Faculty Bibliography

Akt kinases are key signaling components in proliferation-competent and post-mitotic cells. Here, we sought to create a conditionally-inducible form of active Akt for both in vitro and in vivo applications. We fused a ligand-responsive Destabilizing Domain (DD) derived from E. coli dihydrofolate reductase to a constitutively active mutant form of Akt1, Akt(E40K). Prior work indicated that such fusion proteins may be stabilized and induced by a ligand, the antibiotic Trimethoprim (TMP). We observed dose-dependent, reversible induction of both total and phosphorylated/active DD-Akt(E40K) by TMP across several cellular backgrounds in culture, including neurons. Phosphorylation of FoxO4, an Akt substrate, was significantly elevated after DD-Akt(E40K) induction, indicating the induced protein was functionally active. The induced Akt(E40K) protected cells from apoptosis evoked by serum deprivation and was neuroprotective in two cellular models of Parkinson's disease (6-OHDA and MPP+ exposure). There was no significant protection without induction. We also evaluated Akt(E40K) induction by TMP in mouse substantia nigra and striatum after neuronal delivery via an AAV1 adeno-associated viral vector. While there was significant induction in striatum, there was no apparent induction in substantia nigra. To explore the possible basis for this difference, we examined DD-Akt(E40K) induction in cultured ventral midbrain neurons. Both dopaminergic and non-dopaminergic neurons in the cultures showed DD-Akt(E40K) induction after TMP treatment. However, basal DD-Akt(E40K) expression was 3-fold higher for dopaminergic neurons, resulting in a significantly lower induction by TMP in this population. Such findings suggest that dopaminergic neurons may be relatively inefficient in protein degradation, a property that could relate to their lack of apparent DD-Akt(E40K) induction in vivo and to their selective vulnerability in Parkinson's disease. In summary, we generated an inducible, biologically active form of Akt. The degree of inducibility appears to reflect cellular context that will inform the most appropriate applications for this and related reagents.

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival and chemoresistance of cancer cells. The PI3K/Phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (beta) (CaMKK2) is highly expressed in high-grade serous ovarian cancer and investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in the G1/S transition and apoptosis. CaMKK2 knockdown or inhibition, decreased Akt phosphorylation at Thr308 and Ser473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr450, p-PDK1 Ser241 or p-IRS1 Ser636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr308 was significantly inhibited by intracellular Ca2+i -chelation or CaM inhibition. Ionomycin-induced Ca2+-influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.

Background: Impaired 5-HT1A receptor (5-HTR) function can render neurons susceptible to cell death. Because of our findings of increased 5-HT1A expression but reduced neuron density in prefrontal cortex (PFC) of depressed suicides, we hypothesized that neuron viability may be affected by dysregulated 5-HT1AR signaling. To test this, we measured 5-HT1AR-dependent intracellular signaling in PFC of depressed and non-depressed suicides versus matched controls. Methods: Western blotting of brain lysates from 16 triplets examined AKT, pAKT, ERK1/ERK2, pERK1/pERK2, PTEN, BAX, BCL-2 and GSK-3p/pGSK-3p. Actin and GAPDH served as controls. Neuron and glia density was assessed by NeuN immunocytochemistry (neurons), hematoxylin staining (glia) and stereology. Results: pAKT, PTEN, pERK1 and BCL-2 in BA9 were increased in suicides by 21% (p=0.046), 26% (p=0.009), 161% (p=0.035) and 29% (p=0.011), respectively. Glia density did not differ in any region or group (p>0.05; all comparisons). Neuron density did not differ between groups in BA9 and BA47/45. In BA24, non-depressed suicides had increased pAKT/GAPDH ratios (p=0.041) and neuron density (p=0.07) while depressed suicides had lower neuron density (p=0.06) when compared to controls. Conversely, depressed suicides had lower neuron density (p=0.06) when compared to controls. Conclusions: In BA9, changes in 5-HT1AR signaling resulted in significant changes in apoptosis-regulating proteins that in sum may be neuro-protective. In BA24, pAKT was increased only in non-depressed but not depressed suicides, who as a result may be neuro-endangered as predicted from reduced neuron density. These results confirm our hypothesis that biochemical changes in 5-HT1AR signaling in suicide and major depression impinge on neuronal viability

Background Akt kinase plays a key role in neuronal cell function and survival. Biochemical studies on human postmortem brains indicate significant changes in Akt signaling in depression and suicide. AKT1 mutations also are a liability in psychiatric diseases such as schizophrenia. These noteworthy findings suggest an important role for intact Akt signaling in mood regulation. Methods Our studies have examined mouse models of Akt deficiency in anxiety-and depression-like behaviors, and their responsiveness to antidepressants and psychostimulants. In addition to genomic Akt knockout mice for all three Akt isoforms (Akt1, Akt2 and Akt3), we employed Cre-dependent recombination to produce Akt1 deficiency in specific brain regions and neuron populations. Results Following the social defeat regimen, Akt1-deficient mice exhibited increased susceptibility to the detrimental effects of chronic stress. Antidepressant treatment failed to ameliorate depression-like behaviors in the mutants when compared to wild-type controls. Mice with conditional Akt1 deficiency exhibited a spectrum of phenotypes after completing the chronic social defeat paradigm: Akt1 ablation in the frontal cortex significantly decreased resilience, while ablation in other brain areas and specific neuron populations increased resilience. Similarly, ablation of Akt1 in different brain regions produced distinct and opposing results when measuring locomotor responsiveness to cocaine. Discussion Our results in Akt1-mutant mice converge with biochemical and genetic findings in neuropsychiatric patients and confirm the critical requirement for intact Akt signaling in mood regulation. Based on our findings in conditional Akt1 mutant mice, we predict a significant role for frontal cortical Akt signaling in stress resilience and antidepressant response. Our experiments suggest conditional Akt1 mutant mice as a tractable genetic model to investigate brain region-specific contributions of Akt signaling to mood regulation. These mutants aid in understanding how circuit-specific Akt1 deficiencies may become liabilities Conditional Akt1-mutant mice also illustrate specific contributions of Akt signaling to the function and output of distinct neuronal circuits

RhoE/Rnd3 is an atypical member of the Rho family of small GTPases. In addition to regulating actin cytoskeleton dynamics, RhoE is involved in the regulation of cell proliferation, survival, and metastasis. We examined RhoE expression levels during cell cycle and investigated mechanisms controlling them. We show that RhoE accumulates during G1, in contact-inhibited cells, and when the Akt pathway is inhibited. Conversely, RhoE levels rapidly decrease at the G1/S transition and remain low for most of the cell cycle. We also show that the half-life of RhoE is shorter than that of other Rho proteins and that its expression levels are regulated by proteasomal degradation. The expression patterns of RhoE overlap with that of the cell cycle inhibitor p27. Consistently with an involvement of RhoE in cell cycle regulation, RhoE and p27 levels decrease after overexpression of the F-box protein Skp2. We have identified a region between amino acids 231 and 240 of RhoE as the Skp2-interacting domain and Lys(235) as the substrate for ubiquitylation. Based on our results, we propose a mechanism according to which proteasomal degradation of RhoE by Skp2 regulates its protein levels to control cellular proliferation.

BACKGROUND: Tau is a microtubule stabilizing protein and is mainly expressed in neurons. Tau aggregation into oligomers and tangles is considered an important pathological event in tauopathies, such as frontotemporal dementia (FTD) and Alzheimer's disease (AD). Tauopathies are also associated with deficits in synaptic plasticity such as long-term potentiation (LTP), but the specific role of tau in the manifestation of these deficiencies is not well-understood. We examined long lasting forms of synaptic plasticity in JNPL3 (BL6) mice expressing mutant tau that is identified in some inherited FTDs. RESULTS: We found that aged (>12 months) JNPL3 (BL6) mice exhibit enhanced hippocampal late-phase (L-LTP), while young JNPL3 (BL6) mice (age 6 months) displayed normal L-LTP. This enhanced L-LTP in aged JNPL3 (BL6) mice was rescued with the GABAAR agonist, zolpidem, suggesting a loss of GABAergic function. Indeed, we found that mutant mice displayed a reduction in hippocampal GABAergic interneurons. Finally, we also found that expression of mutant tau led to severe sensorimotor-gating and hippocampus-dependent memory deficits in the aged JNPL3 (BL6) mice. CONCLUSIONS: We show for the first time that hippocampal GABAergic function is impaired by pathological tau protein, leading to altered synaptic plasticity and severe memory deficits. Increased understanding of the molecular mechanisms underlying the synaptic failure in AD and FTD is critical to identifying targets for therapies to restore cognitive deficiencies associated with tauopathies.