Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis.

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. the advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

Elevated α5 integrin expression on myeloid cells in motor areas in amyotrophic lateral sclerosis is a therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a fatal disease affecting upper and lower motor neurons. Microglia directly interact with motor neurons and participate in the progression of ALS. Single-cell mass cytometry (CyTOF) analysis revealed prominent expression of α5 integrin in microglia and macrophages in a superoxide dismutase-1 G93A mouse model of ALS (SOD1G93A). In postmortem tissues from ALS patients with various clinical ALS phenotypes and disease duration, α5 integrin is prominent in motor pathways of the central and peripheral nervous system and in perivascular zones associated with the blood-brain barrier. In SOD1G93A mice, administration of a monoclonal antibody against α5 integrin increased survival compared to an isotype control and improved motor function on behavioral testing. Together, these findings in mice and in humans suggest that α5 integrin is a potential therapeutic target in ALS.

Impact of a frequent nearsplice SOD1 variant in amyotrophic lateral sclerosis: optimising SOD1 genetic screening for gene therapy opportunities.

OBJECTIVE: Mutations in superoxide dismutase 1 gene (SOD1), encoding copper/zinc superoxide dismutase protein, are the second most frequent high penetrant genetic cause for amyotrophic lateral sclerosis (ALS) motor neuron disease in populations of European descent. More than 200 missense variants are reported along the SOD1 protein. To limit the production of these aberrant and deleterious SOD1 species, antisense oligonucleotide approaches have recently emerged and showed promising effects in clinical trials. To offer the possibility to any patient with SOD1-ALS to benefit of such a gene therapy, it is necessary to ascertain whether any variant of unknown significance (VUS), detected for example in SOD1 non-coding sequences, is pathogenic. METHODS: We analysed SOD1 mutation distribution after SOD1 sequencing in a large cohort of 470 French familial ALS (fALS) index cases. RESULTS: We identified a total of 27 SOD1 variants in 38 families including two SOD1 variants located in nearsplice or intronic regions of the gene. The pathogenicity of the c.358-10T>G nearsplice SOD1 variant was corroborated based on its high frequency (as the second most frequent SOD1 variant) in French fALS, the segregation analysis confirmed in eight affected members of a large pedigree, the typical SOD1-related phenotype observed (with lower limb onset and prominent lower motor neuron involvement), and findings on postmortem tissues showing SOD1 misaccumulation. CONCLUSIONS: Our results highlighted nearsplice/intronic mutations in SOD1 are responsible for a significant portion of French fALS and suggested the systematic analysis of the SOD1 mRNA sequence could become the method of choice for SOD1 screening, not to miss these specific cases.

Deletion of the inflammatory S100-A9/MRP14 protein does not influence survival in hSOD1G93A ALS mice.

Neuroinflammation is a hallmark of Amyotrophic Lateral Sclerosis (ALS) in hSOD1G93A mouse models where microglial cells contribute to the progressive motor neuron degenerative process. S100-A8 and S100-A9 (also known as MRP8 and MRP14, respectively) are cytoplasmic proteins expressed by inflammatory myeloid cells, including microglia and macrophages. Mainly acting as a heterodimer, S100-A8/A9, when secreted, can activate Toll-like Receptor 4 on immune cells, leading to deleterious proinflammatory cytokine production. Deletion of S100a9 in Alzheimer's disease mouse models showed a positive outcome, reducing pathology. We now assessed its role in ALS. Unexpectedly, our results show that deleting S100a9 in hSOD1G93A ALS mice had no impact on mouse survival, but rather accelerated symptoms with no impact on microglial activation and motor neuron survival, suggesting that blocking S100-A9 would not be a valuable strategy for ALS.

Modifying macrophages at the periphery has the capacity to change microglial reactivity and to extend ALS survival.

Microglia and peripheral macrophages have both been implicated in amyotrophic lateral sclerosis (ALS), although their respective roles have yet to be determined. We now show that macrophages along peripheral motor neuron axons in mouse models and patients with ALS react to neurodegeneration. In ALS mice, peripheral myeloid cell infiltration into the spinal cord was limited and depended on disease duration. Targeted gene modulation of the reactive oxygen species pathway in peripheral myeloid cells of ALS mice, using cell replacement, reduced both peripheral macrophage and microglial activation, delayed symptoms and increased survival. Transcriptomics revealed that sciatic nerve macrophages and microglia reacted differently to neurodegeneration, with abrupt temporal changes in macrophages and progressive, unidirectional activation in microglia. Modifying peripheral macrophages suppressed proinflammatory microglial responses, with a shift toward neuronal support. Thus, modifying macrophages at the periphery has the capacity to influence disease progression and may be of therapeutic value for ALS.

New insights on the disease contribution of neuroinflammation in amyotrophic lateral sclerosis.

PURPOSE OF REVIEW: Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease with a strong neuroinflammatory component. This review summarizes how the connection between neurodegeneration and the immune system is strengthened by new discoveries from ALS genetics and the analysis of subpopulations of immune cells in ALS. RECENT FINDINGS: Recent genes identified in ALS encode for proteins with direct immune roles, which when mutated lead to deregulation of immune functions, potentially influencing the disease. Although neuroinflammation in the central nervous system (CNS) of ALS patients has been well documented, new evidence suggests also direct malfunctions of immune cells in the CNS and at the periphery. Although CD4+ T-regulatory lymphocytes are protective in ALS, their number and function are altered over the disease course. CD8+ T cells are detrimental for motor neurons in the CNS but show some protective roles at the periphery. Similarly, the presence of mast cells in muscles of ALS models and patients and impairments of monocyte functions reveal potential new players in ALS disease progression. SUMMARY: Although motor neuron degeneration is considered the prime event in ALS, dysfunctions in immune processes can impact the disease, highlighting that targeting specific immune components is a strategy for developing biomarkers and ultimately new drugs.

Mitochondrial defect in muscle precedes neuromuscular junction degeneration and motor neuron death in CHCHD10S59L/+ mouse.

Recently, we provided genetic basis showing that mitochondrial dysfunction can trigger motor neuron degeneration, through identification of CHCHD10 encoding a mitochondrial protein. We reported patients, carrying the p.Ser59Leu heterozygous mutation in CHCHD10, from a large family with a mitochondrial myopathy associated with motor neuron disease (MND). Rapidly, our group and others reported CHCHD10 mutations in amyotrophic lateral sclerosis (ALS), frontotemporal dementia-ALS and other neurodegenerative diseases. Here, we generated knock-in (KI) mice, carrying the p.Ser59Leu mutation, that mimic the mitochondrial myopathy with mtDNA instability displayed by the patients from our original family. Before 14 months of age, all KI mice developed a fatal mitochondrial cardiomyopathy associated with enhanced mitophagy. CHCHD10S59L/+ mice also displayed neuromuscular junction (NMJ) and motor neuron degeneration with hyper-fragmentation of the motor end plate and moderate but significant motor neuron loss in lumbar spinal cord at the end stage of the disease. At this stage, we observed TDP-43 cytoplasmic aggregates in spinal neurons. We also showed that motor neurons differentiated from human iPSC carrying the p.Ser59Leu mutation were much more sensitive to Staurosporine or glutamate-induced caspase activation than control cells. These data confirm that mitochondrial deficiency associated with CHCHD10 mutations can be at the origin of MND. CHCHD10 is highly expressed in the NMJ post-synaptic part. Importantly, the fragmentation of the motor end plate was associated with abnormal CHCHD10 expression that was also observed closed to NMJs which were morphologically normal. Furthermore, we found OXPHOS deficiency in muscle of CHCHD10S59L/+ mice at 3 months of age in the absence of neuron loss in spinal cord. Our data show that the pathological effects of the p.Ser59Leu mutation target muscle prior to NMJ and motor neurons. They likely lead to OXPHOS deficiency, loss of cristae junctions and destabilization of internal membrane structure within mitochondria at motor end plate of NMJ, impairing neurotransmission. These data are in favor with a key role for muscle in MND associated with CHCHD10 mutations.