Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy.

The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau.

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

A Mutation in Hnrnph1 That Decreases Methamphetamine-Induced Reinforcement, Reward, and Dopamine Release and Increases Synaptosomal hnRNP H and Mitochondrial Proteins.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.SIGNIFICANCE STATEMENT Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

5' UTR variants in the quantitative trait gene Hnrnph1 support reduced 5' UTR usage and hnRNP H protein as a molecular mechanism underlying reduced methamphetamine sensitivity.

We previously identified a 210 kb region on chromosome 11 (50.37-50.58 Mb, mm10) containing two protein-coding genes (Hnrnph1, Rufy1) that was necessary for reduced methamphetamine-induced locomotor activity in C57BL/6J congenic mice harboring DBA/2J polymorphisms. Gene editing of a small deletion in the first coding exon supported Hnrnph1 as a quantitative trait gene. We have since shown that Hnrnph1 mutants also exhibit reduced methamphetamine-induced reward, reinforcement, and dopamine release. However, the quantitative trait variants (QTVs) that modulate Hnrnph1 function at the molecular level are not known. Nine single nucleotide polymorphisms and seven indels distinguish C57BL/6J from DBA/2J within Hnrnph1, including four variants within the 5' untranslated region (UTR). Here, we show that a 114 kb introgressed region containing Hnrnph1 and Rufy1 was sufficient to cause a decrease in MA-induced locomotor activity. Gene-level transcriptome analysis of striatal tissue from 114 kb congenics vs Hnrnph1 mutants identified a nearly perfect correlation of fold-change in expression for those differentially expressed genes that were common to both mouse lines, indicating functionally similar effects on the transcriptome and behavior. Exon-level analysis (including noncoding exons) revealed decreased 5' UTR usage of Hnrnph1 and immunoblot analysis identified a corresponding decrease in hnRNP H protein in 114 kb congenic mice. Molecular cloning of the Hnrnph1 5' UTR containing all four variants (but none of them individually) upstream of a reporter induced a decrease in reporter signal in both HEK293 and N2a cells, thus, identifying a set of QTVs underlying molecular regulation of Hnrnph1.

TIA1 regulates the generation and response to toxic tau oligomers.

RNA binding proteins (RBPs) are strongly linked to the pathophysiology of motor neuron diseases. Recent studies show that RBPs, such as TIA1, also contribute to the pathophysiology of tauopathy. RBPs co-localize with tau pathology, and reduction of TIA1 protects against tau-mediated neurodegeneration. The mechanism through which TIA1 reduction protects against tauopathy, and whether TIA1 modulates the propagation of tau, are unknown. Previous studies indicate that the protective effect of TIA1 depletion correlates with both the reduction of oligomeric tau and the reduction of pathological TIA1 positive tau inclusions. In the current report, we used a novel tau propagation approach to test whether TIA1 is required for producing toxic tau oligomers and whether TIA1 reduction would provide protection against the spread of these oligomers. The approach used young PS19 P301S tau mice at an age at which neurodegeneration would normally not yet occur and seeding oligomeric or fibrillar tau by injection into hippocampal CA1 region. We find that propagation of exogenous tau oligomers (but not fibrils) across the brain drives neurodegeneration in this model. We demonstrate that TIA1 reduction essentially brackets the pathophysiology of tau, being required for the production of tau oligomers, as well as regulating the response of neurons to propagated toxic tau oligomers. These results indicate that RNA binding proteins modulate the pathophysiology of tau at multiple levels and provide insights into possible therapeutic approaches to reduce the spread of neurodegeneration in tauopathy.

TDP-1/TDP-43 regulates stress signaling and age-dependent proteotoxicity in Caenorhabditis elegans.

TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to Caenorhabditis elegans and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although tdp-1 mutants are stress-sensitive, chronic upregulation of tdp-1 expression is toxic and decreases lifespan. ALS-associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the daf-16-dependent upregulation of tdp-1 expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.

Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress.

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the "c9RAN proteins" thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.

Proximity labeling reveals dynamic changes in the SQSTM1 protein network.

Sequestosome1 (SQSTM1) is an autophagy receptor that mediates degradation of intracellular cargo, including protein aggregates, through multiple protein interactions. These interactions form the SQSTM1 protein network, and these interactions are mediated by SQSTM1 functional interaction domains, which include LIR, PB1, UBA and KIR. Technological advances in cell biology continue to expand our knowledge of the SQSTM1 protein network and of the relationship of the actions of the SQSTM1 protein network in cellular physiology and disease states. Here we apply proximity profile labeling to investigate the SQSTM1 protein interaction network by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including production of SQSTM1 intracellular bodies, binding to known SQSTM1 interacting partners, and capture of novel SQSTM1 protein interactors. Strikingly, aggregated tau protein altered the protein interaction network of SQSTM1 to include many stress-associated proteins. We demonstrate the importance of the PB1 and/or UBA domains for binding network members, including the K18 domain of tau. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 function in cellular physiology and disease state.

Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. A hexanucleotide (GGGGCC) repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of both diseases. The mechanisms by which this repeat expansion causes "c9FTD/ALS" are not definitively known, but RNA-mediated toxicity is a likely culprit. RNA transcripts of the expanded GGGGCC repeat form nuclear foci in c9FTD/ALS, and also undergo repeat-associated non-ATG (RAN) translation resulting in the production of three aggregation-prone proteins. The goal of this study was to examine whether antisense transcripts resulting from bidirectional transcription of the expanded repeat behave in a similar manner. We show that ectopic expression of (CCCCGG)66 in cultured cells results in foci formation. Using novel polyclonal antibodies for the detection of possible (CCCCGG)exp RAN proteins [poly(PR), poly(GP) and poly(PA)], we validated that (CCCCGG)66 is also subject to RAN translation in transfected cells. Of importance, foci composed of antisense transcripts are observed in the frontal cortex, spinal cord and cerebellum of c9FTD/ALS cases, and neuronal inclusions of poly(PR), poly(GP) and poly(PA) are present in various brain tissues in c9FTD/ALS, but not in other neurodegenerative diseases, including CAG repeat disorders. Of note, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell, suggesting these events represent two distinct ways in which the C9ORF72 repeat expansion may evoke neurotoxic effects. These findings provide mechanistic insight into the pathogenesis of c9FTD/ALS, and have significant implications for therapeutic strategies.

Progressive amnestic dementia, hippocampal sclerosis, and mutation in C9ORF72.

The most common cause of familial frontotemporal lobar degeneration with TAR DNA-binding protein-43 pathology (FTLD-TDP) has been found to be an expansion of a hexanucleotide repeat (GGGGCC) in a noncoding region of the gene C9ORF72. Hippocampal sclerosis (HpScl) is a common finding in FTLD-TDP. Our objective was to screen for the presence of C9ORF72 hexanucleotide repeat expansions in a pathologically confirmed cohort of "pure" hippocampal sclerosis cases (n = 33), outside the setting of FTLD-TDP and Alzheimer's disease (AD). Using a recently described repeat-associated non-ATG (RAN) translation (C9RANT) antibody that was found to be highly specific for c9FTD/ALS, we identified a single "pure" HpScl autopsy case with a repeat expansion in C9ORF72 (c9HpScl). Mutation screening was also performed with repeat-primed polymerase chain reaction and further confirmed with Southern blotting. The c9HpScl patient had a 14-year history of a slowly progressive amnestic syndrome and a clinical diagnosis of probable AD. Neuropsychological testing revealed memory impairment, but no deficits in other cognitive domains. Autopsy showed hippocampal sclerosis with TDP-43 immunoreactive neuronal inclusions relatively limited to limbic lobe structures. Neuritic pathology immunoreactive for p62 was more frequent than TDP-43 in amygdala and hippocampus. Frequent p62-positive neuronal inclusions were present in cerebellar granule neurons as is typical of C9ORF72 mutation carriers. There was no significant FTLD or motor neuron disease. C9RANT was found to be sensitive and specific in this autopsy-confirmed series of HpScl cases. The findings in this patient suggest that the clinical and pathologic spectrum of C9ORF72 repeat expansion is wider than frontotemporal dementia and motor neuron disease, including cases of progressive amnestic dementia with restricted TDP-43 pathology associated with HpScl.

Neurotoxic effects of TDP-43 overexpression in C. elegans.

RNA-binding protein TDP-43 has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have engineered pan-neuronal expression of human TDP-43 protein in Caenorhabditis elegans, with the goal of generating a convenient in vivo model of TDP-43 function and neurotoxicity. Transgenic worms with the neuronal expression of human TDP-43 exhibit an 'uncoordinated' phenotype and have abnormal motorneuron synapses. Caenorhabditis elegans contains a single putative ortholog of TDP-43, designated TDP-1, which we show can support alternative splicing of CFTR in a cell-based assay. Neuronal overexpression of TDP-1 also results in an uncoordinated phenotype, while genetic deletion of the tdp-1 gene does not affect movement or alter motorneuron synapses. By using the uncoordinated phenotype as a read-out of TDP-43 overexpression neurotoxicty, we have investigated the contribution of specific TDP-43 domains and subcellular localization to toxicity. Full-length (wild-type) human TDP-43 expressed in C. elegans is localized to the nucleus. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. Interestingly, fusion of TDP-1 C-terminal sequences to TDP-43 missing its C-terminal domain restores normal subnuclear localization and toxicity in C. elegans and CFTR splicing in cell-based assays. Overexpression of wild-type, full-length TDP-43 in mammalian cells (differentiated M17 cells) can also result in cell toxicity. Our results demonstrate that in vivo TDP-43 neurotoxicity can result from nuclear activity of overexpressed full-length protein.

BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain.

Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.

Heavy Metal Neurotoxicants Induce ALS-Linked TDP-43 Pathology.

Heavy metals, such as lead, mercury, and selenium, have been epidemiologically linked with a risk of ALS, but a molecular mechanism proving the connection has not been shown. A screen of putative developmental neurotoxins demonstrated that heavy metals (lead, mercury, and tin) trigger accumulation of TDP-43 into nuclear granules with concomitant loss of diffuse nuclear TDP-43. Lead (Pb) and methyl mercury (MeHg) disrupt the homeostasis of TDP-43 in neurons, resulting in increased levels of transcript and increased splicing activity of TDP-43. TDP-43 homeostasis is tightly regulated, and positively or negatively altering its splicing-suppressive activity has been shown to be deleterious to neurons. These changes are associated with the liquid-liquid phase separation of TDP-43 into nuclear bodies. We show that lead directly facilitates phase separation of TDP-43 in a dose-dependent manner in vitro, possibly explaining the means by which lead treatment results in neuronal nuclear granules. Metal toxicants also triggered the accumulation of insoluble TDP-43 in cultured cells and in the cortices of exposed mice. These results provide novel evidence of a direct mechanistic link between heavy metals, which are a commonly cited environmental risk of ALS, and molecular changes in TDP-43, the primary pathological protein accumulating in ALS.

RNA binding proteins co-localize with small tau inclusions in tauopathy.

The development of insoluble, intracellular neurofibrillary tangles composed of the microtubule-associated protein tau is a defining feature of tauopathies, including Alzheimer's disease (AD). Accumulating evidence suggests that tau pathology co-localizes with RNA binding proteins (RBPs) that are known markers for stress granules (SGs). Here we used proteomics to determine how the network of tau binding proteins changes with disease in the rTg4510 mouse, and then followed up with immunohistochemistry to identify RNA binding proteins that co-localize with tau pathology. The tau interactome networks revealed striking disease-related changes in interactions between tau and a multiple RBPs, and biochemical fractionation studies demonstrated that many of these proteins including hnRNPA0, EWSR1, PABP and RPL7 form insoluble aggregates as tau pathology develops. Immunohistochemical analysis of mouse and human brain tissues suggest a model of evolving pathological interaction, in which RBPs co-localize with pathological phospho-tau but occur adjacent to larger pathological tau inclusions. We suggest a model in which tau initially interacts with RBPs in small complexes, but evolves into isolated aggregated inclusions as tau pathology matures.

Changes in neuronal immunofluorescence in the C- versus N-terminal domains of hnRNP H following D1 dopamine receptor activation.

RNA binding proteins are a diverse class of proteins that regulate all aspects of RNA metabolism. Accumulating studies indicate that heterogeneous nuclear ribonucleoproteins are associated with cellular adaptations in response to drugs of abuse. We recently mapped and validated heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1) as a quantitative trait gene underlying differential behavioral sensitivity to methamphetamine. The molecular mechanisms by which hnRNP H1 alters methamphetamine behaviors are unknown but could involve pre- and/or post-synaptic changes in protein localization and function. Methamphetamine initiates post-synaptic D1 dopamine receptor signaling indirectly by binding to pre-synaptic dopamine transporters and vesicular monoamine transporters of midbrain dopaminergic neurons which triggers reverse transport and accumulation of dopamine at the synapse. Here, we examined changes in neuronal localization of hnRNP H in primary rat cortical neurons that express dopamine receptors that can be modulated by the D1 or D2 dopamine receptor agonists SKF38393 and (-)-Quinpirole HCl, respectively. Basal immunostaining of hnRNP H was localized primarily to the nucleus. D1 dopamine receptor activation induced an increase in hnRNP H nuclear immunostaining as detected by immunocytochemistry with a C-domain directed antibody containing epitope near the glycine-rich domain but not with an N-domain specific antibody. Although there was no change in hnRNP H protein in the nucleus or cytoplasm, there was a decrease in Hnrnph1 transcript following D1 receptor stimulation. Taken together, these results suggest that D1 receptor activation increases availability of the hnRNP H C-terminal epitope, which could potentially reflect changes in protein-protein interactions. Thus, D1 receptor signaling could represent a key molecular post-synaptic event linking Hnrnph1 polymorphisms to drug-induced behavior.

Reducing the RNA binding protein TIA1 protects against tau-mediated neurodegeneration in vivo.

Emerging studies suggest a role for tau in regulating the biology of RNA binding proteins (RBPs). We now show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo protects against neurodegeneration and prolongs survival in transgenic P301S Tau mice. Biochemical fractionation shows co-enrichment and co-localization of tau oligomers and RBPs in transgenic P301S Tau mice. Reducing TIA1 decreased the number and size of granules co-localizing with stress granule markers. Decreasing TIA1 also inhibited the accumulation of tau oligomers at the expense of increasing neurofibrillary tangles. Despite the increase in neurofibrillary tangles, TIA1 reduction increased neuronal survival and rescued behavioral deficits and lifespan. These data provide in vivo evidence that TIA1 plays a key role in mediating toxicity and further suggest that RBPs direct the pathway of tau aggregation and the resulting neurodegeneration. We propose a model in which dysfunction of the translational stress response leads to tau-mediated pathology.

Dioxins and related environmental contaminants increase TDP-43 levels.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative condition that is characterized by progressive loss of motor neurons and the accumulation of aggregated TAR DNA Binding Protein-43 (TDP-43, gene: TARDBP). Increasing evidence indicates that environmental factors contribute to the risk of ALS. Dioxins, related planar polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that activate the aryl hydrocarbon receptor (AHR), a ligand-activated, PAS family transcription factor. Recently, exposure to these toxicants was identified as a risk factor for ALS. METHODS: We examined levels of TDP-43 reporter activity, transcript and protein. Quantification was done using cell lines, induced pluripotent stem cells (iPSCs) and mouse brain. The target samples were treated with AHR agonists, including 6-Formylindolo[3,2-b]carbazole (FICZ, a potential endogenous ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, and benzo(a)pyrene, an abundant carcinogen in cigarette smoke). The action of the agonists was inhibited by concomitant addition of AHR antagonists or by AHR-specific shRNA. RESULTS: We now report that AHR agonists induce up to a 3-fold increase in TDP-43 protein in human neuronal cell lines (BE-M17 cells), motor neuron differentiated iPSCs, and in murine brain. Chronic treatment with AHR agonists elicits over 2-fold accumulation of soluble and insoluble TDP-43, primarily because of reduced TDP-43 catabolism. AHR antagonists or AHR knockdown inhibits agonist-induced increases in TDP-43 protein and TARDBP transcription demonstrating that the ligands act through the AHR. CONCLUSIONS: These results provide the first evidence that environmental AHR ligands increase TDP-43, which is the principle pathological protein associated with ALS. These results suggest novel molecular mechanisms through which a variety of prevalent environmental factors might directly contribute to ALS. The widespread distribution of dioxins, PCBs and PAHs is considered to be a risk factor for cancer and autoimmune diseases, but could also be a significant public health concern for ALS.

Interaction of tau with the RNA-Binding Protein TIA1 Regulates tau Pathophysiology and Toxicity.

Dendritic mislocalization of microtubule associated protein tau is a hallmark of tauopathies, but the role of dendritic tau is unknown. We now report that tau interacts with the RNA-binding protein (RBP) TIA1 in brain tissue, and we present the brain-protein interactome network for TIA1. Analysis of the TIA1 interactome in brain tissue from wild-type (WT) and tau knockout mice demonstrates that tau is required for normal interactions of TIA1 with proteins linked to RNA metabolism, including ribosomal proteins and RBPs. Expression studies show that tau regulates the distribution of TIA1, and tau accelerates stress granule (SG) formation. Conversely, TIA1 knockdown or knockout inhibits tau misfolding and associated toxicity in cultured hippocampal neurons, while overexpressing TIA1 induces tau misfolding and stimulates neurodegeneration. Pharmacological interventions that prevent SG formation also inhibit tau pathophysiology. These studies suggest that the pathophysiology of tauopathy requires an intimate interaction with RNA-binding proteins.