MDMA-induced neurotoxicity of serotonin neurons involves autophagy and rilmenidine is protective against its pathobiology.

Toxicity of 3,4-methylenedioxymethamphetamine (MDMA) towards biogenic amine neurons is well documented and in primate brain predominantly affects serotonin (5-HT) neurons. MDMA induces damage of 5-HT axons and nerve fibres and intracytoplasmic inclusions. Whilst its pathobiology involves mitochondrially-mediated oxidative stress, we hypothesised MDMA possessed the capacity to activate autophagy, a proteostatic mechanism for degradation of cellular debris. We established a culture of ventral pons from embryonic murine brain enriched in 5-HT neurons to explore mechanisms of MDMA neurotoxicity and recruitment of autophagy, and evaluated possible neuroprotective actions of the clinically approved agent rilmenidine. MDMA (100 µM-1 mM) reduced cell viability, like rapamycin (RM) and hydrogen peroxide (H2O2), in a concentration- and time-dependent manner. Immunocytochemistry revealed dieback of 5-HT arbour: MDMA-induced injury was slower than for RM and H2O2, neuritic blebbing occurred at 48 and 72 h and Hoechst labelling revealed nuclear fragmentation with 100 µM MDMA. MDMA effected concentration-dependent inhibition of [3H]5-HT uptake with 500 µM MDMA totally blocking transport. Western immunoblotting for microtubule associated protein light chain 3 (LC3) revealed autophagosome formation after treatment with MDMA. Confocal analyses and immunocytochemistry for 5-HT, Hoechst and LC3 confirmed MDMA induced autophagy with abundant LC3-positive puncta within 5-HT neurons. Rilmenidine (1 µM) protected against MDMA-induced injury and image analysis showed full preservation of 5-HT arbours. MDMA had no effect on GABA neurons, indicating specificity of action at 5-HT neurons. MDMA-induced neurotoxicity involves autophagy induction in 5-HT neurons, and rilmenidine via beneficial actions against toxic intracellular events represents a potential treatment for its pathobiology in sustained usage.

GABAergic striatal neurons exhibit caspase-independent, mitochondrially mediated programmed cell death.

GABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3-morpholinylsydnoneimine (SIN-1), 3-nitropropionic acid (3-NP), NMDA, 3,5-dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow-onset kinetics with time-dependent annexin-V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis-inducing factor and Omi, suggestive of early recruitment of both caspase-dependent and caspase-independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN-1 produced mainly calpain activation, and 3-NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult-dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.

The relationship of cigarette smoking to postoperative complications from dental extractions among female inmates.

OBJECTIVES: The purpose of this study is to assess the contribution of smoking to postoperative complications, including alveolar osteitis (dry socket), after dental extractions. In addition, it attempts to determine the effect of the ban imposed on tobacco use in the prison on postoperative complications. STUDY DESIGN: All inmates having dental extractions at the Federal Correctional Institution in Danbury, CT, during the period January 2004 to April 2005, were included in this study (N = 219; mean age = 37.7 years). Data on postextraction complications were analyzed for association with smoking by using the chi-square test. Significance was set at P < .05. RESULTS: The incidences of overall complications and alveolar osteitis were 19.6% and 5.0%, respectively. It was found that (1) there was a significant difference in overall complications between smokers and nonsmokers (P = .02), (2) there was a significant difference in the incidence of alveolar osteitis between mandibular third molar and other extractions, regardless of smoking status (P = .02), (3) surgical trauma contributed significantly to both an increase in total complications (P = .05) and alveolar osteitis (P = .01), and (4) smoking appeared to be a contributing factor to increased complications among multiple extractions (P = .03). CONCLUSION: In this study, smoking, mandibular third molars, and surgical trauma were significantly associated with the increased incidence of overall complications including alveolar osteitis.

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk.

Excitotoxicity mediated via the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for l-glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up- and downstream of mitochondrial activation. AMPA-mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (deltapsi(m)), early release of cytochrome c (cyt c), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of deltapsi(m), and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke feltapsi(m) as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c, SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis.

Rotenone and MPP+ preferentially redistribute apoptosis-inducing factor in apoptotic dopamine neurons.

Rotenone and 1-methyl-4-phenylpyridinium produce parkinsonian models and we determined whether their mitochondrially mediated actions differentially redistributed the apoptogenic proteins, apoptosis-inducing factor and cytochrome c. Cultured rat mesencephalic dopamine neurons were exposed to rotenone (30 nM) and 1-methyl-4-phenylpyridinium (300 muM, 24 and 48 h) and apoptosis and mitochondrial redistribution of cytochrome c or apoptosis-inducing factor were quantified. Tyrosine hydroxylase-positive dopamine neurons underwent apoptosis (shrinkage, less neurites) and 40% released apoptosis-inducing factor with rotenone (24 h), whereas cytochrome c release reached this value at 48 h when 70% of cells had released apoptosis-inducing factor-positive. 1-Methyl-4-phenylpyridinium produced similar redistribution patterns for both proteins. Preferential redistribution of apoptosis-inducing factor before cytochrome c in dopamine neurons indicates caspase-independent mitochondrial proapoptotic signalling predominates in these parkinsonian models.

Differential effects of human neuromelanin and synthetic dopamine melanin on neuronal and glial cells.

We investigated the effects of neuromelanin (NM) isolated from the human substantia nigra and synthetic dopamine melanin (DAM) on neuronal and glial cell lines and on primary rat mesencephalic cultures. Lactate dehydrogenase (LDH) activity and lipid peroxidation were significantly increased in SK-N-SH cells by DAM but not by NM. In contrast, iron-saturated NM significantly increased LDH activity in SK-N-SH cells, compared with 100 mg/mL ETDA-treated NM containing a low concentration of bound iron. DAM, but not NM, stimulated hydroxyl radical production and increased SK-N-SH cell death via apoptotic-like mechanisms. Neither DAM nor NM induced any changes in the glial cell line U373. 3H-dopamine uptake in primary rat mesencephalic cultures was significantly reduced in DAM-compared with NM-treated cultures, accompanied by increased cell death via an apoptosis-like mechanism. Interestingly, Fenton-induced cell death was significantly decreased in cultures treated with both Fenton reagent and NM, an effect not seen in cultures treated with Fenton reagent plus DAM. These data are suggestive of a protective role for neuromelanin under conditions of high oxidative load. Our findings provide new evidence for a physiological role for neuromelanin in vivo and highlights the caution with which data based upon model systems should be interpreted.

Structural model of the amino propeptide of collagen XI alpha1 chain with similarity to the LNS domains.

Fibrillar collagens are the principal structural molecules of connective tissues. The assembly of collagen fibrils is regulated by quantitatively minor fibrillar collagens, types V and XI. A unique amino-terminal propeptide domain of these collagens has been attributed this regulatory role. The structure of the amino terminal propeptide has yet to be determined. Low sequence similarity necessitated a secondary structure-based method to carry out homology modeling based upon the determined structure of LNS family members, named for a common structure in the laminin LG5 domain, the neurexin 1B domain and the sex hormone binding globulin. Distribution of amino acids within the model suggested glycosaminoglycan interaction and calcium binding. These activities were tested experimentally. Sequence analyses of existing genes for collagens indicate that 16 known collagen alpha chains may contain an LNS domain. A similar approach may prove useful for structure/function studies of similar domains in other collagens with similar domains. This will provide mechanistic details of the organization and assembly of the extracellular matrix and the underlying basis of structural integrity in connective tissues. The absolute requirement for collagen XI in skeletal growth is indicated by collagen XI deficiencies such as chondrodystrophies found in the cho/cho mouse and in humans with Stickler syndrome.

Dietary polyphenols protect dopamine neurons from oxidative insults and apoptosis: investigations in primary rat mesencephalic cultures.

Naturally occurring polyphenols have the potential to prevent oxidative damage in various pathophysiological conditions. Various members of the flavonoid family were investigated to determine if they could protect mesencephalic dopamine (DA) neurones from injury and reduce apoptosis produced by oxidative stressors. Primary mesencephalic cultures were sensitive to oxidative insults (hydrogen peroxide, 4-hydroxynonenal, rotenone, 6-hydroxydopamine and N-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium hydrochloride (MPP+)) which produced concentration-dependent decreases in cellular viability across an apoptotic-necrotic continuum of injury. Flavonoids (catechin, quercetin, chrysin, puerarin, naringenin, genestein) protected mesencephalic cultures from injury by MPP+, which was shown by DNA fragmentation studies and tyrosine hydroxylase (TH) immunocytochemistry of DA neurones to occur by apoptosis. Catechin also reduced injury produced by hydrogen peroxide, 4-hydroxynonenal, rotenone and 6-hydroxydopamine as shown by increases in cellular viability and [3H]DA uptake. When the neuroprotection of catechin against MPP+-induced injury was compared to that produced by the caspase-3 inhibitor, Z-DVED-FMK, both reduced DNA fragmentation and the injury patterns of TH-positive neurones. These data demonstrate the neuroprotective abilities of flavonoids which are able to attenuate the apoptotic injury of mesencephalic DA neurones. Since these DA neurones are under oxidative stress in Parkinsonism, our findings suggest that flavonoids could provide benefits along with other anti-oxidant therapies in Parkinson's disease.

Suppression of MeCP2beta expression inhibits neurite extension in PC12 cells.

Regulation of gene expression is critical to the proper development of neuronal cells. The methyl-CpG binding protein 2 (MeCP2) operates as a transcriptional repressor by facilitating histone deacetylation and DNA methylation-dependent transcriptional silencing. This study examined the importance of MeCP2 in the regulation of neurite formation in PC12 cells. Expression of MeCP2 increased in a time-dependent manner after induction of neuronal differentiation. Expression was assessed at both the transcriptional and translation levels, and reached a maximum at 24 h post-induction. In addition, a marked inhibition of neurite extension and proper localization of a marker for synapse formation, synapsin I, were observed when MeCP2 expression was decreased by the addition of an antisense morpholino oligomer directed to the translational initiation site for MeCP2beta. The removal of the antisense oligomer allowed neurite extension to progress. However, the addition of antisense oligomer to previously differentiated PC12 cells did not affect established neurite processes. Taken collectively, our results indicate a role for MeCP2beta early in the events of neurite formation and that the relative levels of MeCP2alpha and MeCP2beta may be different in early differentiating neurons than is found in the adult brain. In addition, unique functions may exist for the two isoforms of MeCP2. Our results indicate that the inhibition of neurite elaboration caused by a reduction in MeCP2 may be reversible.

A novel role for cholecystokinin: regulation of mesenteric vascular resistance.

The aim of this work was to characterize the vasoactive effect of cholecystokinin on mesenteric vasculature. The mesenteric vascular bed of 3-month-old Sprague-Dawley rats was isolated and perfused at constant flow and changes in perfusion pressure monitored. CCK peptides lacked any direct contractile or relaxing effect on the mesenteric smooth muscle. Transmural nerve stimulation (TNS, 200 mA, 0.2 ms, 8 and 16 Hz) elicited an increase in perfusion pressure reflecting contraction of the bed and CCK inhibited neurogenic contractions elicited by 8 and 16 Hz TNS. The inhibition of neurogenic contractions was blocked by the CCK2 receptor (CCK2R) antagonist, L-365,260 (10 and 100 nM), but not by the CCK1R antagonist, SR-27897. The inhibition of neurogenic contractions was reversed by the non-specific NOS inhibitor, L-NAME as well as by the specific nNOS inhibitor, S-methyl-L-thiocitrulline. In whole-mount segments of mesenteric arteries, CCK2R was detected in the adventitia, in nerve terminals, where it co-localized with synaptophysin and nNOS. CCK-8 immunoreactive fibers were also detected. These results suggest that CCK mediates vasodilatation of the mesenteric vascular bed through the release of NO via its presynaptic CCK2R. Our findings provide, for the first time, a neural mechanism by which CCK may increase mesenteric blood flow.

Cholecystokinin modulates migration of gonadotropin-releasing hormone-1 neurons.

Expression of the brain-gut peptide cholecystokinin (CCK) in the developing olfactory-gonadotropin-releasing hormone-1 (GnRH-1) neuroendocrine systems was characterized, and the function of CCK in these systems was analyzed both in vivo and in vitro. We present novel data demonstrating that CCK transcript and protein are expressed in sensory cells in the developing olfactory epithelium and vomeronasal organ, with both ligand and receptors (CCK-1R and CCK-2R) found on olfactory axons throughout prenatal development. In addition, migrating GnRH-1 neurons in nasal regions express CCK-1R but not CCK-2R receptors. The role of CCK in olfactory-GnRH-1 system development was evaluated using nasal explants, after assessing that the in vivo expression of both CCK and CCK receptors was mimicked in this in vitro model. Exogenous application of CCK (10(-7) m) reduced both olfactory axon outgrowth and migration of GnRH-1 cells. This inhibition was mediated by CCK-1R receptors. Moreover, CCK-1R but not CCK-2R antagonism caused a shift in the location of GnRH-1 neurons, increasing the distance that the cells migrated. GnRH-1 neuronal migration in mice carrying a genetic deletion of either CCK-1R or CCK-2R receptor genes was also analyzed. At embryonic day 14.5, the total number of GnRH-1 cells was identical in wild-type and mutant mice; however, the number of GnRH-1 neurons within forebrain was significantly greater in CCK-1R-/- embryos, consistent with an accelerated migratory process. These results indicate that CCK provides an inhibitory influence on GnRH-1 neuronal migration, contributing to the appropriate entrance of these neuroendocrine cells into the brain, and thus represent the first report of a developmental role for CCK.

Immunolocalization of CCK1R in rat brain using a new anti-peptide antibody.

An antibody directed at the carboxy tail of the cholecystokinin-1 receptor (CCK1R) was characterized by ELISA and Western blotting. Immunohistochemistry established that CCK1R-like immunoreactivity (CCK1R-LI) was widely and topographically distributed through the neuroaxis, appearing relatively higher in rhi- and diencephalon, and intense in both neuronal somata (cytoplasmic) and processes. CCK1R-LI was found in new loci, but also in areas previously identified by receptor autoradiography, electrophysiology and in situ hybridization of CCK1R mRNA. The widespread distribution of CCK1R has implications for the functional roles of these receptors in brain. The high titre and low background seen with this new antiserum makes it of great value for cell and tissue research.

Interaction between amino propeptides of type XI procollagen alpha1 chains.

Type XI collagen is a quantitatively minor yet essential constituent of the cartilage extracellular matrix. The amino propeptide of the alpha1 chain remains attached to the rest of the molecule for a longer period of time after synthesis than the other amino propeptides of type XI collagen and has been localized to the surface of thin collagen fibrils. Yeast two-hybrid system was used to demonstrate that a homodimer of alpha1(XI) amino propeptide (alpha1(XI)Npp) could form in vivo. Interaction was also confirmed using multi-angle laser light scattering, detecting an absolute weight average molar mass ranging from the size of a monomer to the size of a dimer (25,000-50,000 g/mol), respectively. Binding was shown to be saturable by ELISA. An interaction between recombinant alpha1(XI)Npp and the endogenous alpha1(XI)Npp was observed, and specificity for alpha1(XI)Npp but not alpha2(XI)Npp was demonstrated by co-precipitation. The interaction between the recombinant form of alpha1(XI)Npp and the endogenous alpha1(XI)Npp resulted in a stable association during the regeneration of cartilage extracellular matrix by fetal bovine chondrocytes maintained in pellet culture, generating a protein that migrated with an apparent molecular mass of 50-60 kDa on an SDS-polyacrylamide gel.

Cholecystokinin induces cerebral vasodilatation via presynaptic CCK2 receptors: new implications for the pathophysiology of panic.

The authors report that cholecystokinin (CCK), via its subtype 2 receptor (CCK2R) located presynaptically on cerebral arteries, mediates the release of nitric oxide (NO), which induces vasodilatation. Whereas CCK octapeptide and its fragment CCK tetrapeptide (CCK-4) lack a direct effect on the smooth muscle of pial vessels, the authors showed that both CCK peptides modulate the neurogenic responses in bovine cerebral arteries. The neurogenic vasodilatation induced by CCK-4 was blocked by the CCK2R antagonist, L-365,260, and antagonized by neuronal NO synthase (nNOS) inhibitors, but was independent of the endothelium. In whole-mount arteries, CCK2Rs were detected in nerve fibers and colocalized with nNOS and synaptophysin. The findings provide, for the first time, a neural mechanism by which CCK may increase cerebral blood flow.