A positive regulatory loop between mitochondrial activity and nuclear genes such as PGC-1α and PPARδ could be able to coordinate the increase in oxidative metabolism and the expression of slower myosin isoforms.

<p>A stimulation of mitochondrial activity induced by p43, could modulate Ca²⁺ signalling and mitochondrial reactive oxygen species production, which are well known for altering the activity of some transcriptional factors such as NFκB and AP-1. In this way mitochondrial activity could affect nuclear gene expression of proteins such as PGC-1α and PPARδ which are involved in muscle specification and in the regulation of mitochondrial biogenesis. This positive regulatory loop could be deeply involved in the determination of muscle metabolic and contractile phenotypes.</p

p43 overexpression increases mitochondrial transcription and mitochondrial activity.

<p>(A) Relative mRNA expression levels of mitochondrial transcripts (COXII and ND2) in quadriceps muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). **p<0.01. (B) Enzymatic activities of mitochondrial respiratory chain complexes in quadriceps muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). COX: cytochrome c oxydase; CXII: complex II. **p<0.01.</p

p43 increase muscle oxidative fibers.

<p>(A) Hindlimb from transgenic mice of the 86 line compared to wild-type animals at 2 months of age. (B) Relative mRNA expression levels of the four adult MyHC isoforms in the indicated muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). QUA: quadriceps; SOL: soleus. *p<0.05; **p<0.01. (C) Immunohistochemical analysis of gastrocnemius muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age, using antibodies raised against MyHC Type IIa or IIa+IIb. (D) Immunohistochemical analysis of soleus muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age, using antibodies raised against MyHC Type I or IIa. (E) PPARδ protein levels in quadriceps muscle mitochondria from transgenic mice of the 86 line versus wild-type animals, visualized by western-blot using an antibody raised against PPARδ. 50 µg of total protein extracts were analyzed.</p

p43 stimulates mitochondrial biogenesis.

<p>(A) electronic microscopy experiments from longitudinal section taken from quadriceps muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (magnification ×10.000 and ×20.000). Arrows indicate mitochondria. (B) Relative mtDNA content in quadriceps muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). After extraction of muscle DNA, quantitative PCR reactions were performed using ND5 for mtDNA copy estimation, and 18S for the nuclear genome. Ratio ND5/18S was expressed as percent of control value. *p<0.05. (C) Citrate Synthase activity in quadriceps muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). CS: citrate synthase. **p<0.01. (D) Relative mRNA expression levels of the indicated genes from gastrocnemius muscle from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). *p<0.05; **p<0.01; ***p<0.001.</p

p43 overexpression increases body temperature.

<p>(A) Body temperature from transgenic mice of the 86 line compared to wild-type animals at 2 months of age. ***p<0.001. (B) Levels of total T4, total T3 and free T3 in serum from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). T4, total T3 and free T3 were measured using a direct competitive radioimmunoassay from MP-Biomedicals. *p<0.05. (C) Relative mRNA expression levels of type 2 iodothyronine deiodinase (Dio2) in quadriceps from transgenic mice of the 86 line compared to wild-type animals at 2 months of age (n = 8 each group). *p<0.05.</p

Data_Sheet_1_Case report: Thirty-year progression of an EMPF1 encephalopathy due to defective mitochondrial and peroxisomal fission caused by a novel de novo heterozygous DNM1L variant.PDF

Mutations in DNM1L (DRP1), which encode a key player of mitochondrial and peroxisomal fission, have been reported in patients with the variable phenotypic spectrum, ranging from non-syndromic optic atrophy to lethal infantile encephalopathy. Here, we report a case of an adult female patient presenting with a complex neurological phenotype that associates axonal sensory neuropathy, spasticity, optic atrophy, dysarthria, dysphasia, dystonia, and ataxia, worsening with aging. Whole-exome sequencing revealed a heterozygous de novo variant in the GTPase domain of DNM1L [NM_001278464.1: c.176C>A p.(Thr59Asn)] making her the oldest patient suffering from encephalopathy due to defective mitochondrial and peroxisomal fission-1. In silico analysis suggested a protein destabilization effect of the variant Thr59Asn. Unexpectedly, Western blotting disclosed profound decrease of DNM1L expression, probably related to the degradation of DNM1L complexes. A detailed description of mitochondrial and peroxisomal anomalies in transmission electron and 3D fluorescence microscopy studies confirmed the exceptional phenotype of this patient.</p

Data_Sheet_2_Case report: Thirty-year progression of an EMPF1 encephalopathy due to defective mitochondrial and peroxisomal fission caused by a novel de novo heterozygous DNM1L variant.PDF

Mutations in DNM1L (DRP1), which encode a key player of mitochondrial and peroxisomal fission, have been reported in patients with the variable phenotypic spectrum, ranging from non-syndromic optic atrophy to lethal infantile encephalopathy. Here, we report a case of an adult female patient presenting with a complex neurological phenotype that associates axonal sensory neuropathy, spasticity, optic atrophy, dysarthria, dysphasia, dystonia, and ataxia, worsening with aging. Whole-exome sequencing revealed a heterozygous de novo variant in the GTPase domain of DNM1L [NM_001278464.1: c.176C>A p.(Thr59Asn)] making her the oldest patient suffering from encephalopathy due to defective mitochondrial and peroxisomal fission-1. In silico analysis suggested a protein destabilization effect of the variant Thr59Asn. Unexpectedly, Western blotting disclosed profound decrease of DNM1L expression, probably related to the degradation of DNM1L complexes. A detailed description of mitochondrial and peroxisomal anomalies in transmission electron and 3D fluorescence microscopy studies confirmed the exceptional phenotype of this patient.</p

Influence of p43 overexpression on exercise endurance and on muscle contractile activities at 2 and 6 months of age.

<p>(A–B) 2 and 6 month-old transgenic mice (p43) and wild-type controls (WT) with similar body weights (n = 6 for each groups) were subjected to a forced treadmill exercise test. Running distance (A) and running time (B) are indicated. (C–D) Muscle contractile studies of 2 and 6 month-old transgenic mice (p43) and wild-type controls (WT) with similar body weights (n = 6 for each groups). Po (C) and Muscle fatigue index (D) are indicated. *p<0.05, **p<0.01 and ***p<0.001.</p

Influence of p43 overexpression during aging on mitochondrial biogenesis.

<p>(A) electron microscopy experiments from longitudinal sections taken from quadriceps from transgenic mice (p43) and wild-type animals (WT) at 2, 6 and 11 months of age (magnification ×10.000). (B) Relative mtDNA content in quadriceps muscle from transgenic mice (p43) compared to wild-type animals (WT) at 2, 6, 11 and 23 months of age (n = 8 for each group with the exception of 23 month old-group where n = 6). After extraction of muscle DNA, quantitative PCR reactions were performed using ND5 for mtDNA copy estimation, and 18S for the nuclear genome. *p<0.05 and ***p<0.001. Control values at each stage are considered as 100%, and p43 value are expressed in percent of the corresponding control value.</p

Influence of p43 overexpression during aging on mitochondrial activity.

<p>(A–D) Complex I (CXI), Complex II (CXII), Cytochrome oxidase (COX), and Citrate Synthase (CS) activities in gastrocnemius muscle extracts from transgenic mice (p43) and wild-type animals (WT) at 2, 6, 11 and 23 months of age (n = 8 for each group with the exception of 23 month old-group where n = 6). *p<0.05, **p<0.01 and ***p<0.001.</p