Oligodendrocyte pathology exceeds axonal pathology in white matter in human amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. The majority of cases are sporadic (sALS), while the most common inherited form is due to C9orf72 mutation (C9ALS). A high burden of inclusion pathology is seen in glia (including oligodendrocytes) in ALS, especially in C9ALS. Myelin basic protein (MBP) messenger RNA (mRNA) must be transported to oligodendrocyte processes for myelination, a possible vulnerability for normal function. TDP43 is found in pathological inclusions in ALS and is a component of mRNA transport granules. Thus, TDP43 aggregation could lead to MBP loss. Additionally, the hexanucleotide expansion of mutant C9ALS binds hnRNPA2/B1, a protein essential for mRNA transport, causing potential further impairment of hnRNPA2/B1 function, and thus myelination. Using immunohistochemistry for p62 and TDP43 in human post-mortem tissue, we found a high burden of glial inclusions in the prefrontal cortex, precentral gyrus, and spinal cord in ALS, which was greater in C9ALS than in sALS cases. Double staining demonstrated that the majority of these inclusions were in oligodendrocytes. Using immunoblotting, we demonstrated reduced MBP protein levels relative to PLP (a myelin component that relies on protein not mRNA transport) and neurofilament protein (an axonal marker) in the spinal cord. This MBP loss was disproportionate to the level of PLP and axonal loss, suggesting that impaired mRNA transport may be partly responsible. Finally, we show that in C9ALS cases, the level of oligodendroglial inclusions correlates inversely with levels of hnRNPA2/B1 and the number of oligodendrocyte precursor cells. We conclude that there is considerable oligodendrocyte pathology in ALS, which at least partially reflects impairment of mRNA transport. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Disrupted-in-Schizophrenia-1 is essential for normal hypothalamic-pituitary-interrenal (HPI) axis function.

Psychiatric disorders arise due to an interplay of genetic and environmental factors, including stress. Studies in rodents have shown that mutants for Disrupted-In-Schizophrenia-1 (DISC1), a well-accepted genetic risk factor for mental illness, display abnormal behaviours in response to stress, but the mechanisms through which DISC1 affects stress responses remain poorly understood. Using two lines of zebrafish homozygous mutant for disc1, we investigated behaviour and functioning of the hypothalamic-pituitary-interrenal (HPI) axis, the fish equivalent of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we show that the role of DISC1 in stress responses is evolutionarily conserved and that DISC1 is essential for normal functioning of the HPI axis. Adult zebrafish homozygous mutant for disc1 show aberrant behavioural responses to stress. Our studies reveal that in the embryo, disc1 is expressed in neural progenitor cells of the hypothalamus, a conserved region of the vertebrate brain that centrally controls responses to environmental stressors. In disc1 mutant embryos, proliferating rx3+ hypothalamic progenitors are not maintained normally and neuronal differentiation is compromised: rx3-derived ff1b+ neurons, implicated in anxiety-related behaviours, and corticotrophin releasing hormone (crh) neurons, key regulators of the stress axis, develop abnormally, and rx3-derived pomc+ neurons are disorganised. Abnormal hypothalamic development is associated with dysfunctional behavioural and neuroendocrine stress responses. In contrast to wild type siblings, disc1 mutant larvae show altered crh levels, fail to upregulate cortisol levels when under stress and do not modulate shoal cohesion, indicative of abnormal social behaviour. These data indicate that disc1 is essential for normal development of the hypothalamus and for the correct functioning of the HPA/HPI axis.

Sonic hedgehog functions upstream of disrupted-in-schizophrenia 1 (disc1): implications for mental illness.

DISRUPTED-IN-SCHIZOPHRENIA (DISC1) has been one of the most intensively studied genetic risk factors for mental illness since it was discovered through positional mapping of a translocation breakpoint in a large Scottish family where a balanced chromosomal translocation was found to segregate with schizophrenia and affective disorders. While the evidence for it being central to disease pathogenesis in the original Scottish family is compelling, recent genome-wide association studies have not found evidence for common variants at the DISC1 locus being associated with schizophrenia in the wider population. It may therefore be the case that DISC1 provides an indication of biological pathways that are central to mental health issues and functional studies have shown that it functions in multiple signalling pathways. However, there is little information regarding factors that function upstream of DISC1 to regulate its expression and function. We herein demonstrate that Sonic hedgehog (Shh) signalling promotes expression of disc1 in the zebrafish brain. Expression of disc1 is lost in smoothened mutants that have a complete loss of Shh signal transduction, and elevated in patched mutants which have constitutive activation of Shh signalling. We previously demonstrated that disc1 knockdown has a dramatic effect on the specification of oligodendrocyte precursor cells (OPC) in the hindbrain and Shh signalling is known to be essential for the specification of these cells. We show that disc1 is prominently expressed in olig2-positive midline progenitor cells that are absent in smo mutants, while cyclopamine treatment blocks disc1 expression in these cells and mimics the effect of disc1 knock down on OPC specification. Various features of a number of psychiatric conditions could potentially arise through aberrant Hedgehog signalling. We therefore suggest that altered Shh signalling may be an important neurodevelopmental factor in the pathobiology of mental illness.

Genetic and chemical modulation of spastin-dependent axon outgrowth in zebrafish embryos indicates a role for impaired microtubule dynamics in hereditary spastic paraplegia.

Mutations in the SPAST (SPG4) gene, which encodes the microtubule-severing protein spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). Following on from previous work in our laboratory showing that spastin is required for axon outgrowth, we report here that the related microtubule-severing protein katanin is also required for axon outgrowth in vivo. Using confocal time-lapse imaging, we have identified requirements for spastin and katanin in maintaining normal axonal microtubule dynamics and growth cone motility in vivo, supporting a model in which microtubule severing is required for concerted growth of neuronal microtubules. Simultaneous knockdown of spastin and katanin caused a more severe phenotype than did individual knockdown of either gene, suggesting that they have different but related functions in supporting axon outgrowth. In addition, the microtubule-destabilising drug nocodazole abolished microtubule dynamics and growth cone motility, and enhanced phenotypic severity in spast-knockdown zebrafish embryos. Thus, disruption of microtubule dynamics might underlie neuronal dysfunction in this model, and this system could be used to identify compounds that modulate microtubule dynamics, some of which might have therapeutic potential in HSP.

Direct evidence for axonal transport defects in a novel mouse model of mutant spastin-induced hereditary spastic paraplegia (HSP) and human HSP patients.

Mutations in spastin are the most common cause of hereditary spastic paraplegia (HSP) but the mechanisms by which mutant spastin induces disease are not clear. Spastin functions to regulate microtubule organisation, and because of the essential role of microtubules in axonal transport, this has led to the suggestion that defects in axonal transport may underlie at least part of the disease process in HSP. However, as yet there is no direct evidence to support this notion. Here we analysed axonal transport in a novel mouse model of spastin-induced HSP that involves a pathogenic splice site mutation, which leads to a loss of spastin protein. A mutation located within the same splice site has been previously described in HSP. Spastin mice develop gait abnormalities that correlate with phenotypes seen in HSP patients and also axonal swellings containing cytoskeletal proteins, mitochondria and the amyloid precursor protein (APP). Pathological analyses of human HSP cases caused by spastin mutations revealed the presence of similar axonal swellings. To determine whether mutant spastin influenced axonal transport we quantified transport of two cargoes, mitochondria and APP-containing membrane bound organelles, in neurons from mutant spastin and control mice, using time-lapse microscopy. We found that mutant spastin perturbs anterograde transport of both cargoes. In neurons with axonal swellings we found that the mitochondrial axonal transport defects were exacerbated; distal to axonal swellings both anterograde and retrograde transport were severely reduced. These results strongly support a direct role for defective axonal transport in the pathogenesis of HSP because of spastin mutation.

Severe neurological phenotypes of Q129 DRPLA transgenic mice serendipitously created by en masse expansion of CAG repeats in Q76 DRPLA mice.

We herein provide a thorough description of new transgenic mouse models for dentatorubral-pallidoluysian atrophy (DRPLA) harboring a single copy of the full-length human mutant DRPLA gene with 76 and 129 CAG repeats. The Q129 mouse line was unexpectedly obtained by en masse expansion based on the somatic instability of 76 CAG repeats in vivo. The mRNA expression levels of both Q76 and Q129 transgenes were each 80% of that of the endogenous mouse gene, whereas only the Q129 mice exhibited devastating progressive neurological phenotypes similar to those of juvenile-onset DRPLA patients. Electrophysiological studies of the Q129 mice demonstrated age-dependent and region-specific presynaptic dysfunction in the globus pallidus and cerebellum. Progressive shrinkage of distal dendrites of Purkinje cells and decreased currents through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and gamma-aminobutyrate type A receptors in CA1 neurons were also observed. Neuropathological studies of the Q129 mice revealed progressive brain atrophy, but no obvious neuronal loss, associated with massive neuronal intranuclear accumulation (NIA) of mutant proteins with expanded polyglutamine stretches starting on postnatal day 4, whereas NIA in the Q76 mice appeared later with regional specificity to the vulnerable regions of DRPLA. Expression profile analyses demonstrated age-dependent down-regulation of genes, including those relevant to synaptic functions and CREB-dependent genes. These results suggest that neuronal dysfunction without neuronal death is the essential pathophysiologic process and that the age-dependent NIA is associated with nuclear dysfunction including transcriptional dysregulations. Thus, our Q129 mice should be highly valuable for investigating the mechanisms of disease pathogenesis and therapeutic interventions.

Disrupted-in-schizophrenia 1 and neuregulin 1 are required for the specification of oligodendrocytes and neurones in the zebrafish brain.

Schizophrenia may arise from subtle abnormalities in brain development due to alterations in the functions of candidate susceptibility genes such as Disrupted-in-schizophrenia 1 (DISC1) and Neuregulin 1 (NRG1). To provide novel insights into the functions of DISC1 in brain development, we mapped the expression of zebrafish disc1 and set out to characterize its role in early embryonic development using morpholino antisense methods. These studies revealed a critical requirement for disc1 in oligodendrocyte development by promoting specification of olig2-positive cells in the hindbrain and other brain regions. Since NRG1 has well-documented roles in myelination, we also analyzed the roles of nrg1 and ErbB signalling in zebrafish brain development and we observed strikingly similar defects to those seen in disc1 morphant embryos. In addition to their effects on oligodendrocyte development, knock-down of disc1 or nrg1 caused near total loss of olig2-positive cerebellar neurones, but caused no apparent loss of spinal motor neurones. These findings suggest that disc1 and nrg1 function in common or related pathways controlling development of oligodendrocytes and neurones from olig2-expressing precursor cells. Like DISC1 and NRG1, OLIG2 and ERBB4 are promising candidate susceptibility genes for schizophrenia. Hence our findings in the zebrafish embryo suggest that hitherto unappreciated neurodevelopmental connections may exist between key human schizophrenia susceptibility genes. These connections could be investigated in Disc1 and Nrg1 mouse models and in genetically defined groups of patients in order to determine whether they are relevant to the pathobiology of schizophrenia. GenBank accession number for Danio rerio disc1: EU273350.

The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo.

Hereditary spastic paraplegia (HSP) is a collection of neurological disorders characterized by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity. SPG4 mutations are the most common cause of autosomal dominant HSP and Spastin (the SPG4 gene product) is a microtubule severing protein that shares homology with katanin, the microtubule severing activity of which promotes axon growth in cultured neurons. Given the sequence and functional similarity between spastin and katanin, we hypothesized that spastin promotes the dynamic disassembly and remodelling of microtubules required for robust, properly directed motor axon outgrowth. To investigate this hypothesis, we cloned the zebrafish spg4 orthologue and used morpholino antisense oligonucleotides directed against the translation start site and the intron 7-8 splice donor site to knock down spastin function in the developing zebrafish embryo. Reduced spg4 function caused dramatic defects in motor axon outgrowth without affecting the events driving the initial specification of motor neurones. Other neuronal subtypes also exhibited a requirement for spg4 function, since spg4 knock down caused both widespread defects in neuronal connectivity and extensive CNS-specific apoptosis. Our results reveal a critical requirement for spastin to promote axonal outgrowth during embryonic development, and they validate the zebrafish embryo as a novel model system to dissect the pathogenetic mechanisms underlying HSP. Taken together with other recent studies, our findings suggest that axon outgrowth defects may be a common feature of childhood SPG3A and SPG4 cases.

Progressive phenotype and nuclear accumulation of an amino-terminal cleavage fragment in a transgenic mouse model with inducible expression of full-length mutant huntingtin.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized behaviorally by chorea, incoordination, and shortened lifespan and neuropathologically by huntingtin inclusions and neuronal degeneration. In order to facilitate studies of pathogenesis and therapeutics, we have generated a new inducible mouse model of HD expressing full-length huntingtin (Htt) using a tetracycline-regulated promoter. In double transgenic mice Htt was expressed widely in the brain under the control of the tet-transactivator (tTA) driven by the prion promoter PrP (in the absence of doxycycline). Mice expressing full-length mutant Htt, but not full-length normal Htt, displayed a progressive behavioral phenotype, consisting of slowed and irregular voluntary movements, gait ataxia, tremor and jerky movements, incoordination, and weight loss, with a shortened lifespan. Neuropathology included prominent intranuclear inclusions in cortex and striatum as well as cytoplasmic aggregates. This phenotype is very similar to the phenotypes of previous transgenic mice expressing N-terminal fragments of mutant Htt. The current HD-transgenic mice had nuclear accumulation of Htt, particularly an approximately 60-kDa fragment, which appears to represent an N-terminal cleavage product. This fragment is smaller than calpain or caspase-derived cleavage products of Htt, but it is comparable to a product, termed cp-A, which accumulates in nuclei of cells in a previously described cell model. This new mouse model may be useful in the future for pathogenic and preclinical therapeutic studies related to HD. The data suggest that proteolytic processing could be a part of the pathogenesis of HD, potentially representing an attractive therapeutic target.

The cellular and molecular pathology of the motor system in hereditary spastic paraparesis due to mutation of the spastin gene.

Hereditary spastic paraparesis (HSP) is a genetically heterogeneous disorder, the most common cause of which is mutation of the spastin gene. Recent evidence suggests a role for spastin in microtubule dynamics, but the distribution of the protein within the CNS is unknown. The core neuropathology of HSP is distal degeneration of the lateral corticospinal tract and of fasciculus gracilis, but there are few neuropathological studies of cases with a defined mutation. We aimed to determine the distribution of spastin expression in the human CNS and to investigate the cellular pathology of the motor system in HSP due to mutation of the spastin gene. Using an antibody to spastin, we have carried out immunohistochemistry on postmortem brain. We have demonstrated that spastin is a neuronal protein. It is widely expressed in the CNS so that the selectivity of the degeneration in HSP is not due to the normal cellular distribution of the protein. We have identified mutation of the spastin gene in 3 autopsy cases of HSP. Distal degeneration of long tracts in the spinal cord, consistent with a dying back axonopathy, was accompanied by a microglial reaction. The presence of novel hyaline inclusions in anterior horn cells and an alteration in immunostaining for cytoskeletal proteins and mitochondria indicates that long tract degeneration is accompanied by cytopathology in the motor system and may support a role for derangement of cytoskeletal function. All 3 cases also demonstrated evidence of tau pathology outside the motor system, suggesting that the neuropathology is not confined to the motor system in spastin-related HSP.

Hereditary spastic paraparesis: disrupted intracellular transport associated with spastin mutation.

The commonest cause of hereditary spastic paraplegia (HSP) is mutation in the spastin gene. Both the normal function of spastin in the central nervous system and the mechanism by which mutation in spastin causes axonal degeneration are unknown. One hypothesis is that mutant spastin disrupts microtubule dynamics, causing an impairment of organelle transport on the microtubule network, which leads to degeneration in the distal parts of long axons. To study this neuronal and non-neuronal cells were transfected with either wild type or mutant spastin proteins. We demonstrated evidence of a transient interaction of wild-type spastin with microtubules, with resulting disassembly of microtubules, supporting a role for wild-type spastin as a microtubule-severing protein. Mutant spastin demonstrated an abnormal interaction with microtubules, colocalizing with but no longer severing microtubules. The abnormal interaction of mutant spastin with microtubules was demonstrated to be associated with an abnormal perinuclear clustering of mitochondria and peroxisomes, suggestive of an impairment of kinesin-mediated intracellular transport. Our findings indicate that an abnormal interaction of mutant spastin with microtubules, which disrupts organelle transport on the microtubule cytoskeleton, is likely to be the primary disease mechanism in HSP caused by missense mutations in the spastin gene.

Inducible PC12 cell model of Huntington's disease shows toxicity and decreased histone acetylation.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a polyglutamine tract in the huntingtin protein. We have developed PC12 cell lines in which the expression of an N-terminal truncation of huntingtin (N63) with either wild type (23Q) or expanded polyglutamine (148Q) can be induced by the removal of doxycycline. Differentiated PC12 cells induced to express N63-148Q showed cellular toxicity reaching up to 50% at 6 days post-induction. Histone acetyltransferase (HAT) activity and global histone acetylation was significantly decreased in cells expressing truncated huntingtin with mutant but not normal huntingtin. These data suggest that altered chromatin modification via reduction in coactivator activity may cause neuronal transcriptional dysregulation and contribute to cellular toxicity.