Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.

Sex-Dependent Changes to the Intestinal and Hepatic Abundance of Drug Transporters and Metabolizing Enzymes in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at the blood-brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based [tandem mass tag (TMT)] or label-free [data-independent acquisition (DIA)] acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (Padj < 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose [solute carrier (SLC) SLC5A1] and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.

Sphingolipids metabolism alteration in the central nervous system: Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases.

Sphingolipids are complex lipids. They play a structural role in neurons, but are also involved in regulating cellular communication, and neuronal differentiation and maturation. There is increasing evidence to suggest that dysregulated metabolism of sphingolipids is linked to neurodegenerative processes in amyotrophic lateral sclerosis (ALS), Parkinson's disease and Gaucher's disease. In this review, we provide an overview of the role of sphingolipids in the development and maintenance of the nervous system. We describe the implications of altered metabolism of sphingolipids in the pathophysiology of certain neurodegenerative diseases, with a primary focus on ALS. Finally, we provide an update of potential treatments that could be used to target the metabolism of sphingolipids in neurodegenerative diseases.

The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis.

Mutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS.

Stimulation of mTOR-independent autophagy and mitophagy by rilmenidine exacerbates the phenotype of transgenic TDP-43 mice.

Autophagy, which mediates the delivery of cytoplasmic substrates to the lysosome for degradation, is essential for maintaining proper cell homeostasis in physiology, ageing, and disease. There is increasing evidence that autophagy is defective in neurodegenerative disorders, including motor neurons affected in amyotrophic lateral sclerosis (ALS). Restoring impaired autophagy in motor neurons may therefore represent a rational approach for ALS. Here, we demonstrate autophagy impairment in spinal cords of mice expressing mutant TDP-43Q331K or co-expressing TDP-43WTxQ331K transgenes. The clinically approved anti-hypertensive drug rilmenidine was used to stimulate mTOR-independent autophagy in double transgenic TDP-43WTxQ331K mice to alleviate impaired autophagy. Although rilmenidine treatment induced robust autophagy in spinal cords, this exacerbated the phenotype of TDP-43WTxQ331K mice, shown by truncated lifespan, accelerated motor neuron loss, and pronounced nuclear TDP-43 clearance. Importantly, rilmenidine significantly promoted mitophagy in spinal cords TDP-43WTxQ331K mice, evidenced by reduced mitochondrial markers and load in spinal motor neurons. These results suggest that autophagy induction accelerates the phenotype of this TDP-43 mouse model of ALS, most likely through excessive mitochondrial clearance in motor neurons. These findings also emphasise the importance of balancing autophagy stimulation with the potential negative consequences of hyperactive mitophagy in ALS and other neurodegenerative diseases.

Exploring germline recombination in Nestin-Cre transgenic mice using floxed androgen receptor.

The Cre-loxP strategy for tissue selective gene deletion has become a widely employed tool in neuroscience research. The validity of these models is largely underpinned by the temporal and spatial selectivity of recombinase expression under the promoter of the Cre driver line. Ectopic Cre-recombinase expression gives rise to off-target effects which can confound results and is especially detrimental if this occurs in germline cells. The Nestin-Cre transgenic mouse is broadly used for selective gene deletion in neurons of the central and peripheral nervous systems. Here we have crossed this mouse with a floxed androgen receptor (AR) transgenic to generate double transgenic neuronal ARKO mice (ARflox ::NesCre) to study germline deletion in male and female transgenic breeders. In male ARflox ::NesCre breeders, a null AR allele was passed on to 86% of progeny regardless of the inheritance of the NesCre transgene. In female ARflox/wt ::NesCre breeders, a null AR allele was passed on to 100% of progeny where ARflox was expected to be transmitted. This surprisingly high incidence of germline recombination in the Nestin-Cre driver line warrants caution in devising suitable breeding strategies, consideration of accurate genotyping approaches and highlights the need for thorough characterization of tissue-specific gene deletion in this model.

Mutant TDP-43 Expression Triggers TDP-43 Pathology and Cell Autonomous Effects on Primary Astrocytes: Implications for Non-cell Autonomous Pathology in ALS.

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SOD1) is partly non-cell autonomous, involving cellular dysfunction of astrocytes. Whether non-cell autonomous effects occur in other forms of ALS, such as TAR DNA binding protein 43 (TDP-43)-related disease, remains unclear. Here, we characterised the impact of mutant TDP-43 expression on primary astrocytes derived from transgenic TDP-43A315T mice. Mutant TDP-43 astrocytes revealed evidence for TDP-43 pathology, shown by cytoplasmic TDP-43 inclusions and accumulation in insoluble cell fractions which was exacerbated by proteasomal inhibition. L-glutamate uptake, measured using an [3H]D-aspartate assay, was impaired in mutant TDP-43 astrocytes, while ATP accumulation was abnormal, suggesting mutant TDP-43 induced astrocytic dysfunction. Astrocyte activation coupled with spinal and cortical motor neuron loss in transgenic TDP-43A315T mice could imply non-cell autonomous effects of astrocytes in vivo. These data demonstrate mutant TDP-43-mediated cell autonomous effects on astrocytes that may contribute to motor neuron pathology in ALS.

The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice.

The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1G93A mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1G93A mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1G93A mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNß and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1G93A mice.

TDP-43 mutations causing amyotrophic lateral sclerosis are associated with altered expression of RNA-binding protein hnRNP K and affect the Nrf2 antioxidant pathway.

TAR DNA binding protein 43 (TDP-43) is a major disease-associated protein involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Our previous studies found a direct association between TDP-43 and heterogeneous nuclear ribonucleoprotein K (hnRNP K). In this study, utilizing ALS patient fibroblasts harboring a TDP-43M337V mutation and NSC-34 motor neuronal cell line expressing TDP-43Q331K mutation, we show that hnRNP K expression is impaired in urea soluble extracts from mutant TDP-43 cell models. This was confirmed in vivo using TDP-43Q331K and inducible TDP-43A315T murine ALS models. We further investigated the potential pathological effects of mutant TDP-43-mediated changes to hnRNP K metabolism by RNA binding immunoprecipitation analysis. hnRNP K protein was bound to antioxidant NFE2L2 transcripts encoding Nrf2 antioxidant transcription factor, with greater enrichment in TDP-43M337V patient fibroblasts compared to healthy controls. Subsequent gene expression profiling revealed an increase in downstream antioxidant transcript expression of Nrf2 signaling in the spinal cord of TDP-43Q331K mice compared to control counterparts, yet the corresponding protein expression was not up-regulated in transgenic mice. Despite the elevated expression of antioxidant transcripts, we observed impaired levels of glutathione (downstream Nrf2 antioxidant) in TDP-43M337V patient fibroblasts and astrocyte cultures from TDP-43Q331K mice, indicative of elevated oxidative stress and failure of some upregulated antioxidant genes to be translated into protein. Our findings indicate that further exploration of the interplay between hnRNP K (or other hnRNPs) and Nrf2-mediated antioxidant signaling is warranted and may be an important driver for motor neuron degeneration in ALS.

Thiol-Cyanobenzothiazole Ligation for the Efficient Preparation of Peptide-PNA Conjugates.

Antisense oligonucleotide (ASO)-based drugs are emerging with great potential as therapeutic compounds for diseases with unmet medical needs. However, for ASOs to be effective as clinical entities, they should reach their intracellular RNA and DNA targets at pharmacologically relevant concentrations. Over the past decades, various covalently attached delivery vehicles have been utilized for intracellular delivery of ASOs. One such approach is the use of biocompatible cell-penetrating peptides (CPPs) covalently conjugated to ASOs. The stability of the linkage is of paramount importance for maximal intracellular delivery to achieve the desired therapeutic effect. In this study, we have investigated the efficiency and stability of four different bioorthogonal and nonreductive linkages including triazole, thioether, thiosuccinimide thioether and thiazole moieties. Here we have shown that thiazole and thiosuccinimide are the two most efficient and facile approaches for the preparation of peptide-ASO conjugates. The thiazole linkage had a higher stability compared to the thiosuccinimide thioether at physiological conditions (pH 7.4, 37 °C) in the presence of a biologically relevant concentration of glutathione. We have also shown that the peptide-ASO conjugate with a thiosuccinimide linkage has a significantly lower antisense activity compared to the peptide-ASO with the thiazole linkage, which maintains its antisense activity after 24 h of exposure to glutathione. In summary, we have demonstrated that the bioorthogonal thiazole linkage offers the benefits of mild reaction conditions, fast reaction kinetics, absence of any byproducts, and higher stability compared to other conjugation approaches. This facile ligation can be used for the synthesis of a variety of bioconjugates where a stable linkage is required.

SYT1-associated neurodevelopmental disorder: a case series.

Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals' phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.

GAL3 receptor knockout mice exhibit an alcohol-preferring phenotype.

Galanin is a neuropeptide which mediates its effects via three G-protein coupled receptors (GAL1-3 ). Administration of a GAL3 antagonist reduces alcohol self-administration in animal models while allelic variation in the GAL3 gene has been associated with an increased risk of alcohol use disorders in diverse human populations. Based on the association of GAL3 with alcoholism, we sought to characterize drug-seeking behavior in GAL3 -deficient mice for the first time. In the two-bottle free choice paradigm, GAL3 -KO mice consistently showed a significantly increased preference for ethanol over water when compared to wildtype littermates. Furthermore, male GAL3 -KO mice displayed significantly increased responding for ethanol under operant conditions. These differences in alcohol seeking behavior in GAL3 -KO mice did not result from altered ethanol metabolism. In contrast to ethanol, GAL3 -KO mice exhibited similar preference for saccharin and sucrose over water, and a similar preference for a high fat diet over a low fat diet as wildtype littermates. No differences in cognitive and locomotor behaviors were observed in GAL3 -KO mice to account for increased alcohol seeking behavior. Overall, these findings suggest genetic ablation of GAL3 in mice increases alcohol consumption.

Enhancing survival motor neuron expression extends lifespan and attenuates neurodegeneration in mutant TDP-43 mice.

Defects in the RNA-binding proteins survival motor neuron (SMN) and TAR DNA-binding protein 43 (TDP-43) cause progressive motor neuron degeneration in spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), respectively. While low levels of SMN protein in motor neurons result in SMA, recent studies implicate abnormal SMN levels and function in ALS pathogenesis. Here, we determine that SMN protein is upregulated early and progressively in spinal and cortical motor neurons of male transgenic mutant TDP-43A315T mice. Cytoplasmic SMN aggregates that contain TDP-43 and HuR were identified in motor neurons of TDP-43A315T mice, consistent with the incorporation of SMN into stress granules. To test the impact of augmenting SMN levels in TDP-43 proteinopathy, we demonstrate that neuronal overexpression of human SMN in TDP-43A315T mice delayed symptom onset and prolonged survival. SMN upregulation also countered motor neuron degeneration, attenuated activation of astrocytes and microglia and restored AMP kinase activation in spinal cords of TDP-43A315T mice. We also reveal that expression of another factor conferring motor neuron vulnerability, androgen receptor (AR), is reduced in spinal cords of male TDP-43A315T mice. These results establish that SMN overexpression in motor neurons slows disease onset and outcome by ameliorating pathological signs in this model of mutant TDP-43-mediated ALS. Further approaches to augment SMN levels using pharmacological or gene therapy agents may therefore be warranted in ALS. Our data also reinforce a novel potential link between ALS and spinal bulbar muscular atrophy (SBMA), another motor neurodegenerative disease mediated by reduced AR function in motor neurons.

Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment.

The p75 neurotrophin receptor (p75(NTR); also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75(NTR)-derived trophic cell-permeable peptide, c29, to inhibit p75(NTR)-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1(G93A) mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75(NTR) cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75(NTR) in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75(NTR) cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1(G93A) mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo.

Emerging therapies and challenges in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a hereditary neurodegenerative disease with severity ranging from progressive infantile paralysis and premature death (type I) to limited motor neuron loss and normal life expectancy (type IV). Without disease-modifying therapies, the impact is profound for patients and their families. Improved understanding of the molecular basis of SMA, disease pathogenesis, natural history, and recognition of the impact of standardized care on outcomes has yielded progress toward the development of novel therapeutic strategies and are summarized. Therapeutic strategies in the pipeline are appraised, ranging from SMN1 gene replacement to modulation of SMN2 encoded transcripts, to neuroprotection, to an expanding repertoire of peripheral targets, including muscle. With the advent of preliminary trial data, it can be reasonably anticipated that the SMA treatment landscape will transform significantly. Advancement in presymptomatic diagnosis and screening programs will be critical, with pilot newborn screening studies underway to facilitate preclinical diagnosis. The development of disease-modifying therapies will necessitate monitoring programs to determine the long-term impact, careful evaluation of combined treatments, and further acceleration of improvements in supportive care. In advance of upcoming clinical trial results, we consider the challenges and controversies related to the implementation of novel therapies for all patients and set the scene as the field prepares to enter an era of novel therapies. Ann Neurol 2017;81:355-368.

Synapse Dysfunction of Layer V Pyramidal Neurons Precedes Neurodegeneration in a Mouse Model of TDP-43 Proteinopathies.

TDP-43 is a major protein component of pathological neuronal inclusions that are present in frontotemporal dementia and amyotrophic lateral sclerosis. We report that TDP-43 plays an important role in dendritic spine formation in the cortex. The density of spines on YFP+ pyramidal neurons in both the motor and somatosensory cortex of Thy1-YFP mice, increased significantly from postnatal day 30 (P30), to peak at P60, before being pruned by P90. By comparison, dendritic spine density was significantly reduced in the motor cortex of Thy1-YFP::TDP-43A315T transgenic mice prior to symptom onset (P60), and in the motor and somatosensory cortex at symptom onset (P90). Morphological spine-type analysis revealed that there was a significant impairment in the development of basal mushroom spines in the motor cortex of Thy1-YFP::TDP-43A315T mice compared to Thy1-YFP control. Furthermore, reductions in spine density corresponded to mislocalisation of TDP-43 immunoreactivity and lowered efficacy of synaptic transmission as determined by electrophysiology at P60. We conclude that mutated TDP-43 has a significant pathological effect at the dendritic spine that is associated with attenuated neural transmission.

Phosphorylation of hnRNP K by cyclin-dependent kinase 2 controls cytosolic accumulation of TDP-43.

Cytosolic accumulation of TAR DNA binding protein 43 (TDP-43) is a major neuropathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the mechanisms involved in TDP-43 accumulation remain largely unknown. Previously, we reported that inhibitors of cyclin-dependent kinases (CDKs) prevented cytosolic stress granule accumulation of TDP-43, correlating with depletion of heterogeneous ribonucleoprotein (hnRNP) K from stress granules. In the present study, we further investigated the relationship between TDP-43 and hnRNP K and their control by CDKs. Inhibition of CDK2 abrogated the accumulation of TDP-43 into stress granules. Phosphorylated CDK2 co-localized with accumulated TDP-43 and phosphorylated hnRNP K in stress granules. Inhibition of CDK2 phosphorylation blocked phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevented accumulation of TDP-43. Mutation of Ser216 and Ser284 phosphorylation sites on hnRNP K inhibited hnRNP K- and TDP-43-positive stress granule formation in transfected cells. The interaction between hnRNP K and TDP-43 was further confirmed by the loss of TDP-43 accumulation following siRNA-mediated inhibition of hnRNP K expression. A substantial decrease of CDK2 and hnRNP K expression in spinal cord motor neurons in ALS patients demonstrates a potential key role for these proteins in ALS and TDP-43 accumulation, indicating that further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention.

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

Disease Mechanisms in ALS: Misfolded SOD1 Transferred Through Exosome-Dependent and Exosome-Independent Pathways.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neuromuscular degenerative disorder with a poorly defined etiology. ALS patients experience motor weakness, which starts focally and spreads throughout the nervous system, culminating in paralysis and death within a few years of diagnosis. While the vast majority of clinical ALS is sporadic with no known cause, mutations in human copper-zinc superoxide dismutase 1 (SOD1) cause about 20 % of inherited cases of ALS. ALS with SOD1 mutations is caused by a toxic gain of function associated with the propensity of mutant SOD1 to misfold, presenting a non-native structure. The mechanisms responsible for the progressive spreading of ALS pathology have been the focus of intense study. We have shown that misfolded SOD1 protein can seed misfolding and aggregation of endogenous wild-type SOD1 similar to amyloid-ß and prion protein seeding. Our recent observations demonstrate a transfer of the misfolded SOD1 species from cell to cell, modeling the intercellular transmission of disease through the neuroaxis. We have shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell, either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or in association with vesicles which are released into the extracellular environment. Furthermore, once misfolding of wild-type SOD1 has been initiated in a human cell culture, it can induce misfolding in naïve cell cultures over multiple passages of media transfer long after the initial misfolding template is degraded. Herein we review the data on mechanisms of intercellular transmission of misfolded SOD1.