Search

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex, leading to paralysis and eventually to death within 3–5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the endoplasmic reticulum-resident protein cerebral dopamine neurotrophic factor in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and aetiology: (i) the conditional choline acetyltransferase-tTA/TRE-hTDP43-M337V rat model previously described; (ii) the widely used SOD1-G93A mouse model; and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the endoplasmic reticulum stress response in motor neurons, we used three main methods: (i) primary cultures of motor neurons derived from embryonic Day 13 embryos; (ii) immunohistochemical analyses of spinal cord sections with choline acetyltransferase as spinal motor neuron marker; and (iii) quantitative polymerase chain reaction analyses of lumbar motor neurons isolated via laser microdissection. We show that intracerebroventricular administration of cerebral dopamine neurotrophic factor significantly halts the progression of the disease and improves motor behaviour in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. Cerebral dopamine neurotrophic factor rescues motor neurons in vitro and in vivo from endoplasmic reticulum stress-associated cell death and its beneficial effect is independent of genetic disease aetiology. Notably, cerebral dopamine neurotrophic factor regulates the unfolded protein response initiated by transducers IRE1α, PERK and ATF6, thereby enhancing motor neuron survival. Thus, cerebral dopamine neurotrophic factor holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.

Dipeptide repeat proteins (DPRs) occur via repeat-associated non-AUG (RAN) translation. In this issue of Neuron, McEachin et al. (2020) show that the aggregation-prone poly(GA)-rich chimeric DPRs determine divergent poly(GP) mediated pathology between C9ALS/FTD and SCA36.

Functional recovery after stroke is associated with a remapping of neural circuits. This reorganization is often associated with low-frequency, high-amplitude oscillations in the peri-infarct zone in both rodents and humans. These oscillations are reminiscent of sleep slow waves (SW) and suggestive of a role for sleep in brain plasticity that occur during stroke recovery; however, direct evidence is missing. Using a stroke model in male mice, we showed that stroke was followed by a transient increase in NREM sleep accompanied by reduced amplitude and slope of ipsilateral NREM sleep SW. We next used 5 ms optical activation of Channelrhodopsin 2-expressing pyramidal neurons, or 200 ms silencing of Archeorhodopsin T-expressing pyramidal neurons, to generate local cortical UP, or DOWN, states, respectively, both sharing similarities with spontaneous NREM SW in freely moving mice. Importantly, we found that single optogenetically evoked SW (SWopto) in the peri-infarct zone, randomly distributed during sleep, significantly improved fine motor movements of the limb corresponding to the sensorimotor stroke lesion site compared with spontaneous recovery and control conditions, while motor strength remained unchanged. In contrast, SWoptoduring wakefulness had no effect. Furthermore, chronic SWoptoduring sleep were associated with local axonal sprouting as revealed by the increase of anatomic presynaptic and postsynaptic markers in the peri-infarct zone and corresponding contralesional areas to cortical circuit reorganization during stroke recovery. These results support a role for sleep SW in cortical circuit plasticity and sensorimotor recovery after stroke and provide a clinically relevant framework for rehabilitation strategies using neuromodulation during sleep.SIGNIFICANCE STATEMENTBrain stroke is one of the leading causes of death and major disabilities in the elderly worldwide. A better understanding of the pathophysiological mechanisms underlying spontaneous brain plasticity after stroke, together with an optimization of rehabilitative strategies, are essential to improve stroke treatments. Here, we investigate the role of optogenetically induced sleep slow waves in an animal model of ischemic stroke and identify sleep as a window for poststroke intervention that promotes neuroplasticity and facilitates sensorimotor recovery.

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.

Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of multi-domain recombinant Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant CanPI-7 at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression on CanPI-7 feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h). Further, coexpression of H. armigera endogenous PIs with several digestive protease genes were apparent. In addition to the differential expression of endogenous H. armigera PIs, we also observed a distinct novel isoform of endogenous PI in CanPI-7 fed H. armigera larvae. Based on present and earlier studies, we propose potential mechanism of protease regulation in H. armigera and subsequent adaptation strategy to cope with anti-nutritional components of plants.

Mordes et al. (2020) did not detect the survival or motor phenotypes in C9orf72 BAC transgenic mice originally described by Liu et al. (2016). We discuss methodological differences between the Mordes and Liu studies, several additional studies in which survival and motor phenotypes were found, and possible environmental and genetic effects. First, Nguyen et al. (2020) showed robust ALS/FTD phenotypes in C9-BAC versus non-transgenic (NT) mice and that α-GA1 treatment improved survival, behavior, and neurodegeneration. The groups of Gelbard and Saxena also show decreased survival of C9-BAC versus NT mice and neuropathological and behavioral deficits similar to those shown by Liu et al. (2016). Although FVB/N mice can have seizures, increases in seizure severity and death of C9 and NT animals, which may mask C9 disease phenotypes, have been observed in recent C9-500 FVB/NJ-bred cohorts. In summary, we provide an update on phenotypes seen in FVB C9-BAC mice and additional details to successfully use this model. This Matters Arising Response paper addresses the Mordes et al. (2020) Matters Arising paper, published concurrently in Neuron.

Motor neuron–specific microRNA-218 (miR-218) has recently received attention because of its roles in mouse development. However, miR-218 relevance to human motor neuron disease was not yet explored. Here, we demonstrate by neuropathology that miR-218 is abundant in healthy human motor neurons. However, in amyotrophic lateral sclerosis (ALS) motor neurons, miR-218 is down-regulated and its mRNA targets are reciprocally up-regulated (derepressed). We further identify the potassium channel Kv10.1 as a new miR-218 direct target that controls neuronal activity. In addition, we screened thousands of ALS genomes and identified six rare variants in the human miR-218-2 sequence. miR-218 gene variants fail to regulate neuron activity, suggesting the importance of this small endogenous RNA for neuronal robustness. The underlying mechanisms involve inhibition of miR-218 biogenesis and reduced processing by DICER. Therefore, miR-218 activity in motor neurons may be susceptible to failure in human ALS, suggesting that miR-218 may be a potential therapeutic target in motor neuron disease., Target ALS; European Union (European Union); Horizon 2020; European Research Council (ERC), European Union's Seventh Framework Programme (FP7/2007-2013); AFM Telethon; NIH; National Institute of Neurological Disorders and Stroke; NIH/NINDS; United Kingdom, Medical Research Council; Suna and İnan Kıraç Foundation; Legacy Heritage Fund; Bruno and Ilse Frick Foundation for Research on ALS; Teva Pharmaceutical Industries Ltd. as part of the Israeli National Network of Excellence in Neuroscience (NNE); Minna-James-Heineman Stiftung through Minerva; Israel Science Foundation; ALS-Therapy Alliance; Motor Neuron Disease Association (United Kingdom); Thierry Latran Foundation for ALS research; ERA-Net for Research Programmes on Rare Diseases (FP7); IsrALS, Yeda-Sela, Yeda-CEO, Israel Ministry of Trade and Industry; Y. Leon Benoziyo Institute for Molecular Medicine; Kekst Family Institute for Medical Genetics; David and Fela Shapell Family Center for Genetic Disorders Research; Crown Human Genome Center; Nathan, Shirley, Philip and Charlene Vener New Scientist Fund; Julius and Ray Charlestein Foundation; Fraida Foundation; Wolfson Family Charitable Trust; Adelis Foundation; Merck (United Kingdom); ALS Canada Tim E. Noel Postdoctoral Fellowship; Project5 for ALS; Robert Packard Center for ALS Research; University of Missouri Spinal Cord Injury/Disease Research Program; Hope Center for Neurological Disorders; ALS Association ; Biogen; ALS Finding a Cure; Angel Fund; ALS-One; Cellucci Fund; Motor Neurone Disease Association; National Institute for Health Research (NIHR) Biomedical Research Centre

Cerebellar ataxia is a clinically heterogeneous group of disorders, which includes several well-characterized genetic diseases as well as sporadic ataxias. The pathophysiology of ataxia is being understood, and a mechanistic basis for the appearance of these disorders is progressively emerging. Novel genes associated with dominant and recessive ataxias are being steadily identified, and research on their pathomechanisms not only has led to understanding the etiology and underlying cause for the development of ataxia but also has steered the field towards future therapeutic regime, aiming to control and prevent some forms of these diseases. Nevertheless, lack of knowledge for the causation of disease in a sizeable proportion of patient remains, and this issue is further compounded by the rarity of some of these ataxias as well as their restricted geographical distribution. On the other hand, large collaborative studies are providing critical information on the clinical spectrum, progression, and pathophysiology of inherited and sporadic ataxias. In the following sections, we describe the epidemiology, symptoms, pathological progression, and clinical management of various forms of inherited cerebellar ataxias. Finally, we provide a perspective on the challenges faced by the field in translational research and the development of successful therapeutic modalities for patients.

Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1154Q/2Q mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1154Q/2Q mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as preventing oxidative stress-induced DNA / RNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression.

Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron excitability and spinal circuits are pathological hallmarks of ALS, but the underlying molecular mechanisms remain unresolved. Here, we sought to understand whether the expression of mutant SOD1 protein could contribute to altering processes governing motor neuron excitability. We used the conformation specific antibody B8H10 which recognizes a misfolded state of SOD1 (misfSOD1) to longitudinally identify its interactome during early disease stage in SOD1G93A mice. This strategy identified a direct isozyme-specific association of misfSOD1 with Na(+)/K(+)ATPase-α3 leading to the premature impairment of its ATPase activity. Pharmacological inhibition of Na(+)/K(+)ATPase-α3 altered glutamate receptor 2 expression, modified cholinergic inputs and accelerated disease pathology. After mapping the site of direct association of misfSOD1 with Na(+)/K(+)ATPase-α3 onto a 10 amino acid stretch that is unique to Na(+)/K(+)ATPase-α3 but not found in the closely related Na(+)/K(+)ATPase-α1 isozyme, we generated a misfSOD1 binding deficient, but fully functional Na(+)/K(+)ATPase-α3 pump. Adeno associated virus (AAV)-mediated expression of this chimeric Na(+)/K(+)ATPase-α3 restored Na(+)/K(+)ATPase-α3 activity in the spinal cord, delayed pathological alterations and prolonged survival of SOD1G93A mice. Additionally, altered Na(+)/K(+)ATPase-α3 expression was observed in the spinal cord of individuals with sporadic and familial ALS. A fraction of sporadic ALS cases also presented B8H10 positive misfSOD1 immunoreactivity, suggesting that similar mechanism might contribute to the pathology.

Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of multi-domain recombinant Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant CanPI-7 at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression on CanPI-7 feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h). Further, coexpression of H. armigera endogenous PIs with several digestive protease genes were apparent. In addition to the differential expression of endogenous H. armigera PIs, we also observed a distinct novel isoform of endogenous PI in CanPI-7 fed H. armigera larvae. Based on present and earlier studies, we propose potential mechanism of protease regulation in H. armigera and subsequent adaptation strategy to cope with anti-nutritional components of plants.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS‐linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.

Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.

1AbstractSerial block face scanning electron microscopy (SBFSEM) is an increasingly popular method for investigating the three-dimensional ultrastructure of large biological samples. Prior to imaging, samples are typically chemically fixed, stained with osmium and uranyl acetate, and subsequently embedded in resin. The purpose of staining is to provide image contrast and reduce specimen charging under the electron beam, which is detrimental to the quality of imaging. Obtaining, using, and disposing of uranyl acetate is getting increasingly cumbersome in many countries due to new regulations on the handling of radioactive substances. Therefore, we developed an alternative staining procedure that does not rely on the use of uranium or any other radioactive substance. This procedure provides excellent contrast and efficiently reduces specimen charging.

Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

Neurodegenerative diseases selectively target subpopulations of neurons, leading to the progressive failure of defined brain systems, but the basis of such selective neuronal vulnerability has remained elusive. Here, we discuss how a stressor-threshold model of how particular neurons and circuits are selectively vulnerable to disease may underly the etiology of familial and sporadic forms of diseases such as Alzheimer's, Parkinson's, Huntington's, and ALS. According to this model, the intrinsic vulnerabilities of neuronal subpopulations to stressors and specific disease-related misfolding proteins determine neuronal morbidity. Neurodegenerative diseases then involve specific combinations of genetic predispositions and environmental stressors, triggering increasing age-related stress and proteostasis dysfunction in affected vulnerable neurons. Damage to vasculature, immune system, and local glial cells mediates environmental stress, which could drive disease at all stages.

Neurotrophin receptors of the Trk family promote neuronal survival. The signal transduction of Trk receptors is regulated by endosomal trafficking. Monoubiquitination of receptor tyrosine kinases is an established signal for sorting of internalized receptors to late endosomes. The NGF receptor TrkA is sorted to late endosomes and undergoes ubiquitination, indicating a so far undefined regulatory role of proteasomal activity in the trafficking of TrkA. Surprisingly, we found that proteasomal inhibition alters the trafficking of TrkA from the late endosomal sorting pathway to the recycling pathway. Many neurodegenerative diseases are associated with impaired proteasomal activity. Thus, our study suggests that missorting of neurotrophic receptors might contribute to neuronal death in those neurodegenerative diseases that are known to be associated with impaired proteasomal function.

In physiological conditions the maintenance of the cellular proteome is a prerequisite for optimal cell functioning and cell survival. Additionally, cells need to constantly sense and adapt to their changing environment and associated stressors. Cells achieve this via a set of molecular chaperones, protein clearance pathways as well as stress-associated signaling networks which work together to prevent protein misfolding, its aggregation and accumulation in subcellular compartments. These processes together form the proteostasis network which helps in maintaining cellular proteostasis. Imbalance or impairment in this processes is directly linked to ageing associated disorders such as diabetes, cancer, stroke, metabolic disorders, pulmonary fibrosis, inflammation and neurodegenerative diseases. In this review, we provide insights into the proteostasis process and how its failure governs neurodegenerative disorders with a special focus on Amyotrophic lateral sclerosis (ALS). This article is part of a Special Issue entitled SI:ER stress.

Spinocerebellar ataxia type 1 (SCA1), due to the expansion of a polyglutamine repeat within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), the cause of which is poorly understood. Here, we identified the unique proteomic signature of Sca1(154Q/2Q) PCs at an early stage of disease, highlighting extensive alterations in proteins associated with synaptic functioning, maintenance, and transmission. Focusing on Homer-3, a PC-enriched scaffold protein regulating neuronal activity, revealed an early decline in its expression. Impaired climbing fiber-mediated synaptic transmission diminished mTORC1 signaling, paralleling Homer-3 reduction in Sca1(154Q/2Q) PCs. Ablating mTORC1 within PCs or pharmacological inhibition of mTORC1 identified Homer-3 as its downstream target. mTORC1 knockout in Sca1(154Q/2Q) PCs exacerbated and accelerated pathology. Reinstating Homer-3 expression in Sca1(154Q/2Q) PCs attenuated cellular dysfunctions and improved motor deficits. Our work reveals that impaired mTORC1-Homer-3 activity underlies PC susceptibility in SCA1 and presents a promising therapeutic target.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.

Paracrine signalling mediated by cytokine secretion is essential for liver regeneration after hepatic resection, yet the mechanisms of cellular crosstalk between immune and parenchymal cells are still elusive. Interleukin-22 (IL-22) is released by immune cells and mediates strong hepatoprotective functions. However, it remains unclear whether IL-22 is critical for the crosstalk between liver lymphocytes and parenchymal cells during liver regeneration after partial hepatectomy (PH). Here, we found that plasma levels of IL-22 and its upstream cytokine, IL-23, are highly elevated in patients after major liver resection. In a mouse model of PH, deletion of IL-22 was associated with significantly delayed hepatocellular proliferation and an increase of hepatocellular injury and endoplasmic reticulum stress. Using Rag1(-/-) and Rag2(-/-) γc(-/) (-) mice, we show that the main producers of IL-22 post-PH are conventional natural killer cells and innate lymphoid cells type 1. Extracellular adenosine triphosphate (ATP), a potent danger molecule, is elevated in patients immediately after major liver resection. Antagonism of the P2-type nucleotide receptors, P2X1 and P2Y6, significantly decreased IL-22 secretion ex vivo. In vivo, specific inhibition of P2X1 was associated with decreased IL-22 secretion, elevated liver injury, and impaired liver regeneration.This study shows that innate immune cell-derived IL-22 is required for efficient liver regeneration and that secretion of IL-22 in the regenerating liver is modulated by the ATP receptor, P2X1. (Hepatology 2016;63:2004-2017).

Neurotrophins transmit signals retrogradely from synapses to cell bodies by two different types of surface receptors, p75NTR and Trks. Compared to TrkA, the function of p75NTR in nerve growth factor (NGF) endocytosis is less clear, and it is unknown whether p75NTR by itself may internalize other neurotrophins besides NGF. We directly compared TrkA and p75NTR for their ability to internalize NGF, and we also examined the endocytosis of iodinated brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) by p75NTR. Cells expressing solely TrkA internalized NGF more efficiently than cells expressing p75NTR. Surprisingly, cells expressing only p75NTR internalized far more BDNF or NT3 than NGF. Moreover, p75NTR was more important for surface binding than for intracellular accumulation of each neurotrophin. Finally, we established a mechanistic role for the clathrin pathway in p75NTR endocytosis. Our results suggest that p75NTR may have multiple roles in different subcellular locations, functioning both at the cell surface and also within endocytic compartments. © 2004 Elsevier Inc. All rights reserved., This study was supported by a grant from the Swiss National Science Foundation

Neurotrophins are well known for their physiological role as key modulators of neuronal survival, neurite out-growth, and synaptic connectivity during development and into adulthood. Moreover, neurotrophins are potent agents, ameliorating neuronal degeneration in many model systems for neurological diseases. However, a causal role for mutations in neurotrophins or neurotrophin receptors in human neurodegenerative diseases has been largely lacking. As neurotrophin receptors are located at synapses and as their signaling involves the neuronal nucleus, they need to bridge tantalizing distances in order to retrogradely communicate their survival signals. On the other hand, anterogradely transported neurotrophins are released at the synapse and act on postsynaptic cells. Antero- and retrograde signaling and trafficking is an emerging focus of interest in neurotrophin research. Some neurodegenerative diseases are known to affect transport of organelles. Thus, it appears likely that neurodegeneration could be caused by "indirect" effects on neurotrophin trafficking and, hence, signaling. In this review we summarize recent work on neurotrophins in neurodegenerative diseases with special focus on possible implications of disturbed trafficking of organelles and retrograde axonal signaling.

Mechanisms underlying motor neuron subtype-selective endoplasmic reticulum (ER) stress and associated axonal pathology in amyotrophic lateral sclerosis (ALS) remain unclear. Here we show that the molecular environment of the ER between motor neuron subtypes is distinct, with characteristic signatures. We identify cochaperone SIL1, mutated in Marinesco-Sjogren syndrome (MSS), as being robustly expressed in disease-resistant slow motor neurons but not in ER stress-prone fast-fatigable motor neurons. In a mouse model of MSS, we demonstrate impaired ER homeostasis in motor neurons in response to loss of SIL1 function. Loss of a single functional Sil1 allele in an ALS mouse model (SOD1-G93A) enhanced ER stress and exacerbated ALS pathology. In SOD1-G93A mice, SIL1 levels were progressively and selectively reduced in vulnerable fast-fatigable motor neurons. Mechanistically, reduction in SIL1 levels was associated with lowered excitability of fast-fatigable motor neurons, further influencing expression of specific ER chaperones. Adeno-associated virus-mediated delivery of SIL1 to familial ALS motor neurons restored ER homeostasis, delayed muscle denervation and prolonged survival.

During development, targets of outgrowing axons produce neurotrophins. These signaling molecules exert short-distance effects on neuronal growth cones as well as long-distance effects on neuronal cell bodies and nuclei. The retrograde signaling from axon tips to cell bodies is essential for the regulation of neuronal survival during the period of naturally occurring programmed cell death in the nervous system. During this time window, neurons that fail to establish appropriate connections with their target tissues are eliminated by apoptosis. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5) may influence neuronal survival not only via signaling through Trk receptors but also through interactions with p75NTR. Retrograde transport of neurotrophins and their receptors within organelles coined ‘signaling endosomes’ is one potential mechanism contributing to retrograde signaling. However, alternative pathways not involving retrograde transport of neurotrophins are emerging. The current models of retrograde neurotrophin signaling from synapses to cell bodies are discussed, with special focus on potential roles of the enigmatic p75NTR receptor in neurotrophin trafficking.

Antigen-Antibody Interaction Database (AgAbDb) is an immunoinformatics resource developed at the Bioinformatics Centre, University of Pune, and is available online at http://bioinfo.net.in/AgAbDb.htm. Antigen-antibody interactions are a special class of protein-protein interactions that are characterized by high affinity and strict specificity of antibodies towards their antigens. Several co-crystal structures of antigen-antibody complexes have been solved and are available in the Protein Data Bank (PDB). AgAbDb is a derived knowledgebase developed with an objective to compile, curate, and analyze determinants of interactions between the respective antigen-antibody molecules. AgAbDb lists not only the residues of binding sites of antigens and antibodies, but also interacting residue pairs. It also helps in the identification of interacting residues and buried residues that constitute antibody-binding sites of protein and peptide antigens. The Antigen-Antibody Interaction Finder (AAIF), a program developed in-house, is used to compile the molecular interactions, viz. van der Waals interactions, salt bridges, and hydrogen bonds. A module for curating water-mediated interactions has also been developed. In addition, various residue-level features, viz. accessible surface area, data on epitope segment, and secondary structural state of binding site residues, are also compiled. Apart from the PDB numbering, Wu-Kabat numbering and explicit definitions of complementarity-determining regions are provided for residues of antibodies. The molecular interactions can be visualized using the program Jmol. AgAbDb can be used as a benchmark dataset to validate algorithms for prediction of B-cell epitopes. It can as well be used to improve accuracy of existing algorithms and to design new algorithms. AgAbDb can also be used to design mimotopes representing antigens as well as aid in designing processes leading to humanization of antibodies.

The mechanisms underlying disease manifestations in neurodegeneration remain unclear, but their understanding is critical to devising effective therapies. We carry out a longitudinal analysis in vivo of identified motoneurons selectively vulnerable (VUL) or resistant (RES) to motoneuron disease (amyotrophic lateral sclerosis, ALS) and show that subtype-selective endoplasmic reticulum (ER) stress responses influence disease manifestations. VUL motoneurons were selectively prone to ER stress and showed gradually upregulated ER stress markers from birth on in three mouse models of familial ALS (FALS). 25-30 days before the earliest denervations, ubiquitin signals increased in both VUL and RES motoneurons, but an unfolded protein response coupled with microglial activation was initiated selectively in VUL motoneurons. This transition was followed by selective axonal degeneration and spreading stress. The ER stress-protective agent salubrinal attenuated disease manifestations and delayed progression, whereas chronic enhancement of ER stress promoted disease. Thus, whereas all motoneurons are preferentially affected in ALS, ER stress responses in specific motoneuron subtypes influence the progressive manifestations of weakening and paralysis.

Axon degeneration is an active, tightly controlled and versatile process of axon segment self-destruction. Although not involving cell death, it resembles apoptosis in its logics. It involves three distinct steps: induction of competence in specific neurons, triggering of degeneration at defined axon segments of competent neurons, and rapid fragmentation and removal of the segments. The mechanisms that initiate degeneration are specific to individual settings, but the final pathway of pruning is shared; it involves microtubule disassembly, axon swellings, axon fragmentation, and removal of the remnants by locally recruited phagocytes. The tight regulatory properties of axon degeneration distinguish it from passive loss phenomena, and confer significance to processes that involve it. Axon degeneration has prominent roles in development, upon lesions and in disease. In development, it couples the progressive specification of neurons and circuits to the removal of defined axon branches. Competence might involve transcriptional switches, and local triggering can involve axon guidance molecules and synaptic activity patterns. Lesion-induced Wallerian degeneration is inhibited in the presence of Wld(S) fusion protein in neurons; it involves early local, and later, distal degeneration. It has recently become clear that like in other settings, axon degeneration in disease is a rapid and specific process, which should not be confused with a variety of disease-related pathologies. Elucidating the specific mechanisms that initiate axon degeneration should open up new avenues to investigate principles of circuit assembly and plasticity, to uncover mechanisms of disease progression, and to identify ways of protecting synapses and axons in disease.

Neurodegenerative diseases can have long preclinical phases and insidious progression patterns, but the mechanisms of disease progression are poorly understood. Because quantitative accounts of neuronal circuitry affected by disease have been lacking, it has remained unclear whether disease progression reflects processes of stochastic loss or temporally defined selective vulnerabilities of distinct synapses or axons. Here we derive a quantitative topographic map of muscle innervation in the hindlimb. We show that in two mouse models of motoneuron disease (G93A SOD1 and G85R SOD1), axons of fast-fatiguable motoneurons are affected synchronously, long before symptoms appear. Fast-fatigue-resistant motoneuron axons are affected at symptom-onset, whereas axons of slow motoneurons are resistant. Axonal vulnerability leads to synaptic vesicle stalling and accumulation of BC12a1-a, an anti-apoptotic protein. It is alleviated by ciliary neurotrophic factor and triggers proteasome-dependent pruning of peripheral axon branches. Thus, motoneuron disease involves predictable, selective vulnerability patterns by physiological subtypes of axons, episodes of abrupt pruning in the target region and compensation by resistant axons.

NGF binds to two receptors, p75NTR and TrkA. The endosomal trafficking of receptors is of emerging importance for the understanding of their signaling. We compared the endocytic trafficking of the two NGF receptors in PC12 cells. Both p75NTR and TrkA were internalized in response to NGF and colocalized with early endosomes. However, surprisingly, the subsequent endosomal trafficking paths of both NGF receptors diverged: whereas p75NTR recycled back to the surface, TrkA moved to late endosomes and underwent lysosomal degradation. By performing subcellular fractionations of NGF stimulated PC12 cells, tyrosine-phosphorylated TrkA was recovered in fractions corresponding to late endosomes. This implicates these organelles as novel endosomal NGF signaling platforms. Furthermore, the trafficking of NGF receptors could be manipulated by pharmacological means. Disrupting p75NTR recycling diminished TrkA activation in response to low concentrations of NGF, demonstrating a functional role for the recycling of p75NTR. © 2004 Elsevier Inc. All rights reserved., This study was supported by a grant from the Swiss National Science Foundation.

Neuronal survival is dependent on continuous trophic stimulation by neurotrophic factors. Anterograde and retrograde transport of neurotrophic factors and their receptors within neurites is essential for the communication of these survival signals. Lack of neurotrophic input has been proposed as a pathomechanism leading to neurodegenerative disease. The present short review provides a summary of some of the recent data on neurotrophic factors in neurodegenerative disorders and describes how disturbances of axonal trafficking might deprive neurons from trophic input.

We describe the cloning and expression of two novel genes highly similar to the tocopherol-associated protein (hTAP/SEC14L2/SPF). Immunoprecipitation of the three recombinant hTAPs and extraction of their associated lipid-soluble molecules indicates that they bind not just tocopherols, but also phosphatidylinositol, phosphatidylcholine, and phosphatidylglycerol. Ligand competition analysis by isoelectric point mobility shift assay indicates that phosphatidylcholine, tocopherols, and tocopheryl-succinate compete with phosphatidylinositol binding to hTAPs. To investigate a possible function of hTAPs on enzymes involved in phospholipids metabolism, the activity of recombinant phosphatidylinositol 3-kinase (PI3Kgamma/p110gamma) was tested. Recombinant hTAPs reduce in vitro the activity of the recombinant catalytic subunit of PI3Kgamma and stimulate it in the presence of alpha-tocopherol up to 5-fold. Immunoprecipitation of hTAP1 from cells results in co-precipitation of PI3-kinase activity, indicating a physical contact between the two proteins at a cellular level. In summary, hTAPs may modulate, in a tocopherol-sensitive manner, phosphatidylinositol-3-kinase, a central enzyme in signal transduction, cell proliferation, and apoptosis. It is possible that other phosphatidylinositol- and phosphatidylcholine-dependent signaling pathways are modulated by hTAPs and tocopherols, possibly by transporting and presenting these ligands to the corresponding enzymes.

SummaryDelaying clinical disease onset would greatly reduce neurodegenerative disease burden, but the mechanisms influencing early preclinical progression are poorly understood. Here, we show that in mouse models of familial motoneuron (MN) disease, SOD1 mutants specifically render vulnerable MNs dependent on endogenous neuroprotection signaling involving excitability and mammalian target of rapamycin (mTOR). The most vulnerable low-excitability FF MNs already exhibited evidence of pathology and endogenous neuroprotection recruitment early postnatally. Enhancing MN excitability promoted MN neuroprotection and reversed misfolded SOD1 (misfSOD1) accumulation and MN pathology, whereas reducing MN excitability augmented misfSOD1 accumulation and accelerated disease. Inhibiting metabotropic cholinergic signaling onto MNs reduced ER stress, but enhanced misfSOD1 accumulation and prevented mTOR activation in alpha-MNs. Modulating excitability and/or alpha-MN mTOR activity had comparable effects on the progression rates of motor dysfunction, denervation, and death. Therefore, excitability and mTOR are key endogenous neuroprotection mechanisms in motoneurons to counteract clinically important disease progression in ALS.