Rescue from Sexually Dimorphic Neuronal Cell Death by Estradiol and PI3 Kinase Activity.

Responses of primary hippocampal and cortical neurons derived from male and female rats to cellular stressors were studied. It is demonstrated that 17ß-estradiol (E2), a potent neuroprotectant, protected the female neurons but had no effects on the male neurons from CoCl2- and glutamate-induced toxicity. Agonists of the estrogen receptor (ER) subtypes ERα and ERß, DPN and PPT, respectively, had similar effects to E2. By contrast, effects of E2 were abolished by the ER antagonist ICI-182780, further corroborating the neuroprotective role of ERs. In male neurons, CoCl2 predominately activated the apoptosis-inducing factor (AIF)-dependent pathway and AIF translocation from the cytosol to the nucleus. In comparison, CoCl2 activated the caspase pathway and cytochrome c release in female neurons. The inhibitors of these pathways, namely DiQ for AIF and zVAD for caspase, specifically rescued CoCl2-induced cell death in male and female neurons, respectively. When zVAD and ICI-182780, and E2 were applied in combination, it was demonstrated E2 acted on the caspase pathway leading to female-specific neuroprotection. Furthermore, the PI3 kinase (PI3K) inhibitor blocked the rescue effects of DiQ and zVAD on the male and female neurons, respectively, suggesting that PI3K is a common upstream regulator for both pathways. The present study suggested that both sex-specific and nonspecific mechanisms played a role in neuronal responses to stressors and protective reagents.

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons.

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (ΔΨ(m)) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized ΔΨ(m). In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H(2)O(2)-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized ΔΨ(m) and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal ΔΨ(m) and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.

Effect of passive repetitive isokinetic training on cytokines and hormonal changes.

It is well known that muscle strength and power are important factors in exercise. Plyometrics is designed to gain muscle strength and power in a shock method. The passive repetitive isokinetic (PRI) machine is developed for plyometrics. The present study aims to understand the effect of ten-week PRI training in different intensities on human plasma concentration cytokines as well as hormonal changes. Thirty young male subjects were enrolled into the ten-week PRI training program and were divided randomly into traditional, low- and high-intensity PRI training groups. Blood samples were obtained before, during, after and 1-, 2-, 3-, 5- and 7-day (D) post-training. The plasma concentrations of cytokines and hormones were measured by an enzyme-linked immunosorbent assay (ELISA). Elevated plasma IL-2 was found in the subjects in all the training programs. Significant increases of proinflammatory cytokines IL-1beta and TNF-alpha were observed at post 7 D in the high-intensity PRI training (29.5 +/- 4.4 and 515.8 +/- 127.1 pg/ml, respectively). No significance in differences in the plasma concentration of IL-6 was observed in the traditional and low-intensity PRI training. Significant elevation of IL-6 was found at post 5 D in high-intensity PRI training. Higher plasma IL-6 concentration was observed at post 3 and 5 D in high-intensity PRI training compared to low-intensity PRI training (P < 0.05). Significant elevation of plasma IL-15 during (week 6) and after (post 0 D) was observed in low-intensity PRI training. Also, there were differences between low-intensity PRI training and traditional training at post 0, 2, 3, and 5 D. The plasma concentration of cortisol was decreased to the lowest value (118.0 +/- 17.3 ng/ml) at post 0 D in traditional training, then returned to the baseline (220.5 +/- 19.1 ng/ml). In the high-intensity PRI training, but not in the low-intensity PRI training, the cortisol level dropped from 224.9 +/- 25.8 ng/ml at post 0 D down to the 123.2 +/- 22.6 ng/ml at post 1 D. Significant differences were found at post 1 and 5 D between low- and high-intensity PRI training, and post 0, 1, 2, and 3 D between traditional and high-intensity PRI training. Significant increased testosterone was found post 0, 1, 2, and 3 D in traditional training. Higher plasma testosterone was observed during and the recovery period in low-intensity, but not in high-intensity, PRI training. In conclusion, high-intensity PRI training could induce the proinflammatory cytokines, i.e., IL-1beta and TNF-alpha, and decrease plasma cortisol in the recovery period.

Transforming growth factor-ß1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-κB pathways.

BACKGROUND: Transforming growth factor-ß (TGF-ß) and matrix metalloproteinases (MMPs) are the multifunctional factors during diverse physiological and pathological processes including development, wound healing, proliferation, and cancer metastasis. Both TGF-ß and MMPs have been shown to play crucial roles in brain pathological changes. Thus, we investigated the molecular mechanisms underlying TGF-ß1-induced MMP-9 expression in brain astrocytes. METHODS: Rat brain astrocytes (RBA-1) were used. MMP-9 expression was analyzed by gelatin zymography and RT-PCR. The involvement of signaling molecules including MAPKs and NF-κB in the responses was investigated using pharmacological inhibitors and dominant negative mutants, determined by western blot and gene promoter assay. The functional activity of MMP-9 was evaluated by cell migration assay. RESULTS: Here we report that TGF-ß1 induces MMP-9 expression and enzymatic activity via a TGF-ß receptor-activated reactive oxygen species (ROS)-dependent signaling pathway. ROS production leads to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activation of the NF-κB transcription factor. Activated NF-κB turns on transcription of the MMP-9 gene. The rat MMP-9 promoter, containing a NF-κB cis-binding site, was identified as a crucial domain linking to TGF-ß1 action. CONCLUSIONS: Collectively, in RBA-1 cells, activation of ERK1/2- and JNK-NF-κB cascades by a ROS-dependent manner is essential for MMP-9 up-regulation/activation and cell migration induced by TGF-ß1. These findings indicate a new regulatory pathway of TGF-ß1 in regulating expression of MMP-9 in brain astrocytes, which is involved in physiological and pathological tissue remodeling of central nervous system.

The role of Kif5B in axonal localization of Kv1 K(+) channels.

Here we present evidence that the kinesin, Kif5B, is involved in the transportation and axonal targeting of Kv1 channels. We show that a dominant negative variant of Kif5B specifically blocks localization to the axon of expressed, tagged versions of Kv1.3 in cultured cortical slices. In addition, the dominant negative variant of Kif5B blocks axonal localization of endogenous Kv1.1, Kv1.2, and Kv1.4 in cortical neurons in dissociated cultures. We also found evidence that Kif5B interacts with Kv1 channels. Endogenous Kv1.2 colocalized with Kif5B in cortical neurons and coimmunoprecipitated with Kif5B from brain lysate. The T1 domain of Shaker K(+) channels has been shown to play a critical role in targeting the channel to the axon. We have three pieces of evidence to suggest that the T1 domain also mediates interaction between Kv1 channels and Kif5B: Addition of the T1 domain to a heterologous protein, TfR, is sufficient to cause the resulting fusion protein, TfRT1, to colocalize with Kif5B. Also, the T1 domain is necessary for interaction of Kv1.3 with Kif5B in a coimmunoprecipitation assay. Finally, dominant negative variants of Kif5B block axonal targeting of TfRT1, but have no effect on dendritic localization of TfR. Together these data suggest a model where Kif5B interacts with Kv1 channels either directly or indirectly via the T1 domain, causing the channels to be transported to axons.

A role for Kif17 in transport of Kv4.2.

Although kinesins are known to transport neuronal proteins, it is not known what role they play in the targeting of their cargos to specific subcellular compartments in neurons. Here we present evidence that the K+ channel Kv4.2, which is a major regulator of dendritic excitability, is transported to dendrites by the kinesin isoform Kif17. We show that a dominant negative construct against Kif17 dramatically inhibits localization to dendrites of both introduced and endogenous Kv4.2, but those against other kinesins found in dendrites do not. Kv4.2 colocalizes with Kif17 but not with other kinesin isoforms in dendrites of cortical neurons. Native Kv4.2 and Kif17 coimmunoprecipitate from brain lysate, and introduced, tagged versions of the two proteins coimmunoprecipitate from COS cell lysate, indicating that the two proteins interact, either directly or indirectly. The interaction between Kif17 and Kv4.2 appears to occur through the extreme C terminus of Kv4.2 and not through the dileucine motif. Thus, the dileucine motif does not determine the localization of Kv4.2 by causing the channel to interact with a specific motor protein. In support of this conclusion, we found that the dileucine motif mediates dendritic targeting of CD8 independent of Kif17. Together our data show that Kif17 is probably the motor that transports Kv4.2 to dendrites but suggest that this motor does not, by itself, specify dendritic localization of the channel.

The T1 domain of Kv1.3 mediates intracellular targeting to axons.

Shaker K+ channels play an important role in modulating electrical excitability of axons. Recent work has demonstrated that the T1 tetramerization domain of Kv1.2 is both necessary and sufficient for targeting of the channel to the axonal surface [Gu, C., Jan, Y.N. & Jan, L.Y. (2003) Science,301, 646-649]. Here we use a related channel, Kv1.3, as a model to investigate cellular mechanisms that mediate axonal targeting. We show that the T1 domain of Kv1.3 is necessary and sufficient to mediate targeting of the channel to the axonal surface in pyramidal neurons in slices of cortex from neonatal rat. The T1 domain is also sufficient to cause preferential axonal localization of intracellular protein, which indicates that the domain probably does not work through compartment-specific endocytosis or compartment-specific vesicle docking. To determine whether the T1 domain mediates axonal trafficking of transport vesicles, we compared the trafficking of vesicles containing green fluorescent protein-labelled transferrin receptor with those containing the same protein fused with the T1 domain in living cortical neurons. Vesicles containing the wild-type transferrin receptor did not traffic to the axon, in accord with previously published results; however, those containing the transferrin receptor fused to T1 did traffic to the axon. These results are consistent with the T1 domain of Kv1.3 mediating axonal targeting by causing transport vesicles to traffic to axons and they represent the first evidence that such a mechanism might underlie axonal targeting.

Molecular cloning of calcium channel alpha(2)delta-subunits from rat atria and the differential regulation of their expression by IGF-1.

Calcium channels are multimeric proteins consisting of pore-forming (alpha(1)) and auxiliary (alpha(2)delta, beta, gamma) subunits. The auxiliary alpha(2)delta-subunit regulates calcium current density and activation/inactivation kinetics when co-expressed with some, but not all, alpha(1)-subunits. Here we report the differential expression of three alpha(2)delta-subunit cDNAs in rat atria, atrial myocytes and ventricle, and demonstrate that IGF-1 selectively increases the expression of the alpha(2)delta-3 mRNA in the atria. mRNA encoding the alpha(2)delta-1- and alpha(2)delta-2-subunits, but not the alpha(2)delta-3-subunit, is detected in the rat ventricle whereas all three transcripts are found in atrial tissue. Analysis of the rat alpha(2)delta-1 cDNA sequence indicates that the atria express the alpha(2)delta-1e alternatively spliced isoform of this gene. The complete cDNA sequences of the alpha(2)delta-2- and alpha(2)delta-3-subunits from rat atria were determined and found to share 96% and 95% identity, respectively, with their counterparts in mouse. Treatment of acutely cultured atrial myocytes with IGF-1 caused a significant increase of the amount of alpha(2)delta-3, but not alpha(2)delta-1 or alpha(2)delta-2, mRNA. Both L-type and T-type calcium currents are recorded from cardiac tissue although their expression is regionally specific and changes with age and physiological state. Differential regulation of the expression of alpha(2)delta-subunit genes is likely to contribute to alterations in the expression of calcium current in the mammalian heart.

Distribution and relative expression levels of calcium channel beta subunits within the chambers of the rat heart.

Calcium channel beta subunits expressed in rat atria and atrial myocytes are identified and their expression quantified and compared to beta subunit expression in the ventricle. mRNAs encoding all four know beta subunits are expressed in atrial myocytes including the following splice variants: beta1a, beta2b, beta2c, beta2e and beta4d. The specific beta2 splice variants expressed in the atria (beta2b, beta2c, beta2e) differ from those previously reported from rat ventricle. Beta2 isoform is the most abundant beta mRNA expressed in the heart and the amount of both beta2 subunit mRNA and beta2 subunit protein is significantly greater in the ventricles than in the atria. The expression of individual beta2 splice variants varies with age and within different chambers of the heart. The beta2b splice variant appears in both atria and ventricle in both young (4.5 week) and old (16 week) animals, the beta2c isoform is more highly expressed in young as compared to old animals and the beta2e splice variant is robustly expressed only in 4.5 week ventricle. Beta4 mRNA expression is higher in atrial tissue than in ventricles and its expression decreases in older animals. In contrast, the abundance of the beta3 mRNA does not significantly change as a function of postnatal age. Antisense oligonucleotides targeting sequence common to all the beta isoforms as well as that specific for beta2 significantly reduced HVA calcium current density in isolated atrial cells confirming that the beta2 subunits contribute to the regulation of HVA calcium current expression in the rat atria. The complexity of beta isoform expression within the heart may provide a mechanism for functional fine-tuning of the cardiac HVA current.

Calcium channel gamma6 subunits are unique modulators of low voltage-activated (Cav3.1) calcium current.

The calcium channel gamma (gamma) subunit family consists of eight members whose functions include modulation of high voltage-activated (HVA) calcium currents in skeletal muscle and neurons, and regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propanoic acid (AMPA) receptor targeting. Cardiac myocytes express at least three gamma subunits, gamma(4), gamma(6) and gamma(7), whose function(s) in the heart are unknown. Here we compare the effects of the previously uncharacterized gamma(6) subunit with that of gamma(4) and gamma(7) on a low voltage-activated calcium channel (Cav3.1) that is expressed in cardiac myocytes. Co-expression of both the long and short gamma(6) subunit isoforms, gamma(6L) and gamma(6S), with Cav3.1 in HEK-293 cells significantly decreases current density by 49% and 69%, respectively. Two other gamma subunits expressed in cardiac myocytes, gamma(4) and gamma(7), have no significant effect on Cav3.1 current. Neither gamma(6L), gamma(6S), gamma(4) nor gamma(7) significantly affect the voltage dependency of activation or inactivation or the kinetics of Cav3.1 current. Transient expression of gamma(6L) in an immortalized atrial cell line (HL-1) significantly reduces the endogenous low voltage-activated current in these cells by 63%. Green fluorescent protein tagged gamma(6L) is localized primarily in HEK-293 cell surface membranes where it is evenly distributed. Expression of gamma(6L) does not affect the level of Cav3.1 mRNA or the amount of total Cav3.1 protein in transfected HEK-293 cells. These results demonstrate that the gamma(6) subunit has a unique ability to inhibit Cav3.1 dependent calcium current that is not shared with the gamma(4) and gamma(7) isoforms and is thus a potential regulator of cardiac low voltage-activated calcium current.