Zinc enhances autophagic flux and lysosomal function through transcription factor EB activation and V-ATPase assembly.

The stimulation of autophagy or lysosomes has been considered therapeutic for neurodegenerative disorders because the accumulation of misfolded proteins is commonly observed in the brains of individuals with these diseases. Although zinc is known to play critical roles in the functions of lysosomes and autophagy, the mechanism behind this regulatory relationship remains unclear. Therefore, in this study, we examined which mechanism is involved in zinc-mediated activation of autophagy and lysosome. Exposure to zinc at a sub-lethal concentration activated autophagy in a concentration-dependent manner in mRFP-GFP-LC3-expressing H4 glioma cells. Zinc also rescued the blocking of autophagic flux arrested by pharmaceutical de-acidification. Co-treatment with zinc attenuated the chloroquine (CQ)-induced increase in the number and size of mRFP-GFP-LC3 puncta in H4 cells and accumulation of p62 by CQ or ammonium chloride in both H4 and mouse cerebrocortical cultures. Zinc rapidly induced the expression of cathepsin B (CTSB) and cathepsin D (CTSD), representative lysosomal proteases in neurons, which appeared likely to be mediated by transcription factor EB (TFEB). We observed the translocation of TFEB from neurite to nucleus and the dephosphorylation of TFEB by zinc. The addition of cycloheximide, a chemical inhibitor of protein synthesis, inhibited the activity of CTSB and CTSD at 8 h after zinc exposure but not at 1 h, indicating that only late lysosomal activation was dependent on the synthesis of CTSB and CTSD proteins. At the very early time point, the activation of cathepsins was mediated by an increased assembly of V-ATPase on lysosomes and resultant lysosomal acidification. Finally, considering that P301L mutation in tau protein causes frontotemporal dementia through aggressive tau accumulation, we investigated whether zinc reduces the accumulation of protein aggregates in SK-N-BE(2)-C neuroblastoma cells expressing wild-type tau or mutant P301L-tau. Zinc markedly attenuated the levels of phosphorylated tau and total tau as well as p62 in both wild-type and mutant tau-overexpressing cells. We also observed that zinc was more effective than rapamycin at inducing TFEB-dependent CTSB and CTSD expression and V-ATPase-dependent lysosomal acidification and CTSB/CTSD activation. These results suggest that the regulation of zinc homeostasis could be a new approach for developing treatments for neurodegenerative diseases, including Alzheimer's and Parkinson's.

S-Nitrosylation of cathepsin B affects autophagic flux and accumulation of protein aggregates in neurodegenerative disorders.

Protein S-nitrosylation is known to regulate enzymatic function. Here, we report that nitric oxide (NO)-related species can contribute to Alzheimer's disease (AD) by S-nitrosylating the lysosomal protease cathepsin B (forming SNO-CTSB), thereby inhibiting CTSB activity. This posttranslational modification inhibited autophagic flux, increased autolysosomal vesicles, and led to accumulation of protein aggregates. CA-074Me, a CTSB chemical inhibitor, also inhibited autophagic flux and resulted in accumulation of protein aggregates similar to the effect of SNO-CTSB. Inhibition of CTSB activity also induced caspase-dependent neuronal apoptosis in mouse cerebrocortical cultures. To examine which cysteine residue(s) in CTSB are S-nitrosylated, we mutated candidate cysteines and found that three cysteines were susceptible to S-nitrosylation. Finally, we observed an increase in SNO-CTSB in both 5XFAD transgenic mouse and flash-frozen postmortem human AD brains. These results suggest that S-nitrosylation of CTSB inhibits enzymatic activity, blocks autophagic flux, and thus contributes to AD pathogenesis.

Poly(ADP-ribosyl)ation of p53 contributes to TPEN-induced neuronal apoptosis.

Depletion of intracellular zinc by N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

Zinc preconditioning protects against neuronal apoptosis through the mitogen-activated protein kinase-mediated induction of heat shock protein 70.

During brain ischemic preconditioning (PC), mild bursts of ischemia render neurons resistant to subsequent strong ischemic injuries. Previously, we reported that zinc plays a key role in PC-induced neuroprotection in vitro and in vivo. Zinc-triggered p75(NTR) induction transiently activates caspase-3, which cleaves poly(ADP-ribose) polymerase-1 (PARP-1). Subsequently, the PARP-1 over-activation-induced depletion of nicotinamide adenine dinucleotide (NAD(+))/adenosine triphosphate (ATP) after exposures to lethal doses of zinc or N-methyl-D-aspartate is significantly attenuated in cortical neuronal cultures. In the present study, zinc-mediated preconditioning (Zn PC) reduced apoptotic neuronal death that was caused by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), etoposide, or staurosporine in mouse cortical cells. We focused on heat shock protein 70 (HSP70) because NAD(+)/ATP depletion does not directly cause apoptosis, and HSP70 can inhibit the activation of caspase-9 or caspase-3 by preventing apoptosome formation or cytochrome C release. Zn PC-mediated HSP70 induction was required for neuroprotection against neuronal apoptosis, and geldanamycin-induced HSP70 induction sufficiently blocked neuronal apoptotic cell death. Furthermore, Zn PC-mediated HSP70 induction was blocked by chemical inhibitors of extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinase (MAPK) signaling, but not c-Jun N-terminal protein kinase. Similarly, neuroprotection by Zn PC against TPEN-induced apoptosis was almost completely reversed by the blockade of ERK or p38 MAPK signaling. Our findings suggest that the ERK- or p38 MAPK-mediated induction of HSP70 plays a key role in inhibiting caspase-3 activation during Zn PC.