Glucagon-like peptide-1 receptor is involved in learning and neuroprotection.

Glucagon-like peptide-1 (GLP-1) is a gut peptide that, together with its receptor, GLP-1R, is expressed in the brain. Here we show that intracerebroventricular (i.c.v.) GLP-1 and [Ser(2)]exendin(1-9) (HSEGTFTSD; homologous to a conserved domain in the glucagon/GLP-1 family) enhance associative and spatial learning through GLP-1R. [Ser(2)]exendin(1-9), but not GLP-1, is also active when administered peripherally. GLP-1R-deficient mice have a phenotype characterized by a learning deficit that is restored after hippocampal Glp1r gene transfer. In addition, rats overexpressing GLP-1R in the hippocampus show improved learning and memory. GLP-1R-deficient mice also have enhanced seizure severity and neuronal injury after kainate administration, with an intermediate phenotype in heterozygotes and phenotypic correction after Glp1r gene transfer in hippocampal somatic cells. Systemic administration of [Ser(2)]exendin(1-9) in wild-type animals prevents kainate-induced apoptosis of hippocampal neurons. Brain GLP-1R represents a promising new target for both cognitive-enhancing and neuroprotective agents.

Essential role of cytoplasmic cdk5 and Prx2 in multiple ischemic injury models, in vivo.

Recent evidence suggests that abnormal activation of cyclin-dependent kinase 5 (cdk5) is a critical prodeath signal in stroke. However, the mechanism(s) by which cdk5 promotes death is unclear. Complicating the role of cdk5 are the observations that cdk5 can exist in multiple cellular regions and possess both prosurvival and prodeath characteristics. In particular, the critical role of cytoplasmic or nuclear cdk5 in neuronal jury, in vivo, is unclear. Therefore, we determined where cdk5 was activated in models of ischemia and how manipulation of cdk5 in differing compartments may affect neuronal death. Here, we show a critical function for cytoplasmic cdk5 in both focal and global models of stroke, in vivo. Cdk5 is activated in the cytoplasm and expression of DNcdk5 localized to the cytoplasm is protective. Importantly, we also demonstrate the antioxidant enzyme Prx2 (peroxiredoxin 2) as a critical cytoplasmic target of cdk5. In contrast, the role of cdk5 in the nucleus is context-dependent. Following focal ischemia, nuclear cdk5 is activated and functionally relevant while there is no evidence for such activation following global ischemia. Importantly, myocyte enhancer factor 2D (MEF2D), a previously described nuclear target of cdk5 in vitro, is also phosphorylated by cdk5 following focal ischemia. In addition, MEF2D expression in this paradigm ameliorates death. Together, our results address the critical issue of cdk5 activity compartmentalization, as well as define critical substrates for both cytoplasmic and nuclear cdk5 activity in adult models of stroke.

Biodistribution and safety assessment of AAV2-GAD following intrasubthalamic injection in the rat.

BACKGROUND: The steps necessary to translate promising new biological therapies to the clinic are poorly documented. For gene therapy, there are unique aspects that need to be addressed in biodistribution studies. Notably, the spread of the vector beyond the intended target cells or tissue may result in persistent unwanted biological activity or unpredictable biological events; thus, it is critical to evaluate the risks associated with viral vector-mediated gene transfer prior to embarking on human clinical trials. METHODS: In the present study, we conducted a comprehensive assessment of vector biodistribution throughout the brain, blood and major organs of rats that had been injected via the subthalamic nucleus with recombinant adeno-associated virus (AAV) expressing glutamic acid decarboxylase (GAD). In addition, behavioral and histological analyses were also performed. RESULTS: AAV genomes were not detected in blood or cerebrospinal fluid, and did not disseminate to organs outside of the brain in the majority of animals. In the brain, an average of 97.3% of AAV2-GAD genomes were restricted to the area of the ipsilateral subthalamic nucleus (STN). There were no discernable effects of AAV2-GAD on general health, and a behavioral assessment of the animals did not reveal any alteration in general behavior, exploration, locomotion or motor symmetry. CONCLUSIONS: The present study met Food and Drug Administration requirements, in addition to efficacy and toxicity studies in rodents and nonhuman primates, to support and supplement a Phase II clinical trial invloving the gene transfer of AAV2-GAD to the human STN for the potential therapy of Parkinson's disease.

Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates.

Adeno-associated viral (AAV) vectors have become the primary delivery agent for somatic gene transfer into the central nervous system (CNS). To date, AAV-mediated gene delivery to the CNS is based on serotypes 1-9, with efficient gene transfer to neurons only-selective and widespread transduction of glial cells have not been observed. Recently, additional endogenous AAVs have been isolated from nonhuman primate tissues. In this study, transduction obtained with AAV serotypes bb2, cy5, rh20, rh39, and rh43 was compared to that obtained with AAV8, another nonhuman primate isolate previously shown to perform well in mammalian brain. Titer-matched vectors encoding the enhanced green fluorescent protein (EGFP) reporter, driven by the constitutive CAG promoter, were injected into the hippocampus, striatum, or substantia nigra (SN) of adult rats. More widespread neuronal transduction was observed following infusion of cy5, rh20, and rh39 than observed with AAV8. Of interest, preferential transduction of astrocytes was observed with rh43. To optimize glial transduction, vector stocks driven by cell-specific promoters were generated-widespread and targeted transduction of astrocytes and oligodendrocytes was observed using rh43 and AAV8, driven by the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) promoters, expanding the utility of AAV for modeling and treating diseases involving glial cell pathology.

The Parkinson's disease gene DJ-1 is also a key regulator of stroke-induced damage.

Recent evidence has indicated that common mechanisms play roles among multiple neurological diseases. However, the specifics of these pathways are not completely understood. Stroke is caused by the interruption of blood flow to the brain, and cumulative evidence supports the critical role of oxidative stress in the ensuing neuronal death process. DJ-1 (PARK7) has been identified as the gene linked to early-onset familial Parkinson's disease. Currently, our work also shows that DJ-1 is central to death in both in Vitro and in Vivo models of stroke. Loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia, whereas expression of DJ-1 can reverse this sensitivity and indeed provide further protection. Importantly, DJ-1 expression decreases markers of oxidative stress after stroke insult in Vivo, suggesting that DJ-1 protects through alleviation of oxidative stress. Consistent with this finding, we demonstrate the essential role of the oxidation-sensitive cysteine-106 residue in the neuroprotective activity of DJ-1 after stroke. Our work provides an important example of how a gene seemingly specific for one disease, in this case Parkinson's disease, also appears to be central in other neuropathological conditions such as stroke. It also highlights the important commonalities among differing neuropathologies.

Regulation of the postnatal development of dopamine neurons of the substantia nigra in vivo by Akt/protein kinase B.

Following mitosis, specification and migration during embryogenesis, dopamine neurons of the mesencephalon undergo a postnatal naturally occurring cell death event that determines their final adult number, and a period of axonal growth that determines pattern and extent of target contacts. While a number of neurotrophic factors have been suggested to regulate these developmental events, little is known, especially in vivo, of the cell signaling pathways that mediate these effects. We have examined the possible role of Akt/Protein Kinase B by transduction of these neurons in vivo with adeno-associated viral vectors to express either a constitutively active or a dominant negative form of Akt/protein kinase B. We find that Akt regulates multiple features of the postnatal development of these neurons, including the magnitude of the apoptotic developmental cell death event, neuron size, and the extent of target innervation of the striatum. Given the diversity and magnitude of its effects, the regulation of the development of these neurons by Akt may have implications for the many psychiatric and neurologic diseases in which these neurons may play a role.

HSP70 and constitutively active HSF1 mediate protection against CDCrel-1-mediated toxicity.

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.

Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial.

BACKGROUND: Dopaminergic neuronal loss in Parkinson's disease leads to changes in the circuitry of the basal ganglia, such as decreased inhibitory GABAergic input to the subthalamic nucleus. We aimed to measure the safety, tolerability, and potential efficacy of transfer of glutamic acid decarboxylase (GAD) gene with adeno-associated virus (AAV) into the subthalamic nucleus of patients with Parkinson's disease. METHODS: We did an open label, safety and tolerability trial of unilateral subthalamic viral vector (AAV-GAD) injection in 11 men and 1 woman with Parkinson's disease (mean age 58.2, SD=5.7 years). Four patients received low-dose, four medium-dose, and four high-dose AAV-GAD at New York Presbyterian Hospital. Inclusion criteria consisted of Hoehn and Yahr stage 3 or greater, motor fluctuations with substantial off time, and age 70 years or less. Patients were assessed clinically both off and on medication at baseline and after 1, 3, 6, and 12 months at North Shore Hospital. Efficacy measures included the Unified Parkinson's Disease Rating Scale (UPDRS), scales of activities of daily living (ADL), neuropsychological testing, and PET imaging with 18F-fluorodeoxyglucose. The trial is registered with the ClinicalTrials.gov registry, number NCT00195143. FINDINGS: All patients who enrolled had surgery, and there were no dropouts or patients lost to follow-up. There were no adverse events related to gene therapy. Significant improvements in motor UPDRS scores (p=0.0015), predominantly on the side of the body that was contralateral to surgery, were seen 3 months after gene therapy and persisted up to 12 months. PET scans revealed a substantial reduction in thalamic metabolism that was restricted to the treated hemisphere, and a correlation between clinical motor scores and brain metabolism in the supplementary motor area. INTERPRETATION: AAV-GAD gene therapy of the subthalamic nucleus is safe and well tolerated by patients with advanced Parkinson's disease, suggesting that in-vivo gene therapy in the adult brain might be safe for various neurodegenerative diseases.

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus.

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD(+)/H or NADP(+)/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD(+)/H than with NADP(+)/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.

Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Abeta levels.

BACKGROUND: Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-beta peptide (Abeta) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Abeta42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Abeta40, the more prevalent Abeta peptide secreted by cells and a major component of cerebral Abeta deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Abeta42 and Abeta40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. RESULTS: Adeno-associated viral (AAV) vectors encoding BRI-Abeta cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Abeta peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Abeta40 and AAV-BRI-Abeta42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Abeta peptides. BRI-Abeta42 and the combination of BRI-Abeta40+42 overexpression resulted in elevated levels of detergent-insoluble Abeta. No significant increase in detergent-insoluble Abeta was seen in the rats expressing APPsw or BRI-Abeta40. No pathological features were noted in any rats, except the AAV-BRI-Abeta42 rats which showed focal, amorphous, Thioflavin-negative Abeta42 deposits. CONCLUSION: The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Abeta42 alone is sufficient to initiate Abeta deposition, both Abeta40 and Abeta42 may contribute to cognitive deficits.

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.

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.

Promoters and regulatory elements that improve adeno-associated virus transgene expression in the brain.

Since the first demonstration of central nervous system (CNS) transduction with recombinant adeno-associated virus, improvements in vector production and promoter strength have lead to dramatic increases in the number of cells transduced and the level of expression within each cell. The improvements in promoter strength have resulted from a move away from the original cytomegalovirus (CMV) promoter toward the use of hybrid CMV-based promoters and constitutive cellular promoters. This review summarizes and compares different promoters and regulatory elements that have been used with rAAV as a reference toward achieving a high level of rAAV-mediated transgene expression in the CNS.