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1.
Sci Rep ; 8(1): 126, 2018 01 09.
Article in English | MEDLINE | ID: mdl-29317669

ABSTRACT

We previously found that genetic mutants with reduced expression or activity of Scn8a are resistant to induced seizures and that co-segregation of a mutant Scn8a allele can increase survival and seizure resistance of Scn1a mutant mice. In contrast, Scn8a expression is increased in the hippocampus following status epilepticus and amygdala kindling. These findings point to Scn8a as a promising therapeutic target for epilepsy and raise the possibility that aberrant overexpression of Scn8a in limbic structures may contribute to some epilepsies, including temporal lobe epilepsy. Using a small-hairpin-interfering RNA directed against the Scn8a gene, we selectively reduced Scn8a expression in the hippocampus of the intrahippocampal kainic acid (KA) mouse model of mesial temporal lobe epilepsy. We found that Scn8a knockdown prevented the development of spontaneous seizures in 9/10 mice, ameliorated KA-induced hyperactivity, and reduced reactive gliosis. These results support the potential of selectively targeting Scn8a for the treatment of refractory epilepsy.


Subject(s)
Epilepsy, Temporal Lobe/genetics , Epilepsy, Temporal Lobe/metabolism , NAV1.6 Voltage-Gated Sodium Channel/genetics , NAV1.6 Voltage-Gated Sodium Channel/metabolism , Seizures/genetics , Seizures/metabolism , Animals , Disease Models, Animal , Electroencephalography , Epilepsy, Temporal Lobe/diagnosis , Gene Expression Regulation , Gene Knockdown Techniques , Genetic Predisposition to Disease , Hippocampus/metabolism , Immunohistochemistry , Male , Mice , RNA, Small Interfering/genetics , Seizures/diagnosis
2.
Eur J Neurosci ; 40(9): 3351-62, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25156382

ABSTRACT

Chronic restraint stress impairs hippocampal-mediated spatial learning and memory, which improves following a post-stress recovery period. Here, we investigated whether brain-derived neurotrophic factor (BDNF), a protein important for hippocampal function, would alter the recovery from chronic stress-induced spatial memory deficits. Adult male Sprague-Dawley rats were infused into the dorsal hippocampal cornu ammonis (CA)3 region with an adeno-associated viral vector containing the sequence for a short hairpin RNA (shRNA) directed against BDNF or a scrambled sequence (Scr). Rats were then chronically restrained (wire mesh, 6 h/day for 21 days) and assessed for spatial learning and memory using a radial arm water maze (RAWM) either immediately after stressor cessation (Str-Imm) or following a 21-day post-stress recovery period (Str-Rec). All groups learned the RAWM task similarly, but differed on the memory retention trials. Rats in the Str-Imm group, regardless of adeno-associated viral contents, committed more errors in the spatial reference memory domain on the single retention trial during day 3 than did the non-stressed controls. Importantly, the typical improvement in spatial memory following the recovery from chronic stress was blocked with the shRNA against BDNF, as Str-Rec-shRNA performed worse on the RAWM compared with the non-stressed controls or Str-Rec-Scr. The stress effects were specific for the reference memory domain, but knockdown of hippocampal BDNF in unstressed controls briefly disrupted spatial working memory as measured by repeated entry errors on day 2 of training. These results demonstrated that hippocampal BDNF was necessary for the recovery from stress-induced hippocampal-dependent spatial memory deficits in the reference memory domain.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , CA3 Region, Hippocampal/metabolism , Spatial Memory/physiology , Stress, Psychological/metabolism , Animals , Down-Regulation , Male , Rats , Rats, Sprague-Dawley , Restraint, Physical
3.
J Cell Mol Med ; 18(1): 115-24, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24237934

ABSTRACT

The identification of new components implicated in the pathogenesis of osteoarthritis (OA) might improve our understanding of the disease process. Here, we investigated the levels of the survival of motor neuron (SMN) expression in OA cartilage considering the fundamental role of the SMN protein in cell survival and its involvement in other stress-associated pathologies. We report that SMN expression is up-regulated in human OA compared with normal cartilage, showing a strong correlation with the disease severity, a result confirmed in vivo in an experimental model of the disease. We further show that the prominent inflammatory cytokines (IL-1ß, TNF-α) are critical inducers of SMN expression. This is in marked contrast with the reported impaired levels of SMN in spinal muscular atrophy, a single inherited neuromuscular disorder characterized by mutations in the smn gene whereas OA is a complex disease with multiple aetiologies. While the precise functions of SMN during OA remain to be elucidated, the conclusions of this study shed light on a novel pathophysiological pathway involved in the progression of OA, potentially offering new targets for therapy.


Subject(s)
Osteoarthritis, Knee/metabolism , Survival of Motor Neuron 1 Protein/metabolism , Aged , Animals , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Cells, Cultured , Chondrocytes/metabolism , Gene Expression , Humans , Interleukin-1beta/physiology , Osteoarthritis, Knee/pathology , Osteoarthritis, Knee/surgery , Rabbits , Severity of Illness Index , Survival of Motor Neuron 1 Protein/genetics , Tumor Necrosis Factor-alpha/physiology
4.
Eur J Neurosci ; 39(6): 1009-1017, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24354924

ABSTRACT

The nucleus accumbens (NAc) is a critical brain region for the rewarding effects of drugs of abuse. Brain-derived neurotrophic factor (BDNF) can facilitate stress- and drug-induced neuroadaptation in the mesocorticolimbic system. BDNF-containing projections to the NAc originate from the ventral tegmental area (VTA) and the prefrontal cortex, and BDNF release activates tropomyosin-related kinase B (TrkB). In this study, we examined the necessity for BDNF-TrkB signaling in the NAc shell during social defeat stress-induced cross-sensitization to amphetamine. Adeno-associated virus expressing short hairpin RNA directed against TrkB (AAV-shTrkB) was infused bilaterally into the NAc shell to knock down TrkB, whereas AAV-GFP (green fluorescent protein) was used as the control virus. Rats were exposed to intermittent social defeat stress or handling procedures; amphetamine challenge was given at 10 days after the last defeat and locomotor activity was measured. Stressed rats that received the control virus showed cross-sensitization to amphetamine compared with the handled rats. In contrast, NAc TrkB knockdown prevented social defeat stress-induced cross-sensitization. TrkB knockdown in the NAc was found to reduce the level of phospho-extracellular signal-regulated kinase 1 in this region. NAc TrkB knockdown also prevented stress-induced elevation of BDNF and the glutamate receptor type 1 (GluA1) subunit of AMPA receptor in the VTA, as well as ΔFosB expression in the NAc. These findings indicated that BDNF-TrkB signaling in the NAc shell was required for social defeat stress-induced cross-sensitization. NAc TrkB-BDNF signaling also appeared to be involved in the regulation of GluA1 in the VTA, as well as in the NAc ΔFosB accumulation that could trigger cross-sensitization after social defeat stress.


Subject(s)
Amphetamine/pharmacology , Central Nervous System Sensitization , Nucleus Accumbens/metabolism , Receptor, trkB/metabolism , Stress, Psychological/metabolism , Animals , Brain-Derived Neurotrophic Factor/metabolism , Locomotion , Male , Nucleus Accumbens/drug effects , Nucleus Accumbens/physiology , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor, trkB/genetics , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Social Behavior , Stress, Psychological/physiopathology
5.
Neuropsychopharmacology ; 38(11): 2286-96, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23689674

ABSTRACT

Social defeat stress induces persistent cross-sensitization to psychostimulants, but the molecular mechanisms underlying the development of cross-sensitization remain unclear. One candidate is brain-derived neurotrophic factor (BDNF). The present research examined whether ventral tegmental area (VTA) BDNF overexpression would prolong the time course of cross-sensitization after a single social defeat stress, which normally produces transient cross-sensitization lasting <1 week. ΔFosB, a classic molecular marker of addiction, was also measured in mesocorticolimbic terminal regions. Separate groups of intact male Sprague-Dawley rats underwent a single episode of social defeat stress or control handling, followed by amphetamine (AMPH) challenge 3 or 14 days later. AMPH cross-sensitization was apparent 3, but not 14, days after stress. Intra-VTA infusion of adeno-associated viral (AAV-BDNF) vector resulted in a twofold increase of BDNF level in comparison to the group receiving the control virus (AAV-GFP), which lasted at least 45 days. Additionally, overexpression of BDNF in the VTA alone increased ΔFosB in the nucleus accumbens (NAc) and prefrontal cortex. Fourteen days after viral infusions, a separate group of rats underwent a single social defeat stress or control handling and were challenged with AMPH 14 and 24 days after stress. AAV-BDNF rats exposed to stress showed prolonged cross-sensitization and facilitated sensitization to the second drug challenge. Immunohistochemistry showed that the combination of virally enhanced VTA BDNF, stress, and AMPH resulted in increased ΔFosB in the NAc shell compared with the other groups. Thus, elevation of VTA BDNF prolongs cross-sensitization, facilitates sensitization, and increases ΔFosB in mesocorticolimbic terminal regions. As such, elevated VTA BDNF may be a risk factor for drug sensitivity.


Subject(s)
Amphetamine/pharmacology , Brain-Derived Neurotrophic Factor/biosynthesis , Cerebral Cortex/metabolism , Nucleus Accumbens/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Stress, Psychological/metabolism , Ventral Tegmental Area/metabolism , Adenoviridae , Aggression , Animals , Brain-Derived Neurotrophic Factor/administration & dosage , Central Nervous System Sensitization , Central Nervous System Stimulants/pharmacology , Cerebral Cortex/drug effects , Male , Microinjections , Motor Activity/drug effects , Prefrontal Cortex/metabolism , Rats , Time Factors
6.
Horm Behav ; 63(3): 518-26, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23370363

ABSTRACT

Polymorphisms in noncoding regions of the vasopressin 1a receptor gene (Avpr1a) are associated with a variety of socioemotional characteristics in humans, chimpanzees, and voles, and may impact behavior through a site-specific variation in gene expression. The socially monogamous prairie vole offers a unique opportunity to study such neurobiological control of individual differences in complex behavior. Vasopressin 1a receptor (V1aR) signaling is necessary for the formation of the pair bond in males, and prairie voles exhibit greater V1aR binding in the reward-processing ventral pallidum than do asocial voles of the same genus. Diversity in social behavior within prairie voles has been correlated to natural variation in neuropeptide receptor expression in specific brain regions. Here we use RNA interference to examine the causal relationship between intraspecific variation in V1aR and behavioral outcomes, by approximating the degree of naturalistic variation in V1aR expression. Juvenile male prairie voles were injected with viral vectors expressing shRNA sequences targeting Avpr1a mRNA into the ventral pallidum. Down-regulation of pallidal V1aR density resulted in a significant impairment in the preference for a mated female partner and a reduction in anxiety-like behavior in adulthood. No effect on alloparenting was detected. These data demonstrate that within-species naturalistic-like variation in V1aR expression has a profound effect on individual differences in social attachment and emotionality. RNA interference may prove to be a useful technique to unite the fields of behavioral ecology and neurogenetics to perform ethologically relevant studies of the control of individual variation and offer insight into the evolutionary mechanisms leading to behavioral diversity.


Subject(s)
Anxiety/metabolism , Arvicolinae/physiology , Basal Ganglia/metabolism , Pair Bond , Receptors, Vasopressin/metabolism , Sexual Behavior, Animal/physiology , Vasopressins/physiology , Animals , Down-Regulation/physiology , Female , Genetic Vectors/administration & dosage , Individuality , Male , RNA, Messenger/genetics , RNA, Small Interfering/administration & dosage
7.
Blood ; 121(11): 2154-64, 2013 Mar 14.
Article in English | MEDLINE | ID: mdl-23315169

ABSTRACT

Angiogenesis plays an important role in cancer and in many other human diseases. Vascular endothelial growth factor-A (VEGF-A), the best known angiogenic factor, was originally discovered as a potent vascular permeability factor (VPF), suggesting that other vascular permeabilizing agents, such as histamine and serotonin, might also have angiogenic activity. We recently demonstrated that, like VEGF-A, histamine and serotonin up-regulate the orphan nuclear receptor and transcription factor TR3 (mouse homolog Nur77) and that TR3/Nur77 is essential for their vascular permeabilizing activities. We now report that histamine and serotonin are also angiogenic factors that, at low micromolar concentrations, induce endothelial cell proliferation, migration and tube formation in vitro, and angiogenesis in vivo. All of these responses are mediated through specific histamine and serotonin receptors, are independent of VEGF-A, and are directly dependent on TR3/Nur77. Initially, the angiogenic response closely resembled that induced by VEGF-A, with generation of "mother" vessels. However, after ~10 days, mother vessels began to regress as histamine and serotonin, unlike VEGF-A, up-regulated the potent angiogenesis inhibitor thrombospondin-1, thereby triggering a negative feedback loop. Thus, histamine and serotonin induce an angiogenic response that fits the time scale of acute inflammation.


Subject(s)
Histamine/pharmacology , Neovascularization, Physiologic/drug effects , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/physiology , Serotonin/pharmacology , Thrombospondin 1/physiology , Animals , Capillary Permeability/drug effects , Cells, Cultured , Female , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Neovascularization, Physiologic/genetics , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/genetics , Thrombospondin 1/metabolism , Up-Regulation/drug effects , Up-Regulation/genetics
8.
Am J Physiol Gastrointest Liver Physiol ; 304(1): G26-37, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23104559

ABSTRACT

Hepatocyte nuclear factor-4α (HNF4α) is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4α may inhibit hepatocyte proliferation via mechanisms that have yet to be identified. Using a HNF4α knockdown mouse model based on delivery of inducible Cre recombinase via an adeno-associated virus 8 viral vector, we investigated the role of HNF4α in the regulation of hepatocyte proliferation. Hepatocyte-specific deletion of HNF4α resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the downregulated genes were previously known HNF4α target genes involved in hepatic differentiation. Interestingly, ≥500 upregulated genes were associated with cell proliferation and cancer. Furthermore, we identified potential negative target genes of HNF4α, many of which are involved in the stimulation of proliferation. Using chromatin immunoprecipitation analysis, we confirmed binding of HNF4α at three of these genes. Furthermore, overexpression of HNF4α in mouse hepatocellular carcinoma cells resulted in a decrease in promitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4α actively inhibits hepatocyte proliferation by repression of specific promitogenic genes.


Subject(s)
Hepatocyte Nuclear Factor 4/genetics , Hepatocytes/metabolism , Animals , Blotting, Western , Capillary Electrochromatography , Cell Cycle/physiology , Cell Line , Cell Proliferation , Chromatin Immunoprecipitation , Dependovirus/genetics , Flow Cytometry , Fluorescent Antibody Technique , Gene Deletion , Immunohistochemistry , Liver Regeneration , Mice , Mice, Knockout , Microarray Analysis , Mitosis/genetics , Real-Time Polymerase Chain Reaction
9.
J Neurosci Methods ; 212(1): 106-13, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23022696

ABSTRACT

The cholinergic neurons of the basal forebrain (BFCNs) in human and non-human primates are rich in the calcium binding protein calbindin-D(28k) (CB). We have shown a selective loss of CB from BFCNs in the course of normal aging, which appears to predispose these neurons to tangle formation and degeneration in Alzheimer's disease. Our previous preliminary investigation demonstrated that rodent BFCNs are devoid of CB. Here we confirm that rat choline acetyltransferase-rich BFCNs are devoid of CB immunoreactivity. We then describe a method for adeno-associated viral vector (AAV) induced expression of CB in rat BFCNs in vivo. We constructed AAV vectors bearing the CB gene under the control of the CMV promoter, or neuron-specific enolase (NSE) promoter, to bias expression in neurons. Both vectors resulted in CB expression in mouse neuronal cultures, and in rat brain following injections. AAV-NSE-CB resulted in more robust expression in neurons. Injections of 10 µl of AAV-NSE-CB in the BFCNs component located within the internal segment of globus pallidus and internal capsule resulted in expression of CB in 84% of BFCNs. Expression was optimum at 14 days. Injections of AAV-NSE-LacZ resulted in robust ß-galactosidase expression, but no CB immunoreactivity. Our results show that use of NSE promoter leads to high expression of genes in neurons and that the BFCNs can be targeted for expression of genes that are differentially expressed in the rodent and primate brains. These findings have important implications for gene replacement therapy in human BFCNs.


Subject(s)
Cholinergic Neurons/metabolism , Dependovirus/genetics , Gene Expression Regulation/genetics , Prosencephalon/cytology , S100 Calcium Binding Protein G/metabolism , Acyl Carrier Protein/metabolism , Animals , Calbindins , Cells, Cultured , Choline O-Acetyltransferase/metabolism , Genetic Vectors/physiology , Male , Mice , Phosphopyruvate Hydratase/metabolism , Rats , Rats, Sprague-Dawley , S100 Calcium Binding Protein G/genetics , Species Specificity , Transduction, Genetic , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
10.
Mol Med ; 18: 346-58, 2012 May 09.
Article in English | MEDLINE | ID: mdl-22160392

ABSTRACT

Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein [IGFBP]-3 and IGFBP4) while up-regulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 [MAPK/ERK-1/2] and phosphatidylinisitol-3/Akt [PI3K/Akt] signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis.


Subject(s)
Cartilage/metabolism , Chondrocytes/metabolism , Dependovirus/genetics , Insulin-Like Growth Factor I/metabolism , Osteoarthritis/metabolism , Aged , Cell Proliferation , Genetic Vectors , Humans , Insulin-Like Growth Factor Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptor, IGF Type 1/metabolism , Recombination, Genetic
11.
J Neurosci ; 31(45): 16447-57, 2011 Nov 09.
Article in English | MEDLINE | ID: mdl-22072694

ABSTRACT

Chronic exposure to addictive drugs enhances cAMP response element binding protein (CREB)-regulated gene expression in nucleus accumbens (NAc), and these effects are thought to reduce the positive hedonic effects of passive cocaine administration. Here, we used viral-mediated gene transfer to produce short- and long-term regulation of CREB activity in NAc shell of rats engaging in volitional cocaine self-administration. Increasing CREB expression in NAc shell markedly enhanced cocaine reinforcement of self-administration behavior, as indicated by leftward (long-term) and upward (short-term) shifts in fixed ratio dose-response curves. CREB also increased the effort exerted by rats to obtain cocaine on more demanding progressive ratio schedules, an effect highly correlated with viral-induced modulation of BDNF protein in the NAc shell. CREB enhanced cocaine reinforcement when expressed either throughout acquisition of self-administration or when expression was limited to postacquisition tests, indicating a direct effect of CREB independent of reinforcement-related learning. Downregulating endogenous CREB in NAc shell by expressing a short hairpin RNA reduced cocaine reinforcement in similar tests, while overexpression of a dominant-negative CREB(S133A) mutant had no significant effect on cocaine self-administration. Finally, increasing CREB expression after withdrawal from self-administration enhanced cocaine-primed relapse, while reducing CREB levels facilitated extinction of cocaine seeking, but neither altered relapse induced by cocaine cues or footshock stress. Together, these findings indicate that CREB activity in NAc shell increases the motivation for cocaine during active self-administration or after withdrawal from cocaine. Our results also highlight that volitional and passive drug administration can lead to substantially different behavioral outcomes.


Subject(s)
Anesthetics, Local/administration & dosage , CREB-Binding Protein/metabolism , Cocaine/administration & dosage , Gene Expression Regulation/drug effects , Nucleus Accumbens/drug effects , Reinforcement, Psychology , Animals , Brain-Derived Neurotrophic Factor/metabolism , CREB-Binding Protein/genetics , Cocaine/adverse effects , Conditioning, Operant/drug effects , Drug Administration Routes , Extinction, Psychological/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Male , Mutation/genetics , Nucleus Accumbens/metabolism , RNA Interference/physiology , Rats , Rats, Sprague-Dawley , Reinforcement Schedule , Self Administration , Statistics as Topic , Substance Withdrawal Syndrome/physiopathology , Transfection/methods
12.
Neurosci Lett ; 502(3): 192-6, 2011 Sep 20.
Article in English | MEDLINE | ID: mdl-21839142

ABSTRACT

Mesolimbic brain-derived neurotrophic factor (BDNF) is implicated in sustained behavioral changes following chronic social stress, and its depletion may reduce susceptibility to such behavioral alterations. Enhanced mesolimbic BDNF is proposed as pro-depressive and anhedonic, while depleting ventral tegmetal area (VTA) BDNF increases weight by enhancing hedonic eating. Here, we questioned whether depletion of VTA BDNF would alleviate social defeat stress-induced deficits in weight regulation, or affect social behavior in the presence or absence of social stress. Male Sprague-Dawley rats received bilateral intra-VTA infusions of adeno-associated virus (AAV) vectors containing shRNA against BDNF or a control virus. Three weeks later, rats underwent 4 episodes of social defeat stress involving exposure to an aggressive Long-Evans resident rat, or control handling every third day. Depleted VTA BDNF conferred resistance to the deficient weight regulation normally observed during intermittent social defeat stress, and enhanced long-term weight gain regardless of stress history. In addition, social approach and avoidance behavior towards a novel social target were measured 7 weeks after stress. Social defeat stress chronically reduced social behavior, whereas depletion of VTA BDNF chronically increased social behavior. Our results reveal that depletion of VTA BDNF alleviates some consequences of intermittent social defeat stress, enhances social behavior, and may contribute to weight gain. These data implicate VTA BDNF in protracted behavioral responses to stress, social stimuli, and weight regulation.


Subject(s)
Body Weight/genetics , Brain-Derived Neurotrophic Factor/deficiency , Dependovirus/genetics , Social Behavior , Stress, Psychological/physiopathology , Ventral Tegmental Area/metabolism , Animals , Anxiety Disorders/genetics , Anxiety Disorders/physiopathology , Anxiety Disorders/virology , Brain-Derived Neurotrophic Factor/antagonists & inhibitors , Brain-Derived Neurotrophic Factor/genetics , Depressive Disorder/genetics , Depressive Disorder/physiopathology , Depressive Disorder/virology , Disease Models, Animal , Genetic Vectors/physiology , Long-Term Care , Male , RNA, Small Interfering/physiology , Rats , Rats, Long-Evans , Rats, Sprague-Dawley , Stress, Psychological/genetics , Stress, Psychological/virology , Ventral Tegmental Area/physiopathology , Ventral Tegmental Area/virology
13.
J Neurosci ; 30(35): 11735-44, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20810894

ABSTRACT

Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.


Subject(s)
Brain-Derived Neurotrophic Factor/biosynthesis , Brain-Derived Neurotrophic Factor/genetics , Chromatin Assembly and Disassembly/drug effects , Chromatin Assembly and Disassembly/physiology , Cocaine/administration & dosage , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , Reinforcement, Psychology , Animals , Behavior, Addictive/metabolism , Behavior, Addictive/psychology , Cocaine/pharmacology , Cocaine-Related Disorders/metabolism , Cocaine-Related Disorders/psychology , Male , Rats , Rats, Sprague-Dawley , Self Administration , Transcription, Genetic/drug effects , Transcription, Genetic/physiology , Up-Regulation/drug effects , Up-Regulation/physiology
14.
J Exp Med ; 207(6): 1197-208, 2010 Jun 07.
Article in English | MEDLINE | ID: mdl-20513747

ABSTRACT

How proliferative and inhibitory signals integrate to control liver regeneration remains poorly understood. A screen for antiproliferative factors repressed after liver injury identified transducer of ErbB2.1 (Tob1), a member of the PC3/BTG1 family of mito-inhibitory molecules as a target for further evaluation. Tob1 protein decreases after 2/3 hepatectomy in mice secondary to posttranscriptional mechanisms. Deletion of Tob1 increases hepatocyte proliferation and accelerates restoration of liver mass after hepatectomy. Down-regulation of Tob1 is required for normal liver regeneration, and Tob1 controls hepatocyte proliferation in a dose-dependent fashion. Tob1 associates directly with both Caf1 and cyclin-dependent kinase (Cdk) 1 and modulates Cdk1 kinase activity. In addition, Tob1 has significant effects on the transcription of critical cell cycle components, including E2F target genes and genes involved in p53 signaling. We provide direct evidence that levels of an inhibitory factor control the rate of liver regeneration, and we identify Tob1 as a crucial check point molecule that modulates the expression and activity of cell cycle proteins.


Subject(s)
Carrier Proteins/metabolism , Liver Regeneration/physiology , Repressor Proteins/metabolism , Animals , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Carrier Proteins/genetics , Cell Proliferation , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Dependovirus/genetics , Exoribonucleases , Gene Expression Regulation , Gene Regulatory Networks , Hepatectomy , Hepatocytes/metabolism , Hepatocytes/pathology , Intracellular Signaling Peptides and Proteins , Liver/enzymology , Liver/pathology , Mice , Mice, Inbred C57BL , Organ Size , Protein Binding , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleases , Signal Transduction/genetics , Smad Proteins/metabolism , Transcription, Genetic
15.
Biol Psychiatry ; 67(1): 28-35, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19709647

ABSTRACT

BACKGROUND: Previous research has shown that rats reared in an enriched condition (EC) are more sensitive to the acute effects of amphetamine than rats reared in an isolated condition (IC); yet, EC rats self-administer less amphetamine than IC rats. The present study used cocaine to further explore this environmental enrichment behavioral phenotype, as well as the underlying molecular mechanisms involved. METHODS: Enriched condition and IC rats were studied in a broad battery of behavioral tests, including cocaine conditioned place preference (CPP) and self-administration and several measures of anxiety- and depression-related behavior. The involvement of the transcription factor, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), in mediating EC versus IC differences was investigated. RESULTS: Enriched condition rats exhibited less cocaine self-administration, despite showing enhanced cocaine CPP. Enriched condition rats also displayed less depression-like behavior but higher levels of anxiety-like behavior. This behavioral phenotype is consistent with low CREB activity in the nucleus accumbens, a key brain reward region. Indeed, EC rats have less phospho-CREB (the transcriptionally active form of the protein) in the nucleus accumbens than IC rats, and a selective knockdown of CREB in this brain region of normally reared rats, by use of a novel viral vector expressing a short hairpin RNA (shRNA) directed against CREB, reproduced the EC behavioral phenotype. CONCLUSIONS: These studies identify a potential molecular mechanism for how rearing environment-a nonpharmacological, nonsurgical manipulation-can modify a wide range of complex emotional behaviors.


Subject(s)
Behavioral Symptoms , CREB-Binding Protein/metabolism , Environment , Nucleus Accumbens/metabolism , Phenotype , Analysis of Variance , Animals , Animals, Newborn , Anxiety/metabolism , Anxiety/pathology , Behavior, Animal/physiology , Behavioral Symptoms/metabolism , Behavioral Symptoms/pathology , Behavioral Symptoms/physiopathology , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , CREB-Binding Protein/genetics , Cocaine/administration & dosage , Conditioning, Operant/drug effects , Depression/metabolism , Depression/pathology , Disease Models, Animal , Dopamine Uptake Inhibitors/administration & dosage , Dose-Response Relationship, Drug , Food Preferences/physiology , Male , Nucleus Accumbens/drug effects , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Rats , Rats, Sprague-Dawley , Reinforcement Schedule , Self Administration
16.
Blood ; 114(17): 3588-600, 2009 Oct 22.
Article in English | MEDLINE | ID: mdl-19700666

ABSTRACT

Targeting dendritic cell (DC) functions such as migration is a pivotal mechanism used by HIV-1 to disseminate within the host. The HIV-1 envelope protein is the most important of the virally encoded proteins that exploits the migratory capacity of DCs. In the present study, we elucidated the signaling machinery involved in migration of immature DCs (iDCs) in response to HIV-1 envelope protein. We observed that M-tropic HIV-1 glycoprotein 120 (gp120) induces phosphorylation of the nonreceptor tyrosine kinase, Pyk2. Inhibition of Pyk2 activity using a pharmacologic inhibitor, kinase-inactive Pyk2 mutant, and Pyk2-specific small interfering RNA blocked gp120-induced chemotaxis, confirming the role of Pyk2 in iDC migration. In addition, we also illustrated the importance of Pyk2 in iDC migration induced by virion-associated envelope protein, using aldithriol-2-inactivated M-tropic HIV-1 virus. Further analysis of the downstream signaling mechanisms involved in gp120-induced migration revealed that Pyk2 activates p38 mitogen-activated protein kinase, which in turn activates the F-actin-binding protein, leukocyte-specific protein 1, and enhances its association with actin. Taken together, our studies provide an insight into a novel gp120-mediated pathway that regulates DC chemotaxis and contributes to the dissemination of HIV-1 within an infected person.


Subject(s)
Cell Movement , Dendritic Cells/metabolism , Focal Adhesion Kinase 2/metabolism , HIV Envelope Protein gp120/metabolism , Microfilament Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Blotting, Western , Chemotaxis , Flow Cytometry , Focal Adhesion Kinase 2/antagonists & inhibitors , Focal Adhesion Kinase 2/genetics , HIV Envelope Protein gp120/genetics , Humans , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/genetics , Phosphorylation/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Receptors, CCR5/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tyrphostins/pharmacology , p38 Mitogen-Activated Protein Kinases/genetics
17.
J Neurosci ; 29(5): 1312-8, 2009 Feb 04.
Article in English | MEDLINE | ID: mdl-19193878

ABSTRACT

Oxytocin receptors in the nucleus accumbens have been implicated in the regulation of alloparental behavior and pair bond formation in the socially monogamous prairie vole. Oxytocin receptor density in the nucleus accumbens is positively correlated with alloparenting in juvenile and adult female prairie voles, and oxytocin receptor antagonist infused into the nucleus accumbens blocks this behavior. Furthermore, prairie voles have higher densities of oxytocin receptors in the accumbens than nonmonogamous rodent species, and blocking accumbal oxytocin receptors prevents mating-induced partner preference formation. Here we used adeno-associated viral vector gene transfer to examine the functional relationship between accumbal oxytocin receptor density and social behavior in prairie and meadow voles. Adult female prairie voles that overexpress oxytocin receptor in the nucleus accumbens displayed accelerated partner preference formation after cohabitation with a male, but did not display enhanced alloparental behavior. However, partner preference was not facilitated in nonmonogamous meadow voles by introducing oxytocin receptor into the nucleus accumbens. These data confirm a role for oxytocin receptor in the accumbens in the regulation of partner preferences in female prairie voles, and suggest that oxytocin receptor expression in the accumbens is not sufficient to promote partner preferences in nonmonogamous species. These data are the first to demonstrate a direct relationship between oxytocin receptor density in the nucleus accumbens and variation in social attachment behaviors. Thus, individual variation in oxytocin receptor expression in the striatum may contribute to natural diversity in social behaviors.


Subject(s)
Arvicolinae/physiology , Nucleus Accumbens/metabolism , Receptors, Oxytocin/metabolism , Sexual Behavior, Animal/physiology , Social Behavior , Animals , Female , Male , Mating Preference, Animal/physiology , Nucleus Accumbens/chemistry , Nucleus Accumbens/physiology , Pair Bond , Receptors, Oxytocin/physiology
18.
J Biol Chem ; 284(1): 575-584, 2009 Jan 02.
Article in English | MEDLINE | ID: mdl-18927086

ABSTRACT

Strontium ranelate exerts both an anti-catabolic and an anabolic effect on bone cells. To further investigate the molecular mechanism whereby strontium ranelate inhibits bone resorption, we focused our attention on the effects of strontium ranelate on osteoclast apoptosis and on the underlying mechanism(s). Using primary mature rabbit osteoclasts, we demonstrated that strontium (Sro2+) dose-dependently stimulates the apoptosis of mature osteoclasts. As shown previously for calcium (Cao2+), the Sro2+-induced effect on mature osteoclasts is mediated by the Cao2+-sensing receptor, CaR, which in turn stimulates a phospholipase C-dependent signaling pathway and nuclear translocation of NF-kappaB. Unlike Cao2+, however, Sro2+-induced osteoclast apoptosis was shown to depend on PKCbetaII activation and to be independent of inositol 1,4,5-trisphosphate action. As a consequence of these differences in their intracellular signaling pathways, Sro2+ and Cao2+ in combination were shown to exert a greater effect on mature osteoclast apoptosis than did either divalent cation by itself. Altogether, our results show that Sro2+ acts through the CaR and induces osteoclast apoptosis through a signaling pathway similar to but different in certain respects from that of Cao2+. This difference in the respective signaling cascades enables Sro2+ to potentiate Cao2+-induced osteoclast apoptosis and vice versa. In this manner, it is conceivable that Sro2+ and Cao2+ act together to inhibit bone resorption in strontium ranelate-treated patients.


Subject(s)
Apoptosis/drug effects , Bone Density Conservation Agents/pharmacology , Organometallic Compounds/pharmacology , Osteoclasts/metabolism , Receptors, Calcium-Sensing/metabolism , Signal Transduction/drug effects , Thiophenes/pharmacology , Active Transport, Cell Nucleus/drug effects , Animals , Bone Resorption/metabolism , Calcium/metabolism , Cell Nucleus/metabolism , Cells, Cultured , Enzyme Activation/drug effects , NF-kappa B/metabolism , Osteoclasts/cytology , Protein Kinase C/metabolism , Protein Kinase C beta , Rabbits , Type C Phospholipases/metabolism
19.
J Cell Mol Med ; 13(8B): 2476-2488, 2009 Aug.
Article in English | MEDLINE | ID: mdl-18705695

ABSTRACT

Compensating for the loss of extracellular cartilage matrix, as well as counteracting the alterations of the chondrocyte phenotype in osteoarthritis are of key importance to develop effective therapeutic strategies against this disorder. In the present study, we analysed the benefits of applying a potent gene combination to remodel human osteoarthritic (OA) cartilage. We employed the promising recombinant adeno-associated virus (rAAV) vector to deliver the mitogenic fibroblast growth factor 2 (FGF-2) factor, alone or simultaneously with the transcription factor Sox9 as a key activator of matrix synthesis, to human normal and OA articular chondrocytes. We evaluated the effects of single (FGF-2) or combined (FGF-2/SOX9) transgene expression upon the regenerative activities of chondrocytes in three dimensional cultures in vitro and in cartilage explants in situ. Single overexpression of FGF-2 enhanced the survival and proliferation of both normal and OA chondrocytes, without stimulating the matrix synthetic processes in the increased pools of cells. The mitogenic properties of FGF-2 were maintained when SOX9 was co-overexpressed and concomitant with an increase in the production of proteoglycans and type-II collagen, suggesting that the transcription factor was capable of counterbalancing the effects of FGF-2 on matrix accumulation. Also important, expression of type-X collagen, a marker of hypertrophy strongly decreased following treatment by the candidate vectors. Most remarkably, the levels of activities achieved in co-treated human OA cartilage were similar to or higher than those observed in normal cartilage. The present findings show that combined expression of candidate factors in OA cartilage can re-establish key features of normal cartilage and prevent the pathological shift of metabolic homeostasis. These data provide further motivation to develop coupled gene transfer approaches via rAAV for the treatment of human OA.


Subject(s)
Cartilage/physiopathology , Dependovirus/genetics , Fibroblast Growth Factor 2/genetics , Gene Transfer Techniques , Osteoarthritis/physiopathology , SOX9 Transcription Factor/genetics , Genetic Therapy , Genetic Vectors , Humans , Osteoarthritis/genetics , Osteoarthritis/therapy
20.
J Immunol ; 180(8): 5636-44, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18390748

ABSTRACT

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells.


Subject(s)
Endothelial Cells/metabolism , Focal Adhesion Kinase 2/metabolism , Interleukin-8/biosynthesis , Endothelial Cells/drug effects , Endothelial Cells/immunology , Focal Adhesion Kinase 2/immunology , Humans , Interleukin-8/immunology , Lipopolysaccharides/immunology , MAP Kinase Kinase Kinases/immunology , MAP Kinase Kinase Kinases/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/metabolism , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , Tyrphostins/pharmacology
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