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Alterations in microtubule-dependent transport, mitochondrial dysfunction, and autophagic pathology are involved in neurodegeneration observed in sporadic Parkinson's disease. However, the mechanistic link connecting these events remains elusive. We observed that NAD+ metabolism is altered in sporadic Parkinson's disease patient-derived cells, which contributes to Sirtuin-2 activation and subsequent decrease in acetylated-α-tubulin levels. Pharmacological inhibition of sirtuin-2 deacetylase activity selectively enhanced α-tubulin acetylation and facilitated the trafficking and clearance of misfolded proteins. Sirtuin-2 knock-out mice neurons had no alteration in microtubule assembly after exposure to MPP+, allowing the maintenance of a normal autophagic flux. These data were validated using MPTP-treated sirtuin-2 knock-out mice, where no alterations in motor behavior were observed. Biochemical analysis of sporadic Parkinson's disease patient brains supports the in vitro and in vivo data. Our data provide strong evidence that sirtuin-2 controls the functional ability of the autophagic system through acetylation and highlight the association between mitochondrial metabolism and neurodegeneration in sporadic Parkinson's disease.

Objective The aim of this work was to evaluate the role of ubiquitin–proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models. Method We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations. Results We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. Interpretation aSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC.

Cellular homeostasis relies on quality control systems so that damaged biologic structures are either repaired or degraded and entirely replaced by newly formed proteins or even organelles. The clearance of dysfunctional cellular structures in long-lived postmitotic cells, like neurons, is essential to eliminate, per example, defective mitochondria, lipofuscin-loaded lysosomes and oxidized proteins. Short-lived proteins are degraded mainly by proteases and proteasomes whether most long-lived proteins and all organelles are digested by autophagy in the lysosomes. Recently, it an interplay was established between the ubiquitin-proteasome system and macroautophagy, so that both degradative mechanisms compensate for each other. In this article we describe each of these clearance systems and their contribution to neuronal quality control. We will highlight some of the findings that provide evidence for the dysfunction of these systems in Alzheimer's and Parkinson's diseases. Ultimately, we provide an outline on potential therapeutic interventions based on the modulation of cellular degradative systems.

Mitochondrial dysfunction is observed in Alzheimer's disease (AD) brain and peripheral tissues. Amyloid-β (Aβ) peptides are known to interact with several proteins inside the mitochondria, leading to mitochondrial dysfunction. Recent studies have provided substantial evidence that mitochondria serve as direct targets for Aβ-mediated neuronal toxicity. The observations that Aβ progressively accumulates in cortical mitochondria from AD patients and transgenic AD type mouse models suggest the role of mitochondrial Aβ in the pathogenesis or development of AD. Herein, we studied the downstream signaling pathways induced by Aβ-mediated mitochondrial metabolism alterations and its consequences on cellular fate. We found that Aβ peptides induced an increase in NAD+levels and a decrease in ATP levels, which was related with decreases in acetylated tubulin levels and tau hyperphosphorylation. As a result of microtubule disruption, alterations in macroautophagy, like a decrease in autophagossome degradation and altered cellular distribution of LC3B, were found. Taxol, a microtubule stabilizer drug, was able to restore microtubule network and to prevent cell death induced by Aβ peptides. Our data shows for the first time that mitochondrial and cytosolic Aβ oligomers were significantly reduced upon microtubule dynamics re-establishment. These observations point out that an intervention at a microtubule level may be effective as a disease modifying therapy.

When first described by Alois Alzheimer in 1907, AD was seen as a disorder that causes dementia and characterized by two defining neuropathological lesions, later associated with all forms of AD. While the etiology of AD remains largely unclear, there is accumulating evidence suggesting that mitochondrial dysfunction occurs prior to the onset of symptoms in AD. Mitochondria are exceptionally poised to play a crucial role in neuronal cell survival or death because they are regulators of both energy metabolism and apoptotic pathways. This review is mainly focused in the discussion of evidence suggesting a clear association between mitochondrial dysfunction, autophagy impairment and amyloid-beta accumulation in Alzheimer's disease pathophysiology. The knowledge that autophagic insufficiency may compromise the cellular degradation mechanisms that may culminate in the progressive accumulation of dysfunctional mitochondria, aberrant protein aggregates buildup and lysossomal burden shield new insights to the way we address Alzheimer's disease. In line with this knowledge an innovative window for new therapeutic strategies aimed to activate or ameliorate macroautophagy may be opened.

Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function. However, mitochondrial-mediated-calcium homeostasis alterations may lead to its pathologic activation that jeopardizes neuronal structure and function. Here, we provide evidence to support a role for the involvement of calpain 1 in mitochondrial-induced neurodegeneration in a Parkinson's disease (PD) cellular model. We show that dysfunctional mitochondria increases cytosolic calcium, thereby, inducing calpain activation. Interestingly, its inhibition significantly attenuated the accumulation of alpha-synuclein oligomers and contributed to an increase of insoluble alpha-synuclein aggregates, known to be cytoprotective. Moreover, our data corroborate that calpain-1 overactivation in our mitochondrial-deficient cells promote caspase-3 activation. Overall, our findings further clarify the crucial role of dysfunctional mitochondria in the control of molecular mechanisms occurring in PD brain cells, providing a potentially novel correlation between the degradation of calpain substrates suggesting a putative role of calpain and calpain inhibition as a therapeutic tool in PD.

Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Many cellular mechanisms are thought to be involved in the death of these specific neurons in PD, including oxidative stress, changes of intracellular calcium homeostasis, and mitochondrial dysfunction. Since recent studies have revealed that also endoplasmic reticulum (ER) stress in conjunction with abnormal protein degradation can contribute to the PD pathophysiology, we investigated here the molecular mechanisms underlying the interplay between ER and mitochondria and its relevance in the control of neuronal cell death in PD. We observed that MPP+ induced changes in the mitochondrial function, affecting mitochondrial membrane potential and electron transport chain function. Likewise, it was also evident the unfolded protein response activation by an overexpression of GRP78 protein. Moreover, stress stimuli caused the release of Ca2+ from the ER that consistently induced mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Besides, Ca2+ release inhibition prevented MPP+ mediated mitochondria-dependent caspases activation. Our findings show that ER and mitochondria are in a close communication, establishing a dynamic ER-Ca2+-mitochondria interconnection that can play a prominent role in the neuronal cell death induction under particular stressful circumstances of PD pathology.

The cellular mechanisms that may underlie the death of dopaminergic neurons in Parkinson's disease are ubiquitin-proteasomal system (UPS) impairment, mitochondrial dysfunction, and oxidative stress. The goal of this work was to elucidate the correlation between mitochondrial dysfunction and UPS impairment, focusing on the role of oxidative stress. Our data revealed that mitochondria-DNA-depleted cells (ρ0) are compromised at the mitochondrial and UPS levels and also show an alteration of the oxidative status. In parental cells (ρ+), MPP + induced a clear inhibition of complex I activity, as well as an increase in ubiquitinylated protein levels, which was not observed in cells treated with lactacystin. Moreover, MPP + induced a decreased in the 20S chymotrypsin-like and peptidyl-glutamyl peptide hydrolytic-like proteolytic activities after 24 h of exposure. ROS production was increased in ρ+ cells treated with MPP + or lactacystin, at early treatment periods. MPP + induced an increase in carbonyl group formation in ρ+ cells. The results suggest that a mitochondrial alteration leads to an imbalance in the cellular oxidative status, inducing a proteasomal deregulation, which may exacerbate protein aggregation, and consequently degenerative events.

Neurons are exquisitely dependent on quality control systems to maintain a healthy intracellular environment. A permanent assessment of protein and organelle "quality" allows a coordinated action between repair and clearance of damage proteins and dysfunctional organelles. Impairments in the intracellular clearance mechanisms in long-lived postmitotic cells, like neurons, result in the progressive accumulation of damaged organelles and aggregates of aberrant proteins. Using cells bearing Parkinson disease (PD) patients' mitochondria, we demonstrated that aberrant accumulation of autophagosomes in PD, commonly interpreted as an abnormal induction of autophagy, is instead due to defective autophagic clearance. This defect is a consequence of alterations in the microtubule network driven by mitochondrial dysfunction that hinder mitochondria and autophagosome trafficking. We uncover mitochondria and microtubule-directed traffic as main players in the regulation of autophagy in PD.

While the etiology of Parkinson's disease remains largely elusive, there is accumulating evidence suggesting that mitochondrial dysfunction occurs prior to the onset of symptoms in Parkinson's disease. Mitochondria are remarkably primed to play a vital role in neuronal cell survival since they are key regulators of energy metabolism (as ATP producers), of intracellular calcium homeostasis, of NAD+/NADH ratio, and of endogenous reactive oxygen species production and programmed cell death. In this paper, we focus on mitochondrial dysfunction-mediated alpha-synuclein aggregation. We highlight some of the findings that provide proof of evidence for a mitochondrial metabolism control in Parkinson's disease, namely, mitochondrial regulation of microtubule-dependent cellular traffic and autophagic lysosomal pathway. The knowledge that microtubule alterations may lead to autophagic deficiency and may compromise the cellular degradation mechanisms that culminate in the progressive accumulation of aberrant protein aggregates shields new insights to the way we address Parkinson's disease. In line with this knowledge, an innovative window for new therapeutic strategies aimed to restore microtubule network may be unlocked.

Understanding the molecular basis of Parkinson's disease (PD) has proven to be a major challenge in the field of neurodegenerative diseases. Although several hypotheses have been proposed to explain the molecular mechanisms underlying the pathogenesis of PD, a growing body of evidence has highlighted the role of mitochondrial dysfunction and the disruption of the mechanisms of mitochondrial dynamics in PD and other parkinsonian disorders. In this paper, we comment on the recent advances in how changes in the mitochondrial function and mitochondrial dynamics (fusion/fission, transport, and clearance) contribute to neurodegeneration, specifically focusing on PD. We also evaluate the current controversies in those issues and discuss the role of fusion/fission dynamics in the mitochondrial lifecycle and maintenance. We propose that cellular demise and neurodegeneration in PD are due to the interplay between mitochondrial dysfunction, mitochondrial trafficking disruption, and impaired autophagic clearance.

Parkinson’s disease (PD) is associated with perturbed mitochondria function and alpha-synuclein fibrillization. We evaluated potential mechanistic links between mitochondrial dysfunction and alpha-synuclein aggregation. We studied a PD cytoplasmic hybrid (cybrid) cell line in which platelet mitochondria from a PD subject were transferred to NT2 neuronal cells previously depleted of endogenous mitochondrial DNA. Compared to a control cybrid cell line, the PD line showed reduced ATP levels, an increased free/polymerized tubulin ratio, and alpha-synuclein oligomer accumulation. Taxol (which stabilizes microtubules) normalized the PD tubulin ratio and reduced alpha-synuclein oligomerization. A nexus exists between mitochondrial function, cytoskeleton homeostasis, and alpha-synuclein oligomerization. In our model, mitochondrial dysfunction triggers an increased free tubulin, which destabilizes the microtubular network and promotes alpha-synuclein oligomerization.

Mitochondrial metabolism is a highly orchestrated phenomenon in which many enzyme systems cooperate in a variety of pathways to dictate cellular fate. As well as its vital role in cellular energy metabolism (ATP production), mitochondria are powerful organelles that regulate reactive oxygen species production, NAD+/NADH ratio and programmed cell death. In addition, mitochondrial abnormalities have been well recognized to contribute to degenerative diseases, like Parkinson's disease (PD). Particularly a deficiency in the mitochondrial respiratory chain complex I and cristae disruption have been consistently described in PD. Moreover, the products of PD-familial genes, including alpha-synuclein, Parkin, PINK1, DJ-1, LRRK2 and HTR2A, were shown to localize to the mitochondria under certain conditions. It seems that PD has a mitochondrial component so events that would modulate normal mitochondrial functions may compromise neuronal survival. However, it remains an open question whether alterations of these pathways lead to different aspects of PD or whether they converge at a point that is the common denominator of PD pathogenesis. In this review we will focus on mitochondrial metabolic control and its implications on sirtuins activation, microtubule dynamics and autophagic-lysosomal pathway. We will address mitochondrial metabolism modulation as a new promising therapeutic tool for PD.

Mitochondrial dysfunction, oxidative stress, and alpha-synuclein oligomerization occur in Parkinson disease (PD). We used an in vitro PD cybrid approach that models these three phenomena specifically to evaluate the impact of mitochondria-derived oxidative stress on alpha-synuclein oligomerization. Compared with control cybrid cell lines, reactive oxygen species (ROS) production and protein oxidative stress markers were elevated in PD cybrids. The antioxidants CoQ(10) and GSH attenuated changes in PD cybrid peroxide, protein carbonyl, and protein sulfhydryl levels. Elevated PD cybrid alpha-synuclein oligomer levels were also attenuated by CoQ(10) and GSH. In PD cybrids, alpha-synuclein oligomerization was activated via a complex I-mediated increase in the free tubulin/polymerized tubulin ratio. CoQ(10) but not GSH increased complex I activity, restored ATP to control levels, and normalized the PD cybrid free tubulin/polymerized tubulin ratio. Overall, we conclude that two different antioxidants can decrease alpha-synuclein oligomerization whether by improving mitochondrial function or by preventing protein carbonylation or both. We conclude that mitochondrial dysfunction can induce alpha-synuclein oligomerization via ATP depletion-driven microtubule depolymerization and via ROS increase-driven protein oxidation.

The neuroprotective action of a set of new hydroxypyridinone-based (3,4-HP) compounds (A, B and C), which are iron chelators extra-functionalized with a propargylamino group for potential MAO-B inhibition, was evaluated after cell treatment with MPP+ (an in vivo inducer of parkinsonism) and Abeta(1-40) and/or Abeta(1-42) peptides. Our results show that all these compounds improved cell viability in cells treated with MPP+ and Abeta(1-40) peptide or Abeta(1-42) peptide. In order to evaluate the cellular mechanisms underlying the activity of these compounds, we studied their protective role in caspase activation. All compounds tested were able to prevent MPP+ and Brefeldin A induced caspase-2 activation. They also showed quite effective in the inhibition of caspase-4 and caspase-3 activity, an effector caspase in the apoptotic process. Finally, detection of apoptotic-like cell death after cell exposure to MPP+ was also performed by TUNEL assay. Our results demonstrated that all tested compounds prevented DNA fragmentation by decreasing TUNEL positive cells. A, B and C were more effective than DFP (a 3,4-HP iron-chelating agent in clinical use) in MPP+ induced cell death. Therefore, these results evidenced a neuroprotective and antiapoptotic role for the compounds studied.