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Adeno-associated virus (AAV) serotype 8 appears to be the strongest of the natural serotypes reported to date for gene transfer in liver and muscle. In this study, we evaluated AAV8 in the brain by several methods, including biophotonic imaging of green fluorescent protein (GFP). In the adult rat hippocampus, levels of GFP expressed were clearly greater with AAV8 than with AAV2 or AAV5 by Western blot and biophotonic imaging and slightly but significantly greater than AAV1 by Western blot. In the substantia nigra, the GFP expression conferred by AAV8 was toxic to dopamine neurons, although toxicity could be avoided with dose titration. At the low dose at which there was no GFP toxicity from the GFP vector, another AAV8 vector for a disease-related (P301L) form of the microtubule-associated protein tau caused a 78% loss of dopamine neurons and significant amphetamine-stimulated rotational behavior. The AAV8 tau vector-induced cell loss was greater than that from AAV2 or AAV5 tau vectors, demonstrating that the increased gene transfer was functional. While the toxicity observed with GFP expression warrants great caution, the efficient AAV8 is promising for animal models of neurodegenerative diseases and potentially as well for gene therapy of brain diseases.

No abstract

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA A receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA A receptors composed of either ␣ 1 ␤ 2 ␥ 2 L or ␣ 1 ␤ 2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor ␤ 2 subunit that is critical for endocytosis. Internalization of GABA A receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor ␤ 2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA A receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.The GABA A receptor is a ligand-gated chloride channel that, upon activation by GABA 1 (␥-aminobutyric acid), mediates increases in chloride conductance resulting in membrane hyperpolarization and neuronal inhibition (1). The role of these receptors in hyperexcitability states, such as epilepsy and anxiety, is widely recognized. Importantly, GABA A receptors mediate the effects of benzodiazepines and barbiturates, two frequently prescribed classes of therapeutic agents. The GABA A receptor is a pentameric receptor composed of multiple subunits, each containing four membrane-spanning regions (M1-M4) with a large intracellular loop between M3 and M4. A number of subunits exist (␣ 1Ϫ6 , ␤ 1Ϫ3 , ␥ 1Ϫ3 , ␦, , ⑀, ), and receptors composed of ␣ 1 ␤ 2 ␥ 2 L subunits are believed to represent the predominant GABA A receptor subtype in the brain (1).Receptor endocytosis is known to regulate the cell surface expression of neurotransmitter receptors, and such regulation is an important mechanism for controlling the synaptic efficacy of neurotransmitters (2). Although GABA A receptors undergo endocytosis, the mechanism is not well understood. Several lines of evidence indicate that GABA A receptor endocytosis may be clathrin/dynamin-dependent. These include the presence of GABA A receptors in clathrin-coated vesicles isolated from brain (3), the colocalization of the receptor with transferrin receptors (4), and the colocalization and co-immunoprecipitation of hippocampal GABA A receptors with the clathrin adaptor complex AP2 adaptin (5). Additionally, peptides that dis...

The inhibition of ␥-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4␤-phorbol 12-myristate 13-acetate (PMA) was investigated using recombinant human GABA A receptors expressed in Xenopus oocytes. PMA (5 nM) reduced the GABA response in oocytes expressing the ␣1␤2␥2L receptor construct, as measured by the two-electrode voltage-clamp method. GABA responses declined to approximately 25% of their pretreatment value within 45 min. GABA responses in oocytes expressing a receptor construct from which the known PKC phosphorylation sites were absent, ␣1␤2(S410A), were comparably inhibited. Phorbol 12-monomyristate (PMM; 5 nM), which does not activate PKC, did not alter the GABA response in either construct, while the PKC inhibitor calphostin C (0.5 M) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABA A receptor ␣1 subunit/green fluorescent protein (GFP) chimera (␣1GFP) was used to visualize GABA A receptor distribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing ␣1GFP␤2␥2L and ␣1GFP␤2(S410A) receptor constructs. Following PMA treatment, GFP fluorescence in the oocyte plasma membrane was decreased to approximately 45% of the pretreatment values indicating GABA A receptor internalization. This effect of PMA was prevented by calphostin C and was not produced by PMM. Experiments with bd24, a monoclonal antibody which recognizes an extracellular epitope of the ␣1 subunit, were used to demonstrate that PMA, but not PMM, decreases ␣1 subunit immunoreactivity in the plasma membrane of intact oocytes expressing the ␣1␤2␥2L construct, thus confirming the results obtained with the chimeric receptor. It is concluded that, in Xenopus oocytes, PMA induces an internalization of the GABA A receptor through PKC-mediated phosphorylation of an unidentified protein(s) and that this contributes to the decrease in electrophysiological responses to GABA following PKC activation. ␥-Aminobutyric acid (GABA)1 is the principal inhibitory neurotransmitter in the vertebrate central nervous system. The fast inhibitory actions of GABA are mediated by the GABA A receptor, a postsynaptic ligand-gated chloride channel. At least 17 receptor subunit subtypes have been identified (␣ 1-6 , ␤ 1-4 , ␥ 1-4 , ␦, 1,2 ), and the receptor is thought to form a heteropentamer (1). The subunit composition of the receptor determines agonist potency (2, 3), desensitization kinetics (3), phosphorylation (4 -6), and allosteric properties (2, 7, 8).Calcium-phospholipid-dependent protein kinase (PKC) phosphorylates purified GABA A receptors on polypeptides corresponding to ␤ subunits (9), and PKC phosphorylation sites have been identified on a variety of GABA A subunits including ␤1 serine 409 (5, 6), ␥2S serine 327, and ␥2L serine 343/327 (4, 5). Activation of PKC by phorbol esters such as 4␤-phorbol 12-myristate 13-acetate (PMA) inhibits GABA A receptor function in mouse brain cerebellar microsacs (10), cultured cervical ganglion neuro...

Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs is considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast. This is most evident in the treatment of lysosomal storage disorders, cystic fibrosis, and nephrogenic diabetes insipidus, for which proof of principle in humans has been demonstrated.

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GABA (γ-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABA A receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABA A receptor cell surface expression. Forty-two hrs of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GABA-gated chloride currents. In time-course experiments, a 1 hr GABA exposure, followed by a 5 hr incubation in GABA-free medium, was sufficient to increase receptor surface expression. A shorter GABA exposure could be used in HEK 293 cells stably transfected with the GABA transporter GAT-1. In rGAT-1HEK 293 cells, the GABA effect was blocked by the GAT-1 inhibitor NO-711, indicating that GABA was acting intracellularly. The effect of GABA was prevented by brefeldin A (BFA), an inhibitor of early secretory pathway trafficking. Coexpression of GABA A receptors with the GABA synthetic enzyme glutamic acid decarboxylase 67 (GAD67) also resulted in an increase in receptor surface levels. GABA treatment failed to promote the surface expression of GABA binding site mutant receptors, which themselves were poorly expressed at the surface. Consistent with an intracellular action of GABA, we show that GABA does not act by stabilizing surface receptors. Furthermore, GABA treatment rescued the surface expression of a receptor construct that was retained within the secretory pathway. Lastly, the lipophilic competitive antagonist (+)bicuculline promoted receptor surface expression, including the rescue of an secretory pathway-retained receptor. Our results indicate that a neurotransmitter can act as a ligand chaperone in the early secretory pathway to regulate the surface expression of its receptor. This effect appears to rely on binding site occupancy, rather than agonist-induced structural changes, since chaperoning is observed with both an agonist and a competitive antagonist.