RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

PAD2 dysregulation and aberrant protein citrullination feature prominently in reactive astrogliosis and myelin protein aggregation in sporadic ALS.

Alteration in protein citrullination (PC), a common posttranslational modification (PTM), contributes to pathogenesis in various inflammatory disorders. We previously reported that PC and protein arginine deiminase 2 (PAD2), the predominant enzyme isoform that catalyzes this PTM in the central nervous system (CNS), are altered in mouse models of amyotrophic lateral sclerosis (ALS). We now demonstrate that PAD2 expression and PC are altered in human postmortem ALS spinal cord and motor cortex compared to controls, increasing in astrocytes while trending lower in neurons. Furthermore, PC is enriched in protein aggregates that contain the myelin proteins PLP and MBP in ALS. These results confirm our findings in ALS mouse models and suggest that altered PAD2 and PC contribute to neurodegeneration in ALS.

TDP-43 knockdown in mouse model of ALS leads to dsRNA deposition, gliosis, and neurodegeneration in the spinal cord.

Transactive response DNA binding protein 43 kilodaltons (TDP-43) is a DNA and RNA binding protein associated with severe neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), primarily affecting motor neurons in the brain and spinal cord. Partial knockdown of TDP-43 expression in a mouse model (the amiR-TDP-43 mice) leads to progressive, age-related motor dysfunction, as observed in ALS patients. Work in Caenorhabditis elegans suggests that TDP-43 dysfunction can lead to deficits in chromatin processing and double-stranded RNA (dsRNA) accumulation, potentially activating the innate immune system and promoting neuroinflammation. To test this hypothesis, we used immunostaining to investigate dsRNA accumulation and other signs of CNS pathology in the spinal cords of amiR-TDP-43 mice. Compared with wild-type controls, TDP-43 knockdown animals show increases in dsRNA deposition in the dorsal and ventral horns of the spinal cord. Additionally, animals with heavy dsRNA expression show markedly increased levels of astrogliosis and microgliosis. Interestingly, areas of high dsRNA expression and microgliosis overlap with regions of heavy neurodegeneration, indicating that activated microglia could contribute to the degeneration of spinal cord neurons. This study suggests that loss of TDP-43 function could contribute to neuropathology by increasing dsRNA deposition and subsequent innate immune system activation.

Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons.

ALS-linked mutations induce aberrant conformations within the SOD1 protein that are thought to underlie the pathogenic mechanism of SOD1-mediated ALS. Although clinical trials are underway for gene silencing of SOD1, these approaches reduce both wild-type and mutated forms of SOD1. Here, we sought to develop anti-SOD1 nanobodies with selectivity for mutant and misfolded forms of human SOD1 over wild-type SOD1. Characterization of two anti-SOD1 nanobodies revealed that these biologics stabilize mutant SOD1 in vitro. Further, SOD1 expression levels were enhanced and the physiological subcellular localization of mutant SOD1 was restored upon co-expression of anti-SOD1 nanobodies in immortalized cells. In human motor neurons harboring the SOD1 A4V mutation, anti-SOD1 nanobody expression promoted neurite outgrowth, demonstrating a protective effect of anti-SOD1 nanobodies in otherwise unhealthy cells. In vitro assays revealed that an anti-SOD1 nanobody exhibited selectivity for human mutant SOD1 over endogenous murine SOD1, thus supporting the preclinical utility of anti-SOD1 nanobodies for testing in animal models of ALS. In sum, the anti-SOD1 nanobodies developed and presented herein represent viable biologics for further preclinical testing in human and mouse models of ALS.

The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias.

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models.

Increased protein citrullination (PC) and dysregulated protein arginine deiminase (PAD) activity have been observed in several neurodegenerative diseases. PC is a posttranslational modification catalyzed by the PADs. PC converts peptidyl-arginine to peptidyl-citrulline, thereby reducing the positive charges and altering structure and function of proteins. Of the five PADs, PAD2 is the dominant isoform in the central nervous system (CNS). Abnormal PC and PAD dysregulation are associated with numerous pathological conditions, including inflammatory diseases and neurodegeneration. Animal model studies have shown therapeutic efficacy from inhibition of PADs, thus suggesting a role of PC in pathogenesis. To determine whether PC contribute to amyotrophic lateral sclerosis (ALS), a deadly neurodegenerative disease characterized by loss of motor neurons, paralysis, and eventual death, we investigated alterations of PC and PAD2 in two different transgenic mouse models of ALS expressing human mutant SOD1G93A and PFN1C71G, respectively. PC and PAD2 expression are altered dynamically in the spinal cord during disease progression in both models. PC and PAD2 increase progressively in astrocytes with the development of reactive astrogliosis, while decreasing in neurons. Importantly, in the spinal cord white matter, PC accumulates in protein aggregates that contain the myelin proteins PLP and MBP. PC also accumulates progressively in insoluble protein fractions during disease progression. Finally, increased PC and PAD2 expression spatially correlate with areas of the CNS with the most severe motor neuron degeneration. These results suggest that altered PC is an integral part of the neurodegenerative process and potential biomarkers for disease progression in ALS. Moreover, increased PC may contribute to disease-associated processes such as myelin protein aggregation, myelin degeneration, and astrogliosis.

Low-level overexpression of wild type TDP-43 causes late-onset, progressive neurodegeneration and paralysis in mice.

Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed ~30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.

Imaging Net Retrograde Axonal Transport In Vivo: A Physiological Biomarker.

OBJECTIVE: The objective of this study is to develop a novel method for monitoring the integrity of motor neurons in vivo by quantifying net retrograde axonal transport. METHODS: The method uses single photon emission computed tomography to quantify retrograde transport to spinal cord of tetanus toxin fragment C (125 I-TTC) following intramuscular injection. We characterized the transport profiles in 3 transgenic mouse models carrying amyotrophic lateral sclerosis (ALS)-associated genes, aging mice, and SOD1G93A transgenic mice following CRISPR/Cas9 gene editing. Lastly, we studied the effect of prior immunization of tetanus toxoid on the transport profile of TTC. RESULTS: This technique defines a quantitative profile of net retrograde axonal transport of TTC in living mice. The profile is distinctly abnormal in transgenic SOD1G93A mice as young as 65 days (presymptomatic) and worsens with disease progression. Moreover, this method detects a distinct therapeutic benefit of gene editing in transgenic SOD1G93A mice well before other clinical parameters (eg, grip strength) show improvement. Symptomatic transgenic PFN1C71G/C71G ALS mice display gross reductions in net retrograde axonal transport, which is also disturbed in asymptomatic mice harboring a human C9ORF72 transgene with an expanded GGGGCC repeat motif. In wild-type mice, net retrograde axonal transport declines with aging. Lastly, prior immunization with tetanus toxoid does not preclude use of this assay. INTERPRETATION: This assay of net retrograde axonal transport has broad potential clinical applications and should be particularly valuable as a physiological biomarker that permits early detection of benefit from potential therapies for motor neuron diseases. ANN NEUROL 2022;91:716-729.

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice.

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS. Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Slow Intrathecal Injection of rAAVrh10 Enhances its Transduction of Spinal Cord and Therapeutic Efficacy in a Mutant SOD1 Model of ALS.

Mutant SOD1 causes amyotrophic lateral sclerosis (ALS) by a dominant gain of toxicity. Previous studies have demonstrated therapeutic potential of mutant SOD1-RNAi delivered by intrathecal (IT) injection of recombinant adeno-associated virus (rAAV). However, optimization of delivery is needed to overcome the high degree of variation in the transduction efficiency and therapeutic efficacy. Here, on the basis of our previously defined, efficient IT injection method, we investigated the influence of injection speed on transduction efficiency in the central nervous system (CNS). We demonstrate that slow IT injection results in higher transduction of spinal cord and dorsal root ganglia (DRG) while fast IT injection leads to higher transduction of brain and peripheral organs. To test how these effects influence the outcome of RNAi therapy, we used slow and fast IT injection to deliver rAAVrh10-GFP-amiR-SOD1, a rAAV vector that expresses GFP and an artificial miRNA targeting SOD1, in SOD1-G93A mice. Both slow and fast IT injection produced therapeutic efficacy but the slow injection trended slightly toward a better outcome than the fast injection. These results demonstrate that IT injection speed influences the predominance of gene delivery at different CNS sites and should be taken into consideration in future therapeutic trials involving IT injection.

Development of Excipient-Free Freeze-Dryable Unimolecular Hyperstar Polymers for Efficient siRNA Silencing.

We designed a unimolecular hyperstar polymer for efficient small interfering RNA (siRNA) delivery that can be processed under repeated lyophilization and reconstitution without the need of any excipient. The hyperstar polymer contains a biodegradable hyperbranched core and is bound to siRNA through its thousands of cationic arms that radiate from its core. The siRNA/hyperstar complexes showed siRNA transfection efficiency that was superior to that of Lipofectamine2000 in regard to the gene for human Cu, Zn superoxide dismutase 1 (SOD1), whose mutation causes familial amyotrophic lateral sclerosis. More importantly, hyperstar polymers as unimolecular containers minimized the multipolymer cross-interaction during lyophilization, and this maintained the uniquely high transfection efficiency of the siRNA/hyperstar complexes after repeated freeze-drying and reconstitution without the conventional need for excipient.

Creating conditional dual fluorescence labeled transgenic animals for studying function of small noncoding RNAs.

Because the function of most noncoding (nc) RNAs is unknown, Cre-lox transgenic mice are useful tools to determine their functions in a tissue or developmental stage-specific manner. However, the technology faces challenges because expression of ncRNA-transgene lacks protein product. No antibody or peptide-tag can be used to trace ncRNA expression in mouse tissues in real time. Furthermore, transgene integration at different locus or orientations in the genome may result in recombination of genomic fragments in the Cre-lox system. Establishing a reliable method that can be used to determine the precise copy number and orientation of the transgene is critical to the field. We developed a fast and straightforward method to determine ncRNA-transgene copy number, orientation, and insertion site in the genome. Furthermore, upon tissue-specific expression of ncRNA, a Cre-loxP-mediated dual-fluorescence expression system facilitates fluorescence signal switching from green to red, which enables real-time monitoring of ncRNA expression by fluorescence signals. As proof of concept, we demonstrate that after microRNA (miRNA)-Flox mice crossed with Col2a1-Cre mice, miRNA transgene expression could be detected successfully by red fluorescence signals in various cartilaginous tissues. This method of creating small ncRNA transgenic mice facilitates both tissue-specific ncRNA expression and real-time visualization of its expression. It is particularly suitable for in vivo studies of the functional roles and lineage tracing of small ncRNA.

Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity.

Mutations in the profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease caused by the loss of motor neurons leading to paralysis and eventually death. PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how the mutations in PFN1 cause ALS is unclear. To investigate this problem, we have generated transgenic mice expressing either the ALS-associated mutant (C71G) or wild-type protein. Here, we report that mice expressing the mutant, but not the wild-type, protein had relentless progression of motor neuron loss with concomitant progressive muscle weakness ending in paralysis and death. Furthermore, mutant, but not wild-type, PFN1 forms insoluble aggregates, disrupts cytoskeletal structure, and elevates ubiquitin and p62/SQSTM levels in motor neurons. Unexpectedly, the acceleration of motor neuron degeneration precedes the accumulation of mutant PFN1 aggregates. These results suggest that although mutant PFN1 aggregation may contribute to neurodegeneration, it does not trigger its onset. Importantly, these experiments establish a progressive disease model that can contribute toward identifying the mechanisms of ALS pathogenesis and the development of therapeutic treatments.

A Single Injection of Recombinant Adeno-Associated Virus into the Lumbar Cistern Delivers Transgene Expression Throughout the Whole Spinal Cord.

The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here, we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in 60 to 90 % of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells, and endothelial cells. Additionally, the transgene was expressed in some brain areas up to the frontal cortex and the olfactory bulb. The rAAV was distributed predominantly in the spinal cord, where its genome copy was over ten times that of the peripheral organs. Compared with intravenous injection, another method for rAAV delivery to the broad central nervous system (CNS), the intrathecal injection reduced the dosage of rAAV required to achieve similar or higher levels of transgene expression in the CNS by ~100-fold. Finally, the transduced areas were co-localized with the perivascular spaces of Virchow-Robin, from which the rAAV spreads further into the CNS parenchyma, thus suggesting that rAAV penetrated the CNS parenchyma through this pathway. Taken together, we have defined a fast and efficient method to deliver widespread transgene expression in mature spinal cord in mice. This method can be applied to stably overexpress or silence gene expression in the spinal cord to investigate gene functions in mammalian CNS. Additionally, this method can be applied to validate therapeutic targets for spinal cord diseases.

Systemic AAV9 gene transfer in adult GM1 gangliosidosis mice reduces lysosomal storage in CNS and extends lifespan.

GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease where GLB1 gene mutations result in a reduction or absence of lysosomal acid ß-galactosidase (ßgal) activity. ßgal deficiency leads to accumulation of GM1-ganglioside in the central nervous system (CNS). GM1 is characterized by progressive neurological decline resulting in generalized paralysis, extreme emaciation and death. In this study, we assessed the therapeutic efficacy of an adeno-associated virus (AAV) 9-mßgal vector infused systemically in adult GM1 mice (ßGal(-/-)) at 1 × 10(11) or 3 × 10(11) vector genomes (vg). Biochemical analysis of AAV9-treated GM1 mice showed high ßGal activity in liver and serum. Moderate ßGal levels throughout CNS resulted in a 36-76% reduction in GM1-ganglioside content in the brain and 75-86% in the spinal cord. Histological analyses of the CNS of animals treated with 3 × 10(11) vg dose revealed increased presence of ßgal and clearance of lysosomal storage throughout cortex, hippocampus, brainstem and spinal cord. Storage reduction in these regions was accompanied by a marked decrease in astrogliosis. AAV9 treatment resulted in improved performance in multiple tests of motor function and behavior. Also the majority of GM1 mice in the 3 × 10(11) vg cohort retained ambulation and rearing despite reaching the humane endpoint due to weight loss. Importantly, the median survival of AAV9 treatment groups (316-576 days) was significantly increased over controls (250-264 days). This study shows that moderate widespread expression of ßgal in the CNS of GM1 gangliosidosis mice is sufficient to achieve significant biochemical impact with phenotypic amelioration and extension in lifespan.

Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10.

Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood-brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets.

Partial loss of TDP-43 function causes phenotypes of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease that causes motor neuron degeneration, progressive motor dysfunction, paralysis, and death. Although multiple causes have been identified for this disease, >95% of ALS cases show aggregation of transactive response DNA binding protein (TDP-43) accompanied by its nuclear depletion. Therefore, the TDP-43 pathology may be a converging point in the pathogenesis that originates from various initial triggers. The aggregation is thought to result from TDP-43 misfolding, which could generate cellular toxicity. However, the aggregation as well as the nuclear depletion could also lead to a partial loss of TDP-43 function or TDP-43 dysfunction. To investigate the impact of TDP-43 dysfunction, we generated a transgenic mouse model for a partial loss of TDP-43 function using transgenic RNAi. These mice show ubiquitous transgene expression and TDP-43 knockdown in both the periphery and the central nervous system (CNS). Strikingly, these mice develop progressive neurodegeneration prominently in cortical layer V and spinal ventral horn, motor dysfunction, paralysis, and death. Furthermore, examination of splicing patterns of TDP-43 target genes in human ALS revealed changes consistent with TDP-43 dysfunction. These results suggest that the CNS, particularly motor neurons, possess a heightened vulnerability to TDP-43 dysfunction. Additionally, because TDP-43 knockdown predominantly occur in astrocytes in the spinal cord of these mice, our results suggest that TDP-43 dysfunction in astrocytes is an important driver for motor neuron degeneration and clinical phenotypes of ALS.

Oxidative stress and autophagic alteration in brainstem of SOD1-G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease involving both upper and lower motor neurons. The mechanism of motor neuron degeneration is still unknown. Although many studies have been performed on spinal motor neurons, few have been reported on brainstem and its motor nuclei. The aim of this study was to investigate oxidative stress and autophagic changes in the brainstem and representative motor nuclei of superoxide dismutase 1 (SOD1)-G93A mouse model of ALS. The expression levels of cluster of differentiation molecule 11b (CD11b), glial fibrillary acidic protein, glutamate-cysteine ligase catalytic subunit, heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, voltage-dependent anion-selective channel protein 1, Sequestosome 1/p62 (p62), microtubule-associated protein 1 light chain 3B (LC3), and SOD1 proteins in brainstem were examined by Western blot analysis. Immunohistochemistry and immunofluorescence were performed to identify the cellular localization of SOD1, p62, and LC3B, respectively. The results showed that there were progressive asctrocytic proliferation and microglial activation, induction of antioxidant proteins, and increased p62 and LC3II expression in brainstem of SOD1-G93A mice. Additionally, SOD1 and p62 accumulated in hypoglossal, facial, and red nuclei, but not in oculomotor nucleus. Furthermore, electron microscope showed increased autophagic vacuoles in affected brainstem motor nuclei. Our results indicate that brainstem share similar gliosis, oxidative stress, and autophagic changes as the spinal cord in SOD1-G93A mice. Thus, SOD1 accumulation in astrocytes and neurons, oxidative stress, and altered autophagy are involved in motor neuron degeneration in the brainstem, similar to the motor neurons in spinal cord. Therefore, therapeutic trials in the SOD1G93A mice need to target the brainstem in addition to the spinal cord.

Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration and paralysis. No treatment can significantly slow or arrest the disease progression. Mutations in the SOD1 gene cause a subset of familial ALS by a gain of toxicity. In principle, these cases could be treated with RNAi that destroys the mutant mRNA, thereby abolishing the toxic protein. However, no system is available to efficiently deliver the RNAi therapy. Recombinant adenoassociated virus (rAAV) is a promising vehicle due to its long-lasting gene expression and low toxicity. However, ALS afflicts broad areas of the central nervous system (CNS). A lack of practical means to spread rAAV broadly has hindered its application in treatment of ALS. To overcome this barrier, we injected several rAAV serotypes into the cerebrospinal fluid. We found that some rAAV serotypes such as rAAVrh10 and rAAV9 transduced cells throughout the length of the spinal cord following a single intrathecal injection and in the broad forebrain following a single injection into the third ventricle. Furthermore, a single intrathecal injection of rAAVrh10 robustly transduced motor neurons throughout the spinal cord in a non-human primate. These results suggested a therapeutic potential of this vector for ALS. To test this, we injected a rAAVrh10 vector that expressed an artificial miRNA targeting SOD1 into the SOD1G93A mice. This treatment knocked down the mutant SOD1 expression and slowed the disease progression. Our results demonstrate the potential of rAAVs for delivering gene therapy to treat ALS and other diseases that afflict broad areas of the CNS.

TDP-43-The key to understanding amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes motor neuron degeneration leading to progressive muscle atrophy, weakness, paralysis and death. The majority of ALS (>95%) shows intracellular aggregation of transactive response DNA binding protein (TDP-43) as a prominent pathological feature. TDP-43 is normally a nuclear protein. In ALS, TDP-43 accumulates and aggregates in the cytoplasm (thus forming TDP-43 proteinopathy) and is depleted from the nucleus in CNS cells, including motor neurons and glia. While TDP-43 aggregation can harm cells through a gain of toxicity, it can also cause a loss of TDP-43 function in conjunction with its nuclear depletion. TDP-43 regulates its own expression to maintain itself at a constant level. Perturbation of this level by either increasing or decreasing TDP-43 in animal models leads to neurodegeneration and ALS phenotypes. The evidence supports the hypothesis that TDP-43 dysfunction is a critical driver of neurodegeneration in the vast majority of ALS cases.