RESUMO
We have previously shown that intrastriatal injection of Delta RR, the growth-compromised herpes simplex virus type 2 (HSV-2) vector for the antiapoptotic protein ICP10PK, prevents apoptosis caused by the excitotoxin N-methyl-D-aspartate (NMDA) in a mouse model of glutamatergic neuronal cell death (Golembewski et al. [2007] Exp. Neurol. 203:381-393). Because apoptosis regulation is stimulus and cell type specific, our studies were designed to examine the mechanism of Delta RR-mediated neuroprotection in striatal neurons. Organotypic striatal cultures (OSC) that retain much of the synaptic circuitry of the intact striatum were infected with Delta RR or a growth-compromised HSV-2 vector that lacks ICP10PK (Delta PK) and examined for neuroprotection-associated signaling. The mutated ICP10 proteins (p175 and p95) were expressed in 70-80% of neurons from Delta RR- and Delta PK-infected cultures, respectively, as determined by double-immunofluorescent staining with antibodies to ICP10 and NeuN or GAD65. Delta RR- but not Delta PK-treated OSC were protected from NMDA-induced apoptosis, as verified by ethidium homodimer staining, TUNEL, caspase-3 activation, and poly(AD-ribose) polymerase (PARP) cleavage. Neuroprotection was through ICP10PK-mediated activation of the survival pathways MEK/ERK and PI3-K/Akt, up-regulation of the antiapoptotic proteins Bag-1 and Bcl-2, and phosphorylation (inactivation) of the proapoptotic protein Bad. It was blocked by the MEK inhibitor U0126 or the PI3-K inhibitor LY294002, suggesting that either pathway can prevent NMDA-induced apoptosis. The data indicate that Delta RR-delivered ICP10PK stimulates redundant survival pathways that override proapoptotic cascades. Delta RR is a promising gene therapy platform against glutamatergic cell death.
Assuntos
Apoptose/fisiologia , Terapia Genética/métodos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Degeneração Neural/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Ribonucleotídeo Redutases/fisiologia , Animais , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Corpo Estriado/patologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Imunofluorescência , Vetores Genéticos , Herpesvirus Humano 2/genética , Immunoblotting , Marcação In Situ das Extremidades Cortadas , N-Metilaspartato/toxicidade , Neurônios/patologia , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Sprague-Dawley , Ribonucleotídeo Redutases/genética , Células VeroRESUMO
Excessive glutamate receptor activation results in neuronal death, a process known as excitotoxicity. Intrastriatal injection of N-methyl-d-aspartate (NMDA) is a model of excitotoxicity. We used this model to examine whether excitotoxic injury is inhibited by the anti-apoptotic herpes simplex virus type 2 (HSV-2) protein, ICP10PK, delivered by the replication incompetent HSV-2 vector, DeltaRR. Intrastriatal DeltaRR administration (2500 plaque forming units) was nontoxic and did not induce microglial activation 5 days after injection. Intrastriatal injection of DeltaRR with NMDA or 4 h after NMDA injection showed increased neuronal survival and decreased mitochondrial damage compared to injection of NMDA alone. Neuroprotection was due to the inhibition of NMDA-induced apoptosis through ERK activation. DeltaRR-treated mice did not develop NMDA-associated behavioral deficits. The data suggest that DeltaRR is a promising platform for treatment of acute neuronal injury.
Assuntos
Apoptose/efeitos dos fármacos , Aminoácidos Excitatórios/toxicidade , Ácido Glutâmico/toxicidade , N-Metilaspartato/toxicidade , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores , Proteínas Serina-Treonina Quinases/farmacologia , Ribonucleotídeo Redutases/farmacologia , Animais , Apomorfina/farmacologia , Comportamento Animal/efeitos dos fármacos , Chlorocebus aethiops , Corantes , Agonistas de Dopamina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Indicadores e Reagentes , Injeções , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Neostriado , Degeneração Neural/induzido quimicamente , Fenotiazinas , Células VeroRESUMO
Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders is a clinical challenge. We describe a novel paradigm in which the neuroprotective gene is the herpes simplex virus type 2 (HSV-2) antiapoptotic gene ICP10PK and the vector is the growth-compromised HSV-2 mutant DeltaRR. DeltaRR is delivered intranasally. It is not toxic in rats and mice. ICP10PK is expressed in the hippocampus of the DeltaRR-treated animals for at least 42 days in the absence of virus replication and late virus gene expression. Its expression is regulated by an AP-1 amplification loop. Intranasally delivered DeltaRR prevents kainic acid-induced seizures, neuronal loss, and inflammation, in both rats and mice. The data suggest that DeltaRR is a promising therapeutic platform for neurodegenerative diseases.