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.

Brain alpha7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal alpha7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)-derived vectors. In order to determine whether alpha7 receptor-mediated cytotoxicity was dependent on receptor density, rat alpha7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an alpha7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat alpha7 nicotinic receptors increased [3H]MLA binding in this region in wild type and alpha7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no alpha7 receptor response did so following alpha7 receptor gene delivery in wild type mice. Recovery of alpha7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat alpha7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether alpha7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of alpha7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

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.

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.

The accumulation and deposition of the 40-42-amino acid peptide amyloid beta (Abeta) is thought to be a critical event in the pathology of Alzheimer's disease (AD). Both passive and active immunizations against Abeta in amyloid-depositing transgenic mice have reduced Abeta pathology and improved memory-related behavior. Peripheral treatments with other amyloid-binding agents have also reduced Abeta pathology. The present study demonstrates that peripheral delivery of plasmid DNA coding for the amyloid-binding protein plasma gelsolin reduces brain Abeta in two separate amyloid-depositing transgenic mouse models of AD when inter-litter variability is accounted for. The reduction in Abeta pathology observed is accompanied by an apparent increase in activated and reactive microglia and soluble oligomeric forms of amyloid. These findings demonstrate that peripheral expression of plasma gelsolin may be a suitable gene-therapeutic approach for the prevention or treatment of AD.

While activation of α7 nicotinic receptors protects neurons from a variety of apoptotic insults in vitro, little is known about this neuroprotective action in vivo, especially under amyloidogenic conditions that mimic Alzheimer’s disease. We therefore investigated the effects of 4OH-GTS-21, a selective partial agonist for these receptors, on septohippocampal cholinergic and GABAergic neuron survival following fimbria fornix (FFX) lesions in three strains of mice: C57BL/6J wild type mice; human presenilin-1 mutant M146L (PS1) transgenic mice; and mice expressing both mutant PS1 and Swedish mutant K670N/M671L amyloid precursor protein (APP). Initial studies to demonstrated that 4OH-GTS-21 is likely brain permeant based on its ability to improve passive avoidance and Morris water task behaviors in nucleus basalis–lesioned rats. In FFX-lesioned mice, twice per day i.p. injections of 1 mg/kg of 4OH-GTS-21 for 2 weeks promoted the survival and prevented the atrophy of septal cholinergic neurons. Septal parvalbumin-staining GABAergic neurons were not protected by this treatment, although they also express α7 nicotinic receptors, suggesting an indirect, nerve growth factor (NGF)–mediated mechanism. No protection of cholinergic neurons was observed in similarly treated PS1 or APP/PS1 transgenic mice. 4OH-GTS-21 treatment actually reduced cholinergic neuronal size in APP/PS1 mice. Hippocampal amyloid deposition was not affected by FFX lesions or treatment with this α7 nicotinic receptor agonist in APP/PS1 mice under these conditions. These results indicate that brain α7 nicotinic receptors are potential targets for protecting at-risk brain neurons in Alzheimer’s disease, perhaps via their effects on NGF receptors; however, this protection may be sensitive under some conditions to environmental factors such as inhibitory amyloid-peptides.

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.

Transgenic AD model mice overexpressing APPswe develop plaques (with a thioflavine S positive core) and diffuse deposits. We have shown that these amyloid β deposits have a specific time course of development with plaques appearing before diffuse deposits. We hypothesized that local overexpression of APP would cause APP overproduction leading to Aβ pathology, i.e., the mice would show locally increased amyloid deposition. Young adult, C57BL/6 animals were injected in the entorhinal cortex with an adeno–associated virus (AAV) that expresses human APP (with the swedish mutation), and sacrificed 1 and 3 months later. The animals were transcardially perfused, the brains were cut and stained for APP, Aβ and inflammatory markers. In the animals with 1 month of survival following the injection there is a high number of cells expression APP at the injection site, and APP and a few amyloid deposits are present. Mice with 3 months survival demonstrated significantly increased numbers of amyloid deposits, further, some diffuse deposits were present. These results support the hypothesis that amyloid deposits can originate from local brain APP metabolism, independent of (over)expression of mutant or normal protein. Focal expression of mutant APP may overwhelm catabolic or transport mechanisms that normally dispose of amyloidogenic APP fragments.

The gene encoding for the Swedish double mutation (K595N/M596L) of amyloid precursor protein (APP695Swe) was expressed bilaterally in adult rat hippocampus to determine its long-term effects on memory-related behavior as well as amyloid deposition. Recombinant adeno-associated viral serotype 2 (rAAV2) vectors were injected that contained either non-expressing DNA or cDNA encoding for APP695Swe under control of a chicken beta actin/cytomegalovirus promoter/enhancer. Immunolabeling human APP with the antibody 6E10 was observed throughout the cytoplasm of aspiny and, to a lesser extent, spine-bearing hippocampal neurons 6 and 12 months post-injection of the APP695Swe but not control vector. Abeta1-42 immunolabeling was identified in unusual immunoreactive objects within the hilus of the dentate gyrus and in the granule cell layer, proximal to the injection site. At 12 months post-transduction, rats that received the APP695Swe gene also demonstrated significant deficits in the acquisition and probe components of the spatial-memory-related Morris water task compared to control animals. These behavioral deficits occurred in the absence of any amyloid plaques, gliosis, or FluoroJade labeling of dying neurons. In conclusion, prolonged and localized APP695Swe expression in hippocampal neurons is sufficient to produce memory deficits without plaque formation or neuronal loss.

Dysfunctional amyloid catabolism may be causal in AD, and reversing levels of toxic amyloid may be therapeutically effective. Insulin degrading enzyme (IDE) can catabolize monomeric amyloid beta and potentially shift equilibrium away from deposition, and compromised IDE function may contribute to AD. Assess the efficacy of IDE gene therapy for reducing levels of deposited brain amyloid, including possible gender differences. Male and female double transgenic APP/PS1 mice (Jackson Laboratories) approximately 200 days old received unilateral hippocampal injections of 2.4e10 genome particles (gp) of IDE–AAV2/8 or 2.5e10 gp of pGFP–AAV2/82.8x10. Vectors for human IDE or control green fluorescent protein (GFP) expression consisted of coding sequences, chicken beta actin/cytomegalovirus hybrid promoter, between AAV2 inverted repeat sequences, packaged in AAV8 capsids. One month later the mice were sacrificed and the brains were processed for thioflavin S labeling. “Soluble” oligomeric amyloid beta was immunolabeled with primary antibody A11 (Chemicon 9234) and secondary antibody conjugated to quantum dots. Synaptic and potential neuronal degeneration was labeled with FluoroJadeC. Epifluorescence microscopy was used to generate digital video images for treatment–blinded stereological estimation of the fractional area occupation of thioflavin S labeling. Extensive amyloid deposits were present throughout the neocortex and hippocampus. GFP fluorescence and immunoreactivity, and HA–tagged IDE immunoreactivity, revealed transgene expression throughout much of the hippocampus. Oligomer labeling, observed in most neurons, showed no obvious relationship with either vector injections or plaques, but some spherical clusters of dystrophic neurites labeled strongly. FluoroJade C–labeled dystrophic neurites were found in some but not all plaques. Side differences in the thioflavin S–labeled hippocampal but not neocortical tissue area fraction were greater after IDE gene transfer than GFP (p<0.04). Gender was not significant. Localized overexpression of IDE via AAV vector–mediated gene transfer may be effective in reducing brain amyloid accumulation. This material is the result of work supported by AG10485 from NIA and with resources and the use of facilities at the NF/SG VA Medical Center, Gainesville, FL.

alpha 7 Nicotinic receptors are calcium permeant and provide neuroprotection against many insults. We investigated the roles of intracellular calcium ions and downstream calcium channels in this protection. The alpha 7 agonist GTS-21 prevented pheochromocytoma cell death induced by nerve growth factor + serum deprivation over a 3-day interval. This effect was blocked by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in a manner that did not appear to involve changes in receptor density. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked GTS-21-induced protein kinase C activation, a necessary process for protection. The insositol triphosphate calcium-channel blocker xestospongin C and the phospholipases C inhibitor U-73122 blocked protection, ryanodine partially attenuated protection, but the L-type channel antagonist nifedipine had no effect. ERK1/2 but not JNK and p38 were activated by GTS-21, and the ERK phosphorylation inhibitors PD98059 and U0126 blocked protection.

The alpha7-type nicotinic acetylcholine receptor (nAChR) has been recognized as a potential therapeutic target for the treatment of a variety of pathologic conditions, including schizophrenia, Alzheimer's disease, and peripheral inflammation. A unique feature of alpha7 nAChRs that tends to complicate functional assays intended to identify selective drugs for these receptors is the strong concentration-dependent desensitization of their agonist-evoked responses. At low agonist concentrations, voltage-clamp responses are small but tend to closely follow the solution exchange profile, whereas higher agonist concentrations produce responses that peak and then decay very rapidly, usually before the full drug concentration has been achieved. In this article, we report that an alpha7 T245S mutant, which has a point mutation at the sixth position in the alpha7 second transmembrane domain (T6'S), demonstrates a significant gain of function, sustaining current when exposed to relatively high agonist concentrations when expressed in Xenopus laevis oocytes and larger peak currents when expressed in mammalian GH4C1 cells. At the single-channel level, the T6'S mutant has a unitary conductance of 61.7 +/- 5.8 pS, similar to that reported for wild-type alpha7, but a vastly longer average open duration. In addition, channel burst activity indicates a greater than 40% probability of channel re-opening in the sustained presence of 30 muM acetylcholine, consistent with a greater overall open probability relative to wild-type alpha7. Unlike the alpha7 L248T gain-of-function mutant, the T6'S mutant exhibits a pharmacological profile that is remarkably similar to the wild-type alpha7 receptor, implicating it as a potentially useful tool for identifying therapeutic agents.

The hippocampus receives substantial input from the medial septum/diagonal band of broca (MS/DB) via the fibria-fornix (FF). Projections from the MS/DB innervate hippocampal interneurons that express alpha7 nicotinic receptors and regulate excitation in principal cell populations. In the present report we used stereotaxic surgery, whole-cell patch clamping, and immunohistochemical techniques to evaluate the effects of FF and MS/DB lesions on alpha7 nicotinic receptors in stratum radiatum interneurons. Focal somatic application of ACh (1 mM) evoked methyllycaconitine (MLA)-sensitive currents that were markedly reduced following aspirative lesions of the FF. Reductions in current amplitudes were prevented or restored to levels not significantly different from controls following in vivo treatment with the alpha7-selective agonist GTS-21, and GTS-21 treatment did not change current amplitudes measured in tissue from unlesioned animals. MS/DB injections of the selective cholinergic neurotoxin 192 IgG-saporin did not affect alpha7 receptor currents, although MS/DB ChAT and hippocampal AChE immunolabeling were significantly reduced. In contrast, kainic acid lesions of the MS/DB, potentially more selective for GABAergic projection neurons, produced significant reductions in current amplitudes. These findings are the first to show functional changes in alpha7 receptors following hippocampal denervation and suggest that MS/DB hippocampal innervation regulates functional aspects of hippocampal alpha7 receptors. The results confirm hippocampal alpha7 nicotinic receptors as viable therapeutic targets in diseases that involve degradation of the septohippocampal pathway and may indicate that GABAergic MS/DB hippocampal input plays a more substantial role in the regulation of alpha7 nicotinic receptor function than MS/DB hippocampal cholinergic input.

The medial septum-diagonal band (MS/DB) contains primarily cholinergic and GABAergic neurons that project to the hippocampus, and are important for learning and memory. Whole-cell patch clamp methods with brain slices from p11–p20 rats were used to measure MS/DB cell responses to focal somatic application of 1 mM acetylcholine (ACh) and a series of current pulses was applied in order to assess firing frequencies and the presence of hyperpolarization-activated currents (Ih). We identified three types of cells: (1) cells with fast inward currents blocked by methyllycaconitine (MLA) with slow firing rates (3–12 Hz), accommodating action potentials, and no Ih (n = 20); (2) cells with currents that had both fast (MLA-sensitive) and slow components that were blocked with mecamylamine (MEC) that showed fast firing (up to 60 Hz) and slow firing (up to 3 Hz), with accommodating and non-accommodating action potentials (n = 46), 33% of which had Ih; and (3) cells not responsive to ACh with moderate firing rates (10–42 Hz), some with accommodating action potentials and some without (n = 19), of which 92% had Ih. These results are among the first to demonstrate functional nicotinic receptors in the MS/DB. The data suggest that these receptors include α7 and non-α7 subtypes and that the expression of each is correlated with firing frequency and the presence of Ih. Responses to ACh were not affected by tetrodotoxin (TTX) and CdCl2 but were blocked by MLA or MLA and MEC, suggesting that these currents involve direct activation of nicotinic receptors.

Nerve growth factor (NGF) therapy has been proposed to treat cognitive impairments in aged patients including those with Alzheimer's disease. Various viral vectors, including adeno-associated virus serotype 2 (AAV2), have been investigated for their ability to deliver NGF in brain. In this study, hybrid vectors (AAV2/5) consisting of the genome of recombinant AAV2 and the capsid of AAV serotype 5 were evaluated for their ability to deliver NGF and green fluorescent protein (GFP) genes into brain. Compared to AAV2, AAV2/5 consistently led to more septal neurons being transduced with GFP over a wider range of distribution. However, both types of vector provided similar levels of long-term (17 weeks) protection of septal cholinergic neurons from axotomy and led to similar levels of NGF accumulation in this region. These results demonstrate that rAAV-mediated NGF gene delivery is neuroprotective for an extended period of time, but that factors other than transduction efficiency appear to determine transgenic NGF expression in septum.