Search

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles. Prior to the development of these characteristic pathological hallmarks of AD, anterograde axonal transport is impaired. However, the key proteins that initiate these intracellular impairments remain elusive. The collapsin response mediator protein-2 (CRMP-2) plays an integral role in kinesin-1-dependent axonal transport and there is evidence that phosphorylation of CRMP-2 releases kinesin-1. Here, we tested the hypothesis that amyloid-beta (Aβ)-dependent phosphorylation of CRMP-2 disrupts its association with the kinesin-1 (an anterograde axonal motor transport protein) in AD. We found that brain sections and lysates from AD patients demonstrated elevated phosphorylation of CRMP-2 at the T555 site. Additionally, in the transgenic Tg2576 mouse model of familial AD (FAD) that exhibits Aβ accumulation in the brain with age, we found substantial co-localization of pT555CRMP-2 and dystrophic neurites. In SH-SY5Y differentiated neuronal cultures, Aβ-dependent phosphorylation of CRMP-2 at the T555 site was also elevated and this reduced the CRMP-2 association with kinesin-1. The overexpression of an unphosphorylatable form of CRMP-2 in neurons promoted the re-establishment of CRMP-2-kinesin association and axon elongation. These data suggest that Aβ-dependent phosphorylation of CRMP-2 at the T555 site may directly impair anterograde axonal transport protein function, leading to neuronal defects.

Abstract Axo-glial units are highly organised microstructures propagating saltatory conduction and are disrupted during multiple sclerosis (MS). Nogo receptor 1 (NgR1) has been suggested to govern axonal damage during the progression of disease in the MS-like mouse model, experimental autoimmune encephalomyelitis (EAE). Here we have identified that adult ngr1 −/− mice, previously used in EAE and spinal cord injury experiments, display elongated paranodes, and nodes of Ranvier. Unstructured paranodal regions in ngr1 −/− mice are matched with more distributed expression pattern of Caspr. Compound action potentials of optic nerves and spinal cords from naïve ngr1 −/− mice are delayed and reduced. Molecular interaction studies revealed enhanced Caspr cleavage. Our data suggest that NgR1 may regulate axo-myelin ultrastructure through Caspr-mediated adhesion, regulating the electrophysiological signature of myelinated axons of central nervous system (CNS).

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.

We previously identified that ngrl allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngrl preserved axonal integrity, whereas its re-expression in ngr1-/- female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1flx/flx, retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1-/- optic nerves following the re-introduction of NgRl. Moreover, Cre-loxP-mediated axon-specific deletion of ngrl in ngr1flx/flx mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxβ in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxβ displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1-/- mice, when NgRl was reintroduced but was shown to be transported efficiently in the contralateral non-recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngrl. Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1-/- optic nerves. Our data suggest that NgR 1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngrl preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE. [ABSTRACT FROM AUTHOR]

Abstract We have previously reported evidence that Nogo-A activation of Nogo-receptor 1 (NgR1) can drive axonal dystrophy during the neurological progression of experimental autoimmune encephalomyelitis (EAE). However, the B-cell activating factor (BAFF/BlyS) may also be an important ligand of NgR during neuroinflammation. In the current study we define that NgR1 and its homologs may contribute to immune cell signaling during EAE. Meningeal B-cells expressing NgR1 and NgR3 were identified within the lumbosacral spinal cords of ngr1 +/+ EAE-induced mice at clinical score 1. Furthermore, increased secretion of immunoglobulins that bound to central nervous system myelin were shown to be generated from isolated NgR1- and NgR3-expressing B-cells of ngr1 +/+ EAE-induced mice. In vitro BAFF stimulation of NgR1- and NgR3-expressing B cells, directed them into the cell cycle DNA synthesis phase. However, when we antagonized BAFF signaling by co-incubation with recombinant BAFF-R, NgR1-Fc, or NgR3 peptides, the B cells remained in the G0/G1 phase. The data suggest that B cells express NgR1 and NgR3 during EAE, being localized to infiltrates of the meninges and that their regulation is governed by BAFF signaling.

Myelin-associated inhibitory factors within the central nervous system (CNS) are considered to be one of the main obstacles for axonal regeneration following disease or injury. The nogo receptor 1 (NgR1) has been well documented to play a key role in limiting axonal regrowth in the injured and diseased mammalian CNS. However, the role of nogo receptor in immune cell activation during CNS inflammation is yet to be mechanistically elucidated. Microglia/macrophages are immune cells that are regarded as pathogenic contributors to inflammatory demyelinating lesions in multiple sclerosis (MS). In this study, the animal model of MS, experimental autoimmune encephalomyelitis (EAE) was induced in ngr1+/+ and ngr1–/– female mice following injection with the myelin oligodendrocyte glycoprotein (MOG35–55) peptide. A fate-map analysis of microglia/macrophages was performed throughout spinal cord sections of EAE-induced mice at clinical scores of 0, 1, 2 and 3, respectively (increasing locomotor disability) from both genotypes, using the CD11b and Iba1 cell markers. Western immunoblotting using lysates from isolated spinal cord microglia/macrophages, along with immunohistochemistry and flow cytometric analysis, was performed to demonstrate the expression of nogo receptor and its two homologs during EAE progression. Myelin protein engulfment during EAE progression in ngr1+/+ and ngr1–/– mice was demonstrated by western immunblotting of lysates from isolated spinal cord microglia/macrophages, detecting levels of Nogo-A and MOG. The numbers of M1 and M2 microglia/macrophage phenotypes present in the spinal cords of EAE-induced ngr1+/+ and ngr1–/– mice, were assessed by flow cytometric analysis using CD38 and Erg-2 markers. A significant difference in microglia/macrophage numbers between ngr1+/+ and ngr1–/– mice was identified during the progression of the clinical symptoms of EAE, in the white versus gray matter regions of the spinal cord. This difference was unrelated to the expression of NgR on these macrophage/microglial cells. We have identified that as EAE progresses, the phagocytic activity of microglia/macrophages with myelin debris, in ngr1–/– mice, was enhanced. Moreover, we show a modulation from a predominant M1-pathogenic to the M2-neurotrophic cell phenotype in the ngr1–/– mice during EAE progression. These findings suggest that CNS-specific macrophages and microglia of ngr1–/– mice may exhibit an enhanced capacity to clear inhibitory molecules that are sequestered in inflammatory lesions.