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Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.
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Infecções por Vírus Epstein-Barr , Esclerose Múltipla , Animais , Linfócitos B , Moléculas de Adesão Celular Neurônio-Glia , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Camundongos , Proteínas do Tecido NervosoRESUMO
OBJECTIVE: Co-occurring anti-tripartite motif-containing protein 9 and 67 autoantibodies (TRIM9/67-IgG) have been reported in only a very few cases of paraneoplastic cerebellar syndrome. The value of these biomarkers and the most sensitive methods of TRIM9/67-IgG detection are not known. METHODS: We performed a retrospective, multicenter study to evaluate the cerebrospinal fluid and serum of candidate TRIM9/67-IgG cases by tissue-based immunofluorescence, peptide phage display immunoprecipitation sequencing, overexpression cell-based assay (CBA), and immunoblot. Cases in which TRIM9/67-IgG was detected by at least 2 assays were considered TRIM9/67-IgG positive. RESULTS: Among these cases (n = 13), CBA was the most sensitive (100%) and revealed that all cases had TRIM9 and TRIM67 autoantibodies. Of TRIM9/67-IgG cases with available clinical history, a subacute cerebellar syndrome was the most common presentation (n = 7/10), followed by encephalitis (n = 3/10). Of these 10 patients, 70% had comorbid cancer (7/10), 85% of whom (n = 6/7) had confirmed metastatic disease. All evaluable cancer biopsies expressed TRIM9 protein (n = 5/5), whose expression was elevated in the cancerous regions of the tissue in 4 of 5 cases. INTERPRETATION: TRIM9/67-IgG is a rare but likely high-risk paraneoplastic biomarker for which CBA appears to be the most sensitive diagnostic assay. ANN NEUROL 2023;94:1086-1101.
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Proteínas do Tecido Nervoso , Degeneração Paraneoplásica Cerebelar , Humanos , Estudos Retrospectivos , Proteínas do Tecido Nervoso/metabolismo , Biomarcadores/líquido cefalorraquidiano , Autoanticorpos/líquido cefalorraquidiano , Imunoglobulina GRESUMO
Although B cells are implicated in multiple sclerosis (MS) pathophysiology, a predictive or diagnostic autoantibody remains elusive. In this study, the Department of Defense Serum Repository (DoDSR), a cohort of over 10 million individuals, was used to generate whole-proteome autoantibody profiles of hundreds of patients with MS (PwMS) years before and subsequently after MS onset. This analysis defines a unique cluster in approximately 10% of PwMS who share an autoantibody signature against a common motif that has similarity with many human pathogens. These patients exhibit antibody reactivity years before developing MS symptoms and have higher levels of serum neurofilament light (sNfL) compared to other PwMS. Furthermore, this profile is preserved over time, providing molecular evidence for an immunologically active preclinical period years before clinical onset. This autoantibody reactivity was validated in samples from a separate incident MS cohort in both cerebrospinal fluid and serum, where it is highly specific for patients eventually diagnosed with MS. This signature is a starting point for further immunological characterization of this MS patient subset and may be clinically useful as an antigen-specific biomarker for high-risk patients with clinically or radiologically isolated neuroinflammatory syndromes.
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Autoanticorpos , Esclerose Múltipla , Proteínas de Neurofilamentos , Humanos , Esclerose Múltipla/imunologia , Esclerose Múltipla/sangue , Autoanticorpos/sangue , Autoanticorpos/imunologia , Proteínas de Neurofilamentos/sangue , Proteínas de Neurofilamentos/imunologia , Biomarcadores/sangue , Estudos de Coortes , Feminino , Masculino , Adulto , Pessoa de Meia-IdadeRESUMO
Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on measurement of vitamin B12 in the blood, which may not accurately reflect the concentration in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro by depleting its target from the cell surface. Despite a normal serum concentration, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased B12 in the CSF and clinical improvement. Optofluidic screening enabled isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier (BBB). Autoantibodies targeting the same epitope of CD320 were identified in seven other patients with neurologic deficits of unknown etiology, 6% of healthy controls, and 21.4% of a cohort of patients with neuropsychiatric lupus. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we found that the low-density lipoprotein receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.
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Autoanticorpos , Deficiência de Vitamina B 12 , Vitamina B 12 , Humanos , Deficiência de Vitamina B 12/imunologia , Vitamina B 12/sangue , Autoanticorpos/sangue , Autoanticorpos/imunologia , Feminino , Receptores de Superfície Celular/metabolismo , Antígenos CD/metabolismo , Pessoa de Meia-Idade , Doenças Autoimunes/imunologia , Doenças Autoimunes/sangue , Barreira Hematoencefálica/metabolismo , MasculinoRESUMO
Although B cells are implicated in multiple sclerosis (MS) pathophysiology, a predictive or diagnostic autoantibody remains elusive. Here, the Department of Defense Serum Repository (DoDSR), a cohort of over 10 million individuals, was used to generate whole-proteome autoantibody profiles of hundreds of patients with MS (PwMS) years before and subsequently after MS onset. This analysis defines a unique cluster of PwMS that share an autoantibody signature against a common motif that has similarity with many human pathogens. These patients exhibit antibody reactivity years before developing MS symptoms and have higher levels of serum neurofilament light (sNfL) compared to other PwMS. Furthermore, this profile is preserved over time, providing molecular evidence for an immunologically active prodromal period years before clinical onset. This autoantibody reactivity was validated in samples from a separate incident MS cohort in both cerebrospinal fluid (CSF) and serum, where it is highly specific for patients eventually diagnosed with MS. This signature is a starting point for further immunological characterization of this MS patient subset and may be clinically useful as an antigen-specific biomarker for high-risk patients with clinically- or radiologically-isolated neuroinflammatory syndromes.
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[This corrects the article DOI: 10.3389/fneur.2021.728700.].
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OBJECTIVE: To identify the autoantigen in 2 individuals with possible seronegative paraneoplastic neuropathy. METHODS: Serum and CSF were screened by tissue-based assay and panned for candidate autoantibodies by phage display immunoprecipitation sequencing (PhIP-Seq). The candidate antigen was validated by immunostaining knockout tissue and HEK 293T cell-based assay. RESULTS: Case 1 presented with gait instability, distal lower extremity numbness, and paresthesias after a recent diagnosis of serous uterine and fallopian carcinoma. Case 2 had a remote history of breast adenocarcinoma and presented with gait instability, distal lower extremity numbness, and paresthesias that progressed to generalized weakness. CSF and serum from both patients immunostained the axon initial segment (AIS) and node of Ranvier (NoR) of mice and enriched ßIV-spectrin by PhIP-Seq. Patient CSF and serum failed to immunostain NoRs in dorsal root sensory neurons from ßI/ßIV-deficient mice. ßIV-spectrin autoantibodies were confirmed by overexpression of AIS and nodal ßIV-spectrin isoforms Σ1 and Σ6 by a cell-based assay. ßIV-spectrin was not enriched in a combined 4,815 PhIP-Seq screens of healthy and other neurologic disease patients. DISCUSSION: Therefore, ßIV-spectrin autoantibodies may be a marker of paraneoplastic neuropathy. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that ßIV-spectrin antibodies are specific autoantibody biomarkers for paraneoplastic neuropathy.
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Polineuropatia Paraneoplásica , Espectrina , Humanos , Autoanticorpos , Hipestesia , Parestesia , Animais , CamundongosRESUMO
Neuroinvasive infection is the most common cause of meningoencephalitis in people living with human immunodeficiency virus (HIV), but autoimmune etiologies have been reported. We present the case of a 51-year-old man living with HIV infection with steroid-responsive meningoencephalitis whose comprehensive pathogen testing was non-diagnostic. Subsequent tissue-based immunofluorescence with acute-phase cerebrospinal fluid revealed anti-neural antibodies localizing to the axon initial segment (AIS), the node of Ranvier (NoR), and the subpial space. Phage display immunoprecipitation sequencing identified ankyrinG (AnkG) as the leading candidate autoantigen. A synthetic blocking peptide encoding the PhIP-Seq-identified AnkG epitope neutralized CSF IgG binding to the AIS and NoR, thereby confirming a monoepitopic AnkG antibody response. However, subpial immunostaining persisted, indicating the presence of additional autoantibodies. Review of archival tissue-based staining identified candidate AnkG autoantibodies in a 60-year-old woman with metastatic ovarian cancer and seizures that were subsequently validated by cell-based assay. AnkG antibodies were not detected by tissue-based assay and/or PhIP-Seq in control CSF (N = 39), HIV CSF (N = 79), or other suspected and confirmed neuroinflammatory CSF cases (N = 1,236). Therefore, AnkG autoantibodies in CSF are rare but extend the catalog of AIS and NoR autoantibodies associated with neurological autoimmunity.
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The development of autoimmune antibody panels has improved the diagnosis of paraneoplastic neurological disorders (PNDs) of the brain and spinal cord. Here, we present a case of a woman with a history of breast cancer who presented with a subacute sensory ataxia that progressed over 18 months. Her examination and diagnostic studies were consistent with a myelopathy. Metabolic, infectious, and autoimmune testing were non-diagnostic. However, she responded to empirical immunosuppression, prompting further workup for an autoimmune etiology. An unbiased autoantibody screen utilizing phage display immunoprecipitation sequencing (PhIP-Seq) identified antibodies to the anti-Yo antigens cerebellar degeneration related protein 2 like (CDR2L) and CDR2, which were subsequently validated by immunoblot and cell-based overexpression assays. Furthermore, CDR2L protein expression was restricted to HER2 expressing tumor cells in the patient's breast tissue. Recent evidence suggests that CDR2L is likely the primary antigen in anti-Yo paraneoplastic cerebellar degeneration, but anti-Yo myelopathy is poorly characterized. By immunostaining, we detected neuronal CDR2L protein expression in the murine and human spinal cord. This case demonstrates the diagnostic utility of unbiased assays in patients with suspected PNDs, supports prior observations that anti-Yo PND can be associated with isolated myelopathy, and implicates CDR2L as a potential antigen in the spinal cord.
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Retraction notice to: "Remission of Subacute Psychosis in a COVID-19 Patient With an Antineuronal Autoantibody After Treatment With Intravenous Immunoglobulin" by Lindsay S. McAlpine, Brooke Lifland, Joseph R. Check, Gustavo A. Angarita, Thomas T. Ngo, Samuel J. Pleasure, Michael R. Wilson, Serena S. Spudich, Shelli F. Farhadian, and Christopher M. Bartley (Biol Psychiatry 2021; 90:e23-e26); https://doi.org/10.1016/j.biopsych.2021.03.033. This article has been retracted at the request of corresponding author Christopher Bartley, with agreement from all authors and with approval from Biological Psychiatry Editor John H. Krystal, M.D. See Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). After this article was published, the authors determined that two cerebrospinal fluid (CSF) samples were inadvertently confused, resulting in publication of the wrong COVID-19 patient's immunostaining data. The authors determined that the two CSF samples came from COVID-19 patients with sequential case identifiers (i.e., one identifier ended in a "5" and the other in a "6"). To determine whether the published immunostaining results were produced by CSF from another COVID-19 patient, the authors reperformed the mouse brain immunostaining experiments using additional aliquots of stored CSF from the two research participants in question, as well as with the remaining CSF that had been used in the publication. After repeating the immunostaining with these CSF samples, two blinded raters were able to state unequivocally that the CSF samples from the two COVID-19 patients had been confused. Therefore, while the clinical features of the case report are accurate and unaffected, the research data belong to another COVID-19 research participant, not the one described in the published case report. The authors voluntarily informed the Journal of this honest error upon its discovery. All authors agree to retract this paper and sincerely apologize for having allowed the incorrect images to be published with this case report. To avoid misinterpretation of the research findings, both the editors and authors concur that the only proper course of action was to retract this version of the paper. However, this COVID-19 psychosis case remains of clinical interest because of the patient's clear response to immunotherapy. Therefore, the authors are revising the paper, which the Journal will consider further for publication.
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COVID-19 , Transtornos Psicóticos , Autoanticorpos , Humanos , Imunoglobulinas Intravenosas , Transtornos Psicóticos/tratamento farmacológico , SARS-CoV-2RESUMO
Importance: Neuropsychiatric manifestations of COVID-19 have been reported in the pediatric population. Objective: To determine whether anti-SARS-CoV-2 and autoreactive antibodies are present in the cerebrospinal fluid (CSF) of pediatric patients with COVID-19 and subacute neuropsychiatric dysfunction. Design, Setting, and Participants: This case series includes 3 patients with recent SARS-CoV-2 infection as confirmed by reverse transcriptase-polymerase chain reaction or IgG serology with recent exposure history who were hospitalized at the University of California, San Francisco Benioff Children's Hospital and for whom a neurology consultation was requested over a 5-month period in 2020. During this period, 18 total children were hospitalized and tested positive for acute SARS-CoV-2 infection by reverse transcriptase-polymerase chain reaction or rapid antigen test. Main Outcomes and Measures: Detection and characterization of CSF anti-SARS-CoV-2 IgG and antineural antibodies. Results: Of 3 included teenaged patients, 2 patients had intrathecal anti-SARS-CoV-2 antibodies. CSF IgG from these 2 patients also indicated antineural autoantibodies on anatomic immunostaining. Autoantibodies targeting transcription factor 4 (TCF4) in 1 patient who appeared to have a robust response to immunotherapy were also validated. Conclusions and Relevance: Pediatric patients with COVID-19 and prominent subacute neuropsychiatric symptoms, ranging from severe anxiety to delusional psychosis, may have anti-SARS-CoV-2 and antineural antibodies in their CSF and may respond to immunotherapy.
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Anticorpos Antivirais/líquido cefalorraquidiano , Autoanticorpos/líquido cefalorraquidiano , COVID-19/complicações , COVID-19/imunologia , Transtornos Mentais/líquido cefalorraquidiano , Transtornos Mentais/etiologia , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Doenças do Sistema Nervoso/etiologia , Adolescente , Animais , Ansiedade/etiologia , Ansiedade/psicologia , Autoimunidade , Feminino , Humanos , Masculino , Fumar Maconha/imunologia , Camundongos , Transtornos dos Movimentos/etiologia , Exame Neurológico , Fator de Transcrição 4/imunologiaRESUMO
Individuals with coronavirus disease 2019 (COVID-19) frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single-cell RNA sequencing (scRNA-seq) and cytokine analyses of cerebrospinal fluid (CSF) and blood from individuals with COVID-19 with neurological symptoms, we find compartmentalized, CNS-specific T cell activation and B cell responses. All affected individuals had CSF anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are present only during brain infection and not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from an individual with COVID-19 and found that these monoclonal antibodies (mAbs) target antiviral and antineural antigens, including one mAb that reacted to spike protein and neural tissue. CSF immunoglobulin G (IgG) from 5 of 7 patients showed antineural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of individuals with COVID-19 and suggests a role of autoimmunity in neurologic sequelae of COVID-19.
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Periventricular heterotopia (PVH) is a congenital malformation of human cerebral cortex frequently associated with Filamin-A (FLN-A) mutations but the pathogenetic mechanisms remain unclear. Here, we show that the MEKK4 (MAP3K4) pathway is involved in Fln-A regulation and PVH formation. MEKK4(-/-) mice developed PVH associated with breaches in the neuroependymal lining which were largely comprised of neurons that failed to reach the cortical plate. RNA interference (RNAi) targeting MEKK4 also impaired neuronal migration. Expression of Fln was elevated in MEKK4(-/-) forebrain, most notably near sites of failed neuronal migration. Importantly, recombinant MKK4 protein precipitated a complex containing MEKK4 and Fln-A, and MKK4 mediated signaling between MEKK4 and Fln-A, suggesting that MKK4 may bridge these molecules during development. Finally, we showed that wild-type FLN-A overexpression inhibited neuronal migration. Collectively, our results demonstrate a link between MEKK4 and Fln-A that impacts neuronal migration initiation and provides insight into the pathogenesis of human PVH.
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Movimento Celular/fisiologia , Proteínas Contráteis/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MAP Quinase Quinase Quinase 4/fisiologia , Proteínas dos Microfilamentos/biossíntese , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Diferenciação Celular/fisiologia , Movimento Celular/genética , Proteínas Contráteis/genética , Proteínas Contráteis/fisiologia , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroporação , Feminino , Filaminas , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Imuno-Histoquímica , MAP Quinase Quinase Quinase 4/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Neurônios/ultraestrutura , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Fosforilação , Gravidez , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , RNA Interferente Pequeno/genética , Células-Tronco/fisiologiaRESUMO
Postmitotic cortical neurons that fail to initiate migration can remain near their site of origin and form persistent periventricular nodular heterotopia (PH). In human telencephalon, this malformation is most commonly associated with Filamin-A (FLNa) mutations. The lack of genetic animal models that reliably produce PH has delayed our understanding of the underlying molecular mechanisms. This review examines PH pathogenesis using a new mouse model. Although PH have not been observed in Flna-deficient mice generated thus far, the loss of MEKK4, a regulator of Flna, produces striking PH in mice and offers insight into the mechanisms involved in neuronal migration initiation. Elucidating the basic functions of FLNa and associated molecules is crucial for understanding the causes of PH and for developing prevention for at-risk patients.
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Movimento Celular/genética , Córtex Cerebral/crescimento & desenvolvimento , MAP Quinase Quinase Quinase 4/genética , Heterotopia Nodular Periventricular/genética , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Modelos Animais de Doenças , Filaminas , Humanos , MAP Quinase Quinase Quinase 4/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Heterotopia Nodular Periventricular/metabolismo , Heterotopia Nodular Periventricular/patologia , Transdução de Sinais/fisiologiaRESUMO
One third of COVID-19 patients develop significant neurological symptoms, yet SARS-CoV-2 is rarely detected in central nervous system (CNS) tissue, suggesting a potential role for parainfectious processes, including neuroimmune responses. We therefore examined immune parameters in cerebrospinal fluid (CSF) and blood samples from a cohort of patients with COVID-19 and significant neurological complications. We found divergent immunological responses in the CNS compartment, including increased levels of IL-12 and IL-12-associated innate and adaptive immune cell activation. Moreover, we found increased proportions of B cells in the CSF relative to the periphery and evidence of clonal expansion of CSF B cells, suggesting a divergent intrathecal humoral response to SARS-CoV-2. Indeed, all COVID-19 cases examined had anti-SARS-CoV-2 IgG antibodies in the CSF whose target epitopes diverged from serum antibodies. We directly examined whether CSF resident antibodies target self-antigens and found a significant burden of CNS autoimmunity, with the CSF from most patients recognizing neural self-antigens. Finally, we produced a panel of monoclonal antibodies from patients' CSF and show that these target both anti-viral and anti-neural antigens-including one mAb specific for the spike protein that also recognizes neural tissue. This exploratory immune survey reveals evidence of a compartmentalized and self-reactive immune response in the CNS meriting a more systematic evaluation of neurologically impaired COVID-19 patients.
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BACKGROUND: Choroid plexus papillomas (CPPs) are rare benign tumors, and the pigmented subtype is observed even more rarely. CASE DESCRIPTION: We present the case of a 43-year-old woman with complaints of headache and progressive left monocular visual deterioration, whose initial plain computed tomography CT scan showed an ovate high-density tumor located within the insellar region. Magnetic resonance imaging revealed a homogeneously contrast-enhancing tumor extending from the sella turcica to the suprasellar cistern. Single-nostril transsphenoidal endoscopic resection followed by subfrontal subtotal resection was performed in this patient. Postoperative histology revealed that the tumor consisted of hyperchromatic tissue with papillary features. Higher-resolution examination of the tissue revealed this tissue was composed of hyperplastic columnar epithelial cells with hyperchromatic cytoplasmic pigment. Subsequent immunohistochemistry identified the lesion as a pigmented choroid plexus papilloma. Here we review the current literature, discuss the origin of the tumor, the differential diagnosis, and the roles of surgery and radiotherapy. CONCLUSIONS: This case study provides important clinical information for the evaluation, diagnosis, and treatment of pigmented CPP in the sellar region.
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Papiloma do Plexo Corióideo/diagnóstico por imagem , Papiloma do Plexo Corióideo/cirurgia , Pigmentação , Sela Túrcica/diagnóstico por imagem , Sela Túrcica/cirurgia , Adulto , Feminino , HumanosRESUMO
The fragile X mental retardation protein (FMRP) is an mRNA-binding regulator of protein translation that associates with 4-6% of brain transcripts and is central to neurodevelopment. Autism risk genes' transcripts are overrepresented among FMRP-binding mRNAs, and FMRP loss-of-function mutations are responsible for fragile X syndrome, the most common cause of monogenetic autism. It is thought that FMRP-dependent translational repression is governed by the phosphorylation of serine residue 499 (S499). However, recent evidence suggests that S499 phosphorylation is not modulated by metabotropic glutamate receptor class I (mGluR-I) or protein phosphatase 2A (PP2A), two molecules shown to regulate FMRP translational repression. Moreover, the mammalian FMRP S499 kinase remains unknown. We found that casein kinase II (CK2) phosphorylates murine FMRP S499. Further, we show that phosphorylation of FMRP S499 permits phosphorylation of additional, nearby residues. Evidence suggests that these nearby residues are modulated by mGluR-I and PP2A pathways. These data support an alternative phosphodynamic model of FMRP that is harmonious with prior studies and serves as a framework for further investigation.
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Caseína Quinase II/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Animais , Western Blotting , Caseína Quinase II/antagonistas & inibidores , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Células HEK293 , Humanos , Espectrometria de Massas , Camundongos , Naftiridinas/farmacologia , Fenazinas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Biossíntese de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Hyperactive mammalian target of rapamycin (mTOR) is associated with cognitive deficits in several neurological disorders including tuberous sclerosis complex (TSC). The phosphorylation of the mRNA-binding protein FMRP reportedly depends on mTOR complex 1 (mTORC1) activity via p70 S6 kinase 1 (S6K1). Because this phosphorylation is thought to regulate the translation of messages important for synaptic plasticity, we explored whether FMRP phosphorylation of the S6K1-dependent residue (S499) is altered in TSC and states of dysregulated TSC-mTORC1 signaling. Surprisingly, we found that FMRP S499 phosphorylation was unchanged in heterozygous and conditional Tsc1 knockout mice despite significantly elevated mTORC1-S6K1 activity. Neither up- nor down-regulation of the mTORC1-S6K1 axis in vivo or in vitro had any effect on phospho-FMRP S499 levels. In addition, FMRP S499 phosphorylation was unaltered in S6K1-knockout mice. Collectively, these data strongly suggest that FMRP S499 phosphorylation is independent of mTORC1-S6K1 activity and is not altered in TSC.