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Two patients with longitudinally extensive myelopathy were initially biopsied for suspected spinal cord tumors. Both patients were later diagnosed with neuromyelitis optica spectrum disorders (NMOSD) supported by their AQP4-seropositivity. Pathological review of both biopsies revealed demyelinated lesions with thickened vessel walls and tissue rarefaction. Immunohistochemical staining demonstrated findings compatible with acute NMOSD lesions in one case while the other case exhibited findings consistent with chronic NMOSD lesions. A pre-biopsy differential diagnosis of longitudinally extensive spinal cord tumors should include NMOSD. Specific biopsy features, such as cystic changes with vascular wall thickening and astrocyte injury, should raise suspicion for NMOSD.

Neuromyelitis optica (NMO) is an autoimmune disease targeting aquaporin 4 (AQP4), localized mainly at the astrocytic foot processes. Loss of AQP4 and glial fibrillary acidic protein (GFAP) was reported, but the pathological significance of astrocytopathy is still controversial. Here we show that active lesions in NMO display a wide spectrum of pathology even within a single tissue block of an individual patient. We have distinguished six different lesion types. The first reflects complement deposition at the surface of astrocytes, associated with granulocyte infiltration and astrocyte necrosis and followed by demyelination, global tissue destruction and the formation of cystic, necrotic lesions (lesion type 2). Such destructive lesions lead to Wallerian degeneration in lesion-related tracts (lesion type 3). Around active NMO lesions AQP4 may selectively be lost in the absence of aquaporin 1 (AQP1) loss or other structural damage (lesion type 4). Another pattern is characterized by clasmatodendrosis of astrocytes, defined by cytoplasmic swelling and vacuolation, beading and dissolution of their processes and nuclear alterations resembling apoptosis, which was associated with internalization of AQP4 and AQP1 and astrocyte apoptosis in the absence of complement activation. Such lesions give rise to extensive astrocyte loss, which may occur in part in the absence of any other tissue injury, such as demyelination or axonal degeneration (lesion type 5). Finally, lesions with a variable degree of astrocyte clasmatodendrosis are found, which show plaque-like primary demyelination that is associated with oligodendrocyte apoptosis, but with preservation of axons (lesion type 6). In active multiple sclerosis (MS) lesions astrocytes reveal changes of reactive protoplasmatic or fibrillary gliosis. Only in a subset of lesions, in patients with aggressive disease, loss of AQP4 is observed in the initial stage of their formation, which is associated with retraction of astrocyte processes in the absence of complement deposition, granulocyte infiltration or loss of AQP1 or astrocytes. Our data underline the primary assault of astrocytes in NMO lesions, but also indicate that different mechanisms of tissue injury operate in parallel in the same patient and even within the same lesion.

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu(2+)/Zn(2+) superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1(H46R) transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor-alpha (TNFalpha) and monocyte chemoattractant protein-1 (MCP-1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage.

Chondroitin sulfate proteoglycans (CSPGs) are the major components of extracellular matrix in the central nervous system. In the spinal cord under various types of injury, reactive gliosis emerges in the lesion accompanied by CSPG up-regulation. Several types of CSPG core proteins and their side chains have been shown to inhibit axonal regeneration in vitro and in vivo. In the present study, we examined spatiotemporal expression of CSPGs in the spinal cord of transgenic (Tg) rats with His46Arg mutation in the Cu/Zn superoxide dismutase gene, a model of amyotrophic lateral sclerosis (ALS). Immunofluorescence disclosed a significant up-regulation of neurocan, versican, and phosphacan in the ventral spinal cord of Tg rats compared with age-matched controls. Notably, Tg rats showed progressive and prominent accumulation of neurocan even at the presymptomatic stage. Immunoblotting confirmed the distinct increase in the levels of both the full-length neurocan and their fragment isoforms. On the other hand, the up-regulation of versican and phosphacan peaked at the early symptomatic stage, followed by diminishment at the late symptomatic stage. In addition, double immunofluorescence revealed a colocalization between reactive astrocytes and immunoreactivities for neurocan and phosphacan, especially around residual large ventral horn neurons. Thus, reactive astrocytes are suggested to be participants in the CSPG accumulation. Although the possible neuroprotective involvement of CSPG remains to be investigated, the present results suggest that both the reactive astrocytes and the differential accumulation of CSPGs may create a nonpermissive microenvironment for neural regeneration in neurodegenerative diseases such as ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1(H46R)); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1(H46R) transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by selective motor neuron death. We developed a rat model of ALS expressing a human cytosolic copper-zinc superoxide dismutase (SOD1) transgene with two ALS-associated mutations: glycine to alanine at position 93 (G93A) and histidine to arginine at position 46 (H46R). Although the mechanism of ALS is still unclear, there are many hypotheses concerning its cause, including loss of neurotrophic support to motor neurons. Recent evidence suggests that insulin-like growth factors (IGFs) act as neurotrophic factors, and promote the survival and differentiation of neuronal cells including motor neurons. Their ability to enhance the outgrowth of spinal motor neurons suggests their potential as a therapeutic agent for the patients with ALS. In this study, we investigated IGF-II receptor immunoreactivity in the anterior horns of the lumbar level of the spinal cord in SOD1 transgenic rats with the H46R mutation of different ages as well as in normal littermates. The double-immunostaining for IGF-II receptor and glial fibrillary acidic protein (GFAP) demonstrated co-localization on reactive astrocytes ((**)p < 0.001) in the end-stage transgenic rats, whereas it was not evident at the pre-symptomatic stage or at the onset of the disease. Our results demonstrated the IGF-II receptor up-regulation in reactive astrocytes in the spinal cord of transgenic rats, which may reflect a protective response against the loss of IGF-related trophic factors. We suggest that IGF receptors may play a key role in the pathogenesis, and may have therapeutic implications in ALS.

Hepatocyte growth factor (HGF) is one of the most potent survival-promoting factors for motor neurons. We showed that introduction of the HGF gene into neurons of G93A transgenic mice attenuates motor neuron degeneration and increases the lifespan of these mice. Currently, treatment regimens using recombinant protein are closer to clinical application than gene therapy. To examine its protective effect on motor neurons and therapeutic potential we administered human recombinant HGF (hrHGF) by continuous intrathecal delivery to G93A transgenic rats at doses of 40 or 200 microg and 200 microg at 100 days of age (the age at which pathologic changes of the spinal cord appear, but animals show no clinical weakness) and at 115 days (onset of paralysis), respectively, for 4 weeks each. Intrathecal administration of hrHGF attenuates motor neuron degeneration and prolonged the duration of the disease by 63%, even with administration from the onset of paralysis. Our results indicated the therapeutic efficacy of continuous intrathecal administration of hrHGF in transgenic rats and should lead to the consideration for further clinical trials in amyotrophic lateral sclerosis using continuous intrathecal administration of hrHGF.

Mutations in copper-zinc superoxide dismutase gene (SOD1) have been linked to some familial cases of ALS. We report here that rats that express a human SOD1 transgene with two different ALS-associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. By comparing the two transgenic rats with different SOD1 mutations, we demonstrate that the time course in these rats was similar to human SOD1-mediated familial ALS. As in the human disease and transgenic ALS mice, pathological analysis shows selective loss of motor neurons in the spinal cords of these transgenic rats. In addition, typical neuronal Lewy body-like hyaline inclusions as well as astrocytic hyaline inclusions identical to those in human familial ALS are observed in the spinal cords. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral- and cell-mediated therapies).

Advanced pathological and genetic approaches have revealed that mutations in fused in sarcoma/translated in liposarcoma (FUS/TLS), which is pivotal for DNA repair, alternative splicing, translation and RNA transport, cause familial amyotrophic lateral sclerosis (ALS). The generation of suitable animal models for ALS is essential for understanding its pathogenesis and developing therapies. Therefore, we used CRISPR-Cas9 to generate FUS -ALS mutation in the non-classical nuclear localization signal (NLS), H517D (mouse position: H509D) and genome-edited mice. Fus WT/H509D mice showed progressive motor impairment (accelerating rotarod and DigiGait system) with age, which was associated with the loss of motor neurons and disruption of the nuclear lamina and nucleoporins and DNA damage in spinal cord motor neurons. We confirmed the validity of our model by showing that nuclear lamina and nucleoporin disruption were observed in lower motor neurons differentiated from patient-derived human induced pluripotent stem cells (hiPSC-LMNs) with FUS -H517D and in the post-mortem spinal cord of patients with ALS. RNA sequence analysis revealed that most nuclear lamina and nucleoporin-linking genes were significantly decreased in FUS -H517D hiPSC-LMNs. This evidence suggests that disruption of the nuclear lamina and nucleoporins is crucial for ALS pathomechanisms. Combined with patient-derived hiPSC-LMNs and autopsy samples, this mouse model might provide a more reliable understanding of ALS pathogenesis and might aid in the development of therapeutic strategies. [ABSTRACT FROM AUTHOR]

Introduction: Progressive brain atrophy, development of T1-hypointense areas, and T2-fluid-attenuated inversion recovery (FLAIR)-hyperintense lesion formation in multiple sclerosis (MS) are popular volumetric data that are often utilized as clinical outcomes. However, the exact clinical interpretation of these volumetric data has not yet been fully established. Methods: We enrolled 42 consecutive patients with MS who fulfilled the revised McDonald criteria of 2010. They were followed-up for more than 3 years from onset, and cross-sectional brain volumetry was performed. Patients with no brain lesions were excluded in advance from this study. For the brain volumetric data, we evaluated several parameters including age-adjusted gray-matter volume atrophy, age-adjusted white-matter volume atrophy, and T2-FLAIR lesion volume. The numbers of T1-hypointense and T2-FLAIR-hyperintense areas were also measured along the same timeline. The clinical data pertaining to disease duration, expanded disability status scale (EDSS), and MS severity score (MSSS) at the timing of volumetry were collected. Results: Among the 42 patients with MS and brain lesions, the number of T1-hypointensity (rho = 0.51, p<0.001), gray-matter atrophy (rho = 0.40, p<0.01) and white-matter atrophy (rho = 0.49, p<0.001) correlated with the EDSS. T1-hypointensity count divided by FLAIR lesion volume correlated with the MSSS (rho = 0.60, p<0.001). Meanwhile, counts or volumes of FLAIR-hyperintense lesions were associated only with the times of past relapses, and did not correlate with present neurological disability level or ongoing disease activity. These findings were consistent regardless of the presence of spinal cord lesions. Conclusion: Numbers of T1-hypointensities and brain atrophy equally indicated the current neurological disability in MS. The number of T1-hypointensities divided by FLAIR lesion volume represented the clinical severity. The size or number of FLAIR lesions reflected earlier relapses but was not a good indicator of neurological disability or clinical severity. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS). However, these genetic defects occur in only about 20–30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG), in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-κB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-κB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder. [ABSTRACT FROM AUTHOR]

Background: ALS2/alsin is a guanine nucleotide exchange factor for the small GTPase Rab5 and involved in macropinocytosis-associated endosome fusion and trafficking, and neurite outgrowth. ALS2 deficiency accounts for a number of juvenile recessive motor neuron diseases (MNDs). Recently, it has been shown that ALS2 plays a role in neuroprotection against MND-associated pathological insults, such as toxicity induced by mutant Cu/Zn superoxide dismutase (SOD1). However, molecular mechanisms underlying the relationship between ALS2-associated cellular function and its neuroprotective role remain unclear. Methodology/Principal Findings: To address this issue, we investigated the molecular and pathological basis for the phenotypic modification of mutant SOD1-expressing mice by ALS2 loss. Genetic ablation of Als2 in SOD1H46R, but not SOD1G93A, transgenic mice aggravated the mutant SOD1-associated disease symptoms such as body weight loss and motor dysfunction, leading to the earlier death. Light and electron microscopic examinations revealed the presence of degenerating and/or swollen spinal axons accumulating granular aggregates and autophagosome-like vesicles in early- and even pre-symptomatic SOD1H46R mice. Further, enhanced accumulation of insoluble high molecular weight SOD1, polyubiquitinated proteins, and macroautophagy-associated proteins such as polyubiquitin-binding protein p62/SQSTM1 and a lipidated form of light chain 3 (LC3-II), emerged in ALS2-deficient SOD1H46R mice. Intriguingly, ALS2 was colocalized with LC3 and p62, and partly with SOD1 on autophagosome/endosome hybrid compartments, and loss of ALS2 significantly lowered the lysosome-dependent clearance of LC3 and p62 in cultured cells. Conclusions/Significance: Based on these observations, although molecular basis for the distinctive susceptibilities to ALS2 loss in different mutant SOD1-expressing ALS models is still elusive, disturbance of the endolysosomal system by ALS2 loss may exacerbate the SOD1H46R-mediated neurotoxicity by accelerating the accumulation of immature vesicles and misfolded proteins in the spinal cord. We propose that ALS2 is implicated in endolysosomal trafficking through the fusion between endosomes and autophagosomes, thereby regulating endolysosomal protein degradation in vivo. [ABSTRACT FROM AUTHOR]

The progressive patterns of neurological disability in multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) and the significance of clinical relapses to the progressions of neurological disability in these diseases have not been fully elucidated. In this study, to elucidate the impact of relapses to the progression of accumulated neurological disability and to identify the factors to affect the progression of neurological disability in MS and NMOSD, we followed 62 consecutive MS patients and 33 consecutive NMOSD patients for more than 5 years with the clinical symptoms, relapse occurrence, and Expanded Disability Status Scale (EDSS) in the chronic phase. All enrolled MS patients were confirmed to be negative for serum anti-myelin oligodendrocyte glycoprotein antibody. As a result, patients with NMOSD showed significantly severer neurological disability at 5 years from onset than MS patients. Progression in EDSS score was almost exclusively seen after clinical attacks in NMOSD, whereas progression could be observed apart from relapses in MS. Neurological disability did not change without attacks in NMOSD, whereas it sometimes spontaneously improved or deteriorated apart from relapses in MS (p < 0.001). In patients with MS, those with responsible lesions primarily in spinal cord were more likely to show such spontaneous improvement. In conclusion, clinical deterioration in NMOSD patients is irreversible and almost exclusively takes place at the timing of clinical attacks with stepwise accumulation of neurological disability. Meanwhile, changes in EDSS score can be seen apart from relapses in MS patients. Neurological disability in MS patients is partly reversible, and the patients with disease modifying drugs sometimes present spontaneous improvement of the neurological disability. [ABSTRACT FROM AUTHOR]

Abstract Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative syndrome characterized by the systemic loss of motor neurons with prominent astrocytosis and microgliosis in the spinal cord and brain. Astrocytes play an essential role in maintaining extracellular microenvironments that surround motor neurons, and are activated by various insults. Growing evidence points to a non-cell autonomous neurotoxicity caused by chronic and sustained astrocytic activation in patients with neurodegenerative diseases, including ALS. However, the mechanisms that underlie the harmful effects of astrocytosis in patients with ALS remain unresolved. We focused on bone morphogenetic proteins as a major soluble factor that promotes astrocytogenesis and its activation in the adult spinal cord. In a transgenic rat model with ALS-linked mutant Cu/Zn superoxide dismutase gene, BMP4 was progressively up-regulated in reactive astrocytes of the spinal ventral horns, whereas the BMP-antagonist noggin was decreased in association with neuronal degeneration. Continuous intrathecal noggin supplementation after disease onset significantly ameliorated motor dysfunction symptoms, neurogenic muscle atrophy, and extended survival of symptomatic ALS model rats, despite lack of deterrence against neuronal death itself. The exogenous noggin inhibited astrocytic hypertrophy, astrocytogenesis, and neuroinflammation by inactivating both Smad1/5/8 and p38 mitogen-activated protein kinase pathways. Moreover, intrathecal infusion of a Bmp4 -targeted antisense oligonucleotides and provided selective Bmp4 knockdown in vivo, which suppressed astrocyte and microglia activation, reproducing the aforementioned results by noggin treatment. Collectively, we clarified the involvement of BMP4 in the processes of excessive gliosis that exacerbate the disease progression of the ALS model rats. Our study demonstrated that BMP4, with its downstream signaling, might be a novel therapeutic target for disease-modifying therapies in ALS. Highlights • BMP4 increased in astrocytes of a rat model of amyotrophic lateral sclerosis (ALS). • Inhibiting the BMP4 attenuated the disease progression via suppressing gliosis. • The gliosis-promoting BMP4 may become a therapeutic target in ALS. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motor neurons in the brain and spinal cord. Recent studies have shown that mutations in SQSTM1 are linked to ALS. It has also been demonstrated that a systemic loss of SQSTM1 exacerbates disease phenotypes in an ALS mouse model. However, it is still unclear whether and how SQSTM1 in the central nervous system (CNS) specifically regulates ALS-associated disease phenotypes. To address this issue, we generated CNS-specific Sqstm1 deficient SOD1H46R transgenic mice, and conducted gross phenotype analyses as well as the immunohistochemical and biochemical examinations of spinal cord tissues using these mice. CNS-specific SQSTM1 deficiency accelerated the disease onset and shortened the lifespan of SOD1H46R mice. The CNS-specific SQSTM1 ablation also resulted in increased number of ubiquitin-positive aggregates, while their size rather became much smaller. Remarkably, ubiquitin-positive aggregates, which were usually present in extracellular space and/or neuropil in SOD1H46R mice, were preferentially localized to soma and neurites of spinal neurons in CNS-specific SQSTM1 deficient SOD1H46R mice. Next, to further clarify the function of SQSTM1 in neurons, we investigated the contribution of SQSTM1 to the accumulation of polyubiquitinated proteins in relation to the ubiquitin proteasome system (UPS) and the autophagy-endolysosomal system (APELS) in primary cultured motor neurons (PMNs). Loss of SQSTM1 in PMNs resulted in decreased accumulation of insoluble polyubiquitinated proteins, which was induced by simultaneous treatment with proteasome and lysosome inhibitors, suggesting a pivotal role of SQSTM1 in the formation of insoluble protein aggregates. However, SQSTM1 silencing had a limited impact on the susceptibility to proteasome and/or lysosome inhibitor-induced apoptosis in PMNs. Taken together, neuronal SQSTM1, whose functions are associated with both the UPS and APELS, might primarily regulate the distribution and accumulation of misfolded protein aggregates in the CNS, thereby protecting neurons from degeneration in mice. • CNS-specific loss of SQSTM1 exacerbates the onset of ALS mice. • SQSTM1 regulates the distribution of ubiquitin-positive aggregates in spinal cord. • SQSTM1 facilitates the formation of insoluble polyubiquitinated proteins in cells. • SQSTM1 has a limited impact on apoptosis in proteolytically-stressed neurons. • SQSTM1 is implicated in the formation of disease protective protein aggregates. [ABSTRACT FROM AUTHOR]

Abstract: Deficient RNA editing of the AMPA receptor subunit GluR2 at the Q/R site is a primary cause of neuronal death and recently has been reported to be a tightly linked etiological cause of motor neuron death in sporadic amyotrophic lateral sclerosis (ALS). We quantified the RNA editing efficiency of the GluR2 Q/R site in single motor neurons of rats transgenic for mutant human Cu/Zn-superoxide dismutase (SOD1) as well as patients with spinal and bulbar muscular atrophy (SBMA), and found that GluR2 mRNA was completely edited in all the motor neurons examined. It seems likely that the death cascade is different among the dying motor neurons in sporadic ALS, familial ALS with mutant SOD1 and SBMA. [Copyright &y& Elsevier]