Yemiliya Berman

Bax cleavage is mediated by calpain during drug-induced apoptosis

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.

Carboxypeptidase E activity is deficient in mice with the fat mutation: Effect on peptide processing

Carboxypeptidase E (CPE) is involved in the biosynthesis of many peptide hormones and neurotransmitters. Mice with the fat mutation have previously been found to have a point mutation in the cpe gene, and to have greatly reduced levels of CPE-like enzyme activity in the pituitary and pancreatic islets (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). In the present report, we examined CPE-like activity and peptide processing in several tissues of C57BLKS/LtJ-Cpefat/Cpefat mutant (Cpefat/Cpefat) mice. Whereas CPE-like activity is detected in homogenates of Cpefat/Cpefat mouse tissues, the majority of this activity is not due to CPE based on the sensitivity to p-chloromercuriphenyl sulfonate. In addition, the Cpefat/Cpefat activity does not bind to a substrate affinity column under conditions that bind CPE. Furthermore, the enzyme activity and immunoreactive properties of the activity purified from Cpefat/Cpefat brain are distinct from those of CPE. Taken together, these data suggest that CPE is completely inactive in the Cpefat/Cpefat mice, and that all of the CPE-like activity is due to other carboxypeptidases such as carboxypeptidase D. Levels of Leu-enkephalin in Cpefat/Cpefat mouse brain are approximately 5-fold lower than those in control brain. Treatment of the Cpefat/Cpefat brain extract with carboxypeptidase B restores the level of Leu-enkephalin to the level in control brain. Interestingly, the large molecular weight enkephalin-containing peptides are elevated 2-3-fold in Cpefat/Cpefat mouse brain. These data indicate that CPE plays an important role in the processing of peptide hormones in various tissues, but that other carboxypeptidases also contribute to peptide processing. Furthermore, the increase in levels of high molecular weight enkephalin peptides in the Cpefat/Cpefat mouse suggests that CPE is required for efficient peptide processing by the endopeptidases.

Defective prodynorphin processing in mice lacking prohormone convertase PC2.

Abstract: Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A‐8 and a substantial reduction in the levels of Dyn A‐17 and Dyn B‐13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A‐8, Dyn A‐17, and Dyn B‐13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild‐type mice; however, the levels of Dyn peptides in these mice are similar to those of wild‐type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up‐regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild‐type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.

The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila

Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins). These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans. The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies. How methuselah functions in controlling ageing is not clear. A first essential step towards the understanding of methuselah function is to determine the ligands of Methuselah. Here we report the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B. Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress. We conclude that the Stunted–Methuselah system is involved in the control of animal ageing.

Curve-shift analysis of self-stimulation in food-restricted rats: Relationship between daily meal, plasma corticosterone and reward sensitization

Chronic food restriction lowers the threshold for lateral hypothalamic electrical self-stimulation (LHSS). This effect has previously been interpreted to reflect a sensitization of reward. In the present study a curve-shift method was used to explicitly differentiate effects of food restriction on brain stimulation rewarding efficacy and performance. Food restriction consistently shifted rate-frequency curves to the left, lowering the M-50 and Theta-0 parameters of rewarding efficacy. Asymptotic rates of reinforcement and slopes of rate-frequency functions were unaffected, confirming that food restriction does not facilitate LHSS by enhancing performance. In this and previous studies, LHSS in food-restricted rats was measured in the period immediately preceding the daily meal when hunger (i.e., period since last meal) and plasma corticosterone are at peak levels. In the light of evidence that corticosterone may regulate sensitivity of the mesolimbic dopamine pathway and account for the sensitizing effect of stress on psychomotor effects of opiates and stimulants, LHSS and corticosterone were measured in the immediate pre-and post-meal periods. While all food-restricted rats displayed elevated corticosterone levels in the pre-meal period and generally displayed a decline to control levels in the post-meal period, the sensitization of reward was not reversed in the post-meal period. These results indicate that chronic food restriction produces a sensitization of reward that does not depend upon the acute state of hunger that precedes the daily meal and does not vary with dynamic changes in plasma corticosterone level.

Chronic food restriction increases D-1 dopamine receptor agonist-induced phosphorylation of extracellular signal-regulated kinase 1/2 and cyclic AMP response element-binding protein in caudate-putamen and nucleus accumbens.

Results of behavioral and c-fos immunohistochemical studies have suggested that chronic food restriction and maintenance of animals at 75-80% of free-feeding body weight may increase d-1 dopamine (DA) receptor function. The purpose of the present study was to determine whether D-1 DA receptor binding and/or mitogen-activated protein kinase (MAPK) signaling in caudate-putamen (CPu) and nucleus accumbens (NAc) are increased in food-restricted subjects. In the first experiment, saturation binding of the D-1 DA receptor antagonist [3H]SCH-23390 indicated no difference between food-restricted and ad libitum fed rats with regard to density or affinity of d-1 binding sites in CPu or NAc. In the second experiment, activation of extracellular signal-regulated kinases (ERK1/2) and cyclic AMP response element-binding protein (CREB) by i.c.v. injection of the D-1 DA receptor agonist SKF-82958 (20 microg) were markedly greater in food-restricted than ad libitum fed rats. Given a prior finding that SKF-82958 does not differentially stimulate adenylyl cyclase in CPu or NAc of food-restricted versus ad libitum fed subjects, the present results suggest that increased D-1 DA receptor-mediated ERK1/2 MAP kinase signaling may mediate the enhanced downstream activation of CREB, c-fos, and behavioral responses in food-restricted subjects. It is of interest that food restriction also increased the activation of c-Jun N-terminal protein kinase/stress-activated protein kinase, but this effect was no greater in rats injected with SKF-82958 than in those injected with saline vehicle. This represents additional evidence of increased striatal cell signaling in food-restricted subjects, presumably in response to the i.c.v. injection procedure, although the underlying receptor mechanisms remain to be determined. There were no differences between feeding groups in protein levels of the major phosphatases, MKP-2 and PP1. The upregulation of striatal MAP kinase signaling in food-restricted animals may adaptively serve to facilitate associative learning but, at the same time, increase vulnerability to the rewarding and addictive properties of abused drugs.

Effects of an intrahypothalamic injection of antisense oligonucleotides for preproenkephalin mRNA in female rats: evidence for opioid involvement in lordosis reflex

Previous studies in female rats have shown that estrogen increases preproenkephalin (PPE) mRNA levels in the ventrolateral part of the ventromedial nucleus of the hypothalamus (VMHVL), an area implicated in the modulation of sexual behavior. In order to assess the physiological role of hypothalamic opioid expression in lordosis reflex 16-mer oligodeoxynucleotide (ODN) directed towards the PPE mRNA were acutely microinjected above the VMH of estradiol-primed ovariectomized rats. Estradiol-induced lordosis behavior was observed in response to a stud male 2 days thereafter. Antisense (without or with 4 mismatches) ODN injections near the VMHVL resulted in a significant reduction in lordosis quotient compared to control (reverse sense) ODN treatment or to antisense ODN injections targeted anterior or posterior to the VMHVL. In contrast, locomotor activity of these animals in the open-field test was not affected by ODN treatments. Enkephalin immunoreactive levels were determined by radioimmunoassay in the preoptic area, a major terminal field of the VMHVL. Estradiol-induced enkephalin levels were greatly reduced in antisense-treated groups. Using the in situ hybridization technique, PPE mRNA levels in the VMHVL were also determined. A 1.5-2-fold increase in PPE mRNA levels was observed in estradiol-treated rats compared to ovariectomized rats as previously described. This increase in PPE mRNA levels was not affected by ODN treatment, suggesting that the reduction of enkephalin expression was mainly due to physical blockade of PPE mRNA translation and not to its degradation. Taken together, these data further support the behavioral role of PPE expressing VMHVL neurons. They also highlight the in vivo potency of acute administration of antisense phosphorothioate ODNs in blocking neuronal target gene expression.

Chronic food restriction and streptozotocin-induced diabetes differentially alter prodynorphin mRNA levels in rat brain regions

It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative solution hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/microg total RNA), bed nucleus of the stria terminalis (4.18 pg/microg), and in caudate nucleus (3.51 pg/microg). Lower levels were observed in the lateral hypothalamus (1.87 pg/microg) and central nucleus of the amygdala (1.22 pg/microg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need.

Effects of chronic food restriction on prodynorphin-derived peptides in rat brain regions

Chronic food restriction produces a variety of physiological and behavioral adaptations including a potentiation of the reinforcing effect of food, drugs and lateral hypothalamic electrical stimulation. Previous work in this laboratory has revealed that the lowering of self-stimulation threshold by food restriction is reduced by mu- and kappa-selective opioid antagonists. In the present study, the effect of chronic food restriction on levels of three prodynorphin-derived peptides, namely dynorphin A1-17 (A1-17), dynorphin A1-8 (A1-8) and dynorphin B1-13 (B1-13) were measured in eleven brain regions known to be involved in appetite, taste and reward. Food restriction increased levels of A1-17 in dorsal medial (+19.6%), ventral medial (+24.2%) and medial preoptic (+82.9%) hypothalamic areas. Levels of A1-17 decreased in the central nucleus of the amygdala (-35.1%). Food restriction increased levels of A1-8 in nucleus accumbens (+34.4%), bed nucleus of the stria terminalis (+24.5%) and lateral hypothalamus (+41.9%). Food restriction had no effect on levels of B1-13. A1-17 is highly kappa-preferring and the brain regions in which levels increased all have a high ratio of kappa: mu and delta receptors. A1-8 is less discriminating among opioid receptor types and the brain regions in which levels increased have a low ratio of kappa: mu and delta receptors. The present results suggest that food restriction alters posttranslational processing within the dynorphin A domain of the prodynorphin precursor, possibly leading to a change in the balance between kappa and non-kappa opioid receptor stimulation in specific brain regions.

Synthesis, protein levels, activity, and phosphorylation state of tyrosine hydroxylase in mesoaccumbens and nigrostriatal dopamine pathways of chronically food-restricted rats

Chronic food restriction (FR) enhances the rewarding and motor-activating effects of abused drugs, and is accompanied by changes in dopamine (DA) dynamics and increased D-1 DA receptor-mediated cell signaling and transcriptional responses in nucleus accumbens (NAc). However, little is known about effects of FR on DA synthetic activity in the mesoaccumbens and nigrostriatal pathways. In Experiment 1 of the present study, tyrosine hydroxylase (TH) gene expression was measured in ventral tegmental area and substantia nigra, using real-time RT-PCR and in situ hybridization; no differences were observed between FR and ad libitum fed (AL) rats. In Experiment 2, TH protein levels, determined by Western blot, were found to be elevated in NAc and caudate-putamen (CPu) of FR relative to AL rats. In the absence of increased transcription, this may reflect a slowing of TH degradation. In Experiments 3 and 4, DA synthetic activity was assessed by Western blot measurement of TH phosphorylation at Ser40, and HPLC measurement of in vivo tyrosine hydroxylation rate, as reflected by DOPA accumulation following administration of a decarboxylase inhibitor (NSD-1015; 100 mg/kg, i.p.). Basal phospho-(Ser40)-TH levels did not differ between groups but DOPA accumulation was decreased by FR. Decreased DOPA synthesis, despite increased levels of TH protein, may reflect the inhibitory effect of increased DA binding to TH protein or decreased concentrations of cofactor tetrahydrobiopterin. Finally, in response to D-amphetamine (0.5 and 5.0 mg/kg, i.p.), phospho-(Ser40)-TH was selectively decreased in NAc of FR rats. This suggests increased feedback inhibition of DA synthesis-a possible consequence of postsynaptic receptor hypersensitivity, or increased extracellular DA concentration. These results indicate that FR increases TH protein levels, but may decrease the capacity for DA synthesis by decreasing TH activity. According to this scheme, the previously observed upregulation of striatal cell signaling and transcriptional responses to DA receptor agonist administration may include compensatory neuroadaptations.