Mitochondrial augmentation of hematopoietic stem cells in children with single large-scale mitochondrial DNA deletion syndromes.

Patients with single large-scale mitochondrial DNA (mtDNA) deletion syndromes (SLSMDs) usually present with multisystemic disease, either as Pearson syndrome in early childhood or as Kearns-Sayre syndrome later in life. No disease-modifying therapies exist for SLSMDs. We have developed a method to enrich hematopoietic cells with exogenous mitochondria, and we treated six patients with SLSMDs through a compassionate use program. Autologous CD34+ hematopoietic cells were augmented with maternally derived healthy mitochondria, a technology termed mitochondrial augmentation therapy (MAT). All patients had substantial multisystemic disease involvement at baseline, including neurologic, endocrine, or renal impairment. We first assessed safety, finding that the procedure was well tolerated and that all study-related severe adverse events were either leukapheresis-related or related to the baseline disorder. After MAT, heteroplasmy decreased in the peripheral blood in four of the six patients. An increase in mtDNA content of peripheral blood cells was measured in all six patients 6 to 12 months after MAT as compared baseline. We noted some clinical improvement in aerobic function, measured in patients 2 and 3 by sit-to-stand or 6-min walk testing, and an increase in the body weight of five of the six patients suffering from very low body weight before treatment. Quality-of-life measurements as per caregiver assessment and physical examination showed improvement in some parameters. Together, this work lays the ground for clinical trials of MAT for the treatment of patients with mtDNA disorders.

Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit.

Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

Transcriptional regulation of the cholesterol side chain cleavage cytochrome P450 gene (CYP11A1) revisited: binding of GATA, cyclic adenosine 3',5'-monophosphate response element-binding protein and activating protein (AP)-1 proteins to a distal novel cluster of cis-regulatory elements potentiates AP-2 and steroidogenic factor-1-dependent gene expression in the rodent placenta and ovary.

The first and key enzyme controlling the synthesis of steroid hormones is cholesterol side chain cleavage cytochrome P450 (P450scc, CYP11A1). This study sought to elucidate overlooked modes of regulation of P450scc transcription in the rodent placenta and ovary. Transcription of P450scc requires two clusters of cis-regulatory elements: a proximal element (-40) known to bind either activating protein 2 (AP-2) in the placenta, or steroidogenic factor 1 in the ovary, and a distal region of the promoter (-475/-447) necessary for potentiation of the AP-2/steroidogenic factor 1-dependent activity up to 7-fold. In primary cultures of mouse trophoblast giant cells and rat ovarian granulosa cells, binding of trans-factors to the distal regulatory sequences generated transcriptional activity in a tissue-specific pattern: in the placenta, cAMP response element (CRE)-binding protein 1 (CREB-1) and GATA-2 binding generates promoter activity in a cAMP-independent manner, whereas in ovarian cells, CREB-1 and GATA-4 are required for FSH responsiveness. However, as ovarian follicles advance toward ovulation, elevated Fra-2 expression replaces CREB-1 function by binding the same CRE(1/2) motif. Our findings suggest that upon onset of follicular recruitment, CREB-1 mediates FSH/cAMP signaling, which switches to cAMP-independent expression of P450scc in luteinizing granulosa cells expressing Fra-2. In the placenta, the indispensable role of CREB-1 was demonstrated by use of dominant-negative CREB-1 mutant, but neither cAMP nor Ser133 phosphorylation of CREB-1 is required for P450scc transcription. These observations suggest that placental regulation of P450scc expression is subjected to alternative signaling pathway(s) yet to be found.

Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein beta confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models.

Steroidogenic acute regulatory protein (StAR) mediates translocation of cholesterol to the inner membranes of steroidogenic mitochondria, where it serves as a substrate for steroid synthesis. Transcription of StAR in the gonads and adrenal cells is upregulated by trophic hormones, involves downstream signaling pathways and a cohort of trans-factors acting as activators or suppressors of StAR transcription. This study suggests that a 21 basepair long sequence positioned at -81/-61 of the murine StAR promoter is sufficient to confer a robust hormonal activation of transcription in ovarian granulosa cells treated with FSH. We show that recombinant GATA-4 and CCAAT/enhancer-binding protein beta (C/EBPbeta) bind to the promoter at -66/-61 and -81/-70 and activate transcription of a reporter gene when co-expressed in heterologous human embryonic kidney 293 (HEK293) cells. In this cell model, C/EBPbeta and GATA-4 synergize in a sequence dependent manner and p300/CBP further maximizes their joint activities. Inhibitors of the transcriptional activators, such as liver-enriched inhibiting protein (C/EBPbeta-LIP), Friend of GATA-4 (FOG-2) protein and the viral E1A protein abolished the respective factor-dependent activities in HEK293 cells. Binding assays suggest that a dual binding of C/EBPbeta and GATA-4 to the promoter depends on the molar ratio of the factors present while demonstrating GATA-4 predominant association with the promoter DNA. This pattern may reflect on StAR expression at the time of corpus luteum formation when C/EBPbeta levels peak, as does StAR expression.

Infarct-induced steroidogenic acute regulatory protein: a survival role in cardiac fibroblasts.

Steroidogenic acute regulatory protein (StAR) is indispensable for steroid hormone synthesis in the adrenal cortex and the gonadal tissues. This study reveals that StAR is also expressed at high levels in nonsteroidogenic cardiac fibroblasts confined to the left ventricle of mouse heart examined 3 days after permanent ligation of the left anterior descending coronary artery. Unlike StAR, CYP11A1 and 3ß-hydroxysteroid dehydrogenase proteins were not observed in the postinfarction heart, suggesting an apparent lack of de novo cardiac steroidogenesis. Work with primary cultures of rat heart cells revealed that StAR is induced in fibroblasts responding to proapoptotic treatments with hydrogen peroxide or the kinase inhibitor staurosporine (STS). Such induction of StAR in culture was noted before spontaneous differentiation of the fibroblasts to myofibroblasts. STS induction of StAR in the cardiac fibroblasts conferred a marked resistance to apoptotic cell death. Consistent with that finding, down-regulation of StAR by RNA interference proportionally increased the number of STS-treated apoptotic cells. StAR down-regulation also resulted in a marked increase of BAX activation in the mitochondria, an event known to associate with the onset of apoptosis. Last, STS treatment of HeLa cells showed that apoptotic demise characterized by mitochondrial fission, cytochrome c release, and nuclear fragmentation is arrested in individual HeLa cells overexpressing StAR. Collectively, our in vivo and ex vivo evidence suggests that postinfarction expression of nonsteroidogenic StAR in cardiac fibroblasts has novel antiapoptotic activity, allowing myofibroblast precursor cells to survive the traumatized event, probably to differentiate and function in tissue repair at the infarction site.

MTCH2/MIMP is a major facilitator of tBID recruitment to mitochondria.

The BH3-only BID protein (BH3-interacting domain death agonist) has a critical function in the death-receptor pathway in the liver by triggering mitochondrial outer membrane permeabilization (MOMP). Here we show that MTCH2/MIMP (mitochondrial carrier homologue 2/Met-induced mitochondrial protein), a novel truncated BID (tBID)-interacting protein, is a surface-exposed outer mitochondrial membrane protein that facilitates the recruitment of tBID to mitochondria. Knockout of MTCH2/MIMP in embryonic stem cells and in mouse embryonic fibroblasts hinders the recruitment of tBID to mitochondria, the activation of Bax/Bak, MOMP, and apoptosis. Moreover, conditional knockout of MTCH2/MIMP in the liver decreases the sensitivity of mice to Fas-induced hepatocellular apoptosis and prevents the recruitment of tBID to liver mitochondria both in vivo and in vitro. In contrast, MTCH2/MIMP deletion had no effect on apoptosis induced by other pro-apoptotic Bcl-2 family members and no detectable effect on the outer membrane lipid composition. These loss-of-function models indicate that MTCH2/MIMP has a critical function in liver apoptosis by regulating the recruitment of tBID to mitochondria.

Levels of steroidogenic acute regulatory protein and mitochondrial membrane potential in granulosa cells of older poor-responder women.

OBJECTIVE: To compare mitochondrial function in granulosa cells obtained from older (>40 y) low-responder IVF patients with that of young (<35 y) good-responder patients. DESIGN: Prospective laboratory research. SETTING: In vitro fertilization unit in a university hospital. PATIENT(S): Twenty patients undergoing IVF treatment cycles. INTERVENTION(S): Ultrasound guided oocytes pick-up. MAIN OUTCOME MEASURE(S): Mitochondrial function examined by using JC-1 stain for the mitochondrial membrane potential in granulosa cells of both groups and Western blots for assaying and quantification of steroidogenic acute regulatory protein (StAR) and p450scc (side-chain cleavage). RESULT(S): The number of granulosa cells per follicle differed between the two groups, with fewer granulosa cells isolated in the older low-responder women, compared with in the young, normal responders who were the control women. Trypan blue-negative cells showed similar undisturbed mitochondrial membrane potential, and similar ratios of apoptotic granulosa cells were observed in the two groups. In addition, there was no difference in StAR and P450scc protein levels between the two groups. CONCLUSION(S): Our results demonstrate a significant decrease in the number of total aspirated granulosa cells per follicle in older, poor-responder women, which probably explains the reduced hormonal production by those follicles. However, those cells demonstrate normal mitochondrial membrane potential as well as similar levels of StAR, P450scc, and de novo steroid hormone synthesis in the two groups of patients. Our results do not support mitochondrial dysfunction as a main mechanism of reproductive aging.

Transcription of steroidogenic acute regulatory protein in the rodent ovary and placenta: alternative modes of cyclic adenosine 3', 5'-monophosphate dependent and independent regulation.

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.