The role of neurosteroids on Amyloid beta toxcitiy in neuronal cell lines

Introduction: Alzheimer?s disease (AD) is an irreversible, progressive neurodegenarative disorder, which results in memory impairment. AD, the most common cause of dementia among people age 65 and older, takes place in fourth range among the causes of deaths in Western Countries. Many pathophysiological processes play role in its pathogenesis, such as neurofibrillary tangle, inflamation, glutamate toxicitiy, oksidative stress, synaptic damage and lost of cholinergic activity and also some remaining considerations exist about amyloid beta (Aß) toxicity in neural tissue is the basic mechanism. The mechanism of Aß induced toxicity has not known exactly yet.Neuroactive steroid hormones which are biosynthesized in brain or nervous system, are called `neurosteroids? since their extraordinary origins and different functions, their synthesis in neural tissue is able to be induced by many different factors that are consist of Aß peptide. These steroids show dose- or time-dependent protective or toxic effects. However, the relation between Aß toxicity and steroid synthesis has not been clearly identified.Purpose: In this thesis study, it is purposed to determine that the effect of Aß toxicity, forming in two different neuron cell lines, on neurosteroid synthesis and the effects of inhibition of neurosteroid synthesis , in different steps, on neural cell viability. Besides, this work aimed to investigate the effects of pregnenolone (P) and pregnenolone sulfate (PS) ,being well-known among neural steroids, on cell viability in a concentration- and exposure time-dependent manner.Materials and Methods: In our study, using rat pheochromocytoma (PC-12) and human neuroblastoma (SHSY-5Y) cell lines, after the significant toxicity formed in the end of 72 hour with 20/40 ?M Aß25-35 peptide, the change in intracellular P and PS levels were determined by ELISA and HPLC methods. The effects of different concentrations (nM or ?M) of these two steroids on neural cell viability and Aß toxicity were evaluated by MTT reduction, lactate dehydrogenase (LDH) release, flow cytometry methods and microscopic investigation. Moreover, after the inhibition of steroid synthesis in two different steps by using aminoglutethimide (AMG) and 2,4 Dichloro-6-Nitrophenol (DCNP), their effects on neural cell viability were evaluated.Results: When the effect of treatment of PC-12 cells with 20µM and SHSY-5Y cells with 40µM aggregated Aß25?35 peptide on cell viability is evaluated with MTT reduction after 48-72 h exposure a significant decrease is observed compared to control group (p<0.05). It is determined that after 72 h exposure Aß increase P synthesis significantly (p<0.05) and tended to decrease PS synthesis in both neural cell lines.It is determined that external treatment of P and PS in supraphysiological (>1 µM ) concentrations decreased cell viability significantly in both neural cell lines besides in physiological (<1 µM ) concentrations didn?t show toxic effect untill 72 h exposure (p<0.05). Furthermore it is shown that 0.5 µM P had protective effect against Aß-induced cell death and at 50 µM concentrations increased the toxicity of Aß.It is shown that inhibition of steroid synthesis with AMG and DCNP in two different steps resulted in a statistically significant increase in cell viability in both cell line at 72h exposure compared to Aß-treated group.Conclusion: In conclusion, in our study it is shown that Aß is effective on the synthesis of P and the newly synthesized P participated sulfotransferase pathway in restricted manner. Moreover showing that the inhibition of P and PS synthesis, P exposure in low concentration had neuroprotective effects against Aß toxicity would give important clues to prevent Aß related neurodegenerative changes and to understand the mechanisms of neurodegenerative diseases.Key Words: Pregnenolone, pregnenolone sulfate, amyloid beta, Alzheimer?s disease, neurosteroid, neural cell viability