S-Nitrosylation of Divalent Metal Transporter 1 Enhances Iron Uptake to Mediate Loss of Dopaminergic Neurons and Motoric Deficit

Abstract

Elevated iron deposition has been reported in Parkinson's disease (PD). However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe uptake via S-nitrosylation of divalent metal transporter 1 (DMT1). While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in postmortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn or Fe uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry, and DMT1 C23A or C540A substitutions abolished nitric oxide (NO)-mediated DMT1 current increase. To evaluate significance, lipopolysaccharide (LPS) was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral LPS injection resulted in corresponding increase in Fe deposition, JNK activation, dopaminergic neuronal loss and deficit in motoric activity, and these were rescued by the NO synthase inhibitor l-NAME or by the DMT1-selective blocker ebselen. Lentiviral knockdown of DMT1 abolished LPS-induced dopaminergic neuron loss. Neuroinflammation and high cytoplasmic Fe levels have been implicated in the initiation and progression of neurodegenerative diseases. Here, we report the unexpected enhancement of the functional activity of transmembrane divalent metal transporter 1 (DMT1) by S-nitrosylation. We demonstrated that S-nitrosylation increased DMT1-mediated Fe uptake, and two cysteines were identified by mass spectrometry to be the sites for S-nitrosylation and for enhanced iron uptake. One conceptual advance is that while DMT1 activity could be increased by external acidification because the gating of the DMT1 transporter is proton motive, we discovered that DMT1 activity could also be enhanced by S-nitrosylation. Significantly, lipopolysaccharide-induced nitric oxide (NO)-mediated neuronal death in the substantia nigra could be ameliorated by using l-NAME, a NO synthase inhibitor, or by ebselen, a DMT1-selective blocker.

Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)

“…Even if protein samples might have been preheated in most of these publications, they might have observed the right size of the endogenous Fpn1 band as we observed in this study (Figs 4B and 6A ). CiteAb also lists more than 10 publications for DMT1 western blotting using Proteintech, Abcam, or Novus Biologicals anti-DMT1 antibody, only some of which noted that they treated samples with gentle heating at 37°C for 30min [ 47 – 49 ] or 50°C for 15min [ 50 ]. Anti-DMT1 antibodies that have different epitopes from our anti-DMT1 antibody (Cell Signaling Technology, near the N-terminus of human DMT1) may also affect the optimum conditions of protein sample preparation for better resolution of western blotting.…”

Nitric Oxide, Iron and Neurodegeneration

“…Although rodent models are widely used for iron study, it is noteworthy that iron accumulation and cellular storage are different between humans and experimental rodent models. Rodent generally have a lower basal level of iron deposition in the brain, especially in young animals (less than one year old), as shown by several groups (Jeong and David, 2006; Liu et al, 2018).…”

“…The reason for iron accumulation in SN is unclear. Several hypotheses proposed include increased brain-blood-barrier (BBB) permeability (Faucheux et al, 1999; Kortekaas et al, 2005), increased neuroinflammation (Block and Hong, 2005; Conde and Streit, 2006; Machado et al, 2011), increased DMT1 isoform expression or function (Block and Hong, 2005; Salazar et al, 2008; Machado et al, 2011; Liu et al, 2018), and altered transferrin or lactoferrin transport (Faucheux et al, 1995; Mastroberardino et al, 2009). Cellular iron accumulation in PD brain may be caused by elevated influx or decreased efflux.…”

“…Inflammation could contribute to iron accumulation by either increasing DMT1 uptake activity or TfR transport activity. In a mouse model, DMT1 activity was increased to mediate the iron uptake (Salazar et al, 2008), and this increase may be due to direct S-nitrosylation of DMT1 (Liu et al, 2018).…”

“…To inhibit NO-mediated DMT1 functional increase, we used the NOS inhibitor L-name to reduce NO-mediated iron deposition in LPS-evoked mouse inflammatory model. L-NAME significantly ameliorated SN dopaminergic neuron loss and LPS-induced behavior deficit (Liu et al, 2018).…”