Abstract
Neurons are targets of toxicity induced by the human immunodeficiency virus (HIV)-1 protein Tat (transactivator of transcription). Exposure to Tat increases [Ca2+]i in striatal neurons and activates multiple cell death pathways. In earlier studies the authors showed that Tat activated both caspase-3 and endonuclease-G, a caspase-independent effector of apoptosis, and that Tat-induced neurotoxicity was not attenuated by a caspase-3 inhibitor. Because Tat activates multiple, parallel death pathways, the authors attempted to reduce Tat-induced neurotoxicity by manipulating signaling pathways upstream of mitochondrial apoptotic events. PTEN (phosphatase and tensin homolog deleted on chromosome 10), a negative regulator of Akt/PKB (protein kinase B) phosphorylation, was chosen as a target for silencing. Akt/PKB activity directs multiple downstream pathways mediated by GSK3β, BAD, forkhead transcription factors, nuclear factor kappa B (NFκB), and others, in a manner that promotes proliferation and survival. Striatal neurons were nucleofected with short interfering RNA (siRNA) vectors targeting PTEN, or a negative-control siRNA. Although Tat1−86 significantly increased the death of neurons transfected with control construct by 72 h, PTEN-silenced neurons were completely protected. These findings indicate that Akt is a critical intermediary in the direct neurotoxicity induced by HIV-1 Tat, and identify Akt regulation as a possible therapeutic strategy for Tat-induced neurotoxicity in HIV encephalitis (HIVE).
| 1Abbreviations: | ||
| Akt/PKB, | = | Akt1/protein kinase B |
| Bad, | = | bd-2 antagonist of cell death; |
| CMV, | = | cyto-megalovirus; |
| CNS, | = | central nervous system; |
| FACS, | = | fluorescence-activated cell sorting; |
| GFAP, | = | glial fibrillary acidic protein; |
| GFP, | = | green fluorescent protein |
| gp120, | = | glycoprotein 120 |
| GSK, | = | Glycogen synthase kinase |
| HIVE, | = | HIV encephalitis |
| hDNA, | = | hairpin DNA |
| Hpt, | = | bacterial hygromycin B phosphotransferase gene |
| Ikk, | = | I-Kappa-B Kinase |
| Nef, | = | negative regulatory factor |
| NeuN, | = | neuronal nuclei |
| pAkt, | = | phosphorylated Akt |
| PTEN, | = | phosphatase and tensin homolog deleted on chromosome 10 |
| RNAi, | = | RNA interference |
| RT-PCR, | = | reverse transcriptase–polymerase chain reaction |
| siRNA, | = | short interfering RNA |
| SEC, | = | silencing expression cassette |
| Tat, | = | transactivator of transcription |
| Vpr, | = | viral protein R. |
| 1Abbreviations: | ||
| Akt/PKB, | = | Akt1/protein kinase B |
| Bad, | = | bd-2 antagonist of cell death; |
| CMV, | = | cyto-megalovirus; |
| CNS, | = | central nervous system; |
| FACS, | = | fluorescence-activated cell sorting; |
| GFAP, | = | glial fibrillary acidic protein; |
| GFP, | = | green fluorescent protein |
| gp120, | = | glycoprotein 120 |
| GSK, | = | Glycogen synthase kinase |
| HIVE, | = | HIV encephalitis |
| hDNA, | = | hairpin DNA |
| Hpt, | = | bacterial hygromycin B phosphotransferase gene |
| Ikk, | = | I-Kappa-B Kinase |
| Nef, | = | negative regulatory factor |
| NeuN, | = | neuronal nuclei |
| pAkt, | = | phosphorylated Akt |
| PTEN, | = | phosphatase and tensin homolog deleted on chromosome 10 |
| RNAi, | = | RNA interference |
| RT-PCR, | = | reverse transcriptase–polymerase chain reaction |
| siRNA, | = | short interfering RNA |
| SEC, | = | silencing expression cassette |
| Tat, | = | transactivator of transcription |
| Vpr, | = | viral protein R. |
This work was supported by grants P01 DA19398 (PEK, KFH), R01 DA18633 (KFH), R01 NS42089 (PEK), and P20RR015592.