Elsevier

Neurobiology of Disease

Volume 41, Issue 2, February 2011, Pages 508-527
Neurobiology of Disease

Reactive astrocytes and Wnt/β-catenin signaling link nigrostriatal injury to repair in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

https://doi.org/10.1016/j.nbd.2010.10.023Get rights and content

Abstract

Emerging evidence points to reactive glia as a pivotal factor in Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of basal ganglia injury, but whether astrocytes and microglia activation may exacerbate dopaminergic (DAergic) neuron demise and/or contribute to DAergic repair is presently the subject of much debate. Here, we have correlated the loss and recovery of the nigrostriatal DAergic functionality upon acute MPTP exposure with extensive gene expression analysis at the level of the ventral midbrain (VM) and striata (Str) and found a major upregulation of pro-inflammatory chemokines and wingless-type MMTV integration site1 (Wnt1), a key transcript involved in midbrain DAergic neurodevelopment. Wnt signaling components (including Frizzled-1 [Fzd-1] and β-catenin) were dynamically regulated during MPTP-induced DAergic degeneration and reactive glial activation. Activated astrocytes of the ventral midbrain were identified as candidate source of Wnt1 by in situ hybridization and real-time PCR in vitro. Blocking Wnt/Fzd signaling with Dickkopf-1 (Dkk1) counteracted astrocyte-induced neuroprotection against MPP+ toxicity in primary mesencephalic astrocyte–neuron cultures, in vitro. Moreover, astroglial-derived factors, including Wnt1, promoted neurogenesis and DAergic neurogenesis from adult midbrain stem/neuroprogenitor cells, in vitro. Conversely, lack of Wnt1 transcription in response to MPTP in middle-aged mice and failure of DAergic neurons to recover were reversed by pharmacological activation of Wnt/β-catenin signaling, in vivo, thus suggesting MPTP-reactive astrocytes in situ and Wnt1 as candidate components of neuroprotective/neurorescue pathways in MPTP-induced nigrostriatal DAergic plasticity.

Research Highlights

► A major upregulation of Wnt1 was uncovered upon MPTP injury. ► Reactive astrocytes express Wnt1 in the injured midbrain. ► Reactive astrocytes promote DAergic neuroprotection via Wnt1/β-catenin. ► Reactive astrocytes promote adult DA neurogenesis through Wnt/β-catenin signaling. ► Pharmacological activation of Wnt1 signaling promotes DAergic recovery in aged mice.

Introduction

The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of basal ganglia injury recapitulates many of the pathogenetic processes operative in Parkinson's disease (PD), a common neurodegenerative disorder characterized by the progressive loss of dopaminergic (DAergic) neurons in the subtantia nigra pars compacta (SNpc) and astrogliosis (Jackson-Lewis and Przedborski, 2007). The neurotoxin MPTP, converted into its active metabolite,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+) in astrocytes, is selectively transported into striatal DAergic terminals via the DA transporter, DAT, where it induces oxidative stress, the opening of mitochondrial permeability transition pore (mPTP), the release of cytochrome c, and the activation of caspases (Abou-Sleiman et al., 2006, Jackson-Lewis and Przedborski, 2007). In synergy with these early events accounting for approximately 10% of DAergic neuronal death (Wu et al., 2002), glial inflammatory mechanisms are thought to contribute to nigrostriatal DAergic degeneration (Benner et al., 2004; Gao and Hong, 2008, Hu et al., 2008, Hoang et al., 2009, Hirsch and Hunot, 2009).

Astrocytes and microglia normally play neuroprotective roles; however, upon MPTP-mediated neuronal injury, activated microglia produce a panel of cytotoxic mediators including reactive oxygen and nitrogen species as well as proinflammatory cytokines that may perpetuate/exacerbate glial activation, thereby increasing neuronal vulnerability and/or promoting DAergic cell death (McNaught and Jenner, 1999a,b; Vila et al., 2001, Wu et al., 2002, Streit, 2002, Gao et al., 2003, Hauwel et al., 2005, Marchetti and Abbracchio, 2005, Griffiths et al., 2007, Hu et al., 2008, McGeer and McGeer, 2008). Astrocytes are known to secrete both inflammatory and anti-inflammatory, as well as neurotrophic and survival factors, and may play a role in modulating microglial activity, but in severe neurodegenerative conditions, they may lose their neuroprotective functions (McGeer and McGeer, 2008, Sandhu et al., 2009, Chen et al., 2009, L'Episcopo et al., 2010).

In response to MPTP injury, the nigrostriatal DAergic system exhibits compensatory mechanisms, but the degree of plasticity becomes reduced with age (Ricaurte et al., 1987, Hornykiewicz, 1993, Blanchard et al., 1996, Ho and Blum, 1998 Bezard and Gross, 1998, Stanic et al., 2003, Jacowec et al., 2004). Age-related alterations, including increased DAergic neuron vulnerability, increased microglia activation/dysfunction, altered glia–neuron crosstalk, and/or impaired neurogenesis represent potential contributory factors (Butovsky et al., 2006, Miller and Streit, 2007, Griffiths et al., 2009, Streit, 2010, Boger et al., 2010, Njie et al., 2010, Sharpless, 2010).

Recently, the wingless-type MMTV integration site1 (Wnt) pathway has emerged as an essential signaling cascade that regulates multiple processes in developing and adult tissues, including differentiation, neuron survival, axonal extension, synapse formation and plasticity, neurotrophin transcription, and neurogenesis (Patapoutian and Reichardt, 2000, Ciani and Salinas, 2005, Maiese et al., 2008; Inestrosa and Arenas, 2009). The extracellular Wnt molecules signal into the cell via three different pathways: the “canonical” Wnt/β-catenin and “non-canonical” Wnt/planar cell polarity (PCP) and Wnt (Ca2+) pathways (Ciani and Salinas, 2005). Common to all three pathways is binding of the Wnt ligand to the seven-pass transmembrane receptors of the Frizzled (Fzd) family. The hallmark of Wnt/β-catenin pathway is the stabilization of cytosolic β-catenin, which enters the nucleus and activates the transcription of Wnt target genes involved in cell survival, proliferation, and differentiation (Gordon and Nusse, 2006). Recent pieces of evidence clearly indicate that Wnt/β-catenin signaling plays a central role in midbrain DAergic neurodevelopment both in vivo and in vitro (Castelo-Branco et al., 2003, Castelo-Branco et al., 2004; Prakash and Wurst, 2006, Joksimovic et al., 2009), but very little is known on Wnt1/β-catenin signaling in the adult PD midbrain.

To elucidate some of the molecular mechanism(s) underlying MPTP-induced injury and self-recovery, we used extensive gene expression analysis of a total of 92 mRNA species involved in inflammation, immunity, stemness, self-renewal, DAergic development, and DA metabolism and identified a major upregulation of pro-inflammatory transcripts and Wnt1. We herein provide pieces of evidence indicating Wnt1/β-catenin signaling and MPTP-reactive astrocytes as candidate components of neurorescue pathways involved in nigrostriatal DAergic plasticity.

Section snippets

Mice and treatments

Eight- to ten-week-old male C57Bl/6 (Charles River, Calco, Italy) (body weight 24–26 g) received n = 4 intraperitoneal (i.p.) injections of vehicle (saline, 10 ml/kg) or MPTP–HCl (20 mg kg−1 free base; Sigma) dissolved in saline, 2 h apart in 1 day, according to the acute MPTP injection paradigm (Jackson-Lewis et al., 1995). Mice were sacrificed at either early (e.g., 3, 6, 24, 72 h), mid (e.g., 5, 7, and 14 days), and late (e.g., 21, 28, 35, 42 days) time points upon MPTP administration (Table 1). MPTP

The model

In order to elucidate some of the molecular mechanism(s) underlying the MPTP-induced injury of nigrostriatal DA neurons, we first confirmed that the acute MPTP-dose regimen resulted in the expected markers of DAergic toxicity and glia activation. Spatiotemporal analyses in both striatum (Str) and ventral midbrain (VM) supported the early and profound nigrostriatal impairment followed by a substantial recovery. Hence, after a significant motor deficit assessed with the rotarod (Fig. 1A),

Discussion

We herein unveiled Wnt/β-catenin signaling and MPTP-reactive astrocytes “in situ” as candidate components of neurorescue/repair pathways in MPTP-induced nigrostriatal DAergic plasticity. First, wide gene expression analysis of 92 mRNA identified a major upregulation of pro-inflammatory chemokines and Wnt1 during MPTP-induced DAergic degeneration and self-recovery. Next, spatio-temporal analyses within the injured VM showed a dynamic interplay between prototypical proteins of canonical Wnt

Acknowledgments and Funding

The authors wish to thank the Italian Ministry of Health (Con. no. 82; Ps-CARDIO ex 56, PS-NEURO ex 56 to B.M.; Young Investigator Award 2009 to S.P.), Italian Ministry of Research (Cur. Res. 2008–2010 to B.M.), the Italian Multiple Sclerosis Foundation (FISM, grants 2004/R/15 to S.P. and 2002/R/37 to G.M.), the National Multiple Sclerosis Society (NMSS, partial grants RG-4001-A1 to S.P.; RG 3591-A-1 to G.M.; and RG 3762-A-1), the Italian Ministry of Research and University (MIUR, to B.M.), the

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