Mitofusin 2 Integrates Mitochondrial Network Remodelling, Mitophagy and the Renewal of Respiratory Chain Proteins in Neurons After Oxygen and Glucose Deprivation.

In the efforts to develop effective therapeutic strategies limiting post-ischemic injury, mitochondria emerge as key element in determining the fate of the neurons. Mitochondrial damage can be alleviated by various mechanisms including mitochondrial network remodelling, mitochondrial elimination and mitochondrial protein biogenesis. However, the mechanisms regulating the relationship between these phenomena are poorly understood. Here we hypothesize that mitofusin 2 (Mfn2), a mitochondrial GTPase, involved in mitochondrial fusion, mitochondria trafficking and mitochondria and endoplasmic reticulum (ER) tethering, may act as a linking and regulatory factor in neurons following ischemic insult. To verify this assumption, we performed a temporal oxygen and glucose deprivation (OGD) on rat cortical primary culture to determine whether Mfn2 protein reduction may affect the onset of mitophagy, subsequent mitochondrial biogenesis and thus neuronal survival. In our study we found that Mfn2 knock-down increased the susceptibility of the neurons to the OGD. Mfn2 protein reduction prevented mitochondrial network remodelling and resulted in the prolonged mitophagosomes formation in response to the insult. Further on, Mfn2 protein reduction was accompanied by a reduced level of Parkin protein and an increased Parkin accumulation with mitochondria. As for Mfn2-expressing neurons, the OGD insult was followed by an elevated mtDNA content and an increase in the respiratory chain proteins. Neither of this phenomena were observed for Mfn2-reduced neurons. Collectively, our findings show that Mfn2 in neurons is involved in their response to mild and transient OGD stress, balancing the extent of elimination of defective mitochondria and positively influencing mitochondrial respiratory proteins levels. Our study confirms that Mfn2 is an essential element of the neuronal response to ischemic insult, necessary for the neuronal survival.

Visualizing a Convergence of Three Mitochondrial Molecule mRNAs for Drp1/Mfn2/Ucp4 on Soma of Cerebellar Purkinje Cells by RNAscope

We know little about how mitochondrial dynamics regulates in the Purkinje cells. To explore it, we first set up the Gad2-cre:ZsGreen-tdTomatofl/fl mice where Purkinje cells expressed tdTomato in the cerebellum. Secondly, double stainings verified tdTomato cells were Calbinin (CB)-positive Purkinje cells, but colocalized neither with astrocyte marker GFAP nor with microglia marker Iba1. Thirdly, application of RNAscope in situ hybridization with the identification of mRNAs of mitofusin 2 (Mfn2), calcium transporter (Mcu and Nclx) and uncoupling proteins (Ucp2 and Ucp4) were used onto Purkinje cells for specific spatial analysis. Our findings demonstrated that Mfn2 mRNAs expression was evident in Purkinje cells. And few expressions of Ucp4 mRNAs were presented in dendritic shafts of Purkinje cells. It should be noted that Mcu, Nclx, and Ucp2 mRNAs expression were only scattered on both soma and dendrites in Purkinje cells. The double RNAscope profiling of mitochondrial molecules showed Mfn1 mRNAs are presented only in the soma of the Purkinje cells. Double RNAscope showed none of Drp1 mRNAs were co-localized with Mcu mRNAs, as well as almost none of Ucp2 mRNAs were co-localized with Mfn2 mRNAs. All of these results showed the mitochondrial Drp1/Mfn2/Ucp4 convergence on the Purkinje cells. Finally, present research focuses on developing new and more specific molecules tuning the activity of the Purkinje cells activate or inactivate and opening therapeutic windows for Purkinje cells-related diseases. The molecular identification of potential drug targets, mechanism of action, and structural basis of their activity will crucially enable preclinical development.

MFN1 and MFN2 Are Dispensable for Sperm Development and Functions in Mice

MFN1 (Mitofusin 1) and MFN2 (Mitofusin 2) are GTPases essential for mitochondrial fusion. Published studies revealed crucial roles of both Mitofusins during embryonic development. Despite the unique mitochondrial organization in sperm flagella, the biological requirement in sperm development and functions remain undefined. Here, using sperm-specific Cre drivers, we show that either Mfn1 or Mfn2 knockout in haploid germ cells does not affect male fertility. The Mfn1 and Mfn2 double knockout mice were further analyzed. We found no differences in testis morphology and weight between Mfn-deficient mice and their wild-type littermate controls. Spermatogenesis was normal in Mfn double knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules, indicating that sperm formation was not disrupted upon MFN deficiency. Collectively, our findings reveal that both MFN1 and MFN2 are dispensable for sperm development and functions in mice.

Alterations in Mitochondrial and Inflammasome Homeostasis by 2-Chloroethyl Ethyl Sulfide and their Mitigation by Curcumin: An In vitro Study

The mitochondrion has a substantial role in innate immunity and inflammasome signaling pathways. Sulfur mustard (SM) induces toxicity in cytoplasmic organelles. We aimed to evaluate the potential therapeutic effect of curcumin on the toxicity of SM analog through measuring gene expression levels of mitochondrial dynamics followed by induction of the inflammasome signaling pathway. After the treatment of pulmonary epithelial cell line (A549) by 2-chloroethyl ethyl sulfide (CEES) (2500 mM) for 48h, the transcriptional activity of mitochondrial fission and fusion genes such as dynamin-related protein 1 (Drp1), mitochondrial fission 1 protein (Fis1), mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and Dominant optic atrophy (Opa1) and inflammasome pathway genes including absent in melanoma 2 (AIM2), NLR family containing protein 3 (NLRP3), and Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) was measured. Furthermore, the inhibitory effect of curcumin (160 mM) concurrent with SM analog on the expression level of mitochondria and inflammasome genes was investigated. CEES was able to over-express the fission, fusion (Drp1 ~ 8, Fis1 4.5, Mfn2 15, and Opa1 16-fold) and inflammasome genes (AIM2, NLRP3, 8 and 6-fold, respectively), whereas Mfn1 was significantly decreased (0.5-fold) and a not statistically significant decrease was observed in the ASC gene. Curcumin could modulate the effect of CEES, mitigate the expression of fission, fusion, and inflammasome genes exceedingly. However, a major increase in the repairer fusion gene (Mfn1, 6-fold) and complete suppression of the ASC gene were the outcomes of using the curcumin. In conclusion, we suggest curcumin alleviates the disturbance of mitochondrial dynamics and downregulates the inflammasome genes exposed to the CEES.

20 Profiling circRNA and lncRNA Expression in Key Bovine Metabolic Tissues

CircRNAs and lncRNAs are non-coding RNAs that regulate many biological processes at the cellular level. However, knowledge of their function and expression patterns in cattle remains scarce. We investigated circRNA and lncRNA expression profiles in four key metabolic tissues of beef cattle (rumen, liver, muscle, and subcutaneous adipose) using RNA-Seq. Bioinformatic analysis of 189 samples collected from 48 cattle identified 19,766 circRNAs and 10,615 lncRNAs across all four tissues. PCA revealed the expression profiles of both circ- and lncRNA were clearly separated by tissue, suggesting their expression patterns are tissue dependent. Moreover, both datasets contained large numbers of transcripts that were unique to each tissue, underlining the divergence in function across metabolic sites. Further functional analysis of tissue specific circRNAs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that lysine degradation, peroxisome, insulin resistance, and ubiquitin-mediated proteolysis unique to the rumen, liver, muscle, and adipose tissues, respectively. The same analysis of the lncRNAs revealed that purine metabolism, fatty acid degradation, cAMP signaling pathway, and AGE-RAGE signaling pathway in diabetic complications were uniquely enriched in the rumen, liver, muscle, and subcutaneous adipose tissues, respectively. Together, these results revealed tissue specific metabolic pathways that may be regulated by the multiple non-coding RNAs. More specifically, circDLG1 and lncMSTRG.12092.8 were predicted to regulate the expression of Lymphocyte Antigen 6 Family Member G5B (LY6G5B) and pyruvate kinase M1/2 (PKM), Adenylate Kinase 1 (AK1), and Mitofusin 2 (MFN2), respectively, indicating regulatory roles in host immune response, glycolysis, energy balance control, and mitochondrial fusion, all of which may contribute to regulation of energy metabolism. Our findings show that expression profiles of circRNAs and lncRNAs differ among metabolic tissues, and these transcripts may play crucial roles in regulating metabolism in cattle.

Mitochondrial dysfunction in adult midbrain dopamine neurons triggers an early immune response

Dopamine (DA) neurons of the midbrain are at risk to become affected by mitochondrial damage over time and mitochondrial defects have been frequently reported in Parkinson’s disease (PD) patients. However, the causal contribution of adult-onset mitochondrial dysfunction to PD remains uncertain. Here, we developed a mouse model lacking Mitofusin 2 (MFN2), a key regulator of mitochondrial network homeostasis, in adult midbrain DA neurons. The knockout mice develop severe and progressive DA neuron-specific mitochondrial dysfunction resulting in neurodegeneration and parkinsonism. To gain further insights into pathophysiological events, we performed transcriptomic analyses of isolated DA neurons and found that mitochondrial dysfunction triggers an early onset immune response, which precedes mitochondrial swelling, mtDNA depletion, respiratory chain deficiency and cell death. Our experiments show that the immune response is an early pathological event when mitochondrial dysfunction is induced in adult midbrain DA neurons and that neuronal death may be promoted non-cell autonomously by the cross-talk and activation of surrounding glial cells.

Comparative Untargeted Metabolomic Profiling of Induced Mitochondrial Fusion in Pancreatic Cancer

Mitochondria are dynamic organelles that constantly alter their shape through the recruitment of specialized proteins, like mitofusin-2 (Mfn2) and dynamin-related protein 1 (Drp1). Mfn2 induces the fusion of nearby mitochondria, while Drp1 mediates mitochondrial fission. We previously found that the genetic or pharmacological activation of mitochondrial fusion was tumor suppressive against pancreatic ductal adenocarcinoma (PDAC) in several model systems. The mechanisms of how these different inducers of mitochondrial fusion reduce pancreatic cancer growth are still unknown. Here, we characterized and compared the metabolic reprogramming of these three independent methods of inducing mitochondrial fusion in KPC cells: overexpression of Mfn2, genetic editing of Drp1, or treatment with leflunomide. We identified significantly altered metabolites via robust, orthogonal statistical analyses and found that mitochondrial fusion consistently produces alterations in the metabolism of amino acids. Our unbiased methodology revealed that metabolic perturbations were similar across all these methods of inducing mitochondrial fusion, proposing a common pathway for metabolic targeting with other drugs.