Cerebellar degeneration-related proteins in anti-Yo associated paraneoplastic cerebellar degeneration
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Background: Paraneoplastic neurological syndromes (PNS) are rare immune-mediated diseases triggered by cancer, and characterized by circulating onconeural antibodies directed against antigens expressed by neurons and tumor cells. One of the most common forms of PNS is paraneoplastic cerebellar degeneration (PCD). In patients with PCD and ovarian or breast cancer, the dominant onconeural antibody is anti-Yo, detected in serum and cerebrospinal fluid (CSF). Anti-Yo targets two intracellular antigens, cerebellar degeneration-related protein 2 (CDR2) and CDR2-like (CDR2L), expressed in the nucleus and cytoplasm of Purkinje neurons in the cerebellum, respectively. The interaction between anti-Yo and CDR proteins is thought to mediate Purkinje neuron dysfunction and death, leaving the patients in a severely disabled state. The pathomechanisms underlying PCD, including the cellular functions and molecular mechanisms driven by the CDR proteins, are limited. About ten years ago, CDR2L was identified as a new target associated with PCD, and its specific location and neuronal functions remains elusive. Herein, we aim to draw a clearer picture of the anti-Yo associated neurodegeneration by addressing which onconeural antigen is the major target of Yo antibodies, identifying the subcellular localization of CDR2 and CDR2L and interaction partners. Finally, we evaluate the possibility of including CDR2L as a diagnostic marker when diagnosing patients with PCD. Aims: The overarching aim of this work was to gain in depth knowledge about CDR2 and CDR2L in anti-Yo associated PCD. Materials and methods: In paper I, anti-Yo reactivity towards CDR2 and CDR2L was addressed by staining cerebellar sections and cancer cells, and performing immunoprecipitation and fluorescent immunoblotting analysis. HepG2 cells were transfected to investigate whether Yo antibodies detect recombinant forms of the proteins. In paper II, mass spectrometry based proteomics was used to determine antibody specificity, and to identify CDR2 and CDR2L protein binding partners. Findings were further investigated by co-immunoprecipitation, proximity ligation assay and co-localization studies using super-resolution microscopy. Immunochemistry and super-resolution microscopy enabled determination of the subcellular localizations of CDR2 and CDR2L. In paper III, the potential of including CDR2L as a diagnostic marker in PCD was assessed by developing two in-house techniques, cell-based assay for CDR2L and western blot analysis of recombinant CDR2 and CDR2L. Results: We show that CDR2L is the major Yo antigen and that including CDR2L as a marker in the diagnosis of PCD greatly improves test specificity. Furthermore, the subcellular location of CDR2L is found to be in the cell cytoplasm, more specifically in direct contact with the ribosomal subunit protein, rpS6. CDR2 localizes to the nucleus in contact with nuclear speckle proteins, SON and eIF4A3. Conclusions: Identifying CDR2L as the major Yo antigen is an important finding in the work of increasing test specificity for PCD diagnosis, and is essential for further investigation of PCD pathogenesis. However, a central role of CDR2 in PCD can not be excluded. By determining the subcellular locations of the proteins and binding partners, we are one step closer in understanding the functions of CDR2 and CDR2L in anti-Yo associated PCD.
Består avPaper I: Krakenes, T., Herdlevaer, I., Raspotnig, M., Haugen, M., Schubert, M., & Vedeler, C. A. (2019). CDR2L Is the Major Yo Antibody Target in Paraneoplastic Cerebellar Degeneration. Ann Neurol. doi:10.1002/ana.25511. The article is available at: https://hdl.handle.net/1956/21523
Paper II: Herdlevær, I., Kråkenes, T., Schubert, M., & Vedeler, C. A. (2020). "Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration." Annals of clinical and translational neurology 7(11): 2231-2242. The article is available at: https://hdl.handle.net/11250/2728184
Paper III: Herdlevær, I., Haugen, M., Mazengia K., Totland, C., Vedeler, C. A. (2021). "Paraneoplastic Cerebellar Degeneration: The Importance of Including CDR2L as a Diagnostic Marker." Neurology(R) neuroimmunology & neuroinflammation 8(2). The article is available in the thesis file. The article is also available at: https://doi.org/10.1212/NXI.0000000000000963