Alzheimer's disease (AD), the most common neurodegenerative disease, is defined by β-amyloid plaques and tau neurofibrillary tangles. Tau filaments in AD adopt the "Alzheimer's fold", which is distinct from other tauopathies and highly conserved across sporadic and familial AD. However, to date, structural studies have focused on pure AD, despite the high prevalence of comorbid pathologies. In particular, up to half of AD patients harbor limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). This co-pathology-consisting of mislocalized and aggregated TDP-43, mainly restricted to the medial temporal lobe-is associated with accelerated clinical decline and more severe tau pathology. Whether there is a structural basis for this clinical synergy remains unknown. Here, using cryo-electron microscopy, we determine the structure of tau filaments from three AD patients with LATE-NC. We show that in addition to the expected Alzheimer's fold tau filaments, all cases exhibit a distinct fibril morphology identical to chronic traumatic encephalopathy (CTE) fold tau. Additional sampling revealed CTE neuropathology in one patient, suggestive features without definitive CTE in the second, and no evidence of CTE in the third. These findings raise questions about the relationship between LATE-NC and CTE, the effect of TDP-43 on tau conformation, and the etiology of LATE-NC.

Niemann-Pick type C1 disease is a lysosomal storage disorder caused by mutations in the NPC1 gene, resulting in the accumulation of unesterified cholesterol in multiple tissues. Despite its severity, therapeutic options remain limited. Using codon-optimized Npc1 mRNA delivered by lipid nanoparticles (Co-Npc1:LNPs), we achieved enhanced NPC1 protein expression and prolonged therapeutic activity in vitro, correcting both primary and secondary disease defects. In an Npc1 -/- mouse model, a single intravenous injection of Co-Npc1:LNP restored hepatic NPC1 protein levels, normalized autophagic flux, improved lipid abnormalities, and altered markers of liver injury. To interpret transcriptional changes post-Co-Npc1:LNP administration, we performed bulk RNA sequencing and applied MENTOR, a network-based clustering algorithm, and MENTOR-IA, an unbiased functional annotation tool. We identified transcriptionally restored pathways including cholesterol metabolism, lysosomal and mitochondrial function, and liver homeostasis. Overall, the Npc1 -/- mouse liver showed a transcriptional shift toward a more Npc1 +/+ state post-Co-Npc1:LNP treatment. Integration of single-nucleus RNA sequencing with bulk transcriptomics further revealed cell-type-specific correction of disease-associated gene expression across disease-relevant hepatic cell types. Together, we report the development and in vivo validation of the first mRNA-based therapeutic for Niemann-Pick type C1 disease.

INTRODUCTION:Poor sleep is a symptom and candidate risk factor for Alzheimer's disease (AD). We investigated whether objective and subjective longitudinal sleep measures correlated with AD pathology at brain autopsy. METHODS:Rush Memory and Aging Project (MAP) participants (n = 798; Meanage at baseline = 81.72) underwent annual evaluation with actigraphy along with sleep questionnaires and cognitive assessment. Brain autopsy provided quantitative neuropathologic data. RESULTS:Objective sleep fragmentation was associated with higher amyloid density. This association was only observed among individuals with low AD neuropathologic change or no/mild cognitive impairment. Last-measured sleep fragmentation had a stronger association with amyloid density than did mean sleep fragmentation across the study. Poorer subjective sleep quality was paradoxically linked to lower AD-related neuropathologic burden. DISCUSSION:These findings provide histopathologic evidence linking objectively measured sleep fragmentation to cerebral amyloid deposition and suggest that sleep disruption may be most relevant during earlier stages of AD pathogenesis.

Skeletal muscle pathology is a critical but poorly understood contributor to neuromuscular degeneration in spinal and bulbar muscular atrophy (SBMA), a CAG/polyglutamine (polyQ) expansion disorder caused by mutation in the androgen receptor (AR). Using a gene-targeted SBMA mouse model, we applied single-nucleus RNA sequencing to identify a disease-specific population of skeletal muscle myonuclei that replaced normal myonuclear subtypes. This transition was associated with dysregulation of the pathway governed by PGC-1α, a central regulator of myofiber specification and metabolic identity. PGC-1α dysfunction in SBMA muscle was age, hormone, and polyQ length dependent and was partially rescued by subcutaneous delivery of AR -targeted antisense oligonucleotides. Integrated ChIP-seq and RNA-seq analyses revealed that aberrant PGC-1α activity promoted the expression of a distinct set of myofiber specification genes while downregulating those that define healthy Type IIb and Type IIx myonuclei. We propose a model in which this dysfunction arose downstream of polyQ-mediated sequestration of PGC-1α cofactors MEF2, CREB, and CBP, leading to transcriptional reprogramming and cellular dysfunction. These findings implicated PGC-1α dysregulation as a key event linking AR polyQ expansion to skeletal muscle degeneration and suggested a shared mechanism for polyQ-mediated muscle pathology across related neurodegenerative diseases.

Degeneration of the neuromuscular system is a characteristic feature of spinal and bulbar muscular atrophy (SBMA), a CAG/polyglutamine (polyQ) expansion disorder caused by mutation in the androgen receptor (AR). Using a gene-targeted mouse model of SBMA, AR113Q mice, we demonstrate age-dependent degeneration of the neuromuscular system that initially manifests with muscle weakness and atrophy and progresses to include denervation of neuromuscular junctions and lower motor neuron soma atrophy. Using this model, we tested the hypothesis that therapeutic intervention targeting skeletal muscle during this period of disease progression arrests degeneration of the neuromuscular system. To accomplish this, AR-targeted antisense oligonucleotides were administered subcutaneously to symptomatic AR113Q mice to reduce expression of polyQ AR in peripheral tissues but not in the spinal cord. This intervention rescued muscle atrophy, neuromuscular junction innervation, lower motor neuron soma size, and survival in aged AR113Q mice. Single-nucleus RNA sequencing revealed age-dependent transcriptional changes in the AR113Q spinal cord during disease progression, which were mitigated by peripheral AR gene silencing. Our findings underscore the intricate interplay between peripheral tissues and the central nervous system in SBMA and emphasize the therapeutic effectiveness of peripheral gene knockdown in symptomatic disease.

Spinal and bulbar muscular atrophy (SBMA) is a CAG/polyglutamine (polyQ) repeat expansion disorder in which the mutant androgen receptor (AR) protein triggers progressive degeneration of the neuromuscular system in men. As the misfolded polyQ AR is the proximal mediator of toxicity, therapeutic efforts have focused on targeting the mutant protein, but these prior efforts have met with limited success in SBMA patients. Here, we examine the efficacy of small molecule AR proteolysis-targeting chimera (PROTAC) degraders that rapidly and potently promote AR ubiquitination and degradation by the proteasome. We show that the AR PROTAC degrader ARD-1676 clears polyQ AR in an over-expression system, in patient iPSC-derived induced motor neurons and skeletal muscle cells, and in a gene targeted mouse model of disease. Furthermore, we demonstrate that 24-h treatment with ARD-1676 rescues transcriptional dysregulation in SBMA induced skeletal muscle cells. These data provide evidence of therapeutic efficacy and in vivo target engagement, establishing AR PROTAC degraders as potential therapeutic agents for the treatment of SBMA.

Limited ancestral diversity has impaired our ability to detect risk variants more prevalent in ancestry groups of predominantly non-European ancestral background in genome-wide association studies (GWAS). We construct and analyze a multi-ancestry GWAS dataset in the Alzheimer’s Disease Genetics Consortium (ADGC) to test for novel shared and population-specific late-onset Alzheimer’s disease (LOAD) susceptibility loci and evaluate underlying genetic architecture in 37,382 non-Hispanic White (NHW), 6728 African American, 8899 Hispanic (HIS), and 3232 East Asian individuals, performing within ancestry fixed-effects meta-analysis followed by a cross-ancestry random-effects meta-analysis. We identify 13 loci with cross-population associations including known loci at/near CR1, BIN1, TREM2, CD2AP, PTK2B, CLU, SHARPIN, MS4A6A, PICALM, ABCA7, APOE, and two novel loci not previously reported at 11p12 (LRRC4C) and 12q24.13 (LHX5-AS1). We additionally identify three population-specific loci with genome-wide significance at/near PTPRK and GRB14 in HIS and KIAA0825 in NHW. Pathway analysis implicates multiple amyloid regulation pathways and the classical complement pathway. Genes at/near our novel loci have known roles in neuronal development (LRRC4C, LHX5-AS1, and PTPRK) and insulin receptor activity regulation (GRB14). Using cross-population GWAS meta-analyses, we identify novel LOAD susceptibility loci in/near LRRC4C and LHX5-AS1, both with known roles in neuronal development, as well as several novel population-unique loci. Reflecting the power of diverse ancestry in GWAS, we detect the SHARPIN locus with only 13.7

INTRODUCTION:Spinal and Bulbar Muscular Atrophy (SBMA) is a slowly progressive, X-linked, and sex-limited degenerative disorder affecting lower motor neurons and skeletal muscle which lacks disease-modifying therapies. This disease is caused by a CAG/polyglutamine (polyQ) tract expansion in the androgen receptor (AR) gene, and its pathogenesis is driven by toxic gain-of-function mechanisms. Affected men develop proximal limb and bulbar muscle weakness along with signs of partial androgen insensitivity. AREAS COVERED:Toxicity of the polyQ AR is mediated by protein misfolding and nuclear translocation that follow ligand binding, resulting in the disruption of downstream homeostatic mechanisms. This review highlights what is known about disease pathogenesis and how this has been leveraged to test potential therapeutic approaches. The focus is on strategies that alleviate polyQ AR toxicity in SBMA, including those that alter AR function, diminish the expression of the encoding gene, or promote clearance of the misfolded, mutant protein. EXPERT OPINION:We discuss emerging strategies to mitigate polyQ AR toxicity, including gene editing, RNA targeted therapies, and efforts to harness proteostatic mechanisms. These promising approaches are discussed in the context of challenges for drug discovery efforts that are faced when attempting to treat a rare and slowly progressive neurodegenerative disorder.

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.

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.

The Hsp90 and Hsp70 chaperones act as a protein quality control system for several hundred client proteins, including many implicated in neurodegenerative disorders. Hsp90 and Hsp70 are widely thought to be important drug targets. Although many structurally distinct compounds have been developed to target Hsp90, relatively few are known to target Hsp70 and even fewer have been tested in protein quality control systems. To address this, we describe a high-throughput thermal shift-based screen to find compounds that bind and stabilize Hsp70 and then employ assays with misfolded forms of a well-established client protein, neuronal NO synthase (nNOS), to identify compounds that enhance ubiquitination of client proteins. The ubiquitination assay employed a quantitative ELISA method to measure Hsp70:CHIP-dependent ubiquitination of heme-deficient nNOS, which is a model of a misfolded client, in reaction mixtures containing purified E1, E2, Hsp70, CHIP, and ubiquitin. We screened 44,447 molecules from the Maybridge and ChemDiv libraries and found one compound, protein folding disease compound 15 (PFD-15), that enhanced in vitro nNOS ubiquitination with an EC50 of approximately 8 μM. PFD-15 was tested in human embryonic kidney 293 cells stably transfected with a C331A nNOS, a mutation that makes nNOS a preferred client protein for ubiquitination. In this model, PFD-15 decreased steady-state levels of C331A nNOS, but not the wild-type nNOS, in a time- and concentration-dependent manner by a process attenuated by lactacystin, an inhibitor to the proteasome. PFD-15 appears to enhance binding of Hsp70 and CHIP to client proteins without interference of protein quality control mechanisms, enabling the selective clearance of misfolded proteins. SIGNIFICANCE STATEMENT: There are few treatment options for neurodegenerative diseases, which are widely thought to be caused by formation of toxic misfolded proteins. One novel approach is to enhance the Hsp90/Hsp70 protein quality control machinery to remove these misfolded proteins. Targeting Hsp70 may have advantages over targeting Hsp90, but fewer compounds targeting Hsp70 have been developed relative to those for Hsp90. The current study provides a novel approach to enhance the number of compounds targeting the Hsp70's role in protein quality control.

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 (NPC1(I1061T)) 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 NPC1(I1061T). 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 NPC1(I1061T). Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

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 toward the development of disease-modifying therapies.

Solute carrier family 39, member 8 (SLC39A8), is a transmembrane transporter that mediates the cellular uptake of zinc, iron, and manganese (Mn). Human genetic studies document the involvement of SLC39A8 in Mn homeostasis, brain development, and function. However, the role and pathophysiological mechanisms of SLC39A8 in the central nervous system remain elusive. We generated Slc39a8 neuron-specific knockout (Slc39a8-NSKO) mice to study SLC39A8 function in neurons. The Slc39a8-NSKO mice displayed markedly decreased Mn levels in the whole brain and brain regions, especially the cerebellum. Radiotracer studies using 54Mn revealed that Slc39a8-NSKO mice had impaired brain uptake of Mn. Slc39a8-NSKO cerebellums exhibited morphological defects and abnormal dendritic arborization of Purkinje cells. Reduced neurogenesis and increased apoptotic cell death occurred in the cerebellar external granular layer of Slc39a8-NSKO mice. Brain Mn deficiency in Slc39a8-NSKO mice was associated with motor dysfunction. Unbiased RNA-Seq analysis revealed downregulation of key pathways relevant to neurodevelopment and synaptic plasticity, including cAMP signaling pathway genes. We further demonstrated that Slc39a8 was required for the optimal transcriptional response to the cAMP-mediated signaling pathway. In summary, our study highlighted the essential roles of SLC39A8 in brain Mn uptake and cerebellum development and functions.

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.

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.

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 & UDelta;/Pf4 & UDelta;), 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 & UDelta;/Pf4 & UDelta; 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.