An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy.

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations

Alteration of the late endocytic pathway in Charcot-Marie-Tooth type 2B disease

The small GTPase RAB7A regulates late stages of the endocytic pathway and plays specific roles in neurons, controlling neurotrophins trafficking and signaling, neurite outgrowth and neuronal migration. Mutations in the RAB7A gene cause the autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) disease, an axonal peripheral neuropathy. As several neurodegenerative diseases are caused by alterations of endocytosis, we investigated whether CMT2B-causing mutations correlate with changes in this process. To this purpose, we studied the endocytic pathway in skin fibroblasts from healthy and CMT2B individuals. We found higher expression of late endocytic proteins in CMT2B cells compared to control cells, as well as higher activity of cathepsins and higher receptor degradation activity. Consistently, we observed an increased number of lysosomes, accompanied by higher lysosomal degradative activity in CMT2B cells. Furthermore, we found increased migration and increased RAC1 and MMP-2 activation in CMT2B compared to control cells. To validate these data, we obtained sensory neurons from patient and control iPS cells, to confirm increased lysosomal protein expression and lysosomal activity in CMT2B-derived neurons. Altogether, these results demonstrate that in CMT2B patient-derived cells, the endocytic degradative pathway is altered, suggesting that higher lysosomal activity contributes to neurodegeneration occurring in CMT2B.Peer reviewe

Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.

The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the 'Non-Specific-Lethal' (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF's overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies

Synergistic Effect of Mitochondrial and Lysosomal Dysfunction in Parkinson’s Disease

Crosstalk between lysosomes and mitochondria plays a central role in Parkinson’s Disease (PD). Lysosomal function may be influenced by mitochondrial quality control, dynamics and/or respiration, but whether dysfunction of endocytic or autophagic pathway is associated with mitochondrial impairment determining accumulation of defective mitochondria, is not yet understood. Here, we performed live imaging, western blotting analysis, sequencing of mitochondrial DNA (mtDNA) and senescence-associated beta-galactosidase activity assay on primary fibroblasts from a young patient affected by PD, her mother and a healthy control to analyze the occurrence of mtDNA mutations, lysosomal abundance, acidification and function, mitochondrial biogenesis activation and senescence. We showed synergistic alterations in lysosomal functions and mitochondrial biogenesis, likely associated with a mitochondrial genetic defect, with a consequent block of mitochondrial turnover and occurrence of premature cellular senescence in PARK2-PD fibroblasts, suggesting that these alterations represent potential mechanisms contributing to the loss of dopaminergic neurons

Synergistic Effect of Mitochondrial and Lysosomal Dysfunction in Parkinson\u2019s Disease

Crosstalk between lysosomes and mitochondria plays a central role in Parkinson\u2019s Disease (PD). Lysosomal function may be influenced by mitochondrial quality control, dynamics and/or respiration, but whether dysfunction of endocytic or autophagic pathway is associated with mitochondrial impairment determining accumulation of defective mitochondria, is not yet understood. Here, we performed live imaging, western blotting analysis, sequencing of mitochondrial DNA (mtDNA) and senescence-associated beta-galactosidase activity assay on primary fibroblasts from a young patient affected by PD, her mother and a healthy control to analyze the occurrence of mtDNA mutations, lysosomal abundance, acidification and function, mitochondrial biogenesis activation and senescence. We showed synergistic alterations in lysosomal functions and mitochondrial biogenesis, likely associated with a mitochondrial genetic defect, with a consequent block of mitochondrial turnover and occurrence of premature cellular senescence in PARK2-PD fibroblasts, suggesting that these alterations represent potential mechanisms contributing to the loss of dopaminergic neurons

Characterization of MOF mutants in cells.

<p>(A) Nuclear localization of MOF point mutants after depletion of endogenous MOF. Stable cell lines expressing either MOF-GFP wt or MOF-GFP bearing the indicated point mutants were stained with antibodies against GFP and MSL3 as indicated. DNA was counterstained with DAPI. The GFP staining reveals the localization of the ectopic MOF, whereas MSL3 staining provides a reference for the X chromosome territory and for possible dominant negative effects of MOF mutants. The experiment was carried out in three biological replicates for MOF wt, 7KN and 9KN, 2 biological replicates are shown for 2KN. Scale bars: 10 μm. (B) Quantification of GFP immunofluorescence [data in (A)] using CellProfiler. For each cell line the median GFP signal within the nuclei (segmented on the DAPI staining) is plotted, revealing the levels of transgene expression. Non-transfected S2 cells served as GFP-negative control. The black bar indicates the median signal, the box plot presents the standard deviation. The scaling of the y-axis is logarithmic. (C) Quantification of the localization of MOF-GFP to the X-chromosome [data in (A)]. Log enrichment ratios were calculated as territorial signals computationally segmented on the MSL3 staining and the mean intensity of the nuclei (segmented on the DAPI staining). The black bar indicates average GFP enrichment for each cell line. (D) Quantification of the effect of MOF mutants on the localization of MSL3 to the X-chromosomal territories [data in (A)]. For each staining log enrichment ratios were calculated as territorial signals computationally segmented on the MSL3 staining and the mean intensity of the nuclei (segmented on the DAPI staining). The black bar indicates average MSL3 enrichment for each cell line. (E) Association of C-terminal MOF mutants with the MSL-DCC. Extracts from control cells (S2) or from cell lines stably expressing MOF-GFP-wt or the indicated mutated forms were immunoprecipitated with the GFP-trap. Western blots of input lysates or immunoprecipitates were analyzed with antibodies against MOF, MSL1, MSL2, MSL3, MLE and lamin as indicated. Protein size markers (kDa) are indicated to the left. The experiment was repeated in biological triplicates with the same outcome.</p

Functional viability rescue by MOF mutant enzymes.

<p>(A) Survival of MOF-deficient male flies upon expression of MOF point mutants (KN). Male survival was assayed upon expression of the indicated MOF transgenes in the <i>mof</i>^<i>2</i> male lethal background (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177408#sec012" target="_blank">Methods</a>). Male survival was scored as ratio <i>mof</i>^<i>2</i><i>/y</i> males to <i>mof</i>^<i>2</i><i>/Fm7</i> females resulting from the same cross (relative male survival). Error bars represent the standard error of the mean of three biological replicates. (B) Survival of <i>mof</i>^<i>2</i> males upon expression of MOF mutants (KC) as in (A). Error bars represent the standard error of the mean of five biological replicates.</p

MSL2-mediated MOF ubiquitylation in vitro is inhibited by DNA.

<p>In vitro ubiquitylation assays in presence of DNA. Assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177408#pone.0177408.g001" target="_blank">Fig 1C</a>. A saturating amount of DNA (1 μM) was added to the reactions as indicated. Ubiquitylated proteins were detected using antibodies against ubiquitin (aUb) and MOF (aMOF). Protein size markers (kDa) are indicated to the left. The experiment was repeated 3 times with similar outcome.</p

MSL2 ubiquitylates MOF <i>in vitro</i>.

<p>(A) Schematic representation of MOF protein and the ubiquitylated lysines identified by mass spectrometry in vitro. MOF contains an unstructured N-terminal region, a globular chromobarrel and a histone acetyltransferase domains (light and dark gray box, respectively). MOF comprises a C2HC Zinc finger within the histone acetyltransferase domain (black box). Lysines that are ubiquitylated by MSL2 are indicated. Bold bars mark ubiquitylation found in 3 out of 3 biological replicates, dashed bars point to ubiquitylation only detected in 1 out of 3 experiments. (B) Schematic representation of MOF mutants generated in this study. Black bars indicate K to R mutations. Red asterisks indicate the lysines that are ubiquitylated in vitro as in (A). (C) In vitro ubiquitylation of MOF wt and KN MOF mutants. Ubiquitylation assays contained recombinant E1 and E2 enzymes, his-ubiquitin and ATP. MSL2 and different MOF substrates were added as indicated. Ubiquitylated proteins were detected by Western blotting using antibodies specific for ubiquitin (aUb, top) and MOF (aMOF, bottom). In the absence of substrate protein MSL2 exhibits autoubiquitylation on itself as detected in lane 8. Protein size markers are indicated to the left (kDa). Red asterisks indicate bands that correspond to ubiquitylated forms of MOF. (D) In vitro ubiquitylation as in (C) with KC MOF mutant substrates. (E) In vitro ubiquitylation as in (C) with MOF ΔN substrate. (F) In vitro ubiquitylation in (C) with MOF-Nt substrate.</p

Cloning primer for generation of MOF mutant constructs.

<p>Cloning primer for generation of MOF mutant constructs.</p