Christian T. Sheline

Mitochondrial inhibitor models of Huntington's disease and Parkinson's disease induce zinc accumulation and are attenuated by inhibition of zinc neurotoxicity in vitro or in vivo.

Background: Inhibition of mitochondrial function occurs in many neurodegenerative diseases, and inhibitors of mitochondrial complexes I and II are used to model them. The complex II inhibitor, 3-nitroproprionic acid (3-NPA), kills the striatal neurons susceptible in Huntington’s disease. The complex I inhibitor N-methyl-4-phenylpyridium (MPP⁺) and 6-hydroxydopamine (6-OHDA) are used to model Parkinson’s disease. Zinc (Zn²⁺⁾ accumulates after 3-NPA, 6-OHDA and MPP⁺ in situ or in vivo. Objective: We will investigate the role of Zn²⁺ neurotoxicity in 3-NPA, 6-OHDA and MPP⁺. Methods: Murine striatal/midbrain tyrosine hydroxylase positive, or near-pure cortical neuronal cultures, or animals were exposed to 3-NPA or MPP⁺ and 6-OHDA with or without neuroprotective compounds. Intracellular zinc ([Zn²⁺]_i), nicotinamide adenine dinucleotide (NAD⁺), NADH, glycolytic intermediates and neurotoxicity were measured. Results: We showed that compounds or genetics which restore NAD⁺ and attenuate Zn²⁺ neurotoxicity (pyruvate, nicotinamide, NAD⁺, increased NAD⁺ synthesis, sirtuin inhibition or Zn²⁺ chelation) attenuated the neuronal death induced by these toxins. The increase in [Zn²⁺]_i preceded a reduction in the NAD⁺/NADH ratio that caused a reversible glycolytic inhibition. Pyruvate, nicotinamide and NAD⁺ reversed the reductions in the NAD⁺/NADH ratio, glycolysis and neuronal death after challenge with 3-NPA, 6-OHDA or MPP⁺, as was previously shown for exogenous Zn²⁺. To test efficacy in vivo, we injected 3-NPA into the striatum of rats and systemically into mice, with or without pyruvate. We observed early striatal Zn²⁺ fluorescence, and pyruvate significantly attenuated the 3-NPA-induced lesion and restored behavioral scores. Conclusions: Together, these studies suggest that Zn²⁺ accumulation caused by MPP⁺ and 3-NPA is a novel preventable mechanism of the resultant neurotoxicity.

Spreading Depression and Related Events Are Significant Sources of Neuronal Zn2+ Release and Accumulation:

Spreading depression (SD) involves coordinated depolarizations of neurons and glia that propagate through the brain tissue. Repetitive SD-like events are common following human ischemic strokes, and are believed to contribute to the enlargement of infarct volume. Accumulation of Zn2+ is also implicated in ischemic neuronal injury. Synaptic glutamate release contributes to SD propagation, and because Zn2+ is costored with glutamate in some synaptic vesicles, we examined whether Zn2+ is released by SD and may therefore provide a significant source of Zn2+ in the postischemic period. Spreading depression-like events were generated in acutely prepared murine hippocampal slices by deprivation of oxygen and glucose (OGD), and Zn2+ release was detected extracellularly by a Zn2+-selective indicator FluoZin-3. Deprivation of oxygen and glucose-SD produced large FluoZin-3 increases that propagated with the event, and signals were abolished in tissues from ZnT3 knockout animals lacking synaptic Zn2+. Synaptic Zn2+ release was also maintained with repetitive SDs generated by microinjections of KCl under normoxic conditions. Intracellular Zn2+ accumulation in CA1 neurons, assessed using microinjection of FluoZin-3, showed significant increases following SD that was attributed to synaptic Zn2+ release. These results suggest that Zn2+ is released during SDs and could provide a significant source of Zn2+ that contributes to neurodegeneration in the postischemic period.

Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+.

Trophic deprivation‐mediated neuronal death is important during development, after acute brain or nerve trauma, and in neurodegeneration. Serum deprivation (SD) approximates trophic deprivation in vitro, and an in vivo model is provided by neuronal death in the mouse dorsal lateral geniculate nucleus (LGNd) after ablation of the visual cortex (VCA). Oxidant‐induced intracellular Zn2+ release ([Zn2+]i) from metallothionein‐3 (MT‐III), mitochondria or ‘protein Zn2+’, was implicated in trophic deprivation neurotoxicity. We have previously shown that neurotoxicity of extracellular Zn2+ required entry, increased [Zn2+]i, and reduction of NAD+ and ATP levels causing inhibition of glycolysis and cellular metabolism. Exogenous NAD+ and sirtuin inhibition attenuated Zn2+ neurotoxicity. Here we show that: (1) Zn2+ is released intracellularly after oxidant and SD injuries, and that sensitivity to these injuries is proportional to neuronal Zn2+ content; (2) NAD+ loss is involved – restoration of NAD+ using exogenous NAD+, pyruvate or nicotinamide attenuated these injuries, and potentiation of NAD+ loss potentiated injury; (3) neurons from genetically modified mouse strains which reduce intracellular Zn2+ content (MT‐III knockout), reduce NAD+ catabolism (PARP‐1 knockout) or increase expression of an NAD+ synthetic enzyme (Wlds) each had attenuated SD and oxidant neurotoxicities; (4) sirtuin inhibitors attenuated and sirtuin activators potentiated these neurotoxicities; (5) visual cortex ablation (VCA) induces Zn2+ staining and death only in ipsilateral LGNd neurons, and a 1 mg/kg Zn2+ diet attenuated injury; and finally (6) NAD+ synthesis and levels are involved given that LGNd neuronal death after VCA was dramatically reduced in Wlds animals, and by intraperitoneal pyruvate or nicotinamide. Zn2+ toxicity is involved in serum and trophic deprivation‐induced neuronal death.

Synaptic release and extracellular actions of Zn2+ limit propagation of spreading depression and related events in vitro and in vivo

Cortical spreading depression (CSD) is a consequence of a slowly propagating wave of neuronal and glial depolarization (spreading depolarization; SD). Massive release of glutamate contributes to SD propagation, and it was recently shown that Zn(2+) is also released from synaptic vesicles during SD. The present study examined consequences of extracellular Zn(2+) accumulation on the propagation of SD. SD mechanisms were studied first in murine brain slices, using focal KCl applications as stimuli and making electrical and optical recordings in hippocampal area CA1. Elevating extracellular Zn(2+) concentrations with exogenous ZnCl(2) reduced SD propagation rates. Selective chelation of endogenous Zn(2+) (using TPEN or CaEDTA) increased SD propagation rates, and these effects appeared due to chelation of Zn(2+) derived from synaptic vesicles. Thus, in tissues where synaptic Zn(2+) release was absent [knockout (KO) of vesicular Zn(2+) transporter ZnT-3], SD propagation rates were increased, and no additional increase was observed following chelation of endogenous Zn(2+) in these tissues. The role of synaptic Zn(2+) was then examined on CSD in vivo. ZnT-3 KO animals had higher susceptibility to CSD than wild-type controls as evidenced by significantly higher propagation rates and frequencies. Studies of candidate mechanisms excluded changes in neuronal excitability, presynaptic release, and GABA receptors but left open a possible contribution of N-methyl-d-aspartate (NMDA) receptor inhibition. These results suggest the extracellular accumulation of synaptically released Zn(2+) can serve as an intrinsic inhibitor to limit SD events. The inhibitory action of extracellular Zn(2+) on SD may counteract to some extent the neurotoxic effects of intracellular Zn(2+) accumulation in acute brain injury models.

Dietary Zinc Reduction, Pyruvate Supplementation, or Zinc Transporter 5 Knockout Attenuates β-Cell Death in Nonobese Diabetic Mice, Islets, and Insulinoma Cells

Pancreatic zinc (Zn(2+)) concentrations are linked to diabetes and pancreatic dysfunction, but Zn(2+) is also required for insulin processing and packaging. Zn(2+) released with insulin increases β-cell pancreatic death after streptozotocin toxin exposure in vitro and in vivo. Triosephosphate accumulation, caused by NAD(+) loss and glycolytic enzyme dysfunction, occur in type-1 diabetics (T1DM) and animal models. We previously showed these mechanisms are also involved in Zn(2+) neurotoxicity and are attenuated by nicotinamide- or pyruvate-induced restoration of NAD(+) concentrations, Zn(2+) restriction, or inhibition of Sir2 proteins. We tested the hypothesis that similar Zn(2+)- and NAD(+)-mediated mechanisms are involved in β-cell toxicity in models of ongoing T1DM using mouse insulinoma cells, islets, and nonobese diabetic (NOD) mice. Zn(2+), streptozotocin, and cytokines caused NAD(+) loss and death in insulinoma cells and islets, which were attenuated by Zn(2+) restriction, pyruvate, nicotinamide, NAD(+), and inhibitors of Sir2 proteins. We measured diabetes incidence and mortality in NOD mice and demonstrated that pyruvate supplementation, or genetic or dietary Zn(2+) reduction, attenuated these measures. T-lymphocyte infiltration, punctate Zn(2+) staining, and β-cell loss increased with time in islets of NOD mice. Dietary Zn(2+) restriction or Zn(2+) transporter 5 knockout reduced pancreatic Zn(2+) staining and increased β-cell mass, glucose homeostasis, and survival in NOD mice, whereas Zn(2+) supplementation had the opposite effects. Pancreatic Zn(2+) reduction or NAD(+) restoration (pyruvate or nicotinamide supplementation) are suggested as novel targets for attenuating T1DM.

Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization

Spreading depolarization (SD) is a feed‐forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn2+ release and intracellular accumulation occurs with SD, and elevated intracellular Zn2+ ([Zn2+]i) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn2+]i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn2+]i loading prior to SD, by transient ZnCl2 application with the Zn2+ ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post‐synaptic potentials (fEPSPs) in a concentration‐dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn2+]i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn2+ release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn2+]i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn2+]i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn2+]i accumulation in a range of brain injury models.

Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations.

We examined the impact of intracellular dialysis on fluorescence detection of neuronal intracellular Zn2+ accumulation. Comparison between two dialysis conditions (standard; 20 min, brief; 2 min) by standard whole‐cell clamp revealed a high vulnerability of intracellular Zn2+ buffers to intracellular dialysis. Thus, low concentrations of zinc‐pyrithione generated robust responses in neurons with standard dialysis, but signals were smaller in neurons with short dialysis. Release from oxidation‐sensitive Zn2+ pools was reduced by standard dialysis, when compared with responses in neurons with brief dialysis. The dialysis effects were partly reversed by inclusion of recombinant metallothionein‐3 in the dialysis solution. These findings suggested that extensive dialysis could be exploited for selective detection of transmembrane Zn2+ influx. Different dialysis conditions were then used to probe responses to synaptic stimulation. Under standard dialysis conditions, synaptic stimuli generated significant FluoZin‐3 signals in wild‐type (WT) preparations, but responses were almost absent in preparations lacking vesicular Zn2+ (ZnT3‐KO). In contrast, under brief dialysis conditions, intracellular Zn2+ transients were very similar in WT and ZnT3‐KO preparations. This suggests that both intracellular release and transmembrane flux can contribute to intracellular Zn2+ accumulation after synaptic stimulation. These results demonstrate significant confounds and potential use of intracellular dialysis to investigate intracellular Zn2+ accumulation mechanisms.

Involvement of SIRT1 in Zn 2+ , Streptozotocin, Non-Obese Diabetic, and Cytokine-Mediated Toxicities of β-cells

Zn2+ toxicity is implicated in pancreatic β-cell death that occurs secondarily to: streptozotocin exposure in vitro; and both autoimmune attack or streptozotocin in vivo models of T1DM. This is demonstrated by reduced β-cell death or diabetic incidence in vitro or in NOD mice after treatment with Zn2+ preferring chelators, pyruvate, nicotinamide, a reduced zinc diet, sirtuin inhibitors, or zinc transporter knockout. These therapeutics are also demonstrated to be efficacious against Zn2+ neurotoxicity. AIMS To determine if the sirtuin pathway is involved in Zn2+-, streptozotocin-, or cytokine-mediated β-cell death in vitro, and streptozotocin-, or NOD induced T1DM in vivo. METHODS Sensitivity of MIN6 cells expressing empty vector, sirtuin protein-1 (SIRT1) or its siRNA, to Zn2+, streptozotocin, or cytokines, and effects on NAD+ levels were determined. Covariance of manipulating SIRT1 levels with diabetic incidence was tested in vivo. RESULTS 1) sirtuin pathway inhibition or SIRT1 knockdown attenuated Zn2+-, STZ-, and cytokine-mediated toxicity and NAD+ loss in β-cells, 2) SIRT1 overexpression potentiated these toxicities, 3) young SIRT1 β-cell transgenic mice have improved glucose tolerance under basal conditions, but upon aging showed increased sensitivity to streptozotocin compared to SIRT1 +/- mice, and 4) SIRT1 +/- mice in an NOD background or exposed to streptozotocin trended toward reduced diabetic incidence and mortality compared to wildtype. CONCLUSIONS These results have implicated SIRT1-mediated NAD+ loss in Zn2+, STZ, or cytokine toxicities of MIN6, and in NOD or streptozotocin T1DM animal models. Modulation of β-cell Zn2+ and NAD+ levels, and the sirtuin pathway could be novel therapeutic targets for T1DM.