Targeting 14-3-3θ-mediated TDP-43 pathology in amyotrophic lateral sclerosis and frontotemporal dementia mice.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by cytoplasmic deposition of the nuclear TAR-binding protein 43 (TDP-43). Although cytoplasmic re-localization of TDP-43 is a key event in the pathogenesis of ALS/FTD, the underlying mechanisms remain unknown. Here, we identified a non-canonical interaction between 14-3-3θ and TDP-43, which regulates nuclear-cytoplasmic shuttling. Neuronal 14-3-3θ levels were increased in sporadic ALS and FTD with TDP-43 pathology. Pathogenic TDP-43 showed increased interaction with 14-3-3θ, resulting in cytoplasmic accumulation, insolubility, phosphorylation, and fragmentation of TDP-43, resembling pathological changes in disease. Harnessing this increased affinity of 14-3-3θ for pathogenic TDP-43, we devised a gene therapy vector targeting TDP-43 pathology, which mitigated functional deficits and neurodegeneration in different ALS/FTD mouse models expressing mutant or non-mutant TDP-43, including when already symptomatic at the time of treatment. Our study identified 14-3-3θ as a mediator of cytoplasmic TDP-43 localization with implications for ALS/FTD pathogenesis and therapy.

Methyl donor supplementation reduces phospho-Tau, Fyn and demethylated protein phosphatase 2A levels and mitigates learning and motor deficits in a mouse model of tauopathy.

BACKGROUND: Reduced folate status and elevated levels of circulating homocysteine are modifiable risk factors for cognitive decline and dementia. Disturbances in one-carbon metabolism are associated with the pathological accumulation of phosphorylated tau, a hallmark feature of prevalent dementia, including Alzheimer's disease and subgroups of frontotemporal dementia. METHODS: Here, using transgenic TAU58/2 mouse models of human tauopathy, we tested whether dietary supplementation with L-methylfolate (the active folate form), choline and betaine can reduce tau phosphorylation and associated behavioural phenotypes. RESULTS: TAU58/2 mice fed with the methyl donor-enriched diet showed reduced phosphorylation of tau at the pathological S202 (CP13) and S396/S404 (PHF-1) epitopes and alleviation of associated motor and learning deficits. Compared with mice on the control diet, the decrease in cortical phosphorylated tau levels in mice fed with the methyl donor-enriched diet was associated with enhanced methylation of protein phosphatase 2A, the major brain tau Ser/Thr phosphatase. It also correlated with a reduction in protein levels of Fyn, a tau tyrosine kinase that plays a central role in mediating pathological tau-induced neurodegeneration. Conversely, Fyn expression levels were increased in mice with deficiencies in folate metabolism. CONCLUSIONS: Our findings provide the first experimental evidence that boosting one-carbon metabolism with L-methylfolate, choline and betaine can mitigate key pathological, learning and motor deficits in a tauopathy mouse model. They give support to using a combination of methyl donors as a preventive or disease-modifying strategy for tauopathies.

TDP-43 pathology and functional deficits in wild-type and ALS/FTD mutant cyclin F mouse models.

AIMS: Amyotrophic lateral sclerosis (ALS) is characterised by a progressive loss of upper and lower motor neurons leading to muscle weakness and eventually death. Frontotemporal dementia (FTD) presents clinically with significant behavioural decline. Approximately 10% of cases have a known family history, and disease-linked mutations in multiple genes have been identified in FTD and ALS. More recently, ALS and FTD-linked variants have been identified in the CCNF gene, which accounts for an estimated 0.6% to over 3% of familial ALS cases. METHODS: In this study, we developed the first mouse models expressing either wild-type (WT) human CCNF or its mutant pathogenic variant S621G to recapitulate key clinical and neuropathological features of ALS and FTD linked to CCNF disease variants. We expressed human CCNF WT or CCNFS621G throughout the murine brain by intracranial delivery of adeno-associated virus (AAV) to achieve widespread delivery via somatic brain transgenesis. RESULTS: These mice developed behavioural abnormalities, similar to the clinical symptoms of FTD patients, as early as 3 months of age, including hyperactivity and disinhibition, which progressively deteriorated to include memory deficits by 8 months of age. Brains of mutant CCNF_S621G mice displayed an accumulation of ubiquitinated proteins with elevated levels of phosphorylated TDP-43 present in both CCNF_WT and mutant CCNF_S621G mice. We also investigated the effects of CCNF expression on interaction targets of CCNF and found elevated levels of insoluble splicing factor proline and glutamine-rich (SFPQ). Furthermore, cytoplasmic TDP-43 inclusions were found in both CCNF_WT and mutant CCNF_S621G mice, recapitulating the key hallmark of FTD/ALS pathology. CONCLUSIONS: In summary, CCNF expression in mice reproduces clinical presentations of ALS, including functional deficits and TDP-43 neuropathology with altered CCNF-mediated pathways contributing to the pathology observed.

Loss of LAMP5 interneurons drives neuronal network dysfunction in Alzheimer's disease.

In Alzheimer's disease (AD), where amyloid-ß (Aß) and tau deposits in the brain, hyperexcitation of neuronal networks is an underlying disease mechanism, but its cause remains unclear. Here, we used the Collaborative Cross (CC) forward genetics mouse platform to identify modifier genes of neuronal hyperexcitation. We found LAMP5 as a novel regulator of hyperexcitation in mice, critical for the survival of distinct interneuron populations. Interestingly, synaptic LAMP5 was lost in AD brains and LAMP5 interneurons degenerated in different AD mouse models. Genetic reduction of LAMP5 augmented functional deficits and neuronal network hypersynchronicity in both Aß- and tau-driven AD mouse models. To this end, our work defines the first specific function of LAMP5 interneurons in neuronal network hyperexcitation in AD and dementia with tau pathology.

Rapid initiation of cell cycle reentry processes protects neurons from amyloid-ß toxicity.

Neurons are postmitotic cells. Reactivation of the cell cycle by neurons has been reported in Alzheimer's disease (AD) brains and models. This gave rise to the hypothesis that reentering the cell cycle renders neurons vulnerable and thus contributes to AD pathogenesis. Here, we use the fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology to monitor the cell cycle in live neurons. We found transient, self-limited cell cycle reentry activity in naive neurons, suggesting that their postmitotic state is a dynamic process. Furthermore, we observed a diverse response to oligomeric amyloid-ß (oAß) challenge; neurons without cell cycle reentry activity would undergo cell death without activating the FUCCI reporter, while neurons undergoing cell cycle reentry activity at the time of the oAß challenge could maintain and increase FUCCI reporter signal and evade cell death. Accordingly, we observed marked neuronal FUCCI positivity in the brains of human mutant Aß precursor protein transgenic (APP23) mice together with increased neuronal expression of the endogenous cell cycle control protein geminin in the brains of 3-mo-old APP23 mice and human AD brains. Taken together, our data challenge the current view on cell cycle in neurons and AD, suggesting that pathways active during early cell cycle reentry in neurons protect from Aß toxicity.

Contribution of endogenous antibodies to learning deficits and astrocytosis in human P301S mutant tau transgenic mice.

Antibodies have been explored extensively as a potential therapeutic for Alzheimer's disease, where amyloid-ß (Aß) peptides and the tau protein deposit in patient brains. While the major focus of antibody-based therapy development was on Aß, arguably with limited success in clinical trials, targeting tau has become an emerging strategy, possibly extending therapies to dementias with isolated tau pathology. Interestingly, low titres of autoantibodies to pathological tau have been described in humans and transgenic mouse models, but their pathophysiological relevance remained elusive. Here, we used two independent approaches to deplete the B-cell lineage and hence antibody formation in human P301S mutant tau transgenic mice, TAU58/2. TAU58/2 mice were either crossed with the B-cell-deficient Ighm knockout line (muMT-/-) or treated with anti-CD20 antibodies that target B-cell precursors. In both models, B-cell depletion significantly reduced astrocytosis in TAU58/2 mice. Only when B-cells were absent throughout life, in TAU58/2.muMT-/- mice, were spatial learning deficits moderately aggravated while motor performance improved as compared to B-cell-competent TAU58/2 mice. This was associated with changes in brain region-specific tau solubility. No other relevant behavioural or neuropathological changes were observed in TAU58/2 mice in the absence of B-cells/antibodies. Taken together, our data suggests that the presence of antibodies throughout life contributes to astrocytosis in TAU58/2 mice and limits learning deficits, while other deficits and neuropathological changes appear to be independent of the presence of B-cells/antibodies.

CNS cell type-specific gene profiling of P301S tau transgenic mice identifies genes dysregulated by progressive tau accumulation.

The microtubule-associated protein tau undergoes aberrant modification resulting in insoluble brain deposits in various neurodegenerative diseases, including frontotemporal dementia (FTD), progressive supranuclear palsy, and corticobasal degeneration. Tau aggregates can form in different cell types of the central nervous system (CNS) but are most prevalent in neurons. We have previously recapitulated aspects of human FTD in mouse models by overexpressing mutant human tau in CNS neurons, including a P301S tau variant in TAU58/2 mice, characterized by early-onset and progressive behavioral deficits and FTD-like neuropathology. The molecular mechanisms underlying the functional deficits of TAU58/2 mice remain mostly elusive. Here, we employed functional genomics (i.e. RNAseq) to determine differentially expressed genes in young and aged TAU58/2 mice to identify alterations in cellular processes that may contribute to neuropathy. We identified genes in cortical brain samples differentially regulated between young and old TAU58/2 mice relative to nontransgenic littermates and by comparative analysis with a dataset of CNS cell type-specific genes expressed in nontransgenic mice. Most differentially-regulated genes had known or putative roles in neurons and included presynaptic and excitatory genes. Specifically, we observed changes in presynaptic factors, glutamatergic signaling, and protein scaffolding. Moreover, in the aged mice, expression levels of several genes whose expression was annotated to occur in other brain cell types were altered. Immunoblotting and immunostaining of brain samples from the TAU58/2 mice confirmed altered expression and localization of identified and network-linked proteins. Our results have revealed genes dysregulated by progressive tau accumulation in an FTD mouse model.

Does Stroke Rehabilitation Really Matter? Part B: An Algorithm for Prescribing an Effective Intensity of Rehabilitation.

BACKGROUND: The proportional recovery rule suggests that current rehabilitation practices may have limited ability to influence stroke recovery. However, the appropriate intensity of rehabilitation needed to achieve recovery remains unknown. Similarities between rodent and human recovery biomarkers may allow determination of rehabilitation thresholds necessary to activate endogenous biological recovery processes. OBJECTIVE: We determined the relative influence that clinically relevant biomarkers of stroke recovery exert on functional outcome. These biomarkers were then used to generate an algorithm that prescribes individualized intensities of rehabilitation necessary for recovery of function. METHODS: A retrospective cohort of 593 male Sprague-Dawley rats was used to identify biomarkers that best predicted poststroke change in pellet retrieval in the Montoya staircase-reaching task using multiple linear regression. Prospective manipulation of these factors using endothelin-1-induced stroke (n = 49) was used to validate the model. RESULTS: Rehabilitation was necessary to reliably predict recovery across the continuum of stroke severity. As infarct volume and initial impairment increased, more intensive rehabilitation was required to engage recovery. In this model, we prescribed the specific dose of daily rehabilitation required for rats to achieve significant motor recovery using the biomarkers of initial poststroke impairment and infarct volume. CONCLUSIONS: Our algorithm demonstrates an individualized approach to stroke rehabilitation, wherein imaging and functional performance measures can be used to develop an optimized rehabilitation paradigm for rats, particularly those with severe impairments. Exploring this approach in human patients could lead to an increase in the proportion of individuals experiencing recovery of lost motor function poststroke.