Technical Reference #3219

3219.

Human Immunodeficiency Virus-1 Tat Activates Calpain Proteases via the Ryanodine Receptor to Enhance Surface Dopamine Transporter Levels and Increase Transporter-Specific Uptake and Vmax Seth W. Perry; Justin Barbieri; Ning Tong; Oksana Polesskaya; Santosh Pudasaini; Angela Stout; Rebecca Lu; Michelle Kiebala; Sanjay B. Maggirwar; and Harris A. Gelbard, University of Rochester School of Medicine and Dentistry, Journal of Neuroscience, 30(3219), (2010)
Link To Paper

Abstract:
Human immunodeficiency virus-associated neurological disease (HAND) still causes significant morbidity despite success reducing viral loads with combination antiretroviral therapy. The dopamine (DA) system is particularly vulnerable in HAND.Wehypothesize that early “reversible” DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such aging human immunodeficiency virus (HIV)-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinson’s disease (PD) underscoring the need to understand shared molecular targets inHANDand PD. Previously we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here we further report that Tat disruptsDAtransporter (DAT) activity and function resulting in increased plasma membrane (PM)DAT and increasedDATVmax without changes inKmor totalDATprotein. Tat also increases calpain protease activity at the PM demonstrated by total internal reflection fluorescence microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene the calpain inhibitor calpastatin and by a specific inhibitor of GSK-3 . We conclude that Tat activates RyRs via a calcium- and calpain-mediated mechanism that upregulates DAT trafficking to the PM and is independent of DAT protein synthesis reinforcing the feasibility of RyR and GSK-3 inhibition as clinical therapeutic approaches for HAND. Finally we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients and by demonstrating similar findings in Tat-expressing transgenic mice.

Materials & Methods:
Total internal reflection fluorescence live imaging Total internal reflection fluorescence (TIRF) studies used PC12 cells cultured as above on glass-bottom Petri dishes (MatTek) then transfected with eGFP-hDAT DNA using the Lipofectamine 2000 (Invitrogen) protocol and returned to the incubator for 2 d to maximize expression. Cells were removed from the incubator and placed in Hibernate (BrainBits) media (intended for pH equilibration in ambient CO2 levels) with or without treatment for the imaging procedure. Under these conditions cells are stable at ambient air CO2 concentrations for several hours. On treatment with control or Tat cells were immediately taken to the microscope a random field of eGFP-hDAT-transfected PC12 cells was obtained and images under TIRF microscopy (TIRFM) were taken at a rate of one image (500 ms exposure) per 30 s for 30 min using a QImaging Retiga Exi cooled CCD camera and MetaMorph (Molecular Devices/ MDS Analytical Technologies) software for automated image acquisition. Hardware included 488 laser excitation with Melles Griot tunable argon laser Olympus TIRF illuminator on an Olympus IX70 microscope with Olympus 60 1.45 numerical aperture TIRF objective Chroma z488/10 clean-up filter and dichroic mirror with laser illumination custom-synchronized with camera exposures via the I/O port of the camera. Using MetaMorph software fluorescence intensities were analyzed by placing 100 pixel square regions of interest (ROIs) in random locations throughout each of the transfected cells under study and averaging the mean signal over all ROIs for each image series normalized to the starting fluorescence intensity value for each image series.

Microscopic Technique
Total internal reflection fluorescence (TIRF) live imaging

Cell Type(s)
PC12 cells