title: Investigation of the Effects of SIRT1 and SIRT2 Modulators in Neuroinflammatory Models of Depression
Creators: name: Zhang, Yuqing
Publisher: name: Griffith University
Year: 2021
Subjects: subject: neuroinflammation
subject: depression
subject: novel medications
subject: Sirtuins

description: Depression is regarded as a manifestation of mood disorder and a complex phenomenon. The etiology can be primary and/or secondary to organic diseases. Neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and multiple sclerosis are often accompanied by the development of depression. A high prevalence of depression in populations with neurodegenerative diseases also indicates a close relationship between depression and neurodegeneration. There are several hypotheses of the pathogenesis of depression, including monoamine hypothesis, neurotrophic and the brain-derived neurotrophic factor (BDNF) hypothesis, HPA axis theory and circadian rhythm theory. There are apparently no infectious insults in the initiation of depression. However, depressed patients without apparent physical diseases showed an increased level of inflammatory markers. Evidence has indicated that neuroinflammation participates in the genesis and development of depression and interacts with other factors involved in depression. Excessive inflammatory cytokines can reduce the production of serotonin, fuel glutamate excitotoxicity, disturb neural plasticity, reduce the production of brain-derived neurotrophic factor and eventually prime the brain for neurodegeneration by facilitating inflammation and synaptic damage. The target of current commercially available antidepressants is to increase levels of neurotransmitters including serotonin, noradrenaline and/or dopamine via different therapeutic mechanisms. However, the overall remission rate for the present medication options is under 30% according to the result of the National Institute of Mental Health (NIMH)-funded sequenced treatment alternatives to relieve depression (STAR*D) study. Additionally, they are frequently accompanied by undesirable side-effects and toxic effects in overdoses that limit their application. Therefore, there is an ongoing need to investigate the potential for novel medications that target neuroinflammation to address this underlying feature of depression. Sirtuins are a unique class of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases that can affect multiple downstream targets by deacetylation activity. The sirtuin family consists of SIRT1-7 enzymes that differ in subcellular localization, enzymatic activities, physiological functions and pathological roles. Among these seven members, SIRT1 has been reported to exert neuroprotective effects in aging, oxidative stress, neuronal survival, neurogenesis and neuroinflammation. SIRT2 also participates in numerous aging-related cellular activities including DNA repair, oxidative stress and autophagy. Based on these important roles, the pharmacological effects of SIRT1 and SIRT2 modulators have been investigated in numerous neurodegeneration studies where different models were conducted, and contradictory results were generated. Therefore, this thesis proposed a systematic investigation of the effects of SIRT1 and SIRT2 modulators in neuroinflammation and neurodegeneration models of depression including in vitro and in vivo studies. The pharmacological agents assessed in this study include: SIRT1 activator resveratrol; selective SIRT1 inhibitor EX527; dual SIRT1/SIRT2 inhibitor sirtinol; SIRT2 inhibitor AGK2; and positive controls for depression and inflammation, fluoxetine and ibuprofen, respectively. Microglia are the resident macrophages that are regarded as the prime components of the intrinsic immune system in CNS parenchyma. Therefore, microglia play a dominant role in neuroinflammation. Injury or other pathological insults can result in the activation of microglia which are able to release inflammatory mediators and chemokines such as TNF-α, IL-1β, PGE2 and reactive oxidative species. Thus the overall experimental design was to collect the supernatant from lipopolysaccharide (LPS)-stimulated microglia (HAPI cell line) and transfer it onto neuronal cells (SH-SY5Y cell line). Microglia were pre-treated with SIRT drugs to affect their biological performance under LPS stimulation and investigate the alteration of the subsequent outcome on neuronal cells. To achieve this goal, methodology development was conducted to optimize the cell culture condition and differentiation method for SH-SY5Y cell line, which is key to achieve accurate results for this study. The glucose level in culture media needed to be correctly selected for the cell culture of SH-SY5Y cells in this supernatant transfer-based co-culture model. High glucose in culture media is considered a pre-diabetic condition for neurons. Data has shown that the cell viability of high-glucose cultured SH-SY5Y cells was reduced by treatment of HAPI cells-conditioned supernatant, which indicated the existence of low-glucose shock. Whereas, application of low-glucose media can effectively prevent SH-SY5Y cells from low-glucose shock after the treatment of the supernatant collected from HAPI cells. Moreover, the differentiation method for SH-SY5Y was also optimized. The combination induction treatment of retinoic acid and BDNF can successfully differentiate the SH-SY5Y cells morphologically and genetically. The RNA expression of neuronal gene marker synaptophysin (SYP) and enolase 2 (ENO2) have shown to significantly increase after combination treatment of retinoic acid and BDNF. In the in vitro pharmacological study, the effects of these SIRT1 and SIRT2 drugs on the production of inflammatory mediators including PGE2, TNF-α, IL-1β and IL-10 on LPS (0.005 μg/L) -stimulated microglia were assessed. Resveratrol and sirtinol significantly reduced the TNF-α production in HAPI cells by up to 95% and 93% respectively. This result showed their potent inhibiting effects on TNF-α which can initiate cell death pathways by binding TNF receptors. Resveratrol, sirtinol, EX527 and AGK2 also significantly reduced PGE2 production by up to 97%, 100%, 65% and 69% respectively in microglia under LPS stimulation. These results are significant given that prostaglandins contribute to prolonged acute inflammatory responses. The supernatant from LPS-stimulated HAPI cells was transferred to SH-SY5Y cells. All SIRT drugs pre-treated of HAPI cells were shown to significantly increase the survival of undifferentiated SH-SY5Y cells, following the transfer of the supernatant. Their inhibiting effects on the production of inflammatory mediators including TNF-α and PGE2 might contribute to this protection on SH-SY5Y cells. The SIRT1 activator resveratrol and dual SIRT1/SIRT2 inhibitor sirtinol demonstrated the most potent effect compared to others. Additionally, in undifferentiated cells, resveratrol was protective against apoptosis, as indicated by a reduction in Caspase 3/7 activity. However, in contrast, in differentiated SH-SY5Y cells, only sirtinol and AGK2 pre-treated supernatant were found to exert neuroprotective effects. Moreover, no increase in Caspase 3 activity was observed following treatment with supernatant indicating that protection against apoptosis is not the reason for increased cell survival. In the animal study, the two most effective agents, SIRT1 activator resveratrol and dual SIRT1/2 inhibitor sirtinol, from the in vitro study were assessed in the acute phase of the LPS-induced depression mouse model and compared with fluoxetine. The behavioral parameters including locomotor activity and immobility time in the open field test, forced swim test and tail suspension test were significantly improved by resveratrol, sirtinol and fluoxetine in the acute sickness phase of the depression model. In conclusion, this research has emphasized the necessity of increasing the understanding of the roles that neuroinflammation and neurodegeneration playing in the pathophysiology of depression and the importance of investigating SIRT drugs as a novel class of drugs targeting neuroinflammation. This research has shown the effectiveness of SIRT modulators in inhibiting neuroinflammation and subsequent neurodegeneration through in vitro and in vivo studies. Further studies, including combining SIRT modulators and current antidepressant medications are warranted.