Application of proteasomal inhibitors to mouse sympathetic neurons activates the intrinsic apoptotic pathway

Abstract

Proteasomal dysfunction may play a role in a number of neurodegenerative conditions, and in particular Parkinson's disease (PD) and related Lewy body (LB) diseases. Application of proteasomal inhibitors to neuronal cell culture systems is associated with survival-promoting effects or with cell death depending on the model system. We have applied pharmacological proteasomal inhibitors to cultured neonatal mouse sympathetic neurons in order to investigate whether these catecholaminergic neurons, which are affected in PD, are sensitive to proteasomal inhibition and, if so, which cell death pathway is activated. We report here that proteasomal inhibition leads to apoptotic death of mouse sympathetic neurons. This death is accompanied by caspase 3 activation and cytochrome c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochrome c release and caspase 3 activation, and also provided complete protection against proteasomal inhibitioninduced death. Bcl-2 overexpression achieved a similar survival-promoting effect. There was no change in Bax levels following proteasomal inhibition, suggesting that Bax itself is not regulated by the proteasome in this cell culture system, and that a primary increase in Bax is unlikely to account for death. In contrast, levels of the BH3-only protein, Bim, increased with proteasomal inhibition. We conclude that proteasomal inhibition of mouse sympathetic neurons activates the intrinsic apoptotic pathway involving bcl-2 family members and the mitochondria. The proteasome is a multicatalytic complex that is involved in the degradation of many cellular proteins, including those that are rapidly turning over, oxidized, or misfolded. As a general rule, in order to be targeted to the proteasome, proteins need to be ubiquitinated through a series of enzymatic steps that involve the attachment of chains of a small molecule, ubiquitin, to lysine residues (Ciechanover 1998). In addition, some proteins that have a natively unfolded structure may not need ubiquitination to be targeted for degradation at the proteasome (Tofaris et al. 2001). Proteasomal dysfunction has been linked increasingly to neurodegeneration, and in particular to Parkinson's disease (PD) and related Lewy body (LB) diseases (Chung et al. 2001;McNaught et al. 2001;Lang-Rollin et al. 2003). For example, expression of mutant forms of a-synuclein that lead to PD in certain families is associated with proteasomal inhibition (Tanaka et al. 2001;Stefanis et al. 2001;Petrucelli et al. 2002).The availability of selective pharmacological proteasomal inhibitors has led to a number of studies examining the effects of proteasomal inhibition on cultured neuronal cells (Sadoul et al. 1996;Lopes et al. 1997;Lin et al. 1998;Canu et al. 2000;Pasquini et al. 2000;Qiu et al. 2000;Rideout et al. 2001a;McNaught et al. 2002;Petrucelli et al. 2002;Rideout and Stefanis 2002

Proteasome inhibition induces selective motor neuron death in organotypic slice cultures

“…Aging, oxidative stress, and the existence of mutant proteins could induce proteasome dysfunction Urushitani et al 2002). Proteasome impairment induces mitochondria dysfunction (Sullivan et al 2004) and oxidative stress , and causes accumulation of proapoptotic signals (Lang-Rollin et al 2004). The results of the present study indicate that, among many cascades, proteasome dysfunction is a major factor for motor neurons to decide cellular fate.…”

Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations

“…Moreover, several pieces of evidence suggest that PrP may function as a trophic receptor that leads to activation of a neuroprotective state (54 -56). Interestingly, neuronal death induced by proteasome inhibitors is associated with increased generation of free radicals, and decreased glutathione levels, as well as with activation of Bax-mediated apoptosis (57,58), suggesting that the protective effect of PrP against the toxicity of the inhibitors might be caused by the capacity of the protein to act on one or more of these pathways. If this were the case, then our observation that PG14 and D177N PrP exert a neuroprotective effect comparable to that of wild-type PrP (Figs.…”

Cytoplasmic Inclusions of Htt Exon1 Containing an Expanded Polyglutamine Tract Suppress Execution of Apoptosis in Sympathetic Neurons

“…Cultured mouse SCG neurons die apoptotically during proteasome inhibition, showing Bax activation, cyt c release, and caspase activation (Lang-Rollin et al, 2004). However, under similar conditions, rat SCG neurons may survive NGF deprivation during acute withdrawal (Sadoul et al, 1996) but ultimately die nonapoptotically (with some apoptotic features) (Lang-Rollin et al, 2008).…”