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Loss of TAR DNA-binding protein 43 kDa (TDP-43) splicing repression is well-documented in postmortem tissues of amyotrophic lateral sclerosis (ALS), yet whether this abnormality occurs during early-stage disease remains unresolved. Cryptic exon inclusion reflects functional loss of TDP-43, and thus detection of cryptic exon-encoded peptides in cerebrospinal fluid (CSF) could reveal the earliest stages of TDP-43 dysregulation in patients. Here, we use a newly characterized monoclonal antibody specific to a TDP-43-dependent cryptic epitope (encoded by the cryptic exon found inHDGFL2) to show that loss of TDP-43 splicing repression occurs inC9ORF72-associated ALS, including pre-symptomatic mutation carriers. In contrast to neurofilament light and heavy chain proteins, cryptic HDGFL2 accumulates in CSF at higher levels during early stages of disease. Our findings indicate that loss of TDP-43 splicing repression occurs early in disease progression, even pre-symptomatically, and that detection of HDGFL2’s cryptic neoepitope may serve as a prognostic test for ALS which should facilitate patient recruitment and measurement of target engagement in clinical trials.

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell–mediated autoimmunity. IBM muscle biopsies display nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative diseases, where nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43–mediated splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of subjects with IBM. Of 119 muscle biopsies tested, RT-PCR–mediated detection of cryptic exon inclusion was able to diagnose IBM with 84% sensitivity and 99% specificity. To determine the role of T cells in pathogenesis, we generated a xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from subjects with IBM displayed robust regeneration of human myofibers and recapitulated both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts showed invasion by human, oligoclonal CD8 + T cells and exhibited MHC-I up-regulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, associated with cryptic exon inclusion. Reduction of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppressed MHC-I up-regulation. However, rimmed vacuoles and loss of TDP-43 function persisted. These data suggest that T cell depletion does not alter muscle degenerative pathology in IBM.

It is unknown whether specific microglia are selectively induced by amyloid-β(Aβ), tau pathologies, or both in combination. To address this, we use single-cell RNA-sequencing to profile mice bearing both Aβ and tau pathologies during Alzheimer's disease (AD). We identify novel microglia subtypes induced in a disease stage-specific manner. We show that during early-stage disease, interferon signaling induces a subtype of microglia termed EADAM. During late-stage disease, a second microglia subtype termed LADAM is detected. While EADAM and LADAM-like microglia are observed in other neurodegenerative models, the magnitude and composition of subtype markers are distinct from microglia observed with AD-like pathology. The pattern of EADAM- and LADAM-associated gene expression is observed in microglia from human AD, during the early and late stages of disease, respectively. Furthermore, we observe that several siglec genes are selectively expressed in either EADAM or LADAM. Siglecg is expressed in white-matter-associated LADAM, and expression of the human orthologue of Siglecg is progressively elevated in AD-stage-dependent manner but not shown in non-AD tauopathy. Our findings imply that both Aβ and tau pathologies are required for disease stage-specific induction of EADAM and LADAM.

Nuclear depletion of TDP-43, an essential RNA binding protein, may underlie neurodegeneration in amyotrophic lateral sclerosis (ALS). As several functions have been ascribed to this protein, the critical role(s) of TDP-43 in motor neurons that may be compromised in ALS remains unknown. We show here that TDP-43 mediated splicing repression, which serves to protect the transcriptome by preventing aberrant splicing, is central to the physiology of motor neurons. Expression in Drosophila TDP-43 knockout models of a chimeric repressor, comprised of the RNA recognition domain of TDP-43 fused to an unrelated splicing repressor, RAVER1, attenuated motor deficits and extended lifespan. Likewise, AAV9-mediated delivery of this chimeric rescue repressor to mice lacking TDP-43 in motor neurons delayed the onset, slowed the progression of motor symptoms, and markedly extended their lifespan. In treated mice lacking TDP-43 in motor neurons, aberrant splicing was significantly decreased and accompanied by amelioration of axon degeneration and motor neuron loss. This AAV9 strategy allowed long-term expression of the chimeric repressor without any adverse effects. Our findings establish that splicing repression is a major function of TDP-43 in motor neurons and strongly support the idea that loss of TDP-43-mediated splicing fidelity represents a key pathogenic mechanism underlying motor neuron loss in ALS.

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell-mediated autoimmunity. IBM muscle biopsies exhibit nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative disease, and nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43 splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of IBM patients. Out of 119 muscle biopsies tested, RT-PCR-mediated detection of cryptic exon expression is 84% sensitive and 99% specific for diagnosing IBM, indicating utility as a functional and diagnostic biomarker. To determine the role of T cells in pathogenesis, we generated a novel xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from IBM patients display robust regeneration of human myofibers and recapitulate both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts are invaded by human, oligoclonal CD8+ T cells and exhibit MHC-I upregulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, resulting in expression of cryptic exons. Depletion of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppresses MHC-I upregulation, but rimmed vacuoles and loss of TDP-43 function persist. These data suggest that myofiber degeneration occurs independent of T cells, and muscle cell-intrinsic mechanisms, such as loss of TDP-43 splicing repression, drive IBM pathogenesis.One Sentence SummaryDepletion of T cells in a xenograft model of sporadic inclusion body myositis suppresses inflammation but not TDP-43 pathology or muscle degeneration.

A shared neuropathological hallmark in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is nuclear clearance and cytoplasmic aggregation of TARDBP/TDP-43 (TAR DNA binding protein). We previously showed that the ability of TARDBP to repress nonconserved cryptic exons was impaired in brains of patients with ALS and FTD, suggesting that its nuclear depletion contributes to neurodegeneration. However, the critical pathways impacted by the failure to repress cryptic exons that may contribute to neurodegeneration remain undefined. Here, we report that transcriptome analysis of TARDBP-deficient neurons revealed downregulation of ATG7, a critical gene required for macroautophagy/autophagy. Mouse and Drosophila models lacking TARDBP/TBPH in motor neurons exhibiting age-dependent neurodegeneration and motor deficits showed reduction of ATG7 and accumulation of SQSTM1/p62 inclusions. Importantly, genetic upregulation of the autophagy pathway improved motor function and survival in TBPH-deficient flies. Together with our observation that ATG7 is reduced in ALS-FTD brain tissues, these findings identify the autophagy pathway as one key effector of nuclear depletion of TARDBP that contributes to neurodegeneration. We thus suggest that the autophagy pathway is a therapeutic target for ALS-FTD and other disorders exhibiting TARDBP pathology. Abbreviations: ALS: amyotrophic lateral sclerosis; ANOVA: analysis of variance; ChAT: choline acetyltransferase; CTSD: cathepsin D; FTD: frontotemporal dementia; LAMP1: lysosomal associated membrane protein 1; NMJ: neuromuscular junction; RBFOX3/NeuN: RNA binding fox-1 homolog 3; SQSTM1: sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein 43.

Background TDP-43 proteinopathy is a prominent pathological feature that occurs in a number of human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and inclusion body myositis (IBM). Our recent finding that TDP-43 represses nonconserved cryptic exons led us to ask whether cell type-specific cryptic exons could exist to impact unique molecular pathways in brain or muscle. Methods In the present work, we investigated TDP-43’s function in various mouse tissues to model disease pathogenesis. We generated mice to conditionally delete TDP-43 in excitatory neurons or skeletal myocytes and identified the cell type-specific cryptic exons associated with TDP-43 loss of function. Results Comparative analysis of nonconserved cryptic exons in various mouse cell types revealed that only some cryptic exons were common amongst stem cells, neurons, and myocytes; the majority of these nonconserved cryptic exons were cell type-specific. Conclusions Our results suggest that in human disease, TDP-43 loss of function may impair cell type-specific pathways. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0144-x) contains supplementary material, which is available to authorized users.

Missense mutations in ubiquilin 2 (UBQLN2) cause ALS with frontotemporal dementia (ALS-FTD). Animal models of ALS are useful for understanding the mechanisms of pathogenesis and for preclinical investigations. However, previous rodent models carrying UBQLN2 mutations failed to manifest any sign of motor neuron disease. Here, we show that lines of mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have cognitive deficits, shortened lifespans, and develop motor neuron disease, mimicking the human disease. Neuropathologic analysis of the mice with end-stage disease revealed the accumulation of ubiquitinated inclusions in the brain and spinal cord, astrocytosis, a reduction in the number of hippocampal neurons, and reduced staining of TAR-DNA binding protein 43 in the nucleus, with concomitant formation of ubiquitin+ inclusions in the cytoplasm of spinal motor neurons. Moreover, both lines displayed denervation muscle atrophy and age-dependent loss of motor neurons that correlated with a reduction in the number of large-caliber axons. By contrast, two mouse lines expressing WT UBQLN2 were mostly devoid of clinical and pathological signs of disease. These UBQLN2 mouse models provide valuable tools for identifying the mechanisms underlying ALS-FTD pathogenesis and for investigating therapeutic strategies to halt disease.

A central question in Alzheimer's Disease (AD) is whether the neuritic plaque is necessary and sufficient for the development of tau pathology. Hyperphosphorylation of tau is found within dystrophic neurites surrounding β-amyloid deposits in AD mouse models but the pathological conversion of tau is absent. Likewise, expression of a human tau repeat domain in mice is insufficient to drive the pathological conversion of tau. Here we developed an Aβ-amyloidosis mouse model that expresses the human tau repeat domain and show that in these mice, the neuritic plaque facilitates the pathological conversion of wild-type tau. We show that this tau fragment seeds the neuritic plaque-dependent pathological conversion of wild-type tau that spreads from the cortex and hippocampus to the brain stem. These results establish that in addition to the neuritic plaque, a second determinant is required to drive the conversion of wild-type tau., Alzheimer's disease (AD) is pathologically characterized by the accumulation of neuritic plaques and neurofibrillary tangles but it is not known whether the neuritic plaque is necessary to drive the conversion of wild-type tau. Here the authors developed a mouse model in which wild-type tau is converted into pathological tau in a neuritic plaque-dependent manner.

Inactivation of FoxO proteins by phosphorylation is the result of a number of stimuli, including the insulin/IGF pathway. We were interested in the consequence of blunting this pathway by employing transgenic mice with tetracycline-controllable conditional expression of a constitutively active allele of FOXO3 under the control of the forebrain-specific CaMKIIα promoter. Although transgene-expressing mice were viable, brain weight was reduced by 30% in adult animals. Brains showed an isocortex compression with normal cortical layering, and a size reduction in regions known to depend on adult neurogenesis, i.e., the olfactory bulbs and the dentate gyrus. On postnatal activation of the transgene, adult neurogenesis was also severely affected. Investigating the molecular basis of this phenotype, we observed enhanced apoptosis starting from embryonic day E10.5 and a subsequent loss of progenitors in the ventricular/subventricular zones, but not in the isocortex or the striatum of adult mice. The enhanced apoptosis was accompanied by increased expression of PIK3IP1, which we identified as a direct transcriptional target of FOXO3. Transfection of Pik3ip1 into differentiating neural progenitors resulted in a significant reduction of viable cells. We therefore conclude that neural progenitors are particularly vulnerable to FOXO3-induced apoptosis, which is mediated by PIK3IP1, a negative PI3 kinase regulator.

Summary The free radical theory of aging postulates that the production of mitochondrial reactive oxygen species is the major determinant of aging and lifespan. Its role in aging of the connective tissue has not yet been established, even though the incidence of aging-related disorders in connective tissue-rich organs is high, causing major disability in the elderly. We have now addressed this question experimentally by creating mice with conditional deficiency of the mitochondrial manganese superoxide dismutase in fibroblasts and other mesenchyme-derived cells of connective tissues in all organs. Here, we have shown for the first time that the connective tissue-specific lack of superoxide anion detoxification in the mitochondria results in reduced lifespan and premature onset of aging-related phenotypes such as weight loss, skin atrophy, kyphosis (curvature of the spine), osteoporosis and muscle degeneration in mutant mice. Increase in p16INK4a, a robust in vivo marker for fibroblast aging, may contribute to the observed phenotype. This novel model is particularly suited to decipher the underlying mechanisms and to develop hopefully novel connective tissue-specific anti-aging strategies.

The molecular motor dynein and its associated regulatory subunit dynactin have been implicated in several neurodegenerative conditions of the basal ganglia, such as Huntington's disease (HD) and Perry syndrome, an atypical Parkinson-like disease. This pathogenic role has been largely postulated from the existence of mutations in the dynactin subunit p150(Glued). However, dynactin is also able to act independently of dynein, and there is currently no direct evidence linking dynein to basal ganglia degeneration. To provide such evidence, we used here a mouse strain carrying a point mutation in the dynein heavy chain gene that impairs retrograde axonal transport. These mice exhibited motor and behavioural abnormalities including hindlimb clasping, early muscle weakness, incoordination and hyperactivity. In vivo brain imaging using magnetic resonance imaging showed striatal atrophy and lateral ventricle enlargement. In the striatum, altered dopamine signalling, decreased dopamine D1 and D2 receptor binding in positron emission tomography SCAN and prominent astrocytosis were observed, although there was no neuronal loss either in the striatum or substantia nigra. In vitro, dynein mutant striatal neurons displayed strongly impaired neuritic morphology. Altogether, these findings provide a direct genetic evidence for the requirement of dynein for the morphology and function of striatal neurons. Our study supports a role for dynein dysfunction in the pathogenesis of neurodegenerative disorders of the basal ganglia, such as Perry syndrome and HD.

Transgenic mice expressing human mutated superoxide dismutase 1 (SOD1) linked to familial forms of amyotrophic lateral sclerosis are frequently used as a disease model. We used the SOD1G93A mouse in a cross-breeding strategy to study the function of physiological prion protein (Prp). SOD1G93APrp-/- mice exhibited a significantly reduced life span, and an earlier onset and accelerated progression of disease, as compared with SOD1G93APrp+/+ mice. Additionally, during disease progression, SOD1G93APrp-/- mice showed impaired rotarod performance, lower body weight, and reduced muscle strength. Histologically, SOD1G93APrp-/- mice showed reduced numbers of spinal cord motor neurons and extended areas occupied by large vacuoles early in the course of the disease. Analysis of spinal cord homogenates revealed no differences in SOD1 activity. Using an unbiased proteomic approach, a marked reduction of glial fibrillary acidic protein and enhanced levels of collapsing response mediator protein 2 and creatine kinase were detected in SOD1G93APrp-/- versus SOD1G93A mice. In the course of disease, Bcl-2 decreases, nuclear factor-kappaB increases, and Akt is activated, but these changes were largely unaffected by Prp expression. Exclusively in double-transgenic mice, we detected a significant increase in extracellular signal-regulated kinase 2 activation at clinical onset. We propose that Prp has a beneficial role in the SOD1G93A amyotrophic lateral sclerosis mouse model by influencing neuronal and/or glial factors involved in antioxidative defense, rather than anti-apoptotic signaling.

In neurons, cytoplasmic dynein functions as a molecular motor responsible for retrograde axonal transport. An impairment of axonal transport is thought to play a key role in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis, the most frequent motor neuron disease in the elderly. In this regard, previous studies described two heterozygous mouse strains bearing missense point mutations in the dynein heavy chain 1 gene that were reported to display late-onset progressive motor neuron degeneration. Here we show, however, that one of these mutant strains, the so-called Cra mice does not suffer from motor neuron loss, even in aged animals. Consistently, we did not observe electrophysiological or biochemical signs of muscle denervation, indicative of motor neuron disease. The "hindlimb clasping" phenotype of Cra mice could rather be due to the prominent degeneration of sensory neurons associated with a loss of muscle spindles. Altogether, these findings show that dynein heavy chain mutation triggers sensory neuropathy rather than motor neuron disease.

In recent years, magnetic resonance imaging (MRI) has emerged as a preferred tool for the diagnosis of amyotrophic lateral sclerosis (ALS) in humans. A widely used animal model for human ALS is the G93A-superoxide dismutase 1 (G93A-SOD1) transgenic mouse model. However, the mechanisms for the selective degeneration of motor neurons in the brainstem and spinal cord are still uncertain. In our study, we applied MRI at 4.7 Tesla to non-invasively evaluate pathological alterations in the brainstem of this animal model and to follow the progression of the disease. Extending previous investigation, we used the relaxation parameter T 2 as a suitable measure for the progression of ALS, and evaluated the potential agreement with histological evaluation and behavioural data of open-field tests. In the brainstem of G93A-SOD1 mice, T 2 values were significantly increased in the motor nuclei Nc. V, Nc. VII and Nc. XII, as early as Day 80, i.e. before the average disease onset at about Day 90. Moreover, this increase is associated with a progressive development of vacuoles in the brainstem motor nuclei and a significantly decreased performance in behavioural tests. Overall, MRI is a very sensitive tool to obtain correlates for neuronal degeneration in vivo. Furthermore, MRI enables us to investigate a follow up at different time points of the disease. These advantages are especially useful for therapeutic studies with respect to survival rates of motor neurons using mouse models. Finally, our data suggest that MRI does not only resemble the findings of behavioural tests, but is potentially superior to behavioural studies.

The dynactin p150glued subunit, encoded by the gene DCTN1 is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport. This subunit is a candidate modifier for neurodegenerative diseases, in particular motoneuron and extrapyramidal diseases. Based on an extensive screening effort of all 32 exons in more than 2,500 ALS/MND patients, patients suffering from Parkinsonian Syndromes and controls, we investigated 24 sequence variants of p150 in cell-based studies. We used both non-neuronal cell lines and primary rodent spinal motoneurons and report on cell biological abnormalities in five of these sequence alterations and also briefly report on the clinical features. Our results suggest the presence of biological changes caused by some p150 mutants pointing to a potential pathogenetic significance as modifier of the phenotype of the human disease.

In recent years, magnetic resonance imaging (MRI) has emerged as a preferred tool for the diagnosis of amyotrophic lateral sclerosis (ALS) in humans. A widely used animal model for human ALS is the G93A-superoxide dismutase 1 (G93A-SOD1) transgenic mouse model. However, the mechanisms for the selective degeneration of motor neurons in the brainstem and spinal cord are still uncertain. In our study, we applied MRI at 4.7 Tesla to non-invasively evaluate pathological alterations in the brainstem of this animal model and to follow the progression of the disease. Extending previous investigation, we used the relaxation parameter T(2) as a suitable measure for the progression of ALS, and evaluated the potential agreement with histological evaluation and behavioural data of open-field tests. In the brainstem of G93A-SOD1 mice, T(2) values were significantly increased in the motor nuclei Nc. V, Nc. VII and Nc. XII, as early as Day 80, i.e. before the average disease onset at about Day 90. Moreover, this increase is associated with a progressive development of vacuoles in the brainstem motor nuclei and a significantly decreased performance in behavioural tests. Overall, MRI is a very sensitive tool to obtain correlates for neuronal degeneration in vivo. Furthermore, MRI enables us to investigate a follow up at different time points of the disease. These advantages are especially useful for therapeutic studies with respect to survival rates of motor neurons using mouse models. Finally, our data suggest that MRI does not only resemble the findings of behavioural tests, but is potentially superior to behavioural studies.

The free radical theory of aging postulates that the production of mitochondrial reactive oxygen species is the major determinant of aging and lifespan. Its role in aging of the connective tissue has not yet been established, even though the incidence of aging-related disorders in connective tissue-rich organs is high, causing major disability in the elderly. We have now addressed this question experimentally by creating mice with conditional deficiency of the mitochondrial manganese superoxide dismutase in fibroblasts and other mesenchyme-derived cells of connective tissues in all organs. Here, we have shown for the first time that the connective tissue-specific lack of superoxide anion detoxification in the mitochondria results in reduced lifespan and premature onset of aging-related phenotypes such as weight loss, skin atrophy, kyphosis (curvature of the spine), osteoporosis and muscle degeneration in mutant mice. Increase in p16(INK4a) , a robust in vivo marker for fibroblast aging, may contribute to the observed phenotype. This novel model is particularly suited to decipher the underlying mechanisms and to develop hopefully novel connective tissue-specific anti-aging strategies.