Phylogeography of Parnassius citrinarius Based on Mitochondrial DNA Reveals Large Differences in Genetic Structure between the Eastern and Western Japan.

The Japanese Archipelago hosts a rich butterfly fauna, and elucidating the genetic structures of multiple species is necessary to clarify their formation processes. This study aimed to reveal the genetic structure and distribution formation process of Parnassius citrinarius, which is widely distributed across the Japanese Archipelago from Hokkaido to Shikoku, through phylogeographic analysis based on the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence. Thirty haplotypes were revealed from 311 individuals from 47 sites, indicating significant differences in the genetic structures between the eastern and western parts of the Japanese Archipelago. In Eastern Japan, multiple genetic clusters were found, with some sites harboring two clusters. The divergence times among populations in Eastern Japan were relatively recent, and no genetic differentiation was observed between regions, including between Hokkaido and Honshu, which are separated by a narrow strait. In contrast, in Western Japan, including Shikoku, unique genetic clusters were observed in each region. The phylogenetic relationships among populations were regionally clustered, and the divergence times were relatively ancient. The distribution and genetic structure of P. citrinarius in the Japanese Archipelago have been significantly influenced by temperature fluctuations and the presence of geographical barriers during the Pleistocene glacial-interglacial cycles, including the potential formation of refugia in Western Japan.

The predictive value of peripheral blood cell mitochondrial gene expression in identifying the prognosis in pediatric sepsis at preschool age.

Background: Sepsis represents a severe manifestation of infection often accompanied by metabolic disorders and mitochondrial dysfunction. Notably, mitochondrial DNA copy number (mtDNA-CN) and the expression of specific mitochondrial genes have emerged as sensitive indicators of mitochondrial function. To investigate the utility of mitochondrial gene expression in peripheral blood cells for distinguishing severe infections and predicting associated outcomes, we conducted a prospective cohort study. Methods: We established a prospective cohort comprising 74 patients with non-sepsis pneumonia and 67 cases of sepsis induced by respiratory infections, aging from 2 to 6 years old. We documented corresponding clinical data and laboratory information and collected blood samples upon initial hospital admission. Peripheral blood cells were promptly isolated, and both total DNA and RNA were extracted. We utilized absolute quantification PCR to assess mtDNA-CN, as well as the expression levels of mt-CO1, mt-ND1, and mt-ATP6. Subsequently, we extended these comparisons to include survivors and non-survivors among patients with sepsis using univariate and multivariate analyses. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic potential. Results: The mtDNA-CN in peripheral blood cells was significantly lower in the sepsis group. Univariate analysis revealed a significant reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 in patients with sepsis. However, multivariate analysis did not support the use of mitochondrial function in peripheral blood cells for sepsis diagnosis. In the comparison between pediatric sepsis survivors and non-survivors, univariate analysis indicated a substantial reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 among non-survivors. Notably, total bilirubin (TB), mt-CO1, mt-ND1, and mt-ATP6 levels were identified as independent risk factors for sepsis-induced mortality. ROC curves were then established for these independent risk factors, revealing areas under the curve (AUCs) of 0.753 for TB (95% CI 0.596-0.910), 0.870 for mt-CO1 (95% CI 0.775-0.965), 0.987 for mt-ND1 (95% CI 0.964-1.000), and 0.877 for mt-ATP6 (95% CI 0.793-0.962). Conclusion: MtDNA-CN and mitochondrial gene expression are closely linked to the severity and clinical outcomes of infectious diseases. Severe infections lead to impaired mitochondrial function in peripheral blood cells. Notably, when compared to other laboratory parameters, the expression levels of mt-CO1, mt-ND1, and mt-ATP6 demonstrate promising potential for assessing the prognosis of pediatric sepsis.

Combining distribution modelling and phylogeography to understand present, past and future of an endangered spider.

BACKGROUND: Understanding how endangered species respond to climatic changes is fundamental for their conservation. Due to its restricted geographic range, its sensitivity to the ongoing global warming and its continuing decline, the Southwestern-Alpine endemic wolf spider Vesubia jugorum is currently classified as Endangered in the IUCN Red List. Here, we combined species distribution modelling (SDM) and phylogeographic inference to describe the present, the past and the future of this species in light of the mtDNA genetic structure of extant populations. RESULTS: Phylogenetic and network analyses show a high level of genetic differentiation and a strong genetic structure of the populations, likely explicable by a long history of isolation and survival in separate refugia. The SDM projection into past climatic conditions supports these results by showing a smaller distribution range compared to present, mostly restricted to the Maritime and Ligurian Alps, which possibly served as main refugium. Future forecast shows a significant shift in the bioclimatic range towards higher altitudes and latitudes, with a drastic decrease of habitat suitability in the central and south-eastern parts of the range, with consequent general loss of haplotype diversity. CONCLUSION: SDM and phylogeographic inference support the hypothesis that the current distribution and the genetic structure of the extant populations mirror the survival in situ of Vesubia jugorum across repeated glacial and interglacial phases, in line with the 'long-term stability hypothesis'. Future predictions show a significant shift in the bioclimatic range that V. jugorum will be likely unable to track, with profound impact on its long-term survival and its genetic diversity. Our considerations have implication for conservation genetics, highlighting the pivotal role of the transboundary protected areas of the SW-Alps in promoting conservation efforts for this species.

Gasdermin-E-mediated pyroptosis drives immune checkpoint inhibitor-associated myocarditis via cGAS-STING activation.

Immune checkpoint inhibitor (ICI)-induced myocarditis involves intensive immune/inflammation activation; however, its molecular basis is unclear. Here, we show that gasdermin-E (GSDME), a gasdermin family member, drives ICI-induced myocarditis. Pyroptosis mediated by GSDME, but not the canonical GSDMD, is activated in myocardial tissue of mice and cancer patients with ICI-induced myocarditis. Deficiency of GSDME in male mice alleviates ICI-induced cardiac infiltration of T cells, macrophages, and monocytes, as well as mitochondrial damage and inflammation. Restoration of GSDME expression specifically in cardiomyocytes, rather than myeloid cells, in GSDME-deficient mice reproduces ICI-induced myocarditis. Mechanistically, quantitative proteomics reveal that GSDME-dependent pyroptosis promotes cell death and mitochondrial DNA release, which in turn activates cGAS-STING signaling, triggering a robust interferon response and myocardial immune/inflammation activation. Pharmacological blockade of GSDME attenuates ICI-induced myocarditis and improves long-term survival in mice. Our findings may advance the understanding of ICI-induced myocarditis and suggest that targeting the GSDME-cGAS-STING-interferon axis may help prevent and manage ICI-associated myocarditis.

Evolution and maintenance of mtDNA gene content across eukaryotes.

Across eukaryotes, most genes required for mitochondrial function have been transferred to, or otherwise acquired by, the nucleus. Encoding genes in the nucleus has many advantages. So why do mitochondria retain any genes at all? Why does the set of mtDNA genes vary so much across different species? And how do species maintain functionality in the mtDNA genes they do retain? In this review, we will discuss some possible answers to these questions, attempting a broad perspective across eukaryotes. We hope to cover some interesting features which may be less familiar from the perspective of particular species, including the ubiquity of recombination outside bilaterian animals, encrypted chainmail-like mtDNA, single genes split over multiple mtDNA chromosomes, triparental inheritance, gene transfer by grafting, gain of mtDNA recombination factors, social networks of mitochondria, and the role of mtDNA dysfunction in feeding the world. We will discuss a unifying picture where organismal ecology and gene-specific features together influence whether organism X retains mtDNA gene Y, and where ecology and development together determine which strategies, importantly including recombination, are used to maintain the mtDNA genes that are retained.

Mitochondrial DNA copy number and risk of papillary thyroid carcinoma.

BACKGROUND: Mitochondrial DNA (mtDNA) copy number is associated with tumor activity and carcinogenesis. This study was undertaken to investigate mtDNA copy number in papillary thyroid cancer (PTC) tissues and to evaluate the risk of PTC development. The clinicopathological features of patients and mtDNA copy number were correlated. The value of mtDNA copy number was evaluated as a biomarker for PTC. METHOD: DNA was extracted from 105 PTC tissues and 67 control thyroid tissues, and mtDNA copy number mtDNA oxidative damage were determined using qPCR techniques. RESULTS: Overall, the relative mtDNA copy number was significantly higher in PTC patients (p < 0.001). The risk of developing PTC increased significantly across the tertiles of mtDNA copy number (p trend < 0.001). The higher the mtDNA copy number tertile, the greater the risk of developing PTC. Patients with follicular variants had an odds ratio of 2.09 (95% CI: 1.78-2.44) compared to those with classical variants (p < 0.001). The level of mtDNA oxidative damage in PTC was significantly elevated compared to controls (p < 0.001). The ROC analysis of mtDNA copy number indicated an area under the curve (AUC) of 77.7% (95% CI: 0.71 to 0.85, p < 0.001) for the ability of mtDNA copy number z-scores in differentiate between PTC and controls. CONCLUSION: Our results indicated that the augmentation of mtDNA content plays a significant role during the initiation of thyroid cancer, and it might represent a potential biomarker for predicting the risk of PTC.

Multiple unfolded protein response pathways cooperate to link cytosolic dsDNA release to stimulator of interferon gene activation.

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-ß expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-ß induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-ß, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-ß mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-ß via mitochondrial dsDNA release.

Mitochondrial DNA D-loop SNPs unveil molecular signatures of milk production variation in Murrah buffalo.

BACKGROUND: The Murrah buffalo, pivotal in Asian agriculture, faces challenges in maximizing milk production despite significant breeding efforts. Recognizing its economic importance, this study investigates mtDNA D-loop variations in Murrah buffalo as potential indicators of milk production variability, addressing challenges in maximizing yield despite significant breeding efforts. METHODS AND RESULTS: Analyzing mtDNA D-loop sequences from 50 buffaloes, we categorized them into Low (Group 1), Medium (Group 2), and High ECM (Group 3) groups based on milk yields, fat and protein percentage of a 30-day period data. Somatic cell mtDNA D-loop analysis revealed distinct genetic variations, with significant differences among ECM groups. Group 2 showed higher SNP prevalence, group 3 had more insertions/deletions, and Group 1 exhibited the highest transition frequency. Notably, a consistent "C" deletion at the 714th position occurred in Groups 1 and 3, prevalent in 68% of Group 2. A G-A variation at the 93rd position was specific to the medium ECM group. Negative Tajima D values indicated unique variations in each group, with Group 1 having the highest number, and a specific SNP linked to Group 2 was identified. These SNPs in the D-loop region could impact mtDNA replication, influencing mitochondrial content among animals. Our results provide valuable insights into the role of mtDNA D-loop polymorphisms in milk production traits in Murrah buffalo. CONCLUSIONS: Our research highlights the potential for valuable markers of cellular energy efficiency in Murrah buffalo. Exploring diverse cytoplasmic backgrounds opens avenues for mtDNA-based selection strategies, enhancing milk production and optimizing genetic traits for the dairy industry.

Intestinal epithelial damage-derived mtDNA activates STING-IL12 axis in dendritic cells to promote colitis.

Rationale: The treatment of ulcerative colitis (UC) presents an ongoing clinical challenge. Emerging research has implicated that the cGAS-STING pathway promotes the progression of UC, but conflicting results have hindered the development of STING as a therapeutic target. In the current study, we aim to comprehensively elucidate the origins, downstream signaling and pathogenic roles of myeloid STING in colitis and colitis-associated carcinoma (CAC). Methods: Tmem173 fl/fl Lyz2-Cre ert2 mice were constructed for inducible myeloid-specific deletion of STING. RNA-sequencing, flow cytometry, and multiplex immunohistochemistry were employed to investigate immune responses in DSS-induced colitis or AOM/DSS-induced carcinogenesis. Colonic organoids, primary bone marrow derived macrophages and dendritic cells, and splenic T cells were used for in vitro studies. Results: We observed that myeloid STING knockout in adult mice inhibited macrophage maturation, reduced DC cell activation, and suppressed pro-inflammatory Th1 and Th17 cells, thereby protecting against both acute and chronic colitis and CAC. However, myeloid STING deletion in neonatal or tumor-present mice exhibited impaired immune tolerance and anti-tumor immunity. Furthermore, we found that TFAM-associated mtDNA released from damaged colonic organoids, rather than bacterial products, activates STING in dendritic cells in an extracellular vesicle-independent yet endocytosis-dependent manner. Both IRF3 and NF-κB are required for STING-mediated expression of IL-12 family cytokines, promoting Th1 and Th17 differentiation and contributing to excessive inflammation in colitis. Conclusions: Detection of the TFAM-mtDNA complex from damaged intestinal epithelium by myeloid STING exacerbates colitis through IL-12 cytokines, providing new evidence to support the development of STING as a therapeutic target for UC and CAC.

Genetic diversity of a recovering European roller (Coracias garrulus) population from Serbia.

The European Roller (Coracias garrulus), a long-distance migratory bird, faced a considerable decline in breeding pairs throughout Europe at the end of the 20th century. Due to conservation efforts and the installation of nesting boxes, the population of the European Roller in Serbia has made a remarkable recovery. Here, we used the variability of nucleotide sequences of the mitochondrial DNA (mtDNA) control region and 10 microsatellite loci to assess the genetic diversity and structuring, phylogeographic patterns and demographic history of this species using 224 individuals from Serbia. Our results showed moderate level of genetic diversity (HO = 0.392) and a slightly elevated level of inbreeding and homozygosity (FIS = 0.393). Genetic structuring based on microsatellite data indicated three genetic clusters, but without a clear spatial pattern. High haplotype diversity (Hd = 0.987) of the mtDNA control region sequences was detected, and neutrality tests indicated a recent demographic expansion. The phylogeographic analysis, which also included previously published sequences of the mtDNA control region, supported the subdivision into two distinct European and Asian haplogroups (ΦST = 0.712). However, the results of our study showed that a larger number of haplotypes sampled in Serbia are clustered in the Asian haplogroup as compared to previous studies, indicating a historically continuous distribution of this species and possibly a wider distribution of the subspecies Coracias garrulus semenovwi. Our results suggest that the European Roller population in Serbia is genetically stable, with no evidence of recent bottlenecks, and emphasize the importance of artificial nest boxes for promoting and maintaining population dynamics of European Rollers.

mtDNA "nomenclutter" and its consequences on the interpretation of genetic data.

Population-based studies of human mitochondrial genetic diversity often require the classification of mitochondrial DNA (mtDNA) haplotypes into more than 5400 described haplogroups, and further grouping those into hierarchically higher haplogroups. Such secondary haplogroup groupings (e.g., "macro-haplogroups") vary across studies, as they depend on the sample quality, technical factors of haplogroup calling, the aims of the study, and the researchers' understanding of the mtDNA haplogroup nomenclature. Retention of historical nomenclature coupled with a growing number of newly described mtDNA lineages results in increasingly complex and inconsistent nomenclature that does not reflect phylogeny well. This "clutter" leaves room for grouping errors and inconsistencies across scientific publications, especially when the haplogroup names are used as a proxy for secondary groupings, and represents a source for scientific misinterpretation. Here we explore the effects of phylogenetically insensitive secondary mtDNA haplogroup groupings, and the lack of standardized secondary haplogroup groupings on downstream analyses and interpretation of genetic data. We demonstrate that frequency-based analyses produce inconsistent results when different secondary mtDNA groupings are applied, and thus allow for vastly different interpretations of the same genetic data. The lack of guidelines and recommendations on how to choose appropriate secondary haplogroup groupings presents an issue for the interpretation of results, as well as their comparison and reproducibility across studies. To reduce biases originating from arbitrarily defined secondary nomenclature-based groupings, we suggest that future updates of mtDNA phylogenies aimed for the use in mtDNA haplogroup nomenclature should also provide well-defined and standardized sets of phylogenetically meaningful algorithm-based secondary haplogroup groupings such as "macro-haplogroups", "meso-haplogroups", and "micro-haplogroups". Ideally, each of the secondary haplogroup grouping levels should be informative about different human population history events. Those phylogenetically informative levels of haplogroup groupings can be easily defined using TreeCluster, and then implemented into haplogroup callers such as HaploGrep3. This would foster reproducibility across studies, provide a grouping standard for population-based studies, and reduce errors associated with haplogroup nomenclatures in future studies.

Inferring clonal somatic mutations directed by X chromosome inactivation status in single cells.

Analysis of clonal dynamics in human tissues is enabled by somatic genetic variation. Here, we show that analysis of mitochondrial mutations in single cells is dramatically improved in females when using X chromosome inactivation to select informative clonal mutations. Applying this strategy to human peripheral mononuclear blood cells reveals clonal structures within T cells that otherwise are blurred by non-informative mutations, including the separation of gamma-delta T cells, suggesting this approach can be used to decipher clonal dynamics of cells in human tissues.

Causal Relationship between Meat Intake and Biological Aging: Evidence from Mendelian Randomization Analysis.

Existing research indicates that different types of meat have varying effects on health and aging, but the specific causal relationships remain unclear. This study aimed to explore the causal relationship between different types of meat intake and aging-related phenotypes. This study employed Mendelian randomization (MR) to select genetic variants associated with meat intake from large genomic databases, ensuring the independence and pleiotropy-free nature of these instrumental variables (IVs), and calculated the F-statistic to evaluate the strength of the IVs. The validity of causal estimates was assessed through sensitivity analyses and various MR methods (MR-Egger, weighted median, inverse-variance weighted (IVW), simple mode, and weighted mode), with the MR-Egger regression intercept used to test for pleiotropy bias and Cochran's Q test employed to evaluate the heterogeneity of the results. The findings reveal a positive causal relationship between meat consumers and DNA methylation PhenoAge acceleration, suggesting that increased meat intake may accelerate the biological aging process. Specifically, lamb intake is found to have a positive causal effect on mitochondrial DNA copy number, while processed meat consumption shows a negative causal effect on telomere length. No significant causal relationships were observed for other types of meat intake. This study highlights the significant impact that processing and cooking methods have on meat's role in health and aging, enhancing our understanding of how specific types of meat and their preparation affect the aging process, providing a theoretical basis for dietary strategies aimed at delaying aging and enhancing quality of life.

Mitochondrial dysfunction in kidney stones and relief of kidney stones after reducing mtROS.

Mitochondria are essential organelles because they generate the energy required for cellular functions. Kidney stones, as one of the most common urological diseases, have garnered significant attention. In this study, we first collected peripheral venous blood from patients with kidney stones and used qRT-PCR to detect mitochondrial DNA (mtDNA) copy number as a means of assessing mitochondrial function in these patients. Subsequently, through Western blotting, qPCR, immunofluorescence, immunohistochemistry, and transmission electron microscopy, we examined whether calcium oxalate crystals could cause mitochondrial dysfunction in the kidney in both in vitro and in vivo. We then examined the intersection of the DEGs obtained by transcriptome sequencing of the mouse kidney stone model with mitochondria-related genes, and performed KEGG and GO analyses on the intersecting genes. Finally, we administered the mitochondrial ROS scavenger Mito-Tempo in vivo and observed its effects. Our findings revealed that patients with kidney stones had a reduced mtDNA copy number in their peripheral venous blood compared to the control group, suggesting mitochondrial dysfunction in this population. This conclusion was further validated through in vitro and in vivo experiments. Enrichment analyses revealed that the intersecting genes were closely related to metabolism. We observed that after mitochondrial function was preserved, the deposition of calcium oxalate crystals decreased, and the kidney damage and inflammation caused by them were also alleviated. Our research indicates that kidney stones can cause mitochondrial dysfunction. After clearing mtROS, the damage and inflammation caused by kidney stones are reversed, providing new insights into the prevention and treatment of kidney stones.

Elucidating the sustainability of 700 y of Inuvialuit beluga whale hunting in the Mackenzie River Delta, Northwest Territories, Canada.

Beluga whales play a critical role in the subsistence economies and cultural heritage of Indigenous communities across the Arctic, yet the effects of Indigenous hunting on beluga whales remain unknown. Here, we integrate paleogenomics, genetic simulations, and stable δ13C and δ15N isotope analysis to investigate 700 y of beluga subsistence hunting in the Mackenzie Delta area of northwestern Canada. Genetic identification of the zooarchaeological remains, which is based on radiocarbon dating, span three time periods (1290 to 1440 CE; 1450 to 1650 CE; 1800 to 1870 CE), indicates shifts across time in the sex ratio of the harvested belugas. The equal number of females and males harvested in 1450 to 1650 CE versus more males harvested in the two other time periods may reflect changes in hunting practices or temporal shifts in beluga availability. We find temporal shifts and sex-based differences in δ13C of the harvested belugas across time, suggesting historical adaptability in the foraging ecology of the whales. We uncovered distinct mitochondrial diversity unique to the Mackenzie Delta belugas, but found no changes in nuclear genomic diversity nor any substructuring across time. Our findings indicate the genomic stability and continuity of the Mackenzie Delta beluga population across the 700 y surveyed, indicating the impact of Inuvialuit subsistence harvests on the genetic diversity of contemporary beluga individuals has been negligible.

Real-Time Monitoring of mtDNA Aggregation and Mitophagy Induced by a Fluorescent Platinum Complex in Living Cells.

Mitochondrial DNA (mtDNA) is pivotal for mitochondrial morphology and function. Upon mtDNA damage, mitochondria undergo quality control mechanisms, including fusion, fission, and mitophagy. Real-time monitoring of mtDNA enables a deeper understanding of its effect on mitochondrial function and morphology. Controllable induction and real-time tracking of mtDNA dynamics and behavior are of paramount significance for studying mitochondrial function and morphology, facilitating a deeper understanding of mitochondria-related diseases. In this work, a fluorescent platinum complex was designed and developed that not only induces mitochondrial DNA (mtDNA) aggregation but also triggers mitochondrial autophagy (mitophagy) through the MDV pathway for damaged mtDNA clearance in living cells. Additionally, this complex allows for the real-time monitoring of these processes. This complex may serve as a valuable tool for studying mitochondrial microautophagy and holds promise for broader applications in cellular imaging and disease research.

Heterogeneous brain region-specific responses to astrocytic mitochondrial DNA damage in mice.

Astrocytes display context-specific diversity in their functions and respond to noxious stimuli between brain regions. Astrocytic mitochondria have emerged as key players in governing astrocytic functional heterogeneity, given their ability to dynamically adapt their morphology to regional demands on ATP generation and Ca2+ buffering functions. Although there is reciprocal regulation between mitochondrial dynamics and mitochondrial Ca2+ signaling in astrocytes, the extent of this regulation in astrocytes from different brain regions remains unexplored. Brain-wide, experimentally induced mitochondrial DNA (mtDNA) loss in astrocytes showed that mtDNA integrity is critical for astrocyte function, however, possible diverse responses to this noxious stimulus between brain areas were not reported in these experiments. To selectively damage mtDNA in astrocytes in a brain-region-specific manner, we developed a novel adeno-associated virus (AAV)-based tool, Mito-PstI expressing the restriction enzyme PstI, specifically in astrocytic mitochondria. Here, we applied Mito-PstI to two brain regions, the dorsolateral striatum and dentate gyrus, and we show that Mito-PstI induces astrocytic mtDNA loss in vivo, but with remarkable brain-region-dependent differences on mitochondrial dynamics, Ca2+ fluxes, and astrocytic and microglial reactivity. Thus, AAV-Mito-PstI is a novel tool to explore the relationship between astrocytic mitochondrial network dynamics and astrocytic mitochondrial Ca2+ signaling in a brain-region-selective manner.