Investigation of the biochemical parameters in pre-symptomatic and symptomatic SOD1 mutated rat model of ALS

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease and the number of affected people by ALS is increasing worldwide. 90-95% of ALS cases are sporadic (sALS), where 5-10% of cases account for familial ALS (fALS) and there is no effective treatment or drug to cure people with ALS. Two approved drugs named edaravone, and riluzole are used for slowing the progression of the disease but not treating ALS. Clinical diagnosis of ALS is usually difficult and takes time since there are no definitive prognostic biomarkers or pathognomonic tests are existing, however, riluzole and edaravone are only effective at the early stages of ALS. There is an urgent need for the evaluation of possible biomarkers in the blood and serum for the rapid and accurate diagnosis of ALS. Additionally, development of the new therapeutic approaches is required to cure people with ALS and improve their life quality. In this study, SOD1-G93A mutated (SOD1G93A) albino female and male rats expressing human SOD1G93A were purchased from Taconic with the number NTac: SD-Tg(SOD1G93A) L26H. Transgenic rats were inbred and after genotyping, SOD1WT and SOD1G93A male rats were determined and used for the experiments. We have divided male rats into 5 groups based on the age as 0 (40-45 days old), A (70-75 days old), B (90-95 days old), C (110-115 days old), and D (130-135 days old), following that each group is divided into two subgroups according to their mutation status respectively as SOD1 wild type (SOD1WT) and SOD1 G93A mutation (SOD1G93A). Groups 0, A and B were determined as the pre-symptomatic stages of ALS, where group C accounts for the early stage of ALS and group D refers to the late stage of ALS. First, we have investigated different biomarkers in the blood and serum, by comparison, their levels between SOD1G93A and SOD1WT rat groups by eliminating the effects of aging. Thus, we aimed to evaluate possible biomarkers in the blood and serum which could be used for the ALS prognosis and progression in the future. Alkaline phosphates (ALKP), alanine aminotransferase (ALT), amylase, calcium, cholesterol, creatinine, glucose, phosphate, total bilirubin (TBIL), total protein (TP), blood urea nitrogen (BUN), and globulin levels were evaluated as serum biomarkers. Our data showed that cholesterol, creatinine, TP, TBIL, BUN, glucose, phosphate, and calcium can be used as promising biomarkers for the prognosis and progression of ALS since levels of these biomarkers are impaired in the SOD1G93A rats in comparison with SOD1WT rats at the pre-symptomatic and symptomatic stages. Furthermore, white blood cell (WBC), lymphocyte (LYM), mid-range absolute count (MID), granulocyte (GRAN), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width with standard deviation (RDW-SD), red cell distribution width with the coefficient of variation (RDW-CV), platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), procalcitonin (PCT), and platelet-large cell ratio (P-LCR) levels were evaluated in the blood samples as potential biomarkers in the blood as well. Since WBC, LYM, GRAN, RBC, HGB, HCT, and MCH levels are impaired in the SOD1G93A rats compared to the SOD1WT ones, we suggest that indicated parameters can be used as potential biomarkers addressing neuroinflammation and impaired immune system as contributors to the ALS pathogenesis. On the other hand, first time in the literature we have found that MCV, MCH, MCHC, RDW-SD, RDW-CV, PLT, MPV, PDW, PCT, and P-LCR levels significantly changed between SOD1G93A and SOD1WT rats addressing impaired blood metabolism in ALS and further studies can be conducted in human cohort to validate these parameters as potential biomarkers in the ALS prognosis and progression. Glucagon, c-peptide, glucose, peptide YY (PYY), and Monocyte chemoattractant protein-1 (MCP-1) levels were also impaired in the SOD1G93A rats even at the pre-symptomatic stages of the diseases as indicators as altered glucose and energy metabolism. Serum iron (Fe), zinc (Zn), calcium (Ca), potassium (K), magnesium (Mg), and sodium (Na) levels are also impaired in the SOD1G93A rats addressing impaired mineral and trace element metabolism. Histopathological changes indicating tissue damage have been observed in the lung, spinal cord, brain, and liver tissues of SOD1G93A rats and the number of OLIG2 positive cells responsible for the repairment and remyelination in the central nervous system (CNS) has changed in the brain and spinal cord samples between SOD1G93A and SOD1WT groups, however, demyelination has been detected neither spinal cord nor brain tissues upon luxol fast blue staining. Oxidative stress, pentose phosphate pathway (PPP), and glycolysis metabolisms were evaluated by measuring antioxidant enzymes including glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6-PGD), glutathione reductase (GR), glutathione s-transferase (GST), catalase (CAT), superoxide dismutase-1 (SOD1), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), and isocitrate dehydrogenase 3 (IDH3) in both cytosol and mitochondria. Impaired antioxidant defence, increased levels of oxidative stress, impairment in the glycolysis and pentose phosphate pathway (PPP) metabolisms have been detected in both cytosol and mitochondria of the brain, spinal cord, liver, and lung tissues. Monomeric SOD1G93A proteins have been detected in the SOD1G93A rats in all groups as a potential cause of impairment in the indicated metabolisms. Additionally, altered acetylcholine metabolism is described by the decreased levels of acetylcholinesterase (ACHE) and increased levels of choline acetyltransferase (ChAT) in the brain of SOD1G93A rats. Altogether, first time in the literature we have investigated possible biomarkers in the blood and serum by eliminating the effects of aging in the prognosis and progression of ALS disease. We have also proven that monomeric SOD1G93A aggregates are formed in the cytosol and mitochondria of the brain, spinal cord, liver, lung, and kidney tissues of SOD1G93A rats accompanied by impaired glycolysis, oxidative stress, and PPP metabolisms as common pathogenesis in the indicated tissues. Additionally, tissues with higher antioxidant enzyme activities involving in the antioxidant and PPP metabolism were affected less from the disease progression according to the histopathological examination. To conclude, our findings can be used as potential biomarkers in the serum and blood of ALS patients for rapid and accurate diagnosis in the future. Additionally, new therapeutic approaches can be developed targeting the improved glycolysis, PPP, and oxidative stress metabolisms to cure people with ALS.