Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles

AbstractPhosphorylation and nitration of protein tyrosine residues are thought to play a role in signaling pathways at the nerve terminal and to affect functional properties of proteins involved in the synaptic vesicle (SV) exo-endocytotic cycle. We previously demonstrated that the tyrosine residues in the C-terminal domain of the SV protein Synaptophysin (SYP) are targets of peroxynitrite (PN). Here, we have characterized the association between SYP and c-src tyrosine kinase demonstrating that phosphorylation of Tyr273 in the C-terminal domain of SYP is crucial in mediating SYP binding to and activation of c-src. SYP forms a complex with Dynamin I (DynI), a GTPase required for SV endocytosis, which may be regulated by tyrosine phosphorylation of SYP. We here report that, in rat brain synaptosomes treated with PN, the formation of SYP/DynI complex was impaired. Noteworthy, we found that DynI was also modified by PN. DynI tyrosine phosphorylation was down-regulated in a dose-dependent manner, while DynI tyrosine nitration increased. Using mass spectrometry analysis, we identified Tyr354 as one nitration site in DynI. In addition, we tested DynI self-assembly and GTPase activity, which are enhanced by c-src-dependent tyrosine phosphorylation of DynI, and found that both were inhibited by PN. Our results suggest that the site-specific tyrosine residue modifications may modulate the association properties of SV proteins and serve as a regulator of DynI function via control of self-assembly, thus influencing the physiology of the exo-endocytotic cycle

Curcumin protects against NMDA-induced toxicity: A possible role for NR2A subunit

PURPOSE. Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDAinduced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). METHODS. NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca 2ϩ ] i . RESULTS. Curcumin dose-and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDAinduced [Ca 2ϩ ] i rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca 2ϩ ] i . The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. CONCLUSIONS. These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs. (Invest Ophthalmol Vis Sci

Activation of Phosphotyrosine-Mediated Signaling Pathways in the Cortex and Spinal Cord of SOD1G93A, a Mouse Model of Familial Amyotrophic Lateral Sclerosis

Degeneration of cortical and spinal motor neurons is the typical feature of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease for which a pathogenetic role for the Cu/Zn superoxide dismutase (SOD1) has been demonstrated. Mice overexpressing a mutated form of the SOD1 gene (SOD1G93A) develop a syndrome that closely resembles the human disease. The SOD1 mutations confer to this enzyme a “gain-of-function,” leading to increased production of reactive oxygen species. Several oxidants induce tyrosine phosphorylation through direct stimulation of kinases and/or phosphatases. In this study, we analyzed the activities of src and fyn tyrosine kinases and of protein tyrosine phosphatases in synaptosomal fractions prepared from the motor cortex and spinal cord of transgenic mice expressing SOD1G93A. We found that (i) protein phosphotyrosine level is increased, (ii) src and fyn activities are upregulated, and (iii) the activity of tyrosine phosphatases, including the striatal-enriched tyrosine phosphatase (STEP), is significantly decreased. Moreover, the NMDA receptor (NMDAR) subunit GluN2B tyrosine phosphorylation was upregulated in SOD1G93A. Tyrosine phosphorylation of GluN2B subunits regulates the NMDAR function and the recruitment of downstream signaling molecules. Indeed, we found that proline-rich tyrosine kinase 2 (Pyk2) and ERK1/2 kinase are upregulated in SOD1G93A mice. These results point out an involvement of tyrosine kinases and phosphatases in the pathogenesis of ALS

Functional Interaction between Adenosine A_2A and mGlu₅ Receptors Mediates STEP Phosphatase Activation and Promotes STEP/mGlu₅R Binding in Mouse Hippocampus and Neuroblastoma Cell Line

(1) Background: Recently, we found that adenosine A2A receptor (A2AR) stimulation results in an increase in STEP phosphatase activity. In order to delve into the mechanism through which A2AR stimulation induced STEP activation, we investigated the involvement of mGlu5R since it is well documented that A2AR and mGlu5R physically and functionally interact in several brain areas. (2) Methods: In a neuroblastoma cell line (SH-SY5Y) and in mouse hippocampal slices, we evaluated the enzymatic activity of STEP by using a para-nitrophenyl phosphate colorimetric assay. A co-immunoprecipitation assay and a Western blot analysis were used to evaluate STEP/mGlu5R binding. (3) Results: We found that the A2AR-dependent activation of STEP was mediated by the mGlu5R. Indeed, the A2AR agonist CGS 21680 significantly increased STEP activity, and this effect was prevented not only by the A2AR antagonist ZM 241385, as expected, but also by the mGlu5R antagonist MPEP. In addition, we found that mGlu5R agonist DHPG-induced STEP activation was reversed not only by the mGlu5R antagonist MPEP but also by ZM 241385. Finally, via co-immunoprecipitation experiments, we found that mGlu5R and STEP physically interact when both receptors are activated (4) Conclusions: These results demonstrated a close functional interaction between mGlu5 and A2A receptors in the modulation of STEP activity

Primary Retinal Cultures as a Tool for Modeling Diabetic Retinopathy: An Overview

Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conducted in vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabetic milieu on retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight

Megalencephalic Leukoencephalopathy with Subcortical Cysts Disease-Linked MLC1 Protein Favors Gap-Junction Intercellular Communication by Regulating Connexin 43 Trafficking in Astrocytes

Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions