Yang-Hee Kim

Impaired autophagic flux and lysosomal dysfunction contribute critically to the accumulation of pathological protein aggregates in Alzheimer’s disease (AD). Emerging evidence suggests that intracellular zinc dynamics regulate lysosomal function by modulating processes such as acidification and lysosomal biogenesis. We previously identified 1H10 as an AMP-activated protein kinase (AMPK) inhibitor and subsequently demonstrated its zinc-binding capacity and ability to regulate intracellular zinc homeostasis. Building on our prior findings that intra-lysosomal zinc promotes acidification and activates transcription factor EB (TFEB), we investigated whether 1H10 enhances lysosomal function through zinc mobilization in neurons, thereby improving autophagy and reducing pathological protein accumulation. In primary cortical neurons, 1H10 increased lysosomal abundance and enhanced lysosomal degradative capacity in a zinc-dependent manner, as demonstrated by increased cathepsin B activity and DQ-BSA degradation. It alleviated lysosomal dysfunction induced by v-ATPase inhibition and promoted autophagic flux, leading to reduced accumulation of amyloid-β (Aβ) and tau in neuronal models. In 5XFAD mice, 1H10 treatment showed trends toward improved spatial learning in the Morris water maze, reduced tau phosphorylation at Thr205 and Ser214, normalized LC3-II levels, and restored autophagic–lysosomal homeostasis, without significant changes in extracellular amyloid plaque burden. These findings indicate that zinc-mediated lysosomal activation by 1H10 enhances the autophagy-lysosomal pathway and attenuates tau pathology in AD models, suggesting that targeting lysosomal function may represent a potential therapeutic strategy for neurodegenerative disorders characterized by impaired proteostasis.

Dysregulation of autophagy and lysosomal function is central to Parkinson's disease (PD), yet the upstream mechanisms leading to lysosomal failure remain unclear. Across primary mouse cortical neurons, MT‐3 deficient primary mouse astrocytes, human iPSC‐derived midbrain dopaminergic neurons, and Rho ⁰ CHO cells lacking mitochondrial respiration, we investigated how mitochondrial stress perturbs zinc (Zn ²⁺ ) homeostasis and lysosomal integrity. We identify intracellular zinc as a critical mediator linking mitochondrial dysfunction to lysosomal membrane permeabilization (LMP) and neuronal death. Inhibition of mitochondrial complex I by 1‐methyl‐4‐phenylpyridinium (MPP ⁺ ) elevated reactive oxygen species (ROS) and intracellular zinc, jointly driving LMP. Blocking either ROS or zinc markedly attenuated lysosomal damage and cell death, demonstrating that both act upstream of LMP. To define zinc regulation, we examined metallothionein‐3 (MT‐3), a brain‐enriched zinc‐binding protein. MT‐3‐deficient astrocytes were more vulnerable to MPP ⁺ and zinc overload (ZnCl ₂ ) but paradoxically resistant to hydrogen peroxide (H ₂ O ₂ ), suggesting that MT‐3 buffers cytosolic zinc during mitochondrial injury or extracellular zinc influx yet can release bound zinc under oxidative conditions. Using Rho ⁰ cells, we show that MPP ⁺ toxicity depends on mitochondrial ROS, as loss of mitochondrial function nearly abolished cell death. However, Rho ⁰ cells were highly sensitive to ZnCl ₂ and H ₂ O ₂ and exhibited markedly reduced lysosomal abundance, indicating limited capacity to sequester zinc and increased susceptibility to zinc‐mediated injury. These findings support a coordinated system in which lysosomes and zinc‐binding proteins maintain zinc homeostasis. When cytosolic zinc rises, its accumulation within lysosomes induces LMP and accelerates cell death. Collectively, our results identify intracellular zinc as an upstream trigger of lysosomal dysfunction and neurodegeneration. Zinc‐mediated LMP provides a mechanistic link between mitochondrial injury, impaired autophagic flux, and α‐synuclein pathology in PD. Enhancing zinc homeostasis and lysosomal resilience may offer promising therapeutic strategies. image

In the context of acute brain injuries, where zinc neurotoxicity and oxidative stress are acknowledged contributors to neuronal damage, we investigated the pivotal role of lysosomes as a potential protective mechanism. Our research commenced with an exploration of epidermal growth factor (EGF) and its impact on lysosomal dynamics, particularly its neuroprotective potential against zinc-induced cytotoxicity. Using primary mouse cerebrocortical cultures, we observed the rapid induction of EGFR endocytosis triggered by EGF, resulting in a transient increase in lysosomal vesicles. Furthermore, EGF stimulated lysosomal biogenesis, evident through elevated expression of lysosomal-associated membrane protein 1 (LAMP-1) and the induction and activation of prominent lysosomal proteases, particularly cathepsin B (CTSB). This process of EGFR endocytosis was found to promote lysosomal augmentation, thus conferring protection against zinc-induced lysosomal membrane permeabilization (LMP) and subsequent neuronal death. Notably, the neuroprotective effects and lysosomal enhancement induced by EGF were almost completely reversed by the inhibition of clathrin-mediated and caveolin-mediated endocytosis pathways, along with the disruption of retrograde trafficking. Furthermore, tyrosine kinase inhibition of EGFR nullified EGFR endocytosis, resulting in the abrogation of EGF-induced lysosomal upregulation and neuroprotection. An intriguing aspect of our study is the successful replication of EGF’s neuroprotective effects through the overexpression of LAMP-1, which significantly reduced zinc-induced LMP and cell death, demonstrated in both primary mouse cerebrocortical neuronal cultures and human embryonic kidney (HEK) cells. Our research extended beyond zinc-induced neurotoxicity, as we observed EGF’s protective effects against other oxidative stressors linked to intracellular zinc release, including hydrogen peroxide (H₂O₂) and 1-methyl-4-phenylpyridinium ion (MPP⁺). Collectively, our findings unveil the intricate interplay between EGF-triggered EGFR endocytosis, lysosomal upregulation, an increase in the regulatory capacity for zinc homeostasis, and the subsequent alleviation of zinc-induced neurotoxicity. These results present promising avenues for therapeutic interventions to enhance neuroprotection by targeting lysosomal augmentation.

In the context of acute brain injuries, where zinc neurotoxicity and oxidative stress are acknowledged contributors to neuronal damage, we investigated the pivotal role of lysosomes as a potential protective mechanism. Our research commenced with an exploration of epidermal growth factor (EGF) and its impact on lysosomal dynamics, particularly its neuroprotective potential against zinc-induced cytotoxicity. Using primary mouse cerebrocortical cultures, we observed the rapid induction of EGFR endocytosis triggered by EGF, resulting in a transient increase in lysosomal vesicles. Furthermore, EGF stimulated lysosomal biogenesis, evident through elevated expression of lysosomal-associated membrane protein 1 (LAMP-1) and the induction and activation of prominent lysosomal proteases, particularly cathepsin B (CTSB). This process of EGFR endocytosis was found to promote lysosomal augmentation, thus conferring protection against zinc-induced lysosomal membrane permeabilization (LMP) and subsequent neuronal death. Notably, the neuroprotective effects and lysosomal enhancement induced by EGF were almost completely reversed by the inhibition of clathrin-mediated and caveolin-mediated endocytosis pathways, along with the disruption of retrograde trafficking. Furthermore, tyrosine kinase inhibition of EGFR nullified EGFR endocytosis, resulting in the abrogation of EGF-induced lysosomal upregulation and neuroprotection. An intriguing aspect of our study is the successful replication of EGF’s neuroprotective effects through the overexpression of LAMP-1, which significantly reduced zinc-induced LMP and cell death, demonstrated in human embryonic kidney (HEK) cells. Our research extended beyond zinc-induced neurotoxicity, as we observed EGF’s protective effects against other oxidative stressors linked to intracellular zinc release, including hydrogen peroxide (H₂O₂) and 1-methyl-4-phenylpyridinium ion (MPP⁺). Collectively, our findings unveil the intricate interplay between EGF-triggered EGFR endocytosis, lysosomal upregulation, an increase in the regulatory capacity for zinc homeostasis, and the subsequent alleviation of zinc-induced neurotoxicity. These results present promising avenues for therapeutic interventions to enhance neuroprotection by targeting lysosomal augmentation.

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.

AMP-activated protein kinase (AMPK) is necessary for maintaining a positive energy balance and essential cellular processes such as glycolysis, gene transcription, glucose uptake, and several other biological functions. However, brain injury-induced energy and metabolic stressors, such as cerebral ischemia, increase AMPK phosphorylation. Phosphorylated AMPK contributes to excitotoxicity, oxidative, and metabolic problems. Furthermore, brain disease-induced release of zinc from synaptic vesicles contributes to neuronal damage via mechanisms including ROS production, apoptotic cell death, and DNA damage. For this reason, we hypothesized that regulating zinc accumulation and AMPK phosphorylation is critical for protection against global cerebral ischemia (GCI). Through virtual screening based on the structure of AMPK subunit alpha 2, we identified a novel compound, 2G11. In this study, we verified that 2G11 administration has neuroprotective effects via the blocking of zinc translocation and AMPK phosphorylation after GCI. As a result, we demonstrated that 2G11 protected hippocampal neurons against GCI and OGD/R-derived cellular damage. In conclusion, we propose that AMPK inhibition and zinc chelation by 2G11 may be a promising tool for preventing GCI-induced hippocampal neuronal death.

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.

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.

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 μg/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.

A number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, share intra- and/or extracellular deposition of protein aggregates as a common core pathology. While the species of accumulating proteins are distinct in each disease, an increasing body of evidence indicates that defects in the protein clearance system play a crucial role in the gradual accumulation of protein aggregates. Among protein degradation systems, the endosome-autophagosome-lysosome pathway (EALP) is the main degradation machinery, especially for large protein aggregates. Lysosomal dysfunction or defects in fusion with vesicles containing cargo are commonly observed abnormalities in proteinopathic neurodegenerative diseases. In this review, we discuss the available evidence for a mechanistic connection between components of the EALP-especially lysosomes-and neurodegenerative diseases. We also focus on lysosomal pH regulation and its significance in maintaining flux through the EALP. Finally, we suggest that raising cAMP and free zinc levels in brain cells may be beneficial in normalizing lysosomal pH and EALP flux.