Biomedical subjects

Matthew J During

Publications and source records attributed to Matthew J During.

At least 19 recordsLinked to original sources

PubMed2006

Phosphorylation-dependent degradation of transgenic CREB protein initiated by heterodimerization.

The transcription factor CREB (cyclic AMP response element binding protein) is implicated in diverse brain functions and represents a prospective target in gene therapy for human disorders. However, the transgenic expression and stability of exogenously expressed CREB within the cell remains poorly characterized. Here we found that transient expression of a CREB dominant interfering mutant A-CREB or the inducible cAMP early repressor, ICER, led to the dramatic decrease of exogenously co-expressed CREB in 293 human embryonic kidney cells. Elevation of protein kinase A activity within the cells restored CREB protein levels. A-CREB did not effect the transient expression of a truncated CREB lacking the leucine zipper domain demonstrating a specific effect of heterodimerization on CREB protein stability. Somatic gene transfer into the rat brain using a recombinant adeno-associated virus vector provided robust expression of both transgenic CREB and ICER mRNAs under the control of a constitutive neuron specific enolase (NSE) promoter. In contrast to ICER, the expression of the transgenic CREB mRNA did not result in elevation of CREB protein levels within dentate granule cells of the hippocampus, suggesting its prompt degradation under basal conditions. However, following tetanization of the perforant pathway, which is known to induce CREB phosphorylation, there was a significant increase in the amount of transgenic CREB protein within dentate granule cells. Hence, heterodimerization of unphosphorylated CREB with either A-CREB or ICER triggers CREB protein degradation, whereas phosphorylation prevents CREB from such degradation both in vitro and in vivo.

Animals↗

PubMed2006

Oncoprotein Akt/PKB induces trophic effects in murine models of Parkinson's disease.

Despite promising preclinical studies, neurotrophic factors have not yet achieved an established role in the treatment of human neurodegenerative diseases. One impediment has been the difficulty in providing these macromolecules in sufficient quantity and duration at affected sites. An alternative approach is to directly activate, by viral vector transduction, intracellular signaling pathways that mediate neurotrophic effects. We have evaluated this approach in dopamine neurons of the substantia nigra, neurons affected in Parkinson's disease, by adeno-associated virus 1 transduction with a gene encoding a myristoylated, constitutively active form of the oncoprotein Akt/PKB. Adeno-associated virus Myr-Akt has pronounced trophic effects on dopamine neurons of adult and aged mice, including increases in neuron size, phenotypic markers, and sprouting. Transduction confers almost complete protection against apoptotic cell death in a highly destructive neurotoxin model. Activation of intracellular neurotrophic signaling pathways by vector transfer is a feasible approach to neuroprotection and restorative treatment of neurodegenerative disease.

Animals↗

PubMed2006

Combined deletion of Y1, Y2, and Y4 receptors prevents hypothalamic neuropeptide Y overexpression-induced hyperinsulinemia despite persistence of hyperphagia and obesity.

Neuropeptide Y (NPY) is a key regulator of energy homeostasis and is implicated in the development of obesity and type 2 diabetes. Whereas it is known that hypothalamic administration of exogenous NPY peptides leads to increased body weight gain, hyperphagia, and many hormonal and metabolic changes characteristic of an obesity syndrome, the Y receptor(s) mediating these effects is disputed and unclear. To investigate the role of different Y receptors in the NPY-induced obesity syndrome, we used recombinant adeno-associated viral vector to overexpress NPY in mice deficient of selective single or multiple Y receptors (including Y1, Y2, and Y4). Results from this study demonstrated that long-term hypothalamic overexpression of NPY lead to marked hyperphagia, hypogonadism, body weight gain, enhanced adipose tissue accumulation, hyperinsulinemia, and other hormonal changes characteristic of an obesity syndrome. NPY-induced hyperphagia, hypogonadism, and obesity syndrome persisted in all genotypes studied (Y1(-/-), Y2(-/-), Y2Y4(-/-), and Y1Y2Y4(-/-) mice). However, triple deletion of Y1, Y2, and Y4 receptors prevented NPY-induced hyperinsulinemia. These findings suggest that Y1, Y2, and Y4 receptors under this condition are not crucially involved in NPY's hyperphagic, hypogonadal, and obesogenic effects, but they are responsible for the central regulation of circulating insulin levels by NPY.

Adipose Tissue↗

PubMed2006

Subthalamic glutamic acid decarboxylase gene therapy: changes in motor function and cortical metabolism.

Parkinson's disease (PD) is associated with increased excitatory activity within the subthalamic nucleus (STN). We sought to inhibit STN output in hemiparkinsonian macaques by transfection with adeno-associated virus (AAV) containing the gene for glutamic acid decarboxylase (GAD). In total, 13 macaques were rendered hemiparkinsonian by right intracarotid 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injection. Seven animals were injected with AAV-GAD into the right STN, and six received an AAV gene for green fluorescent protein (GFP). Videotaped motor ratings were performed in a masked fashion on a weekly basis over a 55-week period. At 56 weeks, the animals were scanned with (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Histological examination was performed at the end of the study. No adverse events were observed after STN gene therapy. We found that the clinical rating scores for the two treatment groups had different patterns of change over time (group x time interaction, P<0.001). On FDG PET, the GAD animals exhibited an increase in glucose utilization in the right motor cortex relative to GFP controls (P<0.001). Metabolism in this region correlated with clinical ratings at end point (P<0.01). Histology confirmed GAD expression in treated animals. These findings suggest that STN AAV-GAD is well tolerated and potentially effective in a primate model of PD. The changes in motor cortical glucose utilization observed after gene therapy are consistent with the modulation of metabolic brain networks associated with this disorder.

Animals↗

PubMed2006

Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.

Glutamatergic signaling and intracellular calcium mobilization in the spinal cord are crucial for the development of nociceptive plasticity, which is associated with chronic pathological pain. Long-form Homer proteins anchor glutamatergic receptors to sources of calcium influx and release at synapses, which is antagonized by the short, activity-dependent splice variant Homer1a. We show here that Homer1a operates in a negative feedback loop to regulate the excitability of the pain pathway in an activity-dependent manner. Homer1a is rapidly and selectively upregulated in spinal cord neurons after peripheral inflammation in an NMDA receptor-dependent manner. Homer1a strongly attenuates calcium mobilization as well as MAP kinase activation induced by glutamate receptors and reduces synaptic contacts on spinal cord neurons that process pain inputs. Preventing activity-induced upregulation of Homer1a using shRNAs in mice in vivo exacerbates inflammatory pain. Thus, activity-dependent uncoupling of glutamate receptors from intracellular signaling mediators is a novel, endogenous physiological mechanism for counteracting sensitization at the first, crucial synapse in the pain pathway. Furthermore, we observed that targeted gene transfer of Homer1a to specific spinal segments in vivo reduces inflammatory hyperalgesia. Thus, Homer1 function is crucially involved in pain plasticity and constitutes a promising therapeutic target for the treatment of chronic inflammatory pain.

Animals↗

PubMed2006

Somatic gene transfer of cAMP response element-binding protein attenuates memory impairment in aging rats.

cAMP response element-binding protein (CREB) is important for the formation and facilitation of long-term memory in diverse models. However, to our knowledge, involvement of CREB in age-associated memory impairment has not been reported. Here, we use a recombinant adeno-associated virus vector to obtain stable transgenic expression of CREB as well as the inducible cAMP early repressor (ICER) in the hippocampus of adult rats. In a longitudinal study, we show that somatic gene transfer of both CREB and ICER does not alter long-term memory in young (3-month-old) rats. However, at 15 months of age, the same CREB-transduced rats show significantly better long-term memory in spatial-navigation and passive-avoidance tasks compared with their equally aged control littermates, and a threshold effect is evident. In contrast, the aged ICER-transduced rats demonstrate significantly reduced memory in comparison with the same control group. Hippocampal CREB gene transfer prevented the aging-related decrease in long-term memory found in the control rats. These data suggest that elevation of CREB protein levels in a subset of hippocampal neurons as achieved by somatic cell gene transfer might compensate for general deficits in molecular mechanisms underlying age-related memory loss in rats and, therefore, attenuate long-term-memory impairment during normal aging.

Adenoviridae↗

PubMed2006

Role of noradrenergic signaling by the nucleus tractus solitarius in mediating opiate reward.

Norepinephrine (NE) is widely implicated in opiate withdrawal, but much less is known about its role in opiate-induced locomotion and reward. In mice lacking dopamine beta-hydroxylase (DBH), an enzyme critical for NE synthesis, we found that NE was necessary for morphine-induced conditioned place preference (CPP; a measure of reward) and locomotion. These deficits were rescued by systemic NE restoration. Viral restoration of DBH expression in the nucleus tractus solitarius, but not in the locus coeruleus, restored CPP for morphine. Morphine-induced locomotion was partially restored by DBH expression in either brain region. These data suggest that NE signaling by the nucleus tractus solitarius is necessary for morphine reward.

Animals↗

PubMed2006

Local dopamine production in the dorsal striatum restores goal-directed behavior in dopamine-deficient mice.

To determine whether dopamine signaling in the dorsal striatum is sufficient for performance of goal-directed behaviors, local dopamine production was restored in the dorsal striatum of dopamine-deficient (DD) mice through viral-mediated gene therapy. Virally rescued DD (vrDD) mice were tested for learning of an appetitive T-maze task designed to measure goal-directed behavior. The results indicate that in contrast with the performance of DD mice that have dysregulated dopamine signaling, vrDD mice were able to perform the T-maze task and reverse their behavior as well as sham-operated control mice. The authors conclude that finely tuned dopaminergic signaling within the dorsal striatum is sufficient for performance of goal-directed behavior.

Animals↗

PubMed2006

Differential actions of NPY on seizure modulation via Y1 and Y2 receptors: evidence from receptor knockout mice.

PURPOSE: Neuropeptide Y (NPY) has been shown to modulate seizure activities. To provide further understanding of the involvement of two of the most abundantly expressed NPY receptors, Y1 and Y2, we assessed the effect of Y1 and Y2 gene deletion on systemic kainic acid-induced seizures. We also examined the effect of rAAV-mediated hippocampal NPY overexpression on seizure susceptibility in these receptor knockout mice. METHODS: Recombinant adeno-associated viral vector overexpressing NPY (rAAV-NPY) or an empty vector control (rAAV-Empty) was injected into the hippocampus of adult C57BL/6-129/SvJ wild-type male mice and mice deficient of Y1 or Y2 receptors on the same background. Four weeks after vector injection, mice were subjected to systemic kainic acid-induced seizures, and the seizure behaviors were scored. RESULTS: The rAAV-mediated hippocampal overexpression of NPY led to a twofold reduction in seizures induced by systemic kainic acid in wild-type mice and Y1 receptor knockout mice but not in mice deficient of Y2 receptors. A differential action by the receptors was observed in the seizure-induced mortality rate, with increased fatality in Y2-/- mice. In addition, although NPY overexpression did not significantly affect the mortality rate in Y2-/- and wild-type mice, it abolished KA-induced mortality in Y1-/-mice. CONCLUSIONS: This study shows for the first time an altered susceptibility to chemically induced seizures in Y1 and Y2 knockout mice and demonstrates a differential seizure modulation mediated by these receptors via a genetic approach.

Animals↗

PubMed2006

VEGF, a mediator of the effect of experience on hippocampal neurogenesis.

Rodents housed in an enriched environment, exercise by running or perform learning and memory tasks show an increase in hippocampal neurogenesis. We show that both environmental enrichment, as well as performance in the Morris water maze, a hippocampal-dependent learning task, leads to an increase in local VEGF expression in rats. We genetically recreated this situation by somatic cell gene transfer using recombinant adeno-associated virus (AAV) vectors. Genetically increasing hippocampal VEGF in adult rats resulted in a approximately 2 fold increase in neurogenesis associated with improved cognition. In contrast, gene transfer of placental growth factor (PGF) which signals through Flt1, but not KDR receptors had negative effects on neurogenesis and inhibited learning, although it similarly increased endothelial cell proliferation. Expression of a dominant negative, mKDR, inhibited basal neurogenesis and impaired learning. Co-expression of mKDR antagonized VEGF-enhanced neurogenesis and learning without inhibiting endothelial cell proliferation. Furthermore, inhibition of VEGF expression by RNA interference completely blocked the environmental induction of neurogenesis. These data support a model whereby VEGF acting via KDR is a mediator of the effect of the environment on neurogenesis and cognition [1].

Animals↗

PubMed2005

Distinct roles for different Homer1 isoforms in behaviors and associated prefrontal cortex function.

Homer1 mutant mice exhibit behavioral and neurochemical abnormalities that are consistent with an animal model of schizophrenia. Because the Homer1 gene encodes both immediate early gene (IEG) and constitutively expressed (CC) gene products, we used the local infusion of adeno-associated viral vectors carrying different Homer1 transcriptional variants into the prefrontal cortex (PFC) to distinguish between the roles for IEG and CC Homer1 isoforms in the "schizophrenia-like" phenotype of Homer1 mutant mice. PFC overexpression of the IEG Homer1 isoform Homer1a reversed the genotypic differences in behavioral adaptation to repeated stress, whereas overexpression of the constitutively expressed Homer1 isoform Homer1c reversed the genotypic differences in sensorimotor and cognitive processing, as well as cocaine behavioral sensitivity. Homer1a overexpression did not influence PFC basal glutamate content but blunted the glutamate response to cocaine in wild-type mice. In contrast, Homer1c overexpression reversed the genotypic difference in PFC basal glutamate content and enhanced cocaine-induced elevations in glutamate. These data demonstrate active and distinct roles for Homer1a and Homer1c isoforms in the PFC in the mediation of behavior, in the maintenance of basal extracellular glutamate, and in the regulation of PFC glutamate release relevant to schizophrenia and stimulant abuse comorbidity.

Animals↗

PubMed2005

A novel role of circadian transcription factor DBP in hippocampal plasticity.

In neurons, a variety of extracellular stimuli are capable of inducing transcriptional events that underlie complex processes ranging from learning to disease. The mechanisms linking these long-lasting cellular modifications to behavior remain to be established. Here, we show by microarray analysis that hippocampal activation of glucagon-like peptide-1 receptor (GLP-1R), which is associated with improved learning and neuroprotection, results in suppression of the transcription factor DBP (albumin D-site-binding protein). Recombinant adeno-associated virus (rAAV) based gene expression of DBP in the hippocampus of adult rats caused upregulation of mRNAs encoding constituents of the molecular clock, and the DBP target gene, pyridoxal kinase. Behaviorally, DBP over expression inhibited spatial learning but not memory, and enhanced susceptibility to kainate-induced seizures. This phenotype was paralleled by the activation of MAP kinase in dendritic regions of hippocampal neurons in vivo. These data suggest that DBP may represent an important transcriptional link between GLP-1R activation and neuroplasticity in the hippocampus.

Animals↗

PubMed2005

Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo.

The mechanisms involving neuronal death after ischemic/hypoxic insult are complex, involving both rapid (excitotoxic) and delayed (apoptotic-like) processes. Recent evidence suggests that cell cycle regulators such as cyclin-dependent kinases are abnormally activated in neuropathological conditions, including stroke. However, the function of this activation is unclear. Here, we provide evidence that inhibition of the cell cycle regulator, Cdk4, and its activator, cyclinD1, plays critical roles in the delayed death component of ischemic/hypoxic stress by regulating the tumor suppressor retinoblastoma protein. In contrast, the excitotoxic component of ischemia/hypoxia is predominately regulated by Cdk5 and its activator p35, components of a cyclin-dependent kinase complex associated with neuronal development. Hence, our data both characterize the functional significance of the cell cycle Cdk4 and neuronal Cdk5 signals as well as define the pathways and circumstances by which they act to control ischemic/hypoxic damage.

Animals↗

PubMed2005

AAV-mediated hippocampal expression of short and long Homer 1 proteins differentially affect cognition and seizure activity in adult rats.

Homer proteins mediate molecular rearrangements leading to changes in spine morphology. This points to a role of Homer in learning and memory. Homer 1c features both the ligand binding domain and a coiled-coiled domain for self-multimerization. Homer 1a lacks the coiled-coiled domain. Here, we report a new isoform which we termed 1g, lacking the Homer ligand binding domain. We dissected the functional roles of the individual Homer 1 domains, encoded by Homer 1a, 1c, and 1g, in vivo. Recombinant adeno-associated virus (AAV)-mediated overexpression of these forms in the hippocampus of adult rats has opposing effects on learning behavior. Increased levels of Homer 1a impaired hippocampal-dependent memory, while Homer 1g and 1c slightly enhanced memory performance. Homer 1g induced anxiety. Moreover, AAV-Homer 1a animals showed attenuation of electrographic seizures in a model of status epilepticus. These results suggest that Homer 1 proteins play an active role in behavioral plasticity.

Animals↗

PubMed2005

Restoration of aspartoacylase activity in CNS neurons does not ameliorate motor deficits and demyelination in a model of Canavan disease.

Canavan disease is an early onset leukodystrophy associated with psychomotor retardation, seizures, and premature death. This disorder is caused by mutations in the gene encoding the enzyme aspartoacylase (ASPA). Normally, ASPA is enriched in oligodendrocytes and ASPA deficiency results in elevated levels of its substrate molecule, N-acetylaspartate (NAA), brain edema, and dysmyelination. Using adeno-associated virus, we permanently expressed ASPA in CNS neurons of the tremor rat, a genetic model of Canavan disease, and examined the efficacy of the treatment by monitoring NAA metabolism, myelination, motor behavior, and seizures. Assessment of ASPA protein and enzyme activity in whole brain hemispheres showed restoration to normal levels as long as 6 months after treatment. This finding correlated with a reduction of NAA levels, along with a rescue of the seizure phenotype. However, gross brain pathology, such as dilated ventricles and spongiform vacuolization, was unchanged. Moreover, hypomyelination and motor deficits were not resolved by ASPA gene transfer. Our data suggest that NAA-mediated neuronal hyperexcitation but not oligodendrocyte dysfunction can be compensated for by neuronal ASPA expression.

Amidohydrolases↗

PubMed2004

Effect of altering titer, serotype, and promoter in recombinant adenoassociate virus gene therapy expression of spinal cord neurons and astrocytes.

STUDY DESIGN: Descriptive histologic analysis of spinal cord gene therapy. OBJECTIVE: To maximize protein expression in rat spinal cord using recombinant adenoassociate virus viral vector. SUMMARY OF BACKGROUND DATA: There are few reports of spinal cord genetic transfer. There have been no reports that compare techniques to increase protein expression through genetic alterations or have illustrated successful genetic transfer to spinal cord astrocytes. METHODS: Adenoassociate virus constructs were packaged using three separate plasmids: a cis plasmid with the expression cassettes (pAM/neuron-specific enolase/green fluorescent protein/woodchuck posttranscriptional regulatory element/simian virus 40/polyadenylase or pAM/glial fibrillary acid protein/green fluorescent protein/woodchuck posttranscriptional regulatory element/simian virus 40/polyadenylase), the Ad-adenoassociate virus helper trans plasmid, and the essential region from the adenovirus genome (pFDelta6). The adenoassociate virus 2/5 capsid gene replaces the adenoassociate virus 2 capsid region in the trans construct, resulting in a different cellular tropism. Thirty-two adult (300-375 g) male Sprague-Dawley rats underwent L1 laminectomies. A total volume of 6 microL was injected directly into the spinal cord parenchyma at a rate of 600 nL/min with adenoassociate virus 2/glial fibrillary acid protein/green fluorescent protein, adenoassociate virus 2/neuron-specific enolase/green fluorescent protein, adenoassociate virus 2/5/glial fibrillary acid protein/green fluorescent protein, or adenoassociate virus 2/5/neuron-specific enolase/green fluorescent protein and either a low- (4 x 10(8)) or high-titer (1 x 10(10)) viral solution. RESULTS: The gene expression (green fluorescent protein reporter) was present in the cell bodies and axonal processes of all adenoassociate virus/green fluorescent protein constructs. However, a greater spread of virus was observed in rats injected with adenoassociate virus 2/5 compared with adenoassociate virus 2. In addition, more neurons were transduced with adenoassociate virus 2/5 than adenoassociate virus 2, and green fluorescent protein expression in neurons transduced with adenoassociate virus 2/5 appeared more intense compared with adenoassociate virus 2 neurons. The difference observed between adenoassociate virus 2 and adenoassociate virus 2/5 at 4 x 10(8) genomic particles/mL was not as profound when the virus titer was raised to 1 x 10(10) genomic particles/mL. Green fluorescent protein expression was observed in astrocytes following injection of rat spinal cords with either adenoassociate virus 2 or adenoassociate virus 2/5 carrying the glial fibrillary acid protein/green fluorescent protein construct. However, unlike neuron-specific enolase-driven expression, there was less overall expression, but a substantial increase in green fluorescent protein expression was observed with adenoassociate virus 2/5 compared with adenoassociate virus 2 with high virus titers. Furthermore, unlike the neuron-specific enolase promoter, glial fibrillary acid protein-driven expression of green fluorescent protein was not restricted to astrocytes alone. The glial fibrillary acid protein construct was able to transfect glial cells and maintain glial expression. CONCLUSION: Adenoassociate virus can readily transduce spinal cord neurons and is an efficient nonpathologic vector to deliver expression cassettes. Increased titers and the adenoassociate virus 2/5 serotype appeared to maximize expression.

Adenoviridae↗

PubMed2004

Dysregulation of striatal dopamine signaling by amphetamine inhibits feeding by hungry mice.

Amphetamine (AMPH) releases monoamines, transiently stimulates locomotion, and inhibits feeding. Using a genetic approach, we show that mice lacking dopamine (DA-deficient, or DD, mice) are resistant to the hypophagic effects of a moderate dose of AMPH (2 microg/g) but manifest normal AMPH-induced hypophagia after restoration of DA signaling in the caudate putamen by viral gene therapy. By contrast, AMPH-induced hypophagia in response to the same dose of AMPH is not blunted in mice lacking the ability to make norepinephrine and epinephrine (Dbh(-/-)), dopamine D(2) receptors (D2r(-/-)), dopamine D(1) receptors (D1r(-/-)), serotonin 2C receptors (Htr2c(-/Y)), neuropeptide Y (Npy(-/-)), and in mice with compromised melanocortin signaling (A(y)). We suggest that, at this moderate dose of AMPH, dysregulation of striatal DA is the primary cause of AMPH-induced hypophagia and that regulated striatal dopaminergic signaling may be necessary for normal feeding behaviors.

Amphetamine↗

PubMed2004

A role for dopamine in feeding responses produced by orexigenic agents.

Dopamine-deficient (DD) mice become hypophagic and die of starvation by 3 to 4 weeks of age unless dopamine is restored by daily treatment with l-3-4-dihydroxyphenylalanine (l-dopa). We demonstrate here that DD mice mount qualitatively normal counter-regulatory blood glucose responses to insulin and 2-deoxy-d-glucose (2-DG). However, unlike control mice, DD mice fail to eat in response to acute glucoprivation induced by insulin or 2-DG. They also have a severely blunted response to central administration of peptide YY (PYY). Viral-mediated restoration of dopamine synthesis to the central caudate putamen (CPu) of DD mice rescues feeding and survival. However, this treatment fails to restore insulin- and 2-DG-induced feeding despite normalizing feeding in response to food deprivation and PYY. Since dopamine signaling in the CPu is not sufficient for glucoprivation-induced feeding, we propose that this feeding behavior may be mediated by dopamine in an anatomically distinct brain region.

Adenoviridae↗