Effect of Neem Oil on Sperm Mitochondrial Activity

It is a known fact that neem oil has some effect on motility of sperm. Motility of sperm depends on mitochondrial activity present in mid-piece of sperm. In the present study, the mitochondrial activity of sperm was evaluated after treating semen with the different quantities of neem oil. The mitochondrial activity was also evaluated after subjecting the semen samples for different incubation periods keeping the quantity of semen as well as that of neem oil same. Tests were done on thirty normozoospermic semen samples with motile score more than 75%. It was found that as the quantity of neem oil increases, the mitochondrial activity decreases significantly (P < 0.001). Similar results were found, when same quantity of neem oil was treated with same quantity of semen, but incubating for different time durations. The mitochondrial activity decreases significantly (P < 0.001) from one minute to twenty minutes. So, it indicates that as the contact period between neem oil and semen increases the mitochondrial activity decreases significantl

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

Mitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1) function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX), leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA) pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D) prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target

LETM1-Mediated K+ and Na+ Homeostasis Regulates Mitochondrial Ca2+ Efflux

HIGHLIGHTS \u2022 Monovalent cation homeostasis is dysregulated upon LETM1 depletion \u2022 K+/H+ exchange activity is decreased in LETM1 knockdown cells \u2022 LETM1 depletion results in K+ accumulation in the mitochondrial matrix \u2022 LETM1 knockdown does not affect expression of major mitochondrial Ca2+ transport modulators \u2022 LETM1-regulated mitochondrial Ca2+ fluxes are dependent on Na+ Ca2+ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. It is therefore tightly regulated. It has been recently proposed that LETM1\u2014an IMM protein with a crucial role in mitochondrial K+/H+ exchange and volume homeostasis\u2014also acts as a Ca2+/H+ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K+/H+ and Na+/H+ exchange. Decreased exchange activity resulted in matrix K+ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na+/Ca2+ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca2+ Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca2+ fluxes may reflect those on Na+/H+ exchange activity

The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson\u27s disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check

Changes in neuronal CycD/Cdk4 activity affect aging, neurodegeneration, and oxidative stress.

Mitochondrial dysfunction has been implicated in human diseases, including cancer, and proposed to accelerate aging. The Drosophila Cyclin-dependent protein kinase complex cyclin D/cyclin-dependent kinase 4 (CycD/Cdk4) promotes cellular growth by stimulating mitochondrial biogenesis. Here, we examine the neurodegenerative and aging consequences of altering CycD/Cdk4 function in Drosophila. We show that pan-neuronal loss or gain of CycD/Cdk4 increases mitochondrial superoxide, oxidative stress markers, and neurodegeneration and decreases lifespan. We find that RNAi-mediated depletion of the mitochondrial transcription factor, Tfam, can abrogate CycD/Cdk4's detrimental effects on both lifespan and neurodegeneration. This indicates that CycD/Cdk4's pathological consequences are mediated through altered mitochondrial function and a concomitant increase in reactive oxygen species. In support of this, we demonstrate that CycD/Cdk4 activity levels in the brain affect the expression of a set of 'oxidative stress' genes. Our results indicate that the precise regulation of neuronal CycD/Cdk4 activity is important to limit mitochondrial reactive oxygen species production and prevent neurodegeneration

OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L

OPA1, a dynamin-related guanosine triphosphatase mutated in dominant optic atrophy, is required for the fusion of mitochondria. Proteolytic cleavage by the mitochondrial processing peptidase generates long isoforms from eight messenger RNA (mRNA) splice forms, whereas further cleavages at protease sites S1 and S2 generate short forms. Using OPA1-null cells, we developed a cellular system to study how individual OPA1 splice forms function in mitochondrial fusion. Only mRNA splice forms that generate a long isoform in addition to one or more short isoforms support substantial mitochondrial fusion activity. On their own, long and short OPA1 isoforms have little activity, but, when coexpressed, they functionally complement each other. Loss of mitochondrial membrane potential destabilizes the long isoforms and enhances the cleavage of OPA1 at S1 but not S2. Cleavage at S2 is regulated by the i-AAA protease Yme1L. Our results suggest that mammalian cells have multiple pathways to control mitochondrial fusion through regulation of the spectrum of OPA1 isoforms

The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase.

The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH2-terminal amphipathic alpha-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100. The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria

Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons.

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer's disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons

Cardiac-specific Conditional Knockout of the 18-kDa Mitochondrial Translocator Protein Protects from Pressure Overload Induced Heart Failure.

Heart failure (HF) is characterized by abnormal mitochondrial calcium (Ca2+) handling, energy failure and impaired mitophagy resulting in contractile dysfunction and myocyte death. We have previously shown that the 18-kDa mitochondrial translocator protein of the outer mitochondrial membrane (TSPO) can modulate mitochondrial Ca2+ uptake. Experiments were designed to test the role of the TSPO in a murine pressure-overload model of HF induced by transverse aortic constriction (TAC). Conditional, cardiac-specific TSPO knockout (KO) mice were generated using the Cre-loxP system. TSPO-KO and wild-type (WT) mice underwent TAC for 8 weeks. TAC-induced HF significantly increased TSPO expression in WT mice, associated with a marked reduction in systolic function, mitochondrial Ca2+ uptake, complex I activity and energetics. In contrast, TSPO-KO mice undergoing TAC had preserved ejection fraction, and exhibited fewer clinical signs of HF and fibrosis. Mitochondrial Ca2+ uptake and energetics were restored in TSPO KO mice, associated with decreased ROS, improved complex I activity and preserved mitophagy. Thus, HF increases TSPO expression, while preventing this increase limits the progression of HF, preserves ATP production and decreases oxidative stress, thereby preventing metabolic failure. These findings suggest that pharmacological interventions directed at TSPO may provide novel therapeutics to prevent or treat HF