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Autophagy dysregulation and lysosomal dysfunction are critical in Parkinson’s disease. However, the cause and pathogenic signaling of the lysosomal functional deficiency is unknown. Here, we report on the role of zinc as a link between mitochondrial damage and lysosomal depletion. A mitochondrial toxin, 1-methyl-4 phenylpyridinium (MPP⁺), increased reactive oxygen species (ROS) and intracellular zinc ([Zn2+]i), causing lysosomal membrane permeabilization (LMP) and cell death. Supporting this, antioxidant or zinc chelator significantly reduced MPP⁺-induced LMP and neuronal death, whereas lysosomal protease inhibitors attenuated neuronal death but not ROS and [Zn2+]i. Whereas H₂O₂ toxicity was almost completely attenuated in Metallothionein-3 (MT-3) knock-out (KO) astrocytes, zinc overload- or MPP⁺-induced toxicity increased in MT-3 KO astrocyte cultures, suggesting that MT-3 modulates excessive zinc rather than providing a source of zinc after MPP⁺ treatment. Next, mitochondria-deficient Rho 0 cells were used to determine whether mitochondria are a source of zinc. No increase in ROS, [Zn2+]i, LMP, or MPP⁺ toxicity was observed in Rho 0 cells compared to wild-type cells, suggesting that increased ROS and [Zn2+]i by MPP⁺ originated from mitochondria. Taken together, we suggest that LMP is induced by the release of zinc after mitochondrial damage, eventually leading to neuronal death and lysosomal deficiency. Conduct of future studies will be needed to determine whether zinc is involved in MPP+-induced blocking of autophagic flux and accumulation of α-synuclein.

We recently reported that AMP-activated protein kinase (AMPK) contributes to zinc-induced neuronal death by inducing Bim, a pro-apoptotic Bcl-2 homology domain 3-only protein, in a liver kinase B1 (LKB1)-dependent manner. Current data suggest AMPK plays key roles in excitotoxicity and ischemic brain injury, with zinc neurotoxicity representing at least one mechanism of ischemic neuronal death. Inhibition of AMPK could be a viable therapeutic strategy to prevent ischemic brain injury following stroke. This prompted our search for novel inhibitors of AMPK activity and zinc-induced neuronal death using cultured mouse cortex and a rat model of brain injury after middle cerebral artery occlusion (MCAO). In structure-based virtual screening, 118 compounds were predicted to bind the active site of AMPK α2, and 40 showed in vitro AMPK α2 inhibitory activity comparable to compound C (a well-known, potent AMPK inhibitor). In mouse cortical neuronal cultures, 7 of 40 compound reduced zinc-induced neuronal death at levels comparable to compound C. Ultimately, only agents 2G11 and 1H10 significantly attenuated various types of neuronal death, including oxidative stress, excitotoxicity, and apoptosis. When administered as intracerebroventricular injections prior to permanent MCAO in rats, 2G11 and 1H10 reduced brain infarct volumes, whereas compound C did not. Therefore, these novel AMPK inhibitors could be drug development candidates to treat stroke.

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.

Over the last 20 years, it has been shown that complex signaling cascades are involved in zinc excitotoxicity. Free zinc rapidly induces PKC activation, which causes reactive oxygen species (ROS) production at least in part through NADPH oxidase. It also promotes neuronal nitric oxide synthase, thereby increasing nitric oxide (NO) production. Extracellular signal-regulated kinase activation and Egr-1 transcription factor activity were quickly induced by zinc, too. These concurrent actions of kinases consequently produce oxygen free radical, ROS, and NO, which may cause severe DNA damage. Following the excessive activity of poly(ADP-ribose) polymerase-1 depletes NAD+/ATP in the cells. Zinc excitotoxicity exhibits distinct characteristics of apoptosis, too. Activation of caspase-3 is induced by liver kinase B1 (LKB1)-AMP-activated kinase (AMPK)-Bim cascade signaling and induction of p75NTR receptors and p75NTR-associated Death Executor. Thus, zinc excitotoxicity is a mechanism of neuronal cell death showing various cell death patterns. In addition to the above signaling cascades, individual intracellular organelles also play a crucial role in zinc excitotoxicity. Mitochondria and lysosomes function as zinc reservoirs, and as such, are capable of regulating zinc concentration in the cytoplasm. However, when loaded with too much zinc, they may undergo mitochondrial permeability transition pore (mPTP) opening, and lysosomal membrane permeabilization (LMP), both of which are well-established mechanisms of cell death. Since zinc excitotoxicity has been reported to be associated with acute brain injuries, including stroke, trauma, and epilepsy, we performed to find the novel AMPK inhibitors as therapeutic agents for these diseases. Since we thought acute brain injury has complicated neuronal death pathways, we tried to see the neuroprotection against zinc excitotoxicity, calcium-overload excitotoxicity, oxidative damage, and apoptosis. We found that two chemicals showed significant neuroprotection against all cellular neurotoxic models we tested. Finally, we observed the reduction of infarct volume in a rat model of brain injury after middle cerebral artery occlusion (MCAO). In this review, we introduced the AMPK-mediated cell death mechanism and novel strategy for the development of stroke therapeutics. The hope is that this understanding would provide a rationale for acute brain injury and eventually find new therapeutics.

Previous studies in our lab revealed that chemical zinc chelation or zinc transporter 3 (ZnT3) gene deletion suppresses the clinical features and neuropathological changes associated with experimental autoimmune encephalomyelitis (EAE). In addition, although protective functions are well documented for AMP-activated protein kinase (AMPK), paradoxically, disease-promoting effects have also been demonstrated for this enzyme. Recent studies have demonstrated that AMPK contributes to zinc-induced neurotoxicity and that 1H10, an inhibitor of AMPK, reduces zinc-induced neuronal death and protects against oxidative stress, excitotoxicity, and apoptosis. Here, we sought to evaluate the therapeutic efficacy of 1H10 against myelin oligodendrocyte glycoprotein 35-55-induced EAE. 1H10 (5 &mu, g/kg) was intraperitoneally injected once per day for the entire experimental course. Histological evaluation was performed three weeks after the initial immunization. We found that 1H10 profoundly reduced the severity of the induced EAE and that there was a remarkable suppression of demyelination, microglial activation, and immune cell infiltration. 1H10 also remarkably inhibited EAE-associated blood-brain barrier (BBB) disruption, MMP-9 activation, and aberrant synaptic zinc patch formation. Furthermore, the present study showed that long-term treatment with 1H10 also reduced the clinical course of EAE. Therefore, the present study suggests that zinc chelation and AMPK inhibition with 1H10 may have great therapeutic potential for the treatment of multiple sclerosis.

Due to the involvement in the ischemic damage in the brain, 5'-adenosine monophosphate-activated protein kinase subunit α2 (AMPK2) serves as a promising target for the development of new medicines for stroke. Despite such a pharmaceutical importance, only a few small-molecule inhibitors have been reported so far. We aim in this study to identify a new class of AMPK2 inhibitors based on the structure-based virtual screening with docking simulations. To take advantage of and supplement the deficiencies of force field-based and empirical scoring functions, a consensus scoring method is employed to select the putative inhibitors by the combined use of AutoDock and FlexX programs. Prior to the virtual screening with docking simulations, both scoring functions are modified by implementing the molecular solvation free energy term to enhance the accuracy in estimating the protein-ligand binding affinity. As a consequence of the consensus virtual screening with the two modified scoring functions, we find seven structurally diverse AMPK2 inhibitors with micromolar inhibitory activity. Detailed binding mode analyses indicate that all these inhibitors can be stabilized in the ATP-binding pocket through the simultaneous establishment of the multiple hydrogen bonds and hydrophobic interactions. It is also found that a high inhibitory activity can be achieved by the reduction of desolvation cost for the inhibitor as well as by the strengthening of the enzyme-inhibitor interactions. Thus, the results of the present study demonstrate the outperformance of consensus scoring with the force field-based and empirical scoring functions that are modified to include the effects of ligand solvation on protein-ligand docking.

Background We reported that zinc neurotoxicity, a key mechanism of ischemic neuronal death, was mediated by poly ADP-ribose polymerase (PARP) over-activation following NAD+/ATP depletion in cortical cultures. Because AMP-activated protein kinase (AMPK) can be activated by ATP depletion, and AMPK plays a key role in excitotoxicity and ischemic neuronal death, we examined whether AMPK could be involved in zinc neurotoxicity in mouse cortical neuronal cultures. Results Compound C, an AMPK inhibitor, significantly attenuated zinc-induced neuronal death. Activation of AMPK was detected beginning 2 h after a 10-min exposure of mouse cortical neurons to 300 μM zinc, although a significant change in AMP level was not detected until 4 h after zinc treatment. Thus, AMPK activation might not have been induced by an increase in intracellular AMP in zinc neurotoxicity. Furthermore, we observed that liver kinase B1 (LKB1) but not Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), was involved in AMPK activation. Although STO-609, a chemical inhibitor of CaMKKβ, significantly attenuated zinc neurotoxicity, zinc-induced AMPK activation was not affected, which suggested that CaMKKβ was not involved in AMPK activation. Knockdown of LKB1 by siRNA significantly reduced zinc neurotoxicity, as well as zinc-induced AMPK activation, which indicated a possible role for LKB1 as an upstream kinase for AMPK activation. In addition, mRNA and protein levels of Bim, a pro-apoptotic Bcl-2 family member, were noticeably increased by zinc in an AMPK-dependent manner. Finally, caspase-3 activation in zinc-induced neuronal death was mediated by LKB1 and AMPK activation. Conclusions The results suggested that AMPK mediated zinc-induced neuronal death via up-regulation of Bim and activation of caspase-3. Rapid activation of AMPK was detected after exposure of cortical neuronal cultures to zinc, which was induced by LKB1 activation but not increased intracellular AMP levels or CaMKKβ activation. Hence, blockade of AMPK in the brain may protect against zinc neurotoxicity, which is likely to occur after acute brain injury.