The endoplasmic reticulum acetyltransferases ATase1/NAT8B and ATase2/NAT8 are differentially regulated to adjust engagement of the secretory pathway.

Nε-lysine acetylation of nascent glycoproteins within the endoplasmic reticulum (ER) lumen regulates the efficiency of the secretory pathway. The ER acetylation machinery consists of the membrane transporter, acetyl-CoA transporter 1 (AT-1/SLC33A1), and two acetyltransferases, ATase1/NAT8B and ATase2/NAT8. Dysfunctional ER acetylation is associated with severe neurological diseases with duplication of AT-1/SLC33A1 being associated with autism spectrum disorder, intellectual disability, and dysmorphism. Neuron-specific AT-1 over-expression in the mouse alters neuron morphology and function, causing an autism-like phenotype, indicating that ER acetylation plays a key role in neurophysiology. As such, characterizing the molecular mechanisms that regulate the acetylation machinery could reveal critical information about its biology. By using structure-biochemistry approaches, we discovered that ATase1 and ATase2 share enzymatic properties but differ in that ATase1 is post-translationally regulated via acetylation. Furthermore, gene expression studies revealed that the promoters of AT-1, ATase1, and ATase2 contain functional binding sites for the neuron-related transcription factors cAMP response element-binding protein and the immediate-early genes c-FOS and c-JUN, and that ATase1 and ATase2 exhibit additional modes of transcriptional regulation relevant to aging and Alzheimer's disease. In vivo rodent gene expression experiments revealed that Atase2 is specifically induced following activity-dependent events. Finally, over-expression of either ATase1 or ATase2 was sufficient to increase the engagement of the secretory pathway in PC12 cells. Our results indicate important regulatory roles for ATase1 and ATase2 in neuron function with induction of ATase2 expression potentially serving as a critical event that adjusts the efficiency of the secretory pathway for activity-dependent neuronal functions.

Hippocampal Homer1b/c is necessary for contextual fear conditioning and group I metabotropic glutamate receptor mediated long-term depression.

Coiled-coil forms of Homer1, including Homer1b and c (Homer1b/c) have been shown to play a role in hippocampal learning and memory and synaptic plasticity. We have previously found that overexpression of hippocampal Homer1c is sufficient to rescue learning and memory ability in aged learning impaired rats and in Homer1 knockout (KO) mice, and to rescue group I metabotropic glutamate receptor (mGluR1/5) mediated long-term potentiation in KO mice. Here, to determine if Homer1b/c is necessary for successful learning and memory we have utilized a rAAV5 vector expressing a Homer1b/c-targeting short hairpin RNA to knock down the expression of hippocampal Homer1b/c in adult 4-6-month old male Sprague Dawley rats. We have found that reduced hippocampal Homer1b/c expression elicits significant learning deficits in contextual fear conditioning, but not in the Morris water maze or novel object recognition tasks. Furthermore, we demonstrate that reduced hippocampal Homer1b/c is sufficient to completely block mGluR1/5 mediated long-term depression in the Schaffer collateral pathway. These results support a significant role for Homer1b/c in learning and synaptic plasticity; however, the exact role of each of these two protein isoforms in learning and memory remains elusive.

Improved proteostasis in the secretory pathway rescues Alzheimer's disease in the mouse.

The aberrant accumulation of toxic protein aggregates is a key feature of many neurodegenerative diseases, including Huntington's disease, amyotrophic lateral sclerosis and Alzheimer's disease. As such, improving normal proteostatic mechanisms is an active target for biomedical research. Although they share common pathological features, protein aggregates form in different subcellular locations. Nε-lysine acetylation in the lumen of the endoplasmic reticulum has recently emerged as a new mechanism to regulate the induction of autophagy. The endoplasmic reticulum acetylation machinery includes AT-1/SLC33A1, a membrane transporter that translocates acetyl-CoA from the cytosol into the endoplasmic reticulum lumen, and ATase1 and ATase2, two acetyltransferases that acetylate endoplasmic reticulum cargo proteins. Here, we used a mutant form of α-synuclein to show that inhibition of the endoplasmic reticulum acetylation machinery specifically improves autophagy-mediated disposal of toxic protein aggregates that form within the secretory pathway, but not those that form in the cytosol. Consequently, haploinsufficiency of AT-1/SLC33A1 in the mouse rescued Alzheimer's disease, but not Huntington's disease or amyotrophic lateral sclerosis. In fact, intracellular toxic protein aggregates in Alzheimer's disease form within the secretory pathway while in Huntington's disease and amyotrophic lateral sclerosis they form in different cellular compartments. Furthermore, biochemical inhibition of ATase1 and ATase2 was also able to rescue the Alzheimer's disease phenotype in a mouse model of the disease. Specifically, we observed reduced levels of soluble amyloid-ß aggregates, reduced amyloid-ß pathology, reduced phosphorylation of tau, improved synaptic plasticity, and increased lifespan of the animals. In conclusion, our results indicate that Nε-lysine acetylation in the endoplasmic reticulum lumen regulates normal proteostasis of the secretory pathway; they also support therapies targeting endoplasmic reticulum acetyltransferases, ATase1 and ATase2, for a subset of chronic degenerative diseases.

Reduced juvenile long-term depression in tuberous sclerosis complex is mitigated in adults by compensatory recruitment of mGluR5 and Erk signaling.

Tuberous sclerosis complex (TSC) is a multisystem genetic disease that manifests with mental retardation, tumor formation, autism, and epilepsy. Heightened signaling through the mammalian target of rapamycin (mTOR) pathway is involved in TSC pathology, however it remains unclear how other signaling pathways are perturbed and contribute to disease symptoms. Reduced long-term depression (LTD) was recently reported in TSC mutant mice. We find that although reduced LTD is a feature of the juvenile mutant hippocampus, heightened expression of metabotropic glutamate receptor 5 and constitutively activated Erk signaling in the adult hippocampus drives wild-type levels of LTD. Increased mGluR5 and Erk results in a novel mTOR-independent LTD in CA1 hippocampus of adult mice, and contributes to the development of epileptiform bursting activity in the TSC2(+/-) CA3 region of the hippocampus. Inhibition of mGluR5 or Erk signaling restores appropriate mTOR-dependence to LTD, and significantly reduces epileptiform bursting in TSC2(+/-) hippocampal slices. We also report that adult TSC2(+/-) mice exhibit a subtle perseverative behavioral phenotype that is eliminated by mGluR5 antagonism. These findings highlight the potential of modulating the mGluR5-Erk pathway in a developmental stage-specific manner to treat TSC.

Environmental enrichment improves learning and memory and long-term potentiation in young adult rats through a mechanism requiring mGluR5 signaling and sustained activation of p70s6k.

Previous studies from our lab have demonstrated that mild cognitive impairments identified early in life are predictive of cognitive deficits that develop with age, suggesting that enhancements in cognition at an early age can provide a buffer against age-related cognitive decline. Environmental enrichment has been shown to improve learning and memory in the rodent, but the impact of enrichment on synaptic plasticity and the molecular mechanisms behind enrichment are not completely understood. To address these unresolved issues, we have housed 2-month old rats in environmentally enriched (EE), socially enriched (SE), or standard housing (SC) and conducted tests of learning and memory formation at various time intervals. Here we demonstrate that animals that have been exposed to one month of social or environmental enrichment demonstrate enhanced learning and memory relative to standard housed controls. However, we have found that after 4months EE animals perform better than both SE and SC groups and demonstrate an enhanced hippocampal LTP. Our results demonstrate that this LTP is dependent on mGluR5 signaling, activation of ERK and mTOR signaling cascades, and sustained phosphorylation of p70s6 kinase, thus providing a potential target mechanism for future studies of cognitive enhancement in the rodent.

The role of Homer1c in metabotropic glutamate receptor-dependent long-term potentiation.

Group I metabotropic glutamate receptors (mGluR1/5) play a role in synaptic plasticity and they demonstrate direct interactions with the neuronal Homer1c protein. We have previously shown that Homer1c can restore the plasticity deficits in Homer1 knockout mice (H1-KO). Here, we investigated the role of Homer1c in mGluR-dependent synaptic plasticity in wild-type mice, H1-KO, and H1-KO mice overexpressing Homer1c (KO+H1c). We used a form of plasticity induced by activation of mGluR1/5 that transforms short-term potentiaion (STP) induced by a subthreshold theta burst stimulation into long-term potentiation (LTP). We have shown that although acute hippocampal slices from wild-type animals can induce LTP using this stimulation protocol, H1-KO only show STP. Gene delivery of Homer1c into the hippocampus of H1-KO mice rescued LTP to wild-type levels. This form of synaptic plasticity was dependent on mGluR5 but not mGluR1 activation both in wild-type mice and in KO+H1c. mGluR1/5-dependent LTP was blocked with inhibitors of the MEK-ERK and PI3K-mTOR pathways in KO+H1c mice. Moreover, blocking Homer1c-mGluR5 interactions prevented the maintenance of LTP in acute hippocampal slices from KO+H1c. These data indicate that Homer1c-mGluR5 interactions are necessary for mGluR-dependent LTP, and that mGluR1/5-dependent LTP involves PI3K and ERK activation.

Ablation of Mitochondrial RCC1-L Induces Nigral Dopaminergic Neurodegeneration and Parkinsonian-like Motor Symptoms.

Mitochondrial dysfunction has been linked to both idiopathic and familial forms of Parkinson's disease (PD). We have previously identified RCC1-like (RCC1L) as a protein of the inner mitochondrial membrane important to mitochondrial fusion. Herein, to test whether deficits in RCC1L mitochondrial function might be involved in PD pathology, we have selectively ablated the Rcc1l gene in the dopaminergic (DA) neurons of mice. A PD-like phenotype resulted that includes progressive movement abnormalities, paralleled by progressive degeneration of the nigrostriatal tract. Experimental and control groups were examined at 2, 3-4, and 5-6 months of age. Animals were tested in the open field task to quantify anxiety, exploratory drive, locomotion, and immobility; and in the cylinder test to quantify rearing behavior. Beginning at 3-4 months, both female and male Rcc1l knockout mice show rigid muscles and resting tremor, kyphosis and a growth deficit compared with heterozygous or wild type littermate controls. Rcc1l knockout mice begin showing locomotor impairments at 3-4 months, which progress until 5-6 months of age, at which age the Rcc1l knockout mice die. The progressive motor impairments were associated with progressive and significantly reduced tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta (SNc), and dramatic loss of nigral DA projections in the striatum. Dystrophic spherical mitochondria are apparent in the soma of SNc neurons in Rcc1l knockout mice as early as 1.5-2.5 months of age and become progressively more pronounced until 5-6 months. Together, the results reveal the RCC1L protein to be essential to in vivo mitochondrial function in DA neurons. Further characterization of this mouse model will determine whether it represents a new model for in vivo study of PD, and the putative role of the human RCC1L gene as a risk factor that might increase PD occurrence and severity in humans.

Rescue of synaptic plasticity and spatial learning deficits in the hippocampus of Homer1 knockout mice by recombinant Adeno-associated viral gene delivery of Homer1c.

Homer1 belongs to a family of scaffolding proteins that interact with various post-synaptic density proteins including group I metabotropic glutamate receptors (mGluR1/5). Previous research in our laboratory implicates the Homer1c isoform in spatial learning. Homer1 knockout mice (H1-KO) display cognitive impairments, but their synaptic plasticity properties have not been described. Here, we investigated the role of Homer1 in long-term potentiation (LTP) in the hippocampal CA1 region of H1-KO mice in vitro. We found that late-phase LTP elicited by high frequency stimulation (HFS) was impaired, and that the induction and maintenance of theta burst stimulation (TBS) LTP were reduced in H1-KO. To test the hypothesis that Homer1c was sufficient to rescue these LTP deficits, we delivered Homer1c to the hippocampus of H1-KO using recombinant adeno-associated virus (rAAV). We found that rAAV-Homer1c rescued HFS and TBS-LTP in H1-KO animals. Next, we tested whether the LTP rescue by Homer1c was occurring via mGluR1/5. A selective mGluR5 antagonist, but not an mGluR1 antagonist, blocked the Homer1c-induced recovery of late-LTP, suggesting that Homer1c mediates functional effects on plasticity via mGluR5. To investigate the role of Homer1c in spatial learning, we injected rAAV-Homer1c to the hippocampus of H1-KO. We found that rAAV-Homer1c significantly improved H1-KO performance in the Radial Arm Water Maze. These results point to a significant role for Homer1c in synaptic plasticity and learning.

Nurr1 regulates RET expression in dopamine neurons of adult rat midbrain.

Genesis of midbrain dopamine (DA) neurons depends on Nurr1, a nuclear receptor expressed during development and adulthood in these neurons. Nurr1 is required for the expression of genes of dopaminergic phenotype such as tyrosine hydroxylase and DA transporter. The expression of the tyrosine kinase receptor RET also depends on Nurr1 during development. However, it is unknown whether RET expression is regulated by Nurr1 during adulthood, and the mechanism by which Nurr1 regulates RET expression. Using an adeno-associated vector-delivered anti-Nurr1 ribozyme, we knocked-down Nurr1 expression unilaterally in the substantia nigra (SN) of adult rats. Animals injected with the ribozyme displayed a 57.3% decrease in Nurr1 mRNA in the SN accompanied by decreased DA extracellular levels in the striatum. RET mRNA in the injected SN and RET protein in the ipsilateral striatum decreased 76.9% and 47%, respectively. Tyrosine hydroxylase and DA transporter mRNA did not change in Nurr1 knocked-down SN. Nurr1 induced the transcription of the human RET promoter in cell type and concentration-dependent manner. Nurr1 induction of RET promoter is independent of NBRE elements. These results show that the expression of RET in rat adult SN is regulated by Nurr1 and suggest that RET is a transcriptional target of this nuclear receptor.

Striatal readministration of rAAV vectors reveals an immune response against AAV2 capsids that can be circumvented.

Recombinant adeno-associated virus (rAAV) expresses no viral genes after transduction. In addition, because the brain is relatively immunoprivileged, intracranial rAAV transduction may be immunologically benign due to a lack of antigen presentation. However, preexposure to AAV allows neutralizing antibodies (nAbs) to block brain transduction and rAAV readministration in the brain leads to an inflammatory response in the second-injection site. In this study, we replicate our striatal rAAV2/2-GDNF readministration results and extend this effect to a second transgene, green fluorescent protein (GFP). Unlike rAAV2/2-GDNF readministration, striatal rAAV2/2-GFP readministration leads to a loss of transgene in the second site in the absence of detectable circulating nAbs. In order to determine whether the transgene or the AAV2 capsid is the antigenic stimulus in brain for the immune response in the second site, we readministered rAAV2/2-GFP using two different rAAV serotypes (rAAV2/2 followed by rAAV2/5). In this case, there was no striatal inflammation or transgene loss detected in the second-injection site. In addition, striatal readministration of rAAV2/5-GFP also resulted in no detectable immune response. Furthermore, delaying rAAV2/2 striatal readministration to a 11-week interval abrogated the immune response in the second-injection site. Finally, while striatal readministration of rAAV2/2 leads to significant loss of transgene in the second-injection site, this effect is not due to loss of vector genomes as determined by quantitative real-time PCR. We conclude that intracellular processing of AAV capsids after transduction is the immunogenic antigen and capsid serotypes that are processed more quickly than rAAV2/2 are less immunogenic.

Tight Long-term dynamic doxycycline responsive nigrostriatal GDNF using a single rAAV vector.

Glial cell line-derived neurotrophic factor (GDNF) gene transfer is being developed as a treatment for Parkinson's disease (PD). Due to the potential for side effects, external transgene regulation should enhance this strategy's safety profile. Here, we demonstrate dynamic control during long-term expression of GDNF using a recombinant adeno-associated virus (rAAV)-based bicistronic tetracycline (tet)-off construct. Nigrostriatal GDNF overexpression induces body weight alterations in rodents, enabling longitudinal in vivo tracking of GDNF expression after nigral vector delivery. Regulated GDNF expression was highly sensitive to dietary doxycycline (DOX), displaying undetectable striatal GDNF levels at serum DOX levels below those required for antimicrobial activity. However, in the absence of DOX, striatal GDNF levels exceeded levels required for efficacy in PD models. We also demonstrate the absence of a series of known GDNF-associated side effects when using direct intrastriatal vector delivery. Therefore, this single rAAV vector system meets most of the requirements for an experimental reagent for treatment of PD.

Induction of Metabotropic Glutamate Receptor-Mediated Long-Term Depression in the Hippocampal Schaffer Collateral Pathway of Aging Rats.

Scientific progress in the understanding of the molecular mechanisms of aging in the brain is essential for the identification of novel targets for the treatment and prevention of age-associated cognitive disorders. Electrophysiological analysis of synaptic plasticity using extracellular field recordings in rodent hippocampal slices is a well-established method for investigating molecular mechanisms of learning and memory. These methods can be applied to the study of aging in the brain by utilizing hippocampal slices from aged animals. However, this application can be challenging as it is difficult to ensure and maintain the health of slices originating from aged animals. The technique described in this chapter outlines the procedure for measuring metabotropic glutamate receptor-mediated long-term depression in hippocampal slices using extracellular field recordings and includes specific details for the application of this technique in the study of neuronal aging.

Genome-wide analysis of aging and learning-related genes in the hippocampal dentate gyrus.

We have previously described the transcriptional changes that occur in the hippocampal CA1 field of aged rats following a Morris Water Maze (MWM) training paradigm. In this report we proceed with the analysis of the dentate region from the same animals. Animals were first identified as age learning-impaired or age-superior learners when compared to young rats based on their performance in the MWM. Messenger RNA was isolated from the dentate gyrus of each animal to interrogate Affymetrix RAE 230A rat genome microarrays. Microarray profiling identified 1129 genes that were differentially expressed between aged and young rats as a result of aging, and independent of their behavioral training (p<0.005). We applied Ingenuity Pathway Analysis (IPA) algorithms to identify the significant biological processes underlying age-related changes in the dentate gyrus. The most significant functions, as calculated by IPA, included cell movement, cell growth and proliferation, nervous system development and function, cellular assembly and organization, cell morphology and cell death. These significant processes are consistent with age-related changes in neurogenesis, and the neurogenic markers were generally found to be downregulated in senescent animals. In addition, statistical analysis of the different experimental groups of aged animals recognized 85 genes (p<0.005) that were different in the dentate gyrus of aged rats that had learned the MWM when compared to learning impaired and a number of controls for stress, exercise and non-spatial learning. The list of learning-related genes expressed in the dentate adds to the set of genes we previously described in the CA1 region. This long list of genes constitutes a starting tool to elucidating the molecular pathways involved in learning and memory formation.

Long-term consequences of human alpha-synuclein overexpression in the primate ventral midbrain.

Overexpression of human alpha-synuclein (alpha-syn) using recombinant adeno-associated viral (rAAV) vectors provides a novel tool to study neurodegenerative processes seen in Parkinson's disease and other synucleinopathies. We used a pseudotyped rAAV2/5 vector to express human wild-type (wt) alpha-syn, A53T mutated alpha-syn, or the green fluorescent protein (GFP) in the primate ventral midbrain. Twenty-four adult common marmosets (Callithrix jacchus) were followed with regular behavioural tests for 1 year after transduction. alpha-Syn overexpression affected motor behaviour such that all animals remained asymptomatic for at least 9 weeks, then motor bias comprising head position bias and full body rotations were seen in wt-alpha-syn expressing animals between 15 and 27 weeks; in the later phase, the animals overexpressing the A53T alpha -syn, in particular, showed a gradual worsening of motor performance, with increased motor coordination errors. Histological analysis from animals overexpressing either the wt or A53T alpha -syn showed prominent degeneration of dopaminergic fibres in the striatum. In the ventral midbrain, however, the dopaminergic neurodegeneration was more prominent in the A53T group than in the WT group suggesting differential toxicity of these two proteins in the primate brain. The surviving cell bodies and their processes in the substantia nigra were stained by antibodies to the pathological form of alpha-syn that is phosphorylated at Ser position 129. Moreover, we found, for the first time, ubiquitin containing aggregates after overexpression of alpha-syn in the primate midbrain. There was also a variable loss of oligodendroglial cells in the cerebral peduncle. These histological and behavioural data suggest that this model provides unique opportunities to study progressive neurodegeneration in the dopaminergic system and deposition of alpha-syn and ubiquitin similar to that seen in Parkinson's disease, and to test novel therapeutic targets for neuroprotective strategies.

Environmental enrichment improves hippocampal function in aged rats by enhancing learning and memory, LTP, and mGluR5-Homer1c activity.

Previous studies from our laboratory have shown that environmental enrichment (EE) in young rats results in improved learning ability and enhanced metabotropic glutamate receptor-dependent long-term potentiation (mGluR-dependent LTP) resulting from sustained activation of p70S6 kinase. Here, we investigated whether 1-month EE is sufficient to improve hippocampus-dependent learning and memory and enhance hippocampal LTP in 23-24 month-old Fischer 344 male rats. Aged rats were housed in environmentally enriched, socially enriched, or standard housing conditions. We find that aged rats exposed to 1-month of EE demonstrate enhanced learning and memory relative to standard housed controls when tested in the Morris water maze and novel object recognition behavioral tasks. Furthermore, we find that environmentally enriched rats perform significantly better than socially enriched or standard housed rats in the radial-arm water maze and display enhanced mGluR5-dependent hippocampal LTP. Enhanced hippocampal function results from activity-dependent increases in the levels of mGluR5, Homer1c, and phospho-p70S6 kinase. These findings demonstrate that a short exposure of EE to aged rats can have significant effects on hippocampal function.

Neuroinflammatory challenges compromise neuronal function in the aging brain: Postoperative cognitive delirium and Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that targets memory and cognition, and is the most common form of dementia among the elderly. Although AD itself has been extensively studied, very little is known about early-stage preclinical events and/or mechanisms that may underlie AD pathogenesis. Since the majority of AD cases are sporadic in nature, advancing age remains the greatest known risk factor for AD. However, additional environmental and epigenetic factors are thought to accompany increasing age to play a significant role in the pathogenesis of AD. Postoperative cognitive delirium (POD) is a behavioral syndrome that primarily occurs in elderly patients following a surgical procedure or injury and is characterized by disruptions in cognition. Individuals that experience POD are at an increased risk for developing dementia and AD compared to normal aging individuals. One way in which cognitive function is affected in cases of POD is through activation of the inflammatory cascade following surgery or injury. There is compelling evidence that immune challenges (surgery and/or injury) associated with POD trigger the release of pro-inflammatory cytokines into both the periphery and central nervous system. Thus, it is possible that cognitive impairments following an inflammatory episode may lead to more severe forms of dementia and AD pathogenesis. Here we will discuss the inflammation associated with POD, and highlight the advantages of using POD as a model to study inflammation-evoked cognitive impairment. We will explore the possibility that advancing age and immune challenges may provide mechanistic evidence correlating early life POD with AD. We will review and propose neural mechanisms by which cognitive impairments occur in cases of POD, and discuss how POD may augment the onset of AD.

Continuous low-level glial cell line-derived neurotrophic factor delivery using recombinant adeno-associated viral vectors provides neuroprotection and induces behavioral recovery in a primate model of Parkinson's disease.

The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) for Parkinson's disease is likely to depend on sustained delivery of the appropriate amount to the target areas. Recombinant adeno-associated viral vectors (rAAVs) expressing GDNF may be a suitable delivery system for this purpose. The aim of this study was to define a sustained level of GDNF that does not affect the function of the normal dopamine (DA) neurons but does provide anatomical and behavioral protection against an intrastriatal 6-hydroxydopamine (6-OHDA) lesion in the common marmoset. We found that unilateral intrastriatal injection of rAAV resulting in the expression of high levels of GDNF (14 ng/mg of tissue) in the striatum induced a substantial bilateral increase in tyrosine hydroxylase protein levels and activity as well as in DA turnover. Expression of low levels of GDNF (0.04 ng/mg of tissue), on the other hand, produced only minimal effects on DA synthesis and only on the injected side. In addition, the low level of GDNF provided approximately 85% protection of the nigral DA neurons and their projections to the striatum in the 6-OHDA-lesioned hemisphere. Furthermore, the anatomical protection was accompanied by a complete attenuation of sensorimotor neglect, head position bias, and amphetamine-induced rotation. We conclude that when delivered continuously, a low level of GDNF in the striatum (approximately threefold above baseline) is sufficient to provide optimal functional outcome.

Hypothalamic rAAV-mediated GDNF gene delivery ameliorates age-related obesity.

Intraventricular delivery of glial cell line-derived neurotrophic factor (GDNF) results in weight loss. We hypothesized that this effect of GDNF was likely mediated via its effects on dopaminergic neurons in the hypothalamus. Continuous rAAV-mediated GDNF expression in the hypothalamus of young and senescent rats resulted in weight loss compared to controls. However, GDNF-induced weight loss was unrelated to alterations in hypothalamic dopamine levels. The weight loss was associated with decreased food intake and increased energy expenditure, but these effects were not mediated by changes in hypothalamic NPY or POMC expression. Moreover, uncoupling protein 1 levels were unchanged in brown adipose tissue (BAT). The reduction in weight and adiposity were as great or greater in the aged rats even though aged rats are generally resistant to weight loss therapies. In summary, central GDNF gene delivery reduces weight and adiposity in young and aged rats through decreased food intake and increased energy expenditure. Our observations in aged rats suggest that GDNF may be especially effective in reducing obesity in aged obese rats.

Reversal of dyskinesias in an animal model of Parkinson's disease by continuous L-DOPA delivery using rAAV vectors.

Dyskinesias are a major complication of long-term l-3,4-dihydroxyphenylalanine (L-DOPA) treatment in Parkinson's disease, and are believed to result from the intermittent and pulsatile supply of L-DOPA. Daily injections of L-DOPA can prime similar abnormal involuntary movements of the limb, orolingual and axial muscles in rats rendered parkinsonian by destruction of the nigrostriatal dopamine (DA) neurons. In this study we used 33 rats with severe nigrostriatal dopamine depletion and showed that in vivo gene transfer of the DA-synthetic enzymes tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) using recombinant adeno-associated virus vectors can provide a constant source of DOPA production locally in the striatum, at a level that is effective in reducing L-DOPA-induced dyskinesias by >85%, and reverse lesion-induced motor impairments. Furthermore, the abnormal expression of DeltaFosB, prodynorphin and preproenkephalin mRNA within the striatal projection neurons normally seen in dyskinetic animals was completely reversed by TH-GCH1 gene transfer. These findings form a strong basis for replacing, or supplementing, conventional systemic L-DOPA therapy by continuous intrastriatal DOPA using in vivo gene transfer in the treatment of patients with advanced Parkinson's disease.

Small-Scale Recombinant Adeno-Associated Virus Purification.

Recombinant adeno-associated virus (rAAV) vectors have become increasingly popular in research and clinical trials due to their efficient gene transfer and long-term expression in tissues including brain. In addition, rAAV has demonstrated an impressive safety profile in gene therapy trials. The emergence of rAAV serotypes with different cell tropisms and distribution properties has allowed scientists to tailor serotypes to specific experimental needs. AAV does not have a cytopathic effect; therefore, purification methods require extraction of the viral vector from the cell. This involves gradient ultracentrifugation of the cellular extract sometimes followed by chromatography. This chapter describes a small-scale production method for rAAV purification from ten to twenty 15 cm plates of human embryonic kidney-derived 293B cells (HEK 293) cells that can yield approximately 300 µl of a 5 × 10(12) to 1 × 10(13) genome copies/ml viral preparation final concentration.