Endosomal-dependent mitophagy coordinates mitochondrial nucleoid and mtDNA elimination.

Mitophagy and its variants are considered important salvage pathways to remove dysfunctional mitochondria. Non-canonical mitophagy, independent of autophagosome formation and including endosomal-dependent mitophagy, operate upon specific injury. In a recent paper, we describe a new mechanism where, upon mtDNA damage, mitochondrial nucleoids are eliminated via an endosomal-mitophagy pathway. Using proximity proteomics, we identified the proteins required for elimination of mutated mitochondrial nucleoids from the mitochondrial matrix. Among them, ATAD3 and SAMM50 control cristae architecture and nucleoid interaction, necessary for mtDNA extraction. In the mitochondrial outer membrane, SAMM50 coordinates with the retromer protein VPS35 to sequester mtDNA in endosomes and guide them toward elimination, thus avoiding the activation of an exacerbated immune response. Here, we summarize our findings and examine how this newly described pathway contributes to our understanding of mtDNA quality control.

Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.

Understanding the mechanisms governing selective turnover of mutation-bearing mtDNA is fundamental to design therapeutic strategies against mtDNA diseases. Here, we show that specific mtDNA damage leads to an exacerbated mtDNA turnover, independent of canonical macroautophagy, but relying on lysosomal function and ATG5. Using proximity labeling and Twinkle as a nucleoid marker, we demonstrate that mtDNA damage induces membrane remodeling and endosomal recruitment in close proximity to mitochondrial nucleoid sub-compartments. Targeting of mitochondrial nucleoids is controlled by the ATAD3-SAMM50 axis, which is disrupted upon mtDNA damage. SAMM50 acts as a gatekeeper, influencing BAK clustering, controlling nucleoid release and facilitating transfer to endosomes. Here, VPS35 mediates maturation of early endosomes to late autophagy vesicles where degradation occurs. In addition, using a mouse model where mtDNA alterations cause impairment of muscle regeneration, we show that stimulation of lysosomal activity by rapamycin, selectively removes mtDNA deletions without affecting mtDNA copy number, ameliorating mitochondrial dysfunction. Taken together, our data demonstrates that upon mtDNA damage, mitochondrial nucleoids are eliminated outside the mitochondrial network through an endosomal-mitophagy pathway. With these results, we unveil the molecular players of a complex mechanism with multiple potential benefits to understand mtDNA related diseases, inherited, acquired or due to normal ageing.

Mitochondrial DNA mutations induce mitochondrial biogenesis and increase the tumorigenic potential of Hodgkin and Reed-Sternberg cells.

Functioning mitochondria are crucial for cancer metabolism, but aerobic glycolysis is still considered to be an important pathway for energy production in many tumor cells. Here we show that two well established, classic Hodgkin lymphoma (cHL) cell lines harbor deleterious variants within mitochondrial DNA (mtDNA) and thus exhibit reduced steady-state levels of respiratory chain complexes. However, instead of resulting in the expected bioenergetic defect, these mtDNA variants evoke a retrograde signaling response that induces mitochondrial biogenesis and ultimately results in increased mitochondrial mass as well as function and enhances proliferation in vitro as well as tumor growth in mice in vivo. When complex I assembly was impaired by knockdown of one of its subunits, this led to further increased mitochondrial mass and function and, consequently, further accelerated tumor growth in vivo. In contrast, inhibition of mitochondrial respiration in vivo by the mitochondrial complex I inhibitor metformin efficiently slowed down growth. We conclude that, as a new mechanism, mildly deleterious mtDNA variants in cHL cancer cells cause an increase of mitochondrial mass and enhanced function as a compensatory effect using a retrograde signaling pathway, which provides an obvious advantage for tumor growth.

Epidermal glucocorticoid and mineralocorticoid receptors act cooperatively to regulate epidermal development and counteract skin inflammation.

Endogenous and synthetic glucocorticoids (GCs) regulate epidermal development and combat skin inflammatory diseases. GC actions can be mediated through the GC receptor (GR) and/or the mineralocorticoid receptor (MR), highly homologous ligand-activated transcription factors. While the role of GR as a potent anti-inflammatory mediator is well known, that of MR is not as clear, nor is whether these receptors cooperate or antagonize each other in the epidermis. To address this, we generated mice with epidermal-specific loss of both receptors (double knockout, DKO), and analyzed the phenotypical and functional consequences relative to single KOs or controls (CO). At birth, DKO epidermis displayed a phenotype of defective differentiation and inflammation, which was more severe than in either single KO, featuring neutrophil-containing infiltrates, and gene dysregulation characteristic of human psoriatic lesions. This phenotype resolved spontaneously. However, in adulthood, single or combined loss of GC receptors increased susceptibility to inflammation and hyperproliferation triggered by phorbol ester which, different to CO, was not effectively counteracted by GC treatment. Also, DKOs were more susceptible to imiquimod-induced psoriasis than CO showing severe defective epidermal differentiation and microabcesses while single KOs showed an intermediate response. Immortalized DKO keratinocytes featured increased proliferation kinetics and reduced cell size, a unique phenotype relative to single KO cells. The lack of GR and MR in keratinocytes, individual or combined, caused constitutive increases in p38 and ERK activities, which were partially reversed upon reinsertion of receptors into DKO cells. DKO keratinocytes also displayed significant increases in AP-1 and NF-κB transcriptional activities, which were partially rescued by ERK and p38 inhibition, respectively. Reinsertion of GR and MR in DKO keratinocytes resulted in physical and cooperative functional interactions that restored the transcriptional response to GCs. In conclusion, our data have revealed that epidermal GR and MR act cooperatively to regulate epidermal development and counteract skin inflammation.

Mineralocorticoid receptor blockade improves glucocorticoid-induced skin atrophy but partially ameliorates anti-inflammatory actions in an irritative model in human skin explants.

We recently demonstrated that blockade of the mineralocorticoid receptor (MR) effectively ameliorated GC-induced skin atrophy in healthy human skin explants and epidermal MR knockout mice. However, whether MR blockade improves the therapeutic index of glucocorticoids (GCs) in skin pathology was not investigated. We assessed the effects of GCs, MR antagonists (MRA) or both, in SDS-treated human skin explants. All treatments restored SDS-augmented epidermal thickness but only GC plus MRA restored the expression of COL1A1. However, MRA alone or in combination with GCs may exert a dual role in regulating inflammatory cytokines. Thus, although combined treatment may be beneficial to improve irritative skin, extensive in vivo testing is required to establish whether the anti-inflammatory effects of GCs are maintained in the presence of MRA.

Primary aldosteronism patients show skin alterations and abnormal activation of glucocorticoid receptor in keratinocytes.

Primary aldosteronism (PA) is a disease characterized by high aldosterone levels caused by benign adrenal tumors being the most frequent cause of secondary hypertension. Aldosterone plays vital physiological roles through the mineralocorticoid receptor (MR) but in certain cell types, it can also activate the glucocorticoid (GC) receptor (GR). Both MR and GR are structurally and functionally related and belong to the same family of ligand-dependent transcription factors that recognize identical GC regulatory elements (GREs) on their target genes. GCs play key roles in skin pathophysiology acting through both GR and MR; however, the effects of aldosterone and the potential association of PA and skin disease were not previously addressed. Skin samples from PA revealed histopathological alterations relative to control subjects, featuring epidermal hyperplasia, impaired differentiation, and increased dermal infiltrates, correlating with increased NF-κB signaling and up-regulation of TNF-A and IL-6 cytokines. PA skin samples also showed significantly higher expression of MR, GR, and HSD11B2. In cultured keratinocytes, aldosterone treatment increased GRE transcriptional activity which was significantly inhibited by co-treatment with GR- and MR-antagonists. This study demonstrates that high levels of aldosterone in PA patients correlate with skin anomalies and inflammatory features associated with abnormal GR/MR activation in epidermal keratinocytes.

Epidermal Mineralocorticoid Receptor Plays Beneficial and Adverse Effects in Skin and Mediates Glucocorticoid Responses.

Glucocorticoids (GCs) regulate skin homeostasis and combat cutaneous inflammatory diseases; however, adverse effects of chronic GC treatments limit their therapeutic use. GCs bind and activate the GC receptor and the mineralocorticoid receptor (MR), transcription factors that recognize identical hormone responsive elements. Whether epidermal MR mediates beneficial or deleterious GC effects is of great interest for improving GC-based skin therapies. MR epidermal knockout mice exhibited increased keratinocyte proliferation and differentiation and showed resistance to GC-induced epidermal thinning. However, crucially, loss of epidermal MR rendered mice more sensitive to inflammatory stimuli and skin damage. MR epidermal knockout mice showed increased susceptibility to phorbol 12-myristate 13-acetate-induced inflammation with higher cytokine induction. Likewise, cultured MR epidermal knockout keratinocytes had increased phorbol 12-myristate 13-acetate-induced NF-κB activation, highlighting an anti-inflammatory function for MR. GC-induced transcription was reduced in MR epidermal knockout keratinocytes, at least partially due to decreased recruitment of GC receptor to hormone responsive element-containing sequences. Our results support a role for epidermal MR in adult skin homeostasis and demonstrate nonredundant roles for MR and GC receptor in mediating GC actions.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes.

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.