Neuronal PINCH is regulated by TNF-α and is required for neurite extension

Abstract

During HIV infection of the CNS, neurons are damaged by viral proteins, such as Tat and gp120, or by inflammatory factors, such as TNF-α, that are released from infected and/or activated glial cells. Host responses to this damage may include the induction of survival or repair mechanisms. In this context, previous studies report robust expression of a protein called particularly interesting new cysteine histidine-rich protein (PINCH), in the neurons of HIV patients' brains, compared with nearly undetectable levels in HIV-negative individuals (Rearden et al., J Neurosci Res 86:2535-2542, 2008), suggesting PINCH's involvement in neuronal signaling during HIV infection of the brain. To address potential triggers for PINCH induction in HIV patients' brains, an in vitro system mimicking some aspects of HIV infection of the CNS was utilized. We investigated neuronal PINCH expression, subcellular distribution, and biological consequences of PINCH sequestration upon challenge with Tat, gp120, and TNF-α. Our results indicate that in neurons, TNF-α stimulation increases PINCH expression and changes its subcellular localization. Furthermore, PINCH mobility is required to maintain neurite extension upon challenge with TNF-α. PINCH may function as a neuron-specific host-mediated response to challenge by HIV-related factors in the CNS.

Fig. 1. PINCH expression by neurons in the human frontal cortex of an HIVE patient correlates with loss of synaptic vesicles and dendrites

a Double immunolabeling with anti-PINCH antibody (red) and anti-SV2A antibody specific for synaptic vesicles (green) shows abundant peri-nuclear PINCH immunoreactivity (arrow) in a neuron with decreased SV2A, indicating loss of synaptic vesicles. Compare with the adjacent neuron with well-preserved synapses (arrowhead) and no PINCH immunoreactivity. b Double immunolabeling with anti-PINCH antibody (red) and anti-MAP2 antibody (green) showing abundant peri-nuclear PINCH immunoreactivity (arrow, red) of a neuron with decreased dendritic complexity (asterisk). Compare with the adjacent neuron with well-preserved dendritic complexity (arrowhead, green) and no PINCH immunoreactivity. Nuclei are labeled with DAPI (blue). Scale bar = 10 µm

Fig. 2. Exposure to TNF-α and Tat, but not gp120, increases PINCH expression in mouse primary neurons

a Representative Western blot of untreated (Con), neurons exposed to 100 nM full length Tat, 100 ng/ml TNFα, or 25 nM gp120 for 72 h and reacted with anti-PINCH antibody. b Upon exposure to Tat or TNF-α, PINCH protein levels increased significantly above untreated control (*p<0.005, **p<0.001, respectively, by one-way ANOVA with Bonferroni’s multiple comparison). gp120 showed insignificant changes in PINCH protein compared with control. Graphed data represents three independent experiments. c PINCH levels in mouse primary neurons treated for 24, 48, or 72 h with TNF-α or with TNF-receptor neutralizing antibody (TNFR) for 30 min followed by TNF-α for 72 h

Fig. 3. PINCH/ILK co-expression in neurons increases in HIVE and upon TNF-α treatment

a, b Double immunofluorescent labeling of PINCH (green, arrow) and ILK (red) in frontal cortex from HIVE patients showing colocalization of perinuclear PINCH and ILK (arrowheads). c Untreated (Con) and TNF-α treated (72 h) neurons in vitro immunolabeled for PINCH (green) and ILK (red, arrowheads). Nuclei are labeled with DAPI (blue). Magnification, ×100

Fig. 4. TNF-α induced changes in PINCH and ILK localization

a Representative Western blot of PINCH and ILK in nuclear/cytoplasmic fractionation of mouse primary neurons±TNF-α. Tubulin and Lamin A/C were used as loading controls for cytoplasmic and nuclear fractions, respectively. The same blot was stripped and re-probed with each antibody shown. b, c Graphic representation of combined results from Western analyses showing fold changes in PINCH and ILK expression in both the cytoplasm and nucleus in untreated (C) and TNF-α treated neurons, n=3, *p≤0.05, by one-way ANOVA

Fig. 5. Immunoprecipitation with anti-PINCH antibody confirms greater levels of PINCH in the nucleus and a nuclear ILK doublet

a Immunoprecipitation and Western analyses with anti-PINCH antibody reveal greater nuclear levels of PINCH at 37-kDa (arrowhead) compared with cytoplasmic. b Immunoprecipitation with anti-PINCH antibody and Western analyses with anti-ILK antibody reveals an ILK doublet in the nucleus at approximately 60-kDa (arrowheads)

Fig. 6. Chariot-mediated delivery of anti-PINCH antibody into live neurons results in perinuclear accumulation of PINCH and retraction of processes upon exposure to TNF-α

a, c, e Representative HT22 neurons from treatment conditions labeled with anti-PINCH (green), anti-MAP2 (red), and nuclei with DAPI (blue). b, d, f Phase contrast images of HT22 neurons from each treatment condition. a, b Chariot without conjugated anti-PINCH antibody was delivered into live neurons followed by 48-h exposure to TNF-α. c, d Chariot-mediated delivery of anti-PINCH antibody in the absence of TNF-α. e, f Chariot-mediated delivery of anti-PINCH antibody followed by 48-h TNF-α treatment. g Percentage of neurons with processes, and h, average lengths of processes from untreated, TNF-α treated, Chariot delivered without anti-PINCH antibody conjugated followed by 48-h TNF-α treatment (Chariot+TNF-α), Chariot-mediated delivery of anti-PINCH antibody in the absence of TNF-α (Chariot-PINCH), and Chariot-mediated delivery of anti-PINCH antibody followed by 48-h TNF-α treatment (Chariot-PINCH+TNF-α). Results from at least three independent experiments are expressed as percentage±SEM. *p<0.001 by one-way ANOVA with Tukey–Kramer multiple post hoc