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The genetic disease tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by loss of function mutations in either TSC1 (hamartin) or TSC2 (tuberin), which serve as negative regulators of mechanistic target of rapamycin complex 1 (mTORC1) activity. TSC patients exhibit developmental brain abnormalities and tuber formations that are associated with neuropsychological and neurocognitive impairments, seizures and premature death. Mechanistically, TSC1 and TSC2 loss of function mutations result in abnormally high mTORC1 activity. Thus, the development of a strategy to inhibit abnormally high mTORC1 activity may have therapeutic value in the treatment of TSC. mTORC1 is a master regulator of growth processes, and its activity can be reduced by withdrawal of growth factors, decreased energy availability, and by the immunosuppressant rapamycin. Recently, glutamine has been shown to alter mTORC1 activity in a TSC1-TSC2 independent manner in cells cultured under amino acid- and serum-deprived conditions. Since starvation culture conditions are not physiologically relevant, we examined if glutamine can regulate mTORC1 in non-deprived cells and in a murine model of TSC. Our results show that glutamine can reduce phosphorylation of S6 and S6 kinase, surrogate indicators of mTORC1 activity, in both deprived and non-deprived cells, although higher concentrations were required for non-deprived cultures. When administered orally to TSC2 knockout mice, glutamine reduced S6 phosphorylation in the brain and significantly prolonged their lifespan. Taken together, these studies suggest that glutamine supplementation can be used as a potential treatment for TSC.

The transcription factor nuclear factor E2-related factor 2 (Nrf2) regulates the expression of multiple cytoprotective genes that have been shown to offer protection in response to a number of insults. The present study describes a novel strategy to increase expression of Nrf2-responsive genes in brain injured mice. Under normal conditions, the adapter protein Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and promotes its proteosomal degradation in the cytoplasm. The amino acid sequence DEETGE, located at amino acid 77–82 of Nrf2, is critical for Nrf2-Keap1 interaction, and synthetic peptides containing this sequence can be used to disrupt the complex in vitro. We observed that intracerebroventricular (i.c.v.) infusion of a peptide containing the DEETGE sequence along with the cell transduction domain of the HIV-TAT protein (TAT-DEETGE) into brain-injured mice did not increase the mRNA levels for Nrf2-driven genes. However, when a calpain cleavage sequence was introduced between the TAT sequence and the DEETGE sequence, the new peptide (TAT-CAL-DEETGE) increased the mRNA levels of these genes. Increased gene expression was not observed when the TAT-CAL-DEETGE peptide was injected into uninjured animals. Furthermore, injection of TAT-CAL-DEETGE peptides before or after brain injury reduced blood-brain barrier compromise, a prominent secondary pathology that negatively influences outcome. The present strategy to increase Nrf2-responsive gene expression can be adapted to treat other insults or diseases based on their underlying mechanism(s) of cellular damage.

The gamma-secretase complex, a membrane-bound aspartyl protease, hydrolyzes the transmembrane domains of several integral membrane proteins including the key signaling molecules amyloid precursor protein (APP), Notch, deleted in colorectal cancer (DCC), and N- and E-cadherins. The proteolysis processing of these proteins is critical for generation of signaling molecules that may participate in neuronal communication and plasticity. Using a potent gamma-secretase inhibitor, L-685,458, we examined if blockade of its activity in the hippocampus can influence contextual and spatial memory in rats. Surprisingly, we observed that post-training blockade of gamma-secretase activity leads to enhanced long-term memory in two hippocampus-dependent tasks. This suggests that a signaling molecule(s) generated by gamma-secretase activity may have a negative influence on long-term memory formation.

Rho GTPases acting through effector proteins regulate actin dynamics and cytoskeletal structure. In Hermissenda Csp24 is a cytoskeletal-related protein that contributes to the development of intermediate-term memory, and is homologous to other β-thymosin-like repeat proteins containing multiple actin-binding domains. We have examined the role of Rho GTPase activity and its downstream target ROCK, and cyclin-dependent kinase 5 (Cdk5) on the phosphorylation of Csp24 using 32PO4 labeling of proteins separated with 2-D PAGE. The ROCK inhibitor Y-27632 significantly increased Csp24 phosphorylation, and the Rho activator lysophosphatidic acid (LPA) or the Cdk5 inhibitor butyrolactone significantly decreased Csp24 phosphorylation. Pretreatment with Y-27632 before LPA application significantly reduced the decreased phosphorylation of Csp24 normally detected in nervous systems exposed to LPA. Using a pull-down assay we found that LPA treatments activated Rho and exposure to 5-HT decreased Rho activity. Our results indicate that the Rho/ROCK and Cdk5 signaling pathways contribute to the regulation of Csp24 phosphorylation.

Morphological changes, including changes in size, shape, and number of synapses, in neurons have been observed in many species and are thought to be critical for long-term memory storage. Actin filaments are intimately involved in neuronal morphology and regulation of their dynamics can influence memory. Rho GTPase plays a prominent role in this process and has been implicated in both pre- and post-synaptic morphological changes. Therefore, we examined the effect of hippocampal manipulation of Rho and ROCK activity on performance in a spatial memory task. Post-training intrahippocampal infusion of an inhibitor of the downstream effector kinase p160ROCK impaired long-term memory. Furthermore, post-training activation of Rho using lysophosphatidic acid (LPA) enhanced long-term spatial memory. This memory enhancing effect of LPA was not mediated via the Erk cascade, as no change in Erk phosphorylation was observed as a result of its administration. Our results demonstrate a role for the Rho-ROCK pathway in hippocampus-dependent spatial memory.

Abnormal processing of the amyloid precursor protein (APP) is thought to contribute to the formation of amyloid plaques in Alzheimer's disease. Several studies suggest that inflammation, and possibly the cytokines released during the inflammatory process, may participate in the plaque formation. We have utilized a cell culture system to examine the effects of the cytokine interleukin-1 beta (IL-1 beta) on the processing of APP. We present data to show that IL-1 beta increases the maturation of APP and causes enhanced processing of the full length APP isoforms. In addition, as reported previously in HUVEC cells, IL-1 beta increases the secretion of APP in PC12 cells. Indomethacin and NDGA, reported inhibitors of the cyclooxygenase and lipoxygenase pathways, respectively, block these effects, suggesting the involvement of prostaglandins and leukotrienes in IL-1 beta mediated APP processing.