Acute manganese treatment restores defective autophagic cargo loading in Huntington's disease cell lines.

The molecular etiology linking the pathogenic mutations in the Huntingtin (Htt) gene with Huntington's disease (HD) is unknown. Prior work suggests a role for Htt in neuronal autophagic function and mutant HTT protein disrupts autophagic cargo loading. Reductions in the bioavailability of the essential metal manganese (Mn) are seen in models of HD. Excess cellular Mn impacts autophagic function, but the target and molecular basis of these changes are unknown. Thus, we sought to determine if changes in cellular Mn status impact autophagic processes in a wild-type or mutant Htt-dependent manner. We report that the HD genotype is associated with reduced Mn-induced autophagy and that acute Mn exposure increases autophagosome induction/formation. To determine if a deficit in bioavailable Mn is mechanistically linked to the autophagy-related HD cellular phenotypes, we examined autophagosomes by electron microscopy. We observed that a 24 h 100 uM Mn restoration treatment protocol attenuated an established HD 'cargo-recognition failure' in the STHdh HD model cells by increasing the percentage of filled autophagosomes. Mn restoration had no effect on HTT aggregate number, but a 72 h co-treatment with chloroquine (CQ) in GFP-72Q-expressing HEK293 cells increased the number of visible aggregates in a dose-dependent manner. As CQ prevents autophagic degradation this indicates that Mn restoration in HD cell models facilitates incorporation of aggregates into autophagosomes. Together, these findings suggest that defective Mn homeostasis in HD models is upstream of the impaired autophagic flux and provide proof-of-principle support for increasing bioavailable Mn in HD to restore autophagic function and promote aggregate clearance.

Manganese Acts upon Insulin/IGF Receptors to Phosphorylate AKT and Increase Glucose Uptake in Huntington's Disease Cells.

Perturbations in insulin/IGF signaling and manganese (Mn2+) uptake and signaling have been separately reported in Huntington's disease (HD) models. Insulin/IGF supplementation ameliorates HD phenotypes via upregulation of AKT, a known Mn2+-responsive kinase. Limited evidence both in vivo and in purified biochemical systems suggest Mn2+ enhances insulin/IGF receptor (IR/IGFR), an upstream tyrosine kinase of AKT. Conversely, Mn2+ deficiency impairs insulin release and associated glucose tolerance in vivo. Here, we test the hypothesis that Mn2+-dependent AKT signaling is predominantly mediated by direct Mn2+ activation of the insulin/IGF receptors, and HD-related impairments in insulin/IGF signaling are due to HD genotype-associated deficits in Mn2+ bioavailability. We examined the combined effects of IGF-1 and/or Mn2+ treatments on AKT signaling in multiple HD cellular models. Mn2+ treatment potentiates p-IGFR/IR-dependent AKT phosphorylation under physiological (1 nM) or saturating (10 nM) concentrations of IGF-1 directly at the level of intracellular activation of IGFR/IR. Using a multi-pharmacological approach, we find that > 70-80% of Mn2+-associated AKT signaling across rodent and human neuronal cell models is specifically dependent on IR/IGFR, versus other signaling pathways upstream of AKT activation. Mn2+-induced p-IGFR and p-AKT were diminished in HD cell models, and, consistent with our hypothesis, were rescued by co-treatment of Mn2+ and IGF-1. Lastly, Mn2+-induced IGF signaling can modulate HD-relevant biological processes, as the reduced glucose uptake in HD STHdh cells was partially reversed by Mn2+ supplementation. Our data demonstrate that Mn2+ supplementation increases peak IGFR/IR-induced p-AKT likely via direct effects on IGFR/IR, consistent with its role as a cofactor, and suggests reduced Mn2+ bioavailability contributes to impaired IGF signaling and glucose uptake in HD models.

Phosphatidylinositol 3 kinase (PI3K) modulates manganese homeostasis and manganese-induced cell signaling in a murine striatal cell line.

In a recent study, we found that blocking the protein kinase ataxia telangiectasia mutated (ATM) with the small molecule inhibitor (SMI) KU-55933 can completely abrogate Mn-induced phosphorylation of p53 at serine 15 (p-p53) in human induced pluripotent stem cell (hiPSC)-differentiated striatal neuroprogenitors. However, in the immortalized mouse striatal progenitor cell line STHdhQ7/Q7, a concentration of KU55933 far exceeding its IC50 for ATM was required to inhibit Mn-induced p-p53. This suggested an alternative signaling system redundant with ATM kinase for activating p53 in this cell line- one that was altered by KU55933 at these higher concentrations (i.e. mTORC1, DNApk, PI3K). To test the hypothesis that one or more of these signaling pathways contributed to Mn-induced p-p53, we utilized a set of SMIs (e.g. NU7441 and LY294002) known to block DNApk, PI3K, and mTORC1 at distinct concentrations. We found that the SMIs inhibit Mn-induced p-p53 expression near the expected IC50s for PI3K, versus other known targets. We hypothesized that inhibiting PI3K reduces intracellular Mn and thereby decreases activation of p53 by Mn. Using the cellular fura-2 manganese extraction assay (CFMEA), we determined that KU55933/60019, NU7441, and LY294002 (at concentrations near their IC50s for PI3K) all decrease intracellular Mn (∼50%) after a dual, 24-h Mn and SMI exposure. Many pathways are activated by Mn aside from p-p53, including AKT and mTOR pathways. Thus, we explored the activation of these pathways by Mn in STHdh cells as well as the effects of other pathway inhibitors. p-AKT and p-S6 activation by Mn is almost completely blocked upon addition of NU7441(5µM) or LY294002(7µM), supporting PI3K's upstream role in the AKT/mTOR pathway. We also investigated whether PI3K inhibition blocks Mn uptake in other cell lines. LY294002 exposure did not reduce Mn uptake in ST14A, Neuro2A, HEK293, MEF, or hiPSC-derived neuroprogenitors. Next, we sought to determine whether inhibition of PI3K blocked p53 phosphorylation by directly blocking an unknown PI3K/p53 interaction or indirectly reducing intracellular Mn, decreasing p-p53 expression. In-Cell Western and CFMEA experiments using multiple concentrations of Mn exposures demonstrated that intracellular Mn levels directly correlated with p-p53 expression with or without addition of LY294002. Finally, we examined whether PI3K inhibition was able to block Mn-induced p-p53 activity in hiPSC-derived striatal neuroprogenitors. As expected, LY294002 does not block Mn-induced p-p53 as PI3K inhibition is unable to reduce Mn net uptake in this cell line, suggesting the effect of LY294002 on Mn uptake is relatively specific to the STHdh mouse striatal cell line.