Page 149 - ebook

Parkin mediates neuroprotection through activation of

Notch1 signaling

Eun-Hye Jo, Mi-Yeon Kim, So-I Noh, Hyung-Ju Lee and Hee-Sae Park *
School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea

Abstract Figure 1. Parkin activates Notch1-IC Figure 4. Parkin reduces neuronal apoptosis
transcriptional activity. through the Notch1 signaling pathway.
Parkin, an E3 ubiquitin ligase, is the most frequently mutated
gene in hereditary Parkinson’s disease. Inactivation of Parkin
leads to impairment of the ubiquitin–proteasome system,
resulting in the accumulation of misfolded or aggregated proteins
and ensuing neurodegeneration. In this study, we show that
Parkin positively regulates the Notch1 signaling pathway.
Overexpression of Parkin stabilized Notch1-IC protein levels,
whereas knockdown of Parkin decreased Notch1-IC protein
stability. Notably, overexpression of Parkin disrupted oxidative
stress-induced apoptosis in neuronal cells. However, knockdown
of Notch1 inhibited Parkin-induced neuronal cell survival.
Together, these results indicate that Parkin is a novel regulator of
the Notch1 signaling pathway, (a) Intracellular reactive oxygen species (ROS) generation was
analyzed by staining with 10 μM DCFHDA for 15 min in SH-SY5Y
cells transfected with His-Parkin and shNotch1-IC and treated
with 2 mM H2O2 for 20 min as indicated. (b) Apoptosis assay of
the SH-SY5Y cells transfected with His-Parkin and shNotch1-IC
(a–c) Luciferase assay of HEK293 cells transiently transfected and treated with 2 mM H2O2 for 20 min Apoptotic cells were
Introduction with plasmids expressing Parkin and Notch1-IC. Transcriptional stained with 5 μl annexin V-FITC for 5 min in the dark. Results of
activity of Notch1-IC was measured using luciferase reporter three independent experiments are presented as mean ±SD. (c) A
plasmids, (a) 4xCSL-Luc, (b) Hes1-Luc, and (c) Hes5-Luc. (d)
Parkinson’s disease (PD) is the most common movement Transcriptional activity of Notch1-IC in HEK293 cells co- working model of Notch1 activation by Parkin. Parkin interacts
disorder in humans and is characterized by the loss of dopamine- with Fbw7 and induces its ubiquitination and degradation.
producing neurons in the substantia nigra, a region in the transfected with Notch1-IC and either Parkin WT or R42P PD Degradation of Fbw7 stabilizes Notch1-IC and stimulates the
pathogenic mutant. (e) Luciferase assay of HEK293 cells
midbrain, which is implicated in motor control. Among the genes transiently transfected with plasmids expressing Notch1-IC and Notch1 signaling pathway, thus accelerating neuronal survival.
that are responsible for monogenic familial variants of PD, Parkin
appears to play a prominent role, accounting for the majority of Parkin shRNA. (f) Western blot analysis of Parkin using lysates of
HEK293 cells dose dependently transfected with shParkin.
autosomal recessive PD cases. Parkin is an E3 ubiquitin protein
ligase targeting substrate proteins for proteasomal degradation. Results of three independent experiments are presented as mean
±SD.
Mutations in Parkin, which disrupt its catalytic activity, lead to the
accumulation of toxic substrates that damage dopaminergic Figure 5. Parkin significantly stimulates
neurons, consequently causing autosomal recessive PD[1]. neuronal protection under oxidative stress by a
Parkin is transcriptionally upregulated under various stress
conditions including mitochondrial and endoplasmic reticulum Figure 2. Parkin stimulates the interaction Notch1-mediated pathway.
stress, proteotoxic stress, excitotoxicity, proapoptotic between Notch1-IC and RBP-Jκ.
stimulation, as well as overexpression of toxic, misfolded protein
species, and shows wide neuroprotective activity in cellular and
animal models[2–4]. Parkin promotes neuroprotection by
regulating various biological mechanisms, including
mitochondrial integrity, proteasomal degradation of toxic
substances, and nondegradative ubiquitin signaling within cell
death or viability pathways [5]. Parkin is responsible for proper
mitochondrial function and promotes autophagosomal
engulfment of damaged mitochondria in a process termed
mitophagy. Upon mitochondrial depolarization, Parkin cooperates (c) A working model of Notch1 activation by Parkin. Parkin
with mitochondrial PTEN-induced putative kinase 1 (PINK1). interacts with Fbw7 and induces its ubiquitination and
PINK1 expression was found to be necessary for the recruitment degradation. Degradation of Fbw7 stabilizes Notch1-IC and
of Parkin to depolarized mitochondria. Under stress conditions, stimulates the Notch1 signaling pathway, thus accelerating
Parkin stimulates mitochondrial biogenesis and cell survival of neuronal survival.
dopaminergic neurons through ubiquitination of its target (a) Co-IP of Notch1-IC and RBP-Jκ in HEK293 cells transiently
proteins. Furthermore, Parkin can also influence other cell death transfected with Myc-Notch1-IC, Flag-RBP-Jκ, and His-Parkin
and viability pathways through a nondegradative mechanism by expression vectors as indicated. Myc-Notch1-IC, Flag-RBP-Jκ,
modulating ubiquitin signaling in the nuclear factor kappa B (NF- and His-Parkin expression was analyzed using specific
κB) pathway, activation of c-Jun N-terminal kinase, p53 antibodies, respectively. (b) Endogenous co-IP of Notch1-IC and
expression, and mitochondrial translocation of Bax [6–8]. RBP-Jκ in HEK293 cells transiently transfected with shControl
Abnormal activation of the Notch1 pathway is associated with and shParkin. Notch1-IC, RBP-Jκ, and Parkin expressions were
several neurodegenerative diseases including amyotrophic analyzed using anti-Notch1, anti-RBP-Jκ, and anti-Parkin Conclusion
lateral sclerosis, Down’s syndrome, Alzheimer’s, and Pick’s antibodies, respectively. Results of three independent
disease. Notch1 promotes cell survival in the developing nervous experiments are presented as mean ±SD.
system . Although the Notch1 signaling pathway is known to be Previously, it was shown that the Notch1 signaling pathway
important is important for neural stem cell maintenance and proper
during neuronal degeneration, its regulatory mechanism remains neurogenesis in the developing mammalian nervous
to be determined. In this study, we identified Parkin as an Figure 3. Parkin induces Notch1-IC protein system and in the adult brain. Importantly, Notch1 also
activator of Notch1 signaling, indicating that Parkin mediates its plays a role in neuronal differentiation and neuronal
neuroprotective effect by modulating Fbw7-mediated protein stability in an Fbw7-dependent manner. survival. These data indicate that Notch1 is required not
degradation of Notch1. only for the formation but also for the maintenance of
neurons. In the present study, we focused on Parkin-
mediated activation of Notch1 signaling, leading to
neuronal survival. Loss of Parkin E3 ligase activity leads to
References impairment of the ubiquitin–proteasome system, resulting
in the accumulation of misfolded proteins and ensuing
neurodegeneration. Parkin ubiquitinated and degraded the
1 Dawson TM, Dawson VL. The role of parkin in familial and
sporadic target proteins of numerous signaling pathways. Parkin
Parkinson’s disease. Mov Disord 2010; 25 (Suppl 1):S32–S39. ubiquitinates the proapoptotic factor Bax both in vivo and
2 Yang YX, Muqit MM, Latchman DS. Induction of parkin in vitro. Indeed, Parkin also ubiquitinated NF-κB essential
expression in the modulator resulting in protection from stress-induced cell
presence of oxidative stress. Eur J Neurosci 2006; 24:1366–1372. death. Here, we provided the correlation between Parkin
3 LaVoie MJ, Cortese GP, Ostaszewski BL, Schlossmacher MG. and the Notch1 signaling pathway (Fig. 4c). Notch1-IC
The effects of oxidative stress on parkin and other E3 ligases. protein stability is regulated by ubiquitination. Fbw7, an F-
J Neurochem 2007; 103:2354–2368. box protein, is known to be a nuclear ubiquitin E3 ligase
4 Wong ES, Tan JM, Wang C, Zhang Z, Tay SP, Zaiden N, et al. and regulates Notch1-IC proteasome-dependent
Relative sensitivity of parkin and other cysteine-containing degradation. We confirmed that Fbw7 is necessary for
enzymes to stress-induced solubility alterations. J Biol Chem
2007; 282:12310–12318. (a)Western blot analysis using antibodies against Flag and Myc in lysates of Parkinmediated regulation of Notch1-IC protein stability.
5 Winklhofer KF. Parkin and mitochondrial quality control: toward HEK293 cells dose-dependently expressing Flag-Notch1-IC and Myc-Parkin. (b) Parkin inhibited the interaction between Notch1-IC and
Cycloheximide (CHX) chase assay for the half-life of Notch1-IC of SH-SY5Y
assembling the puzzle. Trends Cell Biol 2014; 24:332–341. cells transfected with shControl and shParkin. Cells were treated with either Fbw7, resulting in reduced ubiquitination of Notch1-IC.
6 da Costa CA, Sunyach C, Giaime E, West A, Corti O, Brice A, et 100 μM DMSO or 100 μM CHX for the indicated times. The lysates were These findings reveal a novel mechanism for Parkin-
al. Transcriptional repression of p53 by parkin and impairment immunoblotted using an anti-Notch1-IC and anti-β-actin antibody. (c) Western mediated regulation of the Notch1 signaling pathway.
by mutations associated with autosomal recessive juvenile blot analysis, using antibodies against Flag and Myc, of lysates from HEK293
Parkinson’s disease. Nat Cell Biol 2009; 11:1370–1375. cells dose-dependently expressing Flag-Fbw7 and Myc-Parkin. (d)Westernblot
analysis of Myc-Notch1-IC of HEK293 cells cotransfected with Myc-Notch1-IC,
7 Cha GH, Kim S, Park J, Lee E, Kim M, Lee SB, et al. Parkin Flag-Fbw7, and Myc-Parkin. (e) Co-IP of Notch1-IC and RBP-Jκ in HEK293 cells
negatively regulates JNK pathway in the dopaminergic neurons transiently transfected with Myc-Notch1-IC, Flag-Fbw7, and His-Parkin
of Drosophila. Proc Natl Acad Sci USA 2005; 102:10345–10350. expression vectors as indicated. (f) Endogenous co-IP of Notch1-IC and Fbw7
8 Henn IH, Bouman L, Schlehe JS, Schlierf A, Schramm JE, in HEK293 cells transiently transfected with shControl and shParkin. (g)
Wegener E, et al. Parkin mediates neuroprotection through Luciferase assay of HEK293 cells transiently transfected with plasmids
expressing Myc-Notch1-IC, Fbw7 shRNA, and Parkin shRNA as indicated.
activation of IkappaB kinase/nuclear factor-kappaB signaling. J Results of three independent experiments are presented as mean ±SD.
Neurosci 2007; 27:1868–1878.