1% Triton X PBS for 15 minutes. The cells were rehy drated by 3 washes of PBS and 5 washes of 0. 5% bovine serum albumin. After blocking with 2% BSA for 1 hour the neurons were incubated with primary Rapamycin mTOR anti bodies against microtubule associated protein 2, B III tubulin and glial fibrillary acidic protein overnight at 4 C. The cells were washed 5 times with 0. 5% BSA and were further incu bated with Alexa Flour 488 goat anti mouse IgG, anti rabbit Cy3. After 5 washes with 0. 5% BSA and 5 times with PBS the nuclei were stained with Hoechst 33342 for 30 seconds. The slides were mounted and staining was checked under microscope. Treatment of neurons with specific neutralizing antibodies against proinflammatory cytokines Primary neurons were exposed to supernatants from HIV 1wt, HIV 1Vpr and mock infected MDMs collected on day 8 or day 12 as well as recombinant IL 1B, IL 8 and TNF.

The cells Inhibitors,Modulators,Libraries were replenished with media containing neutralizing antibodies against IL 1B, IL 8 and TNF. The neuronal apoptosis was mea sured 24 48 hours after infection. Annexin V FITC staining Analysis of apoptosis was carried out using the apoptosis detection kit as per the manufacturers instructions. Briefly, neurons were exposed to HIV 1wt, HIV 1Vpr, mock infected MDM supernatants and recombinant cyto kines for 24 48 hours. To detect apoptosis, infected media were removed, the cells were washed twice with 1X PBS and then once with 1X Annexin V binding buf fer. Neurons were stained with Annexin V FITC diluted in 1X binding buffer for 15 minutes at room temperature in the dark, then washed once with 1X binding buffer.

The apoptotic neurons were quanti fied by nuclei staining Inhibitors,Modulators,Libraries with Hoechst 33342 Inhibitors,Modulators,Libraries and analyzed by microscopy. Neuronal apoptosis was calculated from the percentage of cells stained with Annexin V. MTT assay The effect of HIV 1Vpr mediated proinflammatory fac tors on inducing neuronal death was also Inhibitors,Modulators,Libraries assessed using MTT assay. Briefly, 1 �� 105 primary neurons in each well of a 96 well plate were exposed with the supernatants from mock, HIV 1wt and HIV 1Vpr infected MDMs as well as recombinant cyto kines and incubated at 37 C in 5% CO2 for 24 hours. The neurons were incubated with 1. 2 mM of MTT solu tion for 4 hours at 37 C. The formazan crystals were solubilized in 100 ul DMSO and by shaking the plate for 10 minutes. The absorbance Inhibitors,Modulators,Libraries was measured at 540 nm.

Mock infected cells produced the highest O. D reflecting the highest cell survival and cells treated with toward DMSO were used as a negative control. W Caspase 3 7 activities were measured using Caspase GloW 3 7 Assay kit according to manufacturers instructions. Briefly, 5 �� 104 primary neurons differentiated in each well of a 96 well plate were exposed to supernatants of mock, HIV 1wt and HIV 1Vpr infected MDMs for 24 48 hours.