Pathogenesis Of Paraneoplastic Cerebellar Degeneration Research Articles

Multi-omics profiling reveals dysregulated ribosome biogenesis and impaired cell proliferation following knockout of CDR2L

BackgroundCerebellar degeneration-related (CDR) proteins are associated with paraneoplastic cerebellar degeneration (PCD) – a rare, neurodegenerative disease caused by tumour-induced autoimmunity against neural antigens resulting in degeneration of Purkinje neurons in the cerebellum. The pathogenesis of PCD is unknown, in large part due to our limited understanding of the functions of CDR proteins. To this end, we performed an extensive, multi-omics analysis of CDR-knockout cells focusing on the CDR2L protein, to gain a deeper understanding of the properties of the CDR proteins in ovarian cancer.MethodsOvarian cancer cell lines lacking either CDR1, CDR2, or CDR2L were analysed using RNA sequencing and mass spectrometry-based proteomics to assess changes to the transcriptome, proteome and secretome in the absence of these proteins.ResultsFor each knockout cell line, we identified sets of differentially expressed genes and proteins. CDR2L-knockout cells displayed a distinct expression profile compared to CDR1- and CDR2-knockout cells. Knockout of CDR2L caused dysregulation of genes involved in ribosome biogenesis, protein translation, and cell cycle progression, ultimately causing impaired cell proliferation in vitro. Several of these genes showed a concurrent upregulation at the transcript level and downregulation at the protein level.ConclusionsOur study provides the first integrative multi-omics analysis of the impact of knockout of the CDR genes, providing both new insights into the biological properties of the CDR proteins in ovarian cancer, and a valuable resource for future investigations into the CDR proteins.

GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1β and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.

Debate on the pathogenesis of paraneoplastic cerebellar degeneration with anti‐Yo antibody: T‐cell mediated or antibody mediated?

AbstractIt is widely believed that paraneoplastic neurological syndromes with antibodies against intracellular antigens are a cytotoxic T‐cell‐mediated disease triggered in the process of cancer immunity. During this process, coactivation of a humoral immune mechanism is elicited, and autoantibodies against onconeural antigens are produced. These onconeural proteins are usually located in the neuronal cytoplasm or nucleus, and are not targets of antibody‐mediated neuronal damage. Greenlee et al. asserted that the anti‐Yo antibody had a direct effect on Purkinje cell apoptosis, which conflicts with the commonly believed view.

Intrathecal injection of P/Q type voltage-gated calcium channel antibodies from paraneoplastic cerebellar degeneration cause ataxia in mice

The role of antibodies against the P/Q type voltage-gated calcium channels (VGCC-ab) in the pathogenesis of paraneoplastic cerebellar degeneration (PCD) and lung cancer is unclear. We evaluated in mice the effect of intrathecal injection of IgG purified from serum of a patient with both PCD and Lambert-Eaton myasthenic syndrome (LEMS), and from another patient with isolated LEMS. Mice injected with PCD/LEMS IgG developed marked, reversible ataxia compared with those injected with LEMS or control IgG. These findings suggest that P/Q-type VGCC-ab may play a role in the pathogenesis of ataxia in patients with PCD and SCLC.

Purkinje Cell Death After Uptake of Anti-Yo Antibodies in Cerebellar Slice Cultures

Paraneoplastic cerebellar degeneration accompanying gynecological and breast cancers is characteristically accompanied by a serum and cerebrospinal fluid (CSF) antibody response, termed "anti-Yo," which reacts with cytoplasmic proteins of cerebellar Purkinje cells. Because these antibodies interact with cytoplasmic rather than cell surface membrane proteins, their role in causing Purkinje cell death has been questioned. To address this issue, we studied the interaction of anti-Yo antibodies with Purkinje cells in slice (organotypic) cultures of rat cerebellum. We incubated cultures with immunoglobulin G (IgG)-containing anti-Yo antibodies using titers of anti-Yo antibody equivalent to those found in CSF of affected patients. Cultures were then studied in real time and after fixation for potential uptake of antibody and induction of cell death. Anti-Yo antibodies delivered in serum, CSF, or purified IgG were taken up by viable Purkinje cells, accumulated intracellularly, and were associated with cell death. Normal IgG was also taken up by Purkinje cells but did not accumulate and did not affect cell viability. These findings indicate that autoantibodies directed against intracellular Purkinje cell proteins can be taken up to cause cell death and suggest that anti-Yo antibody may be directly involved in the pathogenesis of paraneoplastic cerebellar degeneration.

Functional analysis of CD8 + T cell responses to the onconeural self protein cdr2 in patients with paraneoplastic cerebellar degeneration

Paraneoplastic cerebellar degeneration (PCD) is linked to an immune response against cerebellar degeneration related antigen 2 (cdr2) co-expressed in tumor and Purkinje neurons. Here, comprehensive immune-assessment assays were used to analyze CD8 + T cells from 7 PCD patients, but no evidence was found of CD8 + T cells specific for either of two previously described cdr2 epitopes (cdr2-1 and cdr2-2). In contrast, viral-specific CD8 + T cells from healthy volunteers and PCD patients were measurable. These findings are inconsistent with an obligate role for cdr2-1- or cdr2-2-specific CD8 + T cells in the pathogenesis of PCD.

Effect of a paraneoplastic cerebellar degeneration-associated neural protein on B-myb promoter activity

In this study, we have shown that a paraneoplastic cerebellar degeneration (PCD)-associated antigen, pcd17, binds to a cell cycle-related protein, MRG15. MRG15 derepresses the E2F-responsive B-myb promoter. The pcd17 antigen inhibits the derepression of the B-myb transcriptional activity by MRG15, and, as a result, pcd17 represses the promoter. Delivery of anti-Purkinje cell antibodies (anti-Yo) into the cells inhibits the repression of B-myb promoter activity by pcd17. Because derepression of the B-myb promoter has been implicated in neuronal death, the results suggest the possible role of the antibodies in the pathogenesis of PCD.

Reduction of P/Q-type calcium channels in the postmortem cerebellum of paraneoplastic cerebellar degeneration with Lambert-Eaton myasthenic syndrome.

The aim of this study was to clarify whether autoimmunity against P/Q-type voltage-gated calcium channels (VGCCs) in the cerebellum was associated with the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome (LEMS). We used human autopsy cerebellar tissues from three PCD-LEMS patients and six other disease patients including one with LEMS as the controls. We compared cerebellar P/Q-type VGCC in these patients and controls for the amount and ratio of autoantibody-channel complex using an 125I-omega-conotoxin MVIIC-binding assay with Scatchard analysis, and their distribution using autoradiography. The quantity of cerebellar P/Q-type VGCC measured by Scatchard analysis were reduced in PCD-LEMS patients (63.0 +/- 7.0 fmol/mg, n = 3), compared with the controls (297.8 +/- 38.9 fmol/mg, n = 6). The ratio of autoantibody-VGCC complexes to total P/Q-type VGCCs measured by immunoprecipitation assay were increased in PCD-LEMS patients. We analysed cerebellar specimens by autoradiography using (125)I-omega-conotoxin MVIIC, which specifically binds to P/Q-type VGCCs. In PCD-LEMS cerebellum, the toxin binding sites of P/Q-type VGCCs were markedly reduced compared with controls, especially in the molecular layer, which is the richest area of P/Q-type VGCCs in the normal cerebellum. This suggests that P/Q-type VGCCs of the cerebellar molecular layer is the immunological target in developing PCD-LEMS.

Anti-Yo Positive Paraneoplastic Cerebellar Degeneration Associated with Ovarian Carcinoma: Case Report and Review of the Literature

Paraneoplastic cerebellar degeneration (PCD) is a rare nonmetastatic neurological complication in cancer patients. Anti-Yo is one of the anti-onconeural antibodies found in PCD patients. It is believed that anti-Yo occurs almost always in women and is most likely associated with gynecologic or breast cancers, although exceptions exist. Here we report a PCD patient with ovarian cancer having high-titer anti-Yo. The acute onset of her PCD symptoms mimicked that of a stroke. Her ovarian cancer tissue contained abundant plasma cells and lymphocytes. After a thorough review of the literature, we propose a schematic hypothesis for the autoimmune pathogenesis of PCD. Despite anecdotal case reports of neurological improvement with different combinations of treatment, including IVIg, there is still no definitely effective treatment for PCD. Further research on the pathogenesis of PCD may lead to more effective therapies.

Involvement of amyloid precursor protein in synaptogenesis

The role of antibodies against the P/Q type voltage-gated calcium channels (VGCC-ab) in the pathogenesis of paraneoplastic cerebellar degeneration (PCD) and lung cancer is unclear. We evaluated in mice the effect of intrathecal injection of IgG purified from serum of a patient with both PCD and Lambert-Eaton myasthenic syndrome (LEMS), and from another patient with isolated LEMS. Mice injected with PCD/LEMS IgG developed marked, reversible ataxia compared with those injected with LEMS or control IgG. These findings suggest that P/Q-type VGCC-ab may play a role in the pathogenesis of ataxia in patients with PCD and SCLC.

Uptake of systemically administered human anticerebellar antibody by rat Purkinje cells following blood-brain barrier disruption.

Paraneoplastic cerebellar degeneration accompanying gynecological or breast malignancies is frequently associated with an autoantibody response, termed "type I" or "anti-Yo" directed against cytoplasmic antigens of cerebellar Purkinje cells. The role of this antibody response in the pathogenesis of paraneoplastic cerebellar degeneration is unknown; however, it is also not known whether anti-Purkinje cell antibodies from the systemic circulation bind to target Purkinje cell antigens under the conditions of brain inflammation and blood-brain barrier disruption, which are frequently present at the onset of cerebellar symptoms. Inbred Lewis rats received intraperitoneal injections of type I or normal IgG in the setting of blood-brain barrier disruption induced by adoptive transfer of experimental allergic encephalomyelitis (EAE) and were killed after 24, 48, and 96 h. Brains of these animals were studied histologically for evidence of EAE and immunohistochemically for binding of human or endogenous rat IgG to target neurons. Rat IgG was detected around vessels and in Purkinje cells of all animals studied. Human IgG was detected around vessels of all animals. In animals examined 96h after receiving type I human IgG, human IgG was identified within processes of Purkinje cells and within occasional Purkinje cell bodies. Uptake of type I IgG by other cell types was not observed, and neuronal uptake of IgG was not seen in brains of animals receiving normal human IgG. Our data demonstrate that circulating type I IgG is internalized by cerebellar Purkinje cells in the setting of blood-brain barrier disruption and suggest a mechanism by which an antibody response directed against cytoplasmic antigens of Purkinje cells may reach target antigens at the onset of paraneoplastic cerebellar degeneration.

Internalization of plasma proteins by cerebellar Purkinje cells

Animal studies suggest that Purkinje cells internalize proteins from the blood and CSF. This process may relate to the pathogenesis of paraneoplastic cerebellar degeneration in patients with anti-Purkinje cell antibodies. To determine if human Purkinje cells may also internalize plasma proteins, cerebellar tissue was taken from routine autopsies of eight patients without neurologic or neoplastic disease. Several plasma proteins including IgG, IgA, IgM, transferrin, albumin and alpha-2-macroglobulin were detected by immunohistochemistry within the cytoplasm of Purkinje cells. Internalized proteins frequently filled the entire soma and major dendrites, sparing the nucleus. Vascular structures were also immunolabeled, while glia internalized plasma proteins differentially, with oligodendrocytes selectively internalizing transferrin. Purkinje cells were the most numerous and heavily labeled neuronal cell type in spite of their small numerical representation in the cerebellar neuronal population. Our results are compatible with previous animal studies, and suggest that internalization of specific antibodies could contribute to the pathogenesis of Purkinje cell loss in paraneoplastic cerebellar degeneration.

Partial characterization of the Purkinje cell antigens in paraneoplastic cerebellar degeneration.

Serum from seven patients with paraneoplastic cerebellar degeneration contained anti-Purkinje cell antibodies. The samples were examined by immunoblotting to determine whether they recognized common antigens in isolated human Purkinje cell neurons. Two groups of antigens were detected by all seven sera with Mr 62/64 kd and 34 to 38 kd, both of which contributed to the Purkinje cell antigens detected immunohistochemically. These reactivities were absent from all controls tested. These antibodies may play a role in the pathogenesis of paraneoplastic cerebellar degeneration.