EFFECTS OF UCH‐L1 INHIBITION IN IMMORTALIZED DOPAMINERGIC NEURONS

Abstract

In Parkinson disease (PD), a debilitating neurodegenerative disorder characterized by a host of motor and non‐motor symptoms, neurons in the substantia nigra pars compacta (SN) progressively degenerate. The effect of this degeneration is the loss of striatal dopamine (DA), which famously results in bradykinesia, postural instability, shuffling gait, and resting tremor. Animal and cellular models only partially recapitulate the progression of the disease, since much is still not known about the etiology of PD. Using cell lines affords a method in which to manipulate parameters in a homogeneous population whereby we can study phenomena occurring specifically in dopaminergic neurons. The dopaminergic cell line, MN9D, was generated by fusing mouse neuroblastoma cells with primary mouse mesencephalon neurons. A key feature of these cells is their ability to be differentiated using sodium butyrate in the culture medium, which halts their cell cycle and makes them look morphologically like neurons. Expression of the dopamine transporter and tyrosine hydroxylase, markers of dopamine neurons, are also highly expressed after differentiation of MN9D cells. In these cells, we have studied components of the ubiquitin proteasome system (UPS) such as parkin and UCH‐L1, which are two genes found mutated in familial PD. The latter, UCH‐L1 (ubiquitin carboxy‐terminal hydrolase L1), is a neuron‐specific, highly expressed de‐ubiquitinating enzyme thought to play a role in the susceptibility of mouse SN neurons exposed to the toxicant MPTP (active metabolite is MPP+). UCH‐L1 acts to remove monomeric ubiquitin from protein substrates, regulating proteasome activity and replenishing the pool of monomeric ubiquitin to be tagged on to new substrates. In MN9D cells with MPP+ treatment, we have found that intracellular dopamine, intracellular DOPAC, and UCH‐L1 protein all decrease. In addition, when we treated cells with LDN‐57444 (a UCH‐L1 inhibitor), DA but not DOPAC decreased in these cells, highlighting a possible link between the loss of DA and loss of function of UCH‐L1. These studies will help shed light on the dynamics between UPS function and dopamine synthesis and turnover in MN9D cells as an in vitro model for isolated SN neurons.Support or Funding InformationT32 Pre‐Doctoral Training Grant Institute for Integrative Toxicology; PIs Grant: 1R01 NS065338‐01A2