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Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins. ispartof: NEUROBIOLOGY OF AGING vol:92 ispartof: location:United States status: published

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease primarily afflicting motor neurons (MNs) of the spinal cord, brainstem, and motor cortex, leading to paralysis and eventually death within 3 to 5 years of diagnosis. No cure or effective therapy to halt ALS progression is available. The role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of ALS, as well as a potential drug target, has received increasing attention. Here, we investigated the therapeutic effect of the ER resident protein cerebral dopamine neurotrophic factor (CDNF) in preclinical models of ALS harboring different genetic mutations. We identify that intracerebroventricular (i.c.v.) administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of ALS. CDNF rescues MNs in vitro and in vivo from ER stress associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for ALS. One sentence summary Cerebral dopamine neurotrophic factor (CDNF) was found to substantially attenuate amyotrophic lateral sclerosis (ALS)-associated pathology in three preclinical rodent models via modulation of the endoplasmic reticulum (ER) stress, highlighting its potential therapeutic use in this motor neuron disease.

Neurotrophin signaling via receptor tyrosine kinases is essential for the development and function of the nervous system in vertebrates. TrkB activation and signaling show substantial differences to other receptor tyrosine kinases of the Trk family that mediate the responses to nerve growth factor and neurotrophin-3. Growing evidence suggests that TrkB cell surface expression is highly regulated and determines the sensitivity of neurons to brain-derived neurotrophic factor (BDNF). This translocation of TrkB depends on co-factors and modulators of cAMP levels, N-glycosylation, and receptor transactivation. This process can occur in very short time periods and the resulting rapid modulation of target cell sensitivity to BDNF could represent a mechanism for fine-tuning of synaptic plasticity and communication in complex neuronal networks. This review focuses on those modulatory mechanisms in neurons that regulate responsiveness to BDNF via control of TrkB surface expression.

We aimed to test the hypothesis that in spontaneously hypertensive stroke-prone rats (SHRSP), non-amyloid cerebral small vessel disease/hypertensive arteriopathy (HA) results in vessel wall injury that may promote cerebral amyloid angiopathy (CAA). Our study comprised 21 male SHRSP (age 17-44 weeks) and 10 age- and sex-matched Wistar control rats, that underwent two-photon (2PM) imaging of the arterioles in the parietal cortex using Methoxy-X04, Dextran and cerebral blood flow (CBF) measurements. Our data suggest that HA in SHRSP progresses in a temporal and age-dependent manner, starting from small vessel wall damage (stage 1A), proceeding to CBF reduction (stage 1B), non-occlusive (stage 2), and finally, occlusive thrombi (stage 3). Wistar animals also demonstrated small vessel wall damage, but were free of any of the later HA stages. Nearly half of all SHRSP additionally displayed vascular Methoxy-X04 positivity indicative of cortical CAA. Vascular β-amyloid deposits were found in small vessels characterized by thrombotic occlusions (stage 2 or 3). Post-mortem analysis of the rat brains confirmed the findings derived from intravital 2PM microscopy. Our data thus overall suggest that advanced HA may play a role in CAA development with the two small vessel disease entities might be related to the same pathological spectrum of the aging brain.

The retinal pigment epithelium (RPE)-dependent visual cycle provides 11-cis-retinal to opsins in the photoreceptor outer segments to generate functional visual pigments that initiate phototransduction in response to light stimuli. Both RPE65 isomerase of the visual cycle and the rhodopsin visual pigment have recently been identified as critical players in mediating light-induced retinal degeneration. These findings suggest that the expression and function of RPE65 and rhodopsin need to be coordinately controlled to sustain normal vision and to protect the retina from photodamage. However, the mechanism controlling the development of the retinal visual system remains poorly understood. Here, we show that deficiency in ciliary neurotrophic factor (CNTF) up-regulates the levels of rod and cone opsins accompanied by an increase in the thickness of the outer nuclear layers and the lengths of cone and rod outer segments in the mouse retina. Moreover, retinoid isomerase activity, expression levels of RPE65 and lecithin:retinol acyltransferase (LRAT), which synthesizes the RPE65 substrate, were also significantly increased in the Cntf(−/−) RPE. Rod a-wave and cone b-wave amplitudes of electroretinograms were increased in Cntf(−/−) mice, but rod b-wave amplitudes were unchanged compared with those in WT mice. Up-regulated RPE65 and LRAT levels accelerated both the visual cycle rate and recovery rate of rod light sensitivity in Cntf(−/−) mice. Of note, rods and cones in Cntf(−/−) mice exhibited hypersusceptibility to light-induced degeneration. These results indicate that CNTF is a common extracellular factor that prevents excessive production of opsins, the photoreceptor outer segments, and 11-cis-retinal to protect rods and cones from photodamage.

Spinal muscular atrophy (SMA), the predominant form of motoneuron disease in children and young adults is caused by loss of function of the SMN protein. On the basis of a disrupted splice acceptor site in exon 7, transcripts from a second SMN gene in humans called SMN2 cannot give rise to SMN protein at sufficient levels for maintaining function of motoneurons and motor circuits. First clinical trials with Spinraza/Nusinersen, a drug that counteracts disrupted splicing of SMN2 transcripts, have shown that elevating SMN levels can successfully interfere with motoneuron dysfunction. This review summarizes current knowledge about the pathophysiological alterations in Smn-deficient motoneurons, which lead to defective neuromuscular transmission and altered spinal circuit formation. Both pathological mechanisms are important targets for therapeutic intervention. However, the developmental time window when therapeutic interventions ideally should start is not known. Endogenous SMN expression both from SMN1 and SMN2 genes is high at early developmental stages and declines progressively in humans and mice. Thus, therapeutic SMN upregulation should start just before SMN declines below a critical threshold, and before irreversible defects occur at neuromuscular junctions and in spinal circuits. Previous results indicate that loss of Smn function leads to synaptic dysfunction during a stage of neuromuscular development when synaptic strength determines which synapses are maintained or not. This time window appears as an important target for therapy, which possibly could be supported by additional strategies that strengthen synaptic transmission.

Alpha-motoneurons and muscle fibres are structurally and functionally interdependent. Both cell types particularly rely on endoplasmic reticulum (ER/SR) functions. Mutations of the ER proteins VAPB, SigR1 and HSP27 lead to hereditary motor neuron diseases (MNDs). Here, we determined the expression profile and localization of these ER proteins/chaperons by immunohistochemistry and immunoblotting in biopsy and autopsy muscle tissue of patients with amyotrophic lateral sclerosis (ALS) and other neurogenic muscular atrophies (NMAs) and compared these patterns to mouse models of neurogenic muscular atrophy. Postsynaptic neuromuscular junction staining for VAPB was intense in normal human and mouse muscle and decreased in denervated Nmd2J mouse muscle fibres. In contrast, VAPB levels together with other chaperones and autophagy markers were increased in extrasynaptic regions of denervated muscle fibres of patients with MNDs and other NMAs, especially at sites of focal myofibrillar disintegration (targets). These findings did not differ between NMAs due to ALS and other causes. G93A-SOD1 mouse muscle fibres showed a similar pattern of protein level increases in denervated muscle fibres. In addition, they showed globular VAPB-immunoreactive structures together with misfolded SOD1 protein accumulations, suggesting a primary myopathic change. Our findings indicate that altered expression and localization of these ER proteins and autophagy markers are part of the dynamic response of muscle fibres to denervation. The ER is particularly prominent and vulnerable in both muscle fibres and alpha-motoneurons. Thus, ER pathology could contribute to the selective build-up of degenerative changes in the neuromuscular axis in MNDs.

Insulin-like growth factor 1 (IGF-1) is a pluripotent growth factor with multiple functions in the peripheral and central nervous system. It supports neuronal survival and axon growth, and also acts on myelinating Schwann cells and oligodendroglia. The biological functions of IGF-1 are modulated by IGF-binding proteins (IGFBPs). Expression of IGF-1 and its corresponding IGF-1 receptor (IGF-1R) are dysregulated in patients with diabetes and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). IGFBP5, an inhibitory binding protein for IGF-1, is also substantially increased in nerve biopsies of patients with sensorimotor diabetic neuropathy (DNP). We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which IGF-1R was conditionally depleted in motoneurons, indicating that reduced activity of IGF-1 on IGF-1R in motoneurons is responsible for the observed effect. The upregulation of IGFBP5 has possibly contributed to the lack of efficacy found in previous clinical trials with systemically administered IGF-1 in patients with other forms of motoneuron disease such as ALS. Thus, strategies aiming at circumventing these inhibitory effects could be of benefit for development of new therapies for ALS and DNP. However, these strategies have to be built on a better understanding of the metabolic processes that contribute to neurodegeneration, and on the role of IGF-1 in these metabolic processes that go beyond protection from axonal degeneration and cell death.

Amytrophic lateral sclerosis (ALS) is a neurodegenerative disorder that has been associated with multiple genetic lesions, including mutations in the gene FUS (Fused in Sarcoma), an RNA/DNA-binding protein. Expression of the ALS-associated human FUS in C. elegans results in mislocalization and aggregation of FUS outside the nucleus, and leads to impaired neuromuscular behaviors. However, the mechanisms by which mutant FUS disrupts neuronal health and function remain partially understood. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. Expression of ALS-associated FUS impairs synaptic vesicle docking at neuromuscular junctions, and leads to the emergence of a population of large and electron-dense filament-filled endosomes. Electrophysiological recording of neuromuscular transmission revealed reduced transmission from motor neurons to muscles. Together, these results suggest a potential direct or indirect role of human FUS in the organization of synaptic vesicles, and reduced transmission from motor neurons to muscles.Summary statementAn ALS-associated mutation in a trafficking protein disrupts the organization of the C. elegans neuromuscular junction.

Reply : Adult-onset distal spinal muscular atrophy: a new phenotype associated with KIF5A mutations

Synapse-associated protein 1 (Syap1) is the mammalian homologue of synapse-associated protein of 47 kDa (Sap47) in Drosophila. Genetic deletion of Sap47 leads to deficiencies in short-term plasticity and associative memory processing in flies. In mice, Syap1 is prominently expressed in the nervous system, but its function is still unclear. We have generated Syap1 knockout mice and tested motor behaviour and memory. These mice are viable and fertile but display distinct deficiencies in motor behaviour. Locomotor activity specifically appears to be reduced in early phases when voluntary movement is initiated. On the rotarod, a more demanding motor test involving control by sensory feedback, Syap1-deficient mice dramatically fail to adapt to accelerated speed or to a change in rotation direction. Syap1 is highly expressed in cerebellar Purkinje cells and cerebellar nuclei. Thus, this distinct motor phenotype could be due to a so far unknown function of Syap1 in cerebellar sensorimotor control. The observed motor defects are highly specific since other tests in the modified SHIRPA test, as well as cognitive tasks like novel object recognition, Pavlovian fear conditioning, and anxiety-like behaviour in open field, dark-light transition, and elevated plus maze, do not appear to be strongly affected in Syap1 knockout mice.

Background Cytokines are signaling molecules operating within complex cascade patterns and having exceptional modulatory functions. They impact various physiological processes such as neuroendocrine and metabolic interactions, neurotrophins’ metabolism, neuroplasticity, and may affect behavior and cognition. In our previous study, we found that sex and Cytomegalovirus (CMV)-serostatus may modulate levels of circulating pro- and anti-inflammatory cytokines, metabolic factors, immune cells, and cognitive performance, as well as associations between them. Results In the present study, we used a graph-theoretical approach to investigate the network topology dynamics of 22 circulating biomarkers and 11 measures of cognitive performance in 161 older participants recruited to undergo a six-months training intervention. For network construction, we applied coefficient of determination (R2) that was calculated for all possible pairs of variables (N = 33) in four groups (CMV− men and women; CMV+ men and women). Network topology has been evaluated by clustering coefficient (CC) and characteristic path length (CPL) as well as local (Elocal) and global (Eglobal) efficiency, showing the degree of network segregation (CC and Elocal) and integration (CPL and Eglobal). We found that networks under consideration showed small-world networks properties with more random characteristics. Mean CC, as well as local and global efficiency were highest and CPL shortest in CMV− males (having lowest inflammatory status and highest cognitive performance). CMV− and CMV+ females did not show any significant differences. Modularity analyses showed that the networks exhibit in all cases highly differentiated modular organization (with Q-value ranged between 0.397 and 0.453). Conclusions In this work, we found that segregation and integration properties of the network were notably stronger in the group with balanced inflammatory status. We were also able to confirm our previous findings that CMV-infection and sex modulate multiple circulating biomarkers and cognitive performance and that balanced inflammatory and metabolic status in elderly contributes to better cognitive functioning. Thus, network analyses provide a useful strategy for visualization and quantitative description of multiple interactions between various circulating pro- and anti-inflammatory biomarkers, hormones, neurotrophic and metabolic factors, immune cells, and measures of cognitive performance and can be in general applied for analyzing interactions between different physiological systems.

The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P-TEFb. Release of P-TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P-TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly important for neuron development and maintenance and it can thus be hypothesized that 7SK is at the center of many signaling pathways contributing to neuron function. 7SK activates neuronal gene expression programs that are key for terminal differentiation of neurons. Proteomics studies revealed a complex protein interactome of 7SK that includes several RNA-binding proteins. Some of these novel 7SK subcomplexes exert non-canonical cytosolic functions in neurons by regulating axonal mRNA transport and fine-tuning spliceosome production in response to transcription alterations. Thus, a picture emerges according to which 7SK acts as a multi-functional RNA scaffold that is integral for neuron homeostasis.

This study shows that mice with an ALS-linked heterozygous Tbk1 deletion are normal until at least 200 d of age. However, proteostatic and neuroinflammatory challenge by additional expression of mutant SOD1 unmasks a two-edged role of TBK1 in motoneuron disease., Heterozygous loss-of-function mutations of TANK-binding kinase 1 (TBK1) cause familial ALS, yet downstream mechanisms of TBK1 mutations remained elusive. TBK1 is a pleiotropic kinase involved in the regulation of selective autophagy and inflammation. We show that heterozygous Tbk1 deletion alone does not lead to signs of motoneuron degeneration or disturbed autophagy in mice during a 200-d observation period. Surprisingly, however, hemizygous deletion of Tbk1 inversely modulates early and late disease phases in mice additionally overexpressing ALS-linked SOD1G93A, which represents a “second hit” that induces both neuroinflammation and proteostatic dysregulation. At the early stage, heterozygous Tbk1 deletion impairs autophagy in motoneurons and prepones both the clinical onset and muscular denervation in SOD1G93A/Tbk1+/− mice. At the late disease stage, however, it significantly alleviates microglial neuroinflammation, decelerates disease progression, and extends survival. Our results indicate a profound effect of TBK1 on brain inflammatory cells under pro-inflammatory conditions and point to a complex, two-edged role of TBK1 in SOD1-linked ALS., Graphical Abstract

Significance Neurons are highly polarized cells. RNA-binding proteins contribute to this polarization by generating diverse subcellular transcriptomes. The RNA-binding protein hnRNP R is essential for axon growth in motoneurons. This study reports the RNA interactome for hnRNP R. The main interacting RNA of hnRNP R was the noncoding RNA 7SK. Depletion of 7SK from primary motoneurons disturbed axon growth. This effect was dependent on the interaction of 7SK with hnRNP R. Both hnRNP R and 7SK localize to axons. Loss of 7SK led to a similar depletion of axonal transcripts as loss of hnRNP R. Our data suggest that 7SK, in addition to its role in transcriptional regulation, acts in concert with hnRNP R to sort specific transcripts into axons., Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance.

Spinal muscular atrophy (SMA), one of the most frequent and devastating genetic disorders causing neuromuscular degeneration, has reached the forefront of clinical translation. The quite unique genetic situation of SMA patients, who lack functional SMN1 but carry the misspliced SMN2 copy gene, creates the possibility of correcting SMN2 splicing by antisense oligonucleotides or drugs. Both strategies showed impressive results in pre-clinical trials and are now in Phase II-III clinical trials. SMN gene therapy approaches using AAV9-SMN vectors are also highly promising and have entered a Phase I clinical trial. However, careful analysis of SMA animal models and patients has revealed some limitations that need to be taken very seriously, including: i) a limited time-window for successful therapy delivery, making neonatal screening of SMA mandatory; ii) multi-organ impairment, requiring systemic delivery of therapies; and iii) a potential need for combined therapies that both increase SMN levels and target pathways that preserve/rescue motor neuron function over the lifespan. Meeting these challenges will likely be crucial to cure SMA, instead of only ameliorating symptoms, particularly in its most severe form. This review discusses therapies currently in clinical trials, the hopes for SMA therapy, and the potential limitations of these new approaches.

The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. In particular, the CB1 receptor is highly expressed in the basal ganglia, mostly on terminals of medium-sized spiny neurons, where it plays a key neuromodulatory function. The CB1 receptor also confers neuroprotection in various experimental models of striatal damage. However, the assessment of the physiological relevance and therapeutic potential of the CB1 receptor in basal ganglia-related diseases is hampered, at least in part, by the lack of knowledge of the precise mechanism of CB1 receptor neuroprotective activity. Here, by using an array of pharmacological, genetic and pharmacogenetic (designer receptor exclusively activated by designer drug) approaches, we show that (1) CB1 receptor engagement protects striatal cells from excitotoxic death via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin complex 1 pathway, which, in turn, (2) induces brain-derived neurotrophic factor (BDNF) expression through the selective activation of BDNF gene promoter IV, an effect that is mediated by multiple transcription factors. To assess the possible functional impact of the CB1/BDNF axis in a neurodegenerative-disease context in vivo, we conducted experiments in the R6/2 mouse, a well-established model of Huntington's disease, in which the CB1 receptor and BDNF are known to be severely downregulated in the dorsolateral striatum. Adeno-associated viral vector-enforced re-expression of the CB1 receptor in the dorsolateral striatum of R6/2 mice allowed the re-expression of BDNF and the concerted rescue of the neuropathological deficits in these animals. Collectively, these findings unravel a molecular link between CB1 receptor activation and BDNF expression, and support the relevance of the CB1/BDNF axis in promoting striatal neuron survival.

Regulated release of neurotransmitter depends on the orchestrated function of a large number of proteins in the presynaptic compartment. When synaptic vesicles fuse with the plasma membrane, these membranes and the attached proteins are endocytosed and either recycled or degraded. This turnover needs to be tightly regulated in a timely and spatially confined manner. Increasing evidence suggests that these mechanisms do not only serve for the removal of defective synaptic vesicles or structural proteins of the active zone but also contribute to pathways regulating synaptic strength. The corresponding presynaptic autophagy system thus appears also important for synaptic maintenance and plasticity. Exciting new studies provide evidence how the autophagy machinery recognizes and degrades synaptic components and lay the ground to understand how autophagy in the presynaptic compartment contributes to modulation and maintenance of synaptic function.

Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease., Accumulating evidence suggests that disruption of autophagy is associated with neurodegeneration. Here the authors show that Plekhg5 acts as a GEF for Rab26, a small GTPase that promotes the autophagy of synaptic vesicles in neurons; mice lacking Plekgh5 develop late-onset motoneuron degeneration.

In highly polarized cells such as neurons, most RNA molecules are not randomly distributed but sorted into different compartments. So far, methods to analyze the transcriptome in distinct subcellular compartments are not well established. Here, we first describe the culturing of primary motoneurons in compartmentalized chambers to separate the axons from the somatodendritic compartment. Second, we introduce a method for whole transcriptome amplification followed by high-throughput sequencing to analyze the RNA composition of these two different compartments in neuronal cells.

We have previously shown higher-than-expected rates of schizophrenia in relatives of patients with amyotrophic lateral sclerosis (ALS), suggesting an aetiological relationship between the diseases. Here, we investigate the genetic relationship between ALS and schizophrenia using genome-wide association study data from over 100,000 unique individuals. Using linkage disequilibrium score regression, we estimate the genetic correlation between ALS and schizophrenia to be 14.3% (7.05–21.6; P=1 × 10−4) with schizophrenia polygenic risk scores explaining up to 0.12% of the variance in ALS (P=8.4 × 10−7). A modest increase in comorbidity of ALS and schizophrenia is expected given these findings (odds ratio 1.08–1.26) but this would require very large studies to observe epidemiologically. We identify five potential novel ALS-associated loci using conditional false discovery rate analysis. It is likely that shared neurobiological mechanisms between these two disorders will engender novel hypotheses in future preclinical and clinical studies., Relatives of patients with amyotrophic lateral sclerosis have an unexpectedly high incidence of schizophrenia. Here, the authors show a genetic link between the two conditions, suggesting shared neurobiological mechanisms.

In highly polarized cells such as neurons, most RNA molecules are not randomly distributed but sorted into different compartments. So far, methods to analyze the transcriptome in distinct subcellular compartments are not well established. Here, we first describe the culturing of primary motoneurons in compartmentalized chambers to separate the axons from the somatodendritic compartment. Second, we introduce a method for whole transcriptome amplification followed by high-throughput sequencing to analyze the RNA composition of these two different compartments in neuronal cells.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, which result in reduced levels of functional SMN protein. Biochemical studies have linked the ubiquitously expressed SMN protein to the assembly of pre-mRNA processing U snRNPs, raising the possibility that aberrant splicing is a major defect in SMA. Accordingly, several transcripts affected upon SMN deficiency have been reported. A second function for SMN in axonal mRNA transport has also been proposed that may likewise contribute to the SMA phenotype. The underlying etiology of SMA, however, is still not fully understood. Here, we have used a combination of genomics and live Ca2+ imaging to investigate the consequences of SMN deficiency in a zebrafish model of SMA. In a transcriptome analyses of SMN-deficient zebrafish, we identified neurexin2a (nrxn2a) as strongly down-regulated and displaying changes in alternative splicing patterns. Importantly, the knock-down of two distinct nrxn2a isoforms phenocopies SMN-deficient fish and results in a significant reduction of motor axon excitability. Interestingly, we observed altered expression and splicing of Nrxn2 also in motor neurons from the Smn−/−;SMN2+/+ mouse model of SMA, suggesting conservation of nrxn2 regulation by SMN in mammals. We propose that SMN deficiency affects splicing and abundance of nrxn2a. This may explain the pre-synaptic defects at neuromuscular endplates in SMA pathophysiology.

In motoneuron disease and other neurodegenerative disorders, the loss of synapses and axon branches occurs early but is compensated by sprouting of neighboring axon terminals. Defective local axonal signaling for maintenance and dynamics of the axonal microtubule and actin cytoskeleton plays a central role in this context. The molecular mechanisms that lead to defective cytoskeleton architecture in two mouse models of motoneuron disease are summarized and discussed in this manuscript. In the progressive motor neuropathy (pmn) mouse model of motoneuron disease that is caused by a mutation in the tubulin-specific chaperone E gene, death of motoneuron cell bodies appears as a consequence of axonal degeneration. Treatment with bcl-2 overexpression or with glial-derived neurotrophic factor prevents loss of motoneuron cell bodies but does not influence the course of disease. In contrast, treatment with ciliary neurotrophic factor (CNTF) significantly delays disease onset and prolongs survival of pmn mice. This difference is due to the activation of Stat-3 via the CNTF receptor complex in axons of pmn mutant motoneurons. Most of the activated Stat-3 protein is not transported to the nucleus to activate transcription, but interacts locally in axons with stathmin, a protein that destabilizes microtubules. This interaction plays a major role in CNTF signaling for microtubule dynamics in axons. In Smn-deficient mice, a model of spinal muscular atrophy, defects in axonal translocation of β-actin mRNA and possibly other mRNA species have been observed. Moreover, the regulation of local protein synthesis in response to signals from neurotrophic factors and extracellular matrix proteins is altered in motoneurons from this model of motoneuron disease. These findings indicate that local signals are important for maintenance and plasticity of axonal branches and neuromuscular endplates, and that disturbances in these signaling mechanisms could contribute to the pathophysiology of motoneuron diseases.

Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor, and the mechanisms by which CNTF expression could be increased in the brain are poorly understood. Acetylsalicylic acid (aspirin) is one of the most widely used analgesics. Interestingly, aspirin increased mRNA and protein expression of CNTF in primary mouse and human astrocytes in a dose- and time-dependent manner. Aspirin induced the activation of protein kinase A (PKA) but not protein kinase C (PKC). H-89, an inhibitor of PKA, abrogated aspirin-induced expression of CNTF. The activation of cAMP-response element-binding protein (CREB), but not NF-κB, by aspirin, the abrogation of aspirin-induced expression of CNTF by siRNA knockdown of CREB, the presence of a consensus cAMP-response element in the promoter of CNTF, and the recruitment of CREB and CREB-binding protein to the CNTF promoter by aspirin suggest that aspirin increases the expression of the Cntf gene via the activation of CREB. Furthermore, we demonstrate that aspirin-induced astroglial CNTF was also functionally active and that supernatants of aspirin-treated astrocytes of wild type, but not Cntf null, mice increased myelin-associated proteins in oligodendrocytes and protected oligodendrocytes from TNF-α insult. These results highlight a new and novel myelinogenic property of aspirin, which may be of benefit for multiple sclerosis and other demyelinating disorders.

Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., To elucidate the genetic architecture of amyotrophic lateral sclerosis (ALS) and find associated loci, we assembled a custom imputation reference panel from whole-genome-sequenced patients with ALS and matched controls (n = 1,861). Through imputation and mixed-model association analysis in 12,577 cases and 23,475 controls, combined with 2,579 cases and 2,767 controls in an independent replication cohort, we fine-mapped a new risk locus on chromosome 21 and identified C21orf2 as a gene associated with ALS risk. In addition, we identified MOBP and SCFD1 as new associated risk loci. We established evidence of ALS being a complex genetic trait with a polygenic architecture. Furthermore, we estimated the SNP-based heritability at 8.5%, with a distinct and important role for low-frequency variants (frequency 1-10%). This study motivates the interrogation of larger samples with full genome coverage to identify rare causal variants that underpin ALS risk.

Progressive forms of multiple sclerosis lead to chronic disability, substantial decline in quality of life and reduced longevity. It is often suggested that they occur independently of inflammation. Here we investigated the disease progression in mouse models carrying PLP1 point mutations previously found in patients displaying clinical features of multiple sclerosis. These mouse models show loss-of-function of PLP1 associated with neuroinflammation; the latter leading to clinically relevant axonal degeneration, neuronal loss and brain atrophy as demonstrated by inactivation of the recombination activating gene 1. Moreover, these pathological hallmarks were substantially amplified when we attenuated immune regulation by inactivation of the programmed cell death-1 gene. Our observations support the view that primary oligodendroglial abnormalities can evoke pathogenically relevant neuroinflammation that drives neurodegeneration, as observed in some forms of multiple sclerosis but also in other, genetically-mediated neurodegenerative disorders of the human nervous system. As many potent immunomodulatory drugs have emerged during the last years, it is tempting to consider immunomodulation as a treatment option not only for multiple sclerosis, but also for so far non-treatable, genetically-mediated disorders of the nervous system accompanied by pathogenic neuroinflammation.

Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.

α-, β-, and γ-actin differentially regulate cytoskeletal dynamics and stability in axons of motoneurons. Locally translated α-actin contributes to stable actin filaments in axonal branches, whereas β- and γ-actin give rise to highly dynamic filaments that modulate growth cone dynamics., Axonal branching and terminal arborization are fundamental events during the establishment of synaptic connectivity. They are triggered by assembly of actin filaments along axon shafts giving rise to filopodia. The specific contribution of the three actin isoforms, Actα, Actβ, and Actγ, to filopodia stability and dynamics during this process is not well understood. Here, we report that Actα, Actβ, and Actγ isoforms are expressed in primary mouse motoneurons and their transcripts are translocated into axons. shRNA-mediated depletion of Actα reduces axonal filopodia dynamics and disturbs collateral branch formation. Knockdown of Actβ reduces dynamic movements of growth cone filopodia and impairs presynaptic differentiation. Ablation of Actβ or Actγ leads to compensatory up-regulation of the two other isoforms, which allows maintenance of total actin levels and preserves F-actin polymerization. Collectively, our data provide evidence for specific roles of different actin isoforms in spatial regulation of actin dynamics and stability in axons of developing motoneurons.

Published: May 2016 Animal models point towards a key role of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in mediating exercise-induced structural and functional changes in the hippocampus. Recently, also platelet derived growth factor-C (PDGF-C) has been shown to promote blood vessel growth and neuronal survival. Moreover, reductions of these neurotrophic and angiogenic factors in old age have been related to hippocampal atrophy, decreased vascularization and cognitive decline. In a 3-month aerobic exercise study, forty healthy older humans (60 to 77 years) were pseudo-randomly assigned to either an aerobic exercise group (indoor treadmill, n=21) or to a control group (indoor progressive-muscle relaxation/stretching, n=19). As reported recently, we found evidence for fitness-related perfusion changes of the aged human hippocampus that were closely linked to changes in episodic memory function. Here, we test whether peripheral levels of BDNF, IGF-I, VEGF or PDGF-C are related to changes in hippocampal blood flow, volume and memory performance. Growth factor levels were not significantly affected by exercise, and their changes were not related to changes in fitness or perfusion. However, changes in IGF-I levels were positively correlated with hippocampal volume changes (derived by manual volumetry and voxel-based morphometry) and late verbal recall performance, a relationship that seemed to be independent of fitness, perfusion or their changes over time. These preliminary findings link IGF-I levels to hippocampal volume changes and putatively hippocampus-dependent memory changes that seem to occur over time independently of exercise. We discuss methodological shortcomings of our study and potential differences in the temporal dynamics of how IGF-1, VEGF and BDNF may be affected by exercise and to what extent these differences may have led to the negative findings reported here. (C) 2015 The Authors. Published by Elsevier Inc. German Center for Neurodegenerative Diseases Otto-von-Guericke University (IKND) Swedish Research Council Formas Swedish Research Council for Health, Working Life, and Welfare Swedish Brain Power, an Alexander von Humboldt Research Award

STAT3, an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, has a distinct cytoplasmic function in inhibiting microtubule depolymerization in axons of motoneurons., Axonal maintenance, plasticity, and regeneration are influenced by signals from neighboring cells, in particular Schwann cells of the peripheral nervous system. Schwann cells produce neurotrophic factors, but the mechanisms by which ciliary neurotrophic factor (CNTF) and other neurotrophic molecules modify the axonal cytoskeleton are not well understood. In this paper, we show that activated signal transducer and activator of transcription-3 (STAT3), an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, acts locally in axons of motoneurons to modify the tubulin cytoskeleton. Specifically, we show that activated STAT3 interacted with stathmin and inhibited its microtubule-destabilizing activity. Thus, ectopic CNTF-mediated activation of STAT3 restored axon elongation and maintenance in motoneurons from progressive motor neuronopathy mutant mice, a mouse model of motoneuron disease. This mechanism could also be relevant for other neurodegenerative diseases and provide a target for new therapies for axonal degeneration.

Insulin-like growth factor I (IGF-I) has been successfully tested in the SOD1-G93A mouse model of familial amyotrophic lateral sclerosis (ALS) and proposed for clinical treatment. However, beneficial effects required gene therapy or intrathecal application. Circumventing the dosing issues we recently found that polyethylene glycol (PEG) modified IGF-I (PEG-IGF-I) modulated neuromuscular function after systemic application, and protected against disease progression in a motor neuron disease model. Here we investigated its effects in two SOD1-G93A mouse lines, the G1L with a milder and the G1H with a more severe phenotype. Results showed that in G1L mice, PEG-IGF-I treatment significantly improved muscle force, motor coordination and animal survival. In contrast, treatment of G1H mice with PEG-IGF-I or IGF-I even at high doses did not beneficially affect survival or functional outcomes despite increased signalling in brain and spinal cord by both agents. In conclusion, the data point towards further investigation of the therapeutic potential of PEG-IGF-I in ALS patients with less severe clinical phenotypes.

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of β-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the β-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of β-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that β-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the β-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of β-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn −/− ;SMN2). Taken together our findings suggest that local translation of β-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.

While some cases of familial ALS can be entirely attributed to known inherited variation, the majority (∼ 90%) are sporadic, where the cause(s) are not entirely understood. Both genetic and environmental factors may contribute to susceptibility. Mitochondrial damage, a common feature of neurodegenerative disease, is observed in most patients and inherited polymorphism in the mitochondrial genome has been suggested as a contributing factor. We used an economic and efficient method to test whether such involvement is probable. We genotyped 22 mtDNA coding region SNPs and sequenced the mtDNA hypervariable region 1 to determine the position of each mitochondrial genome within the genealogy of mitochondrial haplotypes in samples of ALS patients (n = 700) and controls (n = 462) from two European populations. We compared haplotype and haplogroup distribution in cases and controls drawn from the same populations. No statistical difference was observed between cases and controls at either the haplogroup or haplotype level (p = ≥ 0.2). In conclusion, it is unlikely that common, shared genetic variants in the mitochondrial genome contribute substantially to ALS. Combining the data with other studies will allow meta-analysis to look for variants with modest effect sizes. The sequencing of complete mitochondrial genomes will be required to assess the role of rare mutations.

To clarify the physiological role of Na+-d-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1−/− mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1−/− mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1−/− mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose–free diet. In wild-type mice, passage of d-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1−/− mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

The microtubule associated tumor suppressor gene 1 (MTUS1) is a recently published tumor suppressor gene, which has also been shown to act as an early component in the growth inhibitory signaling cascade of the angiotensin II type 2 receptor (AT2R). In this study we report the generation of MTUS1 knock-out (KO) mice, which develop normally but reveal higher body weights and slightly decreased blood pressure levels. Twenty-eight percent of the studied MTUS1 KO mice also developed heart hypertrophy and 12% developed nephritis, independent of blood pressure levels. Forty-three percent of the MTUS1 KO mice revealed lymphoid hyperplasia affecting spleen (20%), kidney (37%), lung (23%), lymph nodes (17%), and liver (17%) accompanied with leukocytosis, lymphocytosis, and mild anemia. One animal (3%) developed a marginal zone B-cell lymphoma affecting submandibular salivary gland and regional lymph nodes. The symptoms of all mentioned animals are consistent with a B-cell lymphoproliferative disease with features of systemic lupus erythematosus. In addition, body weight of the MTUS1 KO mice was significantly increased and isolated skin fibroblasts showed increased cell proliferation and decreased cell size, compared to wild-type (WT) fibroblasts in response to depleted FCS concentration and lack of growth factors. In conclusion we herein report the first generation of a MTUS1 KO mouse, developing spontaneous heart hypertrophy and increased cell proliferation, confirming once more the anti-proliferative effect of MTUS1, and a SLE-like lymphoproliferative disease suggesting crucial role in regulation of inflammation. These MTUS1 KO mice can therefore serve as a model for further investigations in cardiovascular disease, autoimmune disease and carcinogenesis.

Amyotrophic lateral sclerosis (ALS) is a genetically diverse disease. At least 15 ALS-associated gene loci have so far been identified, and the causative gene is known in approximately 30% of familial ALS cases. Less is known about the factors underlying the sporadic form of the disease. The molecular mechanisms of motor neuron degeneration are best understood in the subtype of disease caused by mutations in superoxide dismutase 1, with a current consensus that motor neuron injury is caused by a complex interplay between multiple pathogenic processes. A key recent finding is that mutated TAR DNA-binding protein 43 is a major constituent of the ubiquitinated protein inclusions in ALS, providing a possible link between the genetic mutation and the cellular pathology. New insights have also indicated the importance of dysregulated glial cell-motor neuron crosstalk, and have highlighted the vulnerability of the distal axonal compartment early in the disease course. In addition, recent studies have suggested that disordered RNA processing is likely to represent a major contributing factor to motor neuron disease. Ongoing research on the cellular pathways highlighted in this Review is predicted to open the door to new therapeutic interventions to slow disease progression in ALS.

A workshop was held in Zurich (Switzerland) to develop standard procedures and protocols for pre-clinical efficacy studies in mouse models of spinal muscular atrophy (SMA). The workshop was organized by the Network of Excellence TREAT-NMD (Translational Research in Europe – Assessment and Treatment of Neuromuscular Disorders), launched 2007, that addresses the fragmentation currently existing in the evaluation, diagnosis and care of neuromuscular diseases on one side, and in the procedures of drug development from research to the design of clinical trials on the other side. Within this frame, one aspect that needs particular care is the design and performance of pre-clinical animal studies that should predict the efficacy of a new treatment in patients. In the absence of commonly accepted guidelines and standardized protocols, data from different laboratories are often not comparable and the result is a duplication of efforts. The workshop was attended by 30 key researchers from Europe and North America, as well as industry representatives (Santhera Pharmaceuticals, Psychogenics, Genyzme, Trophos) and patient organisations (SMA Foundation, AFM-Association Franc aise contre les Myopathies). The goals of the workshop were (1) to select endpoints that best assess drug efficacy in mouse

Tropomyosin-related kinase (Trk) receptors modulate neuronal structure and function both during development and in the mature nervous system. Interestingly, TrkB and TrkC are expressed as full-length and as truncated splice variants. The cellular function of the kinase-lacking isoforms remains so far unclear. We investigated the role of the truncated receptor TrkB.T1 in the hippocampus of transgenic mice overexpressing this splice variant by analyzing both neuronal structure and function. We observed an impairment in activity-dependent synaptic plasticity as indicated by deficits in long-term potentiation and long-term depression in acute hippocampal slices of transgenic TrkB.T1 mice. In addition, dendritic complexity and spine density were significantly altered in TrkB.T1-overexpressing CA1 neurons. We found that the effect of TrkB.T1 overexpression differs between subgroups of CA1 neurons. Remarkably, overexpression of p75 NTR and its activation by chemical induction of long-term depression in slice cultures rescued the TrkB.T1-dependent morphological alterations specifically in one of the two subgroups observed. These findings suggest that the TrkB.T1 and p75 NTR receptor signaling systems might be cross-linked. Our findings demonstrate that TrkB.T1 regulates the function and the structure of mature pyramidal neurons. In addition, we showed that the ratio of expression levels of p75 NTR and TrkB.T1 plays an important role in modulating dendritic architecture and synaptic plasticity in the adult rodent hippocampus, and, indeed, that the endogenous expression patterns of both receptors change reciprocally over time. We therefore propose a new function of TrkB.T1 as being dominant-negative to p75 NTR .

Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival. Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects. We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease. In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed. In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection. Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection.

Proximal spinal muscular atrophy (SMA) is the predominant form of motor neuron disease in children and young adults. In contrast to other neurodegenerative disorders, SMA is a genetically homozygous autosomal recessive disease that is caused by deficiency of the survival motor neuron (SMN) protein. This homogeneity should in principle facilitate therapy development. Previous therapy approaches have focused on upregulation of SMN expression from a second SMN (SMN2) gene that gives rise to low amounts of functional SMN protein. Drug development to target disease-specific mechanisms at cellular and physiological levels is in its early stages, as the pathophysiological processes that underlie the main disease symptoms are still not fully understood. Mouse models have helped to make conceptual progress in the disease mechanism, but their suitability in the search for therapeutic agents remains to be validated--an issue that is ubiquitous to the translational therapeutic research of other neurodegenerative diseases. Human induced pluripotent stem cell technology for generation of large numbers of human motor neurons could help to fill this gap and advance the power of drug screening. In parallel, advances in oligonucleotide technologies for engineering SMN2 pre-mRNA splicing are approaching their first clinical trials, whose success depends on improved technologies for drug delivery to motor neurons. If this obstacle can be overcome, this could boost therapy development, not only for SMA but also for other neurodegenerative disorders.

Phosphatase and tensin homolog (PTEN), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, is widely involved in the regulation of protein synthesis. Here we show that the PTEN protein is enriched in cell bodies and axon terminals of purified motor neurons. We explored the role of the PTEN pathway by manipulating PTEN expression in healthy and diseased motor neurons. PTEN depletion led to an increase in growth cone size, promotion of axonal elongation and increased survival of these cells. These changes were associated with alterations of downstream signaling pathways for local protein synthesis as revealed by an increase in pAKT and p70S6. Most notably, this treatment also restores beta-actin protein levels in axonal growth cones of SMN-deficient motor neurons. Furthermore, we report here that a single injection of adeno-associated virus serotype 6 (AAV6) expressing siPTEN into hind limb muscles at postnatal day 1 in SMNDelta7 mice leads to a significant PTEN depletion and robust improvement in motor neuron survival. Taken together, these data indicate that PTEN-mediated regulation of protein synthesis in motor neurons could represent a target for therapy in spinal muscular atrophy.

Recurrent mutations in chromatin modifiers are specifically prevalent in adolescent or adult patients with Sonic hedgehog-associated medulloblastoma (SHH MB). Here, we report that mutations in the acetyltransferase CREBBP have opposing effects during the development of the cerebellum, the primary site of origin of SHH MB. Our data reveal that loss of Crebbp in cerebellar granule neuron progenitors (GNPs) during embryonic development of mice compromises GNP development, in part by downregulation of brain-derived neurotrophic factor (Bdnf). Interestingly, concomitant cerebellar hypoplasia was also observed in patients with Rubinstein-Taybi syndrome, a congenital disorder caused by germline mutations of CREBBP. By contrast, loss of Crebbp in GNPs during postnatal development synergizes with oncogenic activation of SHH signaling to drive MB growth, thereby explaining the enrichment of somatic CREBBP mutations in SHH MB of adult patients. Together, our data provide insights into time-sensitive consequences of CREBBP mutations and corresponding associations with human diseases.

Axonal transport and translation of beta-actin mRNA plays an important role for axonal growth and presynaptic differentiation in many neurons including hippocampal, cortical and spinal motor neurons. Several beta-actin mRNA-binding and transport proteins have been identified, including ZBP1, ZBP2 and hnRNP-R. hnRNP-R has been found as an interaction partner of the survival motor neuron protein that is deficient in spinal muscular atrophy. Little is known about the function of hnRNP-R in axonal beta-actin translocation. hnRNP-R and beta-actin mRNA are colocalized in axons. Recombinant hnRNP-R interacts directly with the 3'-UTR of beta-actin mRNA. We studied the role of hnRNP-R in motor neurons by knockdown in zebrafish embryos and isolated mouse motor neurons. Suppression of hnRNP-R in developing zebrafish embryos results in reduced axon growth in spinal motor neurons, without any alteration in motor neuron survival. ShRNA-mediated knockdown in isolated embryonic mouse motor neurons reduces beta-actin mRNA translocation to the axonal growth cone, which is paralleled by reduced axon elongation. Dendrite growth and neuronal survival were not affected by hnRNP-R depletion in these neurons. The loss of beta-actin mRNA in axonal growth cones of hnRNP-R-depleted motor neurons resembles that observed in Smn-deficient motor neurons, a model for the human disease spinal muscular atrophy. In particular, hnRNP-R-depleted motor neurons also exhibit defects in presynaptic clustering of voltage-gated calcium channels. Our data suggest that hnRNP-R-mediated axonal beta-actin mRNA translocation plays an essential physiological role for axon growth and presynaptic differentiation.

Although brain-derived neurotrophic factor (BDNF) is linked with an increasing number of conditions causing brain dysfunction, its role in the postnatal CNS has remained difficult to assess. This is because thebdnf-null mutation causes the death of the animals before BDNF levels have reached adult levels. In addition, the anterograde axonal transport of BDNF complicates the interpretation of area-specific gene deletion. The present study describes the generation of a new conditional mouse mutant essentially lacking BDNF throughout the CNS. It shows that BDNF is not essential for prolonged postnatal survival, but that the behavior of such mutant animals is markedly altered. It also reveals that BDNF is not a major survival factor for most CNS neurons and for myelination of their axons. However, it is required for the postnatal growth of the striatum, and single-cell analyses revealed a marked decreased in dendritic complexity and spine density. In contrast, BDNF is dispensable for the growth of the hippocampus and only minimal changes were observed in the dendrites of CA1 pyramidal neurons in mutant animals. Spine density remained unchanged, whereas the proportion of the mushroom-type spine was moderately decreased. In line with thesein vivoobservations, we found that BDNF markedly promotes the growth of cultured striatal neurons and of their dendrites, but not of those of hippocampal neurons, suggesting that the differential responsiveness to BDNF is part of a neuron-intrinsic program.

Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75(NTR)-antibody-based panning technique, which highly enriches motoneurons within

Valproic acid (VPA), an antiepileptic drug and HDAC inhibitor, has been identified as a drug candidate for spinal muscular atrophy (SMA), a motoneuron disorder for which currently no effective therapy is available. Based on its potential to up-regulate SMN expression from the SMN2 gene in fibroblasts and lymphoblastoid cell lines from SMA patients, we analysed the effects of VPA in isolated motoneurons from Smn −/− ;SMN2 mice, a model for SMA type I. Treatment with VPA increased Smn expression but unexpectedly also led to reduced growth cone size and reduced excitability in axon terminals of mutant motoneurons. Analysis of Ca 2+ currents and distribution of voltage-gated Ca 2+ channels revealed an inhibitory function of VPA on voltage-gated Ca 2+ channels and possibly also other ion channels that contribute to presynaptic excitability of motoneurons. Our data indicate effects of VPA which might aggravate disease-specific symptoms in SMA patients.