Estradiol affects spinophilin protein differently in gonadectomized males and females.

Estrogen (E) treatment of ovariectomized animals increases dendritic spines and/or synaptic protein expression in the hippocampus of female rats [J Neurosci 12 (1992) 2549; Endocrinology 142 (2001) 1284; Endocrinol Rev 20 (1999) 279; Annu Rev Pharmacol Toxicol 41 (2001) 569], mice [Proc Natl Acad Sci USA 101 (2004) 2185], rhesus monkeys [Proc Natl Acad Sci USA 98 (2001) 8071; Endocrinology 144 (2003) 4734; J Comp Neurol 465 (2003) 540] and hippocampal cells in vitro [J Neurosci 16 (1996) 4059; Neuroscience 124 (2004) 549]. The role of E in hippocampal synaptic structural plasticity in males is less well understood. In the present study, we have used a recently developed technique to count spinophilin immunogold-reactive (Ir) puncta as well as in situ hybridization to compare E effects on spinophilin-Ir and mRNA in gonadectomized female and male rats 48 h after E treatment. Spinophilin is an established spine marker, which interacts with several proteins (including actin and protein phosphatase 1) that are highly enriched in spines [Proc Natl Acad Sci USA 94 (1997) 9956; Proc Natl Acad Sci USA 97 (2000) 9287]. We report that E exerts sex-specific effects on dendritic spinophilin-labeled spines in the CA1 region: E treatment significantly increased spinophilin-Ir puncta, indicative of spines, in females, but led to a decrease in males. Furthermore, while hippocampal spinophilin mRNA changes could have occurred earlier, spinophilin mRNA levels were unchanged after 48 h of E in both males and females. This suggests the possibility that E regulates spinophilin protein expression and or stability within dendrites via post-transcriptional mechanisms.

Estrogen induces phosphorylation of cyclic AMP response element binding (pCREB) in primary hippocampal cells in a time-dependent manner.

Using hippocampal primary cell cultures at 14 days in vitro (div), we have investigated actions of 17-beta estradiol (E; 10 nM) on the phosphorylation of CREB and on signaling pathways that regulate CREB phosphorylation. After demonstrating that 14 div is optimal for these studies, we examined the time course of E induction of CREB phosphorylation (pCREB) at serine residue 133. The induction of pCREB occurs as early as 1 h following E treatment, presumably via a mechanism involving an E-stimulated signal transduction system, which is sustained for at least 24 h but inhibited by 48 h. The early activity may represent an initial signal required for events leading to phosphorylation of CREB while the sustained signal may lead to CREB-mediated gene expression for cell survival and synapse formation. Furthermore, we examined the pathways for E action preceding pCREB induction by blocking three major kinases (protein kinase; mitogen activated protein kinase, MAPK; and calcium-calmodulin kinase II, CaMKII) upstream of pCREB. We found that E stimulates each pathway at 24 h and that phosphorylation of CREB is dependent on both MAPK and CaMK activities, but less dependent on the Akt pathway. Because CREB has been linked to E induction of excitatory spine synapses, we used a spine marker, spinophilin, to establish E effects on spine formation. Spinophilin expression was up-regulated in response to E and this effect was blocked by an inhibitor of (CaMKII). These studies demonstrate the central role played by CaMKII pathway in the actions of E on both transcriptional regulation and structural reorganization in neurons.

Episodic ataxia type-1 mutations in the Kv1.1 potassium channel display distinct folding and intracellular trafficking properties.

Episodic ataxia type 1 (EA-1) is a neurological disorder arising from mutations in the Kv1.1 potassium channel alpha-subunit. EA-1 patients exhibit substantial phenotypic variability resulting from at least 14 distinct EA-1 point mutations. We found that EA-1 missense mutations generate mutant Kv1.1 subunits with folding and intracellular trafficking properties indistinguishable from wild-type Kv1.1. However, the single identified EA-1 nonsense mutation exhibits intracellular aggregation and detergent insolubility. This phenotype can be transferred to co-assembled Kv1 alpha- and Kv beta-subunits associated with Kv1.1 in neurons. These results suggest that as in many neurodegenerative disorders, intracellular aggregation of misfolded Kv1.1-containing channels may contribute to the pathophysiology of EA-1.