Targeting DNM1L/DRP1-FIS1 axis inhibits high-grade glioma progression by impeding mitochondrial respiratory cristae remodeling.

BACKGROUND: The dynamics of mitochondrial respiratory cristae (MRC) and its impact on oxidative phosphorylation (OXPHOS) play a crucial role in driving the progression of high-grade glioma (HGG). However, the underlying mechanism remains unclear. METHODS: In the present study, we employed machine learning-based transmission electron microscopy analysis of 7141 mitochondria from 54 resected glioma patients. Additionally, we conducted bioinformatics analysis and multiplex immunohistochemical (mIHC) staining of clinical glioma microarrays to identify key molecules involved in glioma. Subsequently, we modulated the expression levels of mitochondrial dynamic-1-like protein (DNM1L/DRP1), and its two receptors, mitochondrial fission protein 1 (FIS1) and mitochondrial fission factor (MFF), via lentiviral transfection to further investigate the central role of these molecules in the dynamics of glioblastoma (GBM) cells and glioma stem cells (GSCs). We then evaluated the potential impact of DNM1L/DRP1, FIS1, and MFF on the proliferation and progression of GBM cells and GSCs using a combination of CCK-8 assay, Transwell assay, Wound Healing assay, tumor spheroid formation assay and cell derived xenograft assay employing NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt (NCG) mouse model. Subsequently, we validated the ability of the DNM1L/DRP1-FIS1 axis to remodel MRC structure through mitophagy by utilizing Seahorse XF analysis technology, mitochondrial function detection, MRC abundance detection and monitoring dynamic changes in mitophagy. RESULTS: Our findings revealed that compared to low-grade glioma (LGG), HGG exhibited more integrated MRC structures. Further research revealed that DNM1L/DRP1, FIS1, and MFF played pivotal roles in governing mitochondrial fission and remodeling MRC in HGG. The subsequent validation demonstrated that DNM1L/DRP1 exerts a positive regulatory effect on FIS1, whereas the interaction between MFF and FIS1 demonstrates a competitive inhibition relationship. The down-regulation of the DNM1L/DRP1-FIS1 axis significantly impaired mitophagy, thereby hindering the remodeling of MRC and inhibiting OXPHOS function in glioma, ultimately leading to the inhibition of its aggressive progression. In contrast, MFF exerts a contrasting effect on MRC integrity, OXPHOS activity, and glioma progression. CONCLUSIONS: This study highlights that the DNM1L/DRP1-FIS1 axis stabilizes MRC structures through mitophagy in HGG cells while driving their OXPHOS activity ultimately leading to robust disease progression. The inhibition of the DNM1L/DRP1-FIS1 axis hinders MRC remodeling and suppresses GBM progression. We propose that down-regulation of the DNM1L/DRP1-FIS1 axis could be a potential therapeutic strategy for treating HGG.

NIPSNAP3A regulates cellular homeostasis by modulating mitochondrial dynamics.

Mitochondria are essential for cell metabolism and survival as they produce the majority of cellular ATP through oxidative phosphorylation as well as regulate critical processes such as cell proliferation and apoptosis. NIPSNAP family of proteins are predominantly mitochondrial matrix proteins. However, the molecular and cellular functions of the NIPSNAPs, particularly NIPSNAP3A, have remained elusive. Here, we demonstrated that NIPSNAP3A knockdown in HeLa cells inhibited their proliferation and migration and attenuated apoptosis induced by Actinomycin D (Act-D). These findings suggested a complex relationship between cellular processes and mitochondrial functions, mediated by NIPSNAP3A. Further investigations revealed that NIPSNAP3A knockdown not only inhibited mitochondrial fission through reduction of DRP1-S616, but also suppressed cytochrome c release in apoptosis. Collectively, our findings highlight the critical role of NIPSNAP3A in coordinating cellular processes, likely through its influence on mitochondrial dynamics.

Quality control of mitochondria involves lysosomes in multiple definitive ways.

Mitochondria are crucial organelles in maintaining cellular homeostasis. They are involved in processes such as energy production, metabolism of lipids and glucose, and cell death regulation. Mitochondrial dysfunction can lead to various health issues such as aging, cancer, neurodegenerative diseases, and chronic liver diseases. While mitophagy is the main process for getting rid of excess or damaged mitochondria, there are additional mechanisms for preserving mitochondrial quality. One such alternative mechanism we have discovered is a hybrid organelle called mitochondrial-lysosome-related-organelle (MLRO), which functions independently of the typical autophagy process. More recently, another type of vesicle called vesicle derived from the inner mitochondrial membrane (VDIM) has been identified to break down the inner mitochondrial membrane without involving the standard autophagy pathway. In this article, we will delve into the similarities and differences between MLRO and VDIM, including their structure, regulation, and relevance to human diseases.

SENP1 prevents high fat diet-induced non-alcoholic fatty liver diseases by regulating mitochondrial dynamics.

Mitochondrial dynamics plays a crucial role in the occurrence and development of non-alcoholic fatty liver diseases (NAFLD). SENP1, a SUMO-specific protease, catalyzes protein de-SUMOylation and involves in various physiological and pathological processes. However, the exact role of SENP1 in NAFLD remains unclear. Therefore, we investigated the regulatory role of SENP1 in mitochondrial dynamics during the progression of NAFLD. In the study, the NAFLD in vivo model induced by high fat diet (HFD) and in vitro model induced by free fatty acids (FFA) were established to investigate the role and underlying mechanism of SENP1 through detecting mitochondrial morphology and dynamics. Our results showed that the down-regulation of SENP1 expression and the mitochondrial dynamics dysregulation occurred in the NAFLD, evidenced as mitochondrial fragmentation, up-regulation of p-Drp1 ser616 and down-regulation of MFN2, OPA1. However, over-expression of SENP1 significantly alleviated the NAFLD, rectified the mitochondrial dynamics disorder, reduced Cyt-c release and ROS levels induced by FFA or HFD; moreover, the over-expression of SENP1 also reduced the SUMOylation levels of Drp1 and prevented the Drp1 translocation to mitochondria. Our findings suggest that the possible mechanisms of SENP1 were through rectifying the mitochondrial dynamics disorder, reducing Cyt-c release and ROS-mediated oxidative stress. The findings would provide a novel target for the prevention and treatment of NALFD.

GPRASP2 deficiency contributes to apoptosis in the spiral ganglion cells via the AMPK/DRP1 signaling pathway.

G protein-coupled receptor-associated sorting protein 2 (GPRASP2) deficiency has been implicated in immunological inflammation, cancers, and neurological disorders. Our previous work revealed that the pathogenic mutation in GPRASP2 was responsible for X-linked recessive syndromic hearing loss (SHL). Given the specific high expression of GPRASP2 in the spiral ganglion, GPRASP2 likely contributes to the maintenance and functionality of neurons, potentially playing a role in synaptic transmission. The impact of GPRASP2 deficiency on spiral ganglion cells (SGCs) and their underlying pathogenic mechanisms will be investigated in this study. The primary culture of SGCs obtained from mouse cochleae was treated with Gprasp2-targeting short hairpin RNA (Gprasp2-shRNA) via lentivirus infection. The results showed that GPRASP2 deficiency enhanced SGCs apoptosis and decreased cell viability. Meanwhile, a significant abnormality of mitochondrial morphology and decreased membrane potential were observed in GPRASP2-deficient SGCs. These effects could be mitigated by treatment with the mitochondrial division inhibitor 1 (Mdivi-1). In addition to enhancing SGCs apoptosis and decreasing cell viability, GPRASP2 deficiency also inhibited the development of SGCs in mouse cochlear explant culture. Our study further revealed that this deficiency resulted in increased phosphorylation of AMPK and activation of the AMPK/DRP1 pathway, promoting SGCs apoptosis. These findings provide insight into the pathogenic mechanisms by which GPRASP2 deficiency is implicated in auditory dysfunction.

Patient mutations in DRP1 perturb synaptic maturation of cortical neurons.

With the advent of exome sequencing, a growing number of children are being identified with de novo loss of function mutations in the dynamin 1 like (DNM1L) gene encoding the large GTPase essential for mitochondrial fission, dynamin-related protein 1 (DRP1); these mutations result in severe neurodevelopmental phenotypes, such as developmental delay, optic atrophy, and epileptic encephalopathies. Though it is established that mitochondrial fission is an essential precursor to the rapidly changing metabolic needs of the developing cortex, it is not understood how identified mutations in different domains of DRP1 uniquely disrupt cortical development and synaptic maturation. We leveraged the power of induced pluripotent stem cells (iPSCs) harboring DRP1 mutations in either the GTPase or stalk domains to model early stages of cortical development in vitro. High-resolution time-lapse imaging of axonal transport in mutant DRP1 cortical neurons reveals mutation-specific changes in mitochondrial motility of severely hyperfused mitochondrial structures. Transcriptional profiling of mutant DRP1 cortical neurons during maturation also implicates mutation dependent alterations in synaptic development and calcium regulation gene expression. Disruptions in calcium dynamics were confirmed using live functional recordings of 100 DIV (days in vitro) mutant DRP1 cortical neurons. These findings and deficits in pre- and post-synaptic marker colocalization using super resolution microscopy, strongly suggest that altered mitochondrial morphology of DRP1 mutant neurons leads to pathogenic dysregulation of synaptic development and activity.

Acutely blocking excessive mitochondrial fission prevents chronic neurodegeneration after traumatic brain injury.

Progression of acute traumatic brain injury (TBI) into chronic neurodegeneration is a major health problem with no protective treatments. Here, we report that acutely elevated mitochondrial fission after TBI in mice triggers chronic neurodegeneration persisting 17 months later, equivalent to many human decades. We show that increased mitochondrial fission after mouse TBI is related to increased brain levels of mitochondrial fission 1 protein (Fis1) and that brain Fis1 is also elevated in human TBI. Pharmacologically preventing Fis1 from binding its mitochondrial partner, dynamin-related protein 1 (Drp1), for 2 weeks after TBI normalizes the balance of mitochondrial fission/fusion and prevents chronically impaired mitochondrial bioenergetics, oxidative damage, microglial activation and lipid droplet formation, blood-brain barrier deterioration, neurodegeneration, and cognitive impairment. Delaying treatment until 8 months after TBI offers no protection. Thus, time-sensitive inhibition of acutely elevated mitochondrial fission may represent a strategy to protect human TBI patients from chronic neurodegeneration.

TRPA1 protects against contrast-induced renal tubular injury by preserving mitochondrial dynamics via the AMPK/DRP1 pathway.

Mitochondrial dysfunction and oxidative stress are involved in the development of contrast-induced acute kidney injury (CI-AKI). The present study aimed to reveal the role of transient receptor potential ankyrin 1 (TRPA1), an oxidative sensor, in CI-AKI. Trpa1PT-/- mice with Trpa1 conditionally knocked out in renal proximal tubular (PT) cells, Trpa1 overexpression mice (Trpa1-OE), and TRPA1 agonists and antagonists were used to study its function in a mouse model of iohexol-induced CI-AKI. We found that TRPA1 was functionally expressed in PT cells. Activation of TRPA1 with cinnamaldehyde or overexpression of Trpa1 remarkably ameliorated renal tubular injury and dysfunction in a mouse model of CI-AKI, while CI-AKI was significantly exacerbated in Trpa1PT-/- mice. Proteomics demonstrated that mouse kidneys with CI-AKI had downregulated proteins involved in mitochondrial dynamics and upregulated mitophagy-associated proteins. The beneficial effects of TRPA1 activation/overexpression on CI-AKI were associated with improved mitochondrial function, decreased mitochondrial fission and oxidative stress, enhanced mitophagy, and less apoptosis of renal tubular cells. TRPA1-induced decreases in mitochondrial fission were linked to upregulated fusion-related proteins (mitofusin 1, mitofusin 2 and optic atrophy 1) and downregulated fission mediator, phosphorylated dynamin-related protein 1 (Drp1). Importantly, inhibition of Drp1 with mitochondrial division inhibitor 1 improved CI-AKI. In addition, the decreased mitochondrial fission was also mediated by inactivation of AMP-activated protein kinase which mediates mitochondrial biogenesis. The findings suggest that TRPA1 plays a protective role in CI-AKI through regulating mitochondrial fission/fusion, biogenesis, and dysfunction. Activating TRPA1 may become novel therapeutic strategies for the prevention of CI-AKI.

Bcl-2 Orthologues, Buffy and Debcl, Can Suppress Drp1-Dependent Age-Related Phenotypes in Drosophila.

The relationship of Amyotrophic Lateral Sclerosis, Parkinson's disease, and other age-related neurodegenerative diseases with mitochondrial dysfunction has led to our study of the mitochondrial fission gene Drp1 in Drosophila melanogaster and aspects of aging. Previously, the Drp1 protein has been demonstrated to interact with the Drosophila Bcl-2 mitochondrial proteins, and Drp1 mutations can lead to mitochondrial dysfunction and neuronal loss. In this study, the Dopa decarboxylase-Gal4 (Ddc-Gal4) transgene was exploited to direct the expression of Drp1 and Drp1-RNAi transgenes in select neurons. Here, the knockdown of Drp1 seems to compromise locomotor function throughout life but does not alter longevity. The co-expression of Buffy suppresses the poor climbing induced by the knockdown of the Drp1 function. The consequences of Drp1 overexpression, which specifically reduced median lifespan and diminished climbing abilities over time, can be suppressed through the directed co-overexpression of pro-survival Bcl-2 gene Buffy or by the co-knockdown of the pro-cell death Bcl-2 homologue Debcl. Alteration of the expression of Drp1 acts to phenocopy neurodegenerative disease phenotypes in Drosophila, while overexpression of Buffy can counteract or rescue these phenotypes to improve overall health. The diminished healthy aging due to either the overexpression of Drp1 or the RNA interference of Drp1 has produced novel Drosophila models for investigating mechanisms underlying neurodegenerative disease.

GPx1-ERK1/2-CREB pathway regulates the distinct vulnerability of hippocampal neurons to oxidative stress via modulating mitochondrial dynamics following status epilepticus.

Glutathione peroxidase-1 (GPx1) and cAMP/Ca2+ responsive element (CRE)-binding protein (CREB) regulate neuronal viability by maintaining the redox homeostasis. Since GPx1 and CREB reciprocally regulate each other, it is likely that GPx1-CREB interaction may play a neuroprotective role against oxidative stress, which are largely unknown. Thus, we investigated the underlying mechanisms of the reciprocal regulation between GPx1 and CREB in the male rat hippocampus. Under physiological condition, L-buthionine sulfoximine (BSO)-induced oxidative stress increased GPx1 expression, extracellular signal-regulated kinase 1/2 (ERK1/2) activity and CREB serine (S) 133 phosphorylation in CA1 neurons, but not dentate granule cells (DGC), which were diminished by GPx1 siRNA, U0126 or CREB knockdown. GPx1 knockdown inhibited ERK1/2 and CREB activations induced by BSO. CREB knockdown also decreased the efficacy of BSO on ERK1/2 activation. BSO facilitated dynamin-related protein 1 (DRP1)-mediated mitochondrial fission in CA1 neurons, which abrogated by GPx1 knockdown and U0126. CREB knockdown blunted BSO-induced DRP1 upregulation without affecting DRP1 S616 phosphorylation ratio. Following status epilepticus (SE), GPx1 expression was reduced in CA1 neurons and DGC. SE also decreased CREB activity CA1 neurons, but not DGC. SE degenerated CA1 neurons, but not DGC, accompanied by mitochondrial elongation. These post-SE events were ameliorated by N-acetylcysteine (NAC, an antioxidant), but deteriorated by GPx1 knockdown. These findings indicate that a transient GPx1-ERK1/2-CREB activation may be a defense mechanism to protect hippocampal neurons against oxidative stress via maintenance of proper mitochondrial dynamics.

Drp1 splice variants regulate ovarian cancer mitochondrial dynamics and tumor progression.

Aberrant mitochondrial fission/fusion dynamics are frequently associated with pathologies, including cancer. We show that alternative splice variants of the fission protein Drp1 (DNM1L) contribute to the complexity of mitochondrial fission/fusion regulation in tumor cells. High tumor expression of the Drp1 alternative splice variant lacking exon 16 relative to other transcripts is associated with poor outcome in ovarian cancer patients. Lack of exon 16 results in Drp1 localization to microtubules and decreased association with mitochondrial fission sites, culminating in fused mitochondrial networks, enhanced respiration, changes in metabolism, and enhanced pro-tumorigenic phenotypes in vitro and in vivo. These effects are inhibited by siRNAs designed to specifically target the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the pathophysiological importance of Drp1 alternative splicing, highlight the divergent functions and consequences of changing the relative expression of Drp1 splice variants in tumor cells, and strongly warrant consideration of alternative splicing in future studies focused on Drp1.

Cr(VI) induced hepatocyte apoptosis through the CTH/H2S/Drp1 signaling pathway.

Hexavalent chromium [Cr(VI)] is a highly hazardous heavy metal with multiple toxic effects. Occupational studies indicate that its accumulation in humans can lead to liver damage. However, the exact mechanism underlying Cr(VI)-induced hepatotoxicity remains unknown. In this study, we explored the role of CTH/H2S/Drp1 pathway in Cr(VI)-induced oxidative stress, mitochondrial dysfunction, apoptosis, and liver injury. Our data showed that Cr(VI) triggered apoptosis, accompanied by H2S reduction, reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction in both AML12 cells and mouse livers. Moreover, Cr(VI) reduced cystathionine γ-lyase (CTH) and dynamin related protein 1 (Drp1) S-sulfhydration levels, and elevated Drp1 phosphorylation levels at Serine 616, which promoted Drp1 mitochondrial translocation and Drp1-voltage-dependent anion channel 1 (VDAC1) interactions, ultimately leading to mitochondria-dependent apoptosis. Elevated hydrogen sulfide (H2S) levels eliminated Drp1 phosphorylation at Serine 616 by increasing Drp1 S-sulfhydration, thereby preventing Cr(VI)-induced Drp1-VDAC1 interaction and hepatotoxicity. These findings indicated that Cr(VI) induced mitochondrial apoptosis and hepatotoxicity by inhibiting CTH/H2S/Drp1 pathway and that targeting either CTH/H2S pathway or Drp1 S-sulfhydration could serve as a potential therapy for Cr(VI)-induced liver injury.

Jin-Xin-Kang alleviates heart failure by mitigating mitochondrial dysfunction through the Calcineurin/Dynamin-Related Protein 1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic heart failure (CHF) is a severe consequence of cardiovascular disease, marked by cardiac dysfunction. Jin-Xin-Kang (JXK) is a traditional Chinese herbal formula used for the treatment of CHF. This formula consists of seven medicinal herbs, including Ginseng (Ginseng quinquefolium (L.) Alph.Wood), Astragali Radix (Astragalus membranaceus (Fisch.) Bunge), Salvia miltiorrhiza (Salvia miltiorrhiza Bunge), Descurainiae Semen Lepidii Semen (Descurainia sophia (L.) Webb ex Prantl), Leonuri Herba (Leonurus japonicus Houtt.), Cinnamomi Ramulus (Cinnamomum cassia (L.) J.Presl), and Ilex pubescens (Ilex pubescens Hook. & Arn.). Its clinical efficacy has been validated through prospective randomized controlled studies. However, the specific mechanisms of action for this formula have yet to be elucidated. AIM OF THE STUDY: This study aimed to investigate the effect of JXK on mitochondrial function and its mechanism in the treatment of CHF. METHODS: JXK components were qualitatively analyzed using UPLC-Q-Orbitrap-MS. HF was induced in mice via transverse aortic constriction (TAC). After successful model establishment, lyophilized JXK-L (4.38 g/kg) and JXK-H (13.14 g/kg) were administered for 8 weeks. In vitro, hypertrophic myocardium was induced using angiotensin II (Ang II) for 48 h, followed by JXK-L and JXK-H treatment. Network pharmacology and molecular docking techniques were used to predict the relevant targets of JXK. Cardiac function, serum markers, and histopathological changes were evaluated to assess cardiac function. Immunofluorescence of Tomm20, mitochondrial membrane potential, and ROS were measured to assess mitochondrial dysfunction. Protein expression of calcineurin (CaN) and Drp1 in the myocardium was assessed by Western blot analysis. RESULTS: We detected that the active components of JXK include terpenes, glycosides, flavonoids, amino acids, and alkaloids, among others. In mice with CHF, JXK improved cardiac function and reversed ventricular remodeling. Network pharmacology indicated that JXK can inhibit the calcium signaling pathway. The molecular docking results demonstrated that the active components of JXK effectively bind with CaN. Both in vitro and in vivo experiments confirmed that JXK regulated the CaN/Drp1 pathway and alleviated mitochondrial dysfunction. CONCLUSION: JXK can inhibit the CaN/Drp1 pathway to improve mitochondrial function, and consequently treat CHF.

The Scavenging Activity of Coenzyme Q10 Plus a Nutritional Complex on Human Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are common retinal diseases responsible for most blindness in working-age and elderly populations. Oxidative stress and mitochondrial dysfunction play roles in these pathogenesis, and new therapies counteracting these contributors could be of great interest. Some molecules, like coenzyme Q10 (CoQ10), are considered beneficial to maintain mitochondrial homeostasis and contribute to the prevention of cellular apoptosis. We investigated the impact of adding CoQ10 (Q) to a nutritional antioxidant complex (Nutrof Total®; N) on the mitochondrial status and apoptosis in an in vitro hydrogen peroxide (H2O2)-induced oxidative stress model in human retinal pigment epithelium (RPE) cells. H2O2 significantly increased 8-OHdG levels (p < 0.05), caspase-3 (p < 0.0001) and TUNEL intensity (p < 0.01), and RANTES (p < 0.05), caspase-1 (p < 0.05), superoxide (p < 0.05), and DRP-1 (p < 0.05) levels, and also decreased IL1ß, SOD2, and CAT gene expression (p < 0.05) vs. control. Remarkably, Q showed a significant recovery in IL1ß gene expression, TUNEL, TNFα, caspase-1, and JC-1 (p < 0.05) vs. H2O2, and NQ showed a synergist effect in caspase-3 (p < 0.01), TUNEL (p < 0.0001), mtDNA, and DRP-1 (p < 0.05). Our results showed that CoQ10 supplementation is effective in restoring/preventing apoptosis and mitochondrial stress-related damage, suggesting that it could be a valid strategy in degenerative processes such as AMD or DR.

RIPK3 causes mitochondrial dysfunction and albuminuria in diabetic podocytopathy through PGAM5-Drp1 signaling.

BACKGROUND: Receptor-interacting protein kinase (RIPK)3 is an essential molecule for necroptosis and its role in kidney fibrosis has been investigated using various kidney injury models. However, the relevance and the underlying mechanisms of RIPK3 to podocyte injury in albuminuric diabetic kidney disease (DKD) remain unclear. Here, we investigated the role of RIPK3 in glomerular injury of DKD. METHODS: We analyzed RIPK3 expression levels in the kidneys of patients with biopsy-proven DKD and animal models of DKD. Additionally, to confirm the clinical significance of circulating RIPK3, RIPK3 was measured by ELISA in plasma obtained from a prospective observational cohort of patients with type 2 diabetes, and estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), which are indicators of renal function, were followed up during the observation period. To investigate the role of RIPK3 in glomerular damage in DKD, we induced a DKD model using a high-fat diet in Ripk3 knockout and wild-type mice. To assess whether mitochondrial dysfunction and albuminuria in DKD take a Ripk3-dependent pathway, we used single-cell RNA sequencing of kidney cortex and immortalized podocytes treated with high glucose or overexpressing RIPK3. RESULTS: RIPK3 expression was increased in podocytes of diabetic glomeruli with increased albuminuria and decreased podocyte numbers. Plasma RIPK3 levels were significantly elevated in albuminuric diabetic patients than in non-diabetic controls (p = 0.002) and non-albuminuric diabetic patients (p = 0.046). The participants in the highest tertile of plasma RIPK3 had a higher incidence of renal progression (hazard ratio [HR] 2.29 [1.05-4.98]) and incident chronic kidney disease (HR 4.08 [1.10-15.13]). Ripk3 knockout improved albuminuria, podocyte loss, and renal ultrastructure in DKD mice. Increased mitochondrial fragmentation, upregulated mitochondrial fission-related proteins such as phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp1), and mitochondrial ROS were decreased in podocytes of Ripk3 knockout DKD mice. In cultured podocytes, RIPK3 inhibition attenuated mitochondrial fission and mitochondrial dysfunction by decreasing p-mixed lineage kinase domain-like protein (MLKL), PGAM5, and p-Drp1 S616 and mitochondrial translocation of Drp1. CONCLUSIONS: The study demonstrates that RIPK3 reflects deterioration of renal function of DKD. In addition, RIPK3 induces diabetic podocytopathy by regulating mitochondrial fission via PGAM5-Drp1 signaling through MLKL. Inhibition of RIPK3 might be a promising therapeutic option for treating DKD.

SIRT6 prevent chronic cerebral hypoperfusion induced cognitive impairment by remodeling mitochondrial dynamics in a STAT5-PGAM5-Drp1 dependent manner.

Vascular dementia (VaD) is a prevalent form of dementia resulting from chronic cerebral hypoperfusion (CCH). However, the pathogenic mechanisms of VaD and corresponding therapeutic strategies are not well understood. Sirtuin 6 (SIRT6) has been implicated in various biological processes, including cellular metabolism, DNA repair, redox homeostasis, and aging. Nevertheless, its functional relevance in VaD remains unexplored. In this study, we utilized a bilateral common carotid artery stenosis (BCAS) mouse model of VaD to investigate the role of SIRT6. We detected a significant decrease in neuronal SIRT6 protein expression following CCH. Intriguingly, neuron-specific ablation of Sirt6 in mice exacerbated neuronal damage and cognitive deficits after CCH. Conversely, treatment with MDL-800, an agonist of SIRT6, effectively mitigated neuronal loss and facilitated neurological recovery. Mechanistically, SIRT6 inhibited excessive mitochondrial fission by suppressing the CCH-induced STAT5-PGAM5-Drp1 signaling cascade. Additionally, the gene expression of monocyte SIRT6 in patients with asymptomatic carotid stenosis showed a correlation with cognitive outcomes, suggesting translational implications in human subjects. Our findings provide the first evidence that SIRT6 prevents cognitive impairment induced by CCH, and mechanistically, this protection is achieved through the remodeling of mitochondrial dynamics in a STAT5-PGAM5-Drp1-dependent manner.

Novel Thienopyrimidine-Hydrazinyl Compounds Induce DRP1-Mediated Non-Apoptotic Cell Death in Triple-Negative Breast Cancer Cells.

Apoptosis induction with taxanes or anthracyclines is the primary therapy for TNBC. Cancer cells can develop resistance to anticancer drugs, causing them to recur and metastasize. Therefore, non-apoptotic cell death inducers could be a potential treatment to circumvent apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induced non-apoptotic cell death in TNBC cells. These lead compounds were 15- to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to that of normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death, characterized by the absence of cellular shrinkage and the absence of nuclear fragmentation and apoptotic blebs. Although TPH104c and TPH104m induced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase the levels of cytochrome c and intracellular reactive oxygen species (ROS) and caspase activation, and cell death was not rescued by incubating cells with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). Furthermore, TPH104c and TPH104m significantly downregulated the expression of the mitochondrial fission protein, DRP1, and their levels determined their cytotoxic efficacy. Overall, TPH104c and TPH104m induced non-apoptotic cell death, and further determination of their cell death mechanisms will aid in the development of new potent and efficacious anticancer drugs to treat TNBC.

BRAFV600E/p-ERK/p-DRP1(Ser616) Promotes Tumor Progression and Reprogramming of Glucose Metabolism in Papillary Thyroid Cancer.

Background: Papillary thyroid cancer (PTC) with the BRAFV600E mutation is associated with a poorer prognosis. BRAF inhibitors may demonstrate limited efficacy due to emerging drug resistance. The Warburg effect may have cancer therapeutic implications. It is not known if the BRAFV600E mutation is associated with altered glucose metabolism in PTC. Methods: This study examined the effect of BRAFV600E and dynamin-related protein 1 (DRP1) on various cellular processes in PTC cells, including cell proliferation, migration, invasion, mitochondrial fission, glucose metabolism, reactive oxygen species (ROS) generation, and apoptosis. We used RT-qPCR to assess the expression of key glycolytic enzymes in thyroid cancer tissues. Additionally, the regulatory interaction between BRAFV600E and DRP1 was investigated through Western blot and immunohistochemical staining. We further evaluated the impact of DRP1 in PTC and the inhibitory effects of dabrafenib and 2-deoxy-d-glucose (2-DG) in vitro and in vivo. Results: We found that the BRAFV600E mutation significantly augments aerobic glycolysis while suppressing oxidative phosphorylation in PTC. We identified the BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway as a critical mediator in PTC progression. First, the BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway enhances cell proliferation by upregulating hexokinase 2 expression and thereby increasing aerobic glycolysis. Second, it inhibits apoptosis by promoting mitochondrial fission and reducing ROS levels. Moreover, we demonstrated that the combination therapy of 2-DG and dabrafenib markedly impedes the progression of BRAFV600E-positive PTC. Conclusion: The BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway plays a pivotal role in glucose metabolism reprogramming, contributing to the aggressiveness and progression of BRAFV600E-positive PTC. Our findings suggest that a combined therapeutic approach using 2-DG and dabrafenib has the potential to improve the outcome of PTC patients with BRAFV600E.

Isotoosendanin inhibits triple-negative breast cancer metastasis by reducing mitochondrial fission and lamellipodia formation regulated by the Smad2/3-GOT2-MYH9 signaling axis.

Triple-negative breast cancer (TNBC) is incurable and prone to widespread metastasis. Therefore, identification of key targets for TNBC progression is urgently needed. Our previous study revealed that isotoosendanin (ITSN) reduced TNBC metastasis by targeting TGFßR1. ITSN is currently used as an effective chemical probe to further discover the key molecules involved in TNBC metastasis downstream of TGFßR1. The results showed that GOT2 was the gene downstream of Smad2/3 and that ITSN decreased GOT2 expression by abrogating the activation of the TGF-ß-Smad2/3 signaling pathway through directly binding to TGFßR1. GOT2 was highly expressed in TNBC, and its knockdown decreased TNBC metastasis. However, GOT2 overexpression reversed the inhibitory effect of ITSN on TNBC metastasis both in vitro and in vivo. GOT2 interacted with MYH9 and hindered its binding to the E3 ubiquitin ligase STUB1, thereby reducing MYH9 ubiquitination and degradation. Moreover, GOT2 also enhanced the translocation of MYH9 to mitochondria and thus induced DRP1 phosphorylation, thereby promoting mitochondrial fission and lamellipodia formation in TNBC cells. ITSN-mediated inhibition of mitochondrial fission and lamellipodia formation was associated with reduced GOT2 expression. In conclusion, ITSN prevented MYH9-regulated mitochondrial fission and lamellipodia formation in TNBC cells by enhancing MYH9 protein degradation through a reduction in GOT2 expression, thus contributing to its inhibition of TNBC metastasis.

Developmental disruption of the mitochondrial fission gene drp-1 extends the longevity of daf-2 insulin/IGF-1 receptor mutant.

The dynamic nature of the mitochondrial network is regulated by mitochondrial fission and fusion, allowing for re-organization of mitochondria to adapt to the cell's ever-changing needs. As organisms age, mitochondrial fission and fusion become dysregulated and mitochondrial networks become increasingly fragmented. Modulation of mitochondrial dynamics has been shown to affect longevity in fungi, yeast, Drosophila and C. elegans. Disruption of the mitochondrial fission gene drp-1 drastically increases the already long lifespan of daf-2 insulin/IGF-1 signaling (IIS) mutants. In this work, we determined the conditions required for drp-1 disruption to extend daf-2 longevity and explored the molecular mechanisms involved. We found that knockdown of drp-1 during development is sufficient to extend daf-2 lifespan, while tissue-specific knockdown of drp-1 in neurons, intestine or muscle failed to increase daf-2 longevity. Disruption of other genes involved in mitochondrial fission also increased daf-2 lifespan as did treatment with RNA interference clones that decrease mitochondrial fragmentation. In exploring potential mechanisms involved, we found that deletion of drp-1 increases resistance to chronic stresses. In addition, we found that disruption of drp-1 increased mitochondrial and peroxisomal connectedness in daf-2 worms, increased oxidative phosphorylation and ATP levels, and increased mitophagy in daf-2 worms, but did not affect their ROS levels, food consumption or mitochondrial membrane potential. Disruption of mitophagy through RNA interference targeting pink-1 decreased the lifespan of daf-2;drp-1 worms suggesting that increased mitophagy contributes to their extended lifespan. Overall, this work defined the conditions under which drp-1 disruption increases daf-2 lifespan and has identified multiple changes in daf-2;drp-1 mutants that may contribute to their lifespan extension.