One approach to treatment of negative cognitive effects associated with Alzheimer's disease and schizophrenia may involve activation of neuronal alpha 7 nicotinic acetylcholine receptors (nAChRs). We used the alpha 7-selective partial agonist 3-(4-hydroxy, 2-methoxybenzylidene)anabaseine (4OH-GTS-21), the alpha 7 modulator 5-hydroxyindole (5-HI), and recombinant adeno-associated virus (rAAV)-mediated alpha 7 gene transfer in order to test the hypothesis whether combining these strategies would significantly increase indirect measures of alpha 7 nAChR function, including measures of spontaneous synaptic events in CA1 pyramidal cells. 5-HI (1 mM), and 5-HI (1 mM) + 4OH-GTS-21 (5 mu M) increased the frequency of APV- and NBQX-sensitive currents, while 5-HI + 4OH-GTS-21 increased the frequency and amplitude of bicuculline-sensitive currents. Effects on EPSCs were blocked with tetrodotoxin (TTX) (1 mu M), but not by methyllycaconitine (MLA) (50 nM). Neither TTX nor MLA reduced the potentiation of IPSC frequencies. However, TTX blocked, and in some cases MLA reduced, the potentiation of IPSC amplitudes. These data suggest that effects of 5-HI + 4OH-GTS-21 on EPSC frequency were associated with action potential-dependent transmitter release produced by 5HT, and that potentiation of IPSC amplitudes resulted at least in part, from activation of alpha 7 nAChRs. Finally, rAAV-mediated alpha 7 gene transfer did not alter the magnitude of effects produced by 5-HI or 5-HI + 4OH-GTS-21. Thus, although we previously showed that direct measures of alpha 7 nAChR function were enhanced by alpha 7 gene transfer, indirect measures of alpha 7 nAChRs function were not significantly enhanced by combining alpha 7 gene transfer with either agonist activation or positive allosteric modulation of alpha 7 nAChRs. (c) 2008 Elsevier B.V. All rights reserved.

Human immunodeficiency virus type 1 (HIV-1) causes neuronal degeneration and, at a late stage, creates HIV-associated dementia (HAD) and other neurological abnormalities. Therefore, the need for neuroprotective agents is great. However, therapeutic agents that reduce HIV neurotoxicity are difficult to characterize and develop because rodents are not infected by HIV. This study was undertaken to develop an animal model of HIV neurotoxicity by using the HIV-1 envelope glycoprotein 120 (gp120). Vehicle or gp120 was injected acutely in the striatum of adult rats. gp120 produced loss of nigrostriatal neurons, as shown both by histochemical analysis of brain sections for apoptosis and biochemical determination of dopamine. The neurotrophin brain-derived neurotrophic factor (BDNF) delivered by a recombinant adeno-associated viral vector prevented gp120 toxicity. This study's results support the notion that gp120 produces a widespread neurotoxicity similar to that observed in HIV-positive individuals and that BDNF may be a suitable neuroprotective agent for HAD.

Human immunodeficiency virus type 1 (HIV-1) causes neuronal degeneration and, at a late stage, creates HIV-associated dementia (HAD) and other neurological abnormalities. Therefore, the need for neuroprotective agents is great. However, therapeutic agents that reduce HIV neurotoxicity are difficult to characterize and develop because rodents are not infected by HIV. This study was undertaken to develop an animal model of HIV neurotoxicity by using the HIV-1 envelope glycoprotein 120 (gp120). Vehicle or gp120 was injected acutely in the striatum of adult rats. gp120 produced loss of nigrostriatal neurons, as shown both by histochemical analysis of brain sections for apoptosis and biochemical determination of dopamine. The neurotrophin brain-derived neurotrophic factor (BDNF) delivered by a recombinant adeno-associated viral vector prevented gp120 toxicity. This study's results support the notion that gp120 produces a widespread neurotoxicity similar to that observed in HIV-positive individuals and that BDNF may be a suitable neuroprotective agent for HAD.

Glycoprotein 120 (gp120) from the T-tropic strain of the human immunodeficiency virus type 1 has been shown to cause neuronal apoptosis through activation of the chemokine receptor CXCR4. Therefore, reducing CXCR4 expression may prevent gp120-mediated apoptosis. Brain-derived neurotrophic factor (BDNF) is known to reduce both gp120 neurotoxicity and CXCR4 expression in vitro. The scope of this work is to establish whether BDNF is neuroprotective against gp120 in vivo and, if so, whether this effect correlates with its ability to down-regulate CXCR4. Serotype 2 adeno-associated viral vector encoding for BDNF (rAAV-BDNF) or control vector was microinjected into the striata of adult rats. Two weeks later gp120 was injected into the same striatum, and apoptosis determined. Pretreatment with rAAV-BDNF prior to gp120 microinjection prevented caspase-3 activation as well as in situ terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling in the striatum and substantia nigra. In addition, rAAV-BDNF reversed the loss of tyrosine hydroxylase immunoreactivity induced by gp120 in both areas. CXCR4 expression was then determined by immunohistochemistry and RT-PCR, and found to be decreased in striata of rAAV-BDNF-treated rats. Conversely, BDNF heterozygous mice exhibited an increase in CXCR4 mRNA levels compared to wild-type littermates. Our data suggest that down-regulation of CXCR4 expression may contribute to the neuroprotective activity of BDNF against gp120 toxicity in the basal ganglia.

Cholinergic hypofunction underlies the devastating memory deficits observed in Alzheimer's disease. The effectiveness of traditional cholinergic replacement therapies may be hindered by neuronal nicotinic receptor desensitization. One approach to augment the effectiveness of therapeutic interventions may be to introduce additional neurotransmitter receptors via gene delivery. In order to evaluate the potential for introducing functional nicotinic receptors in vivo, we used rAAV neuron–specific vectors to express rat alpha7–type nicotinic receptors (rAAV– alpha7) in male Sprague Dawley rats. The vector rAAV serotype 8/2 in combination with rAAV +GFP was sterotaxically injected into the hippocampus. Whole cell patch–clamp methods and fluorescence microscopy were used in hippocampal slices to evaluate responses to 1 mM ACh. Two weeks following surgery we detected elevated levels of high affinity [3H]MLA binding (over 1000 fmol/mg protein) and increased immuno–histochemical staining of alpha7 receptors. In normal animals functional alpha7–type receptors are located on the soma of CA1 and dentate gyrus interneurons, and on dentate mossy cells, but not on the soma of pyramidal cells or granule cells. Following injections of rAAV– alpha7 we found: 1) MLA sensitive ACh evoked somatic responses in GFP+ CA1 pyramidal cells and dentate granule cells. 2) The magnitude of MLA sensitive ACh evoked currents in GFP+ CA1 interneurons was ∼ 8x greater than in controls. 3) In the presence of the alpha7 selective agonist 4OH–GTS21 (5 um) and the alpha7 receptor potentiator 5–hydroxyindole (5–OHi) (1 mM), the frequency of spontaneous glutamatergic synaptic events in CA1 pyramidal cells was substantially (4–19 times) increased in 100% of the cells, whereas in control cells this increase was smaller (1.5– 12 times) and was found in only ∼57% of cells. These results show that functional alpha7–subtype nicotinic receptors can be delivered in vivo. They demonstrate that functional receptors can be delivered to cells which do not normally express them, and that the function of these receptors can be significantly increased in cells which do normally express them. In conclusion, the results demonstrate that rAAV–alpha7 gene delivery can enhance synaptic transmission, potentially improving information processing.

This chapter contains sections titled: General Overview of Gene Therapy Anatomical Considerations Naked DNA Non-Viral Vectors Polymer-Based Vectors Introduction Influence of Charge and Size Biodistribution and Gene Expression Lipid-Based Vectors Introduction Influence of Physico-Chemical Properties Biodistribution and Gene Expression Viral Vectors rAAV: Properties rAAV Serotype and Biodistribution Summary References

Homomeric α7 and heteromeric α4β2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in α7 receptors for which the α7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in α4 receptors co-expressed with β2 resulted in α4β2 receptors for which ACh potency was relatively unchanged, although the efficacy of the α7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in α7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both α7 and α4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the α4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.