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A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease.

ABSTRACT: The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling.

SUBMITTER: Tidball AM

PROVIDER: S-EPMC4355025 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease.

Tidball Andrew M AM Bryan Miles R MR Uhouse Michael A MA Kumar Kevin K KK Aboud Asad A AA Feist Jack E JE Ess Kevin C KC Neely M Diana MD Aschner Michael M Bowman Aaron B AB

Human molecular genetics 20141208 7

The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell lin ...[more]

PMID: 25489053

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Project description: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.

Project description:Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor symptoms that are preceded by cognitive deficits and is considered as a disorder that primarily affects forebrain striatal neurons. To gain a better understanding of the molecular and cellular mechanisms associated with disease progression, we analyzed the expression of proteins involved in GABAergic neurotransmission in the striatum of the R6/1 transgenic mouse model. Western blot, quantitative PCR and immunohistochemical analyses were conducted on male R6/1 mice and age-matched wild type littermates. Analyses were performed on 2 and 6 month-old animals, respectively, before and after the onset of motor symptoms. Expression of GAD 67, GAD 65, NL2, or gephyrin proteins, involved in GABA synthesis or synapse formation did not display major changes. In contrast, expression of ?1, ?3 and ?5 GABAAR subunits was increased while the expression of ? was decreased, suggesting a change in tonic- and phasic inhibitory transmission. Western blot analysis of the striatum from 8 month-old Hdh Q111, a knock-in mouse model of HD with mild deficits, confirmed the ?1 subunit increased expression. From immunohistochemical analyses, we also found that ?1 subunit expression is increased in medium-sized spiny projection neurons (MSN) and decreased in parvalbumin (PV)-expressing interneurons at 2 and 6 months in R6/1 mice. Moreover, ?2 subunit labeling on the PV and MSN cell membranes was increased at 2 months and decreased at 6 months. Alteration of gene expression in the striatum and modification of GABAA receptor subtypes in both interneurons and projection neurons suggested that HD mutation has a profound effect on synaptic plasticity at an early stage, before the onset of motor symptoms. These results also indicate that cognitive and other behavioral deficits may be associated with changes in GABAergic neurotransmission that consequently could be a relevant target for early therapeutic treatment.

Dataset Information

A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease.

Publications

A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease.

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