteins upregulated in AF+ microglia. Sheet 3: Pathway overrepresentation testing for proteins upregulated in AF- microglia. https://cdn.elifesciences.org/articles/57495/elife-57495-fig5-data2-v2.xlsx Download elife-57495-fig5-data2-v2.xlsx Figure 5—source data 3 Ingenuity pathway analysis of differentially regulated proteins in AF subsets. Sheet 1: Description of the column headers. Sheet 2: Overlap of differentially regulated proteins with IPA canonical pathways based on input list of 351 proteins with adj. p val <0.01 and fold change > |1.3|. Sheet 3: Upstream regulators identified by IPA based on input list of 351 differentially regulated proteins with adj. p val <0.01 and fold change > |1.3|. https://cdn.elifesciences.org/articles/57495/elife-57495-fig5-data3-v2.xlsx Download elife-57495-fig5-data3-v2.xlsx To systematically explore additional pathways distinguishing AF subsets at the molecular level, Gene Ontology (GO) term enrichment was performed with Panther (Figure 5B and Figure 5—source data 2). DEPs upregulated in AF+ microglia were enriched in pathways related to intracellular vesicle-mediated transport, lysosomal organization, protein transport, lipid catabolic processes and regulation of TOR signaling (Figure 5B,C). DEPs downregulated in AF+ microglia showed enrichment in RNA-related biological processes (transcription, RNA metabolism, splicing) and chromatin silencing. In agreement with GO term enrichment analysis, the top canonical pathways identified by Ingenuity Pathway Analysis (IPA) as enriched in AF+ microglia included phagosome maturation, autophagy, numerous catabolic pathways (amino acid and glutaryl-coA catabolism, ketogenesis and fatty acid β-oxidation), mitochondrial dysfunction, and pathways that were indicative of mTOR deregulation (mTOR, AMPK) (Figure 5D and Figure 5—source data 3). Finally, upstream regulators predicted to explain the distinct proteome displayed by AF+ cells included transcription factors involved in the regulation of cell cycle, senescence and apoptosis (TP53, MYC, CDKN2A), ER stress and unfolded protein response (XBP1), autophagy and lysosomal biogenesis (TFEB) and inflammatory responses (NFKBIA, PPARA) (Figure 5E and Figure 5—source data

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most destructive disease of rice and causes tremendous losses of rice yield worldwide. To explore the molecular mechanisms involved in the rice–M. oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae. This data article contains additional results and analysis of M. oryzae-regulated phosphoproteins in rice leaves [1]. We report the analysis of M. oryzae-regulated phosphoproteins at all time points, including Venn diagram analysis, close-up views, relative intensities, and functional category, and the MS spectra of representative phosphoprotein and representative phosphorylated peptides.

TRPML1 is a cation channel belonging to the transient receptor potential (TRP) superfamily whose localization is commonly assigned to late endosomes and lysosomes due to the presence of the lysosomal localization signals on its C- and N-termini. Because mutations in the TRPML1-coding gene MCOLN1 are responsible for the lysosomal storage disease mucolipidosis type IV (MLIV), the majority of research into this ion channel has been focused on its role in preventing the buildup of undigested material in the endolysosomal pathway. Recent data suggest that TRPML1 contributes to processes beyond the degradative, recycling, and sorting functions of late-endosomes and lysosomes. Indeed, modulation of endolysosomal function using TRPML1 activators and inhibitors promises new approaches to such conditions as cancer, immune disorders, and neurodegenerative diseases. This chapter is focused on processes shown or inferred to depend on TRPML1 and predicting new uses for modulating activity of this interesting channel.

Despite regionally-dependent heterogeneity, microglia subsets in healthy brain have not been identified. Utilizing autofluorescence (AF) as a discriminating parameter, we identified two novel microglia subsets in both mice and non-human primates, termed autofluorescence-positive (AF+) and autofluorescence-negative (AF-). While AF subsets maintained a consistent ratio throughout adulthood, AF within AF+ microglia linearly increased with age. Transmission electron microscopy of AF subsets revealed a selective and age-dependent increase in size and complexity of storage bodies in AF+ cells, correlating with elevated LAMP1 levels and suggesting that storage bodies accounted for AF. AF accumulation was respectively accelerated and impaired by lysosomal and autophagy disruption in Cln3- and Atg5-deficient mice, but unaltered in Trem2-/-. Aging and Cln3-deficiency correlated with oxidative stress, increased apoptosis, and cellular loss of AF+ cells. Post-depletion repopulation kinetics revealed AF- cells as precursors of AF+ microglia via gradual accumulation of storage bodies to a set proportion in the healthy brain.