When zinc ion acts as a signaling molecule to regulate cell function, its concentrations are not static.  Dynamic changes of labile zinc concentrations have been reported as “zinc waves” in mast cells as a result of inflammatoryresponses during extracellular stimulation, zinc elevations can also be induced during T cell receptor activation, cell differentiation or changes of redox homeostasis.  To record such zinc dynamics, specific and sensitive imaging probes are needed to reveal and monitor the real-time kinetics and amplitude of zinc signals in living cells and animals.  The commercially available small molecule zinc sensors have either low sensitivity for zinc or nonspecific subcellular localization, limiting their capacity to reveal the subtle physiological dynamic changes in zinc concentrations within nanomolar range.  For the past five years at DU, we have created a series of genetically encoded zinc sensors based on a single fluorescent protein including GZnP1 (ACS Chem Biol. 2016), GZnP2 (ACS Chem Biol. 2018) and GZnP3 (Nat Commun. 2019) that meet various demanding requirements in terms of brightness, binding affinity, sensitivity and kinetics.  We have utilized these new sensors to provide the first measurement of zinc concentrations in the mitochondrial intermembrane space (ACS Chem Biol. 2018).  In addition, we revealed that lysosomes store a pool of labile zinc that can be released into the cytosol through TRPML1 channel in neurons (Nat Commun. 2019). In addition, we engineered red zinc sensors based on the red fluorescent protein (mApple) (ACS Sens. 2022).  These red-shifted sensors can be used along with the green sensors for multicolor simultaneous imaging of different signals.  Application of red sensors also have more advantages in biological systems in terms of low autofluorescence, low light-scattering and less phototoxicity, making them ideal for deep tissue imaging.  This project was supported by the NIH/NIBIB R00 and NIH/NINDS R01 grant.

Disturbed zinc levels in the brain have been implicated in psychiatric and neurological diseases.  Clinical studies have demonstrated reduction of zinc in patients suffering from psychological disorders such as depression, ADHD, and autism or neurodegenerative diseases such as Parkinson’s diseases.  On the other hand, excess zinc has been linked with neurotoxicity and neurodegeneration in ischemia, seizures, brain injury and Alzheimer’s disease.  However, the roles of zinc in these diseases cannot be simply attributed to the changes in total zinc concentrations.  Instead, the alterations of zinc homeostasis among different subcellular compartments might be associated with the neurotoxicity.  For example, excess mitochondrial zinc is related to ischemic injuries in hippocampal neurons, high zinc accumulated in the lysosomes is found in the cultured fibroblasts derived from patients suffering from lysosomal storage disease mucolipidosis type IV (MLIV), while low lysosomal zinc concentrations were associated with Kufor-Rakeb syndrome (KRS), also referred to as Parkinson’s disease 9, a rare hereditary form of juvenile- onset parkinsonism. 

With the genetic encoding ability of our zinc sensors, we were able to measure labile zinc levels in various subcellular compartments in disease cells.  We found that lysosomal zinc can be released through TRPML1 channels in the hippocampal neurons (Nat Commun. 2019).  Loss-of-function mutations in TRPML1 are the genetic cause of MLIV and the various patient mutations in TRPML1 showed a strong correlation between the channel’s permeability to zinc and disease severity.  Using the fibroblasts derived from the MLIV patients, we found that the cytosolic zinc concentrations are reduced, while the mitochondrial zinc concentrations are increased compared with the normal human fibroblasts.  Moreover, we also discovered that mitochondrial morphology are altered in MLIV fibroblasts.  We will continue with this study to profile changes in subcellular zinc homeostasis in fibroblasts and neurons with defective TRPML1 and determine the causative relationship between high mitochondrial zinc concentrations and mitochondrial malfunction in MLIV.  This project is currently supported by a 5-year NIH/NINDS R01 grant.

We found that the primary cultured hippocampal neurons can fire spontaneous and synchronized zinc spikes during a critical period of developing stages (DIV 14-21).  Blocking of glutamate receptors and calcium influx depleted the zinc spikes, suggesting that zinc spikes are driven by the glutamate-mediated spontaneous neural excitability and calcium spikes that have been characterized in early developing neurons.  Through simultaneous imaging of calcium or pH along with zinc, we uncovered that a downward pH spike is evoked with each zinc spike and this transient cellular acidification occurs downstream of calcium spikes, but upstream of zinc spikes (J Neurochem, 2021). Our results lead us to propose that the calcium influx-induced pH reduction (<pH 7) mediates the signal transfer from calcium spikes to zinc spikes.

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