Neuroprotective effects of violacein in a model of inherited amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.IIBCE (MEC), PEDECIBA y ANII

Modulation of Astrocytic Mitochondrial Function by Dichloroacetate Improves Survival and Motor Performance in Inherited Amyotrophic Lateral Sclerosis

Mitochondrial dysfunction is one of the pathogenic mechanisms that lead to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). Astrocytes expressing the ALS-linked SOD1G93A mutation display a decreased mitochondrial respiratory capacity associated to phenotypic changes that cause them to induce motor neuron death. Astrocyte-mediated toxicity can be prevented by mitochondria-targeted antioxidants, indicating a critical role of mitochondria in the neurotoxic phenotype. However, it is presently unknown whether drugs currently used to stimulate mitochondrial metabolism can also modulate ALS progression. Here, we tested the disease-modifying effect of dichloroacetate (DCA), an orphan drug that improves the functional status of mitochondria through the stimulation of the pyruvate dehydrogenase complex activity (PDH). Applied to astrocyte cultures isolated from rats expressing the SOD1G93A mutation, DCA reduced phosphorylation of PDH and improved mitochondrial coupling as expressed by the respiratory control ratio (RCR). Notably, DCA completely prevented the toxicity of SOD1G93A astrocytes to motor neurons in coculture conditions. Chronic administration of DCA (500 mg/L) in the drinking water of mice expressing the SOD1G93A mutation increased survival by 2 weeks compared to untreated mice. Systemic DCA also normalized the reduced RCR value measured in lumbar spinal cord tissue of diseased SOD1G93A mice. A remarkable effect of DCA was the improvement of grip strength performance at the end stage of the disease, which correlated with a recovery of the neuromuscular junction area in extensor digitorum longus muscles. Systemic DCA also decreased astrocyte reactivity and prevented motor neuron loss in SOD1G93A mice. Taken together, our results indicate that improvement of the mitochondrial redox status by DCA leads to a disease-modifying effect, further supporting the therapeutic potential of mitochondria-targeted drugs in ALS

Molecular Characterization of a Novel Patched-Related Protein in Apis mellifera and Drosophila melanogaster

The molecular identification and characterization of the patched-related (ptr) gene and protein in Apis mellifera and Drosophila melanogaster are reported. Ptr proteins are closely related in predicted topology and domain organization to the protein encoded by the Drosophila segment polarity gene patched. Ptrs have 12 potential transmembrane domains arranged in two sets of 1+5 membrane-spanning segments containing a conserved sterol-sensing domain (SSD) and functional GxxxD and PPXY motifs. Phylogenetic analysis showed that Ptrs belong to a previously uncharacterized class of insect proteins that share a high level of sequence identity. Analysis using quantitative real-time polymerase chain reaction (qPCR) indicates that ptr gene is preferentially expressed during embryo stages of A. mellifera development; interestingly, this pattern of temporal expression was also observed for the D. melanogaster homologue, suggesting that these proteins might be involved in embryo morphogenesis. To understand Ptr function at the molecular level, we investigated the subcellular distribution of DmPtr. We have shown by biochemical analysis that DmPtr protein is tightly associated with membranes. Consistently, Ptr immunoreactivity appears to be localized at the sites of membrane furrow formation during cellularization of D. melanogaster embryos. These studies indicated that Ptrs belong to a previously uncharacterized class of insect transmembrane proteins that share a high level of sequence identity. Our analysis of ptr gene expression and protein localization suggest that Ptr might fulfil a developmental role by participating in processes that require growth and stabilization of plasma membrane.This work was supported by Fondecyt 1050235 (to V.C.). C.B. was supported by fellowships from: CSIC-Programa de recursos humanos (Proyecto 720-contrapartida de convenios), Universidad de la República and AMSUD Pasteur-regional training fellowship program

DCA increases mean survival of SOD1^G93A transgenic mice.

<p>Kaplan-Meyer survival curves from DCA-treated and control SOD1^G93A male (A) and female (B) mice. DCA was administered in drinking water from 70 days of age until death as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034776#s4" target="_blank">materials and methods</a>. 9 animals per group, p<0.05, Kaplan-Meyer log-rank test.</p

DCA prevents SOD1^G93A astrocyte neurotoxicity to motor neurons.

<p>Motor neuron survival 72 h after plating either on non Tg or SOD1^G93A-bearing astrocytes pretreated with DCA or vehicle as indicated. Data are expressed as percentage of non Tg control, mean ± SEM from four independent experiments. *p<0.05, significantly different from non Tg control. **p<0.05, significantly different from SOD1^G93A control.</p

DCA delays loss of grip strength and neuromuscular junction shrinkage in SOD1^G93A mice.

<p>A) Hind-limb grip strength records from non Tg or SOD1^G93A male mice treated with DCA or vehicle as indicated. DCA-treated non Tg animals did not show differences with control ones and data are not shown in order to simplify the graph. Data are mean ± SEM from 9 animals per group. *p<0.05, significantly different from SOD1^G93A control. B) ACh receptors labeled with TMR-BgTx in representative EDL neuromuscular junctions from non Tg (top), SOD1^G93A control (middle) or DCA-treated SOD1^G93A (bottom). Quantification of total TMR-BgTx-stained neuromuscular area in the different groups of animals. Data are expressed as percentage of non Tg control, mean ± SEM from 15–35 neuromuscular junctions from 2–4 animals per group. *p<0.05, significantly different from non Tg control. **p<0.05, significantly different from SOD1^G93A control. Scale bar: 30 µm.</p

DCA recovers mitochondrial respiration rate and controls proliferation in SOD1^G93A astrocytes.

<p>A) Representative immunoblot for PDH-E1α(pSer²⁹³), total PDH-E1α, and β-actin of lysates from non Tg and SOD1^G93A astrocytes after 24 h treatment with DCA or vehicle as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034776#s4" target="_blank">Methods</a>. B) Quantification of the PDH-E1α(pSer²⁹³) to total PDH-E1α ratio between relative densitometric levels normalized against vehicle-treated non Tg astrocytes. C) Calculated respiratory control ratio (RCR) for mitochondria from non Tg or SOD1^G93A-bearing astrocytes treated with DCA or vehicle as indicated. D) Percentage of BrdU immunoreactive nuclei of non Tg and SOD1^G93A astrocytes after 24 h treatment with DCA. Data for panels B, C, and D are expressed as mean ± SEM from three independent experiments performed in duplicate. *p<0.05, significantly different from non Tg control. **p<0.05, significantly different from SOD1^G93A control.</p

Site of action of dichloroacetate.

<p>DCA inhibits the mitochondrial enzyme PDH kinase, thereby maintaining the PDH complex in its unphosphorylated catalytically active state and facilitating the aerobic oxidation of glucose.</p