Astrocytic metabolic switch is a novel etiology for Cocaine and HIV-1 Tat-mediated neurotoxicity

Abstract

Calcium (Ca²⁺) dynamics and oxidative signaling control mitochondrial bioenergetics in the central nervous system, where astrocytes are a major energy source for neurons. Cocaine use exacerbates HIV-associated neurocognitive disorders, but little is known about disruptions in astrocyte metabolism in this context. Our data show that the HIV protein Tat and cocaine induce a metabolic switch from glucose to fatty acid oxidation in astrocytes, thereby limiting lactate transport to neurons. Mechanistic analyses revealed increased Mitochondrial Ca²⁺ Uniporter (MCU)-mediated Ca²⁺ uptake in astrocytes exposed to Tat and cocaine due to oxidation of MCU. Since our data suggest that mitochondrial oxidation is dependent in part on MCU-mediated Ca²⁺ uptake, we targeted MCU to restore glycolysis in astrocytes to normalize extracellular lactate levels. Knocking down MCU in astrocytes prior to Tat and cocaine exposure prevented metabolic switching and protected neurons. These findings identify a novel molecular mechanism underlying neuropathogenesis in HIV and cocaine use.

Fig. 1. Exposure of astrocytes to rTat/cocaine augment mitochondrial respiration.

a Extracellular lactate levels in media from astrocytes exposed to rTat, cocaine, or rTat and cocaine. b Representative Western blot of monocarboxylate transporter 4 (MCT4) levels in astrocytes exposed to rTat, cocaine, or rTat and cocaine. c Quantification of normalized intensity of MCT4 levels from (b) to GAPDH. d Quantification of LDH activity in astrocytes exposed to rTat, cocaine, or rTat and cocaine. e Quantification of intracellular lactate levels in astrocytes exposed to rTat, cocaine, or rTat and cocaine. f Quantification of pyruvate levels in astrocytes exposed to rTat, cocaine, or rTat and cocaine. g Quantification of PDH activity in astrocytes exposed to rTat, cocaine, or rTat and cocaine. h Representative western blot of PDHA1 (phospho S293) and PDH from astrocytes exposed to rTat, cocaine, or rTat and cocaine. i Quantification of normalized PDHA1 (phospho S293) levels to total PDH. j Quantification of Acetyl-CoA levels in astrocytes exposed to rTat, cocaine, or rTat and cocaine. k Measurement of oxygen consumption rate (OCR) in astrocytes exposed to rTat, cocaine, or rTat and cocaine. After basal OCR measurement, oligomycin (A), FCCP (B), and rotenone + Antimycin A (C) were added as indicated. Representative traces of OCR in astrocytes are shown. l, m Quantification of basal OCR (l), maximal OCR (m) in astrocytes exposed to rTat, cocaine, or rTat and cocaine. n Quantification of cellular ATP levels in astrocytes exposed to rTat, cocaine, or rTat and cocaine. o Representative Western blot for electron transport chain complex components (CI, CII, CIII, CVI, CV) in astrocytes exposed to rTat, cocaine, or rTat and cocaine. The outer mitochondrial membrane receptor, TOM20 was used as the loading control. p Quantification of mitochondrial ROS levels in astrocytes exposed to rTat, cocaine, or rTat and cocaine. Data indicate Mean ± SEM; ***P < 0.001, ** P < 0.01, *P < 0.05; n = 12–16

Fig. 2. MCU-mediated Ca²⁺ uptake enhances mitochondrial metabolism in astrocytes exposed to rTat/cocaine.

a, c Representative Δψ_m (a) and [Ca²⁺]_out traces (c). Permeabilized cells were loaded with the Δψ_m indicator JC-1 and extra-mitochondrial Ca²⁺ ([Ca²⁺]_out) indicator Fura2FF to which a series of extra-mitochondrial Ca²⁺ pulses (3 μM) were added (arrowheads) to assess the [Ca²⁺]_out clearance rate. b Quantification of basal Δψ_m before the addition of [Ca²⁺]_out. d–f Quantification of the rate of [Ca²⁺]_m uptake as a function of decrease in [Ca²⁺]_out (d), number of extra-mitochondrial Ca²⁺ pulses handled (e), Total [Ca²⁺]_m taken up by astrocytes treated with rTat, cocaine, or rTat and cocaine (f). g rTat/cocaine-induced oxidative modification of MCU. Representative Western blot of lysates prepared from astrocytes exposed to rTat, cocaine, or rTat and cocaine and expressing MCU-FLAG (AdMCU). The lysates were incubated with mPEG5 (30 mins) and oxidation of MCU was identified using anti-Flag antibody. h Cell lysates prepared from control (Scr siRNA) and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine were assessed by Western blotting with anti-MCU antibody. The outer mitochondrial membrane receptor, TOM20 was used as the loading control. i, j Mean traces of [Ca²⁺]_m (rhod-2) dynamics in control (Scr siRNA) (i) and MCU KD (j) astrocytes exposed to rTat, cocaine, or rTat and cocaine. After measurement of baseline fluorescence, cells were stimulated with glutamate (200 μM, arrowheads) and changes in [Ca²⁺]_m fluorescence were measured. k, l Mean traces of [Ca²⁺]_m (rhod-2) dynamics in control (Scr siRNA) (k) and MCU KD (l) astrocytes exposed to rTat, cocaine, or rTat and cocaine. After measurement of baseline fluorescence, cells were stimulated with Ionomycin (2.5 μM, arrowheads) and changes in [Ca²⁺]_m fluorescence were measured. m Quantification of peak amplitude of rhod-2 fluorescence after glutamate stimulation. n Quantification of peak amplitude of rhod-2 fluorescence after ionomycin stimulation. Data represents Mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001; n = 6-8. Scr siRNA is a Scrambled/non-target siRNA control.

Fig. 3. Knock down of MCU switches astrocytes treated with rTat/cocaine to utilization of fatty acids and facilitates glucose oxidation.

a Quantification of extracellular lactate levels in control (Scr siRNA) and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. b Quantification of intracellular lactate levels in control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. c Quantification of LDH activity in control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. d Quantification of pyruvate levels in control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. e Quantification of Acetyl-CoA levels in control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. g Cell lysates prepared from control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine were assessed by Western blotting with antibodies specific for PDHA1 (phospho S293) and PDH. Quantification of normalized PDHA1 (phospho S293) levels with total PDH (h). i Measurement of OCR in control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. Representative traces of OCR in control (i) and MCU KD astrocytes (j). k, l Quantification of basal OCR (k), maximal OCR (l) in control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. m Quantification of cellular ATP levels in control and MCU KD astrocytes exposed to rTat, cocaine, or rTat and cocaine. Data indicate Mean ± SEM; ***P < 0.001, **P < 0.01, *P < 0.05; n = 24–30

Fig. 4. Mitochondrial fatty acid oxidation (FAO) is increased in astrocytes treated with rTat/cocaine.

a, b Mean traces of [Ca²⁺]_c (Fluo-4) dynamics in control (Scr siRNA) (a) and MCU KD (b) astrocytes exposed to rTat, cocaine, or rTat and cocaine. After measurement of baseline fluorescence, cells were stimulated with glutamate (200 μM, arrowheads) and changes in [Ca²⁺]_c fluorescence were measured. c Cell lysates prepared from control and MCU KD astrocytes treated with rTat, cocaine, or rTat and cocaine were analyzed by Western blot with antibodies specific for p-CAMKII, CAMKII, p-AMPK, AMPK, p-ACC, ACC, FAS, and β-actin. d Cell lysates prepared from control and MCU KD astrocytes treated with rTat, cocaine, or rTat and cocaine were analyzed by Western blot with antibodies specific for CPT1, CPT2, and GAPDH. e, f Quantification of CPT1 (e) and CPT2 levels (f) normalized with GAPDH. g and h Measurement of OCR in control (g) and MCU KD (h) astrocytes with carnitine as a substrate. After basal OCR measurement, oligomycin (A), FCCP (B), and rotenone + antimycin A (C) were added as indicated. i–l Quantification of basal OCR (i), maximal OCR (j), spare capacity (k), and ATP-coupled respiration (l). Data indicate Mean ± SEM; ***P < 0.001, **P < 0.01, *P < 0.05; n = 12–16

Fig. 5. Co-culturing astrocytes knocked down for MCU protects neurons from rTat/cocaine-induced toxicity.

a Quantification of extracellular lactate levels in control (Scr siRNA) and MCU KD astrocytes and neuronal co-cultured media. b Quantification of ATP levels in co-cultured neuronal lysate. c Lysates from neurons co-cultured with astrocytes were Western blotted for Synaptophysin, PSD-95 and MAP2. d–f Quantification of the normalized Synaptophysin (d), PSD-95 (e), and MAP2 (f) protein levels in neurons. g, h Quantification of cytokine levels in media from co-cultures: TNFα (g), and IL6 (h) levels. i Representative Western blot depicts unaltered MCU levels in neurons co-cultured with control (Scr siRNA) or MCU KD astrocytes. The mitochondrial membrane synthase, ATP5A was used as the loading control. j Quantification of normalized MCU levels in neurons co-cultured with control (Scr siRNA) or MCU KD astrocytes. Data represents Mean ± SEM; ***P < 0.001; n = 6–8. k Schematic representation of the proposed astrocytic metabolic switch results in neurodegeneration during rTat/cocaine treatment