Acute focal brain damage alters mitochondrial dynamics and autophagy in axotomized neurons

Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.Mitochondria are essential organelles for cell function and viability, and are central to several processes such as energy production, metabolism, calcium buffering, and life/death decisions.¹ Neurons have a high and constant demand for mitochondrial metabolism, to maintain their functions, and contain many mitochondria throughout the cytoplasm, distributed to axons, presynaptic terminals, and dendritic shafts. Mitochondria are highly dynamic organelles that continuously move and change shape. Their morphology is governed by the dynamic equilibrium between fusion and fission processes, both of which are mediated by evolutionarily conserved members of the dynamin family of large GTPases.² Fusion between the outer mitochondrial membranes (OMMs) is mediated by membrane-anchored mitofusins (Mfn1 and Mfn2), whereas that between inner mitochondrial membranes is controlled by optic atrophy 1.³ Mitochondrial fission is regulated by dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1).⁴ Drp1 is predominantly expressed in the cytoplasm and is recruited to mitochondria, where it associates with Fis1 to form a complex that constricts the inner and outer membranes, allowing mitochondria to divide.^{5, 6}Mitochondrial dynamics are crucial to the maintenance of mitochondrial function and neuron survival, as evidenced by findings that pathological imbalances between fusion and fission events develop in many neurodegenerative disorders and brain trauma.^{7, 8} Moreover, mitochondrial fission regulates organelle shape and mediates mitochondria-dependent cell death.⁹ The release of proapoptotic factors, such as cytochrome c (with consequent formation of the apoptosome and caspase activation), from depolarized mitochondria into the cytosol is a significant event in the induction of apoptosis and is associated with Drp1-mediated fragmentation of the mitochondrial network.¹⁰The elimination of dysfunctional mitochondria is therefore a key process with regard to the viability of neurons (and other cell types). Damaged mitochondria that accelerate cell death are removed through autophagy, an evolutionarily conserved lysosome-mediated degradation pathway that maintains the balance between organelle biogenesis, protein synthesis, and degradation of cellular components.¹¹Mitochondria can be selectively degraded by autophagy – a pathway known as mitophagy.¹² Priming of damaged mitochondria can involve several mechanisms, one of which is triggered by Parkin, a cytosolic E3 ubiquitin ligase that is mutated in familial forms of Parkinson''s disease (PD).¹³ Parkin recruitment to impaired mitochondria requires the kinase activity of PINK1 (PTEN-induced putative kinase 1),^{14, 15, 16} a serine/threonine kinase that is also mutated in other autosomal recessive forms of PD.¹⁷ PINK1 levels are very low in polarized mitochondria, to prevent mitophagy of healthy mitochondria; in contrast, when mitochondria are depolarized, full-length PINK1 accumulates rapidly at damaged organelles, crossing the OMM and acting as cellular sensors of damaged mitochondria.¹⁵ PINK1 then recruits Parkin to the mitochondrial surface, where it ubiquitinates several OMM proteins, which in turn recruit other proteins to initiate mitophagy.¹⁸ Mfn1 is a direct substrate of Parkin and its degradation has been suggested to prevent the fusion of damaged and healthy mitochondria.¹⁹ Drp1-dependent fission of mitochondria is also a crucial event that effects their degradation through mitophagy and the inhibition of fission specifically prevents mitochondrial autophagy.²⁰In this study, we examined mitochondrial function and its relationship with autophagy machinery in an in vivo model of acute focal CNS (central nervous system) lesion, focusing on remote changes that are induced by hemicerebellectomy (HCb). Unilateral HCb is a suitable model in which axonal damage-induced neuronal death mechanisms can be studied.^{21, 22} In this model, neuronal degeneration is caused by target deprivation and axonal damage of contralateral precerebellar nuclei of the inferior olive and pontine nuclei. Remote damage is a multifactorial phenomenon in which many components become active in specific time frames²¹ and is significant in determining the overall clinical outcome in many CNS pathologies, including spinal cord injury and traumatic brain injury.^{23, 24, 25}Recently, we demonstrated that autophagy is activated in axotomized neurons after HCb, subsequent to cytochrome c release from mitochondria.²⁶ Further, rapamycin-enhanced autophagy has neuroprotective effects, reducing neuronal death and improving functional recovery. In this study, we examined mitochondrial dynamics and function in axotomized neurons after HCb in mice and analysed the effects of rapamycin on selective elimination of damaged mitochondria.     

Di-(2-ethylhexyl) phthalate and autism spectrum disorders

ASDs (autism spectrum disorders) are a complex group of neurodevelopment disorders, still poorly understood, steadily rising in frequency and treatment refractory. Extensive research has been so far unable to explain the aetiology of this condition, whereas a growing body of evidence suggests the involvement of environmental factors. Phthalates, given their extensive use and their persistence, are ubiquitous environmental contaminants. They are EDs (endocrine disruptors) suspected to interfere with neurodevelopment. Therefore they represent interesting candidate risk factors for ASD pathogenesis. The aim of this study was to evaluate the levels of the primary and secondary metabolites of DEHP [di-(2-ethylhexyl) phthalate] in children with ASD. A total of 48 children with ASD (male: 36, female: 12; mean age: 11±5 years) and age- and sex-comparable 45 HCs (healthy controls; male: 25, female: 20; mean age: 12±5 years) were enrolled. A diagnostic methodology, based on the determination of urinary concentrations of DEHP metabolites by HPLC-ESI-MS (HPLC electrospray ionization MS), was applied to urine spot samples. MEHP [mono-(2-ethylhexenyl) 1,2-benzenedicarboxylate], 6-OH-MEHP [mono-(2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate], 5-OH-MEHP [mono-(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate] and 5-oxo-MEHP [mono-(2-ethyl-5-oxohexyl) 1,2-benzenedicarboxylate] were measured and compared with unequivocally characterized, pure synthetic compounds (>98%) taken as standard. In ASD patients, significant increase in 5-OH-MEHP (52.1%, median 0.18) and 5-oxo-MEHP (46.0%, median 0.096) urinary concentrations were detected, with a significant positive correlation between 5-OH-MEHP and 5-oxo-MEHP (r_s = 0.668, P<0.0001). The fully oxidized form 5-oxo-MEHP showed 91.1% specificity in identifying patients with ASDs. Our findings demonstrate for the first time an association between phthalates exposure and ASDs, thus suggesting a previously unrecognized role for these ubiquitous environmental contaminants in the pathogenesis of autism.     

Differentiation of single cell derived human mesenchymal stem cells into cells with a neuronal phenotype: RNA and microRNA expression profile

The adult bone marrow contains a subset of non-haematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi-lineage differentiation. The molecular mechanisms governing the self-renewal and differentiation of MSCs remain largely unknown. In a previous paper we demonstrated the ability to induce human clonal MSCs to differentiate into cells with a neuronal phenotype (DMSCs). In the present study we evaluated gene expression profiles by Sequential Analysis of Gene Expression (SAGE) and microRNA expression profiles before and after the neuronal differentiation process. Various tissue-specific genes were weakly expressed in MSCs, including those of non-mesodermal origin, suggesting multiple potential tissue-specific differentiation, as well as stemness markers. Expression of OCT4, KLF4 and c-Myc cell reprogramming factors, which are modulated during the differentiation process, was also observed. Many peculiar nervous tissue genes were expressed at a high level in DMSCs, along with genes related to apoptosis. MicroRNA profiling and correlation with mRNA expression profiles allowed us to identify putative important genes and microRNAs involved in the differentiation of MSCs into neuronal-like cells. The profound difference in gene and microRNA expression patterns between MSCs and DMSCs indicates a real functional change during differentiation from MSCs to DMSCs.     

Uric acid activates NRLP3 inflammasome in an in-vivo model of epithelial to mesenchymal transition in the kidney

Uric acid (UA) has been associated with renal fibrosis and progression of chronic kidney disease. However, the underlying mechanisms of this process have still not been identified. Here, we studied the role of the innate imunity receptor NLRP3/ASC in UA induced epithelial-mesenchymal transition (EMT) in kidney. Wistar rats were fed with oxonic acid 2% and UA 2% (OXA?+?U), OXA?+?U plus allopurinol (ALL) or regular chow (C) for 7 weeks. We analyzed the presence of EMT markers, the expression of NLRP3, ASC, Caspase-1 and Smad 2/3 molecules and the mitochondrial morphological and functional characteristics. High UA induced renal fibrosis, mild chronic inflammation, as well as morphological and biochemical evidence of EMT. High UA also increased the expression of NLRP3/ASC with activation of both inflammasome related caspase-1 and inflammasome unrelated Smad 2/3 pathways. Ultrastructural co-localization of NLRP3 and Smad 2/3 indicated physical interaction between the two molecules. No morphological or functional changes were found between mitochondria exposed to high UA. In conclusion, kidney epithelial NLRP3/ASC expression was increased in high UA state in rats and both inflammasome related caspase-1 and non-inflammasome related P-Smad 2/3 pathways were associated with the observed EMT, inflammation and fibrosis induced by UA in the kidney.     

Remodeling of uterine innervation

This minireview reports current hypotheses concerning the remodeling of sympathetic innervation in rodent and human uterus during the estrous cycle and gestation. Neural modulation in this organ is related to sexual hormone concentrations, and a reduction in nerve density is observed when estrogen levels are high during the estrous cycle. Estrogen receptor alpha is considered to be the major receptor mediating the action of estrogen. In the uterus, the expression of neurotrophins, such as nerve growth factor, which are involved in the survival and growth of nerve fibers, changes in response to steroid levels. Despite much research, further studies are necessary to clarify various aspects of nerve growth control under diverse physiological conditions. These studies could be of importance, since alterations of the biological mechanisms of uterus innervation may play significant roles in various pathologies, such as infertility and spontaneous abortion.     

Induction of oxidative stress by the metabolites accumulating in 3-methylglutaconic aciduria in cerebral cortex of young rats

3-methylglutaconic (MGT), 3-methylglutaric (MGA) and occasionally 3-hydroxyisovaleric (OHIVA) acids accumulate in a group of diseases known as 3-methylglutaconic aciduria (MGTA). Although the clinical presentation of MGTA is mainly characterized by neurological symptoms, the mechanisms of brain damage in this disease are poorly known. In the present study we investigated the in vitro effect of MGT, MGA and OHIVA on various parameters of oxidative stress in cerebral cortex from young rats. Thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence were significantly increased by MGT, MGA and OHIVA, indicating that these metabolites induce lipid oxidative damage. Furthermore, the addition of melatonin, alpha-tocopherol and superoxide dismutase plus catalase fully prevented MGT-induced increase on TBA-RS, suggesting that free radicals were involved in this effect. These metabolites also provoked protein oxidative damage determined by increased carbonyl formation and sulfhydryl oxidation, but did not induce superoxide generation in submitochondrial particles. It was also verified that MGA and MGT significantly decreased the non-enzymatic antioxidant defenses in cerebral cortex supernatants and that melatonin and alpha-tocopherol totally blocked MGA-induced GSH reduction. The data indicate that the metabolites accumulating in MGTA elicit oxidative stress in vitro in the cerebral cortex. It is therefore presumed that this pathomechanism may be involved in the brain damage observed in patients affected by MGTA.     

Differential survival of Euselasia apisaon Dahman (lepidoptera: riodinidae) pupae at understorey plants in the Eucalyptus plantations of Belo Oriente, MG, Brazil

Herbivorous insects may attack eucalyptus causing economic losses. One of these pests is the moth Euselasia apisaon Dahman, a key pest in the basin of middle Rio Doce. Here we studied the survival of pupae of this moth in Eucalyptus and in understorey plants and tested the hypotheses: i) live pupae are more abundant in plants of the understorey than in eucalyptus, ii) there is no difference between the abundance of pupae in different plants of the understorey. We sampled three areas cultivated with eucalyptus in Belo Oriente, MG, and samples were taken in five plots each area, getting five branches of each plant and of five eucalyptus trees that bordered the plot. The proportion of live and dead pupae and the mortality rate were estimated. The abundance of live pupae was higher in the understorey and the mortality rate of pupae was the same among different families of plants of the understorey. It is possible the larger available leaf area of understorey plants justify the greater abundance of live pupae in this habitat, however, avoidance of feeding habitat to finish the life cycle is also a possible explanation. Mortality rate in plants of the understorey points to an equal pressure of natural enemies on the pupae. These appointments help us to understand the dynamics of pests in eucalyptus plantations, providing important information to support actions against pests in natural environments.