ABSTRACT
Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Arginine/metabolism , Cytoplasmic Granules/metabolism , Dipeptides/metabolism , Intrinsically Disordered Proteins/metabolism , Proteins/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Arginine/chemistry , C9orf72 Protein , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cytoplasmic Granules/pathology , DNA Helicases , Dipeptides/chemistry , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , HeLa Cells , Humans , Intrinsically Disordered Proteins/chemistry , Lipid Droplets/metabolism , Phosphorylation , Poly-ADP-Ribose Binding Proteins , Protein Domains , Proteins/chemistry , RNA/metabolism , RNA Helicases , RNA Recognition Motif Proteins , Time Factors , TransfectionABSTRACT
Several neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia (SCA) are caused by non-coding nucleotide repeat expansions. Different pathogenic mechanisms may underlie these non-coding repeat expansion disorders. While gain-of-function mechanisms, such as toxicity associated with expression of repeat RNA or toxicity associated with repeat-associated non-ATG (RAN) products, are most frequently connected with these disorders, loss-of-function mechanisms have also been implicated. We review the different pathways that have been linked to non-coding repeat expansion disorders such as C9ORF72-linked ALS/frontotemporal dementia (FTD), myotonic dystrophy, fragile X tremor/ataxia syndrome (FXTAS), SCA, and Huntington's disease-like 2. We discuss modes of RNA toxicity focusing on the identity and the interacting partners of the toxic RNA species. Using the C9ORF72 ALS/FTD paradigm, we further explore the efforts and different methods used to disentangle RNA vs. RAN toxicity. Overall, we conclude that there is ample evidence for a role of RNA toxicity in non-coding repeat expansion diseases.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Ataxia/pathology , C9orf72 Protein/genetics , DNA Repeat Expansion/genetics , Fragile X Syndrome/pathology , Frontotemporal Dementia/pathology , Myotonic Dystrophy/pathology , Neurodegenerative Diseases/pathology , RNA/toxicity , Tremor/pathology , Amyotrophic Lateral Sclerosis/genetics , Ataxia/genetics , Fragile X Syndrome/genetics , Frontotemporal Dementia/genetics , Humans , Mutation , Myotonic Dystrophy/genetics , Neurodegenerative Diseases/genetics , RNA/genetics , Tremor/geneticsABSTRACT
Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.
Subject(s)
Alzheimer Disease , Amyotrophic Lateral Sclerosis , Neanderthals , Neurodegenerative Diseases , Parkinson Disease , Animals , Humans , Neanderthals/genetics , Neurodegenerative Diseases/genetics , Selection, GeneticABSTRACT
Worldwide, stroke is the main cause of long-term adult disability. After the initial insult, most patients undergo a subacute period with intense plasticity and rapid functional improvements. This period is followed by a chronic phase where recovery reaches a plateau that is only partially modifiable by rehabilitation. After experimental stroke, various subacute rehabilitation paradigms improve recovery. However, in order to reach the best possible outcome, a combination of plasticity-promoting strategies and rehabilitation might be necessary. EphA4 is a negative axonal guidance regulator during development. After experimental stroke, reduced EphA4 levels improve functional outcome with similar beneficial effects upon the inhibition of EphA4 downstream targets. In this study, we assessed the effectiveness of a basic enriched environment in the chronic phase after photothrombotic stroke in mice as well as the therapeutic potential of EphA4 targeted therapy followed by rehabilitation. Our findings show that environmental enrichment in the chronic phase improves functional outcome up to 2 months post-stroke. Although EphA4 levels increase after experimental stroke, subacute EphA4 inhibition followed by environmental enrichment does not further increase recovery. In conclusion, we show that environmental enrichment during the chronic phase of stroke improves functional outcome in mice with no synergistic effects of the used EphA4 targeted therapy.
Subject(s)
Disease Models, Animal , Peptide Fragments/pharmacology , Receptor, EphA4/antagonists & inhibitors , Recovery of Function , Stroke Rehabilitation/methods , Stroke/drug therapy , Animals , Male , Mice , Mice, Inbred C57BL , Molecular Targeted Therapy , Phosphorylation , Stroke/etiology , Stroke/pathologyABSTRACT
OBJECTIVE: The role of the survival of motor neuron (SMN) gene in amyotrophic lateral sclerosis (ALS) is unclear, with several conflicting reports. A decisive result on this topic is needed, given that treatment options are available now for SMN deficiency. METHODS: In this largest multicenter case control study to evaluate the effect of SMN1 and SMN2 copy numbers in ALS, we used whole genome sequencing data from Project MinE data freeze 2. SMN copy numbers of 6,375 patients with ALS and 2,412 controls were called from whole genome sequencing data, and the reliability of the calls was tested with multiplex ligation-dependent probe amplification data. RESULTS: The copy number distribution of SMN1 and SMN2 between cases and controls did not show any statistical differences (binomial multivariate logistic regression SMN1 p = 0.54 and SMN2 p = 0.49). In addition, the copy number of SMN did not associate with patient survival (Royston-Parmar; SMN1 p = 0.78 and SMN2 p = 0.23) or age at onset (Royston-Parmar; SMN1 p = 0.75 and SMN2 p = 0.63). INTERPRETATION: In our well-powered study, there was no association of SMN1 or SMN2 copy numbers with the risk of ALS or ALS disease severity. This suggests that changing SMN protein levels in the physiological range may not modify ALS disease course. This is an important finding in the light of emerging therapies targeted at SMN deficiencies. ANN NEUROL 2021;89:686-697.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Survival of Motor Neuron 1 Protein/genetics , Case-Control Studies , Cohort Studies , Female , Gene Dosage , Humans , Male , Reproducibility of Results , Risk Factors , Severity of Illness Index , Survival of Motor Neuron 2 Protein/genetics , Whole Genome SequencingABSTRACT
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the progressive loss of specific groups of neurons. Due to clinical, genetic and pathological overlap, both diseases are considered as the extremes of one disease spectrum and in a number of ALS and FTD patients, fused in sarcoma (FUS) aggregates are present. Even in families with a monogenetic disease cause, a striking variability is observed in disease presentation. This suggests the presence of important modifying genes. The identification of disease-modifying genes will contribute to defining clear therapeutic targets and to understanding the pathways involved in motor neuron death. In this study, we established a novel in vivo screening platform in which new modifying genes of FUS toxicity can be identified. Expression of human FUS induced the selective apoptosis of crustacean cardioactive peptide (CCAP) neurons from the ventral nerve cord of fruit flies. No defects in the development of these neurons were observed nor were the regulatory CCAP neurons from the brain affected. We used the number of CCAP neurons from the ventral nerve cord as an in vivo read-out for FUS toxicity in neurons. Via a targeted screen, we discovered a potent modifying role of proteins involved in nucleocytoplasmic transport. Downregulation of Nucleoporin 154 and Exportin1 (XPO1) prevented FUS-induced neurotoxicity. Moreover, we show that XPO1 interacted with FUS. Silencing XPO1 significantly reduced the propensity of FUS to form inclusions upon stress. Taken together, our findings point to an important role of nucleocytoplasmic transport proteins in FUS-induced ALS/FTD.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Frontotemporal Dementia/genetics , Karyopherins/genetics , Neuropeptides/genetics , RNA-Binding Protein FUS/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Animals, Genetically Modified/genetics , Apoptosis/genetics , Drosophila/genetics , Drosophila Proteins/genetics , Female , Frontotemporal Dementia/physiopathology , Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics , Humans , Male , Mutation , Neurons/pathology , Nuclear Pore Complex Proteins/genetics , Protein Aggregation, Pathological/genetics , Exportin 1 ProteinABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder of which the progression is influenced by several disease-modifying factors. Here, we investigated ELP3, a subunit of the elongator complex that modifies tRNA wobble uridines, as one of such ALS disease modifiers. ELP3 attenuated the axonopathy of a mutant SOD1, as well as of a mutant C9orf72 ALS zebrafish model. Furthermore, the expression of ELP3 in the SOD1G93A mouse extended the survival and attenuated the denervation in this model. Depletion of ELP3 in vitro reduced the modified tRNA wobble uridine mcm5s2U and increased abundance of insoluble mutant SOD1, which was reverted by exogenous ELP3 expression. Interestingly, the expression of ELP3 in the motor cortex of ALS patients was reduced and correlated with mcm5s2U levels. Our results demonstrate that ELP3 is a modifier of ALS and suggest a link between tRNA modification and neurodegeneration.
Subject(s)
Amyotrophic Lateral Sclerosis , Histone Acetyltransferases , Motor Cortex/metabolism , Nerve Tissue Proteins , RNA, Transfer , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA, Transfer/genetics , RNA, Transfer/metabolism , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , ZebrafishABSTRACT
Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleus post injury identified cathepsin D (CTSD) as the most upregulated gene. In aged GRN-/- cortices, CTSD was also upregulated, but the relative CTSD activity was reduced and improved upon exogenous GRN addition. Moreover, GRN and its C-terminal granulin domain granulinE (GrnE) both stimulated the proteolytic activity of CTSD in vitro. Pull-down experiments confirmed a direct interaction between GRN and CTSD. This interaction was also observed with GrnE and stabilized the CTSD enzyme at different temperatures. Investigating the importance of this interaction for axonal regeneration in vivo we found that, although individually tolerated, a combined reduction of GRN and CTSD synergistically reduced axonal outgrowth. Our data links the neurotrophic effect of GRN and GrnE with a lysosomal chaperone function on CTSD to maintain its proteolytic capacity.
Subject(s)
Cathepsin D/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Animals , Cathepsin D/genetics , Facial Nerve/metabolism , Frontotemporal Dementia/genetics , Granulins , Haploinsufficiency , Humans , Intercellular Signaling Peptides and Proteins/genetics , Mice , Mice, Transgenic , Molecular Chaperones/genetics , Mutation , ProgranulinsSubject(s)
Amyotrophic Lateral Sclerosis/metabolism , DNA-Binding Proteins/metabolism , Inflammation/etiology , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Animals , DNA-Binding Proteins/antagonists & inhibitors , Disease Models, Animal , Humans , Mice , Nucleotides, Cyclic/metabolismABSTRACT
Intravenous immunoglobulin (IVIG) is a potential therapy for chronic inflammatory demyelinating polyneuropathy (CIDP). To investigate the efficacy and safety of the IVIG IgPro10 (Privigen) for treatment of CIDP, results from Privigen Impact on Mobility and Autonomy (PRIMA), a prospective, open-label, single-arm study of IVIG in immunoglobulin (Ig)-naïve or IVIG pre-treated subjects (NCT01184846, n = 28) and Polyneuropathy And Treatment with Hizentra (PATH), a double-blind, randomized study including an open-label, single-arm IVIG phase in IVIG pre-treated subjects (NCT01545076, IVIG restabilization phase n = 207) were analyzed separately and together (n = 235). Efficacy assessments included change in adjusted inflammatory neuropathy cause and treatment (INCAT) score, grip strength and Medical Research Council (MRC) sum score. Adverse drug reactions (ADRs) and ADRs/infusion were recorded. Adjusted INCAT response rate was 60.7% in all PRIMA subjects at Week 25 (76.9% in IVIG pre-treated subjects) and 72.9% in PATH. In the pooled cohort (n = 235), INCAT response rate was 71.5%; median time to INCAT improvement was 4.3 weeks. No clear demographic differences were noticed between early (responding before Week 7, n = 148) and late responders (n = 21). In the pooled cohort, median change from baseline to last observation was -1.0 (interquartile range -2.0; 0.0) point for INCAT score; +8.0 (0.0; 20.0) kPa for maximum grip strength; +3.0 (1.0; 7.0) points for MRC sum score. In the pooled cohort, 271 ADRs were reported in 105 subjects (44.7%), a rate of 0.144 ADRs per infusion. This analysis confirms the efficacy and safety of IgPro10, a recently FDA-approved IVIG for CIDP, in a population of mainly pre-treated subjects with CIDP [Correction added on 14 March 2019 after first online publication: the INCAT response rate has been corrected.].
Subject(s)
Immunoglobulins, Intravenous/pharmacology , Immunologic Factors/pharmacology , Outcome Assessment, Health Care , Polyradiculoneuropathy, Chronic Inflammatory Demyelinating/drug therapy , Adult , Aged , Aged, 80 and over , Double-Blind Method , Europe , Female , Humans , Immunoglobulins, Intravenous/administration & dosage , Immunologic Factors/administration & dosage , Male , Middle Aged , Prospective Studies , Young AdultABSTRACT
Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.
Subject(s)
Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/therapy , Glycine-tRNA Ligase/genetics , Histone Deacetylase 6/metabolism , Mutation/genetics , Animals , Axonal Transport/genetics , Cells, Cultured , Charcot-Marie-Tooth Disease/physiopathology , Disease Models, Animal , Enzyme Inhibitors/therapeutic use , Ganglia, Spinal/cytology , Histone Deacetylase 6/genetics , Hydroxamic Acids/therapeutic use , Indoles/therapeutic use , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Neurons/drug effects , Motor Neurons/metabolism , Neural Conduction/genetics , Neuromuscular Junction/pathology , Neuromuscular Junction/physiopathology , Psychomotor Performance/physiology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Tubulin/metabolismABSTRACT
The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodies may deserve further evaluation as potential therapeutics in disorders in which EphA4-mediated signaling plays a role.
Subject(s)
Antibody Affinity , Receptor, EphA4/immunology , Single-Domain Antibodies/immunology , Animals , Cell Line , Humans , Mice , Protein Domains , Receptor, EphA4/chemistry , Single-Domain Antibodies/chemistryABSTRACT
Amyotrophic lateral sclerosis (ALS) is a late-onset devastating degenerative disease mainly affecting motor neurons. Motor neuron degeneration is accompanied and aggravated by oligodendroglial pathology and the presence of reactive astrocytes and microglia. We studied the role of the Notch signaling pathway in ALS, as it is implicated in several processes that may contribute to this disease, including axonal retraction, microgliosis, astrocytosis, oligodendrocyte precursor cell proliferation and differentiation, and cell death. We observed abnormal activation of the Notch signaling pathway in the spinal cord of SOD1G93A mice, a well-established model for ALS, as well as in the spinal cord of patients with sporadic ALS (sALS). This increased activation was particularly evident in reactive GFAP-positive astrocytes. In addition, one of the main Notch ligands, Jagged-1, was ectopically expressed in reactive astrocytes in spinal cord from ALS mice and patients, but absent in resting astrocytes. Astrocyte-specific inactivation of Jagged-1 in presymptomatic SOD1G93A mice further exacerbated the activation of the Notch signaling pathway and aggravated the course of the disease in these animals without affecting disease onset. These data suggest that aberrant Notch signaling activation contributes to the pathogenesis of ALS, both in sALS patients and SOD1G93A mice, and that it is mitigated in part by the upregulation of astrocytic Jagged-1.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Astrocytes/metabolism , Jagged-1 Protein/metabolism , Receptor, Notch1/metabolism , Signal Transduction/physiology , Adult , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Astrocytes/pathology , Female , Humans , Jagged-1 Protein/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Receptor, Notch1/genetics , Spinal Cord/metabolism , Spinal Cord/pathology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
As cancer is becoming more and more a chronic disease, a large proportion of patients is confronted with devastating side effects of certain anti-cancer drugs. The most common neurological complications are painful peripheral neuropathies. Chemotherapeutics that interfere with microtubules, including plant-derived vinca-alkaloids such as vincristine, can cause these chemotherapy-induced peripheral neuropathies (CIPN). Available treatments focus on symptom alleviation and pain reduction rather than prevention of the neuropathy. The aim of this study was to investigate the potential of specific histone deacetylase 6 (HDAC6) inhibitors as a preventive therapy for CIPN using multiple rodent models for vincristine-induced peripheral neuropathies (VIPN). HDAC6 inhibition increased the levels of acetylated α-tubulin in tissues of rodents undergoing vincristine-based chemotherapy, which correlates to a reduced severity of the neurological symptoms, both at the electrophysiological and the behavioral level. Mechanistically, disturbances in axonal transport of mitochondria is considered as an important contributing factor in the pathophysiology of VIPN. As vincristine interferes with the polymerization of microtubules, we investigated whether disturbances in axonal transport could contribute to VIPN. We observed that increasing α-tubulin acetylation through HDAC6 inhibition restores vincristine-induced defects of axonal transport in cultured dorsal root ganglion neurons. Finally, we assured that HDAC6-inhibition offers neuroprotection without interfering with the anti-cancer efficacy of vincristine using a mouse model for acute lymphoblastic leukemia. Taken together, our results emphasize the therapeutic potential of HDAC6 inhibitors with beneficial effects both on vincristine-induced neurotoxicity, as well as on tumor proliferation.
Subject(s)
Antineoplastic Agents/adverse effects , Histone Deacetylase 6/antagonists & inhibitors , Neoplasms/drug therapy , Neuroprotective Agents/pharmacology , Peripheral Nervous System Diseases/drug therapy , Vincristine/adverse effects , Animals , Antineoplastic Agents/pharmacology , Axonal Transport/drug effects , Cells, Cultured , Disease Models, Animal , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Histone Deacetylase 6/metabolism , Histone Deacetylase Inhibitors/pharmacology , Male , Mice, Inbred NOD , Mice, SCID , Microtubules/drug effects , Microtubules/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Neoplasms/enzymology , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/enzymology , Tubulin/metabolismABSTRACT
Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease characterized by the selective death of motor neurons. Disease pathophysiology is complex and not yet fully understood. Higher gene expression of the inositol 1,4,5-trisphosphate receptor 2 gene (ITPR2), encoding the IP3 receptor 2 (IP3R2), was detected in sporadic ALS patients. Here, we demonstrate that IP3R2 gene expression was also increased in spinal cords of ALS mice. Moreover, an increase of IP3R2 expression was observed in other models of chronic and acute neurodegeneration. Upregulation of IP3R2 gene expression could be induced by lipopolysaccharide (LPS) in murine astrocytes, murine macrophages and human fibroblasts indicating that it may be a compensatory response to inflammation. Preventing this response by genetic deletion of ITPR2 from SOD1G93A mice had a dose-dependent effect on disease duration, resulting in a significantly shorter lifespan of these mice. In addition, the absence of IP3R2 led to increased innate immunity, which may contribute to the decreased survival of the SOD1G93A mice. Besides systemic inflammation, IP3R2 knockout mice also had increased IFNγ, IL-6 and IL1α expression. Altogether, our data indicate that IP3R2 protects against the negative effects of inflammation, suggesting that the increase in IP3R2 expression in ALS patients is a protective response.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Inflammation/genetics , Inositol 1,4,5-Trisphosphate Receptors/genetics , Superoxide Dismutase-1/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Disease Models, Animal , Humans , Inflammation/pathology , Interferon-gamma/biosynthesis , Interleukin-1beta/biosynthesis , Interleukin-6/biosynthesis , Lipopolysaccharides , Male , Mice , Mice, Knockout , Motor Neurons/metabolism , Motor Neurons/pathology , Spinal Cord/metabolism , Spinal Cord/pathologyABSTRACT
The exact mechanism underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) associated with the GGGGCC repeat expansion in C9orf72 is still unclear. Two gain-of-function mechanisms are possible: repeat RNA toxicity and dipeptide repeat protein (DPR) toxicity. We here dissected both possibilities using a zebrafish model for ALS. Expression of two DPRs, glycine-arginine and proline-arginine, induced a motor axonopathy. Similarly, expanded sense and antisense repeat RNA also induced a motor axonopathy and formed mainly cytoplasmic RNA foci. However, DPRs were not detected in these conditions. Moreover, stop codon-interrupted repeat RNA still induced a motor axonopathy and a synergistic role of low levels of DPRs was excluded. Altogether, these results show that repeat RNA toxicity is independent of DPR formation. This RNA toxicity, but not the DPR toxicity, was attenuated by the RNA-binding protein Pur-alpha and the autophagy-related protein p62. Our findings demonstrate that RNA toxicity, independent of DPR toxicity, can contribute to the pathogenesis of C9orf72-associated ALS/FTD.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , C9orf72 Protein/metabolism , RNA/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Animals, Genetically Modified , Axons/metabolism , Axons/pathology , C9orf72 Protein/genetics , DNA Repeat Expansion , Disease Models, Animal , Escherichia coli , Gene Transfer Techniques , Humans , Motor Neurons/metabolism , Motor Neurons/pathology , ZebrafishABSTRACT
Several recent breakthroughs have provided notable insights into the pathogenesis of amyotrophic lateral sclerosis (ALS), with some even shifting our thinking about this neurodegenerative disease and raising the question as to whether this disorder is a proteinopathy, a ribonucleopathy or both. In addition, these breakthroughs have revealed mechanistic links between ALS and frontotemporal dementia, as well as between ALS and other neurodegenerative diseases, such as the cerebellar atrophies, myotonic dystrophy and inclusion body myositis. Here, we summarize the new findings in ALS research, discuss what they have taught us about this disease and examine issues that are still outstanding.
Subject(s)
Amyotrophic Lateral Sclerosis/etiology , Mutation/genetics , RNA-Binding Proteins/genetics , Amyotrophic Lateral Sclerosis/diagnosis , Amyotrophic Lateral Sclerosis/genetics , Animals , Frontotemporal Dementia/diagnosis , Frontotemporal Dementia/genetics , HumansABSTRACT
We investigated the mutation spectrum of the TANK-Binding Kinase 1 (TBK1) gene and its associated phenotypic spectrum by exonic resequencing of TBK1 in a cohort of 2,538 patients with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), or FTD plus ALS, ascertained within the European Early-Onset Dementia Consortium. We assessed pathogenicity of predicted protein-truncating mutations by measuring loss of RNA expression. Functional effect of in-frame amino acid deletions and missense mutations was further explored in vivo on protein level and in vitro by an NFκB-induced luciferase reporter assay and measuring phosphorylated TBK1. The protein-truncating mutations led to the loss of transcript through nonsense-mediated mRNA decay. For the in-frame amino acid deletions, we demonstrated loss of TBK1 or phosphorylated TBK1 protein. An important fraction of the missense mutations compromised NFκB activation indicating that at least some functions of TBK1 are lost. Although missense mutations were also present in controls, over three times more mutations affecting TBK1 functioning were found in the mutation fraction observed in patients only, suggesting high-risk alleles (P = 0.03). Total mutation frequency for confirmed TBK1 LoF mutations in the European cohort was 0.7%, with frequencies in the clinical subgroups of 0.4% in FTD, 1.3% in ALS, and 3.6% in FTD-ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Frontotemporal Dementia/genetics , Protein Serine-Threonine Kinases/genetics , White People/genetics , Aged , Alleles , Amino Acid Substitution , Amyotrophic Lateral Sclerosis/diagnosis , Amyotrophic Lateral Sclerosis/epidemiology , Case-Control Studies , Cohort Studies , Enzyme Activation , Female , Frontotemporal Dementia/diagnosis , Frontotemporal Dementia/epidemiology , Genetic Association Studies , Heterozygote , Humans , Male , Middle Aged , Mutation , NF-kappa B/metabolism , Phenotype , Protein Serine-Threonine Kinases/metabolism , Sequence DeletionABSTRACT
INTRODUCTION: The C9orf72 repeat expansion has been reported as a negative prognostic factor in amyotrophic lateral sclerosis (ALS). We have examined the prognostic impact of the C9orf72 repeat expansion in European subgroups based on gender and site of onset. METHODS: C9orf72 status and demographic/clinical data from 4925 patients with ALS drawn from 3 prospective ALS registers (Ireland, Italy and the Netherlands), and clinical data sets in the UK and Belgium. Flexible parametric survival models were built including known prognostic factors (age, diagnostic delay and site of onset), gender and the presence of an expanded repeat in C9orf72. These were used to explore the effects of C9orf72 on survival by gender and site of onset. Individual patient data (IPD) meta-analysis was used to estimate HRs for results of particular importance. RESULTS: 457 (8.95%) of 4925 ALS cases carried the C9orf72 repeat expansion. A meta-analysis of C9orf72 estimated a survival HR of 1.36 (1.18 to 1.57) for those carrying the expansion. Models evaluating interaction between gender and C9orf72 repeat expansions demonstrated that the reduced survival due to C9orf72 expansion was being driven by spinal onset males (HR 1.56 (95% CI 1.25 to 1.96). CONCLUSIONS: This study represents the largest combined analysis of the prognostic characteristics of the C9orf72 expansion. We have shown for the first time that the negative prognostic implication of this variant is driven by males with spinal onset disease, indicating a hitherto unrecognised gender-mediated effect of the variant that requires further exploration.
ABSTRACT
Genome-wide association studies have been successful in identifying common variants that influence the susceptibility to complex diseases. From these studies, it has emerged that there is substantial overlap in susceptibility loci between diseases. In line with those findings, we hypothesized that shared genetic pathways may exist between multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). While both diseases may have inflammatory and neurodegenerative features, epidemiological studies have indicated an increased co-occurrence within individuals and families. To this purpose, we combined genome-wide data from 4088 MS patients, 3762 ALS patients and 12 030 healthy control individuals in whom 5 440 446 single-nucleotide polymorphisms (SNPs) were successfully genotyped or imputed. We tested these SNPs for the excess association shared between MS and ALS and also explored whether polygenic models of SNPs below genome-wide significance could explain some of the observed trait variance between diseases. Genome-wide association meta-analysis of SNPs as well as polygenic analyses fails to provide evidence in favor of an overlap in genetic susceptibility between MS and ALS. Hence, our findings do not support a shared genetic background of common risk variants in MS and ALS.