P. Hande Özdinler

The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.

Abstract Background The involvement of non-neuronal cells and the cells of innate immunity has been attributed to the initiation and progression of ALS. TDP-43 pathology is observed in a broad spectrum of ALS cases and is one of the most commonly shared pathologies. The potential involvement of the neuroimmune axis in the motor cortex of ALS patients with TDP-43 pathology needs to be revealed. This information is vital for building effective treatment strategies. Methods We investigated the presence of astrogliosis and microgliosis in the motor cortex of ALS patients with TDP-43 pathology. prpTDP-43 A315T -UeGFP mice, corticospinal motor neuron (CSMN) reporter line with TDP-43 pathology, are utilized to reveal the timing and extent of neuroimmune interactions and the involvement of non-neuronal cells to neurodegeneration. Electron microscopy and immunolabeling techniques are used to mark and monitor cells of interest. Results We detected both activated astrocytes and microglia, especially rod-like microglia, in the motor cortex of patients and TDP-43 mouse model. Besides, CCR2+ TMEM119- infiltrating monocytes were detected as they penetrate the brain parenchyma. Interestingly, Betz cells, which normally do not express MCP1, were marked with high levels of MCP1 expression when diseased. Conclusions There is an early contribution of a neuroinflammatory response for upper motor neuron (UMN) degeneration with respect to TDP-43 pathology, and MCP1-CCR2 signaling is important for the recognition of diseased upper motor neurons by infiltrating monocytes. The findings are conserved among species and are observed in both ALS and ALS-FTLD patients.

Mitochondrial defects result in dysregulation of metabolomics and energy homeostasis that are detected in upper motor neurons (UMNs) with TDP-43 pathology, a pathology that is predominantly present in both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). While same mitochondrial problems are present in the UMNs of ALS patients with TDP-43 pathology and UMNs of TDP-43 mouse models, and since pathologies are shared at a cellular level, regardless of species, we first analyzed the metabolite profile of both healthy and diseased motor cortex to investigate whether metabolomic changes occur with respect to TDP-43 pathology. High-performance liquid chromatography, high-resolution mass spectrometry and tandem mass spectrometry (HPLC-MS/MS) for metabolite profiling began to suggest that reduced levels of NAD+ is one of the underlying causes of metabolomic problems. Since nicotinamide mononucleotide (NMN) was reported to restore NAD+ levels, we next investigated whether NMN treatment would improve the health of diseased corticospinal motor neurons (CSMN, a.k.a. UMN in mice). prpTDP-43A315T-UeGFP mice, the CSMN reporter line with TDP-43 pathology, allowed cell-type specific responses of CSMN to NMN treatment to be assessed in vitro. Our results show that metabolomic defects occur early in ALS motor cortex and establishing NAD+ balance could offer therapeutic benefit to UMNs with TDP-43 pathology.

Mitochondrial defects are one of the common underlying causes of neuronal vulnerability in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is the most commonly observed proteinopathy. Disrupted inner mitochondrial membrane (IMM) reported in the upper motor neurons (UMNs) of ALS patients with TDP-43 pathology is recapitulated in the UMNs of well-characterized hTDP-43 mouse model of ALS. The construct validity, such as shared and common cellular pathology in mice and human, offers a unique opportunity to test treatment strategies that may translate to patients. SBT-272 is a well-tolerated brain-penetrant small molecule that stabilizes cardiolipin, a phospholipid found in IMM, thereby restoring mitochondrial structure and respiratory function. We investigated whether SBT-272 can improve IMM structure and health in UMNs diseased with TDP-43 pathology in our well-characterized UMN reporter line for ALS. We found that SBT-272 significantly improved mitochondrial structural integrity and restored mitochondrial motility and function. This led to improved health of diseased UMNs in vitro. In comparison to edaravone and AMX0035, SBT-272 appeared more effective in restoring health of diseased UMNs. Chronic treatment of SBT-272 for sixty days starting at an early symptomatic stage of the disease in vivo led to a significant reduction in astrogliosis, microgliosis, and TDP-43 pathology in the ALS motor cortex. Our results underscore the therapeutic potential of SBT-272, especially within the context of TDP-43 pathology and mitochondrial dysfunction.

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1D83G/D83G mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1D83G/D83G mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1−/−). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.

Objective The aim of this study was to investigate the role of ubiquitin C-terminal hydrolase-L1 (UCHL1) for motor neuron circuitry and especially in spinal motor neuron (SMN) health, function, and connectivity. Methods Since mutations in UCHL1 gene leads to motor dysfunction in patients, we investigated the role of UCHL1 on SMN survival, axon health, and connectivity with the muscle, by employing molecular and cellular marker expression analysis and electrophysiological recordings, in healthy wild-type and Uchl1nm3419 (UCHL1−/−) mice, which lack all UCHL1 function. Results There is pure motor neuropathy with selective degeneration of the motor, but not sensory axons in the absence of UCHL1 function. Neuromuscular junctions (NMJ) are impaired in muscle groups that are innervated by slow-twitch or fast-twitch SMN. However, unlike corticospinal motor neurons, SMN cell bodies remain intact with no signs of elevated endoplasmic reticulum (ER) stress. Interpretation Presence of NMJ defects and progressive retrograde axonal degeneration in the absence of major SMN soma loss suggest that defining pathology as a function of neuron number is misleading and that upper and lower motor neurons utilize UCHL1 function in different cellular events. In line with findings in patients with mutations in UCHL1 gene, our results suggest a unique role of UCHL1, especially for motor neuron circuitry. SMN require UCHL1 to maintain NMJ and motor axon health, and that observed motor dysfunction in the absence of UCHL1 is not due to SMN loss, but mostly due to disintegrated circuitry.

Upper motor neurons (UMNs) are a key component of motor neuron circuitry. Their degeneration is a hallmark for diseases, such as hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), and amyotrophic lateral sclerosis (ALS). Currently there are no preclinical assays investigating cellular responses of UMNs to compound treatment, even for diseases of the UMNs. The basis of UMN vulnerability is not fully understood, and no compound has yet been identified to improve the health of diseased UMNs: two major roadblocks for building effective treatment strategies.Novel UMN reporter models, in which UMNs that are diseased because of misfolded superoxide dismutase protein (mSOD1) toxicity and TDP-43 pathology are labeled with eGFP expression, allow direct assessment of UMN response to compound treatment. Electron microscopy reveals very precise aspects of endoplasmic reticulum (ER) and mitochondrial damage. Administration of NU-9, a compound initially identified based on its ability to reduce mSOD1 toxicity, has profound impact on improving the health and stability of UMNs, as identified by detailed cellular and ultrastructural analyses.Problems with mitochondria and ER are conserved in diseased UMNs among different species. NU-9 has drug-like pharmacokinetic properties. It lacks toxicity and crosses the blood brain barrier. NU-9 improves the structural integrity of mitochondria and ER, reduces levels of mSOD1, stabilizes degenerating UMN apical dendrites, improves motor behavior measured by the hanging wire test, and eliminates ongoing degeneration of UMNs that become diseased both because of mSOD1 toxicity and TDP-43 pathology, two distinct and important overarching causes of motor neuron degeneration.Mechanism-focused and cell-based drug discovery approaches not only addressed key cellular defects responsible for UMN loss, but also identified NU-9, the first compound to improve the health of diseased UMNs, neurons that degenerate in ALS, HSP, PLS, and ALS/FTLD patients.

Amyotrophic lateral sclerosis (ALS) is one of the most complex motor neuron diseases. Even though scientific discoveries are accelerating with an unprecedented pace, to date more than 30 clinical trials have ended with failure and staggering frustration. There are too many compounds that increase life span in mice, but too little evidence that they will improve human condition. Increasing the chances of success for future clinical trials requires advancement of preclinical tests. Recent developments, which enable the visualization of diseased motor neurons, have the potential to bring novel insight. As we change our focus from mice to motor neurons, it is possible to foster a new vision that translates into effective and long-term treatment strategies in ALS and related motor neuron disorders (MND).

Developing effective treatment strategies for neurodegenerative diseases require an understanding of the underlying cellular pathways that lead to neuronal vulnerability and progressive degeneration. To date, numerous mutations in 147 distinct genes are identified to be "associated" with, "modifier" or "causative" of amyotrophic lateral sclerosis (ALS). Protein products of these genes and their interactions helped determine the protein landscape of ALS, and revealed upstream modulators, key canonical pathways, interactome domains and novel therapeutic targets. Our analysis originates from known human mutations and circles back to human, revealing increased PPARG and PPARGC1A expression in the Betz cells of sALS patients and patients with TDP43 pathology, and emphasizes the importance of lipid homeostasis. Downregulation of YWHAZ, a 14-3-3 protein, and cytoplasmic accumulation of ZFYVE27 especially in diseased Betz cells of ALS patients reinforce the idea that perturbed protein communications, interactome defects, and altered converging pathways will reveal novel therapeutic targets in ALS.

Amyotrophic lateral sclerosis (ALS) is a complex disease, that affects the motor neuron circuitry. After consecutive failures in clinical trials for the past 20 years, edaravone was recently approved as the second drug for ALS. This generated excitement in the field revealed the need to improve preclinical assays for continued success. Here, we focus on the importance and relevance of upper motor neuron (UMN) pathology in ALS, and discuss how incorporation of UMN survival in preclinical assays will improve inclusion criteria for clinical trials and expedite the drug discovery effort in ALS and related motor neuron diseases.

There are no effective cures for upper motor neuron (UMN) diseases, such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and hereditary spastic paraplegia. Here, we show UMN loss occurs independent of spinal motor neuron degeneration and that UMNs are indeed effective cellular targets for gene therapy, which offers a potential solution especially for UMN disease patients. UCHL1 (ubiquitin C-terminal hydrolase-L1) is a deubiquitinating enzyme crucial for maintaining free ubiquitin levels. Corticospinal motor neurons (CSMN, a.k.a UMNs in mice) show early, selective, and profound degeneration in Uchl1nm3419 (UCHL1−/−) mice, which lack all UCHL1 function. When UCHL1 activity is ablated only from spinal motor neurons, CSMN remained intact. However, restoring UCHL1 specifically in CSMN of UCHL1−/− mice via directed gene delivery was sufficient to improve CSMN integrity to the healthy control levels. In addition, when UCHL1 gene was delivered selectively to CSMN that are diseased due to misfolded SOD1 toxicity and TDP-43 pathology via AAV-mediated retrograde transduction, the disease causing misfolded SOD1 and mutant human TDP-43 were reduced in hSOD1G93A and prpTDP-43A315T models, respectively. Diseased CSMN retained their neuronal integrity and cytoarchitectural stability in two different mouse models that represent two distinct causes of neurodegeneration in ALS.

Abstract Even though amyotrophic lateral sclerosis (ALS) is a disease of the upper and lower motor neurons, to date none of the compounds in clinical trials have been tested for improving the health of diseased upper motor neurons (UMNs). There is an urgent need to develop preclinical assays that include UMN health as a readout. Since ALS is a complex disease, combinatorial treatment strategies will be required to address the mechanisms perturbed in patients. Here, we describe a novel in vitro platform that takes advantage of an UMN reporter line in which UMNs are genetically labeled with fluorescence and have misfolded SOD1 toxicity. We report that NU-9 , an analog of the cyclohexane-1,3-dione family of compounds, improves the health of UMNs with misfolded SOD1 toxicity more effectively than riluzole or edaravone, -the only two FDA-approved ALS drugs to date-. Interestingly, when NU-9 is applied in combination with riluzole or edaravone, there is an additive effect on UMN health, as they extend longer axons and display enhanced branching and arborization, two important characteristics of healthy UMNs in vitro.

Brain is complex and heterogeneous. Even though numerous independent studies indicate cortical hyperexcitability as a potential contributor to amyotrophic lateral sclerosis (ALS) pathology, the mechanisms that are responsible for upper motor neuron (UMN) vulnerability remain elusive. In an effort to reveal both intrinsic and extrinsic factors that contribute to corticospinal motor neuron (CSMN, a.k.a UMN in mice) vulnerability, we investigated the motor cortex of hSOD1G93A mice at P30 (postnatal day 30), ¬a presymptomatic time point. Glutamate uncaging by laser scanning photostimulation revealed altered dynamics especially within the inhibitory circuitry and more specifically in L2/3 of the cortex, whereas the excitatory microcircuits were unchanged. Observed microcircuitry changes were specific to CSMN in the motor column. Electrophysiological evaluation of the intrinsic properties in response to the microcircuit changes, as well as the exon microarray expression profiles of CSMN isolated from hSOD1G93A and healthy mice at P30 revealed the presence of a very dynamic set of events, ultimately directed to establish, maintain and retain the balance at this early stage. In addition, the expression profile of key voltage gated potassium and sodium channel subunits as well as of the inhibitory GABA receptor subunits and modulatory proteins began to suggest the challenges CSMN face at this early stage. Since neurodegeneration is initiated when neurons can no longer maintain balance, the complex cellular events that occur at this critical time point help reveal how CSMN try cope with challenges of disease manifestation. This information is critically important for proper modulation of upper motor neurons and for developing effective treatment strategies in the near future.

In neurodegenerative diseases, a select set of neuron population displays early vulnerability and undergoes progressive degeneration. The heterogeneity of the cerebral cortex and the heterogeneity of patient populations diagnosed with the same disease offer many challenges for developing effective and long‐term treatment options. Currently, patients who are considered to have a ‘rare’ disease are left with no hopes for cure, and many of the neurodegenerative diseases progress fast without any effective solutions. However, as our understanding of disease mechanisms evolve, we begin to realize that the boundaries between diseases are not as sharp as once believed. There are many patients who develop disease due to common underlying causes and mechanisms. As we move forward with drug discovery effort, it becomes obvious that we will have to shift our focus from finding a cure for a disease, to finding solutions to the disease‐causing cellular mechanisms so that patients can be treated by mechanism‐based strategies. This paradigm shift will lay the foundation for personalized medicine approaches for neurodegenerative disease patients and patients diagnosed with a rare disease.

While much is known about general controls over axon guidance of broad classes of projection neurons (those with long-distance axonal connections), molecular controls over specific axon targeting by distinct neuron subtypes are poorly understood. Corticospinal motor neurons (CSMN) are prototypical and clinically important cerebral cortex projection neurons; they are the brain neurons that degenerate in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases, and their injury is central to the loss of motor function in spinal cord injury. Primary culture of purified immature murine CSMN has been recently established, using either fluorescence-activated cell sorting (FACS) or immunopanning, enabling a previously unattainable level of subtype-specific investigation, but the resulting number of CSMN is quite limiting for standard approaches to study axon guidance. We developed a microfluidic system specifically designed to investigate axon targeting of limited numbers of purified CSMN and other projection neurons in culture. The system contains two chambers for culturing target tissue explants, allowing for biologically revealing axonal growth "choice" experiments. This device will be uniquely enabling for investigation of controls over axon growth and neuronal survival of many types of neurons, particularly those available only in limited numbers.

Abstract Mitochondrial defects are one of the common underlying causes of neuronal vulnerability in motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is the most common proteinopathy in ALS. Disrupted inner mitochondrial membrane (IMM) reported in the upper motor neurons (UMNs) of ALS patients with TDP-43 pathology is recapitulated in the UMNs of well-characterized mutant hTDP-43 mouse models of ALS. The construct validity, such as common cellular pathology in mice and human, offers a unique opportunity to test treatment strategies that may translate. SBT-272 is a well-tolerated brain-penetrant small molecule that stabilizes cardiolipin, a phospholipid found in IMM, thereby restoring mitochondrial structure and respiratory function. We investigated whether SBT-272 can improve IMM structure and health in UMNs diseased with TDP-43 pathology in our well-characterized UMN reporter line for ALS. We found that SBT-272 significantly improved mitochondrial structural integrity and restored mitochondrial motility and function. This led to improved health of diseased UMNs in vitro. In comparison to edaravone and AMX0035, SBT-272 appeared more effective in restoring health of diseased UMNs. Chronic treatment of SBT-272 for sixty days starting at an early symptomatic stage of the disease in vivo led to a reduction in astrogliosis, microgliosis, and retention of UMN degeneration in the ALS motor cortex. Our results underscore the therapeutic potential of SBT-272, especially within the context of TDP-43 pathology and mitochondrial dysfunction. Highlights Early and progressive upper motor neuron (UMN) degeneration defines ALS pathology Mitochondrial defects are prominent and common in UMNs with TDP-43 pathology SBT-272 treatment improves mitochondrial stability, mobility and function SBT-272 treatment reduces astrogliosis, microgliosis and improves UMN health