Molecular mechanisms of ageing in neurodegeneration role of oxidative and endoplamic reticulum stress in different human diseases

Resumo

It is hypothesized that the neurodegenerative diseases (NDDs) could be an accelerated form of aging selective for nervous tissue in specific anatomic locations. Accordingly, the processes observed in the biological basis of aging (oxidative stress, accumulation of highly modified protein aggregates, mitochondrial dysfunction) and the ensuing consequences processes are more intense and premature in these cell populations. The aim of this work was to investigate the potential interplay between oxidative and endoplasmic reticulum (ER) stress and the underling signalling pathways, as a potential mechanism involved in the pathogenesis of the neurodegenerative disorders affecting different locations, and characterized by protein aggregates. We characterized protein oxidative damage, its major contributors and its pathophysiological consequences in the sporadic form of amyotrophic lateral sclerosis (ALS) patients with lumbar onset disease, in the frontotemporal tauopathy PicK's disease (PiD) and in the argyrophilic grain disease (AGD) patients. The results of ALS samples was compared with in vitro models of the disease. After extensive pathological characterization, samples from spinal cords (SC) and frontal cortex (FC) from ALS patients, FC and occipital cortex (OC) from PiD patients, and hippocampus (HC) from AGD patients were analyzed in comparison with age-matched control samples. The concentration of markers for specific pathways of protein oxidative damage (direct oxidation, glycoxidation and lipoxidation) and fatty acid composition were assessed by mass spectrometry. Contributors to protein oxidation (mitochondrial respiratory complexes, antioxidant defence and proteolysis) and its consequences (endoplasmic reticulum stress and/or unfolded protein response (UPR)) were evaluated by western-blot of specific markers. Furthermore, the mitochondrial biogenesis system was assessed by measuring by western-blot the levels of key factors ALS was associated to increased direct oxidative, glycoxidative and lipoxidative damage in SC and, to a lower extent, in FC samples. This was associated to increased lipid peroxidizability, and to impaired neuroprotective responses because of decreased docosahexaenoic content as well as alterations of the mitochondrial respiratory complexes and proteasomal impairment. Endoplasmic reticulum stress was evidenced in SC, but not in FC. Therefore, it could be concluded that sporadic ALS leads to increased oxidative damage in proteins and to ER stress in SC, while FC is less affected, but not preserved. In samples from FC, but not in OC of PiD, there were evidences of ER stress such as activated UPR, associated to specific depletion in ER chaperones. Those findings are related to increased ubiquitination compatible with alteration in ubiquitin-proteasome system. In the same location, evidences for increased direct oxidative and lipoxidative damages targeting antioxidant enzymes were found, with decreased amount of glycoxidation markers. Strinkingly, increases in most of the examined parameters of oxidative stress in morphologically preserved OC of PiD patients were detected as well. The changes registered in PiD could be associated with disturbances in mitochondrial respiratory complexes compatible with diminished mitochondrial biogenesis and lack of antioxidant defence, combined with depletion in the contents of the neuroprotective docosahexaenoic acid observed in FC. In this line, the content of the transcription factors related to antioxidant responses and mitochondrial biogenesis showed significant changes in FC but less marked in OC. In contrast, while OC showed increased oxidative damage, mitochondrial respiratory chain and biogenesis were preserved, a finding associated to increased docosahexaenoic content, suggesting an appropriate response to the generated increase in oxidative stress. Analysis of various oxidative stress biomarkers in HC of AGD revealed significantly decreased levels of the markers of glycoxidation, similarly to PiD, which is compatible with defects in glycolytic potential in this location. Those defects have been previously reported in other NDDs and may be associated to oxidative modifications of glycolytic enzymes also evidenced here. There were no changes in the concentrations direct protein oxidation markers. This suggests a preferential role of other forms of oxidative damage, such as lipoxidation, as evidenced by increased malondialdehyde-lysine levels in this disease. Western blot measurements also revealed increased protein reactive carbonyl groups further supporting elevated oxidative damage in HC of AGD samples, which can be attributed to the mitochondrial dysfunction evidenced by disturbance in the respiratory chain function and reduced mitochondria number. Furthermore, the key molecules critically involved in UPR were found activated, which caused elevation in ER chaperones. Most importantly, despite the reduced number of mitochondria, transcription factors for their biogenesis were not increased, suggesting that impaired mitochondria biogenesis may be implicated in AGD pathogenesis. The described results indicate the implication of oxidative and endoplasmic reticulum stress in sporadic ALS, PiD and AGD suggesting a possible interplay between them through proteolysis dysfunction, with a predominant role of mitochondrial impairment leading to the neurodegenerative process.