Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Here, we show that neurons rescued from NGF withdrawal–induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, we find that the mRNA for BIMEL, a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and that this is reduced by dominant-negative c-Jun. Finally, overexpression of BIMEL in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim−/− knockout mice die more slowly after NGF withdrawal.

Female rodents producing endogenous estrogens are protected from stroke damage in comparison with male counterparts. This natural protection is lost after ovariectomy or reproductive senescence. The aim of this study is to determine whether estrogen reduces early neuronal injury and cell loss after ischemia by increasing the expression of Bcl-2. Male, intact female, ovariectomized, and estrogen-repleted ovariectomized rats were subjected to middle cerebral artery occlusion, and 22 hr later the level and localization of Bcl-2 mRNA and protein were determined. The levels of post-ischemic bcl-2 mRNA and protein were increased exclusively in neurons within the peri-infarct region. Intact females and estrogen-treated castrates demonstrated increased bcl-2 mRNA and protein expression compared with males and estrogen-deficient females, accompanied by a decrease in infarct size. To test the hypothesis that the neuroprotective mechanism of estrogen functions via Bcl-2, we compared ischemic outcome in male, female, and ovariectomized wild-type mice and mice overexpressing Bcl-2 exclusively in neurons. Wild-type female mice sustained smaller infarcts compared with males. Bcl-2 overexpression reduced infarct size in males, but provided no added protection in the female. Moreover, ovariectomy exacerbated infarction in wildtype females, but had no effect in Bcl-2 overexpressors. These data indicate that overexpression of Bcl-2 simulates the protection against ischemic injury conferred by endogenous female sex steroids. We concluded that estrogen rescues neurons after focal cerebral ischemia by increasing the level of Bcl-2 in peri-infarct regions and that estrogen-induced bcl-2 gene expression is an important downstream component of neuronal protection in female stroke.

PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in response to the Rho GTPase Cdc42. Here we show that PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 was shown to interact specifically with LIMK1 in binding assays. Immune complex kinase assays revealed that both wild-type and constitutively active PAK4 phosphorylated LIMK1 even more strongly than PAK1, and activated PAK4 stimulated LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments revealed that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibited the specific cytoskeletal and cell shape changes that were induced in response to a recently characterized constitutively activated PAK4 mutant.

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.

LIM-kinase1 (LIMK1) is a serine-only protein kinase that contains LIM and PDZ protein-protein interaction domains which is highly expressed in neurons. Overexpression of LIMK1 in cultured cells results in accumulation of filamentous (F-) actin. LIMK1 phosphorylates cofilin, an actin depolymerisation factor, which is then unable to bind and depolymerise F-actin. Rac-GTP enhances phosphorylation of LIMK1 and cofilin, which leads to accumulation of F-actin, while Rac-GDP and PMA reduce these effects. LIMK1 is therefore a key component of a signal transduction network that connects extracellular stimuli to changes in cytoskeletal structure. Control of cell morphology and mobility via LIMK1 activity may provide novel approaches to cancer therapy.

The protooncogene bcl-2 inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE-hbcl-2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE-hbcl-2 transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP+. Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE-hbcl-2 heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.

Bcl-2 is a crucial regulator of cell survival and death. We have recently demonstrated that transgenic mice overexpressing the human Bcl-2 protein specifically in their neurons have an increased number of neuronal cells which can survive in tissue culture in the absence of neurotrophic factors. In order to understand why only some neurons can be rescued from developmental and neurotrophic factor deprivation-induced death, we have studied the expression pattern of the transgene during embryonic development an in adulthood. We have demonstrated that transgene expression starts in embryos at E12.5 and that only half of the sensory neurons of the dorsal root ganglia expressed detectable levels of the human Bcl-2. These results may explain why only 40% of the sensory neurons survived in tissue culture in the absence of neurotrophic factors.

We have recently shown that dopamine (DA) can trigger apoptosis, an active program of cellular self-destruction, in various neuronal cultures and proposed that inappropriate activation of apoptosis by DA and or its oxidation products may initiate nigral cell loss in Parkinson's disease (PD). Since DA toxicity may be mediated via generation of oxygen-free radical species, we examined whether DA-induced cell death in PC12 cells may be inhibited by antioxidants. We have found that the thiol containing compounds, reduced glutathione (GSH),N-acetyl-cysteine (NAC), and dithiothreitol (DTT) were markedly protective, while vitamins C and E had lesser or no effect. The thiol antioxidants and vitamin C but not vitamin E, prevented dopamine autooxidation and production of dopamine-melanin. Their protective effect has also manifested by inhibiting DA-induced apoptosis; DNA fragmentation was prevented as was shown histochemically by thein situend-labeled DNA technique (TUNEL). Intracellular GSH and other thiols constitute an important natural defense against oxidative stress. We have found that depletion of cellular GSH by the addition of phoron, a substrate of glutathione transferase, and buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl transpeptidase, significantly enhanced DA toxicity. Cotreatment with NAC rescued the cells from the toxic effect of BSO + DA, and phoron + DA, while addition of GSH provided only partial protection from BSO + DA toxicity. Our data indicate that the thiol family of antioxidants, but not vitamins C and E, are highly effective in rescuing cells from DA-induced apoptosis. Further study of the mechanisms underlying the unique protective capacity of thiol antioxidants may lead to the development of new neuroprotective therapeutic strategies for PD.

LIM-kinase 1 and LIM-kinase 2 (LIMK1 and LIMK2) are members of a novel protein kinase subfamily containing LIM motifs at the N-terminus. There are two isoforms ofLimk2transcripts coding proteins with distinct N-terminal structures: LIMK2a, containing two LIM motifs, and LIMK2b, with one and one-half LIM motifs. Here we report the cDNA and genomic structures of mouse LIMK2. The deduced 638-amino-acid sequence of mouse LIMK2a shows 98% identity with that of rat LIMK2a. The mouseLimk2agene consists of at least 16 exons and spans more than 50 kb. Exon/intron boundaries of the mouseLimk2agene are exactly conserved with those of the mouseLimk1gene. An additional exon encoding theLimk2b-specific 5′-terminal sequence was found to be located between exons 2 and 3, suggesting thatLimk2aand2bmRNAs are transcribed from a singleLimk2gene by an alternative usage of exons near the 5′ end of the gene.Limk2aandLimk2btranscripts were expressed at different ratios in a variety of mouse tissues.

The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life.

The olfactory epithelium is the only neuronal tissue capable of generating new neurons during adult life and hence must express genes responsible for this phenomenon. Therefore, we have used mRNA from immortalized olfactory epithelial cells to search for novel protein tyrosine kinases by polymerase chain reaction, using as primers conserved sequences from the catalytic domain of known kinase genes. A full-length complementary DNA clone corresponding to one such polymerase chain reaction product was isolated and sequenced. This complementary DNA, designated Kiz-1, encodes a protein containing two prominent domains; the NH2-terminal region contains a cysteine/histidine-rich moiety previously identified as a zinc-finger domain in proteins of the LIM family, while the COOH-terminus contains a kinase domain. Kiz-1 is expressed mainly in the brain of adult mice but also in a range of cultured cell lines, regardless of their tissue of origin. Immunohistochemical studies on adult mouse brain demonstrated that Kiz-1 is expressed exclusively in neurons, not in astrocytes or oligodendrocytes. In the developing embryo, however, Kiz-1 is expressed in all tissues. In COS cells transfected with Kiz-1 complementary DNA and in the immortalized olfactory epithelial cells, Kiz-1 was found mainly in the cytoplasm, but in neurons of the adult brain, it resided also in the nucleus. Two Kiz-1 mRNA species are expressed in cell lines as well as in the murine and human brain. One transcript lacks a region of 60 nucleotides, which lies within the catalytic domain of the kinase and is encoded by a separate exon. Our results suggest that Kiz-1 may play distinct roles in dividing cells and in differentiated neurons.

A protein tyrosine kinase (NYK/FLK-1), bearing all the hallmarks of a growth factor receptor, has been isolated from a cDNA library generated from enriched populations of mouse day 10 embryonic neuroepithelium and from day 18 embryonic colon. Sequence analysis of cDNAs covering the entire coding region of this 5.7 kb mRNA predicted the presence of seven immunoglobulin-like domains in the extracellular region of this molecule. This feature, coupled with the detection of an insert domain bisecting the kinase domain of the predicted protein sequence, places NYK/FLK-1 firmly in the Platelet-derived Growth Factor Receptor-related class of molecules. NYK/FLK-1 is expressed at high levels in adult heart, lung, kidney, brain and skeletal muscle, but is also expressed at lower levels in most other adult tissues. In situ hybridization of day 12.5 embryo sections demonstrated NYK/FLK-1 mRNA expression in endothelial cells lining the dorsal aorta and intervertebral veins. In addition, expression was found in cells lining the capillary plexus which surrounds the developing neuroepithelium, and in the endothelial cells which are found within the embryonic lung, spleen, liver and metanephros.