Measurement of proteasomal dysfunction in cell models of dopaminergic degeneration.

Parkinson's disease (PD) is the second most common neurodegenerative diseases, which occurs in both inheritable and sporadic forms. The interplay of the genetic mutations and environmental exposure to disease risk factors contributes to the pathogenic events leading to the demise of dopaminergic neurons in PD. Proteasome is one of the major proteolytic machinery responsible for degrading unwanted and damaged intracellular proteins. Emerging evidence implicates the incomplete proteolysis by ubiquitin-proteasome system (UPS) in PD pathogenesis. Proteasome inhibition recapitulates some of the key features of PD in vivo and in vitro. Varieties of dopaminergic neurotoxins emerge to inhibit proteasomal function. Given that some PD-related gene mutations impair proteolytic function of UPS, it has been well-accepted that both genetic and environmental factors may conspire to compromise the UPS in the initiation and progression of the disease. The enzymatic assays for the proteasomal activities with fluorogenic substrates and western blot analysis of ubiquitinated proteins provide an entry point to determine UPS function in the process of dopaminergic degeneration.

Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility.

Neuronal development, function and repair critically depend on axonal transport of vesicles and protein complexes, which is mediated in part by the molecular motor kinesin-1. Adaptor proteins recruit kinesin-1 to vesicles via direct association with kinesin heavy chain (KHC), the force-generating component, or via the accessory light chain (KLC). Binding of adaptors to the motor is believed to engage the motor for microtubule-based transport. We report that the adaptor protein Sunday Driver (syd, also known as JIP3 or JSAP1) interacts directly with KHC, in addition to and independently of its known interaction with KLC. Using an in vitro motility assay, we show that syd activates KHC for transport and enhances its motility, increasing both KHC velocity and run length. syd binding to KHC is functional in neurons, as syd mutants that bind KHC but not KLC are transported to axons and dendrites similarly to wild-type syd. This transport does not rely on syd oligomerization with itself or other JIP family members. These results establish syd as a positive regulator of kinesin activity and motility.

Neuroproteomics approaches to decipher neuronal regeneration and degeneration.

Given the complexity of brain and nerve tissues, systematic approaches are essential to understand normal physiological conditions and functional alterations in neurological diseases. Mass spectrometry-based proteomics is increasingly used in neurosciences to determine both basic and clinical differential protein expression, protein-protein interactions, and post-translational modifications. Proteomics approaches are especially useful to understand the mechanisms of nerve regeneration and degeneration because changes in axons following injury or in disease states often occur without the contribution of transcriptional events in the cell body. Indeed, the current understanding of axonal function in health and disease emphasizes the role of proteolysis, local axonal protein synthesis, and a broad range of post-translational modifications. Deciphering how axons regenerate and degenerate has thus become a postgenomics problem, which depends in part on proteomics approaches. This review focuses on recent proteomics approaches designed to uncover the mechanisms and molecules involved in neuronal regeneration and degeneration. It emerges that the principal degenerative mechanisms converge to oxidative stress, dysfunctions of axonal transport, mitochondria, chaperones, and the ubiquitin-proteasome systems. The mechanisms regulating nerve regeneration also impinge on axonal transport, cytoskeleton, and chaperones in addition to changes in signaling pathways. We also discuss the major challenges to proteomics work in the nervous system given the complex organization of the brain and nerve tissue at the anatomical, cellular, and subcellular levels.

Mitochondrial accumulation of polyubiquitinated proteins and differential regulation of apoptosis by polyubiquitination sites Lys-48 and -63.

Proteins tagged with lysine (Lys, K) 48 polyubiquitins chains are destined for degradation by the 26S proteasomal system. Impairment of the ubiquitin proteasome system (UPS) function culminates in the accumulation of polyubiquitinated proteins in many neurodegenerative conditions including Parkinson's disease (PD). Nevertheless, the cellular mechanisms underlying cell death induced by an impaired UPS are still not clear. Intriguingly, recent studies indicate that several proteins associated with familial PD are capable of promoting the assembly of Lys-63 polyubiquitin chains. Therefore, the objective of this study was to examine the role of K48 and K63 ubiquitination in mitochondria-mediated apoptosis in in vitro models of dopaminergic degeneration. Exposure of the widely used proteasome inhibitor MG-132 to dopaminergic neuronal cell line (N27) induced a rapid accumulation of polyubiquitinated proteins in the mitochondria. This appears to result in the preferential association of ubiquitin conjugates in the outer membrane and polyubiquitination of outer membrane proteins. Interestingly, the ubiquitin(K48R) mutant effectively rescued cells from MG-132-induced mitochondrial apoptosis without altering the antioxidant status of cells; whereas the ubiquitin(K63R) mutant augmented the proapoptotic effect of MG-132. Herein, we report a novel conclusion that polyubiquitinated proteins, otherwise subjected to proteasomal degradation, preferentially accumulate in the mitochondria during proteolytic stress; and that polyubiquitination of Lys-48 and Lys-63 are key determinants of mitochondria-mediated cell death during proteasomal dysfunction. Together, these findings yield novel insights into a crosstalk between the UPS and mitochondria in dopaminergic neuronal cells.

Apoptosis induced by baicalin involving up-regulation of P53 and bax in MCF-7 cells.

Anticancer effect of baicalin (1) has been well documented. However, the molecular mechanisms underlying the cytotoxicity of baicalin in cancer cells remain unclear. In the present study, we examined the potential roles of p53, bax, and bcl-2 in baicalin-triggered apoptosis in MCF-7 cells, a cell line derived from human breast cancer. The results showed that cell proliferation was significantly inhibited by baicalin in a dose- and time-dependent manner. Flow cytometric analysis also revealed that most of the baicalin-treated MCF-7 cells were arrested in the G(0)/G(1) phase. Significant amount of cells underwent apoptotic cell death 24 h following baicalin treatment. Typical apoptotic characteristics such as chromatin condensation and the formation of apoptotic bodies were noted 48 h following baicalin exposure. Semi-quantitative analysis using RT-PCR revealed dramatic elevation of mRNA levels of proapoptotic molecules p53 and bax, but not the anti-apoptotic bcl-2. Consistently, significant elevation of p53 and bax was substantiated by the western blot. Collectively, the data demonstrated that baicalin-induced apoptotic cell death in the breast cancer cells involves the up-regulation of proapoptotic p53 and bax, implying potential crucial roles of bax and p53 in the baicalin-induced apoptosis.

Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

Environmental neurotoxic chemicals-induced ubiquitin proteasome system dysfunction in the pathogenesis and progression of Parkinson's disease.

Proteolytic degradation of unwanted proteins by the ubiquitin-proteasome system (UPS) is critical for normal maintenance of various cellular functions. Parkinson's disease (PD), one of the most prevalent neurodegenerative disorders, is characterized by prominent and irreversible nigral dopaminergic neuronal loss and intracellular protein aggregations. Epidemiological studies imply both environmental neurotoxins and genetic predisposition as potential risk factors for PD, though mechanisms underlying selective dopaminergic degeneration remain unclear. Studies with experimental PD models and postmortem PD brains have provided explicit evidence for mitochondria dysfunction and oxidative stress in PD pathogenesis. Recent identification of mutants in PINK1, DJ-1, Parkin, and LRRK-2 genes compliments the oxidative stress and mitochondrial dysfunction hypotheses in dopaminergic neuronal degeneration in PD. Mutants of alpha-synuclein, Uch-L1 and Parkin support the involvement of UPS dysfunction in PD. Furthermore, various Parkinsonian toxicants have been shown to impair mitochondrial function, redox balances, and to some extent protein degradation machinery. Because environmental exposure to various neurotoxic agents is considered a dominant risk for development of PD, the interrelationship between neurotoxicant exposures and UPS dysfunction must be clearly understood. Elucidation of this interrelationship will help clarify 2 areas: (i) whether UPS dysfunction in PD is a primary pathogenic factor leading to nigral neuronal death or if it simply occurs as a consequence of oxidative stress and mitochondrial dysfunction and (ii) the interaction of genes and environment in the acceleration of nigral dopaminergic degeneration by targeting UPS. We review the recent evidence for UPS deficits in dopaminergic degeneration triggered by neurotoxins.

Proteasome inhibitor MG-132 induces dopaminergic degeneration in cell culture and animal models.

Impairment in ubiquitin-proteasome system (UPS) has recently been implicated in Parkinson's disease, as demonstrated by reduced proteasomal activities, protein aggregation and mutation of several genes associated with UPS. However, experimental studies with proteasome inhibitors failed to yield consensus regarding the effect of proteasome inhibition on dopaminergic degeneration. In this study, we systematically examined the effect of the proteasome inhibitor MG-132 on dopaminergic degeneration in cell culture and animal models of Parkinson's disease. Exposure of immortalized dopaminergic neuronal cells (N27) to low doses of MG-132 (2-10 microM) resulted in dose- and time-dependent cytotoxicity. Further, exposure to MG-132 (5 microM) for 10 min led to dramatic reduction of proteasomal activity (>70%) accompanied by a rapid accumulation of ubiquitinated proteins in these cells. MG-132 treatment also induced increases in caspase-3 activity in a time-dependent manner, with significant activation occurring between 90 and 150 min. We also noted a 12-fold increase in DNA fragmentation in MG-132 treated N27 cells. Similarly, primary mesencephalic neurons exposed to 5 microM MG-132 also induced >60% loss of TH positive neurons but only a minimal loss of non-dopaminergic cells. Stereotaxic injection of MG-132 (0.4 microg in 4 microl) into the substantia nigra compacta (SNc) in C57 black mice resulted in significant depletion of ipisilateral striatal dopamine and DOPAC content as compared to the vehicle-injected contralateral control sides. Also, we observed a significant decrease in the number of TH positive neurons in the substantia nigra of MG-132-injected compared to the vehicle-injected sites. Collectively, these results demonstrate that the proteasomal inhibitor MG-132 induces dopamine depletion and nigral dopaminergic degeneration in both cell culture and animal models, and suggest that proteasomal dysfunction may promote nigral dopaminergic degeneration in Parkinson's disease.

Dieldrin induces ubiquitin-proteasome dysfunction in alpha-synuclein overexpressing dopaminergic neuronal cells and enhances susceptibility to apoptotic cell death.

Exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). The organochlorine pesticide dieldrin is one of the environmental chemicals potentially linked to PD. Because recent evidence indicates that abnormal accumulation and aggregation of alpha-synuclein and ubiquitin-proteasome system dysfunction can contribute to the degenerative processes of PD, in the present study we examined whether the environmental pesticide dieldrin impairs proteasomal function and subsequently promotes apoptotic cell death in rat mesencephalic dopaminergic neuronal cells overexpressing human alpha-synuclein. Overexpression of wild-type alpha-synuclein significantly reduced the proteasomal activity. Dieldrin exposure dose-dependently (0-70 microM) decreased proteasomal activity, and 30 microM dieldrin inhibited activity by more than 60% in alpha-synuclein cells. Confocal microscopic analysis of dieldrin-treated alpha-synuclein cells revealed that alpha-synuclein-positive protein aggregates colocalized with ubiquitin protein. Further characterization of the aggregates with the autophagosomal marker mondansyl cadaverine and the lysosomal marker and dot-blot analysis revealed that these protein oligomeric aggregates were distinct from autophagosomes and lysosomes. The dieldrin-induced proteasomal dysfunction in alpha-synuclein cells was also confirmed by significant accumulation of ubiquitin protein conjugates in the detergent-insoluble fraction. We found that proteasomal inhibition preceded cell death after dieldrin treatment and that alpha-synuclein cells were more sensitive than vector cells to the toxicity. Furthermore, measurement of caspase-3 and DNA fragmentation confirmed the enhanced sensitivity of alpha-synuclein cells to dieldrin-induced apoptosis. Together, our results suggest that increased expression of alpha-synuclein predisposes dopaminergic cells to proteasomal dysfunction, which can be further exacerbated by environmental exposure to certain neurotoxic compounds, such as dieldrin.