Sensory-motor deficits and neurofilament disorganization in gigaxonin-null mice

International audienceABSTRACT: BACKGROUND: Giant Axonal Neuropathy (GAN) is a fatal neurodegenerative disorder with early onset characterized by a severe deterioration of the peripheral and central nervous system, involving both the motor and sensory tracts and leading to ataxia, speech defect and intellectual disabilities. The broad deterioration of the nervous system is accompanied by a generalized disorganization of the intermediate filaments, including neurofilaments in neurons, but the implication of this defect in disease onset or progression remains unknown. The identification of gigaxonin, the substrate adaptor of an E3 ubiquitin ligase, as the defective protein in GAN allows us to now investigate the crucial role of the gigaxonin-E3 ligase in sustaining neuronal and intermediate filament integrity. To study the mechanisms controlled by gigaxonin in these processes and to provide a relevant model to test the therapeutic approaches under development for GAN, we generated a Gigaxonin-null mouse by gene targeting. RESULTS: We investigated for the first time in Gigaxonin-null mice the deterioration of the motor and sensory functions over time as well as the spatial disorganization of neurofilaments. We showed that gigaxonin depletion in mice induces mild but persistent motor deficits starting at 60 weeks of age in the 129/SvJ-genetic background, while sensory deficits were demonstrated in C57BL/6 animals. In our hands, another gigaxonin-null mouse did not display the early and severe motor deficits reported previously. No apparent neurodegeneration was observed in our knock-out mice, but dysregulation of neurofilaments in proximal and distal axons was massive. Indeed, neurofilaments were not only more abundant but they also showed the abnormal increase in diameter and misorientation that are characteristics of the human pathology. CONCLUSIONS: Together, our results show that gigaxonin depletion in mice induces mild motor and sensory deficits but recapitulates the severe neurofilament dysregulation seen in patients. Our model will allow investigation of the role of the gigaxonin-E3 ligase in organizing neurofilaments and may prove useful in understanding the pathological processes engaged in other neurodegenerative disorders characterized by accumulation of neurofilaments and dysfunction of the Ubiquitin Proteasome System, such as Amyotrophic Lateral Sclerosis, Huntington's, Alzheimer's and Parkinson's diseases

Etude du mécanisme d'action des anticorps neutralisant les isolants primaires du virus de l'immunodéficience humaine de type 1 (VIH-1)

Les essais de vaccination contre le VIH n'ont jusqu'ici pas permis d'induire des anticorps neutralisants (AcN) efficaces contre les isolats primaires (IP) du virus, bien que de tels AcN puissent être détectés dans une faible proportion des sérums de sujets infectés. Mon projet de recherche a consisté à caractériser les AcN présents dans ces échantillons et à étudier leur mode d'action, l'acquisition de ces connaissances fondamentales étant utile pour la mise au point d'immunogènes capables d'induire une réponse humorale neutralisante.L'activité neutralisante de sérums et de plasmas de 32 patients infectés a été recherchée vis-à-vis de 4 IP. La purification des immunoglobulines a permis d'attribuer la majeure partie de ces activités aux IgG, qui ont été purifiées pour l'étude du mécanisme d'action des AcN.Bien que les AcN n'empêchent pas l'attachement des IP aux PBMC, leur fixation sur le virus déjà associé à ces cellules n'a pas permis d'inhiber efficacement sa réplication. Les complexes AcN-IP, obtenus en l'absence de PBMC puis purifiés, ont au contraire un pouvoir infectieux très fortement réduit, ce qui suggère que les épitopes reconnus par les AcN sont accessibles sur le virus libre.Le blocage du site de fixation de la gp120 sur le récepteur CD4 ou la dissociation de la majorité des sous-unités gp120 à la surface du virus inhibent la réplication des IP sans diminuer leur attachement aux PBMC. Celui-ci engagerait donc d'autres récepteur(s) que le CD4 à la surface de ces cellules, et peut-être d'autres structures que la gp120 sur les virions. L'attachement des IP aux PBMC est de fait inhibé en partie après digestion des groupements héparane-sulfate cellulaires.L'étude de la fixation des IgG sur les IP a montré qu'il n'existe pas de corrélation entre celle-ci et la neutralisation. Les résultats obtenus suggèrent de plus que la majorité des IgG de patients pourraient se fixer sur le virus via des épitopes révélés suite à la dissociation de la gp120.HIV vaccination trials have not resulted in the induction of neutralizing antibodies (nAb) that were effective against primary isolates (PI) of the virus, while such nAbs have been detected in a little fraction of sera from infected patients. The aim of my research project was to characterize nAb in such samples and to study their mechanism of action, as the acquisition of this fundamental knowledge could be useful to design immunogens that may induce a neutralizing humoral response.The neutralizing activity of sera and plasmas of 32 infected patients was measured against four PI. The purification of immunoglobulins showed that most of the activities were mediated by IgG, and these Ig were purified for the study of the mechanisms of neutralization.The attachment of PI to PBMC was not inhibited by nAb, although their fixation to the virus that was already associated to the cells did not result in an efficient blocking of its replication. nAb-PI complexes, formed in absence of PBMC and purified, had lost most of their infectivity, suggesting that epitopes that are targeted by nAb are accessible on the free viral particles.The blocking of the gp120-binding site on CD4 or the shedding of the majority of the gp120 associated to the virus resulted in the inhibition of viral replication but did not impair the attachment of PI to PBMC. This early event is thus mediated by alternative receptors on the cell surface, and may also imply structures other than the gp120 on the virus surface. Indeed, heparan sulfate were shown to be partly responsible for the attachment of PI to PBMC.The study of the binding of IgG to PI particles showed that there is no correlation between binding and neutralization. Moreover, our results suggest that a large part of the binding of polyclonal IgG to viral particles may occur through epitopes that are exposed after the dissociation of the gp120.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Polyclonal Immunoglobulin G from Patients Neutralizes Human Immunodeficiency Virus Type 1 Primary Isolates by Binding Free Virions, but without Interfering with an Initial CD4-Independent Attachment of the Virus to Primary Blood Mononuclear Cells

We investigated the relationship between human immunodeficiency virus type 1 (HIV-1) primary isolate (PI) antibody-mediated neutralization and attachment to primary blood mononuclear cells (PBMC). Incubation of PIs with immunoglobulin G (IgG) purified from infected patients did not inhibit attachment of the viruses with PBMC, but partial to complete neutralization was achieved. Neutralization of PIs already fixed on the cells was achieved by some IgG samples only and was of limited intensity compared to the former neutralization protocol. On the contrary, the binding of IgG to free virions was shown to be sufficient to reach potent neutralization, as the infectivity of IgG-PI complexes purified from the bulk of antibodies before addition to PBMC was strongly diminished compared to mock-treated controls. Monoclonal antibodies to the CDR2 domain of CD4 completely inhibited the infection of PBMC without interfering with the attachment of PIs to the cells, suggesting that, under these experimental conditions, the initial attachment of viruses to PBMC involves alternative cellular receptors. This initial interaction may also involve other components of the viral envelope than gp120, as partial depletion of the surface glycoproteins of primary viral particles that resulted in an almost complete loss of infectivity did not impair attachment to PBMC. A limited inhibition of attachment was observed when interfering with putative interactions with cellular heparan sulfate, whereas no effect was observed for cellular CD147 or nucleolin or for virion-incorporated cyclophilin A. Altogether, our results favor a mechanism of neutralization of HIV-1 PIs by polyclonal IgG where antibodies predominantly bind free virions and neutralize without interfering with the attachment to PBMC, which, in this model, is mainly CD4 independent

Exacerbated pathology of viral encephalitis in mice with central nervous system-specific autoantibodies.

We examine here the outcome of viral encephalomyelitis [mouse hepatitis virus (MHV) A59, Theiler's encephalomyelitis virus, and Coxsackievirus B3] in mice with autoantibodies to a central nervous system (CNS)-specific antigen, myelin oligodendrocyte glycoprotein, that usually develop no clinical disease. Morbidity and mortality of the acute viral CNS disease was augmented by the presence of the autoantibodies in all three viral infections. Transfer of serum containing the autoantibodies at the time of infection with MHV was sufficient to reproduce the exacerbated disease. The presence of the autoantibodies was found to result in increased infiltration of mononuclear cells into the brain. Early demyelination was severely augmented in brains and spinal cords of MHV-infected mice with CNS-specific autoantibodies. The antibody-mediated exacerbation was shown to be independent of the complement system but to require expression of Fc receptors, because it was observed in C'-3-deficient but not in Fc receptor-deficient mice. Our study illustrates the possibility that infections can lead to much more profound immunopathology in the presence of an otherwise latent autoimmune condition

Exacerbated Pathology of Viral Encephalitis in Mice with Central Nervous System-Specific Autoantibodies

We examine here the outcome of viral encephalomyelitis [mouse hepatitis virus (MHV) A59, Theiler’s encephalomyelitis virus, and Coxsackievirus B3] in mice with autoantibodies to a central nervous system (CNS)-specific antigen, myelin oligodendrocyte glycoprotein, that usually develop no clinical disease. Morbidity and mortality of the acute viral CNS disease was augmented by the presence of the autoantibodies in all three viral infections. Transfer of serum containing the autoantibodies at the time of infection with MHV was sufficient to reproduce the exacerbated disease. The presence of the autoantibodies was found to result in increased infiltration of mononuclear cells into the brain. Early demyelination was severely augmented in brains and spinal cords of MHV-infected mice with CNS-specific autoantibodies. The antibody-mediated exacerbation was shown to be independent of the complement system but to require expression of Fc receptors, because it was observed in C′-3-deficient but not in Fc receptor-deficient mice. Our study illustrates the possibility that infections can lead to much more profound immunopathology in the presence of an otherwise latent autoimmune condition