Exploring the role of lipids in protein modification and amyotrophic lateral sclerosis / Explorando o papel dos lipídeos na modificação de proteínas e esclerose lateral amiotrófica

Lipids are a diverse and ubiquitous group of compounds, which have several biological functions such as structural components of cell membranes, energy storage, and participation in signaling pathways. Free radicals or reactive oxygen species could attack polyunsaturated fatty acid esterified to phospholipids generating oxidized products. Once oxidized, lipids are able to modify amino acids residues in proteins leading to modulation signaling pathways and cellular redox balance. Furthermore, alteration of lipid homeostasis is also linked to development and progression of neurodegenerative diseases. The purposes of this study were (i) to investigate the role of lipids in protein aggregation, (ii) to investigate the plasma lipidome of an ALS rat model (SOD1G93A rats), and (iii) to investigate the effect of high-fat diet in plasma lipidome of an ALS rat model. In chapters 1 and 2, the interaction between cytochrome c (cytc) and cardiolipin hydroperoxide (CLOOH), as well as cholesterol hydroperoxide (ChOOH) promoted protein aggregation. Mass spectrometry analysis of tryptic peptides from CLOOH-containing reaction revealed K72 and H26 consistently modified by 4- hydroxynonenal (4-HNE). Further, adduction of K27, K73 and K88 were detected with 4- oxynonenal (4-ONE). For the first time, we characterized the dityrosine cross-linked peptides at Y48-Y74, Y48-97 and Y74-Y97 in oligomeric cytc. Similarly, ChOOH-containing reaction showed dityrosine cross-linked peptides at Y48-Y48, Y48-Y74 and Y48-Y97 in dimeric cytc. In accordance to previous studies, the proposed mechanism under covalent protein oligomerization mediated by lipid hydroperoxide could be related to modification of lysine and tyrosine residues. In chapter 3, we characterized the lipid composition of blood plasma in amyotrophic lateral sclerosis (ALS), since dysregulation of lipid metabolism is increasingly associated with neuropathology. Using untargeted lipidomics approach based on liquid chromatography coupled to mass spectrometry, we found main alterations in triglycerides, phospholipids and sphingolipids in symptomatic ALS rats relative to controls. Additionally, for the first time we reported acylceramides species in the plasma. In order to investigate the source of these lipid alterations, we analyzed the lipid content of fractioned lipoproteins. Triglycerides and phospholipids were found in very low-density lipoprotein (VLDL), while acylceramides and hexosylceramides were found enriched in high-density lipoprotein (HDL). In chapter 4, high-fat diet containing lard or high-fish oil as much as 60% of total lipids has both the largest change on plasma lipid composition. Overall survival was not statistically different when compared to control diet. Increased levels of acylceramides, hexosylceramides and acylcarnitines were observed in ALS rats fed a control diet or high-fat diet in comparison to WT controls. Importantly, untargeted lipidomic analysis of blood plasma highlighted acylceramide d18:1/24:1+20:4 as potential biomarkers of ALS progression. Thus, our lipidomic analysis provides a novel insight into the molecular level event driving molecular dysregulation in ALS. Additional research is needed to determine the effect of plasma lipid alteration on motor neuron process and energetic metabolism. Collectively, our findings reinforce the idea that lipids play a relevant role in modulating cellular processes linked to protein aggregation and neurodegeneration

Os lipídeos são moléculas que possuem várias funções biológicas importantes, atuando como componente de membranas celulares, servindo com fonte de reserva de energia e participando de vias de sinalização. Os ácidos graxos poli-insaturados esterificados aos fosfolipídeos, por exemplo, são potenciais alvos para o ataque de radicais livres gerando produtos oxidados que são capazes de modificar resíduos de aminoácidos em proteínas levando a modulação das vias de sinalização e balanço redox. Por outro lado, alteração na homeostase do metabolismo dos lipídeos está relacionada ao desenvolvimento e progressão de doenças neurodegenerativas. Tendo em vista a importância dos lipídeos nos processos biológicos, os objetivos desse estudo foram (i) investigar o papel dos lipídeos na agregação proteica (capítulo 1 e 2), (ii) investigar as alterações na composição lipídica do plasma de rato modelo SOD1G93A de esclerose lateral amiotrófica (ELA) (capítulo 3) e (iii) investigar o efeito da suplementação de dietas hiperlipídicas na composição lipídica do plasma de rato modelo SOD1G93A (capítulo 4). No capítulo 1 e 2, a interação do citocromo c (citc) com hidroperóxido de cardiolipina (CLOOH) e hidroperóxido de colesterol (ChOOH) promove a agregação covalente do citc. Análise por nLC-MS/MS dos peptídeos digeridos identificou resíduos de lisina (K72) e histidina (H26) modificado por 4-hidroxininenal (4-HNE), enquanto os resíduos K27, K73 e K88 foram modificados por 4-oxinonenal (4-ONE). Pela primeira vez, nós caracterizamos ditirosinas (Y48-Y74, Y48-97 e Y74-Y97) na reação do citc com CLOOH. Também foram caracterizadas ditirosinas envolvendo os resíduos Y48-Y48, Y48-Y74 e Y48-Y97 na reação com ChOOH. Esses resultados corroboram com estudos anteriores que sugerem um mecanismo de agregação proteica envolvendo a perda da carga positiva de lisina e formação de ditirosina pela combinação de radicais de tirosil. No capítulo 3, a análise da composição lipídica do plasma de ratos SOD1G93A utilizando LC-MS/MS revelou alterações significativas na composição de triglicérides, glicerofosfolipídeos e esfingolipídeos em ratos sintomáticos comparado com os assintomáticos. É importante destacar que pela primeira vez acilceramidas foram identificadas em plasma de rato modelo para ALS. Análise da composição lipídica de lipoproteínas isoladas, maior fonte de lipídeos circulantes no plasma, mostraram alterações de triglicérides e glicerofosfolipídeos em VLDL. As acilceramidas e as hexosilceramidas, por sua vez, foram encontradas em maior abundância em HDL. No capítulo 4, a suplementação com dietas hiperlipídicas (rica em banha de porco e óleo de peixe) alterou significativamente o perfil lipídico do plasma em relação a doença. Contudo, não foi observado aumento significativo na sobrevida dos ratos ALS comparado com dieta controle. Independente da dieta, a concentração plasmática de acilcarnitina, hexosilceramidas e acilceramidas foram significativamente aumentadas em ratos ALS comparado com WT. A análise do perfil lipídico do plasma mostrou que a acilceramida d18:1/24:1+20:4 pode ser um potencial marcador de progressão da ALS. Dessa forma, os resultados mostrados fornecem uma visão enriquecedora sobre o evento a nível molecular que conduz a desregulação lipídica na ELA. Coletivamente, nossos resultados reforçam a importância dos lipídeos na modulação dos processos celulares ligados a agregação de proteínas e na neurodegeneração

Aged Lewis rats exposed to low and moderate doses of rotenone are a good model for studying the process of protein aggregation and its effects upon central nervous system cell physiology / Ratos Lewis idosos expostos a baixa e moderada doses de rotenona são um bom modelo para estudar o processo de agregação proteica e seus efeitos sobre a fisiologia celular do sistema nervoso central

ABSTRACT Cell physiology is impaired before protein aggregation and this may be more relevant than inclusions themselves for neurodegeneration. The present study aimed to characterize an animal model to enable the analysis of the cell biology before and after protein aggregation. Ten-month-old Lewis rats were exposed either to 1 or 2 mg/kg/day of rotenone, delivered subcutaneously through mini-pumps, for one month. Hyperphosphorylated TAU, alpha-synuclein, amyloid-beta peptide and protein carbonylation (indicative of oxidative stress) were evaluated in the hippocampus, substantia nigra and locus coeruleus through immunohistochemistry or western blot. It was found that 2 mg/kg/day rotenone increased amyloid-beta peptide, hyperphosphorylation of TAU and alpha-synuclein. Rotenone at 1mg/kg/day did not alter protein levels. Protein carbonylation remained unchanged. This study demonstrated that aged Lewis rats exposed to a low dose of rotenone is a useful model to study cellular processes before protein aggregation, while the higher dose makes a good model to study the effects of protein inclusions.

RESUMO A fisiologia celular está prejudicada antes da agregação proteica podendo ser mais importante para a neurodegeneração do que as próprias inclusões. Assim, o objetivo deste estudo é caracterizar um modelo animal para analisar os mecanismos e efeitos da agregação proteica. Ratos Lewis com 10 meses de idade foram expostos a rotenona (1 ou 2 mg/kg/dia), administrada subcutaneamente, utilizando minibombas osmóticas. Os níveis de peptídeo beta-amiloide, TAU hiperfosforilada, alfa-sinucleína e proteínas carboniladas (indicativo de estresse oxidativo) foram avaliados por imunohistoquímica e western blot no hipocampo, substância negra e locus coeruleus. Foi demonstrado que 2 mg/kg/dia de rotenona promoveu aumento do peptídeo beta-amiloide, hiperfosforilação da TAU e alfa-sinucleína. Já 1 mg/kg/dia de rotenona não alterou os níveis dessas proteína nessas regiões. As proteínas carboniladas não se alteraram. Foi demonstrado que ratos Lewis idosos expostos a baixas doses de rotenona são modelo de estudo dos processos celulares antes da agregação proteica, enquanto 2 mg/kg/dia de rotenona permite estudos sobre os efeitos da agregação proteica.

Cataract-causing mutation S228P promotes βB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain

β/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various β/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in βB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of βB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native βB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of βB1-crystallin on βA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native βB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all β/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of β/γ-crystallin domains by protecting the hydrophobic core from solvent access.

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley, Minas Gerais, Brazil, with Chagas disease

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region.

Transcriptional mutagenesis by 8-oxodG in alpha-synuclein aggregation and the pathogenesis of Parkinson's disease

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease associated with selective loss of dopaminergic neurons. The characteristic hallmark of the disease is intracytoplasmic proteinacious inclusion bodies called Lewy bodies, primarily consisting of a presynaptic protein alpha-synuclein. Oxidative stress-mediated damage to macromolecules have been shown to occur frequently in PD. Oxidative damage to DNA in the form of oxidized guanine (8-oxodG) accumulates in both the mitochondrial and nuclear DNA of dopaminergic neurons of the substantia nigra in PD. 8-oxodG-mediated transcriptional mutagenesis has been shown to have the potential to alter phenotype of cells through production of mutant pool of proteins. This review comprehensively summarizes the role of oxidative stress-mediated damage incurred during neurodegeneration, and highlights the scope of transcriptional mutagenesis event in leading to alpha-synuclein aggregation as seen in PD.

Loss of glucocerebrosidase 1 activity causes lysosomal dysfunction and alpha-synuclein aggregation

Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes beta-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When alpha-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of alpha-synuclein aggregates.

Phytomedicines as potential inhibitors of β amyloid aggregation: significance to Alzheimer's disease / 中国天然药物

Throughout the history of drug development, plants have been an important source for the discovery of novel therapeutically active compounds for many diseases. The ethnopharmacological approach has provided several leads to identify potential new drugs from plant sources, including those for memory disorders. For the treatment of Alzheimer's disease the drug discovery focus shifted from cholinesterase inhibitors, to other targets primarily based on two key neuropathological hallmarks, namely the hyperphosphorylation of the tau protein resulting in the formation of neurofibrillary tangles (NFTs), and the increased formation and aggregation of amyloid-beta peptide (Aβ) derived from amyloid precursor protein (APP). The present article aims to provide a comprehensive literature survey of plants and their constituents that have been tested for Aβ aggregation, thus possibly relieving several features of Alzheimer's disease (AD).