Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration.

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.

RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

The Psychiatric Cell Map Initiative: A Convergent Systems Biological Approach to Illuminating Key Molecular Pathways in Neuropsychiatric Disorders.

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A.

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.

Loss of TAX1BP1-Directed Autophagy Results in Protein Aggregate Accumulation in the Brain.

Protein aggregates disrupt cellular homeostasis, causing toxicity linked to neurodegeneration. Selective autophagic elimination of aggregates is critical to protein quality control, but how aggregates are selectively targeted for degradation is unclear. We compared the requirements for autophagy receptor proteins: OPTN, NBR1, p62, NDP52, and TAX1BP1 in clearance of proteotoxic aggregates. Endogenous TAX1BP1 is recruited to and required for the clearance of stress-induced aggregates, whereas ectopic expression of TAX1BP1 increases clearance through autophagy, promoting viability of human induced pluripotent stem cell-derived neurons. In contrast, TAX1BP1 depletion sensitizes cells to several forms of aggregate-induced proteotoxicity. Furthermore, TAX1BP1 is more specifically expressed in the brain compared to other autophagy receptor proteins. In vivo, loss of TAX1BP1 results in accumulation of high molecular weight ubiquitin conjugates and premature lipofuscin accumulation in brains of young TAX1BP1 knockout mice. TAX1BP1 mediates clearance of a broad range of cytotoxic proteins indicating therapeutic potential in neurodegenerative diseases.

Neuronal enhancers are hotspots for DNA single-strand break repair.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

Frontotemporal lobar degeneration targets brain regions linked to expression of recently evolved genes.

In frontotemporal lobar degeneration (FTLD), pathological protein aggregation in specific brain regions is associated with declines in human-specialized social-emotional and language functions. In most patients, disease protein aggregates contain either TDP-43 (FTLD-TDP) or tau (FTLD-tau). Here, we explored whether FTLD-associated regional degeneration patterns relate to regional gene expression of human accelerated regions (HARs), conserved sequences that have undergone positive selection during recent human evolution. To this end, we used structural neuroimaging from patients with FTLD and human brain regional transcriptomic data from controls to identify genes expressed in FTLD-targeted brain regions. We then integrated primate comparative genomic data to test our hypothesis that FTLD targets brain regions linked to expression levels of recently evolved genes. In addition, we asked whether genes whose expression correlates with FTLD atrophy are enriched for genes that undergo cryptic splicing when TDP-43 function is impaired. We found that FTLD-TDP and FTLD-tau subtypes target brain regions with overlapping and distinct gene expression correlates, highlighting many genes linked to neuromodulatory functions. FTLD atrophy-correlated genes were strongly enriched for HARs. Atrophy-correlated genes in FTLD-TDP showed greater overlap with TDP-43 cryptic splicing genes and genes with more numerous TDP-43 binding sites compared with atrophy-correlated genes in FTLD-tau. Cryptic splicing genes were enriched for HAR genes, and vice versa, but this effect was due to the confounding influence of gene length. Analyses performed at the individual-patient level revealed that the expression of HAR genes and cryptically spliced genes within putative regions of disease onset differed across FTLD-TDP subtypes.

LYST deficiency impairs autophagic lysosome reformation in neurons and alters lysosome number and size.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder caused by biallelic mutations in the lysosomal trafficking regulator (LYST) gene. Even though enlarged lysosomes and/or lysosome-related organelles (LROs) are the typical cellular hallmarks of CHS, they have not been investigated in human neuronal models. Moreover, how and why the loss of LYST function causes a lysosome phenotype in cells has not been elucidated. We report that the LYST-deficient human neuronal model exhibits lysosome depletion accompanied by hyperelongated tubules extruding from enlarged autolysosomes. These results have also been recapitulated in neurons differentiated from CHS patients' induced pluripotent stem cells (iPSCs), validating our model system. We propose that LYST ensures the correct fission/scission of the autolysosome tubules during autophagic lysosome reformation (ALR), a crucial process to restore the number of free lysosomes after autophagy. We further demonstrate that LYST is recruited to the lysosome membrane, likely to facilitate the fission of autolysosome tubules. Together, our results highlight the key role of LYST in maintaining lysosomal homeostasis following autophagy and suggest that ALR dysregulation is likely associated with the neurodegenerative CHS phenotype.

An ANXA11 P93S variant dysregulates TDP-43 and causes corticobasal syndrome.

INTRODUCTION: Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization. METHODS: We described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling. RESULTS: ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways. DISCUSSION: This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity. HIGHLIGHTS: ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.

ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell-derived neurons.

Induced pluripotent stem cells (iPSCs) hold promise to revolutionize studies of intracellular transport in live human neurons and to shed new light on the role of dysfunctional transport in neurodegenerative disorders. Here, we describe an approach for live imaging of axonal and dendritic transport in iPSC-derived cortical neurons. We use transfection and transient expression of genetically-encoded fluorescent markers to characterize the motility of Rab-positive vesicles, including early, late and recycling endosomes, as well as autophagosomes and mitochondria in iPSC-derived neurons. Comparing transport parameters of these organelles with data from primary rat hippocampal neurons, we uncover remarkable similarities. In addition, we generated lysosomal-associated membrane protein 1 (LAMP1)-enhanced green fluorescent protein (EGFP) knock-in iPSCs and show that knock-in neurons can be used to study the transport of endogenously labeled vesicles, as a parallel approach to the transient overexpression of fluorescently labeled organelle markers.

A human iPSC-derived inducible neuronal model of Niemann-Pick disease, type C1.

BACKGROUND: Niemann-Pick disease, type C (NPC) is a childhood-onset, lethal, neurodegenerative disorder caused by autosomal recessive mutations in the genes NPC1 or NPC2 and characterized by impaired cholesterol homeostasis, a lipid essential for cellular function. Cellular cholesterol levels are tightly regulated, and mutations in either NPC1 or NPC2 lead to deficient transport and accumulation of unesterified cholesterol in the late endosome/lysosome compartment, and progressive neurodegeneration in affected individuals. Previous cell-based studies to understand the NPC cellular pathophysiology and screen for therapeutic agents have mainly used patient fibroblasts. However, these do not allow modeling the neurodegenerative aspect of NPC disease, highlighting the need for an in vitro system that permits understanding the cellular mechanisms underlying neuronal loss and identifying appropriate therapies. This study reports the development of a novel human iPSC-derived, inducible neuronal model of Niemann-Pick disease, type C1 (NPC1). RESULTS: We generated a null i3Neuron (inducible × integrated × isogenic) (NPC1-/- i3Neuron) iPSC-derived neuron model of NPC1. The NPC1-/- and the corresponding isogenic NPC1+/+ i3Neuron cell lines were used to efficiently generate homogenous, synchronized neurons that can be used in high-throughput screens. NPC1-/- i3Neurons recapitulate cardinal cellular NPC1 pathological features including perinuclear endolysosomal storage of unesterified cholesterol, accumulation of GM2 and GM3 gangliosides, mitochondrial dysfunction, and impaired axonal lysosomal transport. Cholesterol storage, mitochondrial dysfunction, and axonal trafficking defects can be ameliorated by treatment with 2-hydroxypropyl-ß-cyclodextrin, a drug that has shown efficacy in NPC1 preclinical models and in a phase 1/2a trial. CONCLUSION: Our data demonstrate the utility of this new cell line in high-throughput drug/chemical screens to identify potential therapeutic agents. The NPC1-/- i3Neuron line will also be a valuable tool for the NPC1 research community to explore the pathological mechanisms contributing to neuronal degeneration.

Fatal Coronary Artery Vasculitis With Mixed Features.

ABSTRACT: Atherosclerotic cardiovascular disease (ASCVD) is often investigated by medical examiners as a cause of sudden death. Because of the variation in presentation of atherosclerotic cardiovascular disease, the examiner must be cautious when assigning a final diagnosis. The presented case depended upon histologic examination of coronary artery lesions to reach an appropriate final diagnosis of vasculitis with mixed features. Autopsy findings showed hepatosplenic vasculitis with noncaseating granulomas, and multifocal diffuse coronary fibrosis with histologic findings consistent with late-stage polyarteritis nodosa (PAN). However, the patient lacked the hallmark renal involvement observed in PAN. Furthermore, the vasculitis within the liver showed a highly granulomatous appearance, more consistent with IgG4 disease. In these mixed-appearance cases with limited history, exact categorization of the disease may prove difficult to impossible. Herein, we review a differential diagnosis of classic vasculitides with a focus on those that commonly affect the coronary arteries in adults, namely, PAN.

Glyphosate and Polyoxyethyleneamine Ingestion Leading to Renal, Hepatic, and Pulmonary Failure.

ABSTRACT: Glyphosate is an organophosphorus compound and the active ingredient in commonly used herbicides, whereas polyoxyethyleneamine (POEA) is a nonionic surfactant often coupled with glyphosate in these herbicides to increase their efficacy. Cases of glyphosate-POEA ingestion have shown a variety of outcomes, ranging from skin and mucosal surface irritation to death. Here, we report mortality after ingestion of at least 237 mL of an herbicide confirmed to contain both glyphosate and POEA. The decedent's electronic medical record indicates presentation to the emergency department shortly after ingestion and rapid decompensation, with death occurring on the fourth day of admission. The autopsy report showed extensive pulmonary edema and congestion with no alimentary tract abnormalities. Microscopically, airway inflammation, edema, and hemorrhage were shown as well as pericentral necrosis and macrovascular hepatic steatosis. This case is unusual for several reasons including the fatal outcome in a young 30-year-old patient, the large volume of the herbicide consumed, the associated large volume aspirated, and the lung pathology associated with exposure to glyphosate-POEA since inhalation, and in this case, aspiration is an uncommon route of glyphosate-POEA exposure. This report therefore offers rare respiratory tract pathological findings and the clinical course after aspiration of a large volume of glyphosate-POEA.

Development and Comparative Evaluation of Endolysosomal Proximity Labeling-Based Proteomic Methods in Human iPSC-Derived Neurons.

Proximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein-protein and protein-organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network by knocking in (KI) an engineered ascorbate peroxidase (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batches of APEX labeling and different APEX probes. We conducted a comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.

Microglial NFκB-TNFα hyperactivation induces obsessive-compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.

Acanthamoeba spp. and Balamuthia mandrillaris leading to fatal granulomatous amebic encephalitis.

Acanthamoeba spp. and Balamuthia mandrillaris are free-living amebae known to cause disseminated and fatal central nervous system dysfunction which manifests as granulomatous amebic encephalitis (GAE) with exceedingly rare frequency. We report two lethal cases of infection with free-living amebae: an acute case of Acanthamoeba spp. infection in an immunocompromised female and a subacute case of B. mandrillaris in a Hispanic male. The Acanthamoeba spp. infection presented with an atypical lesion in the thalamus that caused rapid deterioration of the patient while the case of B. mandrillaris had a prolonged clinical course with multifocal lesions beginning in the frontal lobe. Cerebrospinal fluid results were non-specific in both cases, however, post-mortem histology demonstrated the presence of trophozoites along a perivascular distribution of necrosis and infiltrate composed primarily of neutrophils. In addition to detailing the clinical presentations of these infrequent amebic infections, we offer insight into the difficulties surrounding their diagnoses in order to aid the clinician in accurate and timely identification.

Reduced contrast sensitivity among older women is associated with increased risk of cognitive impairment.

OBJECTIVE: Several cross-sectional studies have reported an association between visual contrast sensitivity (a functional measure of low contrast vision) and poor cognitive performance or dementia, but no studies have investigated this association prospectively in a population-based cohort with final adjudication of mild cognitive impairment (MCI)/dementia. METHODS: In a prospective, community-based study of aging women (Study of Osteoporotic Fractures), we analyzed whether visual contrast sensitivity was associated with increased risk of MCI or dementia and/or worse performance on various cognitive tests assessed 10 years later. Contrast sensitivity was assessed at baseline in each eye using a VISTECH VCTS 6500 wall chart. MCI/dementia was adjudicated by an expert panel. Multivariate logistic and linear regression models were analyzed. RESULTS: Of 1,352 white (88.2%) and African American (11.8%) women with a mean age of 77.7 years (standard deviation = 3.3), 536 (39.6%) went on to develop MCI/dementia over 10 years. MCI/dementia risk was more than doubled (odds ratio = 2.16, 95% confidence interval = 1.58-2.96) in women with the lowest quartile of contrast sensitivity compared to the highest (p < 0.0001 for the linear trend). Reduced baseline contrast sensitivity was also associated with lower performance on several cognitive measures assessed 10 years later. INTERPRETATION: Among older women, reduced contrast sensitivity is associated with a greater risk of MCI/dementia. These findings suggest that visual system neurodegeneration or dysfunction may parallel or precede dementia-related cortical or subcortical degeneration, and that contrast sensitivity testing may be useful in identifying aging adults at high risk for dementia. Ann Neurol 2018;83:730-738.