(b) Lack of signals derived from degradation products recognized by the M2F6 antibody

(b) Lack of signals derived from degradation products recognized by the M2F6 antibody.(TIF) pone.0125119.s004.tif (233K) GUID:?75F5FC80-5CE2-4492-A38E-7B31ADAA8508 S5 Fig: Losses of f-actin and drebrin occur concomitantly after NMDA treatment. blue-positive cells. The asterisk indicates a statistically significant difference (= 1.56 10C6 by a Tukey-Kramer test) compared with the non-treated (0 h) condition (ns, not significant). The data are represented as the mean standard Febantel deviation of n = 3 replicates.(TIF) pone.0125119.s002.tif (2.3M) GUID:?A0338C4D-FFC8-4296-B8AB-4F879D313EC4 S3 Fig: EGTA and calpain inhibitor-I suppress the excitotoxicity-induced degradation of drebrin in cultured rat hippocampal neurons. (a) Western blot analyses of drebrin A proteolytic fragments in neurons that were pretreated with EGTA for 30 min (a) or calpain inhibitor-I for 1 h (b), and then exposed to NMDA. The samples used in Fig 1C and 1E were reanalyzed using the DAS2 antibody in (a) and (b), respectively. The experiments were repeated a minimum of three times with similar results.(TIF) pone.0125119.s003.tif (110K) GUID:?AD9306F3-235E-4907-865B-8FD7EFEC9384 S4 Fig: The M2F6 antibody detects reduced amounts of full-length drebrin but no degradation products. (a) Detection of NMDA-induced decreases in the levels of drebrin A and E using the M2F6 antibody. The expression level of Hsp90 was used as a loading control. (b) Lack of signals derived from degradation products recognized by the M2F6 antibody.(TIF) pone.0125119.s004.tif (233K) GUID:?75F5FC80-5CE2-4492-A38E-7B31ADAA8508 S5 Fig: Losses of f-actin and drebrin occur concomitantly after NMDA treatment. (a) The signal intensities (AU, arbitrary units) of drebrins A and E (drebrin A/E) and f-actin in circular areas (40 m diameter) that covered the cell soma and proximal dendrites of 100 randomly selected NeuN-positive neurons. Each dot represents one neuron. The thresholds indicated by orange lines are set at 40 percent or 70 percent of median values of drebrin or f-actin in NMDA(-) condition, respectively. (b) Febantel Schematic representation showing the identification of four quadrants (Q1-Q4). (c) Statistical analysis of the numbers of neurons included in each quadrant (Q1-Q4). The data are represented as the mean standard deviation of n = 3 replicates. ***< 0.005 by a Students t-test.(TIF) pone.0125119.s005.tif (2.0M) GUID:?5A1F970A-F35F-4AD1-8A23-EB0354BB6FFA S6 Fig: Localization of drebrins at dendritic spines in the mouse neurons used in this study. Immunostaining of mouse cortical (left panels) and hippocampal neurons (right panels) at 12 days (DIV) using antibodies against drebrin A/E (M2F6) and phalloidin (f-actin). Scale bar: 10 m.(TIF) pone.0125119.s006.tif (4.8M) GUID:?4B881FD6-9DFC-4A36-B02A-7E20FF8E4B40 S7 Fig: Calpain degrades drebrin A directly cleavage assay using crude brain cortical extract as a substrate in the absence or presence of 100M calpain inhibitor-1 (CI-1). The asterisks indicate the degradation products detected specifically in the cleavage assay. The arrow indicates a nonspecific band.(TIF) pone.0125119.s007.tif (691K) GUID:?D14A6737-77B2-4D11-BB99-0391713D0040 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimers disease (AD) and Downs syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a Febantel functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly and [6,7], and is thought to contribute to neuronal loss in both acute neurodegenerative diseases such as stroke [4C10] and chronic neurodegenerative diseases such as AD [11,12]. Memantine, an open-channel blocker that preferentially inhibits overactivated NMDARs, shows significant positive effects on the cognition of patients with moderate to severe AD [13]. In addition, several NMDAR antagonists can protect neurons from ischemic damage in animal models [14C18]. These findings indicate that excitotoxicity caused by overactivation of NMDARs plays a central role in the pathogenesis HNRNPA1L2 of chronic and acute neurodegenerative diseases. To understand the pathogenesis and refine the therapeutic strategies for these diseases, it is crucial to elucidate the cellular responses to overactivation of NMDARs and the molecular basis of the resulting neuronal death..