A spinal and bulbar muscular atrophy (SBMA) disease-specific human embryonic stem cell (hESC) line, UMICHe002-A/UM197-1.

Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked degenerative disorder of the neuromuscular system that is caused by an expanded CAG/polyglutamine (polyQ) tract within the Androgen Receptor (AR) gene. This mutation causes progressive muscle weakness and atrophy in men. Here, we report the establishment of the first SBMA disease-specific human embryonic stem cell (hESC) line in the NIH hESC registry, UM197-1. UM197-1 exhibits pluripotency, the ability to differentiate into three germ layers in vitro, and provides a new cellular model system to study SBMA disease pathogenesis.

Mutant induced neurons and humanized mice enable identification of Niemann-Pick C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step towards the development of disease-modifying therapies.

Human SIRT5 variants with reduced stability and activity do not cause neuropathology in mice.

SIRT5 is a sirtuin deacylase that removes negatively charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal conditions, the phenotypes of SIRT5 deficiency are quite subtle. Here, we identify two homozygous SIRT5 variants in patients suspected to have mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generated a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology, or other gross phenotypes. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, but are likely not by themselves the primary pathogenic cause of the neuropathology observed in the patients.

Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.

Combined cytotoxic and immune-stimulatory gene therapy for primary adult high-grade glioma: a phase 1, first-in-human trial.

BACKGROUND: High-grade gliomas have a poor prognosis and do not respond well to treatment. Effective cancer immune responses depend on functional immune cells, which are typically absent from the brain. This study aimed to evaluate the safety and activity of two adenoviral vectors expressing HSV1-TK (Ad-hCMV-TK) and Flt3L (Ad-hCMV-Flt3L) in patients with high-grade glioma. METHODS: In this dose-finding, first-in-human trial, treatment-naive adults aged 18-75 years with newly identified high-grade glioma that was evaluated per immunotherapy response assessment in neuro-oncology criteria, and a Karnofsky Performance Status score of 70 or more, underwent maximal safe resection followed by injections of adenoviral vectors expressing HSV1-TK and Flt3L into the tumour bed. The study was conducted at the University of Michigan Medical School, Michigan Medicine (Ann Arbor, MI, USA). The study included six escalating doses of viral particles with starting doses of 1×1010 Ad-hCMV-TK viral particles and 1×109 Ad-hCMV-Flt3L viral particles (cohort A), and then 1×1011 Ad-hCMV-TK viral particles and 1×109 Ad-hCMV-Flt3L viral particles (cohort B), 1×1010 Ad-hCMV-TK viral particles and 1×1010 Ad-hCMV-Flt3L viral particles (cohort C), 1×1011 Ad-hCMV-TK viral particles and 1×1010 Ad-hCMV-Flt3L viral particles (cohort D), 1×1010 Ad-hCMV-TK viral particles and 1×1011 Ad-hCMV-Flt3L viral particles (cohort E), and 1×1011 Ad-hCMV-TK viral particles and 1×1011 Ad-hCMV-Flt3L viral particles (cohort F) following a 3+3 design. Two 1 mL tuberculin syringes were used to deliver freehand a mix of Ad-hCMV-TK and Ad-hCMV-Flt3L vectors into the walls of the resection cavity with a total injection of 2 mL distributed as 0·1 mL per site across 20 locations. Subsequently, patients received two 14-day courses of valacyclovir (2 g orally, three times per day) at 1-3 days and 10-12 weeks after vector administration and standad upfront chemoradiotherapy. The primary endpoint was the maximum tolerated dose of Ad-hCMV-Flt3L and Ad-hCMV-TK. Overall survival was a secondary endpoint. Recruitment is complete and the trial is finished. The trial is registered with ClinicalTrials.gov, NCT01811992. FINDINGS: Between April 8, 2014, and March 13, 2019, 21 patients were assessed for eligibility and 18 patients with high-grade glioma were enrolled and included in the analysis (three patients in each of the six dose cohorts); eight patients were female and ten were male. Neuropathological examination identified 14 (78%) patients with glioblastoma, three (17%) with gliosarcoma, and one (6%) with anaplastic ependymoma. The treatment was well-tolerated, and no dose-limiting toxicity was observed. The maximum tolerated dose was not reached. The most common serious grade 3-4 adverse events across all treatment groups were wound infection (four events in two patients) and thromboembolic events (five events in four patients). One death due to an adverse event (respiratory failure) occurred but was not related to study treatment. No treatment-related deaths occurred during the study. Median overall survival was 21·3 months (95% CI 11·1-26·1). INTERPRETATION: The combination of two adenoviral vectors demonstrated safety and feasibility in patients with high-grade glioma and warrants further investigation in a phase 1b/2 clinical trial. FUNDING: Funded in part by Phase One Foundation, Los Angeles, CA, The Board of Governors at Cedars-Sinai Medical Center, Los Angeles, CA, and The Rogel Cancer Center at The University of Michigan.

Apolipoprotein-mimetic nanodiscs reduce lipid accumulation and improve liver function in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a severe lipid storage disorder caused by the diminished activity of the acid sphingomyelinase enzyme. ASMD is characterized by the accumulation of sphingomyelin in late endosomes and lysosomes leading to progressive neurological dysfunction and hepatosplenomegaly. Our objective was to investigate the utility of synthetic apolipoprotein A-I (ApoA-I) mimetics designed to act as lipid scavengers for the treatment of ASMD. We determined the lead peptide, 22A, could reduce sphingomyelin accumulation in ASMD patient skin fibroblasts in a dose dependent manner. Intraperitoneal administration of 22A formulated as a synthetic high-density lipoprotein (sHDL) nanodisc mobilized sphingomyelin from peripheral tissues into circulation and improved liver function in a mouse model of ASMD. Together, our data demonstrates that apolipoprotein mimetics could serve as a novel therapeutic strategy for modulating the pathology observed in ASMD.

Spinal and bulbar muscular atrophy: From molecular pathogenesis to pharmacological intervention targeting skeletal muscle.

The clinical characteristics of SBMA, also known as Kennedy's disease (OMIM 313200), were initially documented by Dr. H Kawahara in the 18th century and a hundred years later by Dr. W. Kennedy. SBMA is a neuromuscular disease caused by expansions of a CAG microsatellite tandem repeat in exon 1 of the androgen receptor (AR) gene located on the X chromosome. These expansions result in the production of AR with an aberrantly expanded polyglutamine (polyQ) tract. In this review, we explore recent advancements in the significance of gene expression changes in skeletal muscle and discuss how pharmacological interventions targeting this aspect of disease pathogenesis can potentially be translated into therapies for SBMA patients.

The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells.

The intracellular cholesterol transporter NPC1 functions in late endosomes and lysosomes to efflux unesterified cholesterol, and its deficiency causes Niemann-Pick disease Type C, an autosomal recessive lysosomal disorder characterized by progressive neurodegeneration and early death. Here, we use single-nucleus RNA-seq on the forebrain of Npc1-/- mice at P16 to identify cell types and pathways affected early in pathogenesis. Our analysis uncovers significant transcriptional changes in the oligodendrocyte lineage during developmental myelination, accompanied by diminished maturation of myelinating oligodendrocytes. We identify upregulation of genes associated with neurogenesis and synapse formation in Npc1-/- oligodendrocyte lineage cells, reflecting diminished gene silencing by H3K27me3. Npc1-/- oligodendrocyte progenitor cells reproduce impaired maturation in vitro, and this phenotype is rescued by treatment with GSK-J4, a small molecule inhibitor of H3K27 demethylases. Moreover, mobilizing stored cholesterol in Npc1-/- mice by a single administration of 2-hydroxypropyl-ß-cyclodextrin at P7 rescues myelination, epigenetic marks, and oligodendrocyte gene expression. Our findings highlight an important role for NPC1 in oligodendrocyte lineage maturation and epigenetic regulation, and identify potential targets for therapeutic intervention.

Niemann-Pick C1 protein regulates platelet membrane-associated calcium ion signaling in thrombo-occlusive diseases in mice.

BACKGROUND: Pathophysiologic platelet activation leads to thrombo-occlusive diseases such as myocardial infarction or ischemic stroke. Niemann-Pick C1 protein (NPC1) is involved in the regulation of lysosomal lipid trafficking and calcium ion (Ca2+) signaling, and its genetic mutation causes a lysosomal storage disorder. Lipids and Ca2+ are key players in the complex orchestration of platelet activation. OBJECTIVES: The present study aimed to determine the impact of NPC1 on Ca2+ mobilization during platelet activation in thrombo-occlusive diseases. METHODS: Using MK/platelet-specific knockout mice of Npc1 (Npc1Pf4∆/Pf4∆), ex vivo and in vitro approaches as well as in vivo models of thrombosis, we investigated the effect of Npc1 on platelet function and thrombus formation. RESULTS: We showed that Npc1Pf4∆/Pf4∆ platelets display increased sphingosine levels and a locally impaired membrane-associated and SERCA3-dependent Ca2+ mobilisation compared to platelets from wildtype littermates (Npc1lox/lox). Further, we observed decreased platelet. CONCLUSION: Our findings highlight that NPC1 regulates membrane-associated and SERCA3-dependent Ca2+ mobilization during platelet activation and that MK/platelet-specific ablation of Npc1 protects against experimental models of arterial thrombosis and myocardial or cerebral ischemia/reperfusion injury.

Defective excitation-contraction coupling and mitochondrial respiration precede mitochondrial Ca2+ accumulation in spinobulbar muscular atrophy skeletal muscle.

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived muscle biopsies, we show that SBMA mice in the presymptomatic stage develop a respiratory defect matching defective expression of genes involved in excitation-contraction coupling (ECC), altered contraction dynamics, and increased fatigue. These processes are followed by stimulus-dependent accumulation of calcium into mitochondria and structural disorganization of the muscle triads. Deregulation of expression of ECC genes is concomitant with sexual maturity and androgen raise in the serum. Consistent with the androgen-dependent nature of these alterations, surgical castration and AR silencing alleviate the early and late pathological processes. These observations show that ECC deregulation and defective mitochondrial respiration are early but reversible events followed by altered muscle force, calcium dyshomeostasis, and dismantling of triad structure.

Bicalutamide and Trehalose Ameliorate Spinal and Bulbar Muscular Atrophy Pathology in Mice.

Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.

The status of digital pathology and associated infrastructure within Alzheimer's Disease Centers.

Digital pathology (DP) has transformative potential, especially for Alzheimer disease and related disorders. However, infrastructure barriers may limit adoption. To provide benchmarks and insights into implementation barriers, a survey was conducted in 2019 within National Institutes of Health's Alzheimer's Disease Centers (ADCs). Questions covered infrastructure, funding sources, and data management related to digital pathology. Of the 35 ADCs to which the survey was sent, 33 responded. Most respondents (81%) stated that their ADC had digital slide scanner access, with the most frequent brand being Aperio/Leica (62.9%). Approximately a third of respondents stated there were fees to utilize the scanner. For DP and machine learning (ML) resources, 41% of respondents stated none was supported by their ADC. For scanner purchasing and operations, 50% of respondents stated they received institutional support. Some were unsure of the file size of scanned digital images (37%) and total amount of storage space files occupied (50%). Most (76%) were aware of other departments at their institution working with ML; a similar (76%) percentage were unaware of multiuniversity or industry partnerships. These results demonstrate many ADCs have access to a digital slide scanner; additional investigations are needed to further understand hurdles to implement DP and ML workflows.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Neuromuscular junction pathology is correlated with differential motor unit vulnerability in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, neuromuscular neurodegenerative disease for which there is no cure. The disease is characterized by a selective decrease in fast-muscle power (e.g., tongue pressure, grip strength) accompanied by a selective loss of fast-twitch muscle fibers. However, the relationship between neuromuscular junction (NMJ) pathology and fast-twitch motor unit vulnerability has yet to be explored. In this study, we used a cross-model comparison of two mouse models of SBMA to evaluate neuromuscular junction pathology, glycolytic-to-oxidative fiber-type switching, and cytoskeletal alterations in pre- and postsynaptic termini of tibialis anterior (TA), gastrocnemius, and soleus hindlimb muscles. We observed significantly increased NMJ and myofiber pathology in fast-twitch, glycolytic motor units of the TA and gastrocnemius compared to slow-twitch, oxidative motor units of the soleus, as seen by decreased pre- and post-synaptic membrane area, decreased pre- and post-synaptic membrane colocalization, increased acetylcholine receptor compactness, a decrease in endplate area and complexity, and deficits in neurofilament heavy chain. Our data also show evidence for metabolic dysregulation and myofiber atrophy that correlate with severity of NMJ pathology. We propose a model in which the dynamic communicative relationship between the motor neuron and muscle, along with the developmental subtype of the muscle, promotes motor unit subtype specific vulnerability, metabolic alterations, and NMJ pathology.

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage.

Niemann-Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.

TDP-43 proteinopathy occurs independently of autophagic substrate accumulation and underlies nuclear defects in Niemann-Pick C disease.

AIMS: Neuronal cytoplasmic inclusions of TAR-DNA binding protein of 43 kDa (TDP-43) are a pathological hallmark of diverse neurodegenerative disorders, yet the processes that mediate their formation and their functional significance remain incompletely understood. Both dysfunction in autophagy and neuroinflammation have been linked to TDP-43 mislocalisation. Here, we investigate TDP-43 proteinopathy in Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disease (LSD) distinguished by the accumulation of unesterified cholesterol within late endosomes and lysosomes. NPC is characterised by neurodegeneration, neuroinflammation and multifocal disruption of the autophagy pathway. METHODS: We utilised immunohistochemistry, confocal microscopy, electron microscopy and biochemical and gene expression studies to characterise TDP-43 pathology and autophagic substrate accumulation in Npc1-deficient mice. RESULTS: In the NPC brain, cytoplasmic TDP-43 mislocalisation was independent of autophagic substrate accumulation. These pathologies occurred in distinct neuronal subtypes, as brainstem cholinergic neurons were more susceptible to TDP-43 mislocalisation, whereas glutamatergic neurons exhibited hallmarks of autophagic dysfunction. Furthermore, TDP-43 mislocalisation did not co-localise with markers of stress granules or progress to ubiquitinated aggregates over months in vivo, indicating a stable, early stage in the aggregation process. Neither microgliosis nor neuroinflammation were sufficient to drive TDP-43 proteinopathy in the NPC brain. Notably, cytoplasmic TDP-43 co-localised with the nuclear import factor importin α, and TDP-43 mislocalised neurons demonstrated nuclear membrane abnormalities and disruption of nucleocytoplasmic transport. CONCLUSION: Our findings highlight the relationship between LSDs and TDP-43 proteinopathy, define its functional importance in NPC by triggering nuclear dysfunction, and expand the spectrum of TDP-43 pathology in the diseased brain.