Tauopathy Model Research Articles

Swallowing Dynamics and Breathing-Swallowing Coordination Are Altered in Patients with Mild Cognitive Impairment

Introduction: Postinspiratory activity, which is essential for laryngeal closure during swallowing to prevent aspiration of food into the airways, is reduced in a mouse model of tauopathy. Therefore, we hypothesized that patients at the stage of mild cognitive impairment (MCI) exhibit alterations in swallowing dynamics and coordination between swallowing and breathing. Methods: We examined breathing-swallowing coordination in patients with MCI. Patients who scored ≥24 on the Mini-Mental State Examination and <26 on the Japanese version of the Montreal Cognitive Assessment were recruited at Sumoto Itsuki Hospital. Parameters associated with breathing-swallowing coordination were assessed using a combination of two sensors: a respiratory flow sensor and a piezoelectric sensor attached to the skin surface of the anterior neck. Results: Nineteen patients met the criteria for MCI; 16 of these patients (79.5 ± 9.1 years old) scored <3 on the 10-item Eating Assessment Tool and were enrolled in the study. Their data were compared with those of an age-matched elderly cohort (79.9 ± 2.9 years old). The frequencies of swallowing during inspiration and swallowing immediately followed by inspiration in patients with MCI were 6.9% and 9.6%, respectively; these frequencies were not significantly different from those of the age-matched elderly cohort. However, the timing of swallowing in the respiratory cycle was significantly delayed in the MCI patients, and both time from the onset to the peak of laryngeal elevation and the duration between the onset of rapid laryngeal elevation and the time when the larynx returned to the resting position were significantly lengthened in this group. Conclusion: At the stage of MCI, breathing-swallowing coordination has already started to decline.

Tauopathy induces degeneration and impairs regeneration of sensory nerves in the cornea

The cornea is transparent and innervated by a dense collection of sensory nerves originating from the ocular branch of the trigeminal nerve. This study was designed to comprehensively analyze alterations of corneal sub-basal nerve plexus in a mouse model of tauopathy (P301L transgenic mice) to test the possibility of using corneal nerves as a biomarker for tauopathy. Corneal sensitivity, thickness and epithelial wound healing were measured non-invasively by aeshesiometer, optical coherence tomography and fluorescein staining, respectively. Tau, corneal nerves and immune cells were examined by immunohistochemistry or Western blot. At the early stage of tauopathy, although corneal sensitivity, thickness and nerve fiber density were not greatly altered, corneal nerve abnormalities were observed in the peripheral region of young P301L mice. With aging, the density of abnormal nerves increased, while corneal sensitivity, epithelial thickness, nerve fiber density and length decreased in middle-aged P301L mice compared with WT mice. After corneal epithelial injury in young mice, no difference in reepithelialization was observed between two groups of mice, however, the regeneration of corneal nerves in P301L mice lagged behind WT mice, which was reflected by delayed recovery of corneal sensitivity, decreased corneal nerve density and length and density of CD45+ dendriform cells in P301L mice. In conclusion, our data provide compelling evidence that corneal nerves were changed in a mouse model of tauopathy in an age-dependent manner. Moreover, tau overexpression impairs corneal nerve regeneration. These results suggest that cornea may serve as a promising ocular site for the early diagnosis of tauopathy.

Implication of type 4 NADPH oxidase (NOX4) in tauopathy

Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.

Effects of interleukin-1 receptor antagonist overexpression in a mouse model of tauopathy.

Innate immune activation is suggested to exacerbate tauopathy. Interluekin-1 (IL-1) is a cytokine that can initiate innate immune activation and has been argued to play a central role in Alzheimer's pathogenesis. The Interleukin-1 receptor antagonist (IL-1RA) is an endogenous agent that regulates IL-1 signaling by interfering with activation of the IL-1 receptor. Anakinra is an FDA approved version of IL-1RA. Mouse IL-1RA was cloned by PCR and demonstrated activity in a HEK cell reporter line stimulated with IL-1beta. Three month old Tg4510 mice were treated with AAV9 expressing either IL-1RA or GFP unilaterally in the hippocampus and anterior cortex. Tissues were collected at 7 mo of age and the anterior brain processed for ELISA measurements and the posterior half prepared for histochemistry. In vitro, IL-1RA protein was secreted into the cell culture media. IL-1 reporter cell lines transfected with IL-1RA greatly reduced reporter expression in response to IL-1 exposure, indicating the gene product had antagonist activity. Studies in Tg4510 mice detected increases in IL-1 RNA at 6 and 8 mo of age, but not at 4 mo. Measures of IL-1RA by ELISA indicated an over 100 fold increased expression in mice injected with AAV-IL-1RA. However, the only significant impact of IL-1RA expression on tauopathy was a slight reduction in the ELISA values for detergent insoluble p-Ser199-tau in the IL-1RA mice. No differences were found in any of the histological measures of tauopathy or ELISA measures of total tau or p-Ser-396tau CONCLUSION: IL-1RA had a minimal impact on the accumulation of tauopathy in the Tg4510 mice. The overexpression of IL-1RA preceded the endogenous increase in IL-1 in this model. Other studies have found that blocking IL-1 production through inflammasome deletion did reduce tauopathy (Ising et al, Nature (2019) 575:669). These authors used a different mouse model with lower overall expression of tau. The minimal effect seen in this study may relate to the model of tauopathy used, the extent of IL-1 signaling reduction, or the possibility that the effects of inflammasome deletion are mediated through effector molecules other than the IL-1 receptor.

The Lonafarnib target, Rhes, is uniquely dysregulated in tauopathies: A human postmortem study

AbstractBackgroundThe farnesyltransferase inhibitor, Lonafarnib, is capable of reducing tau lesions and associated atrophy in tauopathy models through activity on the Ras GTPase, Rhes (Hernandez I et al., 2019). While hinting at a role of Rhes in tau proteinopathic etiology, it is unclear how Rhes activity changes in human tauopathies or other neurodegenerative diseases. As these models reflect rare, familial forms of tauopathy, we sought to detect evidence of Rhes changes in human sporadic tauopathy versus FTLD‐TDP and synucleinopathies.MethodAllocortical and neocortical sections from postmortem cases (Table 1) representing five different tauopathies, FTLD‐TDP, Lewy body disease, and healthy controls (HC) were immunohistochemically stained for Rhes, proteinopathic lesions (i.e. PHF‐1 positive tau inclusions, phospho‐TDP‐43, or α‐synuclein), and NeuN. Single‐nucleus RNA‐sequencing (snRNA‐seq) was also performed in a separate cohort representing HC and early AD.Result40 cases were examined in this study (Table 1). Rhes is widespread in cortical neurons, as detected by immunohistochemical staining and snRNA‐seq (Figure 1). In tauopathies, neurons either present Rhes as diffuse or punctiform, or feature an absence of Rhes (Figure 2). Accordingly, we classified neurons as Rhes type Diffuse, Punctiform, or Absent. The proportion of neurons with PHF‐1 positive tau inclusions significantly differ between the three neuronal Rhes phenotypes (p &lt; 0.0001) with the majority of Absent type neurons harboring PHF‐1 positive tau inclusions and Diffuse type neurons mostly devoid of PHF‐1 positive tau inclusions (Figure 3, Table 2). Punctiform and Absent type neurons were not detected in neurons with phospho‐TDP‐43 inclusions or α‐synuclein inclusions (Figure 4).ConclusionGiven the distribution of PHF‐1 positive tau inclusions across different Rhes‐deliniated neuronal phenotypes, we propose a model by which Rhes takes on a punctiform phenotype then is ultimately cleared from neurons with tau lesions. This postmortem study in humans, together with in vivo and in vitro work done in familial tauopathy models, suggests the presence of a causal relationship between Rhes clearance and tau proteinopathic lesions, but not phospho‐TDP‐43 or α‐synuclein inclusions. As such, Rhes cellular phenotype represents a novel postmortem hallmark of tauopathy and supports to application of Lonafarnib to the treatment of tauopathies.

Disentangling the determinants of transposable element expression in cognitive aging and Alzheimer's disease deficits using the AD-BXD mouse population.

Alzheimer's Disease (AD) is a complex multifaceted disease that is mediated by multiple unidentified genetic and environmental factors. One such factor that has been implicated, but is yet to be fully understood, is the role of transposable elements (TE) in AD. Here, we explore the role and scope of TEs using genetically diverse population of mice carrying familial AD mutations (AD-BXDs) and their non-transgenic littermates (Ntg-BXDs). We quantified the expression levels of all known transposable elements (TEs) in the hippocampus using TEtranscripts (v.0.36) in 28 different mouse strains carrying familial AD mutations (AD-BXDs) and in their non-transgenic littermates (Ntg-BXDs) at 6 and 14 months of age. Differentially abundant TEs as a function of genotype (Ntg vs 5XFAD) and age (6m vs 14m) were assessed using DESeq2. We assessed heritability of TE expression for TEs that were identified as significantly differential expressed from the above comparisons, to obtain an estimate of heritable variation in a genetically diverse population. Lastly, we correlated TE expression to short-term and long-term memory using contextual fear conditioning. Differential expression analysis revealed higher number of differentially expressed TEs in the 14-month age group between AD-BxD and their Ntg littermate controls as compared to the 6-month age group. Majority of these differentially expressed TE are upregulated and belong to the category of LTR transposons. Importantly, we observe high average heritable variation (h2 6m-Ntg =0.65, h2 6m-5xFAD= 0.58, h2 14m-Ntg =0.44, h2 14m-5xFAD =0.79) across our genetically diverse AD-BXD panel. Lastly, we find significant negative correlations among contextual fear conditioning phenotypes and TE expression in the 5XFAD genotype group in both our 6m and 14m age groups. Our findings reveal a role of transposable elements, particularly LTR transposons across our genetically diverse population of mice, specifically in our 14m 5xFAD cohort. This finding is in line with previously published studies of the prevalence of LTR transposons in tauopathy models but is the first to assess the abundance and report on the extent of heritable variation of TE along with their correlation to cognitive behavior in a genetically diverse but translationally relevant mouse population of human AD.

Stem cell models of primary tauopathies reveal defects in synaptic function.

Primary tauopathies are characterized neuropathologically by inclusions containing abnormal forms of the microtubule-associated protein tau (MAPT) and clinically by diverse neuropsychiatric, cognitive, and motor impairments. Autosomal dominant mutations in the MAPT gene cause heterogeneous forms of frontotemporal lobar degeneration with tauopathy (FTLD-Tau). Common and rare variants in the MAPT gene increase the risk for sporadic FTLD-Tau, including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). We generated a collection of fibroblasts from 140 MAPT mutation/risk variant carriers, PSP, CBD, and cognitively normal controls; 31 induced pluripotent stem cell (iPSC) lines from MAPT mutation carriers, non-carrier family members, and autopsy-confirmed PSP patients; 33 genome engineered iPSCs that were corrected or mutagenized; and forebrain neural progenitor cells (NPCs). To begin to identify the genes and pathways that are dysregulated in primary tauopathies, we performed transcriptomic analyses in induced pluripotent stem cell (iPSC)-derived neurons carrying MAPT p.R406W and CRISPR/Cas9-corrected isogenic controls. We found that the expression of the MAPT p.R406W mutation was sufficient to create a significantly different transcriptomic profile compared with that of the isogeneic controls and to cause the differential expression of 328 genes. Sixty-one of these genes were also differentially expressed between MAPT p.R406W carriers and control brains. Twelve of these genes are also differentially expressed between PSP and control brains. Together, these genes are enriched for pathways involved in GABA-mediated signaling and synaptic function, which may contribute to the pathogenesis of FTLD-tau and other primary tauopathies. Here, we present a resource of fibroblasts, iPSCs, and NPCs with comprehensive clinical histories that can be accessed by the scientific community for disease modeling and development of novel therapeutics for tauopathies.

Identification of pharmacodynamics readouts through acute dosing of a small molecule inhibitor of tau self‐association in a tauopathy mouse model

AbstractBackgroundThe accumulation of tau aggregates correlates with and facilitates disease progression in pure and mixed tauopathies such as Progressive Supranuclear Palsy and Alzheimer’s disease. However, a disease‐modifying‐therapeutic targeting tau aggregation has not yet shown success in the clinic. Our target approach is highly differentiated from other programs that are focused on inhibiting tau aggregation and all downstream cascades. We have selected and optimized small molecules capable of targeting tau self‐association (TSA), the first and rate‐limiting step in tau aggregation. We have demonstrated the efficacy of our lead TSA inhibitor in two mouse models of tauopathy (htau mice; tau‐P301L‐JNPL3 mice) in preventive and therapeutic studies. Treatment with the lead TSA inhibitor blocked tau self‐association and formation of large tau aggregates, reduced overall tau accumulation, and rescued impaired motor function in aged mice with pre‐existing tau aggregates. IND‐enabling studies for the lead TSA inhibitor are completing, and a FDA submission is planned for Q1 2022. In this study, we aim to identify acute measures of efficacy in aged homozygous female tau‐P301L‐JNPL3 mice that will be used to model Pharmacokinetics (PK)/Pharmacodynamics (PD) relationship of our TSA inhibitors.MethodsAcute dose range‐finding (ADRF) study was performed using multiple routes of administration. C57BL/6 mice (n=30) were either administered vehicle or a single dose of a selected TSA inhibitor ranging from 2 to 200 mg/kg (n = 3 mice/gender/group). Biospecimens were collected at Cmax. PK studies in C57BL/6 mice will be performed with and without fasting to determine the food effects on bioavailability.ResultsSerum and brain homogenate samples from the ADRF study are being analyzed to evaluate TSA inhibitor concentrations by LC‐MS/MS. These results will be used to select the dose and route of administration for PK studies in 9 months‐old homozygous female JNPL3 mice. To determine the selective dose for PD evaluations, PK studies will be performed using three doses. Some of the PD measures that will be evaluated are tau self‐association, tau hyperphosphorylation, and motor behavior.ConclusionsUnderstanding PK/PD relationship will be crucial for determining appropriate dosing and target engagement biomarkers in subsequent Phase‐1b studies.

Cornel iridoid glycoside protects against STAT1‐dependent synapse and memory deficits by increasing NMDA receptor expression in tau transgenic mice

AbstractBackgroundP301S transgenic mice is an animal model of tauopathy and Alzheimer’s disease (AD), exhibiting tau pathology and synaptic dysfunction. Cornel iridoid glycoside (CIG) is an active ingredient extracted from Cornus officinalis, a traditional Chinese herb. The purpose of the present study was to investigate the effects and mechanisms of CIG on tau pathology and synaptic dysfunction using P301S mice.MethodIntragastric administration of CIG for 3.5 months was applied to P301S mice from 9‐month‐old to 12.5‐month‐old of age. Objective recognition test and Y maze task were used to test the cognitive impairments of P301S mice. Electrophysiological recording was applied to record long‐term potential. Transmission electron microscopy was used to investigate the the ultrastructure and number of synapse. Immunohistochemical staining was applied to detect tau phosphorylation. Western blotting was used to detect the expression levels of related proteins.ResultsIntragastric administration of CIG for 3.5 months improved learning and memory abilities, increased the survival rate of P301S mice. Electrophysiological recordings and transmission Electron microscopy study showed that CIG improved synaptic plasticity, increased the ultrastructure and number of synapse. Moreover, CIG increased the expression of N‐methyl‐D‐aspartate receptors (NMDARs) subunits GluN1, GluN2A and GluN2B, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor (AMPAR) subunit GluA1. The major mechanism of CIG involved in the regulation of synaptic dysfunction was to inhibited the activation of Janus kinase‐2 (JAK2) /signal transducer and activator of transcription 1 (STAT1) signaling pathway and STAT1‐induced suppression of NMDAR expression.ConclusionBased on our findings, CIG might be a promising candidate for the prevention of tauopathy such as AD.

Testing the neuroinflammatory role of tau‐induced transposable elements in tauopathy

AbstractBackgroundNovel mechanisms through which pathogenic tau leads to neuronal toxicity remain largely unexplored as therapeutic targets for tauopathies. Activation of retrotransposons has been identified as a mechanism by which pathogenic tau contributes to neurotoxicity (Guo et al., 2018; Sun et al., 2018). To mobilize, retrotransposons are transcribed into RNA, reverse transcribed into DNA, and reinserted into the genome in a new location. Control of retrotransposons occurs at two levels, 1) transcriptional control via the location of retrotransposons within highly condensed heterochromatin and 2) by post‐transcriptional silencing mechanisms. While activated retrotransposons can create novel mutations via the “copy‐and‐paste” mechanism, double stranded RNAs (dsRNAs) formed from transposable element transcripts trigger the innate immune response, further contributing to cell toxicity (Cuellar et al., 2017; Kassiotis &amp; Stoye, 2016; Roulois et al., 2015). Elevation of dsRNAs is documented in models of AGS and TDP‐43 proteinopathy, where dsRNA derived from repetitive elements can stimulate an interferon response (Ahmad et al., 2018; LaRocca, et al., 2019; Rodriguez et al., 2021).We are currently testing the hypothesis that transposable element‐derived dsRNAs stimulate the immune response in tauopathy.MethodBioinformatic analysis of bidirectional transcription was completed on RNA‐sequencing data from control and tau transgenic Drosophila. dsRNAs were quantified by J2 anti‐dsRNA antibody via immunofluorescence and ELISA. Analysis of inflammatory markers in Drosophila was completed by RNA‐sequencing and with a custom NanoString Gene Expression codeset.ResultWe find that dsRNAs are elevated in the brains of tau transgenic Drosophila, in the cortex of a mouse model of tauopathy, and in post‐mortem brain tissue from human tauopathy. Based on bioinformatic analysis, we identified retrotransposons that are bidirectionally transcribed in tau transgenic Drosophila. RNA sequencing and NanoString analysis of tau transgenic flies reveals elevated levels of innate immune response gene transcripts. Loss of transposon transcriptional silencing results in similar dsRNA production and immune system activation found in tau transgenic Drosophila.ConclusionOur goal is to determine if RNA intermediates from tau‐induced transposable elements are a therapeutic target for tauopathies. Overall, our findings support a model where dsRNA production induced by loss of transcriptional transposon silencing contributes to inflammation in tauopathy

Functional study in the young rTg4510 mouse model of tauopathy

AbstractBackgroundTau hyperphosphorylation (pTau) is considered a key pathogenic event in the development of tauopathies. Nevertheless, we recently demonstrated that at the very early disease stage, pTau promotes neuroprotection by preventing seizure‐like activity.MethodTo further support the notion that very early pTau is not detrimental, the present work evaluated the young rTg4510 mouse model of tauopathy as a case study. Thus, in mice at one month of age (p30‐35), we studied the increase of pTau within the hippocampal area as well as hippocampal and locomotor function.ResultOur results showed that the very young rTg4510 model has no detectable changes in hippocampal dependent tasks, such as spontaneous alternation and nesting, or in locomotor activity. However, at this very early stage the hippocampal neurons from p30‐35 rTg4510 mice accumulate pTau protein and exhibit changes in hippocampal oscillatory activity. Moreover, we found an increased number of dendrites per cell in granule neurons.ConclusionAltogether, this study provides new insights into the early pathogenesis of tauopathies and provides further evidence that pTau remodels hippocampal function and morphology.

Efficacy of a vectorized anti‐tau antibody using systemic dosing of a blood brain barrier penetrant AAV capsid in mouse models of tauopathies

AbstractBackgroundAnti‐tau immunotherapy is a promising therapy for tauopathies, including Alzheimer's disease (AD), frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP). While passive immunization with anti‐tau monoclonal antibodies has been shown by several laboratories to reduce age‐dependent tau pathology and neurodegeneration in mouse tauopathy models, these studies have typically used repeated high weekly doses of antibody and reported only moderate reduction of tau pathology. To circumvent these limitations, using an adeno‐associated virus (AAV) vector to deliver a tau monoclonal antibody into hippocampus by direct injection to the location have been described. High levels of antibody and marked reduction of tau pathology is achieved by this approach. To further optimize this approach with a lesser invasive administration route, we have previously demonstrated broad distribution and expression of a vectorized anti‐tau antibody in the mouse brain using a blood‐brain barrier (BBB) penetrant AAV capsid administered intravenously (IV). This gene therapy‐based approach has potential advantages, including continuous expression of antibody in the central nervous system (CNS) after a single administration of vector, increased CNS levels of tau antibody relative to passive immunotherapy, and the potential to target intracellular tau aggregates. Here we describe studies characterizing the onset of transduction following dosing with a vectorized tau antibody and its efficacy in mouse models of tauopathy.MethodAAV vectors comprising the BBB‐penetrant AAV capsid and a transgene encoding an anti‐tau monoclonal antibody were administered by IV bolus to mouse models of tauopathy. Biodistribution and cellular tropism in the CNS were evaluated by ELISA and (or) immunostaining. Tau pathology was measured by AT100 immunohistochemistry and AT8 ELISA.ResultWe then investigated efficacy in reduction of tau pathology in mouse tauopathy models. Treatment with our vectorized antibody resulted in durable antibody expression in the CNS and a corresponding reduction in CNS insoluble pathological tau and neurofibrillary tangles.ConclusionOur results indicate that systemic dosing of a vectorized anti‐tau antibody using a BBB‐penetrant AAV capsid results in reduced tau pathology and may represent a new single‐dose therapeutic strategy for treating various tauopathies.

Neuronal NOX4 knockdown alleviates pathological tau-related alterations in a humanized mouse model of tauopathy

Neuronal NOX4 knockdown alleviates pathological tau-related alterations in a humanized mouse model of tauopathy

Impact of MAPT mutations on transcriptomic signatures of FTLD brains and patient‐derived pluripotent cell models

AbstractBackgroundMutations in the microtubule‐associated protein tau (MAPT) cause heterogeneous forms of frontotemporal lobar dementia with tau inclusions (FTLD‐tau). Yet the pathogenic events linked to disease remain poorly understood. This study aimed to identify genes and pathways that lead to FTLD‐tau.MethodTo identify the earliest genes and pathways that are dysregulated in FTLD‐tau, we identified differentially expressed genes in RNA‐seq data generated from induced pluripotent stem cell (iPSC)–derived cortical neurons carrying MAPT R406W, MAPT P301L, and MAPT IVS10+16 and isogenic, controls and brain tissue samples from progressive supranuclear palsy (PSP) and control brains. We then identified pathological pathways and drug targets that were enriched among the differentially expressed genes.ResultsWe identified 275 genes that were differentially expressed in iPSC–derived cortical neurons from MAPT R406W carriers compared to isogenic controls, MAPT IVS10+16 carriers compared to isogenic controls, and MAPT P301L carriers compared with isogenic controls. These commonly dysregulated genes were enriched for pathways involving synaptic function, neuronal development, and endolysosomal function. A subset of these genes were also changed in brains from human subjects with PSP compared to normal control brains. Finally, a subset of genes, enriched in glutamate receptor signaling, were altered across the mutant neurons and significantly changed with tau accumulation in a mouse model of tauopathy (Tau‐P301L mice).ConclusionThe results from this study demonstrate that iPSC‐derived neurons capture molecular processes that occur in human brains and can be used to model disease and point to common molecular pathways driven by 3 distinct MAPT mutations

Biologic TNF-\u03b1 inhibitors reduce microgliosis, neuronal loss, and tau phosphorylation in a transgenic mouse model of tauopathy

BackgroundTumor necrosis factor-α (TNF-α) plays a central role in Alzheimer’s disease (AD) pathology, making biologic TNF-α inhibitors (TNFIs), including etanercept, viable therapeutics for AD. The protective effects of biologic TNFIs on AD hallmark pathology (Aβ deposition and tau pathology) have been demonstrated. However, the effects of biologic TNFIs on Aβ-independent tau pathology have not been reported. Existing biologic TNFIs do not cross the blood–brain barrier (BBB), therefore we engineered a BBB-penetrating biologic TNFI by fusing the extracellular domain of the type-II human TNF-α receptor (TNFR) to a transferrin receptor antibody (TfRMAb) that ferries the TNFR into the brain via receptor-mediated transcytosis. The present study aimed to investigate the effects of TfRMAb-TNFR (BBB-penetrating TNFI) and etanercept (non-BBB-penetrating TNFI) in the PS19 transgenic mouse model of tauopathy.MethodsSix-month-old male and female PS19 mice were injected intraperitoneally with saline (n = 12), TfRMAb-TNFR (1.75 mg/kg, n = 10) or etanercept (0.875 mg/kg, equimolar dose of TNFR, n = 10) 3 days/week for 8 weeks. Age-matched littermate wild-type mice served as additional controls. Blood was collected at baseline and 8 weeks for a complete blood count. Locomotion hyperactivity was assessed by the open-field paradigm. Brains were examined for phosphorylated tau lesions (Ser202, Thr205), microgliosis, and neuronal health. The plasma pharmacokinetics were evaluated following a single intraperitoneal injection of 0.875 mg/kg etanercept or 1.75 mg/kg TfRMAb-TNFR or 1.75 mg/kg chronic TfRMAb-TNFR dosing for 4 weeks.ResultsEtanercept significantly reduced phosphorylated tau and microgliosis in the PS19 mouse brains of both sexes, while TfRMAb-TNFR significantly reduced these parameters in the female PS19 mice. Both TfRMAb-TNFR and etanercept treatment improved neuronal health by significantly increasing PSD95 expression and attenuating hippocampal neuron loss in the PS19 mice. The locomotion hyperactivity in the male PS19 mice was suppressed by chronic etanercept treatment. Equimolar dosing resulted in eightfold lower plasma exposure of the TfRMAb-TNFR compared with etanercept. The hematological profiles remained largely stable following chronic biologic TNFI dosing except for a significant increase in platelets with etanercept.ConclusionBoth TfRMAb-TNFR (BBB-penetrating) and non-BBB-penetrating (etanercept) biologic TNFIs showed therapeutic effects in the PS19 mouse model of tauopathy.

Investigating circular RNA‐mediated neurotoxicity in tauopathies

AbstractBackgroundCircular RNAs (circRNA) are a subclass of non‐coding RNAs with a covalently closed loop structure that are formed via non‐canonical splicing1. CircRNAs are specifically enriched in the brain among different species and further accumulate in the brain as a function of physiological aging2. CircRNA biogenesis is regulated by N6‐methyladenosine (m6A), a post transcriptional RNA modification3. Elevated levels of circRNAs and abnormal m6A modification significantly correlate with clinical diagnosis and development of Alzheimer’s disease, respectively4, 5. Alzheimer’s disease and related “tauopathies” are pathologically defined by various forms of tau aggregates in the brains of affected individuals. We have previously reported that tau‐induced dysfunction of lamin, a nuclear scaffold protein, causes nuclear polymorphisms including invaginations and blebs 6, 7. We have also found that polyadenylated RNA accumulates within nuclear invaginations in the context of tauopathy and that genetic and pharmacologic reduction of RNA export reduce RNA accumulation within invaginations and suppresses tau neurotoxicity8. While dysfunction of the nuclear envelope and consequent aberrant RNA export mediate tau‐induced neurotoxicity, the identity of RNAs that accumulate within nuclear blebs, the role of m6A in circRNA accumulation, and their relationship with aging and tauopathy are currently unknown. Based on the association between nuclear polymorphism and RNA export, alongside the global enrichment of circRNAs and disrupted RNA methylation in Alzheimer’s disease brains, I hypothesize that m6A‐dependent accumulation of circRNAs in tauopathies sequester complementary RNAs and RNA binding proteins into large inclusions that trigger RNA export via nuclear blebbing.MethodsRNA‐seq, digital PCR, TUNEL, electron microscopy and immunofluorescence.ResultsI find that circRNAs accumulate in brains of a Drosophila model of tauopathy and that RNAi‐mediated reduction of mbl, which is particularly enriched in its circular form in the brain, significantly suppresses tau‐induced neurotoxicity. RNAi‐mediated reduction of an m6A writer and reader significantly reduces circMbl biogenesis and neuronal death in tau transgenic Drosophila. I find that circMbl lines the nuclear blebs that contain large inclusions in brains of tau transgenic Drosophila.ConclusionsTau‐induced accumulation of circRNA is mediated by aberrant m6A modification. Presence of circMbl‐lined nuclear buds suggests a potential role of nuclear blebs in circRNA nuclear export.

The effect of microglial genes on network diffusion of pathology in mouse models of tauopathy

AbstractBackgroundMisfolded tau and amyloid, the hallmark pathologies of Alzheimer’s disease, undergo “prion‐like” trans‐synaptic transmission along the brain’s anatomic connectivity network (Frost, et al,2010). Our lab has previously shown that Alzheimer’s neuropathology progression is well‐approximated by a network diffusion model (NDM) (Raj, et al,2012). However, other processes in the brain may affect the neuropathology progression by mediating intraregional clearance/amplification and interregional spread. For instance, microglial activation is a common pathological feature of Alzheimer’s disease, with TREM2‐expressing microglia being integral to the inflammatory response. We show here that the NDM can be extended to include effects of microglial mediation of pathology.MethodWe modified the NDM to an extended NDM (eNDM) with additional model parameters related to microglia‐mediated spread/clearance and applied it to the 426×426 region mouse connectome. The eNDM included datasets of longitudinal spatiotemporal tau pathology in PS19 mice, expression levels of five microglial homeostatic signature genes (Cx3Cr1, C1qa, P2ry12, Fcrls, Olfml3) as well as TREM2 and CD33, microglial surface‐markers and phagocytosis regulators (Li, et al,2018). We compared the fits of the NDM to those of the eNDM using each gene individually as well as the first two principal components of spatial gene expression (Lein, et al,2007) after optimizing model parameters using a constrained nonlinear optimization algorithm.ResultsInclusion of microglial gene expression improved the predictive power of the model beyond that of the NDM (R2=0.46). Using the principal components of gene expression generally provided better model fits than individual genes. Notably, exclusion of TREM2 before performing PCA had a significant effect on model fit (p&lt;0.001, R2=0.5) (Table 1, Figure 1), increasing the values of the optimal diffusion and spread parameters.ConclusionAlthough network spread models capture well the spread of pathology between brain regions, there are other factors that mediate this process. We demonstrated that an eNDM with microglial genes had an improved model fit even after accounting for differences in model parsimony. We also showed that exclusion of TREM2 from the model resulted in higher diffusivity parameter values, thus supporting the biological observation of TREM2‐knockout resulting in increased spread of pathology, putatively by affecting microglial phagocytic potential (Griciuc, et al, 2019).

HCN channelopathy couples disease‐associated tau to synaptic dysfunction

AbstractBackgroundDeficits in synaptic function and neurite connectivity are early correlates of tauopathies. Altered tau processing and mis‐localization coincide with reductions in synapse density and synaptic function, suggesting a causal role for tau in disease pathogenesis. In parallel, altered activity of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels, involved in synaptic integration, is implicated in disease progression in animal models of tauopathy. However, the mechanisms underlying these pathological events are not fully understood.MethodsWe performed histological and biochemical analyses of hippocampal tissues from Alzheimer’s disease (AD), age‐matched controls and Tau35 mice, in which a human tauopathy‐associated tau fragment is minimally expressed. Image analysis and quantification were performed on antibody‐labelled human and mouse hippocampal sections (Visiopharm). Brain tissue from Tau35 and wild‐type (WT) mice was also examined on western blots, by Golgi‐Cox staining and transmission electron microscopy. Primary hippocampal neurons from Tau35 and WT mice were transfected with a plasmid expressing enhanced green fluorescent protein (eGFP) and imaged up to 14 days in vitro. Three‐dimensional digital reconstruction of dendrites and spines enabled morphological analysis of eGFP‐expressing neurons. In parallel, patch‐clamp recordings were performed to assess functional changes and HCN channel activity in Tau35 neurons.ResultsHere we show that HCN channels are functionally linked to tau abnormalities. Alterations in HCN channel expression are detected in both Tau35 mouse and human post‐mortem AD brain. Tau35 neurons exhibit altered synaptic cytoarchitecture, including progressive reductions in dendritic branching, pre‐synaptic vesicles, spine density and synaptic markers, along with the development of tau pathology. These changes are accompanied by functional abnormalities in network activity, including increased HCN‐dependent sag voltage, reduced frequency and slower kinetics of spontaneous excitatory postsynaptic currents.ConclusionsOur findings are consistent with the hypothesis that disease‐associated tau species impact on HCN channels to drive network‐wide structural and functional synaptic deficits, with significant therapeutic relevance for tauopathies.

Modeling tauopathy using human-like pathogenic tau seeds.

Compared with traditional transgenic models of tauopathy, there is no confounding effect from the insertion of the exogenous gene and overexpression of the human mutant tau in the tau spreading models. By strategically choosing the location and time of the inoculation, we can spatiotemporally determine the starting point of tau pathology in the model mimicking the development of tauopathy. Moreover, they recapitulate the strain-specific features of tau pathology. Regardless of these advantages, human-like tau spreading models are very much confined due to the limitation on quantity and quality of tau seeds. In vitro seeding assay has shown its great potential of amplifying the pathogenic tau seeds to expand their quantity and increase their quality. In the study, we used biochemically enriched, human-derived, tau aggregates as seeds to template recombinant tau (rT40) in vitro. Using biochemistry and electron microscopy, we examined the biophysical properties of the amplified materials and the human-derived tau seeds. We also interrogated the pathogenicity of the amplified human-like tau strains with orthogonal spreading models of tauopathy. As a result, we observed elongated tau filaments with recombinant T40 attached to the seeds. Compared with the seeds control, the amplified tau strains showed increased pathogenic tau species after seeding. The amplified tau strains recapitulate the pathogenicity and biophysical property of the original seeds. Our data suggest that the in vitro seeded recombinant tau can be recruited onto the seeds and converted into the confirmation of pathological tau seeds in a strain-dependent manner. Along with the conversion, the pathogenicity of different tau strains can be conferred and conserved into the recombinant tau. The study provides an authentic way of amplifying tau strains in vitro as well as a tool to expand our ability to understand tau fibrillization and transmission.

Elucidating cellular contributions to tau-mediated neurodegeneration using drosophila and single-cell transcriptomics.

Alzheimer's Dementia (AD) is neuropathologically defined by aggregations of amyloid-beta plaques and tau neurofibrillary tangles in the brain, with tau pathology being most strongly correlated with cognitive decline. Systems biology approaches profiling post-mortem human AD brain tissue have highlighted involvement of key processes such as innate immunity and translational regulation. However, interpretation of cross-sectional post-mortem gene expression data is confounded by co-morbidities and mixed brain pathologies at autopsy. Moreover, characterizing longitudinal, age-related changes in Alzheimer's dementia (AD) is of particular importance because clinical manifestation of AD is a decades-long process, and age is the strongest known risk factor. We previously completed RNA-sequencing and proteomics of several highly tractable, longitudinal, Drosophila models of tauopathy to characterize tau- and age-specific perturbations. However, cellular heterogeneity confounds interpretation of bulk tissue data. We thus leverage single cell RNA sequencing (scRNA-seq) to explore cell-specific contributions to tau- and age-mediated gene expression. Whole brains of adult Drosophila pan-neuronally expressing a mutant variant of human Tau (R406W) and controls were dissociated for scRNA-seq (10x Genomics Chromium) at 1-, 10-, and 20- days of age. Perturbations in cellular composition and gene expression were quantified. To address cross-species conservation of tau-mediated scRNAseq signatures, we leverage existing human AD brain scRNA-seq data. As expected, we highlight evidence for tau-mediated degeneration of Kenyon cells, a neuronal population in the Drosophila "mushroom body" that is required for learning and memory, along with a concomitant increase in glial cell numbers (e.g. gliosis). Gene set enrichment analysis reveal overrepresentation of acetylcholine receptor regulation pathways in vulnerable mushroom body neurons. Interestingly, regularized regression analyses highlight innate immune activity as predictive of tau-related neuronal reduction. Cluster-specific transcriptional signatures between flies and humans demonstrate strong correlations among inferred cell identities. Our results comprise a powerful single cell transcriptomic resource for studying tau-mediated disruption of gene expression and dynamic age-dependent changes at cellular resolution in a tractable model system that is amenable for high throughput functional validation.