PAK6 is a member of the group B family of PAK serine/threonine kinases, and is highly expressed in the brain. The group B PAKs, including PAK4, PAK5, and PAK6, were first identified as effector proteins for the Rho GTPase Cdc42. They have important roles in filopodia formation, the extension of neurons, and cell survival. Pak4 knockout mice die in utero, and the embryos have several abnormalities, including a defect in the development of motor neurons. In contrast, Pak5 knockout mice do not have any noticeable abnormalities. So far nothing is known about the biological function of Pak6. To address this, we have deleted the Pak6 gene in mice. Since Pak6 and Pak5 are both expressed in the brain, we also generated Pak5/Pak6 double knockout mice. These mice were viable and fertile, but had several locomotor and behavioral deficits. Our results indicate that Pak5 and Pak6 together are not required for viability, but are required for a normal level of locomotion and activity as well as for learning and memory. This is consistent with a role for the group B PAKs in the nervous system.

As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary post-mitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By qPCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19 ARF ) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19 ARF , Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity. Keywords E2F1; HIV-associated dementia; transcription factorHuman immunodeficiency virus (HIV)-associated dementia (HAD) is a common neurological disorder associated with HIV infection. Pathologic studies of the brains of patients with HAD suggest an inflammatory mechanism in the progression of this disease, as evidenced by astrogliosis, microgliosis, and perivascular macrophage infiltration (1-3). Although neuronal death, dendritic loss and synaptic loss are features of HAD, there is little evidence of direct HIV infection of neurons. Instead, neuronal dysfunction and death likely result from the release of various neurotoxic factors from activated macrophages and microglia, such as reactive oxygen species and excitatory amino acids (4-6). Correlative * Corresponding Author Kelly L. Jordan-Sciutto, Ph.D., Department of Pathology, University of Pennsylvania, 240 S. 40 th St, Rm 312 Levy Bldg, Philadelphia, PA 19104-6030, Jordan@path.dental.upenn.edu, phone: 215-898-4196, fax: 215-573-2050. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. E2F1 is a member of the E2F family of transcription factors, which play...

Aberrant expression and activation of the cell cycle protein E2F1 in neurons has been implicated in many neurodegenerative diseases. As a transcription factor regulating G1 to S phase progression in proliferative cells, E2F1 is often upregulated and activated in models of neuronal death. However, despite its well studied functions in neuronal death, little is known regarding the role of E2F1 in the mature brain. In the present study, we used a combined approach to study the effect of E2F1 gene disruption on mouse behavior and brain biochemistry. We identified significant age-dependent olfactory and memory related deficits in E2f1 mutant mice. In addition, we found that E2F1 exhibits punctated staining and localizes closely to the synapse. Furthermore, we found a mirroring age-dependent loss of postsynaptic protein-95 in the hippocampus and olfactory bulb as well as a global loss of several other synaptic proteins. Coincidently, E2F1 expression is significantly elevated at the ages in which behavioral and synaptic perturbations were observed. Lastly, we show that deficits in adult neurogenesis persist late in aged E2f1 mutant mice which may partially contribute to the behavior phenotypes. Taken together, our data suggest that the disruption of E2F1 function leads to specific age-dependent behavioral deficits and synaptic perturbations.

The transcription factor E2F1 activates gene targets required for G1-S phase progression and for apoptosis, and exhibits increased expression levels in neurons in several CNS diseases including HIV encephalitis, Alzheimer disease, and Parkinson Disease. While E2F1 is known to regulate cell viability through activation of caspases, here we present evidence supporting the involvement of E2F1 in NMDA receptor-dependent, HIV-induced neuronal death mediated by calpains. Using an in vitro model of HIV-induced neurotoxicity that is dependent on NMDA receptor and calpain activation, we have shown that cortical neurons lacking functional E2F1 are less susceptible to neuronal death. Additionally, we report that neuronal E2F1 is cleaved by calpain to a stable 55-kiloDalton fragment following NR2B-dependent NMDA receptor stimulation. This cleavage of E2F1 is protein conformation-dependent and involves at least two cleavage events, one at each terminus of the protein. Intriguingly, the stabilized E2F1 cleavage product is produced in postmitotic neurons of all ages, but fails to be stabilized in cycling cells. Finally, we show that a matching E2F1 cleavage product is produced in human fetal neurons, suggesting that calpain cleavage of E2F1 may be produced in human cortical tissue. These results suggest neuronal E2F1 is processed in a novel manner in response to NMDA receptor-mediated toxicity, a mechanism implicated in HAND pathogenesis as well as several other diseases of the CNS.

E2F1 is a transcription factor classically known to regulate G/G to S phase progression in the cell cycle. In addition, E2F1 also regulates a wide range of apoptotic genes and thus has been well studied in the context of neuronal death and neurodegenerative diseases. However, its function and regulation in the mature central nervous system are not well understood. Alternative splicing is a well-conserved post-transcriptional mechanism common in cells of the CNS and is necessary to generate diverse functional modifications to RNA or protein products from genes. Heretofore, physiologically significant alternatively spliced E2F1 transcripts have not been reported. In the present study, we report the identification of two novel alternatively spliced E2F1 transcripts: E2F1b, an E2F1 transcript retaining intron 5, and E2F1c, an E2F1 transcript excluding exon 6. These alternatively spliced transcripts are observed in the brain and neural cell types including neurons, astrocytes, and undifferentiated oligodendrocytes. The expression of these E2F1 transcripts is distinct during maturation of primary hippocampal neuroglial cells. Pharmacologically-induced global translation inhibition with cycloheximide, anisomycin or thapsigargin lead to significantly reduced expression of E2F1a, E2F1b and E2F1c. Conversely, increasing neuronal activity by elevating the concentration of potassium chloride selectively increased the expression of E2F1b. Furthermore, experiments expressing these variants in vitro show the transcripts can be translated to generate a protein product. Taken together, our data suggest that the alternatively spliced E2F1 transcript behave differently than the E2F1a transcript, and our results provide a foundation for future investigation of the function of E2F1 splice variants in the CNS.

Necrosis and apoptosis are well established as two primary cell death pathways. Mixed neuroglial cultures are commonly used to study cell death mechanisms in neural cells. However, the ages of these cultures vary across studies and little attention has been paid to how cell death processes may change as the cultures mature. To clarify whether neuroglial culture age affects cell death mechanisms, we treated 1- and 3-week-old neuroglial cultures with either the excitotoxic stimulus, N-methyl-d-aspartate (NMDA), or with the oxidative stressor, hydrogen peroxide (H2O2). Although NMDA is known to be toxic only in cultures that are at least 2 weeks old, H2O2 is toxic in cultures of all ages. Here, we confirm that, in 1-week-old neuroglial cultures, NMDA does not induce toxicity, whereas H2O2 induces both calpain-mediated and caspase-mediated neuronal death. In 3-week-old cultures, both NMDA and H2O2 trigger calpain-mediated, but not caspase-mediated, neuronal death. Further, we observed a decrease in caspase-3 levels and an increase in calpain levels in untreated neuroglial cultures as they aged. The findings presented here show that neuronal cell death mechanisms vary with culture age and highlight the necessity of considering culture age when interpreting neural cell culture data.