Developmental regulation of zinc homeostasis in differentiating oligodendrocytes.

Oligodendrocytes develop through sequential stages and understanding pathways regulating their differentiation remains an important area of investigation. Zinc is required for the function of enzymes, proteins and transcription factors, including those important in myelination and mitosis. Our previous studies using the ratiometric zinc sensor chromis-1 demonstrated a reduction in intracellular free zinc concentrations in mature MBP+ oligodendrocytes compared with earlier stages (Bourassa et al., 2018). We performed a more detailed developmental study to better understand the temporal course of zinc homeostasis across the oligodendrocyte lineage. Using chromis-1, we found a transient increase in free zinc after O4+,O1- pre-oligodendrocytes were switched from proliferation medium into terminal differentiation medium. To gather other evidence for dynamic regulation of free zinc during oligodendrocyte development, qPCR was used to evaluate mRNA expression of major zinc storage proteins metallothioneins (MTs) and metal regulatory transcription factor 1 (MTF1), which controls expression of MTs. MT1, MT2 and MTF1 mRNAs were increased several fold in mature oligodendrocytes compared to oligodendrocytes in proliferation medium. To assess the depth of the zinc buffer, we assayed zinc release from intracellular stores using the oxidizing thiol reagent 2,2'-dithiodipyridine (DTDP). Exposure to DTDP resulted in âˆ¼ 100% increase in free zinc in pre-oligodendrocytes but, paradoxically more modest âˆ¼ 60% increase in mature oligodendrocytes despite increased expression of MTs. These results suggest that zinc homeostasis is regulated during oligodendrocyte development, that oligodendrocytes are a useful model for studying zinc homeostasis in the central nervous system, and that regulation of zinc homeostasis may be important in oligodendrocyte differentiation.

Copper induces neuron-sparing, ferredoxin 1-independent astrocyte toxicity mediated by oxidative stress.

Copper is an essential enzyme cofactor in oxidative metabolism, anti-oxidant defenses, and neurotransmitter synthesis. However, intracellular copper, when improperly buffered, can also lead to cell death. Given the growing interest in the use of copper in the presence of the ionophore elesclomol (CuES) for the treatment of gliomas, we investigated the effect of this compound on the surround parenchyma-namely neurons and astrocytes in vitro. Here, we show that astrocytes were highly sensitive to CuES toxicity while neurons were surprisingly resistant, a vulnerability profile that is opposite of what has been described for zinc and other toxins. Bolstering these findings, a human astrocytic cell line was similarly sensitive to CuES. Modifications of cellular metabolic pathways implicated in cuproptosis, a form of copper-regulated cell death, such as inhibition of mitochondrial respiration or knock-down of ferredoxin 1 (FDX1), did not block CuES toxicity to astrocytes. CuES toxicity was also unaffected by inhibitors of apoptosis, necrosis or ferroptosis. However, we did detect the presence of lipid peroxidation products in CuES-treated astrocytes, indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Indeed, treatment with anti-oxidants mitigated CuES-induced cell death in astrocytes indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Lastly, prior induction of metallothioneins 1 and 2 in astrocytes with zinc plus pyrithione was strikingly protective against CuES toxicity. As neurons express high levels of metallothioneins basally, these results may partially account for their resistance to CuES toxicity. These results demonstrate a unique toxic response to copper in glial cells which contrasts with the cell selectivity profile of zinc, another biologically relevant metal.

Developmental regulation of zinc homeostasis in differentiating oligodendrocytes.

Oligodendrocytes develop through well characterized stages and understanding pathways regulating their differentiation remains an active area of investigation. Zinc is required for the function of many enzymes, proteins and transcription factors, including those important in myelination and mitosis. Our previous studies using the ratiometric zinc sensor chromis-1 demonstrated a reduction in intracellular free zinc concentrations in mature oligodendrocytes compared with earlier stages (Bourassa et al., 2018). We performed a more detailed developmental study to better understand the temporal course of zinc homeostasis across the oligodendrocyte lineage. Using chromis-1, we found a transient increase in free zinc after developing oligodendrocytes were switched into differentiation medium. To gather other evidence for dynamic regulation of free zinc during oligodendrocyte development, qPCR was used to evaluate mRNA expression of the major zinc storage proteins metallothioneins (MTs), and metal regulatory transcription factor 1 (MTF-1) which controls expression of MTs. MT-1, MT-2 and MTF1 mRNAs were all increased several fold in mature oligodendrocytes compared to developing oligodendrocytes. To assess the depth of the zinc buffer, we assayed zinc release from intracellular stores using the oxidizing thiol reagent 2,2'-dithiodipyridine (DTDP). Exposure to DTDP resulted in a ∼100% increase in free zinc in developing oligodendrocytes but, paradoxically more modest ∼60% increase in mature oligodendrocytes despite the increased expression of MTs. These results suggest that zinc homeostasis is regulated during oligodendrocyte development, that oligodendrocytes are a useful model for studying zinc homeostasis in the central nervous system, and that regulation of zinc homeostasis may be important in oligodendrocyte differentiation.

GluA1-Shank3 interaction decreases in response to chronic neuronal depolarization.

Interactions between AMPA receptors and synaptic scaffolding proteins are key regulators of synaptic receptor density and, thereby, synapse strength. Shank3 is one such scaffolding protein with high clinical relevance, as genetic variants and deletions of this protein have been linked to autism spectrum disorder. Shank3 acts as a master regulator of the postsynaptic density of glutamatergic synapses, interacting with ionotropic and metabotropic glutamate receptors and cytoskeletal elements to modulate synaptic structure. Notably, Shank3 has been shown to interact directly with the AMPAR subunit GluA1, and Shank3 knockout animals show deficits in AMPAR-mediated synaptic transmission. In this study, we sought to characterize the stability of GluA1-Shank3 interaction in response to chronic stimuli using a highly sensitive and specific proximity ligation assay. We found that GluA1-Shank3 interactions decrease in response to prolonged neuronal depolarization induced by elevated extracellular potassium, and that this reduced interaction is blocked by NMDA receptor antagonism. These results firmly establish the close interaction of GluA1 and Shank3 in cortical neurons in vitro, and that this select interaction is subject to modulation by depolarization.

Neurodevelopmental Consequences of Dietary Zinc Deficiency: A Status Report.

Zinc is a tightly regulated trace mineral element playing critical roles in growth, immunity, neurodevelopment, and synaptic and hormonal signaling. Although severe dietary zinc deficiency is relatively uncommon in the United States, dietary zinc deficiency is a substantial public health concern in low- and middle-income countries. Zinc status may be a key determinant of neurodevelopmental processes. Indeed, limited cohort studies have shown that serum zinc is lower in people diagnosed with autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), and depression. These observations have sparked multiple studies investigating the mechanisms underlying zinc status and neurodevelopmental outcomes. Animal models of perinatal and adult dietary zinc restriction yield distinct behavioral phenotypes reminiscent of features of ASD, ADHD, and depression, including increased anxiety and immobility, repetitive behaviors, and altered social behaviors. At the cellular and molecular level, zinc has demonstrated roles in neurogenesis, regulation of cellular redox status, transcription factor trafficking, synaptogenesis, and the regulation of synaptic architecture via the Shank family of scaffolding proteins. Although mechanistic questions remain, the current evidence suggests that zinc status is important for adequate neuronal development and may be a yet overlooked factor in the pathogenesis of several psychiatric conditions. This review aims to summarize current knowledge of the role of zinc in the neurophysiology of the perinatal period, the many cellular targets of zinc in the developing brain, and the potential consequences of alterations in zinc homeostasis in early life.

Intracellular zinc signaling influences NMDA receptor function by enhancing the interaction of ZnT1 with GluN2A.

Zinc, loaded into glutamate-containing presynaptic vesicles and released into the synapse in an activity-dependent manner, modulates neurotransmission through its actions on postsynaptic targets, prominently via high-affinity inhibition of GluN2A-containing NMDA receptors. Recently, we identified a postsynaptic transport mechanism that regulates endogenous zinc inhibition of NMDARs. In this new model of zinc regulation, the postsynaptic transporter ZnT1 mediates zinc inhibition of NMDARs by binding to GluN2A. Through this interaction, ZnT1, a transporter that moves zinc from the cytoplasm to the extracellular domain, generates a zinc microdomain that modulates NMDAR-mediated neurotransmission. As ZnT1 expression is transcriptionally driven by the metal-responsive transcription factor 1 (MTF-1), we found that intracellular zinc strongly drives MTF-1 in cortical neurons in vitro and increases the number of GluN2A-ZnT1 interactions, thereby enhancing tonic zinc inhibition of NMDAR-mediated currents. Importantly, this effect is absent when the interaction between GluN2A and ZnT1 is disrupted by a cell-permeable peptide. These results suggest that zinc-regulated gene expression can dynamically regulate NMDAR-mediated synaptic processes.

A randomized controlled trial investigating the impact of maternal dietary supplementation with pomegranate juice on brain injury in infants with IUGR.

Animal studies have demonstrated the therapeutic potential of polyphenol-rich pomegranate juice. We recently reported altered white matter microstructure and functional connectivity in the infant brain following in utero pomegranate juice exposure in pregnancies with intrauterine growth restriction (IUGR). This double-blind exploratory randomized controlled trial further investigates the impact of maternal pomegranate juice intake on brain structure and injury in a second cohort of IUGR pregnancies diagnosed at 24-34 weeks' gestation. Ninety-nine mothers and their eligible fetuses (n = 103) were recruited from Brigham and Women's Hospital and randomly assigned to 8 oz pomegranate (n = 56) or placebo (n = 47) juice to be consumed daily from enrollment to delivery. A subset of participants underwent fetal echocardiogram after 2 weeks on juice with no evidence of ductal constriction. 57 infants (n = 26 pomegranate, n = 31 placebo) underwent term-equivalent MRI for assessment of brain injury, volumes and white matter diffusion. No significant group differences were found in brain volumes or white matter microstructure; however, infants whose mothers consumed pomegranate juice demonstrated lower risk for brain injury, including any white or cortical grey matter injury compared to placebo. These preliminary findings suggest pomegranate juice may be a safe in utero neuroprotectant in pregnancies with known IUGR warranting continued investigation.Clinical trial registration: NCT04394910, https://clinicaltrials.gov/ct2/show/NCT04394910 , Registered May 20, 2020, initial participant enrollment January 16, 2016.