Ubiquitylome profiling of Parkin-null brain reveals dysregulation of calcium homeostasis factors ATP1A2, Hippocalcin and GNA11, reflected by altered firing of noradrenergic neurons

Neurobiol Dis. 2019 Jul;127:114-130. doi: 10.1016/j.nbd.2019.02.008. Epub 2019 Feb 11.

Abstract

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear how selective neural circuits become vulnerable and finally undergo atrophy. We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein targets of Parkin activity, an ubiquitin E3 ligase. Thus, we now used aged Parkin-knockout (KO) mouse brain for a global quantification of ubiquitylated peptides by mass spectrometry (MS). This approach confirmed the most abundant substrate to be VDAC3, a mitochondrial outer membrane porin that modulates calcium flux, while uncovering also >3-fold dysregulations for neuron-specific factors. Ubiquitylation decreases were prominent for Hippocalcin (HPCA), Calmodulin (CALM1/CALML3), Pyruvate Kinase (PKM2), sodium/potassium-transporting ATPases (ATP1A1/2/3/4), the Rab27A-GTPase activating protein alpha (TBC1D10A) and an ubiquitin ligase adapter (DDB1), while strong increases occurred for calcium transporter ATP2C1 and G-protein subunits G(i)/G(o)/G(Tr). Quantitative immunoblots validated elevated abundance for the electrogenic pump ATP1A2, for HPCA as neuron-specific calcium sensor, which stimulates guanylate cyclases and modifies axonal slow afterhyperpolarization (sAHP), and for the calcium-sensing G-protein GNA11. We assessed if compensatory molecular regulations become insufficient over time, leading to functional deficits. Patch clamp experiments in acute Parkin-KO brain slices indeed revealed alterations of the electrophysiological properties in aged noradrenergic locus coeruleus (LC) neurons. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in sAHP and shortening of action potential duration, without modulation of KCNQ potassium currents. These findings indicate altered calcium-dependent excitability in a PARK2 model of PD, mediated by diminished turnover of potential Parkin targets such as ATP1A2 and HPCA. The data also identified further novel Parkin substrate candidates like SIRT2, OTUD7B and CUL5. Our elucidation of neuron-specific mechanisms of PD pathogenesis helps to explain the known exceptional susceptibility of noradrenergic and dopaminergic projections to alterations of calcium homeostasis and its mitochondrial buffering.

Keywords: Calcium; Firing frequency; Mitochondria; Parkin; Parkinson's disease; Ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenergic Neurons / metabolism*
  • Animals
  • Brain / metabolism*
  • GTP-Binding Protein alpha Subunits / metabolism*
  • Hippocalcin / metabolism*
  • Mass Spectrometry
  • Mice
  • Mice, Knockout
  • Mitochondria / metabolism
  • Mitochondrial Membrane Transport Proteins / metabolism
  • Patch-Clamp Techniques
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Voltage-Dependent Anion Channels / metabolism

Substances

  • GNA11 protein, mouse
  • GTP-Binding Protein alpha Subunits
  • Mitochondrial Membrane Transport Proteins
  • Vdac3 protein, mouse
  • Voltage-Dependent Anion Channels
  • Hippocalcin
  • Ubiquitin-Protein Ligases
  • parkin protein
  • Atp1a2 protein, mouse
  • Sodium-Potassium-Exchanging ATPase