Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Mapping the Genetic Landscape of Human Cells.

Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.

A neo-substrate that amplifies catalytic activity of parkinson's-disease-related kinase PINK1.

Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III, and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP, and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function.

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.

Mouse midbrain dopaminergic neurons survive loss of the PD-associated mitochondrial protein CHCHD2.

Mutations in the mitochondrial protein CHCHD2 cause autosomal dominant Parkinson's disease characterized by the preferential loss of substantia nigra dopamine (DA) neurons. Therefore, understanding the function of CHCHD2 in neurons may provide vital insights into how mitochondrial dysfunction contributes to neurodegeneration in PD. To investigate the normal requirement and function of CHCHD2 in neurons, we first examined CHCHD2 levels and showed that DA neurons have higher CHCHD2 levels than other neuron types, both in vivo and in co-culture. We then generated mice with either a targeted deletion of CHCHD2 in DA neurons or a deletion in the brain or total body. All three models were viable, and loss of CHCHD2 in the brain did not cause degeneration of DA neurons. Mice lacking CHCHD2 in DA neurons did display sex-specific changes to locomotor activity, but we did not observe differences in assays of muscle strength, exercise endurance or motor coordination. Furthermore, mitochondria derived from mice lacking CHCHD2 did not display abnormalities in OXPHOS function. Lastly, resilience to CHCHD2 deletion could not be explained by functional complementation by its paralog CHCHD10, as deletion of both CHCHD10 and CHCHD2 did not cause degeneration of DA neurons in the midbrain. These findings support the hypothesis that pathogenic CHCHD2 mutations cause PD through a toxic gain-of-function, rather than loss-of-function mechanism.

Fewer systematic prostate core biopsies in clinical stage T1c prostate cancer leads to biochemical recurrence after brachytherapy as monotherapy.

BACKGROUND: After brachytherapy, fewer prostate biopsy cores at diagnosis can underestimate the pathological characteristics of prostate cancer (PCa) with lower concordance, resulting in improper treatment, particularly in patients with low-risk nonpalpable cT1c PCa. The aim of this study was to assess the relationship between the number of biopsy cores at diagnosis and long-term clinical outcomes after brachytherapy for cT1c PCa. METHODS: We reviewed 516 patients with localized cT1c PCa with Gleason scores of 3 + 3 = 6 or 3 + 4 = 7 who underwent brachytherapy as monotherapy without hormonal therapy between January 2005 and September 2014 at our institution. Clinical staging was based on the American Joint Committee on Cancer manual for staging. Thus, the cT1c category is based solely on digital rectal examination. The primary outcome was biochemical recurrence (BCR). Based on the optimized cutoff value for biopsy core number obtained from receiver operating characteristic analysis, patients were divided into the biopsy cores ≤8 (N = 123) and ≥9 (N = 393) groups. The BCR-free survival rate was compared between the groups. Prognostic factors for BCR were evaluated, including age, initial prostate-specific antigen (PSA) level, Gleason score, positive core rate, PSA density, prostate magnetic resonance imaging findings, and biopsy core number. RESULTS: The median patient age was 66.0 years (interquartile range [IQR]: 61.0-71.0 years), and the median follow-up time was 11.1 years (IQR: 9.5-13.3 years). The median number of core biopsies was 12 (IQR: 9-12). The area under the curve was 0.637 (95% confidence interval [CI]: 0.53-0.75), and the optimal biopsy core cutoff value for BCR prediction was 8.5 (sensitivity = 43.5%, specificity = 77.1%). Although fewer patients had Gleason scores of 3 + 4 = 7 (19/123 [15%] vs. 125/393 [32%], p < 0.02) in the biopsy cores ≤8 group, the 10-year BCR-free survival rate was significantly lower in the biopsy cores ≤8 group than in the biopsy cores ≥9 group (93.8% vs. 96.3%, p < 0.05). Multivariate analysis revealed that a lower biopsy core number (hazard ratio: 0.828, 95% CI: 0.71-0.97, p < 0.03) and a Gleason score of 3 + 4 = 7 (hazard ratio: 3.26, 95% CI: 1.37-7.73, p < 0.01) significantly predicted BCR. CONCLUSIONS: A low number of prostate core biopsies results in worse BCR-free survival after brachytherapy as monotherapy in patients with cT1c PCa.

Smooth Muscle Cell Klf4 Expression Is Not Required for Phenotype Modulation or Aneurysm Formation in Marfan Syndrome Mice-Brief Report.

BACKGROUND: Smooth muscle cell (SMC) phenotypic reprogramming toward a mixed synthetic-proteolytic state is a central feature of aortic root aneurysm in Marfan syndrome (MFS). Previous work identified Klf4 as a potential mediator of SMC plasticity in MFS. METHODS: MFS (Fbn1C1041G/+) mouse strains with an inducible vascular SMC fluorescent reporter (MFSSMC) with or without SMC-specific deletion of Klf4 exons 2 to 3 (MFSSMC-Klf4Δ) were generated. Simultaneous SMC tracing and Klf4 loss-of-function (Klf4Δ mice) was induced at 6 weeks of age. Aneurysm growth was assessed via serial echocardiography (4-24 weeks). Twenty-four-week-old mice were assessed via histology, RNA in situ hybridization, and aortic single-cell RNA sequencing. RESULTS: MFS mice demonstrated progressive aortic root dilatation compared with control (WTSMC) mice regardless of Klf4 genotype (P<0.001), but there was no difference in aneurysm growth in MFSSMC-Klf4Δ versus MFSSMC (P=0.884). Efficient SMC Klf4 deletion was confirmed via lineage-stratified genotyping, RNA in situ hybridization, and immunohistochemistry. Single-cell RNA sequencing of traced SMCs revealed a highly similar pattern of phenotype modulation marked by loss of contractile markers (eg, Myh11, Cnn1) and heightened expression of matrix genes (eg, Col1a1, Fn1) between Klf4 genotypes. Pseudotemporal quantitation of SMC dedifferentiation confirmed that Klf4 deletion did not alter the global extent of phenotype modulation, but reduced expression of 23 genes during this phenotype transition in MFSSMC-Klf4Δmice, including multiple chondrogenic genes expressed by only the most severely dedifferentiated SMCs (eg, Cytl1, Tnfrsf11b). CONCLUSIONS: Klf4 is not required to initiate SMC phenotype modulation in MFS aneurysm but may exert regulatory control over chondrogenic genes expressed in highly dedifferentiated SMCs.

Lineage-Specific Induced Pluripotent Stem Cell-Derived Smooth Muscle Cell Modeling Predicts Integrin Alpha-V Antagonism Reduces Aortic Root Aneurysm Formation in Marfan Syndrome Mice.

BACKGROUND: The role of increased smooth muscle cell (SMC) integrin αv signaling in Marfan syndrome (MFS) aortic aneurysm remains unclear. Herein, we examine the mechanism and potential efficacy of integrin αv blockade as a therapeutic strategy to reduce aneurysm progression in MFS. METHODS: Induced pluripotent stem cells (iPSCs) were differentiated into aortic SMCs of the second heart field (SHF) and neural crest (NC) lineages, enabling in vitro modeling of MFS thoracic aortic aneurysms. The pathological role of integrin αv during aneurysm formation was confirmed by blockade of integrin αv with GLPG0187 in Fbn1C1039G/+ MFS mice. RESULTS: iPSC-derived MFS SHF SMCs overexpress integrin αv relative to MFS NC and healthy control SHF cells. Furthermore, integrin αv downstream targets (FAK [focal adhesion kinase]/AktThr308/mTORC1 [mechanistic target of rapamycin complex 1]) were activated, especially in MFS SHF. Treatment of MFS SHF SMCs with GLPG0187 reduced p-FAK/p-AktThr308/mTORC1 activity back to control SHF levels. Functionally, MFS SHF SMCs had increased proliferation and migration compared to MFS NC SMCs and control SMCs, which normalized with GLPG0187 treatment. In the Fbn1C1039G/+ MFS mouse model, integrin αv, p-AktThr308, and downstream targets of mTORC1 proteins were elevated in the aortic root/ascending segment compared to littermate wild-type control. Mice treated with GLPG0187 (age 6-14 weeks) had reduced aneurysm growth, elastin fragmentation, and reduction of the FAK/AktThr308/mTORC1 pathway. GLPG0187 treatment reduced the amount and severity of SMC modulation assessed by single-cell RNA sequencing. CONCLUSIONS: The integrin αv-FAK-AktThr308 signaling pathway is activated in iPSC SMCs from MFS patients, specifically from the SHF lineage. Mechanistically, this signaling pathway promotes SMC proliferation and migration in vitro. As biological proof of concept, GLPG0187 treatment slowed aneurysm growth and p-AktThr308 signaling in Fbn1C1039G/+ mice. Integrin αv blockade via GLPG0187 may be a promising therapeutic approach to inhibit MFS aneurysmal growth.

Safe and promising outcomes of in-hospital preoperative rehabilitation for coronary artery bypass grafting after an acute coronary syndrome.

OBJECTIVE: In patients with stable hemodynamic status after an acute coronary syndrome (ACS), coronary artery bypass grafting (CABG) after preoperative investigations can provide outcomes comparable to those of emergency surgery. However, no established guidelines exist regarding the preparation period before surgery. We report the results of the use of an inpatient cardiac rehabilitation program followed by CABG after an ACS to improve post-operative outcomes and prognosis after discharge. METHODS: From 2005 to 2017, 471 patients underwent either isolated or combined CABG at our institution, and of those, the 393 who received isolated CABG were included in the analysis. Twenty-seven patients (6.9%) were admitted with ACS and underwent preoperative rehabilitation before undergoing CABG, with a subsequent review of surgical morbidity and mortality rates. Propensity score matching yielded a cohort of 26 patients who underwent preoperative rehabilitation (group A) and 26 controls (group B). Preoperative characteristics were similar between groups. RESULTS: The completion rate of the rehabilitation program was 96.3%. All programs were conducted with inpatients, with an average length of stay of 23 ± 12 days. All patients completed in-bed exercises, and 85% completed out-of-bed exercises. The 30-day postoperative mortality was 0% in both groups A and B, and the rate of postoperative major adverse cardiac or cerebrovascular events at 12 months did not differ significantly between groups (7.7% vs 3.9%, respectively; p = 1.0). The duration of mechanical ventilation (1.3 ± 0.3 vs 1.5 ± 0.3 days, respectively; p = 0.633), length of intensive care unit stay (4.4 ± 2.1 vs 4.8 ± 2.3 days, respectively; p = 0.584) and length of hospital stay (25 ± 13 vs 22 ± 9 days, respectively; p = 0.378) did not differ significantly between groups. CONCLUSIONS: No complications of preoperative rehabilitation were observed, suggesting that it is an acceptable option for patients who experience ACS and undergo CABG. These results are promising in offering more robust designs of future trials.

Embryologic Origin Influences Smooth Muscle Cell Phenotypic Modulation Signatures in Murine Marfan Syndrome Aortic Aneurysm.

BACKGROUND: Aortic root smooth muscle cells (SMC) develop from both the second heart field (SHF) and neural crest. Disparate responses to disease-causing Fbn1 variants by these lineages are proposed to promote focal aortic root aneurysm formation in Marfan syndrome (MFS), but lineage-stratified SMC analysis in vivo is lacking. METHODS: We generated SHF lineage-traced MFS mice and performed integrated multiomic (single-cell RNA and assay for transposase-accessible chromatin sequencing) analysis stratified by embryological origin. SMC subtypes were spatially identified via RNA in situ hybridization. Response to TWIST1 overexpression was determined via lentiviral transduction in human aortic SMCs. RESULTS: Lineage stratification enabled nuanced characterization of aortic root cells. We identified heightened SHF-derived SMC heterogeneity including a subset of Tnnt2 (cardiac troponin T)-expressing cells distinguished by altered proteoglycan expression. MFS aneurysm-associated SMC phenotypic modulation was identified in both SHF-traced and nontraced (neural crest-derived) SMCs; however, transcriptomic responses were distinct between lineages. SHF-derived modulated SMCs overexpressed collagen synthetic genes and small leucine-rich proteoglycans while nontraced SMCs activated chondrogenic genes. These modulated SMCs clustered focally in the aneurysmal aortic root at the region of SHF/neural crest lineage overlap. Integrated RNA-assay for transposase-accessible chromatin analysis identified enriched Twist1 and Smad2/3/4 complex binding motifs in SHF-derived modulated SMCs. TWIST1 overexpression promoted collagen and SLRP gene expression in vitro, suggesting TWIST1 may drive SHF-enriched collagen synthesis in MFS aneurysm. CONCLUSIONS: SMCs derived from both SHF and neural crest lineages undergo phenotypic modulation in MFS aneurysm but are defined by subtly distinct transcriptional responses. Enhanced TWIST1 transcription factor activity may contribute to enriched collagen synthetic pathways SHF-derived SMCs in MFS.

Apolipoprotein A2 isoforms associated with exocrine pancreatic insufficiency in early chronic pancreatitis.

BACKGROUND AND AIM: Apolipoprotein A2 (apoA2) isoforms have been reported to undergo the aberrant processing in pancreatic cancer and pancreatic risk populations compared with that in healthy subjects. This study aimed to clarify whether apoA2 isoforms were as useful as N-benzoyl-p-aminobenzoic acid (BT-PABA) test for exocrine pancreatic dysfunction markers in patients with early chronic pancreatitis (ECP). METHODS: Fifty consecutive patients with functional dyspepsia with pancreatic enzyme abnormalities (FD-P) (n = 18), with ECP (n = 20), and asymptomatic patients with pancreatic enzyme abnormalities (AP-P) (n = 12) based on the Rome IV classification and the Japan Pancreatic Association were enrolled in this study. The enrolled patients were evaluated using endoscopic ultrasonography and endoscopic ultrasonography elastography. Five pancreatic enzymes were estimated. Pancreatic exocrine function was analyzed using the BT-PABA test. Lighter and heavier apoA2 isoforms, AT and ATQ levels were measured by enzyme-linked immunosorbent assay methods. RESULTS: There were no significant differences in clinical characteristics such as age, gender, body mass index, alcohol consumption and smoking among patients with AP-P, FD-P, and ECP. The BT-PABA test and lighter apoA2 isoform, AT level in the enrolled patients had a significant correlation (P < 0.01). The BT-PABA test in patients with ECP was significantly lower (P = 0.04) than that in AP-P. ApoA2-AT level in patients with ECP was lower than that in AP-P, albeit, insignificantly. Interestingly, apo A2-AT level was significantly (P = 0.041) associated with exocrine pancreatic insufficiency by multiple logistic regression analysis. CONCLUSIONS: ApoA2-AT level is a useful tool to evaluate exocrine pancreatic insufficiency in the early stage of chronic pancreatitis.

Comparison of pancreatic enzyme abnormalities and protease-activated receptor-2-positive eosinophils in the duodenum of patients with functional dyspepsia-irritable bowel syndrome overlap with functional dyspepsia alone in Asian populations.

BACKGROUND AND AIM: Some patients with functional gastrointestinal disorders exhibit pancreatic dysfunctions and pancreatic enzyme abnormalities. Thus, we aimed to clarify whether significant differences in clinical characteristics, prevalence of pancreatic enzyme abnormalities, duodenal inflammation, and protease-activated receptor 2 (PAR2) expression levels related to hypersensitivity exist between functional dyspepsia (FD) alone and FD-irritable bowel syndrome (IBS) overlap group. METHODS: Ninety-three patients based on the Rome IV criteria, FD alone (n = 44) and FD overlapped with IBS (n = 49) group were enrolled. The patients scored their own clinical symptoms after consuming high-fat meals. Serum trypsin, PLA2, lipase, p-amylase, and elastase-1 levels were measured. PAR2, eotaxin-3, and TRPV4 mRNA levels in duodenum were determined using real-time polymerase chain reaction methods. PRG2- and PAR2 in the duodenum were evaluated using immunostaining. RESULTS: FD score and global GSRS in patients with FD-IBS overlap were significantly higher than FD alone. Although the prevalence of pancreatic enzyme abnormalities in patients with FD alone was significantly (P < 0.01) higher than that in FD-IBS overlap, the ratio of aggravation of clinical symptoms following high-fat intake in patients with FD-IBS overlap was significantly higher (P = 0.007) than that in patients with FD alone. PAR2- and PRG2-double positive cells were localized in the degranulated eosinophils in the duodenum of patients with FD-IBS overlap. The number of PAR2- and PRG2-double positive cells in FD-IBS overlap was significantly (P < 0.01) higher than FD alone. CONCLUSIONS: Pancreatic enzyme abnormalities and PAR2 expression on degranulated eosinophils infiltrations in the duodenum may be associated with the pathophysiology of patients with FD-IBS overlap in Asian populations.

[Staged Hybrid Repair of Extensive Thoracic Aortic Aneurysm with Right Aortic Arch and Isolated Left Subclavian Artery:Report of a Case].

A 69-year-old male was referred to our hospital due to extensive thoracic aortic aneurysm with right aortic arch and isolated left subclavian artery. We chose staged hybrid repair to avoid the risk of spinal cord injury and bilateral recurrent nerve paralysis. First, ascending aorta replacement with elephant trunk and reconstruction of the cervical branches were underwent. Second, we performed the thoracic endovascular aortic repair for aortic arch and descending aortic aneurysm. The postoperative course was satisfactory and, computed tomography (CT) showed successful aortic repair without any type of endoleak. He was discharged on the ninth day after the second operation. To our knowledge, this is the first report of surgical repair for an aortic aneurysm with right aortic arch and isolated subclavian artery.

Biallelic pathogenic variants in COX11 are associated with an infantile-onset mitochondrial encephalopathy.

Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q10 (CoQ10 ) supplementation. This finding is surprising since COX11 has no known role in CoQ10 biosynthesis. Here, we report a novel gene-disease association by identifying biallelic pathogenic variants in COX11 associated with infantile-onset mitochondrial encephalopathies in two unrelated families using trio genome and exome sequencing. Functional studies showed that mutant COX11 fibroblasts had decreased ATP levels which could be rescued by CoQ10 . These results not only suggest that COX11 variants cause defects in energy production but reveal a potential metabolic therapeutic strategy for patients with COX11 variants.

Mitochondrial fission is a critical modulator of mutant APP-induced neural toxicity.

Alterations in mitochondrial fission may contribute to the pathophysiology of several neurodegenerative diseases, including Alzheimer's disease (AD). However, we understand very little about the normal functions of fission or how fission disruption may interact with AD-associated proteins to modulate pathogenesis. Here we show that loss of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) in CA1 and other forebrain neurons markedly worsens the learning and memory of mice expressing mutant human amyloid precursor protein (hAPP) in neurons. In cultured neurons, Drp1KO and hAPP converge to produce mitochondrial Ca2+ (mitoCa2+) overload, despite decreasing mitochondria-associated ER membranes (MAMs) and cytosolic Ca2+. This mitoCa2+ overload occurs independently of ATP levels. These findings reveal a potential mechanism by which mitochondrial fission protects against hAPP-driven pathology.

Loss of HIPK2 Protects Neurons from Mitochondrial Toxins by Regulating Parkin Protein Turnover.

Mitochondria are important sources of energy, but they are also the target of cellular stress, toxin exposure, and aging-related injury. Persistent accumulation of damaged mitochondria has been implicated in many neurodegenerative diseases. One highly conserved mechanism to clear damaged mitochondria involves the E3 ubiquitin ligase Parkin and PTEN-induced kinase 1 (PINK1), which cooperatively initiate the process called mitophagy that identifies and eliminates damaged mitochondria through the autophagosome and lysosome pathways. Parkin is a mostly cytosolic protein, but is rapidly recruited to damaged mitochondria and target them for mitophagy. Moreover, Parkin interactomes also involve signaling pathways and transcriptional machinery critical for survival and cell death. However, the mechanism that regulates Parkin protein level remains poorly understood. Here, we show that the loss of homeodomain interacting protein kinase 2 (HIPK2) in neurons and mouse embryonic fibroblasts (MEFs) has a broad protective effect from cell death induced by mitochondrial toxins. The mechanism by which Hipk2-/- neurons and MEFs are more resistant to mitochondrial toxins is in part due to the role of HIPK2 and its kinase activity in promoting Parkin degradation via the proteasome-mediated mechanism. The loss of HIPK2 leads to higher cytosolic Parkin protein levels at basal conditions and upon exposure to mitochondrial toxins, which protects mitochondria from toxin-induced damage. In addition, Hipk2-/- neurons and MEFs show increased expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1), a Parkin downstream target that can provide additional benefits via transcriptional activation of mitochondrial genes. Together, these results reveal a previously unrecognized avenue to target HIPK2 in neuroprotection via the Parkin-mediated pathway.SIGNIFICANCE STATEMENT In this study, we provide evidence that homeodomain interacting protein kinase 2 (HIPK2) and its kinase activity promote Parkin degradation via the proteasome-mediated pathway. The loss of HIPK2 increases cytosolic and mitochondrial Parkin protein levels under basal conditions and upon exposure to mitochondrial toxins, which protect mitochondria from toxin-induced damage. In addition, Hipk2-/- neurons and mouse embryonic fibroblasts also show increased expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1), a Parkin downstream target that can provide additional benefits via transcriptional activation of mitochondrial genes. These results indicate that targeting HIPK2 and its kinase activity can have neuroprotective effects by elevating Parkin protein levels.

A high-throughput screen of real-time ATP levels in individual cells reveals mechanisms of energy failure.

Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.

Single-Cell Transcriptomic Profiling of Vascular Smooth Muscle Cell Phenotype Modulation in Marfan Syndrome Aortic Aneurysm.

OBJECTIVE: To delineate temporal and spatial dynamics of vascular smooth muscle cell (SMC) transcriptomic changes during aortic aneurysm development in Marfan syndrome (MFS). Approach and Results: We performed single-cell RNA sequencing to study aortic root/ascending aneurysm tissue from Fbn1C1041G/+ (MFS) mice and healthy controls, identifying all aortic cell types. A distinct cluster of transcriptomically modulated SMCs (modSMCs) was identified in adult Fbn1C1041G/+ mouse aortic aneurysm tissue only. Comparison with atherosclerotic aortic data (ApoE-/- mice) revealed similar patterns of SMC modulation but identified an MFS-specific gene signature, including plasminogen activator inhibitor-1 (Serpine1) and Kruppel-like factor 4 (Klf4). We identified 481 differentially expressed genes between modSMC and SMC subsets; functional annotation highlighted extracellular matrix modulation, collagen synthesis, adhesion, and proliferation. Pseudotime trajectory analysis of Fbn1C1041G/+ SMC/modSMC transcriptomes identified genes activated differentially throughout the course of phenotype modulation. While modSMCs were not present in young Fbn1C1041G/+ mouse aortas despite small aortic aneurysm, multiple early modSMCs marker genes were enriched, suggesting activation of phenotype modulation. modSMCs were not found in nondilated adult Fbn1C1041G/+ descending thoracic aortas. Single-cell RNA sequencing from human MFS aortic root aneurysm tissue confirmed analogous SMC modulation in clinical disease. Enhanced expression of TGF-ß (transforming growth factor beta)-responsive genes correlated with SMC modulation in mouse and human data sets. CONCLUSIONS: Dynamic SMC phenotype modulation promotes extracellular matrix substrate modulation and aortic aneurysm progression in MFS. We characterize the disease-specific signature of modSMCs and provide temporal, transcriptomic context to the current understanding of the role TGF-ß plays in MFS aortopathy. Collectively, single-cell RNA sequencing implicates TGF-ß signaling and Klf4 overexpression as potential upstream drivers of SMC modulation.