Abstract
Mitochondria-dependent apoptosis plays an important role in the embryonic development of the midbrain dopaminergic system as well as in Parkinson’s disease. Central to mitochondria-dependent apoptosis is the Bcl2 family of apoptosis-regulating proteins. However, it was unclear which Bcl2 proteins are important for the survival of dopaminergic neurons. Here, we identify Mcl1 as a critical Bcl2 pro-survival factor in midbrain dopaminergic neurons. Using a chemical biology approach to inhibit various components of the apoptotic machinery in the dopaminergic MN9D cell line or the control neuroblastoma N2A cell line, we find that functional inhibition of Mcl1 with the high affinity small molecule inhibitor UMI-77 results in a rapid and dose-dependent loss of viability, selectively in dopaminergic cells. In-depth analysis of the apoptotic signaling pathway reveals that chemical inhibition of Mcl1 results in the activation of Bax, activation of cleaved caspase-3 and finally cell death. The dependence of mouse dopaminergic midbrain neurons on Mcl1 was confirmed using ex vivo slice cultures from Pitx3GFP/+ and wildtype mice. In mouse dopaminergic midbrain neurons positive for the midbrain dopaminergic marker Pitx3, or tyrosine hydroxylase, UMI-77 treatment caused a dramatic increase in cleaved caspase 3, indicating that Mcl1 activity is required for basal neuronal survival. Overall, our results suggest that Mcl1 is of critical importance to dopaminergic neurons and is a weak link in the chain controlling cellular survival. Boosting the pro-survival function of Mcl1 should be pursued as a therapeutic approach to augment the resilience of midbrain dopaminergic neurons to apoptotic stress in Parkinson’s disease.
Highlights
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease[1]
Microarray data[6] and RT-quantitative PCR (qPCR) data performed on sorted dopamine midbrain neurons from Pitx3GFP/+ heterozygous mice suggest that Mcl[1] is one of the most highly expressed pro-survival Bcl[2] factors in dopamine neurons
To test the relative importance of Bcl[2], Bcl-xL, and Mcl[1], we used a small molecule chemical screen coupled with readouts for apoptotic cell death in two cell lines, the dopaminergic MN9D and the more commonly used non-dopaminergic neuroblastoma N2A cell line
Summary
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease[1]. Its clinical symptoms arise due to the progressive death of dopamine neurons in the substantia nigra pars compacta (SNpc) resulting in the loss of dopaminergic input to the striatum. At the onset of clinical symptoms it is estimated that more than 60% of the dopamine neurons have died[1]. Attenuating the loss of Mitochondria-dependent apoptosis is executed by the formation of a proteolipid pore in the outer mitochondrial membrane by oligomers of Bax and/or Bak[4]. The formation of these proteo-lipid pores allows components such as Cytochrome C, Apoptosis Inducing Factor (AIF), and Endonuclease G to leak out from the mitochondrial intermembrane space into the cytosol. Following mitochondrial outer membrane permeabilization (MOMP), mitochondrial contents spill into the cytosol to activate caspases resulting in the apoptotic phenotype[4]
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