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.

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.

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.

Genetic approaches to the treatment of inherited neuromuscular diseases.

Inherited neuromuscular diseases are a heterogeneous group of developmental and degenerative disorders that affect motor unit function. Major challenges toward developing therapies for these diseases include heterogeneity with respect to clinical severity, age of onset and the primary cell type that is affected (e.g. motor neurons, skeletal muscle and Schwann cells). Here, we review recent progress toward the establishment of genetic therapies to treat inherited neuromuscular disorders that affect both children and adults with a focus on spinal muscular atrophy, Charcot-Marie-Tooth disease and spinal and bulbar muscular atrophy. We discuss clinical features, causative mutations and emerging approaches that are undergoing testing in preclinical models and in patients or that have received recent approval for clinical use. Many of these efforts employ antisense oligonucleotides to alter pre-mRNA splicing or diminish target gene expression and use viral vectors to replace expression of mutant genes. Finally, we discuss remaining challenges for optimizing the delivery and effectiveness of these approaches. In sum, therapeutic strategies for neuromuscular diseases have shown encouraging results, raising hope that recent strides will translate into significant clinical benefits for patients with these disorders.

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.

Targeting Hsp70 facilitated protein quality control for treatment of polyglutamine diseases.

The polyglutamine (polyQ) diseases are a group of nine fatal, adult-onset neurodegenerative disorders characterized by the misfolding and aggregation of mutant proteins containing toxic expansions of CAG/polyQ tracts. The heat shock protein 90 and 70 (Hsp90/Hsp70) chaperone machinery is a key component of cellular protein quality control, playing a role in the regulation of folding, aggregation, and degradation of polyQ proteins. The ability of Hsp70 to facilitate disaggregation and degradation of misfolded proteins makes it an attractive therapeutic target in polyQ diseases. Genetic studies have demonstrated that manipulation of Hsp70 and related co-chaperones can enhance the disaggregation and/or degradation of misfolded proteins in models of polyQ disease. Therefore, the development of small molecules that enhance Hsp70 activity is of great interest. However, it is still unclear if currently available Hsp70 modulators can selectively enhance disaggregation or degradation of misfolded proteins without perturbing other Hsp70 functions essential for cellular homeostasis. This review discusses the multifaceted role of Hsp70 in protein quality control and the opportunities and challenges Hsp70 poses as a potential therapeutic target in polyQ disease.

MEF2 impairment underlies skeletal muscle atrophy in polyglutamine disease.

Polyglutamine (polyQ) tract expansion leads to proteotoxic misfolding and drives a family of nine diseases. We study spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder of the neuromuscular system caused by the polyQ androgen receptor (AR). Using a knock-in mouse model of SBMA, AR113Q mice, we show that E3 ubiquitin ligases which are a hallmark of the canonical muscle atrophy machinery are not induced in AR113Q muscle. Similarly, we find no evidence to suggest dysfunction of signaling pathways that trigger muscle hypertrophy or impairment of the muscle stem cell niche. Instead, we find that skeletal muscle atrophy is characterized by diminished function of the transcriptional regulator Myocyte Enhancer Factor 2 (MEF2), a regulator of myofiber homeostasis. Decreased expression of MEF2 target genes is age- and glutamine tract length-dependent, occurs due to polyQ AR proteotoxicity, and is associated with sequestration of MEF2 into intranuclear inclusions in muscle. Skeletal muscle from R6/2 mice, a model of Huntington disease which develops progressive atrophy, also sequesters MEF2 into inclusions and displays age-dependent loss of MEF2 target genes. Similarly, SBMA patient muscle shows loss of MEF2 target gene expression, and restoring MEF2 activity in AR113Q muscle rescues fiber size and MEF2-regulated gene expression. This work establishes MEF2 impairment as a novel mechanism of skeletal muscle atrophy downstream of toxic polyglutamine proteins and as a therapeutic target for muscle atrophy in these disorders.

Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann-Pick diseases.

BACKGROUND: Niemann-Pick disease type C is a fatal and progressive neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes. We sought to develop new therapeutics for this disorder by harnessing the body's endogenous cholesterol scavenging particle, high-density lipoprotein (HDL). METHODS: Here we design, optimize, and define the mechanism of action of synthetic HDL (sHDL) nanoparticles. RESULTS: We demonstrate a dose-dependent rescue of cholesterol storage that is sensitive to sHDL lipid and peptide composition, enabling the identification of compounds with a range of therapeutic potency. Peripheral administration of sHDL to Npc1 I1061T homozygous mice mobilizes cholesterol, reduces serum bilirubin, reduces liver macrophage size, and corrects body weight deficits. Additionally, a single intraventricular injection into adult Npc1 I1061T brains significantly reduces cholesterol storage in Purkinje neurons. Since endogenous HDL is also a carrier of sphingomyelin, we tested the same sHDL formulation in the sphingomyelin storage disease Niemann-Pick type A. Utilizing stimulated Raman scattering microscopy to detect endogenous unlabeled lipids, we show significant rescue of Niemann-Pick type A lipid storage. CONCLUSIONS: Together, our data establish that sHDL nanoparticles are a potential new therapeutic avenue for Niemann-Pick diseases.

Inkjet-printed micro-calibration standards for ultraquantitative Raman spectral cytometry.

Herein we report the development of a cytometric analysis platform for measuring the contents of individual cells in absolute (picogram) scales; this study represents the first report of Raman-based quantitation of the absolute mass - or the total amount - of multiple endogenous biomolecules within single-cells. To enable ultraquantitative calibration, we engineered single-cell-sized micro-calibration standards of known composition by inkjet-printer deposition of biomolecular components in microarrays across the surface of silicon chips. We demonstrate clinical feasibility by characterizing the compositional phenotype of human skin fibroblast and porcine alveolar macrophage cell populations in the respective contexts of Niemann-Pick disease and drug-induced phospholipidosis: two types of lipid storage disorders. We envision this microanalytical platform as the foundation for many future biomedical applications, ranging from diagnostic assays to pathological analysis to advanced pharmaco/toxicokinetic research studies.

The human brainome: network analysis identifies HSPA2 as a novel Alzheimer’s disease target.

Our hypothesis is that changes in gene and protein expression are crucial to the development of late-onset Alzheimer’s disease. Previously we examined how DNA alleles control downstream expression of RNA transcripts and how those relationships are changed in late-onset Alzheimer’s disease. We have now examined how proteins are incorporated into networks in two separate series and evaluated our outputs in two different cell lines. Our pipeline included the following steps: (i) predicting expression quantitative trait loci; (ii) determining differential expression; (iii) analysing networks of transcript and peptide relationships; and (iv) validating effects in two separate cell lines. We performed all our analysis in two separate brain series to validate effects. Our two series included 345 samples in the first set (177 controls, 168 cases; age range 65–105; 58% female; KRONOSII cohort) and 409 samples in the replicate set (153 controls, 141 cases, 115 mild cognitive impairment; age range 66–107; 63% female; RUSH cohort). Our top target is heat shock protein family A member 2 (HSPA2), which was identified as a key driver in our two datasets. HSPA2 was validated in two cell lines, with overexpression driving further elevation of amyloid-β40 and amyloid-β42 levels in APP mutant cells, as well as significant elevation of microtubule associated protein tau and phosphorylated-tau in a modified neuroglioma line. This work further demonstrates that studying changes in gene and protein expression is crucial to understanding late onset disease and further nominates HSPA2 as a specific key regulator of late-onset Alzheimer’s disease processes.10.1093/brain/awy215_video1awy215media15824729224001.

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.

Disease Affects Bdnf Expression in Synaptic and Extrasynaptic Regions of Skeletal Muscle of Three SBMA Mouse Models.

Spinal bulbar muscular atrophy (SBMA) is a slowly progressive, androgen-dependent neuromuscular disease in men that is characterized by both muscle and synaptic dysfunction. Because gene expression in muscle is heterogeneous, with synaptic myonuclei expressing genes that regulate synaptic function and extrasynaptic myonuclei expressing genes to regulate contractile function, we used quantitative PCR to compare gene expression in these two domains of muscle from three different mouse models of SBMA: the "97Q" model that ubiquitously expresses mutant human androgen receptor (AR), the 113Q knock-in (KI) model that expresses humanized mouse AR with an expanded glutamine tract, and the "myogenic" model that overexpresses wild-type rat AR only in skeletal muscle. We were particularly interested in neurotrophic factors because of their role in maintaining neuromuscular function via effects on both muscle and synaptic function, and their implicated role in SBMA. We confirmed previous reports of the enriched expression of select genes (e.g., the acetylcholine receptor) in the synaptic region of muscle, and are the first to report the synaptic enrichment of others (e.g., glial cell line-derived neurotrophic factor). Interestingly, all three models displayed comparably dysregulated expression of most genes examined in both the synaptic and extrasynaptic domains of muscle, with only modest differences between regions and models. These findings of comprehensive gene dysregulation in muscle support the emerging view that skeletal muscle may be a prime therapeutic target for restoring function of both muscles and motoneurons in SBMA.

Genetic and pharmacological evidence implicates cathepsins in Niemann-Pick C cerebellar degeneration.

Niemann-Pick C1 (NPC) disease, an autosomal recessive lipid trafficking disorder caused by loss-of-function mutations in the NPC1 gene, is characterized by progressive neurodegeneration resulting in cognitive impairment, ataxia and early death. Little is known about the cellular pathways leading to neuron loss. Here, we studied the effects of diminishing expression of cystatin B, an endogenous inhibitor of cathepsins B, H and L, on the development of NPC neuropathology. We show that decreased expression of cystatin B in patient fibroblasts enhances cathepsin activity. Deletion of the encoding Cstb gene in Npc1-deficient mice resulted in striking deleterious effects, particularly within the cerebellum where diffuse loss of Purkinje cells was observed in young mice. This severe pathology occurred through cell autonomous mechanisms that triggered Purkinje cell death. Moreover, our analyses demonstrated the mislocalization of lysosomal cathepsins within the cytosol of Npc1-deficient Purkinje cells. We provide evidence that this may be a consequence of damage to lysosomal membranes by reactive oxygen species (ROS), leading to the leakage of lysosomal contents that culminates in apoptotic cell death. Consistent with this notion, toxicity from ROS was attenuated in an NPC cell model by cystatin B over-expression or pharmacological inhibition of cathepsin B. The observation that Npc1 and Cstb deletion genetically interact to potently enhance the degenerative phenotype of the NPC cerebellum provides strong support for the notion that lysosomal membrane permeabilization contributes to cerebellar degeneration in NPC disease.

Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases.

Currently available therapies for adult onset neurodegenerative diseases provide symptomatic relief but do not modify disease progression. Here we explore a new neuroprotective approach based on drugs targeting chaperone-directed protein quality control. Critical target proteins that unfold and aggregate in these diseases, such as the polyglutamine androgen receptor in spinal and bulbar muscular atrophy, huntingtin in Huntington's disease, α-synuclein in Parkinson's disease, and tau in Alzheimer's disease, are client proteins of heat shock protein 90 (Hsp90), and their turnover is regulated by the protein quality control function of the Hsp90/Hsp70-based chaperone machinery. Hsp90 and Hsp70 have opposing effects on client protein stability in protein quality control; Hsp90 stabilizes the clients and inhibits their ubiquitination, whereas Hsp70 promotes ubiquitination dependent on CHIP (C terminus of Hsc70-interacting protein) and proteasomal degradation. We discuss how drugs that modulate proteostasis by inhibiting Hsp90 function or promoting Hsp70 function enhance the degradation of the critical aggregating proteins and ameliorate toxic symptoms in cell and animal disease models.

Polyglutamine androgen receptor-mediated neuromuscular disease.

An expanded polyglutamine (polyQ) tract at the amino-terminus of the androgen receptor (AR) confers toxic properties responsible for neuronal and non-neuronal degeneration in spinal and bulbar muscular atrophy (SBMA), one of nine polyQ expansion diseases. Both lower motor neurons and peripheral tissues, including skeletal muscle, are affected, supporting the notion that SBMA is not a pure motor neuron disease but a degenerative disorder of the neuromuscular system. Here, we review experimental evidence demonstrating both nerve and muscle degeneration in SBMA model systems and patients. We propose that polyQ AR toxicity targets these components in a time-dependent fashion, with muscle pathology predominating early and motor neuron loss becoming more significant at late stages. This model of pathogenesis has important therapeutic implications, suggesting that symptoms arising from degeneration of nerve or muscle predominate at different points and that directed interventions targeting these components will be variably effective depending upon disease progression.

A murine Niemann-Pick C1 I1061T knock-in model recapitulates the pathological features of the most prevalent human disease allele.

Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol-sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1(I1061T), encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1(-/-) mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1(-/-) mouse, this Npc1(tm(I1061T)Dso) model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1(I1061T) protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.

Transcriptional activation of TFEB/ZKSCAN3 target genes underlies enhanced autophagy in spinobulbar muscular atrophy.

Spinobulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in exon 1 of the androgen receptor (AR) gene. SBMA demonstrates androgen-dependent toxicity due to unfolding and aggregation of the mutant protein. There are currently no disease-modifying therapies, but of increasing interest for therapeutic targeting is autophagy, a highly conserved cellular process mediating protein quality control. We have previously shown that genetic manipulations inhibiting autophagy diminish skeletal muscle atrophy and extend the lifespan of AR113Q knock-in mice. In contrast, manipulations inducing autophagy worsen muscle atrophy, suggesting that chronic, aberrant upregulation of autophagy contributes to pathogenesis. Since the degree to which autophagy is altered in SBMA and the mechanisms responsible for such alterations are incompletely defined, we sought to delineate autophagic status in SBMA using both cellular and mouse models. Here, we confirm that autophagy is induced in cellular and knock-in mouse models of SBMA and show that the transcription factors transcription factor EB (TFEB) and ZKSCAN3 operate in opposing roles to underlie these changes. We demonstrate upregulation of TFEB target genes in skeletal muscle from AR113Q male mice and SBMA patients. Furthermore, we observe a greater response in AR113Q mice to physiological stimulation of autophagy by both nutrient starvation and exercise. Taken together, our results indicate that transcriptional signaling contributes to autophagic dysregulation and provides a mechanistic framework for the pathologic increase of autophagic responsiveness in SBMA.

Post-Mortem evaluation of amyloid-dopamine terminal positron emission tomography dementia classifications.

Clinical classification of early dementia and mild cognitive impairment (MCI) is imprecise. We reported previously that molecular imaging classification of early dementia and MCI with dual amyloid and dopamine terminal positron emission tomography differs significantly from expert clinical classification. We now report pathological diagnoses in a substantial subset of our previously imaged subjects. Among 36 subjects coming to autopsy, imaging classifications and pathological diagnosis were concordant in 33 cases (κ = 0.85). This approach enhanced specificity of Alzheimer's disease diagnosis. The strong concordance of imaging-based classifications and pathological diagnoses suggests that this imaging approach will be useful in establishing more accurate and convenient classification biomarkers for dementia research.