ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles

Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS

Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation

Background Sizing of GGGGCC hexanucleotide repeat expansions within the C9ORF72 locus, which account for approximately 10% of all amyotrophic lateral sclerosis (ALS) cases, is urgently required to answer fundamental questions about mechanisms of pathogenesis in this important genetic variant. Currently employed PCR protocols are limited to discrimination between the presence and absence of a modified allele with more than 30 copies of the repeat, while Southern hybridisation-based methods are confounded by the somatic heterogeneity commonly present in blood samples, which might cause false-negative or ambiguous results. Results We describe an optimised Southern hybridisation-based protocol that allows confident detection of the presence of a C9ORF72 repeat expansion alongside independent assessment of its heterogeneity and the number of repeat units. The protocol can be used with either a radiolabeled or non-radiolabeled probe. Using this method we have successfully sized the C9ORF72 repeat expansion in lymphoblastoid cells, peripheral blood, and post-mortem central nervous system (CNS) tissue from ALS patients. It was also possible to confidently demonstrate the presence of repeat expansion, although of different magnitude, in both C9ORF72 alleles of the genome of one patient. Conclusions The suggested protocol has sufficient advantages to warrant adoption as a standard for Southern blot hybridisation analysis of GGGGCC repeat expansions in the C9ORF72 locu

Peripheral sensory neurons survive in the absence of α- and γ-synucleins

Physiological functions of α-synuclein, a protein implicated in certain types of neurodegeneration, and two other members of the same family, β-synuclein and γ-synuclein, are not clearly understood. It has been suggested that synucleins are involved in intracellular processes associated with survival of neurons and their response to stress, and that changes of synuclein ratio might have deteriorating effects on neurons. In wild-type mice, sensory neurons of the peripheral nervous system express α-synuclein and notably high levels of γ-synuclein, but targeted inactivation of either of these genes has no effect on these neurons. Here we produced double, α-synuclein/γ-synuclein null mutant mice, which develop normally, are fertile, and show no obvious signs of pathology in adulthood. Survival of α/γ-synuclein-deficient peripheral sensory neurons in vivo and in primary tissue culture is indistinguishable from survival of wild-type neurons. The absence of two synucleins does not lead to expression in sensory neurons of the third member of the family, β-synuclein. Therefore, our results demonstrate that neurons with normally high levels of synuclein(s) can develop and survive normally in the absence of any of these proteins. This suggests that other intraneuronal mechanisms and pathways effectively compensate the loss of synuclein function in null mutant animals. Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever reserved

Neurotrophin-regulated signalling pathways

Neurotrophins are a family of closely related proteins that were identified initially as survival factors for sensory and sympathetic neurons, and have since been shown to control many aspects of survival, development and function of neurons in both the peripheral and the central nervous systems. Each of the four mammalian neurotrophins has been shown to activate one or more of the three members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB and TrkC). In addition, each neurotrophin activates p75 neurotrophin receptor (p75NTR), a member of the tumour necrosis factor receptor superfamily. Through Trk receptors, neurotrophins activate Ras, phosphatidyl inositol-3 (PI3)-kinase, phospholipase C-γ1 and signalling pathways controlled through these proteins, such as the MAP kinases. Activation of p75NTR results in activation of the nuclear factor-κB (NF-κB) and Jun kinase as well as other signalling pathways. Limiting quantities of neurotrophins during development control the number of surviving neurons to ensure a match between neurons and the requirement for a suitable density of target innervation. The neurotrophins also regulate cell fate decisions, axon growth, dendrite growth and pruning and the expression of proteins, such as ion channels, transmitter biosynthetic enzymes and neuropeptide transmitters that are essential for normal neuronal function. Continued presence of the neurotrophins is required in the adult nervous system, where they control synaptic function and plasticity, and sustain neuronal survival, morphology and differentiation. They also have additional, subtler roles outside the nervous system. In recent years, three rare human genetic disorders, which result in deleterious effects on sensory perception, cognition and a variety of behaviours, have been shown to be attributable to mutations in brain-derived neurotrophic factor and two of the Trk receptors

Cytokines promote the survival of mouse cranial sensory neurones at different developmental stages

To investigate when the neurotrophic cytokines ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin-M (OSM), interleukin-6 (IL-6) and cardiotrophin-1 (CT-1) act on developing sensory neurones and whether they co-operate with neurotrophins in regulating neuronal survival, we studied the in vitro trophic effects of these factors on two well-characterized populations of cranial sensory neurones at closely staged intervals throughout embryonic development. The cutaneous sensory neurones of the trigeminal ganglion, which show an early, transient survival response to BDNF and NT3 before becoming NGF-dependent, were supported by CNTF, LIF, OSM and CT-1 during the late fetal period, several days after the neurones become NGF-dependent. At this stage of development, these cytokines promoted the survival of a subset of NGF-responsive neurones. The enteroceptive neurones of the nodose ganglion, which retain dependence on BDNF throughout fetal development, were supported throughout their development by CNTF, LIF, OSM and CT-1, and displayed an additional survival response to IL-6 in the late fetal period, These findings indicate that populations of sensory neurones display different developmental patterns of cytokine responsiveness and show that embryonic trigeminal neurones pass through several phases of differing neurotrophic factor survival requirements

Substitution of Met-38 to Ile in γ-synuclein found in two patients with amyotrophic lateral sclerosis induces aggregation into amyloid

α-, β-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson’s disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member