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Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis.

ABSTRACT: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by myelin and axonal pathology. In a viral model of MS, we tested whether axonopathy initiation and development are based on an impaired transport of neurofilaments. Spinal cords of Theiler's murine encephalomyelitis virus (TMEV)-infected and mock-infected mice and TMEV infected neuroblastoma N1E-115 cells were analyzed by microarray analysis, light microscopy and electron and laser confocal microscopy. In vivo axonal accumulation of non-phosphorylated neurofilaments after TMEV infection revealed a temporal development caused by the impairments of the axonal traffic consisting of the downregulation of kinesin family member 5A, dynein cytoplasmic heavy chain 1, tau-1 and β-tubulin III expression. In addition, alterations of the protein metabolism were also noticed. In vitro, the TMEV-infected N1E-115 cells developed tandem-repeated swellings similar to in vivo alterations. Furthermore, the hypothesis of an underlying axonal self-destruction program involving nicotinamide adenine dinucleotide depletion was supported by molecular findings. The obtained data indicate that neurofilament accumulation in TME is mainly the result of dysregulation of their axonal transport machinery and impairment of neurofilament phosphorylation and protein metabolism. The present findings allow a more precise understanding of the complex interactions responsible for initiation and development of axonopathies in inflammatory degenerative diseases.

SUBMITTER: Kreutzer M

PROVIDER: S-EPMC8092950 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis.

Kreutzer Mihaela M Seehusen Frauke F Kreutzer Robert R Pringproa Kidsadagorn K Kummerfeld Maren M Claus Peter P Deschl Ulrich U Kalkul Arno A Beineke Andreas A Baumgärtner Wolfgang W Ulrich Reiner R

Brain pathology (Zurich, Switzerland) 20111117 4

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by myelin and axonal pathology. In a viral model of MS, we tested whether axonopathy initiation and development are based on an impaired transport of neurofilaments. Spinal cords of Theiler's murine encephalomyelitis virus (TMEV)-infected and mock-infected mice and TMEV infected neuroblastoma N1E-115 cells were analyzed by microarray analysis, light microscopy and electron and laser confocal microscopy. In viv ...[more]

PMID: 21988534

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Project description:BackgroundDue to limited access to brain tissue, the precise mechanisms underlying neuro-axonal dysfunction in neurological disorders such as multiple sclerosis (MS) are largely unknown. In that context, profiling DNA methylation, which is a stable and cell type-specific regulatory epigenetic mark of genome activity, offers a unique opportunity to characterize the molecular mechanisms underpinning brain pathology in situ. We examined DNA methylation patterns of neuronal nuclei isolated from post-mortem brain tissue to infer processes that occur in neurons of MS patients.ResultsWe isolated subcortical neuronal nuclei from post-mortem white matter tissue of MS patients and non-neurological controls using flow cytometry. We examined bulk DNA methylation changes (total n = 29) and further disentangled true DNA methylation (5mC) from neuron-specific DNA hydroxymethylation (5hmC) (n = 17), using Illumina Infinium 450K arrays. We performed neuronal sub-type deconvolution using glutamate and GABA methylation profiles to further reduce neuronal sample heterogeneity. In total, we identified 2811 and 1534 significant (genome-wide adjusted P value < 0.05) differentially methylated and hydroxymethylated positions between MS patients and controls. We found striking hypo-5mC and hyper-5hmC changes occurring mainly within gene bodies, which correlated with reduced transcriptional activity, assessed using published RNAseq data from bulk brain tissue of MS patients and controls. Pathway analyses of the two cohorts implicated dysregulation of genes involved in axonal guidance and synaptic plasticity, with meta-analysis confirming CREB signalling as the most highly enriched pathway underlying these processes. We functionally investigated DNA methylation changes of CREB signalling-related genes by immunohistofluoresence of phosphorylated CREB in neurons from brain sections of a subcohort of MS patients and controls (n = 15). Notably, DNA methylation changes associated with a reduction of CREB activity in white matter neurons of MS patients compared to controls.ConclusionsOur data demonstrate that investigating 5mC and 5hmC modifications separately allows the discovery of a substantial fraction of changes occurring in neurons, which can escape traditional bisulfite-based DNA methylation analysis. Collectively, our findings indicate that neurons of MS patients acquire sustained hypo-5mC and hyper-5hmC, which may impair CREB-mediated neuro-axonal integrity, in turn relating to clinical symptoms.

Project description:Multiple sclerosis involves demyelination and axonal degeneration of the central nervous system. The molecular mechanisms of axonal degeneration are relatively unexplored in both multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis. We previously reported that targeting the axonal growth inhibitor, Nogo-A, may protect against neurodegeneration in experimental autoimmune encephalomyelitis; however, the mechanism by which this occurs is unclear. We now show that the collapsin response mediator protein 2 (CRMP-2), an important tubulin-associated protein that regulates axonal growth, is phosphorylated and hence inhibited during the progression of experimental autoimmune encephalomyelitis in degenerating axons. The phosphorylated form of CRMP-2 (pThr555CRMP-2) is localized to spinal cord neurons and axons in chronic-active multiple sclerosis lesions. Specifically, pThr555CRMP-2 is implicated to be Nogo-66 receptor 1 (NgR1)-dependent, since myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced NgR1 knock-out (ngr1(-)(/)(-)) mice display a reduced experimental autoimmune encephalomyelitis disease progression, without a deregulation of ngr1(-)(/)(-) MOG(35-55)-reactive lymphocytes and monocytes. The limitation of axonal degeneration/loss in experimental autoimmune encephalomyelitis-induced ngr1(-)(/)(-) mice is associated with lower levels of pThr555CRMP-2 in the spinal cord and optic nerve during experimental autoimmune encephalomyelitis. Furthermore, transduction of retinal ganglion cells with an adeno-associated viral vector encoding a site-specific mutant T555ACRMP-2 construct, limits optic nerve axonal degeneration occurring at peak stage of experimental autoimmune encephalomyelitis. Therapeutic administration of the anti-Nogo(623-640) antibody during the course of experimental autoimmune encephalomyelitis, associated with an improved clinical outcome, is demonstrated to abrogate the protein levels of pThr555CRMP-2 in the spinal cord and improve pathological outcome. We conclude that phosphorylation of CRMP-2 may be downstream of NgR1 activation and play a role in axonal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis. Blockade of Nogo-A/NgR1 interaction may serve as a viable therapeutic target in multiple sclerosis.

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Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis.

Publications

Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis.

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