TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A.

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.

Dysregulated copper transport in multiple sclerosis may cause demyelination via astrocytes.

Demyelination is a key pathogenic feature of multiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS.

Transcriptomic analysis of frontotemporal lobar degeneration with TDP-43 pathology reveals cellular alterations across multiple brain regions.

Frontotemporal lobar degeneration (FTLD) is a group of heterogeneous neurodegenerative disorders affecting the frontal and temporal lobes of the brain. Nuclear loss and cytoplasmic aggregation of the RNA-binding protein TDP-43 represents the major FTLD pathology, known as FTLD-TDP. To date, there is no effective treatment for FTLD-TDP due to an incomplete understanding of the molecular mechanisms underlying disease development. Here we compared postmortem tissue RNA-seq transcriptomes from the frontal cortex, temporal cortex, and cerebellum between 28 controls and 30 FTLD-TDP patients to profile changes in cell-type composition, gene expression and transcript usage. We observed downregulation of neuronal markers in all three regions of the brain, accompanied by upregulation of microglia, astrocytes, and oligodendrocytes, as well as endothelial cells and pericytes, suggesting shifts in both immune activation and within the vasculature. We validate our estimates of neuronal loss using neuropathological atrophy scores and show that neuronal loss in the cortex can be mainly attributed to excitatory neurons, and that increases in microglial and endothelial cell expression are highly correlated with neuronal loss. All our analyses identified a strong involvement of the cerebellum in the neurodegenerative process of FTLD-TDP. Altogether, our data provides a detailed landscape of gene expression alterations to help unravel relevant disease mechanisms in FTLD.

The Fabp5/calnexin complex is a prerequisite for sensitization of mice to experimental autoimmune encephalomyelitis.

We previously showed that calnexin (Canx)-deficient mice are desensitized to experimental autoimmune encephalomyelitis (EAE) induction, a model that is frequently used to study inflammatory demyelinating diseases, due to increased resistance of the blood-brain barrier to immune cell transmigration. We also discovered that Fabp5, an abundant cytoplasmic lipid-binding protein found in brain endothelial cells, makes protein-protein contact with the cytoplasmic C-tail domain of Canx. Remarkably, both Canx-deficient and Fabp5-deficient mice commonly manifest resistance to EAE induction. Here, we evaluated the importance of Fabp5/Canx interactions on EAE pathogenesis and on the patency of a model blood-brain barrier to T-cell transcellular migration. The results demonstrate that formation of a complex comprised of Fabp5 and the C-tail domain of Canx dictates the permeability of the model blood-brain barrier to immune cells and is also a prerequisite for EAE pathogenesis.

Distinct responses of neurons and astrocytes to TDP-43 proteinopathy in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by motor neuron loss, resulting in muscle wasting, paralysis and eventual death. A key pathological feature of ALS is cytoplasmically mislocalized and aggregated TDP-43 protein in >95% of cases, which is considered to have prion-like properties. Historical studies have predominantly focused on genetic forms of ALS, which represent ∼10% of cases, leaving the remaining 90% of sporadic ALS relatively understudied. Additionally, the role of astrocytes in ALS and their relationship with TDP-43 pathology is also not currently well understood. We have therefore used highly enriched human induced pluripotent stem cell (iPSC)-derived motor neurons and astrocytes to model early cell type-specific features of sporadic ALS. We first demonstrate seeded aggregation of TDP-43 by exposing human iPSC-derived motor neurons to serially passaged sporadic ALS post-mortem tissue (spALS) extracts. Next, we show that human iPSC-derived motor neurons are more vulnerable to TDP-43 aggregation and toxicity compared with their astrocyte counterparts. We demonstrate that these TDP-43 aggregates can more readily propagate from motor neurons into astrocytes in co-culture paradigms. We next found that astrocytes are neuroprotective to seeded aggregation within motor neurons by reducing (mislocalized) cytoplasmic TDP-43, TDP-43 aggregation and cell toxicity. Furthermore, we detected TDP-43 oligomers in these spALS spinal cord extracts, and as such demonstrated that highly purified recombinant TDP-43 oligomers can reproduce this observed cell-type specific toxicity, providing further support to a protein oligomer-mediated toxicity hypothesis in ALS. In summary, we have developed a human, clinically relevant, and cell-type specific modelling platform that recapitulates key aspects of sporadic ALS and uncovers both an initial neuroprotective role for astrocytes and the cell type-specific toxic effect of TDP-43 oligomers.

Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis.

Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

Production of IL-27 in multiple sclerosis lesions by astrocytes and myeloid cells: Modulation of local immune responses.

The mechanisms whereby human glial cells modulate local immune responses are not fully understood. Interleukin-27 (IL-27), a pleiotropic cytokine, has been shown to dampen the severity of experimental autoimmune encephalomyelitis, but it is still unresolved whether IL-27 plays a role in the human disease multiple sclerosis (MS). IL-27 contribution to local modulation of immune responses in the brain of MS patients was investigated. The expression of IL-27 subunits (EBI3 and p28) and its cognate receptor IL-27R (the gp130 and TCCR chains) was elevated within post-mortem MS brain lesions compared with normal control brains. Moreover, astrocytes (GFAP(+) cells) as well as microglia and macrophages (Iba1(+) cells) were important sources of IL-27. Brain-infiltrating CD4 and CD8 T lymphocytes expressed the IL-27R specific chain (TCCR) implying that these cells could respond to local IL-27 sources. In primary cultures of human astrocytes inflammatory cytokines increased IL-27 production, whereas myeloid cell inflammatory M1 polarization and inflammatory cytokines enhanced IL-27 expression in microglia and macrophages. Astrocytes in postmortem tissues and in vitro expressed IL-27R. Moreover, IL-27 triggered the phosphorylation of the transcription regulator STAT1, but not STAT3 in human astrocytes; indeed IL-27 up-regulated MHC class I expression on astrocytes in a STAT1-dependent manner. These findings demonstrated that IL-27 and its receptor were elevated in MS lesions and that local IL-27 can modulate immune properties of astrocytes and infiltrating immune cells. Thus, therapeutic strategies targeting IL-27 may influence not only peripheral but also local inflammatory responses within the brain of MS patients.

In vitro prion-like behaviour of TDP-43 in ALS.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and >95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded 'prion-like' protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.

Fingolimod may support neuroprotection via blockade of astrocyte nitric oxide.

OBJECTIVE: Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFκB pathway and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration. Here we investigated the role of the crosstalk between sphingosine-1-phosphate (S1P) and cytokine signaling cascades in astrocyte activation and inflammation-mediated neurodegeneration, and addressed the effects of fingolimod on astrocyte-neuron interaction and NO synthesis in vivo. METHODS: Immunohistochemistry, immunofluorescence, and confocal microscopy were used to detect S1P receptors, interleukin (IL) 1R, IL17RA, and nitrosative stress in multiple sclerosis (MS) plaques, experimental autoimmune encephalomyelitis (EAE) spinal cord, and the spinal cord of fingolimod-treated EAE mice. An in vitro model was established to study the effects of S1P, IL1, and IL17 stimulation on NFkB translocation and NO production in astrocytes, on spinal neuron survival, and on astrocyte-neuron interaction. Furthermore, fingolimod efficacy in blocking astrocyte-mediated neurodegeneration was evaluated. RESULTS: We found coordinated upregulation of IL1R, IL17RA, S1P1, and S1P3 together with nitrosative markers in astrocytes within MS and EAE lesions. In vitro studies revealed that S1P, IL17, and IL1 induced NFκB translocation and NO production in astrocytes, and astrocyte conditioned media triggered neuronal death. Importantly, fingolimod blocked the 2 activation events evoked in astrocytes by either S1P or inflammatory cytokines, resulting in inhibition of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of fingolimod to EAE mice hampered astrocyte activation and NO production. INTERPRETATION: A neuroprotective effect of fingolimod in vivo may result from its inhibitory action on key astrocyte activation steps.

Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

The mechanisms whereby immune cells infiltrating the CNS in multiple sclerosis patients contribute to tissue injury remain to be defined. CD4 T cells are key players of this inflammatory response. Myelin-specific CD4 T cells expressing CD56, a surrogate marker of NK cells, were shown to be cytotoxic to human oligodendrocytes. Our aim was to identify NK-associated molecules expressed by human CD4 T cells that confer this oligodendrocyte-directed cytotoxicity. We observed that myelin-reactive CD4 T cell lines, as well as short-term PHA-activated CD4 T cells, can express NKG2C, the activating receptor interacting with HLA-E, a nonclassical MHC class I molecule. These cells coexpress CD56 and NKG2D, have elevated levels of cytotoxic molecules FasL, granzyme B, and perforin compared with their NKG2C-negative counterparts, and mediate significant in vitro cytotoxicity toward human oligodendrocytes, which upregulated HLA-E upon inflammatory cytokine treatment. A significantly elevated proportion of ex vivo peripheral blood CD4 T cells, but not CD8 T cells or NK cells, from multiple sclerosis patients express NKG2C compared with controls. In addition, immunohistochemical analyses showed that multiple sclerosis brain tissues display HLA-E(+) oligodendrocytes and NKG2C(+) CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4 T cells contribute to tissue injury in multiple sclerosis.

Full-length and fragmented netrin-1 in multiple sclerosis plaques are inhibitors of oligodendrocyte precursor cell migration.

Oligodendrocytes exhibit a limited capacity to remyelinate in multiple sclerosis. Factors present in multiple sclerosis lesions are thought to inhibit oligodendrocyte precursor cell migration, limiting their recruitment to axons requiring remyelination; however, few inhibitors have been identified. A candidate inhibitor is netrin-1, a secreted protein that repels migrating oligodendrocyte precursor cells during neural development and is expressed by myelinating oligodendrocytes in the mature rodent central nervous system. Herein, we examined the distribution of netrin-1 in adult human white matter and multiple sclerosis lesions. We detected full-length netrin-1 protein and shorter netrin-1 fragments in samples of normal white matter and of multiple sclerosis lesions from adult human brain. We demonstrate that peptides corresponding to amino terminal domains VI and V of netrin-1 repel migrating oligodendrocyte precursor cells, but lack the chemoattractant activity of full-length netrin-1. Furthermore, recombinant domains VI-V of netrin-1 disrupt the chemoattractant activity of full-length netrin-1, consistent with a competitive mechanism of action. These findings indicate that full-length and fragmented forms of netrin-1, found in multiple sclerosis lesions, have the capacity to inhibit oligodendrocyte precursor migration, identifying netrin-1 as a potential target for therapies that promote remyelination.

Nav1.5 sodium channels in macrophages in multiple sclerosis lesions.

BACKGROUND: Macrophages are dynamic participants in destruction of white matter in active multiple sclerosis (MS) plaques. Regulation of phagocytosis and myelin degradation along endosomal pathways in macrophages is highly-orchestrated and critically-dependent upon acidification of endosomal lumena. Evidence from in vitro studies with macrophages and THP-1 cells suggests that sodium channel Nav1.5 is present in the limiting membrane of maturing endosomes where it plays a prominent role in the accumulation of protons. However, a contribution of the Nav1.5 channel to macrophage-mediated events in vivo has not been demonstrated. METHOD: We examined macrophages within active MS lesions by immunohistochemistry to determine whether Nav1.5 is expressed in these cells in situ and, if expressed, whether it is localized to specific compartments along the endocytic pathway. RESULTS: Our results demonstrate that Nav1.5 is expressed within macrophages in active MS lesions, and that it is preferentially expressed in late endosomes and phagolysosomes (Rab7(+), LAMP-1(+)), and sparsely expressed in early (EEA-1(+)) endosomes. Triple-immunolabeling studies showed localization of Nav1.5 within Rab7(+) endosomes containing proteolipid protein, a myelin marker, in macrophages within active MS plaques. CONCLUSIONS: These observations support the suggestion that Nav1.5 contributes to the phagocytic pathway of myelin degradation in macrophages in vivo within MS lesions.

STING-Triggered CNS Inflammation in Human Neurodegenerative Diseases.

BACKGROUND: Some neurodegenerative diseases have an element of neuroinflammation that is triggered by viral nucleic acids, resulting in the generation of type I interferons. In the cGAS-STING pathway, microbial and host-derived DNA bind and activate the DNA sensor cGAS, and the resulting cyclic dinucleotide, 2'3-cGAMP, binds to a critical adaptor protein, stimulator of interferon genes (STING), which leads to activation of downstream pathway components. However, there is limited work demonstrating the activation of the cGAS-STING pathway in human neurodegenerative diseases. METHODS: Post-mortem CNS tissue from donors with multiple sclerosis (n = 4), Alzheimer's disease (n = 6), Parkinson's disease (n = 3), amyotrophic lateral sclerosis (n = 3) and non-neurodegenerative controls (n = 11) were screened by immunohistochemistry for STING and relevant protein aggregates (e.g., amyloid-ß, α-synuclein, TDP-43). Human brain endothelial cells were cultured and stimulated with the STING agonist palmitic acid (1-400 µM) and assessed for mitochondrial stress (release of mitochondrial DNA into cytosol, increased oxygen consumption), downstream regulator factors, TBK-1/pIRF3 and inflammatory biomarker interferon-ß release and changes in ICAM-1 integrin expression. RESULTS: In neurodegenerative brain diseases, elevated STING protein was observed mainly in brain endothelial cells and neurons, compared to non-neurodegenerative control tissues where STING protein staining was weaker. Interestingly, a higher STING presence was associated with toxic protein aggregates (e.g., in neurons). Similarly high STING protein levels were observed within acute demyelinating lesions in multiple sclerosis subjects. To understand non-microbial/metabolic stress activation of the cGAS-STING pathway, brain endothelial cells were treated with palmitic acid. This evoked mitochondrial respiratory stress up to a ~2.5-fold increase in cellular oxygen consumption. Palmitic acid induced a statistically significant increase in cytosolic DNA leakage from endothelial cell mitochondria (Mander's coefficient; p < 0.05) and a significant increase in TBK-1, phosphorylated transcription factor IFN regulatory factor 3, cGAS and cell surface ICAM. In addition, a dose response in the secretion of interferon-ß was observed, but it failed to reach statistical significance. CONCLUSIONS: The histological evidence shows that the common cGAS-STING pathway appears to be activated in endothelial and neural cells in all four neurodegenerative diseases examined. Together with the in vitro data, this suggests that the STING pathway might be activated via perturbation of mitochondrial stress and DNA leakage, resulting in downstream neuroinflammation; hence, this pathway may be a target for future STING therapeutics.

IL-21 and IL-21 receptor expression in lymphocytes and neurons in multiple sclerosis brain.

IL-17-producing CD4(+) T cells (Th-17) contribute to the pathogenesis of experimental autoimmune encephalomyelitis and are associated with active disease in multiple sclerosis (MS). In addition to IL-17, Th-17 cells can also express IL-21, IL-22, and IL-6 under Th-17-polarizing conditions (IL-6 and transforming growth factor-ß). In this study we investigated IL-21 and IL-21 receptor (IL-21R) expression in MS lesions by in situ hybridization and immunohistochemistry. We detected strongly IL-21(+) infiltrating cells predominantly in acute but also in chronic active white matter MS lesions in which IL-21 expression was restricted to CD4(+) cells. In contrast, IL-21R was much more broadly distributed on CD4(+), CD19(+), and CD8(+) lymphocytes but not major histocompatibility complex class-II(+) macrophages/microglia. Interestingly, in cortical areas we detected both IL-21 and IL-21R expression by neurons. These findings suggest role(s) for IL-21 in both the acute and chronic stages of MS via direct effects on T and B lymphocytes and, demonstrated for the first time, also on neurons.

Clonally expanded mitochondrial DNA deletions within the choroid plexus in multiple sclerosis.

Mitochondrial DNA deletions (∆-mtDNA) have been implicated in the pathogenesis of Alzheimer's disease (AD), multiple sclerosis (MS) and Parkinson's disease (PD), as well as ageing. Clonal expansion of ∆-mtDNA is the process by which a mutant mtDNA molecule increases to high levels within a single cell containing both wild-type and mutant mtDNA. Unlike in AD and PD, the diffuse inflammatory process in MS involves the choroid plexus, and mitochondria are exposed to reactive oxygen and nitrogen species over a prolonged period. We determined the extent of respiratory enzyme deficiency and ∆-mtDNA at a single cell level within choroid plexus epithelial cells in MS as well as in AD, PD and controls. The respiratory enzyme-deficient (lacking complex IV and with intact complex II activity) cells were more prevalent within the choroid plexus in AD, MS and PD compared with controls. The main catalytic subunit of complex IV (subunit-I of cytochrome c oxidase) was lacking in significantly more respiratory enzyme-deficient cells in MS compared with AD, PD and controls. The single cell analysis showed a fourfold increase in the percentage of respiratory enzyme-deficient choroid plexus epithelial cells harbouring clonally expanded ∆-mtDNA in MS. Our findings establish clonal expansion of ∆-mtDNA as a feature relatively more prominent within the choroid plexus epithelium in MS than AD, PD or controls. We propose clonal expansion of ∆-mtDNA as a molecular link between inflammation and part of a delayed cellular energy failure in MS.

Contribution of astrocyte-derived IL-15 to CD8 T cell effector functions in multiple sclerosis.

The contribution of local factors to the activation of immune cells infiltrating the CNS of patients with multiple sclerosis (MS) remains to be defined. The cytokine IL-15 is pivotal in the maintenance and activation of CD8 T lymphocytes, a prominent lymphocyte population found in MS lesions. We investigated whether astrocytes are a functional source of IL-15 sufficient to enhance CD8 T lymphocyte responses and whether they provide IL-15 in the inflamed CNS of patients with MS. We observed that human astrocytes in primary cultures increased surface IL-15 levels upon activation with combinations of proinflammatory cytokines. Expanded human myelin autoreactive CD8 T lymphocytes cultured with such activated astrocytes displayed elevated lytic enzyme content, NKG2D expression, and Ag-specific cytotoxicity. These functional enhancements were abrogated by anti-IL-15-blocking Abs. Immunohistochemical analysis of brain tissue sections obtained from patients with MS demonstrated colocalization for IL-15 and the astrocyte marker glial fibrillary acidic protein within white matter lesions. The majority of astrocytes (80-90%) present in demyelinating MS lesions expressed IL-15, whereas few astrocytes in normal control brain sections had detectable IL-15. IL-15 could be detected in the majority of Iba-1-expressing microglia in the control sections, albeit in lower numbers when compared with microglia/macrophages in MS lesions. Furthermore, infiltrating CD8 T lymphocytes in MS lesions were in close proximity to IL-15-expressing cells. Astrocyte production of IL-15 resulting in the activation of CD8 T lymphocytes ascribes a role for these cells as contributors to the exacerbation of tissue damage during MS pathogenesis.

Acid-sensing ion channel 1 is involved in both axonal injury and demyelination in multiple sclerosis and its animal model.

Although there is growing evidence for a role of excess intracellular cations, particularly calcium ions, in neuronal and glial cell injury in multiple sclerosis, as well as in non-inflammatory neurological conditions, the molecular mechanisms involved are not fully determined. We previously showed that the acid-sensing ion channel 1 which, when activated under the acidotic tissue conditions found in inflammatory lesions opens to allow influx of sodium and calcium ions, contributes to axonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, the extent and cellular distribution of acid-sensing ion channel 1 expression in neurons and glia in inflammatory lesions is unknown and, crucially, acid-sensing ion channel 1 expression has not been determined in multiple sclerosis lesions. Here we studied acute and chronic experimental autoimmune encephalomyelitis and multiple sclerosis spinal cord and optic nerve tissues to describe in detail the distribution of acid-sensing ion channel 1 and its relationship with neuronal and glial damage. We also tested the effects of amiloride treatment on tissue damage in the mouse models. We found that acid-sensing ion channel 1 was upregulated in axons and oligodendrocytes within lesions from mice with acute experimental autoimmune encephalomyelitis and from patients with active multiple sclerosis. The expression of acid-sensing ion channel 1 was associated with axonal damage as indicated by co-localization with the axonal injury marker beta amyloid precursor protein. Moreover, blocking acid-sensing ion channel 1 with amiloride protected both myelin and neurons from damage in the acute model, and when given either at disease onset or, more clinically relevant, at first relapse, ameliorated disability in mice with chronic-relapsing experimental autoimmune encephalomyelitis. Together these findings suggest that blockade of acid-sensing ion channel 1 has the potential to provide both neuro- and myelo-protective benefits in multiple sclerosis.

A comparative clinical, pathological, biochemical and genetic study of fused in sarcoma proteinopathies.

Neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration are rare diseases characterized by ubiquitin-positive inclusions lacking transactive response DNA-binding protein-43 and tau. Recently, mutations in the fused in sarcoma gene have been shown to cause familial amyotrophic lateral sclerosis and fused in sarcoma-positive neuronal inclusions have subsequently been demonstrated in neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration with ubiquitinated inclusions. Here we provide clinical, imaging, morphological findings, as well as genetic and biochemical data in 14 fused in sarcoma proteinopathy cases. In this cohort, the age of onset was variable but included cases of young-onset disease. Patients with atypical frontotemporal lobar degeneration with ubiquitinated inclusions all presented with behavioural variant frontotemporal dementia, while the clinical presentation in neuronal intermediate filament inclusion disease was more heterogeneous, including cases with motor neuron disease and extrapyramidal syndromes. Neuroimaging revealed atrophy of the frontal and anterior temporal lobes as well as the caudate in the cases with atypical frontotemporal lobar degeneration with ubiquitinated inclusions, but was more heterogeneous in the cases with neuronal intermediate filament inclusion disease, often being normal to visual inspection early on in the disease. The distribution and severity of fused in sarcoma-positive neuronal cytoplasmic inclusions, neuronal intranuclear inclusions and neurites were recorded and fused in sarcoma was biochemically analysed in both subgroups. Fused in sarcoma-positive neuronal cytoplasmic and intranuclear inclusions were found in the hippocampal granule cell layer in variable numbers. Cortical fused in sarcoma-positive neuronal cytoplasmic inclusions were often 'Pick body-like' in neuronal intermediate filament inclusion disease, and annular and crescent-shaped inclusions were seen in both conditions. Motor neurons contained variable numbers of compact, granular or skein-like cytoplasmic inclusions in all fused in sarcoma-positive cases in which brainstem and spinal cord motor neurons were available for study (five and four cases, respectively). No fused in sarcoma mutations were found in any cases. Biochemically, two major fused in sarcoma species were found and shown to be more insoluble in the atypical frontotemporal lobar degeneration with ubiquitinated inclusions subgroup compared with neuronal intermediate filament inclusion disease. There is considerable overlap and also significant differences in fused in sarcoma-positive pathology between the two subgroups, suggesting they may represent a spectrum of the same disease. The co-existence of fused in sarcoma-positive inclusions in both motor neurons and extramotor cerebral structures is a characteristic finding in sporadic fused in sarcoma proteinopathies, indicating a multisystem disorder.