The aging brain - molecular and metabolic changes

Aging is a complex set of events that involves the whole body. However, disruption of the central nervous system (CNS) function is the aspect of aging that elderly people worry about most. Aging has different effects on different aspects of neurological function. Our knowledge of the basic molecular mechanism of brain aging has significantly improved over the past few decades. The rate of aging is not fixed, but is plastic and subject to modifications. The environmental factor proven to be very potent in modulating aging is reduced dietary intake. Dietary restriction (DR) is a vigorous nongenetic and nonpharmacological intervention that is known to delay ageing and increase an active and healthy lifespan in diverse species, from yeast to mammals. Additionally, DR can improve various brain functions, including learning and memory, synaptic plasticity and neurogenesis.Biologia Serbica (2017), 39(1): 26-3

Different levels of epidermal growth factor signaling modifies the differentiation of specific cell types in mouse postnatal retina

Epidermal growth factor (EGF) signaling has been implicated in the regulation of the differentiation and proliferation of retinal progenitors. We assessed how different levels of EGF signaling, achieved either by increasing receptor expression or via addition of the exogenous ligand, or an increase in both, can affect the differentiation of progenitors in the first week of postnatal retinal development in the model system of retinal explants (REs). Proliferating progenitor cells in REs were infected with either the control CLV3/ESR-related peptide family (CLE)-green fluorescent protein (GFP)-or with EGF receptor (EGFR)-GFP-expressing retrovirus, and grown in the control medium or in the presence of exogenous EGF (10 ng/mL). The differentiation of infected cells into Muller glia (Sox9+), rod photoreceptors (rhodopsin+) and horizontal cells (calbindin+) was analyzed. In all the examined conditions, infected cells differentiated into Muller glia and rod photoreceptors that normally develop postnatally. Horizontal cells finished their development during the embryonic stages and progenitors infected with control-GFP virus did not differentiate into GFP+/calbindin-in either control or EGFsupplemented medium, however, cells infected with EGFR-GFP differentiated into horizontal cells (GFP+/calbindin+) in both culture conditions. These results imply that altering the levels of EGFR and/or the amount of the EGF ligand can overcome progenitor competence restriction

Expression profiles of cholesterol metabolism-related genes are altered during development of experimental autoimmune encephalomyelitis in the rat spinal cord

Increased evidence suggests that dysregulation of cholesterol metabolism may be a key event contributing to progression of multiple sclerosis (MS). Using an experimental autoimmune encephalomyelitis (EAE) model of MS we revealed specific changes in the mRNA and protein expression of key molecules involved in the maintaining of cholesterol homeostasis in the rat spinal cord: 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR), apolipoprotein E (ApoE) and cholesterol 24-hydroxylase (CYP46A1) during the course of disease. The presence of myelin lipid debris was seen only at the peak of EAE in demyelination loci being efficiently removed during the recovery period. Since CYP46A1 is responsible for removal of cholesterol excess, we performed a detailed profiling of CYP46A1 expression and revealed regional and temporal specificities in its distribution. Double immunofluorescence staining demonstrated CYP46A1 localization with neurons, infiltrated macrophages, microglia and astrocytes in the areas of demyelination, suggesting that these cells play a role in cholesterol turnover in EAE. We propose that alterations in the regulation of cholesterol metabolism at the onset and peak of EAE may add to the progression of disease, while during the recovery period may have beneficial effects contributing to the regeneration of myelin sheath and restoration of neuronal function.Scientific Reports (2017), 7(1): 270

Comparative analysis of expression of angiogenic factors and CD44 gene in human glioma and neuroblastoma cell lines in vitro

Angiogenesis is essential for tumor growth and relies on the production of angiogenic factors. By comparative analysis using RT-PCR method of angiogenic growth factors: VEGF, bFGF, PDGF-A, angiogenin- 1 and IL-8 we established the level of expression of these genes necessary for angiogenesis in glioma and neuroblastoma cell lines. Our analyses were also extended to CD44 gene, which plays an important role in cascade of metastasis and progression of brain tumors. Significant differences in the level of gene expression of angiogenic factors and CD44 gene between the two cell lines observed throughout this study can be used as a prognostic marker for predicting clinical outcome in human brain tumors at the time of the initial staging.Angiogeneza je neophodna za rast tumora i zahteva proizvodnju angiogenih trofičkih faktora koji učestvuju u tumorogenezi. Uporednom analizom angiogenih trofičkih faktora: VEGF, bFGF, PDGF-A, angiogenina-1 i IL-8 pomoću metode RT-PCR utvrdili smo nivo ekspresije ovih gena uključenih u proces angiogeneze u ćelijskim linijama glioma i neuroblastoma. Takođe smo proširili analize i na CD44 gen koji igra važnu ulogu u kaskadi nastanka i progresiji metastaza tumora mozga. Dobijeni rezultati ukazuju na značajnu razliku u nivou genske ekspresije angiogenih faktora i CD44 gena u ove dve ćelijske linije čije se poreklo razlikuje ne samo po nastanku već i po mestu rasejavnja metastaza. Rezultati bi mogli da posluže kao prognostički faktor u prekliničkim i kliničkim istraživanjima tumora mozga od inicijalnih do terminalnih stupnjeva nastanka i terapije

The Expression of Major Facilitator Superfamily Domain-Containing Protein2a (Mfsd2a) and Aquaporin 4 Is Altered in the Retinas of a 5xFAD Mouse Model of Alzheimer’s Disease

Cerebral amyloid angiopathy (CAA) is characterized by amyloid β (Aβ) accumulation in the blood vessels and is associated with cognitive impairment in Alzheimer’s disease (AD). The increased accumulation of Aβ is also present in the retinal blood vessels and a significant correlation between retinal and brain amyloid deposition was demonstrated in living patients and animal AD models. The Aβ accumulation in the retinal blood vessels can be the result of impaired transcytosis and/or the dysfunctional ocular glymphatic system in AD and during aging. We analyzed the changes in the mRNA and protein expression of major facilitator superfamily domain-containing protein2a (Mfsd2a), the major regulator of transcytosis, and of Aquaporin4 (Aqp4), the key player implicated in the functioning of the glymphatic system, in the retinas of 4- and 12-month-old WT and 5xFAD female mice. A strong decrease in the Mfsd2a mRNA and protein expression was observed in the 4 M and 12 M 5xFAD and 12 M WT retinas. The increase in the expression of srebp1-c could be at least partially responsible for the Mfsd2a decrease in the 4 M 5xFAD retinas. The decrease in the pericyte (CD13+) coverage of retinal blood vessels in the 4 M and 12 M 5xFAD retinas and in the 12 M WT retinas suggests that pericyte loss could be associated with the Mfsd2a downregulation in these experimental groups. The observed increase in Aqp4 expression in 4 M and 12 M 5xFAD and 12 M WT retinas accompanied by the decreased perivascular Aqp4 expression is indicative of the impaired glymphatic system. The findings in this study reveal the impaired Mfsd2a and Aqp4 expression and Aqp4 perivascular mislocalization in retinal blood vessels during physiological (WT) and pathological (5xFAD) aging, indicating their importance as putative targets for the development of new treatments that can improve the regulation of transcytosis or the function of the glymphatic system

Loss of Cathepsin B and L Leads to Lysosomal Dysfunction, NPC-Like Cholesterol Sequestration and Accumulation of the Key Alzheimer's Proteins

Proper function of lysosomes is particularly important in neurons, as they cannot dilute accumulated toxic molecules and aggregates by cell division. Thus, impairment of lysosomal function plays an important role in neuronal degeneration and in the pathogenesis of numerous neurodegenerative diseases. In this work we analyzed how inhibition and/or loss of the major lysosomal proteases, the cysteine cathepsins B and L (CtsB/L), affects lysosomal function, cholesterol metabolism and degradation of the key Alzheimer's disease (AD) proteins. Here, we show that cysteine CtsB/L, and not the aspartyl cathepsin D (CtsD), represent a major lysosomal protease(s) that control lysosomal function, intracellular cholesterol trafficking and AD-like amyloidogenic features. Intriguingly, accumulation of free cholesterol in late endosomes/lysosomes upon CtsB/L inhibition resembled a phenotype characteristic for the rare neurodegenerative disorder Niemann-Pick type C (NPC). CtsB/L inhibition and not the inhibition of CtsD led to lysosomal impairment assessed by decreased degradation of EGF receptor, enhanced LysoTracker staining and accumulation of several lysosomal proteins LC3II, NPC1 and NPC2. By measuring the levels of NPC1 and ABCA1, the two major cholesterol efflux proteins, we showed that CtsB/L inhibition or genetic depletion caused accumulation of the NPC1 in lysosomes and downregulation of ABCA1 protein levels and its expression. Furthermore, we revealed that CtsB/L are involved in degradation of the key Alzheimer’s proteins: amyloid-β peptides (Aβ) and C-terminal fragments of the amyloid precursor protein (APP) and in degradation of β-secretase (BACE1). Our results imply CtsB/L as major regulators of lysosomal function and demonstrate that CtsB/L may play an important role in intracellular cholesterol trafficking and in degradation of the key AD proteins. Our findings implicate that enhancing the activity or levels of CtsB/L could provide a promising and a common strategy for maintaining lysosomal function and for preventing and/or treating neurodegenerative diseases

6-hydroxydopamine lesions of the striatum lead to the alterations of dopamine receptor mrna in parkinsonian rats

The effects of four-site intrastriatal 6-hydroxydopamine (6-OHDA) lesions were examined in adult male rats. Five days after the lesions the animals were checked for specific rotational behavior induced by middle dose of amphetamine and the results confirmed the effectiveness of the lesions. The RNAs from the striatum were isolated at different time points after the lesion, and the RT-PCR analyse were performed for the D1 and D2 receptor mRNA. The results show a decline in the D2 receptor mRNA level (40%) at 6 h and 24 h points while this change was not observed seven days after the lesion. In contrast, no statistically significant changes in the level of the D1 receptor mRNA after the lesion at any time point were found.Ispitivani su efekti četiri ubodne 6-hidroksidopaminske (6-OHDA) lezije striatuma kod odraslih mužjaka pacova. Pet dana nakon lezije, životinje su testirane na specifično rotaciono ponaš anje pod uticajem srednje doze amfetamina i rezultati su potvrdili efikasnost lezije. RNK iz striatuma su izolovane u različitim vremenskim tačkama nakon lezije i urađena je RT-PCR analiza iRNK za D1 i D2 dopaminske receptore. Rezultati pokazuju smanjivanje nivoa iRNK za D2 receptor (40%) 6 h i 24 h nakon lezije, dok sedam dana nakon lezije nema promena. Za razliku od ovih rezultata, u nivou iRNK za D1 receptor ne postoje statistički značajne razlike u bilo kojoj vremenskoj tački

Changes in expression of GFAP, ApoE and APP mRNA and protein levels in the adult rat brain following cortical injury

The recovery period following cortical injury (CI) is characterized by a dynamic and highly complex interplay between beneficial and detrimental events. The aim of this study was to examine the expressions of Glial Fibrillary Acidic Protein (GFAP), Apolipoprotein E (ApoE) and Amyloid Precursor Protein (APP), all of which are involved in brain plasticity and neurodegeneration. Our results reveal that CI strongly influenced GFAP, ApoE and APP mRNA expression, as well as GFAP and ApoE protein expression. Considering the pivotal role of these proteins in the brain, the obtained results point to their potential contribution in neurodegeneration and consequent Alzheimer's disease development.Projekat ministarstva br. 17305

The presymptomatic treatment with 3HFWC nanosubstance decreased plaque load in 5XFAD mouse model of Alzheimer's disease

Introduction In the present study, we assessed the effects of the hyper-harmonized-hydroxylated fullerene–water complex (3HFWC) on Alzheimer's disease (AD) neuropathological hallmarks in 5XFAD mice, an AD animal model. Methods The 3-week-old 5XFAD mice were exposed to 3HFWC water solution ad libitum for 3 months in the presymptomatic phase of pathology. The functional effects of the treatment were confirmed through near-infrared spectroscopy (NIRS) analysis through machine learning (ML) using artificial neural networks (ANNs) to classify the control and 3HFWC-treated brain tissue samples. The effects of 3HFWC treatment on amyloid-β (Aβ) accumulation, plaque formation, gliosis, and synaptic plasticity in cortical and hippocampal tissue were assessed. Results The 3HFWC treatment significantly decreased the amyloid-β plaque load in specific parts of the cerebral cortex. At the same time, 3HFWC treatment did not induce the activation of glia (astrocytes and microglia) nor did it negatively affect synaptic protein markers (GAP-43, synaptophysin, and PSD-95). Conclusion The obtained results point to the potential of 3HFWC, when applied in the presymptomatic phase of AD, to interfere with amyloid plaque formation without inducing AD-related pathological processes such as neuroinflammation, gliosis, and synaptic vulnerability

Apoptotic clearance in rabbits with experimental pulmonary emphysema

In order to better understand pathogenesis of pulmonary emphysema, the model of experimentally induced pulmonary emphysema in Chinchilla rabbits was used for the estimation of apoptotic clearance of pulmonary tissue. Bronchoalveolar lavage was performed in three groups of animals: experimental group-E on hypercholesterolemic diet (4% edible oil solution of crystalline cholesterol), control group-C1 on standard diet for that animal species and animals on oily diet-C2. Apoptotic detection in cytocentrifuge preparations of lung washings was evaluated by in situ TUNEL. The property of alveolar macrophages to engulf apoptotic cells was estimated by light microscopy including 300 features (related subsequent steps: adsorption, internalization and intracellular processing of free apoptotic bodies) and was evaluated by scoring and indexing method. Internalization of apoptotic bodies by alveolar macrophages, as well as free apoptotic bodies were decreased in E compared to both C1 and C2 group (p<0.01 and p<0.05 respectively). Intracellular processing of apoptotic bodies by alveolar macrophages is significantly decreased in C2 in comparison with E (p<0.05) and C1 group (p<0.01). Apoptotic capacity of pulmonary tissue is significantly decreased in C2 in comparison with C1 group (p<0.01). The results implicate that immuno-metabolic competence of pulmonary tissue might be essentially associated with tissue remodeling in pulmonary emphysema.U cilju boljeg razumevanja patogeneze plućnog emfizema, u radu je korišć en eksperimentalni model emfizema pluća na činčila kunićima za procenu apoptotskog kapaciteta plućnoga tkiva. Bronholaveolarna lavaža je urađena na tri grupe životinja: eksperimentalnoj grupi-E na hiperholesterolskoj dijeti (4% uljani rastvor kristalnog holesterola), kontrolnoj grupi-C1 na standardnoj dijeti za tu životinjsku vrstu i grupi životinja na uljanoj dijeti-C2. Određivanje apoptotskih parametara cito-centrifužnih preparata bronhoalveolarnog lavata vršeno je posle bojenja preparata TUNEL in situ citohemijskim metodom. Sposobnost alveolarnih makrofaga da odstrane apoptotske ćelije fagocitozom procenjivana je svetlosnom mikroskopijom na 300 prikaza po preparatu (prikazi uključuju: adsorpciju, internalizaciju i intracelularno procesiranje apoptotskih tela) i evaluirana metodom indeksiranja i skora. Internalizacija apoptotskih tela alveolarnim makrofazima, kao i relativni procenat slobodnih apoptotskih tela, bili su signifikantno smanjeni u E grupi poredeći sa C1 (p<0.01) i C2 grupom (p<0.05). Intracelularno procesiranje apoptotskih tela alveolarnim makrofazima bilo je signifikantno smanjeno u C2 u odnosu na E (p<0.05) i C1 grupu (p<0.01). Apoptotski kapacitet tkiva pluća bio je signifikantno smanjen u C2 u poređenju sa C1 grupom (p<0.01). Ovi rezultati ukazuju da imuno-metabolička kompetentnost plućnog tkiva može biti suštinski povezana sa remodelovanjem tkiva pluća u eksperimentalnom emfizemu pluća.nul