Aging Triggers Cytoplasmic Depletion and Nuclear Translocation of the E3 Ligase Mahogunin: A Function for Ubiquitin in Neuronal Survival.

A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination.

Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells.

Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III-IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.

[MGRN1 affects the mitophagy of spermatogonial stem cells in mice].

Objective: To study the effect of mahogunin ring finger-1 (MGRN1) on the mitophagy of the spermatogonial stem cells (SSC) in mice. METHODS: SSCs cultured in vitro were divided into three groups: empty vector control, MGRN1 (MGRN1 in SSCs knocked down by RNAi), and MGRN1 + FCCP (inducing mitophagy with carbonyl cyanide p-trifluoromethoxyphenylhydrazone ï¼»FCCPï¼½ in the SSCs with down-regulated MGRN1). The expressions of mitochondrial function-related proteins (Cytochromo c and COX IV) and mitophagy-related proteins (LC3, P62, FUNDC1 and CK2) and the phosphorylation of FUNDC1 were detected by Western blot. Mitochondria and mitochondrial autophagosomes in the SSCs were observed under the electron microscope. RESULTS: Compared with the empty vector control group, the MGRN1 and MGRN1 + FCCP groups showed significantly down-regulated expressions of Cytochromo c, Cox IV, LC3 and P62, increased phosphorylation level of FUNDC1, and up-regulated expression of CK2 in the SSCs (P < 0.05). No statistically significant differences were found in the expressions of Cytochromo c, Cox IV, LC3, P62 and CK2 or in the phosphorylation level of FUNDC1 between the MGRN1 and MGRN1 + FCCP groups (P > 0.05). Electron microscopy manifested increased mitochondrial damage and reduced mitochondrial autophagosomes in the SSCs in the MGRN1 and MGRN1 + FCCP groups compared with those in the control group. CONCLUSIONS: MGRN1 affects mitophagy in the SSCs of mice, which may be associated with the effect of CK2 on the phosphorylation of FUNDC1, and its molecular mechanism needs to be further studied.

MGRN1-mediated ubiquitination of α-tubulin regulates microtubule dynamics and intracellular transport.

MGRN1-mediated ubiquitination of α-tubulin regulates microtubule stability and mitotic spindle positioning in mitotic cells. This study elucidates the effect of MGRN1-mediated ubiquitination of α-tubulin in interphase cells. Here, we show that MGRN1-mediated ubiquitination regulates dynamics of EB1-labeled plus ends of microtubules. Intracellular transport of mitochondria and endosomes are affected in cultured cells where functional MGRN1 is depleted. Defects in microtubule-dependent organellar transport are evident in cells where noncanonical K6-mediated ubiquitination of α-tubulin by MGRN1 is compromised. Loss of MGRN1 has been previously correlated with late-onset spongiform neurodegeneration. Mislocalised cytosolically exposed PrP (Ctm PrP) interacts with MGRN1 leading to its loss of function. Expression of Ctm PrP generating mutants of PrP[PrP(A117V) and PrP(KHII)] lead to decrease in MGRN1-mediated ubiquitination of α-tubulin and intracellular transport defects. Brain lysates from PrP(A117V) transgenic mice also indicate loss of tubulin polymerization as compared to non-transgenic controls. Depletion of MGRN1 activity may hamper physiologically important processes like mitochondrial movement in neuronal processes and intracellular transport of ligands through the endosomal pathway thereby contributing to the pathogenesis of neurodegeneration in certain types of prion diseases.

The Role of gp91phox and the Effect of Tranexamic Acid Administration on Hair Color in Mice.

We observed that on long-term breeding, gp91phox-knockout (gp91phox-/-) mice developed white hair. Here, we investigate the origin of this hitherto unexplained phenomenon. Moreover, we investigated the effect of tranexamic acid administration on the hair color in gp91phox-/- mice. We administered tranexamic acid (about 12 mg/kg/day) orally to 9-week-old C57BL/6j (control) and gp91phox-/- mice, thrice a week for 12 months. Compared to control mice, gp91phox-/- mice showed more white hair. However, the concentrations of reactive oxygen species and the levels of interleukin (IL)-1ß and transforming growth factor (TGF)-ß in the skin were lower than those in the control group. Furthermore, increase in white hair was observed in the control mice upon administration of the IL-1ß antagonist. On the other hand, administration of tranexamic acid led to brown colored hair on gp91phox-/- mice. Although tranexamic acid treatment did not alter the expression levels of melanocortin receptor 1 and agouti signaling protein on hair follicles, it increased the expression of mahogunin ring finger protein 1 (MGRN1) and collagen XVII. These results suggested that retention of black hair requires the gp91phox/ROS/IL-1ß/TGF-ß pathway and that elevated levels of MGRN1 and collagen XVII lead to brown hair in gp91phox-/- mice.

Oligodendroglial deletion of ESCRT-I component TSG101 causes spongiform encephalopathy.

BACKGROUND INFORMATION: Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery. As loss of MGRN1 is predicted to cause partial TSG101 loss-of-function, we hypothesised that CNS vacuolation in Mgrn1 null mice may be caused by the accumulation of multi-cisternal endosome-like 'class E' vacuolar protein sorting (vps) compartments similar to those observed in Tsg101-depleted cells in culture. RESULTS: To test this hypothesis, Tsg101 was deleted from mature oligodendroglia in vivo. This resulted in severe spongiform encephalopathy, histopathologically similar to that observed in Mgrn1 null mutant mice but with a more rapid onset. Vacuoles in the brains of Tsg101-deleted and Mgrn1 mutant mice labelled with endosomal markers, consistent with an endosomal origin. Vacuoles in the brains of mice inoculated with Rocky Mountain Laboratory (RML) prions did not label with these markers, indicating a different origin, consistent with previously published studies that indicate RML prions have a primary effect on neurons and cause vacuolation in an MGRN1-independent manner. Oligodendroglial deletion of Rab7, which mediates late endosome-to-lysosome trafficking and autophagosome-lysosome fusion, did not cause spongiform change. CONCLUSIONS: Our data suggest that the formation of multi-cisternal 'class E' vps endosomal structures in oligodendroglia leads to vacuolation. SIGNIFICANCE: This work provides the first evidence that disrupting multi-vesicular body formation in oligodendroglia can cause white matter vacuolation and demyelination. HIV is known to hijack the endosomal sorting machinery, suggesting that HIV infection of the CNS may also act through this pathway to cause encephalopathy.

Pleiotropic effects of coat colour-associated mutations in humans, mice and other mammals.

The characterisation of the pleiotropic effects of coat colour-associated mutations in mammals illustrates that sensory organs and nerves are particularly affected by disorders because of the shared origin of melanocytes and neurocytes in the neural crest; e.g. the eye-colour is a valuable indicator of disorders in pigment production and eye dysfunctions. Disorders related to coat colour-associated alleles also occur in the skin (melanoma), reproductive tract and immune system. Additionally, the coat colour phenotype of an individual influences its general behaviour and fitness. Mutations in the same genes often produce similar coat colours and pleiotropic effects in different species (e.g., KIT [reproductive disorders, lethality], EDNRB [megacolon] and LYST [CHS]). Whereas similar disorders and similar-looking coat colour phenotypes sometimes have a different genetic background (e.g., deafness [EDN3/EDNRB, MITF, PAX and SNAI2] and visual diseases [OCA2, RAB38, SLC24A5, SLC45A2, TRPM1 and TYR]). The human predilection for fancy phenotypes that ignore disorders and genetic defects is a major driving force for the increase of pleiotropic effects in domestic species and laboratory subjects since domestication has commenced approximately 18,000 years ago.

The effect of mahogunin gene mutant on reproduction in male mice: a new sight for infertility?

Mahogunin is an important mediator of chromogenesis and neurodegeneration. Mahoganoid is a mutation of the mahogunin gene, which causes a pleiotropic phenotype that includes suppression of obesity, spongiform neurodegeneration and improvement of insulin sensitivity. Our previous research found that mahoganoid widely expressed in the male rat reproductive system, and mahoganoid-deficient mice have reduced embryonic viability. But the reproductive change in mahogunin knockout (md(nc) ) male mice has not been reported previously. Here, we report that the mahogunin mRNA also widely exists in reproductive system of male mice, and its mRNA expression in the testis was in accordance with the first spermatogenesis wave cycle. Moreover, we find that md(nc) male mice were able to mate with females but no pups are delivered. Besides, the sperms' active progressive motility and hormone secretion (E2, FSH, LH, PRL) were obviously decreased while abnormal sperm rate showed no significant difference in md(nc) compared to wild-type (WT) male mice. This study indicates the mahogunin deficiency results in the infertility of male mice, disruption of hormones secretion and impaired active progressive motility, which may additionally illuminate the aetiology of male infertility in human.

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker.

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.

Mahogunin Ring Finger 1 Is Required for Genomic Stability and Modulates the Malignant Phenotype of Melanoma Cells.

The mouse mahoganoid mutation abrogating Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1-knockout melanocytes with genetically matched controls and melan-md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger γH2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea-induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16-F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1-KO B16-F10 cells had lower mitotic indices, fewer Ki67-positive cells and showed a trend towards smaller size. In short-term lung colonization assays Mgrn1-KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.

E3 ligase mahogunin (MGRN1) influences amyloid precursor protein maturation and secretion.

Altered processing of the Amyloid Precursor Protein (APP) is a well-recognized central pathogenic mechanism in Alzheimer's Disease (AD), and regulation of APP processing is a major focus of research in the AD field. However, how age-associated cellular and molecular changes contribute to changes in the amyloidogenic processing of APP have not been extensively clarified so far. We here provide evidence that the processing of APP is influenced by the e3 ubiquitin ligase Mahogunin (MGRN1), a neuroprotective molecule whose levels decrease with aging. Specifically, the expression of MGRN1 inhibits the maturation of APP by sequestering it in the secretory pathway. This sequestration significantly delayed the proteolytic processing of APP, resulting in a reduced ß-amyloid (Aß) peptide release into the extracellular environment. Accordingly, a reduction of MGRN1 levels in hippocampal neurons, as it occurs during physiological aging, leads to an increased Aß40 and Aß42 release. We therefore propose that age contributes to the amyloidogenic processing of APP by altering its intracellular trafficking along the secretory pathway due in part to the down-regulation of MGRN1.

RML prions act through Mahogunin and Attractin-independent pathways.

While the conversion of the normal form of prion protein to a conformationally distinct pathogenic form is recognized to be the primary cause of prion disease, it is not clear how this leads to spongiform change, neuronal dysfunction and death. Mahogunin ring finger-1 (Mgrn1) and Attractin (Atrn) null mutant mice accumulate vacuoles throughout the brain that appear very similar to those associated with prion disease, but they do not accumulate the protease-resistant scrapie form of the prion protein or become sick. A study demonstrating an interaction between cytosolically-exposed prion protein and MGRN1 suggested that disruption of MGRN1 function may contribute to prion disease pathogenesis, but we recently showed that neither loss of MGRN1 nor MGRN1 overexpression influences the onset or progression of prion disease following intracerebral inoculation with Rocky Mountain Laboratory prions. Here, we show that loss of ATRN also has no effect on prion disease onset or progression and discuss possible mechanisms that could cause vacuolation of the central nervous system in Mgrn1 and Atrn null mutant mice and whether the same pathways might contribute to this intriguing phenotype in prion disease.

Functional conservation between mammalian MGRN1 and plant LOG2 ubiquitin ligases.

Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.