Supplementary Material for: 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

<b><i>Background:</i></b> 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<sup>+</sup>) and 6-hydroxydopamine (6-OHDA) are used to model Parkinson’s disease. Zinc (Zn<sup>2+)</sup> accumulates after 3-NPA, 6-OHDA and MPP<sup>+</sup> in situ or in vivo. <b><i>Objective:</i></b> We will investigate the role of Zn<sup>2+</sup> neurotoxicity in 3-NPA, 6-OHDA and MPP<sup>+</sup>. <b><i>Methods:</i></b> Murine striatal/midbrain tyrosine hydroxylase positive, or near-pure cortical neuronal cultures, or animals were exposed to 3-NPA or MPP<sup>+</sup> and 6-OHDA with or without neuroprotective compounds. Intracellular zinc ([Zn<sup>2+</sup>]<sub>i</sub>), nicotinamide adenine dinucleotide (NAD<sup>+</sup>), NADH, glycolytic intermediates and neurotoxicity were measured. <b><i>Results:</i></b> We showed that compounds or genetics which restore NAD<sup>+</sup> and attenuate Zn<sup>2+</sup> neurotoxicity (pyruvate, nicotinamide, NAD<sup>+</sup>, increased NAD<sup>+</sup> synthesis, sirtuin inhibition or Zn<sup>2+</sup> chelation) attenuated the neuronal death induced by these toxins. The increase in [Zn<sup>2+</sup>]<sub>i</sub> preceded a reduction in the NAD<sup>+</sup>/NADH ratio that caused a reversible glycolytic inhibition. Pyruvate, nicotinamide and NAD<sup>+</sup> reversed the reductions in the NAD<sup>+</sup>/NADH ratio, glycolysis and neuronal death after challenge with 3-NPA, 6-OHDA or MPP<sup>+</sup>, as was previously shown for exogenous Zn<sup>2+</sup>. 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 Zn<sup>2+</sup> fluorescence, and pyruvate significantly attenuated the 3-NPA-induced lesion and restored behavioral scores. <b><i>Conclusions:</i></b> Together, these studies suggest that Zn<sup>2+</sup> accumulation caused by MPP<sup>+</sup> and 3-NPA is a novel preventable mechanism of the resultant neurotoxicity.