Miro proteins prime mitochondria for Parkin translocation and mitophagy.

The Parkinson's disease-associated protein kinase PINK1 and ubiquitin ligase Parkin coordinate the ubiquitination of mitochondrial proteins, which marks mitochondria for degradation. Miro1, an atypical GTPase involved in mitochondrial trafficking, is one of the substrates tagged by Parkin after mitochondrial damage. Here, we demonstrate that a small pool of Parkin interacts with Miro1 before mitochondrial damage occurs. This interaction does not require PINK1, does not involve ubiquitination of Miro1 and also does not disturb Miro1 function. However, following mitochondrial damage and PINK1 accumulation, this initial pool of Parkin becomes activated, leading to the ubiquitination and degradation of Miro1. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagic removal of mitochondria. Moreover, we demonstrate that Miro1 EF-hand domains control Miro1's ubiquitination and Parkin recruitment to damaged mitochondria, and they protect neurons from glutamate-induced mitophagy. Together, our results suggest that Miro1 functions as a calcium-sensitive docking site for Parkin on mitochondria.

Mitochondrial biogenesis is required for axonal growth.

During early development, neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Rapid growth requires a large quantity of building materials, efficient intracellular transport and also a considerable amount of energy. To produce this energy, the neuron should first generate new mitochondria because the pre-existing mitochondria are unlikely to provide a sufficient acceleration in ATP production. Here, we demonstrate that mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons. We also demonstrate that growth signals activating the CaMKKß, LKB1-STRAD or TAK1 pathways also co-activate the AMPK-PGC-1α-NRF1 axis leading to the generation of new mitochondria to ensure energy for upcoming growth. In conclusion, our results suggest that neurons are capable of signalling for upcoming energy requirements. Earlier activation of mitochondrial biogenesis through these pathways will accelerate the generation of new mitochondria, thereby ensuring energy-producing capability for when other factors for axonal growth are synthesized.

Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome.

Deficiency of the protein Wolfram syndrome 1 (WFS1) is associated with multiple neurological and psychiatric abnormalities similar to those observed in pathologies showing alterations in mitochondrial dynamics. The aim of this study was to examine the hypothesis that WFS1 deficiency affects neuronal function via mitochondrial abnormalities. We show that down-regulation of WFS1 in neurons leads to dramatic changes in mitochondrial dynamics (inhibited mitochondrial fusion, altered mitochondrial trafficking, and augmented mitophagy), delaying neuronal development. WFS1 deficiency induces endoplasmic reticulum (ER) stress, leading to inositol 1,4,5-trisphosphate receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn, alters mitochondrial dynamics. Importantly, ER stress, impaired Ca2+ homeostasis, altered mitochondrial dynamics, and delayed neuronal development are causatively related events because interventions at all these levels improved the downstream processes. Our data shed light on the mechanisms of neuronal abnormalities in Wolfram syndrome and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.

Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-ß Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/ß-Catenin Pathway.

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid ß (Aß) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aß toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aß-mediated suppression of neurogenic and Akt/Wnt/ß-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·ß-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·ß-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3ß. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·ß-catenin signaling.

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways.

The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell's compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.

Principles of the mitochondrial fusion and fission cycle in neurons.

Mitochondrial fusion-fission dynamics play a crucial role in many important cell processes. These dynamics control mitochondrial morphology, which in turn influences several important mitochondrial properties including mitochondrial bioenergetics and quality control, and they appear to be affected in several neurodegenerative diseases. However, an integrated and quantitative understanding of how fusion-fission dynamics control mitochondrial morphology has not yet been described. Here, we took advantage of modern visualisation techniques to provide a clear explanation of how fusion and fission correlate with mitochondrial length and motility in neurons. Our main findings demonstrate that: (1) the probability of a single mitochondrion splitting is determined by its length; (2) the probability of a single mitochondrion fusing is determined primarily by its motility; (3) the fusion and fission cycle is driven by changes in mitochondrial length and deviations from this cycle serves as a corrective mechanism to avoid extreme mitochondrial length; (4) impaired mitochondrial motility in neurons overexpressing 120Q Htt or Tau suppresses mitochondrial fusion and leads to mitochondrial shortening whereas stimulation of mitochondrial motility by overexpressing Miro-1 restores mitochondrial fusion rates and sizes. Taken together, our results provide a novel insight into the complex crosstalk between different processes involved in mitochondrial dynamics. This knowledge will increase understanding of the dynamic mitochondrial functions in cells and in particular, the pathogenesis of mitochondrial-related neurodegenerative diseases.

Endoplasmic reticulum stress and its role in various neurodegenerative diseases.

The Endoplasmic reticulum (ER), a critical cellular organelle, maintains cellular homeostasis by regulating calcium levels and orchestrating essential functions such as protein synthesis, folding, and lipid production. A pivotal aspect of ER function is its role in protein quality control. When misfolded proteins accumulate within the ER due to factors like protein folding chaperone dysfunction, toxicity, oxidative stress, or inflammation, it triggers the Unfolded protein response (UPR). The UPR involves the activation of chaperones like calnexin, calreticulin, glucose-regulating protein 78 (GRP78), and Glucose-regulating protein 94 (GRP94), along with oxidoreductases like protein disulphide isomerases (PDIs). Cells employ the Endoplasmic reticulum-associated degradation (ERAD) mechanism to counteract protein misfolding. ERAD disruption causes the detachment of GRP78 from transmembrane proteins, initiating a cascade involving Inositol-requiring kinase/endoribonuclease 1 (IRE1), Activating transcription factor 6 (ATF6), and Protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathways. The accumulation and deposition of misfolded proteins within the cell are hallmarks of numerous neurodegenerative diseases. These aberrant proteins disrupt normal neuronal signalling and contribute to impaired cellular homeostasis, including oxidative stress and compromised protein degradation pathways. In essence, ER stress is defined as the cellular response to the accumulation of misfolded proteins in the endoplasmic reticulum, encompassing a series of signalling pathways and molecular events that aim to restore cellular homeostasis. This comprehensive review explores ER stress and its profound implications for the pathogenesis and progression of neurodegenerative diseases.

Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy.

Parkinson disease is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. α-Synuclein accumulation in turn leads to compensatory effects that may include the up-regulation of autophagy. Another common feature of Parkinson disease (PD) is mitochondrial dysfunction. Here, we provide evidence that the overactivation of autophagy may be a link that connects the intracellular accumulation of α-synuclein with mitochondrial dysfunction. We found that the activation of macroautophagy in primary cortical neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial destruction and loss, which is associated with a bioenergetic deficit and neuronal degeneration. No mitochondrial removal or net loss was observed when we suppressed the targeting of mitochondria to autophagosomes by silencing Parkin, overexpressing wild-type Mitofusin 2 and dominant negative Dynamin-related protein 1 or blocking autophagy by silencing autophagy-related genes. The inhibition of targeting mitochondria to autophagosomes or autophagy was also partially protective against mutant A53T α-synuclein-induced neuronal cell death. These data suggest that overactivated mitochondrial removal could be one of the contributing factors that leads to the mitochondrial loss observed in PD models.

Negative feedback system to maintain cell ROS homeostasis: KEAP1-PGAM5 complex senses mitochondrially generated ROS to induce mitophagy.

If cellular reactive oxygen species (ROS) production surpasses the intracellular antioxidant capacity, thus altering the ROS homeostasis, the cell needs to eradicate faulty mitochondria responsible for these excessive ROS. We have shown that even moderate ROS production breaks the KEAP1-PGAM5 complex, inhibiting the proteasomal removal of PGAM5. This leads to an accumulation of PGAM5 interfering with PINK1 processing that sensitizes mitochondria to autophagic removal. We propose that such a negative feedback system maintains cell ROS homeostasis.

Molecular Mechanisms and Regulation of Mammalian Mitophagy.

Mitochondria in the cell are the center for energy production, essential biomolecule synthesis, and cell fate determination. Moreover, the mitochondrial functional versatility enables cells to adapt to the changes in cellular environment and various stresses. In the process of discharging its cellular duties, mitochondria face multiple types of challenges, such as oxidative stress, protein-related challenges (import, folding, and degradation) and mitochondrial DNA damage. They mitigate all these challenges with robust quality control mechanisms which include antioxidant defenses, proteostasis systems (chaperones and proteases) and mitochondrial biogenesis. Failure of these quality control mechanisms leaves mitochondria as terminally damaged, which then have to be promptly cleared from the cells before they become a threat to cell survival. Such damaged mitochondria are degraded by a selective form of autophagy called mitophagy. Rigorous research in the field has identified multiple types of mitophagy processes based on targeting signals on damaged or superfluous mitochondria. In this review, we provide an in-depth overview of mammalian mitophagy and its importance in human health and diseases. We also attempted to highlight the future area of investigation in the field of mitophagy.

A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy.

When ROS production exceeds the cellular antioxidant capacity, the cell needs to eliminate the defective mitochondria responsible for excessive ROS production. It has been proposed that the removal of these defective mitochondria involves mitophagy, but the mechanism of this regulation remains unclear. Here, we demonstrate that moderate mitochondrial superoxide and hydrogen peroxide production oxidates KEAP1, thus breaking the interaction between this protein and PGAM5, leading to the inhibition of its proteasomal degradation. Accumulated PGAM5 interferes with the processing of the PINK1 in the mitochondria leading to the accumulation of PINK1 on the outer mitochondrial membrane. In turn, PINK1 promotes Parkin recruitment to mitochondria and sensitizes mitochondria for autophagic removal. We also demonstrate that inhibitors of the KEAP1-PGAM5 protein-protein interaction (including CPUY192018) mimic the effect of mitochondrial ROS and sensitize mitophagy machinery, suggesting that these inhibitors could be used as pharmacological regulators of mitophagy. Together, our results show that KEAP1/PGAM5 complex senses mitochondrially generated superoxide/hydrogen peroxide to induce mitophagy.

Mitochondrial transport proteins RHOT1 and RHOT2 serve as docking sites for PRKN-mediated mitophagy.

The Parkinson disease-associated proteins PINK1 and PRKN coordinate the ubiquitination of mitochondrial outer membrane proteins to tag them either for degradation or for autophagic clearance of the mitochondrion. The proteins include the mitochondrial trafficking proteins RHOT1 and RHOT2, the removal of which may be required for immobilization of mitochondria prior to mitophagy. Here, we demonstrate that RHOT1 and RHOT2 are not only substrates for PINK1-PRKN-dependent degradation but that they also play an active role in the process of mitophagy. RHOT1, and likely also RHOT2, may act as a docking site for inactive PRKN prior to mitochondrial damage, thus keeping PRKN in close proximity to its potential substrates and thereby facilitating mitophagy. We also show that RHOT1 functions as a calcium-sensing docking site for PRKN, and we suggest that calcium binding to RHOT is a key step in the calcium-dependent activation of mitophagy machinery.

Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species.

Free heme is very toxic because it generates highly reactive hydroxyl radicals ((.)OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H(2)O(2) and (.)OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. (.)OH scavengers such as mannitol, as well as the spin trap alpha-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.

Molecular characterization and localization of Plasmodium falciparum choline kinase.

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.

Apoptosis in liver during malaria: role of oxidative stress and implication of mitochondrial pathway.

Hepatic dysfunction is a common clinical complication in malaria, although its pathogenesis remains largely unknown. Using a variety of in vivo and ex vivo approaches, we have shown for the first time that malarial infection induces hepatic apoptosis through augmentation of oxidative stress. Apoptosis in hepatocyte has been confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin-nick-end labeling assay (TUNEL) and caspase-3 activation. Gene expression analysis using RT-PCR indicates the significant down-regulation of Bcl-2 and up-regulation of Bax expression in liver of malaria infected mice suggesting the involvement of mitochondrial pathway of apoptosis. The levels of Fas expression and caspase-8 activity in infected liver were same as that of uninfected control mice indicating death receptor (Fas) pathway did not contribute to liver apoptosis during malarial infection. Moreover, evidence has been presented by confocal microscopy to show the translocation of Bax from cytosol to mitochondria in apoptotic hepatocyte, resulting in opening of permeability transition pores, which in turn decreases mitochondrial membrane potential and induces cytochrome c release into cytosol. Malarial infection induces the generation of hydroxyl radical (*OH) in liver, which may be responsible for the induction of oxidative stress and apoptosis as administration of *OH specific antioxidant as well as spin trap, alpha-phenyl-tert-butyl-nitrone in malaria-infected mice significantly inhibits the development of oxidative stress as well as induction of apoptosis. Thus, results suggest the implication of oxidative stress induced-mitochondrial pathway of apoptosis in the pathophysiology of hepatic dysfunction in malaria.

Antimalarial drugs inhibiting hemozoin (beta-hematin) formation: a mechanistic update.

Digestion of hemoglobin in the food vacuole of the malaria parasite produces very high quantities of redox active toxic free heme. Hemozoin (beta-hematin) formation is a unique process adopted by Plasmodium sp. to detoxify free heme. Hemozoin formation is a validated target for most of the well-known existing antimalarial drugs and considered to be a suitable target to develop new antimalarials. Here we discuss the possible mechanisms of free heme detoxification in the malaria parasite and the mechanistic details of compounds, which offer antimalarial activity by inhibiting hemozoin formation. The chemical nature of new antimalarial compounds showing antimalarial activity through the inhibition of hemozoin formation has also been incorporated, which may help to design future antimalarials with therapeutic potential against multi-drug resistant malaria.

BECN1 is involved in the initiation of mitophagy: it facilitates PARK2 translocation to mitochondria.

The autophagy protein BECN1/Beclin 1 is known to play a central role in autophagosome formation and maturation. The results presented here demonstrate that BECN1 interacts with the Parkinson disease-related protein PARK2. This interaction does not require PARK2 translocation to mitochondria and occurs mostly in cytosol. However, our results suggest that BECN1 is involved in PARK2 translocation to mitochondria because loss of BECN1 inhibits CCCP- or PINK1 overexpression-induced PARK2 translocation. Our results also demonstrate that the observed PARK2-BECN1 interaction is functionally important. Measurements of the level of MFN2 (mitofusin 2), a PARK2 substrate, demonstrate that depletion of BECN1 prevents PARK2 translocation-induced MFN2 ubiquitination and loss. BECN1 depletion also rescues the MFN2 loss-induced suppression of mitochondrial fusion. In sum, our results demonstrate that BECN1 interacts with PARK2 and regulates PARK2 translocation to mitochondria as well as PARK2-induced mitophagy prior to autophagosome formation.

PGC-1{alpha} and PGC-1{beta} regulate mitochondrial density in neurons.

Recent studies indicate that regulation of cellular oxidative capacity through enhancing mitochondrial biogenesis may be beneficial for neuronal recovery and survival in human neurodegenerative disorders. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to be a master regulator of mitochondrial biogenesis and cellular energy metabolism in muscle and liver. The aim of our study was to establish whether PGC-1alpha and PGC-1beta control mitochondrial density also in neurons and if these coactivators could be up-regulated by deacetylation. The results demonstrate that PGC-1alpha and PGC-1beta control mitochondrial capacity in an additive and independent manner. This effect was observed in all studied subtypes of neurons, in cortical, midbrain, and cerebellar granule neurons. We also observed that endogenous neuronal PGC-1alpha but not PGC-1beta could be activated through its repressor domain by suppressing it. Results demonstrate also that overexpression of SIRT1 deacetylase or suppression of GCN5 acetyltransferase activates transcriptional activity of PGC-1alpha in neurons and increases mitochondrial density. These effects were mediated exclusively via PGC-1alpha, since overexpression of SIRT1 or suppression of GCN5 was ineffective where PGC-1alpha was suppressed by short hairpin RNA. Moreover, the results demonstrate that overexpression of PGC-1beta or PGC-1alpha or activation of the latter by SIRT1 protected neurons from mutant alpha-synuclein- or mutant huntingtin-induced mitochondrial loss. These evidences demonstrate that activation or overexpression of the PGC-1 family of coactivators could be used to compensate for neuronal mitochondrial loss and suggest that therapeutic agents activating PGC-1 would be valuable for treating neurodegenerative diseases in which mitochondrial dysfunction and oxidative damage play an important pathogenic role.