Paraneoplastic cerebellar degeneration: Characterizing the Yo antibody antigens

Background: The pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Yo-antibodies is unclear. The disease is generally accepted as immune-mediated, but whether the Yo antibodies themselves are pathogenic or if T cells are responsible for the neurodegeneration is not known. Yo antibodies are, nevertheless, good biomarkers for the disease. The primary target of Yo antibodies was until recently thought to be CDR2. We showed, however, that these antibodies bind to CDR2L and not CDR2. CDR2L is present on both bound and free ribosomes in the cytoplasm of cerebellar Purkinje neurons as well as other cells types, but the cellular function and spatial conformation remains unknown. Objective: Paper I: To determine the major antigen of Yo antibodies in PCD patients. Paper II: To define the subcellular location of both CDR2 and CDR2L and potential interaction partners. Paper III: To generate a protocol for Purkinje neuron culture from both embryonic and postnatal rats that can be used for further characterization of PCD pathogenesis. Methods: Paper I: Patient samples (serum and CSF), cerebellar tissue (human and rat), cancer cell cultures (OvCar3 and HepG2), immunostaining, immunoprecipitation, fluorescent immunoblotting and recombinant DNA transfection. Paper II: Patient samples (serum and CSF), cerebellar tissue (human), cancer cell cultures (OvCar3 and HepG2), Purkinje neuron cultures (rat), mass spectrometry-based proteomics, immunostaining, proximity ligation assay, super-resolution microscopy and immunoprecipitation. Paper III: Culturing of dissociated rat cerebellar tissue, immunostaining, Purkinje neuron counting, dendritic branch analysis, lentiviral transfection and micro-electrode array recordings. Results: Paper I: We demonstrated that CDR2L, and not CDR2, is the major antigen target of Yo antibodies. These antibodies do, however, bind recombinant CDR2. Paper II: We found that CDR2L is predicted to interact with several ribosomal proteins and that it indeed does interact with the ribosomal protein rpS6. Interaction partners of CDR2 included the nuclear speckle proteins eIF4A3, SON and SRSF2. Paper III: We found that a support layer, pH stability and co-factor supplements were essential to generate rat cerebellar cell cultures with high yield of mature Purkinje neurons. Conclusions: Paper I: The finding that Yo antibodies bind endogenous CDR2L, and not CDR2, allows us to rethink the mechanisms involved in Yo-mediated PCD. The binding of recombinant CDR2 suggests that these proteins have common epitopes which is not surprising considering their 45% amino acid sequence identity. Furthermore, test assays using CDR2L instead of CDR2 could be more sensitive, reducing the large amounts of false-positive results obtained today. Paper II: Previous studies suggested that Yo antibodies bind a ribosomal target, but the locations of CDR2 and CDR2L were unknown. Our finding that CDR2L interacts specifically with ribosomal proteins, while CDR2 interacts with nuclear speckle proteins, adds further support for CDR2L being the primary Yo antibody target. Since one of the interaction partners of CDR2, eIF4A3, translocates from the nucleus to the ribosome, where it interacts with rpS6, this also adds an indirect link between CDR2L and CDR2. Whether CDR2L and CDR2 have similar roles or are involved in related processes in protein transcription and translation remains to be resolved. Paper III: We established a robust primary culture protocol that gave high yields of mature Purkinje neurons from both embryonic and postnatal rats. These cultures were well suited to high-throughput screening, genetic manipulation and electrophysiological recordings and will be useful for exploring both neurodegenerative and regenerative mechanisms.Doktorgradsavhandlin

Development and Optimization of a Multilayer Rat Purkinje Neuron Culture

Elucidation of the mechanisms involved in neurodegenerative diseases of the cerebellum has been hampered by the lack of robust single cell models to study Purkinje neurons and replicate at the same time in vivo features. Cerebellar Purkinje neurons are difficult to grow in dispersed cell culture, and only limited work has been done using rat cells. We developed a refined protocol for growing rat Purkinje neurons from embryonic and postnatal tissue ex vivo that results in well-developed, mature, functional, and synaptically active neurons. The rat Purkinje neurons generated are responsive to paracrine factors and genetic manipulation, allowing great experimental flexibility at the single-cell level. This ex vivo model can be used to investigate disease mechanisms that disturb Purkinje neuron morphology, function, and communication in high- and low-throughput screening formats.publishedVersio

CDR2L Is the Major Yo Antibody Target in Paraneoplastic Cerebellar Degeneration

The pathogenesis of Yo‐mediated paraneoplastic cerebellar degeneration (PCD) is unclear. We applied cerebrospinal fluid and serum from PCD patients as well as CDR2 and CDR2L antibodies to neuronal tissue, cancer cell lines, and cells transfected with recombinant CDR2 and CDR2L to elucidate which is the major antigen of Yo antibodies. We found that Yo antibodies bound endogenous CDR2L, but not endogenous CDR2. However, Yo antibodies can bind the recombinant CDR2 protein used in routine clinical testing for these antibodies. Because Yo antibodies only bind endogenous CDR2L, we conclude that CDR2L is the major antigen of Yo antibodies in PCD.publishedVersio

Safety and Clinical Efficacy of Mesenchymal Stem Cell Treatment in Traumatic Spinal Cord Injury, Multiple Sclerosis and Ischemic Stroke – A Systematic Review and Meta-Analysis

Background: Mesenchymal 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). Methods: A 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. Findings: Of 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. Interpretation: MSC-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.publishedVersio

Multiple brain abscesses and ventriculitis secondary to chronic periodontitis

Chronic marginal periodontitis is a common oral disease, but can in rare cases cause severe intracranial infection. This case illustrates that poor dental status can be life threatening, in particular for immunocompromised patients

Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

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

CDR2L Is the Major Yo Antibody Target in Paraneoplastic Cerebellar Degeneration

The pathogenesis of Yo‐mediated paraneoplastic cerebellar degeneration (PCD) is unclear. We applied cerebrospinal fluid and serum from PCD patients as well as CDR2 and CDR2L antibodies to neuronal tissue, cancer cell lines, and cells transfected with recombinant CDR2 and CDR2L to elucidate which is the major antigen of Yo antibodies. We found that Yo antibodies bound endogenous CDR2L, but not endogenous CDR2. However, Yo antibodies can bind the recombinant CDR2 protein used in routine clinical testing for these antibodies. Because Yo antibodies only bind endogenous CDR2L, we conclude that CDR2L is the major antigen of Yo antibodies in PCD

Safety and Clinical Efficacy of Mesenchymal Stem Cell Treatment in Traumatic Spinal Cord Injury, Multiple Sclerosis and Ischemic Stroke – A Systematic Review and Meta-Analysis

Background: Mesenchymal 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). Methods: A 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. Findings: Of 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. Interpretation: MSC-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

The neuroprotective potential of mesenchymal stem cells from bone marrow and human exfoliated deciduous teeth in a murine model of demyelination.

IntroductionMultiple sclerosis (MS) is characterized by chronic inflammation, demyelination, and axonal degeneration within the central nervous system (CNS), for which there is no current treatment available with the ability to promote neuroprotection or remyelination. Some aspects of the progressive form of MS are displayed in the murine cuprizone model, where demyelination is induced by the innate immune system without major involvement of the adaptive immune system. Mesenchymal stem cells (MSCs) are multipotent cells with immunomodulatory and neuroprotective potential. In this study, we aimed to assess the neuroprotective potential of MSCs from bone marrow (BM-MSCs) and stem cells from human exfoliated deciduous teeth (SHED) in the cuprizone model.MethodsHuman BM-MSCs and SHED were isolated and characterized. Nine-week-old female C57BL/6 mice were randomized to receive either human BM-MSCs, human SHED or saline intraperitoneally. Treatments were administered on day -1, 14 and 21. Outcomes included levels of local demyelination and inflammation, and were assessed with immunohistochemistry and histology.ResultsBM-MSCs were associated with increased myelin content and reduced microglial activation whereas mice treated with SHED showed reduced microglial and astroglial activation. There were no differences between treatment groups in numbers of mature oligodendrocytes or axonal injury. MSCs were identified in the demyelinated corpus callosum in 40% of the cuprizone mice in both the BM-MSC and SHED group.ConclusionOur results suggest a neuroprotective effect of MSCs in a toxic MS model, with demyelination mediated by the innate immune system