Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.

Complementary interacting molecules on myelin and axons are required for long-term axon-myelin stability. Their disruption results in axon degeneration, contributing to the pathogenesis of demyelinating diseases. Myelin-associated glycoprotein (MAG), a minor constituent of central and peripheral nervous system myelin, is a member of the Siglec family of sialic acid-binding lectins and binds to gangliosides GD1a and GT1b, prominent molecules on the axon surface. Mice lacking the ganglioside biosynthetic gene Galgt1 fail to express complex gangliosides, including GD1a and GT1b. In the current studies, CNS and PNS histopathology and behavior of Mag-null, Galgt1-null, and double-null mice were compared on the same mouse strain background. When back-crossed to >99% C57BL/6 strain purity, Mag-null mice demonstrated marked CNS, as well as PNS, axon degeneration, in contrast to prior findings using mice of mixed strain background. On the same background, Mag- and Galgt1-null mice exhibited quantitatively and qualitatively similar CNS and PNS axon degeneration and nearly identical decreases in axon diameter and neurofilament spacing. Double-null mice had qualitatively similar changes. Consistent with these findings, Mag- and Galgt1-null mice had similar motor behavioral deficits, with double-null mice only modestly more impaired. Despite their motor deficits, Mag- and Galgt1-null mice demonstrated hyperactivity, with spontaneous locomotor activity significantly above that of wild type mice. These data demonstrate that MAG and complex gangliosides contribute to axon stability in both the CNS and PNS. Similar neuropathological and behavioral deficits in Galgt1-, Mag-, and double-null mice support the hypothesis that MAG binding to gangliosides contributes to long-term axon-myelin stability.

By unknown mechanisms, the symptoms of amyotrophic lateral sclerosis (ALS) seem to spread along neuroanatomical pathways to engulf the motor nervous system. The rate at which symptoms spread is one of the primary drivers of disease progression. One mechanism by which ALS symptoms could spread is by a prion-like propagation of a toxic misfolded protein from cell to cell along neuroanatomic pathways. Proteins that can transmit toxic conformations between cells often can also experimentally transmit disease between individual organisms. To survey the ease with which motor neuron disease (MND) can be transmitted, we injected spinal cord homogenates prepared from paralyzed mice expressing mutant superoxide dismutase 1 (SOD1-G93A and G37R) into the spinal cords of genetically vulnerable SOD1 transgenic mice. From the various models we tested, one emerged as showing high vulnerability. Tissue homogenates from paralyzed G93A mice induced MND in 6 of 10 mice expressing low levels of G85R-SOD1 fused to yellow fluorescent protein (G85R-YFP mice) by 3-11 months, and produced widespread spinal inclusion pathology. Importantly, second passage of homogenates from G93A → G85R-YFP mice back into newborn G85R-YFP mice induced disease in 4 of 4 mice by 3 months of age. Homogenates from paralyzed mice expressing the G37R variant were among those that transmitted poorly regardless of the strain of recipient transgenic animal injected, a finding suggestive of strain-like properties that manifest as differing abilities to transmit MND. Together, our data provide a working model for MND transmission to study the pathogenesis of ALS.

A hallmark feature of amyotrophic lateral sclerosis (ALS) is that symptoms appear to spread along neuroanatomical pathways to engulf the motor nervous system, suggesting a propagative toxic entity could be involved in disease pathogenesis. Evidence for such a propagative entity emerged recently in studies using mice that express G85R-SOD1 mutant protein fused to YFP (G85R-SOD1: YFP). Heterozygous G85R-SOD1:YFP transgenic mice do not develop ALS symptoms out to 20 months of age. However, when newborns are injected with spinal homogenates from paralyzed mutant SOD1 mice, the G85R-SOD1:YFP mice develop paralysis as early as 6 months of age. We now demonstrate that injecting spinal homogenates from paralyzed mutant SOD1 mice into the sciatic nerves of adult G85R-SOD1:YFP mice produces a spreading motor neuron disease within 3.0 ± 0.2 months of injection. The formation of G85R-SOD1:YFP inclusion pathology spreads slowly in this model system; first appearing in the ipsilateral DRG, then lumbar spinal cord, before spreading rostrally up to the cervical cord by the time mice develop paralysis. Reactive astrogliosis mirrors the spread of inclusion pathology and motor neuron loss is most severe in lumbar cord. G85R-SOD1:YFP inclusion pathology quickly spreads to discrete neurons in the brainstem and midbrain that are synaptically connected to spinal neurons, suggesting a trans-synaptic propagation of misfolded protein. Taken together, the data presented here describe the first animal model that recapitulates the spreading phenotype observed in patients with ALS, and implicates the propagation of misfolded protein as a potential mechanism for the spreading of motor neuron disease.

Evidence of misfolded wild-type superoxide dismutase 1 (SOD1) has been detected in spinal cords of sporadic ALS (sALS) patients, suggesting an etiological relationship to SOD1-associated familial ALS (fALS). Given that there are currently a number of promising therapies under development that target SOD1, it is of critical importance to better understand the role of misfolded SOD1 in sALS. We previously demonstrated the permissiveness of the G85R-SOD1:YFP mouse model for MND induction following injection with tissue homogenates from paralyzed transgenic mice expressing SOD1 mutations. This prompted us to examine whether WT SOD1 can self-propagate misfolding of the G85R-SOD1:YFP protein akin to what has been observed with mutant SOD1. Using the G85R-SOD1:YFP mice, we demonstrate that misfolded conformers of recombinant WT SOD1, produced in vitro, induce MND with a distinct inclusion pathology. Furthermore, the distinct pathology remains upon successive passages in the G85R-SOD1:YFP mice, strongly supporting the notion for conformation-dependent templated propagation and SOD1 strains. To determine the presence of a similar misfolded WT SOD1 conformer in sALS tissue, we screened homogenates from patients diagnosed with sALS, fALS, and non-ALS disease in an organotypic spinal cord slice culture assay. Slice cultures from G85R-SOD1:YFP mice exposed to spinal homogenates from patients diagnosed with ALS caused by the A4V mutation in SOD1 developed robust inclusion pathology, whereas spinal homogenates from more than 30 sALS cases and various controls failed. These findings suggest that mutant SOD1 has prion-like attributes that do not extend to SOD1 in sALS tissues.