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.

TDP-43 and Inflammation: Implications for Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

The abnormal mislocalisation and ubiquitinated protein aggregation of the TAR DNA binding protein 43 (TDP-43) within the cytoplasm of neurons and glia in the central nervous system (CNS) is a pathological hallmark of early-onset neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathomechanisms underlying abnormal mislocalisation and aggregation of TDP-43 remain unknown. However, there is a growing body of evidence implicating neuroinflammation and immune-mediated mechanisms in the pathogenesis of neurodegeneration. Importantly, most of the evidence for an active role of immunity and inflammation in the pathogenesis of ALS and FTD relates specifically to TDP-43, posing the question as to whether immune-mediated mechanisms could hold the key to understanding TDP-43's underlying role in neurodegeneration in both diseases. Therefore, this review aims to piece together key lines of evidence for the specific association of TDP-43 with key immune and inflammatory pathways to explore the nature of this relationship and the implications for potential pathomechanisms underlying neurodegeneration in ALS and FTD.

Syntaxins 6 and 8 facilitate tau into secretory pathways.

Tau pathology initiates in defined brain regions and is known to spread along neuronal connections as symptoms progress in Alzheimer's disease (AD) and other tauopathies. This spread requires the release of tau from donor cells, but the underlying molecular mechanisms remained unknown. Here, we established the interactome of the C-terminal tail region of tau and identified syntaxin 8 (STX8) as a mediator of tau release from cells. Similarly, we showed the syntaxin 6 (STX6), part of the same SNARE family as STX8 also facilitated tau release. STX6 was previously genetically linked to progressive supranuclear palsy (PSP), a tauopathy. Finally, we demonstrated that the transmembrane domain of STX6 is required and sufficient to mediate tau secretion. The differential role of STX6 and STX8 in alternative secretory pathways suggests the association of tau with different secretory processes. Taken together, both syntaxins, STX6 and STX8, may contribute to AD and PSP pathogenesis by mediating release of tau from cells and facilitating pathology spreading.

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.

Onset of hippocampal network aberration and memory deficits in P301S tau mice are associated with an early gene signature.

Hyperphosphorylation and deposition of tau in the brain characterizes frontotemporal dementia and Alzheimer's disease. Disease-associated mutations in the tau-encoding MAPT gene have enabled the generation of transgenic mouse models that recapitulate aspects of human neurodegenerative diseases, including tau hyperphosphorylation and neurofibrillary tangle formation. Here, we characterized the effects of transgenic P301S mutant human tau expression on neuronal network function in the murine hippocampus. Onset of progressive spatial learning deficits in P301S tau transgenic TAU58/2 mice were paralleled by long-term potentiation deficits and neuronal network aberrations during electrophysiological and EEG recordings. Gene-expression profiling just prior to onset of apparent deficits in TAU58/2 mice revealed a signature of immediate early genes that is consistent with neuronal network hypersynchronicity. We found that the increased immediate early gene activity was confined to neurons harbouring tau pathology, providing a cellular link between aberrant tau and network dysfunction. Taken together, our data suggest that tau pathology drives neuronal network dysfunction through hyperexcitation of individual, pathology-harbouring neurons, thereby contributing to memory deficits.

Neurodegeneration and Motor Deficits in the Absence of Astrogliosis upon Transgenic Mutant TDP-43 Expression in Mature Mice.

Amyotrophic lateral sclerosis is a rapidly progressing and fatal disease characterized by muscular atrophy due to loss of upper and lower motor neurons. Pathogenic mutations in the TARDBP gene encoding TAR DNA binding protein-43 (TDP-43) have been identified in familial amyotrophic lateral sclerosis. We have previously reported transgenic mice with neuronal expression of human TDP-43 carrying the pathogenic A315T mutation (iTDP-43A315T mice) using a tetracycline-controlled inducible promotor system. Constitutive expression of transgenic TDP-43A315T in the absence of doxycycline resulted in pronounced early-onset and progressive neurodegeneration, and motor and memory deficits. Here, delayed transgene expression of TDP-43A315T by oral doxycycline treatment of iTDP-43A315T mice from birth till weaning was analyzed. After doxycycline withdrawal, transgenic TDP-43A315T expression gradually increased and resulted in cytoplasmic TDP-43, widespread ubiquitination, and cortical and hippocampal atrophy. In addition, these mice developed motor and gait deficits with underlying muscle atrophy, similar to that observed in the constitutive iTDP-43A315T mice. Surprisingly, in contrast to the constitutive iTDP-43A315T mice, these mice did not develop astrogliosis. In summary, delayed activation coupled with gradual increase in TDP-43A315T expression in the central nervous system of mature mice resulted in progressive functional deficits with neuron and muscle loss, but in the absence of a glial response. This suggests that astrocytosis does not contribute to functional deficits and neuronal loss upon TDP-43A315T expression in mature mice.

Developmental Expression of Mutant PFN1 in Motor Neurons Impacts Neuronal Growth and Motor Performance of Young and Adult Mice.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with limited treatment and no cure. Mutations in profilin 1 were identified as a cause of familial ALS (fALS) in 2012. We investigated the functional impact of mutant profilin 1 expression in spinal cords during mouse development. We developed a novel mouse model with the expression of profilin 1 C71G under the control of the Hb9 promoter, targeting expression to α-motor neurons in the spinal cord during development. Embryos of transgenic mice showed evidence of a significant reduction of brachial nerve diameter and a loss of Mendelian inheritance. Despite the lack of transgene expression, adult mice presented with significant motor deficits. Transgenic mice had a significant reduction in the number of motor neurons in the spinal cord. Further analysis of these motor neurons in aged transgenic mice revealed reduced levels of TDP-43 and ChAT expression. Although profilin 1 C71G was only expressed during development, adult mice presented with some ALS-associated pathology and motor symptoms. This study highlights the effect of profilin 1 during neurodevelopment and the impact that this may have in later ALS.

Selective Spatiotemporal Vulnerability of Central Nervous System Neurons to Pathologic TAR DNA-Binding Protein 43 in Aged Transgenic Mice.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing and fatal disease characterized by muscular atrophy because of loss of upper and lower motor neurons. Histopathologically, most patients with ALS have abnormal cytoplasmic accumulation and aggregation of the nuclear RNA-regulating protein TAR DNA-binding protein 43 (TDP-43). Pathogenic mutations in the TARDBP gene that encode TDP-43 have been identified in familial ALS. We have previously reported transgenic mice with neuronal expression of human TDP-43 carrying the pathogenic A315T mutation (iTDP-43A315T mice), presenting with early-onset motor deficits in adolescent animals. Here, we analyzed aged iTDP-43A315T mice, focusing on the spatiotemporal profile and progression of neurodegeneration in upper and lower motor neurons. Magnetic resonance imaging and histologic analysis revealed a differential loss of upper motor neurons in a hierarchical order as iTDP-43A315T mice aged. Furthermore, we report progressive gait problems, profound motor deficits, and muscle atrophy in aged iTDP-43A315T mice. Despite these deficits and TDP-43 pathologic disorders in lower motor neurons, stereological analysis did not show cell loss in spinal cords. Taken together, neuronal populations in aging iTDP-43A315T mice show differential susceptibility to the expression of human TDP-43A315T.

Tau exacerbates excitotoxic brain damage in an animal model of stroke.

Neuronal excitotoxicity induced by aberrant excitation of glutamatergic receptors contributes to brain damage in stroke. Here we show that tau-deficient (tau-/-) mice are profoundly protected from excitotoxic brain damage and neurological deficits following experimental stroke, using a middle cerebral artery occlusion with reperfusion model. Mechanistically, we show that this protection is due to site-specific inhibition of glutamate-induced and Ras/ERK-mediated toxicity by accumulation of Ras-inhibiting SynGAP1, which resides in a post-synaptic complex with tau. Accordingly, reducing SynGAP1 levels in tau-/- mice abolished the protection from pharmacologically induced excitotoxicity and middle cerebral artery occlusion-induced brain damage. Conversely, over-expression of SynGAP1 prevented excitotoxic ERK activation in wild-type neurons. Our findings suggest that tau mediates excitotoxic Ras/ERK signaling by controlling post-synaptic compartmentalization of SynGAP1.Excitotoxicity contributes to neuronal injury following stroke. Here the authors show that tau promotes excitotoxicity by a post-synaptic mechanism, involving site-specific control of ERK activation, in a mouse model of stroke.

Site-specific phosphorylation of tau inhibits amyloid-ß toxicity in Alzheimer's mice.

Amyloid-ß (Aß) toxicity in Alzheimer's disease (AD) is considered to be mediated by phosphorylated tau protein. In contrast, we found that, at least in early disease, site-specific phosphorylation of tau inhibited Aß toxicity. This specific tau phosphorylation was mediated by the neuronal p38 mitogen-activated protein kinase p38γ and interfered with postsynaptic excitotoxic signaling complexes engaged by Aß. Accordingly, depletion of p38γ exacerbated neuronal circuit aberrations, cognitive deficits, and premature lethality in a mouse model of AD, whereas increasing the activity of p38γ abolished these deficits. Furthermore, mimicking site-specific tau phosphorylation alleviated Aß-induced neuronal death and offered protection from excitotoxicity. Our work provides insights into postsynaptic processes in AD pathogenesis and challenges a purely pathogenic role of tau phosphorylation in neuronal toxicity.

No Overt Deficits in Aged Tau-Deficient C57Bl/6.Mapttm1(EGFP)Kit GFP Knockin Mice.

Several mouse lines with knockout of the tau-encoding MAPT gene have been reported in the past; they received recent attention due to reports that tau reduction prevented Aß-induced deficits in mouse models of Alzheimer's disease. However, the effects of long-term depletion of tau in vivo remained controversial. Here, we used the tau-deficient GFP knockin line Mapttm1(EGFP)kit on a pure C57Bl/6 background and subjected a large cohort of males and females to a range of motor, memory and behavior tests and imaging analysis, at the advanced age of over 16 months. Neither heterozygous nor homozygous Mapttm1(EGFP)kit mice presented with deficits or abnormalities compared to wild-type littermates. Differences to reports using other tau knockout models may be due to different genetic backgrounds, respective gene targeting strategies or other confounding factors, such as nutrition. To this end, we report no functional or morphological deficits upon tau reduction or depletion in aged mice.

Disinhibition-like behavior in a P301S mutant tau transgenic mouse model of frontotemporal dementia.

Frontotemporal dementia (FTD) presents clinically with behavioral changes including disinhibition. Mutations in the tau-encoding MAPT gene identified in familial cases of FTD have been used to generate transgenic mouse models of the human condition. Here, we report behavioral changes in a recently developed P301S mutant tau transgenic mouse, including disinhibition-like behavior in the elevated plus maze and hyperactivity in the open field arena. Furthermore, histological analysis revealed the amygdala as a primary and early site of pathological tau deposition in these mice. Taken together, neuropathological and behavioral changes in P301S tau transgenic mice resemble features of human FTD.

Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.

The nuclear transactive response DNA-binding protein 43 (TDP-43) undergoes relocalization to the cytoplasm with formation of cytoplasmic deposits in neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Pathogenic mutations in the TDP-43-encoding TARDBP gene in familial ALS as well as non-mutant human TDP-43 have been utilized to model FTD/ALS in cell culture and animals, including mice. Here, we report novel A315T mutant TDP-43 transgenic mice, iTDP-43(A315T), with controlled neuronal over-expression. Constitutive expression of human TDP-43(A315T) resulted in pronounced early-onset and progressive neurodegeneration, which was associated with compromised motor performance, spatial memory and disinhibition. Muscle atrophy resulted in reduced grip strength. Cortical degeneration presented with pronounced astrocyte activation. Using differential protein extraction from iTDP-43(A315T) brains, we found cytoplasmic localization, fragmentation, phosphorylation and ubiquitination and insolubility of TDP-43. Surprisingly, suppression of human TDP-43(A315T) expression in mice with overt neurodegeneration for only 1 week was sufficient to significantly improve motor and behavioral deficits, and reduce astrogliosis. Our data suggest that functional deficits in iTDP-43(A315T) mice are at least in part a direct and transient effect of the presence of TDP-43(A315T). Furthermore, it illustrates the compensatory capacity of compromised neurons once transgenic TDP-43 is removed, with implications for future treatments.

The responses of osteoblasts, osteoclasts and endothelial cells to zirconium modified calcium-silicate-based ceramic.

In this study we have developed Ca(3)ZrSi(2)O(9) (Baghdadite) ceramics by incorporating Zirconium in Ca-Si system and determined their biological properties. Ca(3)ZrSi(2)O(9) ceramics possess apatite-formation ability in simulated body fluid, indicating their potential bioactivity. The response of human osteoblast like cells (HOB), osteoclast and endothelial cells when cultured on Ca(3)ZrSi(2)O(9) ceramics was investigated. Scanning electron microscopy and immunofluorescence studies demonstrated that this material supports HOB cell attachment with organized cytoskeleton structure. Compared to CaSiO(3), Ca(3)ZrSi(2)O(9) ceramics induced increased HOB proliferation and differentiation as shown by increased methyltetrazidium salt (MTS), alkaline phosphatase activity, and mRNA expression levels of bone-related genes (Collagen type I, alkaline phosphatase, Bone Sialoprotein, receptor activator of NF-kappaB ligand and osteoprotegerin). Ca(3)ZrSi(2)O(9) ceramics supported the fusion of monocytes to form functional osteoclasts with their characteristic features of f-actin ring structures and the expression of alpha(v)beta(3) integrin consistent with functional activity. Osteoclasts cultured on Ca(3)ZrSi(2)O(9) expressed increased levels of osteoclast-related genes; Cathepsin K, Carbonic Anhydrase II, Matrix metalloproteinase-9, receptor activator of NF-kappaB and Calcitonin Receptor, consistent with the formation of functional osteoclasts. In addition to HOB and osteoclasts, Ca(3)ZrSi(2)O(9) supported the attachment of endothelial cells, which expressed the endothelial cell markers; ZO-1 and VE-Cadherin. Results presented here indicate that Ca(3)ZrSi(2)O(9) ceramics have the potential for applications in bone tissue regeneration.