Glycans in Sera of Amyotrophic Lateral Sclerosis Patients and Their Role in Killing Neuronal Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by degeneration of upper and lower motor neurons. To date, glycosylation patterns of glycoproteins in fluids of ALS patients have not been described. Moreover, the aberrant glycosylation related to the pathogenesis of other neurodegenerative diseases encouraged us to explore the glycome of ALS patient sera. We found high levels of sialylated glycans and low levels of core fucosylated glycans in serum-derived N-glycans of patients with ALS, compared to healthy volunteer sera. Based on these results, we analyzed the IgG Fc N297-glycans, as IgG are major serum glycoproteins affected by sialylation or core fucosylation and are found in the motor cortex of ALS patients. The analyses revealed a distinct glycan, A2BG2, in IgG derived from ALS patient sera (ALS-IgG). This glycan increases the affinity of IgG to CD16 on effector cells, consequently enhancing Antibody-Dependent Cellular Cytotoxicity (ADCC). Therefore, we explore whether the Fc-N297-glycans of IgG may be involved in ALS disease. Immunostaining of brain and spinal cord tissues revealed over-expression of CD16 and co-localization of intact ALS-IgG with CD16 and in brain with activated microglia of G93A-SOD1 mice. Intact ALS-IgG enhanced effector cell activation and ADCC reaction in comparison to sugar-depleted or control IgG. ALS-IgG were localized in the synapse between brain microglia and neurons of G93A-SOD1 mice, manifesting a promising in vivo ADCC reaction. Therefore, glycans of ALS-IgG may serve as a biomarker for the disease and may be involved in neuronal damage

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons. A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons. A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons

“ALS reversals”: demographics, disease characteristics, treatments, and co-morbidities

<p><i>Objective</i>: To identify differences in demographics, disease characteristics, treatments, and co-morbidities between patients with “amyotrophic lateral sclerosis (ALS) reversals” and those with typically progressive ALS. <i>Methods:</i> Cases of possible ALS reversals were found in prior publications, in the Duke ALS clinic, through self-referral or referral from other Neurologists, and on the internet. Of 89 possible reversals identified, 36 cases were included because chart or literature review confirmed their diagnosis and a robust, sustained improvement in at least one objective measure. Controls were participants in the Pooled Resource Open-Access ALS Clinical Trials database and the National ALS Registry. Cases and controls were compared using descriptive statistics. <i>Results</i>: ALS reversals were more likely to be male, have limb onset disease, and initially progress faster. The prevalences of myasthenia gravis (MG) and purely lower motor neuron disease in cases were higher than estimates of these prevalences in the general population. The odds of taking curcumin, luteolin, cannabidiol, azathioprine, copper, glutathione, vitamin D, and fish oil were greater for cases than controls. <i>Conclusions:</i> When compared to patients with typically progressive ALS, patients with reversals differed in their demographics, disease characteristics, and treatments. While some of these patients may have had a rare antibody-mediated ALS mimicker, such as atypical myasthenia gravis, details of their exams, EMGs and family histories argue that this was unlikely. Instead, our data suggest that ALS reversals warrant evaluation for mechanisms of disease resistance and that treatments associated with multiple ALS reversals deserve further study.</p

Killing human neuroblastoma or mouse NSC34 cells through the ADCC pathway.

<p>ADCC was performed using human neuroblastoma as target cells, PBMCs as effector cells, and pools of serum samples of ALS, healthy control, inflammatory bowel disease patients, and multiple sclerosis patients as IgG sources. The controls contain: neuroblastoma cells incubated with IgG pools from the different serum sources and co-cultures of neuroblastoma cells and PBMCs (<b>A</b>). FACS results from PBMCs pre-treated with anti-CD16 antibodies (<b>B</b>, left) and the heavy chain of ALS-IgG before and after PNGase-F treatment in SDS-PAGE and Western blot using ECL lectin (<b>B</b>, right); ADCC mediated by CD16-blocked effector cells or by ALS-IgG after PNGase-F treatment, as compared to the ADCC against neuroblastoma mediated by unblocked effector cells and untreated ALS-IgG (<b>C</b>); Killing of NSC34 cells by ADCC as described above was mediated by intact (untreated) ALS-IgG, PNGase-F treated ALS-IgG and by IgG from healthy controls (<b>D</b>). Neuroblastoma lysis by CD32- and CD64-positive THP1 cells was mediated by ALS-IgG, IgG of healthy controls, and in serum free of IgG (<b>E</b>). Spontaneous lysis was measured in neuroblastoma or NSC34 cultures. Triple staining of NeuN, Iba1, and ALS-IgG by anti-human IgGs conjugated to FITC demonstrates the localization of intact ALS-IgG in immune synapse (arrow) amongst microglia and neurons (<b>F</b>). Data represent the mean ± SD of triplicate measurements from triplicate independent experiments. Pools of healthy and patient samples contained a mixture of four individual serum samples with similar glycan amounts represented in peaks 12 and 13. Statistical significance, ** p<0.01, * p<0.05 and NS (Not significant) is represented versus the appropriate controls in each panel.</p

Statistic analyses comparing N-glycans derived from 19 ALS patient sera and from 24 healthy candidate sera.

<p>Fold of change: (+) assigns that N-glycan amounts in ALS sera are higher relative to glycans from healthy candidate sera and (−) assigns the contrary.</p

ALS-IgG co-localized with CD16 and microglia cells in brain and spinal-cord tissues of G93A-SOD1 mice.

<p>Representative confocal microscopic images of brain cortex and spinal cord slices taken from 130-day old G93A-SOD1 mice and age-matched littermates stained for CD16, hIgG, Iba1, NeuN (neurons) and counterstained with nuclear DAPI. Localization of ALS-IgG before and after PNGase-F treatment and of healthy control-IgG in wild-type and mSOD1 brain tissues. The boxed area is a high magnification of CD16- and intact ALS-IgG-positive cell (<b>A</b>); Quantity of ALS-IgG accumulation before and after PNGase-F treatment and of IgG from healthy control in mSOD1 brain slices (<b>B</b>); Quantity of ALS-IgG before and after PNGase-F treatment and of IgG from healthy control co-localized with CD16 in wild-type and mSOD1 brain tissues (<b>C</b>); Co-localization of intact ALS-IgG with microglia was detected in mSOD1 brain tissue (<b>D</b>), and co-localization of intact ALS-IgG with NeuN in mSOD1 brain and spinal cord tissues (<b>E</b>). The measurements were performed on five fields from 3–4 sections per mouse. Error bars indicate means ± SD. Asterisks denote the significance of differences relative to deglycosylated ALS-IgG or control-IgG in mSOD1 sections or ALS-IgG in non-SOD1 littermates, *** p<0.005 represents a comparison using ANOVA t-test.</p