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Objective: Identify the subcellular location and potential binding partners of two cerebellar degeneration-related proteins, CDR2L and CDR2, associated with anti-Yo-mediated paraneoplastic cerebellar degeneration. Methods: Cancer cells, rat Purkinje neuron cultures, and human cerebellar sections were exposed to cerebrospinal fluid and serum from patients with paraneoplastic cerebellar degeneration with Yo antibodies and with several antibodies against CDR2L and CDR2. We used mass spectrometry-based proteomics, super-resolution microscopy, proximity ligation assay, and co-immunoprecipitation to verify the antibodies and to identify potential binding partners. Results: We confirmed the CDR2L specificity of Yo antibodies by mass spectrometry-based proteomics and found that CDR2L localized to the cytoplasm and CDR2 to the nucleus. CDR2L co-localized with the 40S ribosomal protein S6, while CDR2 co-localized with the nuclear speckle proteins SON, eukaryotic initiation factor 4A-III, and serine/ arginine-rich splicing factor 2. Interpretation: We showed that Yo antibodies specifically bind to CDR2L in Purkinje neurons of PCD patients where they potentially interfere with the function of the ribosomal machinery resulting in disrupted mRNA translation and/or protein synthesis. Our findings demonstrating that CDR2L interacts with ribosomal proteins and CDR2 with nuclear speckle proteins is an important step toward understanding PCD pathogenesis.

The inability to reliably replicate mitochondrial DNA (mtDNA) by mitochondrial DNA polymerase gamma (POLG) leads to a subset of common mitochondrial diseases associated with neuronal death and depletion of neuronal mtDNA. Defining disease mechanisms remains difficult due to the limited access to human tissue. Astrocytes are highly abundant in the brain, playing a crucial role in the support and modulation of neuronal function. Astrocytes also respond to insults affecting the brain. Following damage to the center neural system, which can be hypoxia, inflammation or neurodegeneration, astrocytes become activated, lose their supportive role and gain toxic functions that induce rapid death of neurons and oligodendrocytes. The role of astrocyte reactivation and the consequences this has for neuronal homeostasis in mitochondrial diseases has not been explored. Here, using patient cells carrying POLG mutations, we generated iPSCs and then differentiated into astrocytes. We demonstrated that POLG-astrocytes exhibited both mitochondrial dysfunctions, including loss of mitochondrial membrane potential, energy failure, complex I and IV defects, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG-astrocytes exhibited a toxic effect leading to the death of neurons. Our findings demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. Our studies provide a robust astroglia-neuron interaction model for future investigation of mitochondrial involvement in neurogenesis and neurodegenerative diseases.

BackgroundMesenchymal stem cells (MSCs) is an attractive candidate in regenerative research and clinical trials have assessed their therapeutic potential in different neurological conditions with disparate etiologies. In this systematic review, we aimed to assess safety and clinical effect of MSC treatment in traumatic spinal cord injury (TSCI), multiple sclerosis (MS) and ischemic stroke (IS).MethodsA systematic search was performed 2021-12-10 in MEDLINE, EMBASE, Web of Science and Cochrane where clinical studies assessing MSC treatment in TSCI, MS or IS were included. Studies without control group were excluded for efficacy analysis, but included in the safety analysis. For efficacy, AIS score, EDSS score and mRS were used as clinical endpoints and assessed in a meta-analysis using the random effects model.FindingsOf 5,548 identified records, 54 studies were included. Twenty-six studies assessed MSC treatment in TSCI, 14 in MS and nine in IS, of which seven, seven and five studies were controlled, respectively. There were seven serious adverse events (SAEs), of which four were related to the surgical procedure and included one death due to complications following the implantation of MSCs. Three SAEs were considered directly related to the MSC treatment and all these had a transient course. In TSCI, a meta-analysis showed no difference in conversion from AIS A to C and a trend toward more patients treated with MSCs improving from AIS A to B as compared to controls (p = 0.05). A subgroup analysis performed per protocol, showed more MSC treated patients improving from AIS A to C in studies including patients within 8 weeks after injury (p = 0.04). In MS and IS, there were no significant differences in clinical outcomes between MSC treated patients and controls as measured by EDSS and mRS, respectively.InterpretationMSC-treatment is safe in patients with TSCI, MS and IS, although surgical implantation of MSC led to one fatal outcome in TSCI. There was no clear clinical benefit of MSC treatment, but this is not necessarily a proof of inefficacy due to the low number of controlled studies. Future studies assessing efficacy of MSC treatment should aim to do this in randomized, controlled studies.

Cerebellar degeneration related protein 1 (CDR1) is expressed in the cerebellum, and CDR1 antibodies have been associated with paraneoplastic cerebellar degeneration (PCD). In this study, we examined CDR1 expression in cerebellum and in ovarian and breast tumors, as well as the intracellular localization of CDR1 in cancer cells in culture. CDR1 was strongly expressed in the cytosol and dendrites of Purkinje cells and in interneurons of the molecular layer in cerebellum. CDR1 was also present in ovarian and breast tumors, as well as in ovarian and breast cancer cell lines, but was not present in normal breast or ovarian tissue. In cells overexpressing CDR1, CDR1 localized close to the plasma membrane in a polarized pattern at one edge. CDR1 was strongly expressed on the outer surface, apparently in filopodias or lamellipodias, in cells endogenously expressing CDR1. Overexpression of CDR1 showed a 37 and a 45 kDa band in western blot. The 37-kDa isoform was present in 16 ovarian cancer lysates, while the 45-kDa isoform was only found in three ovarian cancer patients. The presence of CDR1 in ovarian cancer was not associated with PCD. CDR1 antibodies were only found in serum from one patient with PCD and ovarian tumor with metastases. Therefore, CDR1 is probably not a marker for PCD. However, CDR1 may be associated with cell migration and differentiation.