Andrew L. Mammen

Characterization of Multiple Phosphorylation Sites on the AMPA Receptor GluR1 Subunit

We have characterized the phosphorylation of the glutamate receptor subunit GluR1, using biochemical and electrophysiological techniques. GluR1 is phosphorylated on multiple sites that are all located on the C-terminus of the protein. Cyclic AMP-dependent protein kinase specifically phosphorylates SER-845 of GluR1 in transfected HEK cells and in neurons in culture. Phosphorylation of this residue results in a 40% potentiation of the peak current through GluR1 homomeric channels. In addition, protein kinase C specifically phosphorylates Ser-831 of GluR1 in HEK-293 cells and in cultured neurons. These results are consistent with the recently proposed transmembrane topology models of glutamate receptors, in which the C-terminus is intracellular. In addition, the modulation of GluR1 by PKA phosphorylation of Ser-845 suggests that phosphorylation of this residue may underlie the PKA-induced potentiation of AMPA receptors in neurons.

Phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole4-propionic acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II.

Modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) receptors in the brain by protein phosphorylation may play a crucial role in the regulation of synaptic plasticity. Previous studies have demonstrated that calmodulin (CaM) kinase II can phosphorylate and modulate AMPA receptors. However, the sites of CaM kinase phosphorylation have not been unequivocally identified. In the current study, we have generated two phosphorylation site-specific antibodies to analyze the phosphorylation of the glutamate receptor GluR1 subunit. These antibodies recognize GluR1 only when it is phosphorylated on serine residues 831 or 845. We have used these antibodies to demonstrate that serine 831 is specifically phosphorylated by CaM kinase II in transfected cells expressing GluR1 as well as in hippocampal slice preparations. Two-dimensional phosphopeptide mapping experiments indicate that Ser-831 is the major site of CaM kinase II phosphorylation on GluR1. In addition, treatment of hippocampal slice preparations with phorbol esters and forskolin increase the phosphorylation of serine 831 and 845, respectively, indicating that protein kinase C and protein kinase A phosphorylate these residues in hippocampal slices. These results identify the site of CaM kinase phosphorylation of the GluR1 subunit and demonstrate that GluR1 is multiply phosphorylated by protein kinase A, protein kinase C, and CaM kinase II in situ.

Autoantibodies against 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase in patients with statin‐associated autoimmune myopathy

OBJECTIVE In addition to inducing a self-limited myopathy, statin use is associated with an immune-mediated necrotizing myopathy (IMNM), with autoantibodies that recognize ∼200-kd and ∼100-kd autoantigens. The purpose of this study was to identify these molecules to help clarify the disease mechanism and facilitate diagnosis. METHODS The effect of statin treatment on autoantigen expression was addressed by immunoprecipitation using sera from patients. The identity of the ∼100-kd autoantigen was confirmed by immunoprecipitation of in vitro-translated 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) protein. HMGCR expression in muscle was analyzed by immunofluorescence. A cohort of myopathy patients was screened for anti-HMGCR autoantibodies by enzyme-linked immunosorbent assay and genotyped for the rs4149056 C allele, a predictor of self-limited statin myopathy. RESULTS Statin exposure induced expression of the ∼200-kd/∼100-kd autoantigens in cultured cells. HMGCR was identified as the ∼100-kd autoantigen. Competition experiments demonstrated no distinct autoantibodies recognizing the ∼200-kd protein. In muscle biopsy tissues from anti-HMGCR-positive patients, HMGCR expression was up-regulated in cells expressing neural cell adhesion molecule, a marker of muscle regeneration. Anti-HMGCR autoantibodies were found in 45 of 750 patients presenting to the Johns Hopkins Myositis Center (6%). Among patients ages 50 years and older, 92.3% had taken statins. The prevalence of the rs4149056 C allele was not increased in patients with anti-HMGCR. CONCLUSION Statins up-regulate the expression of HMGCR, the major target of autoantibodies in statin-associated IMNM. Regenerating muscle cells express high levels of HMGCR, which may sustain the immune response even after statins are discontinued. These studies demonstrate a mechanistic link between an environmental trigger and the development of sustained autoimmunity. Detection of anti-HMGCR autoantibodies may facilitate diagnosis and direct therapy.

A novel autoantibody recognizing 200-kd and 100-kd proteins is associated with an immune-mediated necrotizing myopathy

OBJECTIVE Myofiber necrosis without prominent inflammation is a nonspecific finding in patients with dystrophies and toxic or immune-mediated myopathies. However, the etiology of a necrotizing myopathy is often obscure, and the question of which patients would benefit from immunosuppression remains unanswered. The aim of this study was to identify novel autoantibodies in patients with necrotizing myopathy. METHODS Muscle biopsy specimens and serum samples were available for 225 patients with myopathy. Antibody specificities were determined by performing immunoprecipitations from (35)S-methionine-labeled HeLa cell lysates. Selected biopsy specimens were stained for membrane attack complex, class I major histocompatibility complex (MHC), and endothelial cell marker CD31. RESULTS Muscle biopsy specimens from 38 of 225 patients showed predominantly myofiber necrosis. Twelve of these patients had a known autoantibody association with or other etiology for their myopathy. Sixteen of the remaining 26 sera immunoprecipitated 200-kd and 100-kd proteins; this specificity was observed in only 1 of 187 patients without necrotizing myopathy. Patients with the anti-200/100 autoantibody specificity had proximal weakness (100%), high creatine kinase levels (mean maximum 10,333 IU/liter), and an irritable myopathy on electromyography (88%). Sixty-three percent of these patients had been exposed to statins prior to the onset of weakness. All patients responded to immunosuppressive therapy, and many experienced a relapse of weakness when the medication was tapered. Immunohistochemical studies showed membrane attack complex on small blood vessels in 6 of 8 patients and on the surface of non-necrotic myofibers in 4 of 8 patients. Five of 8 patients had abnormal capillary morphology, and 4 of 8 patients expressed class I MHC on the surface of non-necrotic myofibers. CONCLUSION An anti-200/100-kd specificity defines a subgroup of patients with necrotizing myopathy who previously were considered to be autoantibody negative. We propose that these patients have an immune-mediated myopathy that is frequently associated with prior statin use and should be treated with immunosuppressive therapy.

Dermatomyositis and polymyositis

Dermatomyositis (DM) and polymyositis (PM) are autoimmune myopathies characterized clinically by proximal muscle weakness, muscle inflammation, extramuscular manifestations, and frequently, the presence of autoantibodies. Although there is some overlap, DM and PM are separate diseases with different pathophysiological mechanisms. Furthermore, unique clinical phenotypes are associated with each of the myositis‐specific autoantibodies (MSAs) associated with these disorders. This review will focus on the clinical features, pathology, and immunogenetics of PM and DM with an emphasis on the importance of autoantibodies in defining unique phenotypes and, perhaps, as clues to help elucidate the mechanisms of disease.

Most patients with cancer-associated dermatomyositis have antibodies to nuclear matrix protein NXP-2 or transcription intermediary factor 1γ.

OBJECTIVE Since dermatomyositis (DM) is associated with an increased risk of malignancy, accurate identification of patients likely to harbor cancers is important. Using immunoprecipitations from radiolabeled cell lysates, several groups recently showed that anti-transcription intermediary factor 1γ (anti-TIF-1γ) antibodies are associated with malignancy in DM. We undertook this study to develop sensitive, specific assays to detect antibodies against TIF-1γ and nuclear matrix protein NXP-2 and to evaluate their association with malignancy in DM. METHODS To detect anti-TIF-1γ antibodies, immunoprecipitations were performed using lysates made from HeLa cells overexpressing TIF-1γ, with detection by immunoblotting. Anti-NXP-2 antibodies were assayed by immunoprecipitation using (35) S-methionine-labeled NXP-2 generated by in vitro transcription/translation. We analyzed patient sera from DM cohorts seen at the Stanford University Dermatology Clinic (n = 111) and the Johns Hopkins Myositis Center (n = 102). RESULTS A total of 17% and 38% of patients had antibodies against NXP-2 and TIF-1γ, respectively. Reactivity against either NXP-2 or TIF-1γ identified 83% of patients with cancer-associated DM. In addition to older age and male sex, cancer was associated with antibodies to NXP-2 or TIF-1γ on multivariate analysis (odds ratio 3.78 [95% confidence interval 1.33-10.8]). Stratification by sex revealed that anti-NXP-2 was specifically associated with cancer in males (odds ratio 5.78 [95% confidence interval 1.35-24.7]). CONCLUSION These studies demonstrate that anti-NXP-2 and anti-TIF-1γ antibodies are frequent DM specificities (found in 55% of patients) and are present in most patients with cancer-associated DM.

Rituximab therapy for myopathy associated with anti–signal recognition particle antibodies: A case series

The myopathy associated with anti–signal recognition particle (anti‐SRP) is a severe necrotizing immune‐mediated disease characterized by rapidly progressive proximal muscle weakness, markedly elevated serum creatine kinase (CK) levels, and poor responsiveness to traditional immunosuppressive therapies. Reports on the efficacy of B cell depletion therapy for anti‐SRP–associated myopathy are mixed. We describe 8 patients with anti‐SRP–associated myopathy and their response to treatment with the anti‐CD20 monoclonal antibody rituximab.

Antibody levels correlate with creatine kinase levels and strength in anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase-associated autoimmune myopathy

OBJECTIVE Autoantibodies recognizing 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) are found in patients with statin-associated immune-mediated necrotizing myopathy and, less commonly, in statin-unexposed patients with autoimmune myopathy. The main objective of this study was to define the association of anti-HMGCR antibody levels with disease activity. METHODS Anti-HMGCR levels, creatine kinase (CK) levels, and strength were assessed in anti-HMGCR-positive patients. Associations of antibody level with CK level and strength at visit 1 were analyzed in 55 patients, 40 of whom were exposed to statins. In 12 statin-exposed and 5 statin-unexposed patients with serum from 5 serial visits, the evolution of antibody levels, CK levels, and strength was investigated. RESULTS Antibody levels were associated with CK levels (P < 0.001), arm strength (P < 0.05), and leg strength (P < 0.05) at visit 1, but these associations were only significant among statin-exposed patients in stratified analyses. With immunosuppressive treatment over 26.2 ± 12.6 months (mean ± SD), antibody levels declined (P < 0.05) and arm abduction strength improved (P < 0.05) in the 17 patients followed up longitudinally. The separate analysis showed that statin-exposed patients developed decreased antibody levels (P < 0.01), decreased CK levels (P < 0.001), improved arm strength (P < 0.05), and improved hip flexion strength (P < 0.05) with treatment. Anti-HMGCR antibody levels did not normalize in any patient. CONCLUSION In the entire cohort, initial anti-HMGCR levels correlated with indicators of disease activity; with immunosuppressive treatment, antibody levels declined and arm strength improved. Statin-exposed patients had significant improvements in CK levels and strength whereas statin-unexposed patients did not, suggesting a phenotypic difference between statin-exposed and statin-unexposed anti-HMGCR-positive patients.

Rarity of anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies in statin users, including those with self-limited musculoskeletal side effects.

Statins, among the most commonly prescribed medications, are associated with a wide range of musculoskeletal side effects. These include a progressive autoimmune myopathy with anti–3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (anti‐HMGCR) antibodies that requires immunosuppression. However, it remains unknown whether these antibodies are found in statin users with and without self‐limited musculoskeletal side effects; this limits their diagnostic utility. The current work assessed the prevalence of anti‐HMGCR antibodies in these groups of statin users.

Neurotransmitter release regulated by nitric oxide in PC-12 cells and brain synaptosomes

BACKGROUND Nitric oxide is a messenger molecule of the nervous system, which is produced by the enzyme nitric oxide synthase, which may regulate cyclic guanosine monophosphate levels and which has been implicated in the control of neurotransmitter release. PC-12 pheochromocytoma cells differentiate to form neuronal cells in culture when they are exposed to nerve growth factor. The levels of cyclic guanosine monophosphate in the cells and their ability to release acetylcholine in response to K(+)-depolarization are both maximal after eight days of treatment with nerve growth factor. We set out to assess a possible role for nitric oxide in the processes that occur in differentiating PC-12 cells. RESULTS Nitric oxide synthase is first evident in differentiating PC-12 cells eight days after beginning treatment with nerve growth factor, coinciding with the marked increase in K(+)-depolarization-induced release of acetylcholine. The release of both acetylcholine and dopamine in response to K(+)-depolarization is blocked by inhibitors of nitric oxide synthase and by hemoglobin, which binds nitric oxide. Providing l-arginine, a precursor required for nitric oxide synthesis, reverses the effects of the inhibitors. In synaptosomal preparations from the corpus striatum, inhibitors of nitric oxide synthase prevent the release of glutamate in response to the glutamate derivative N-methyl-d-aspartate but not in response to K(+)-depolarization. CONCLUSION Nitric oxide may mediate the release of acetylcholine and dopamine in response to K(+)-depolarization in PC-12 cells and the release of glutamate in response to N-methyl-d-aspartate in striatal synaptosomes. Nitric oxide synthase expression is induced after eight days of treating PC-12 cells with nerve growth factor, coinciding with a marked enhancement of the release of neurotransmitters in response to K(+)-depolarization.