The findings of the present study showed that LPS impaired motor performance through increase in α-synuclein and TLR4, and insulin signaling dysfunction in the striatum, all of which improved by insulin, alone and with TAK24. Administration of S961 had no effect on these behavioral and molecular deficits, confirming the important role of insulin signaling pathways in LPS-induced PD pathology.
LPS is widely used to study the neuroinflammation model of PD, both in vitro and in vivo. In animals, LPS can be injected into the striatum, SN or globus pallidus, or be systemically administered by an intraperitoneal injection (Liu and Bing 2011). It has been demonstrated that systemic inflammation induced by LPS is correlated with neuroinflammation, neurodegeneration and development of PD (Oliynyk, Marynchenko et al. 2021). LPS is a potent agonist for TLR4 on microglial cells, and therefore produces some pathological features of PD, like dopaminergic neurons loss in SNpc, by extensive microglial activation and releasing pro-inflammatory mediators (Liu and Bing 2011). Astrocytes also highly express TLR4, and are activated after exposure to LPS (La Vitola, Balducci et al. 2021). In vitro studies have shown that LPS treatment increases the expression of TLR4 at both mRNA and protein levels in primary murine and rat astrocytes cells (Bowman, Rasley et al. 2003, Li, Zhang et al. 2016), and lithium reduces astrocytes activation through inhibition of TLR4 expression (Li, Zhang et al. 2016). An in vitro study has indicated that LPS treatment increases TLR4 gene expression, and inflammatory factors like IL-1β, IL-6 and TNF as well. Insulin-like growth factor-I (IGF-I) treatment has been shown to reduce LPS-induced TLR4 overexpression (Bellini, Hereñú et al. 2011). Exogenous IGF-I and its gene deliver to primary astrocytes from mice cerebral cortex which could decrease TLR4 expression, and eventually counteract the LPS-induced neuroinflammation (Bellini, Hereñú et al. 2011). Consistent with these studies, we also indicated overexpression of TLR4 in the striatum of rats receiving LPS, confirming the induction of neuroinflammation, which was attenuated by insulin.
Insulin through IRS1/PI3K/Akt pathway regulates microglial activation and pro-inflammatory cytokines production (Yang, Hsieh et al. 2017). Microglial cells, in response to inflammatory insults, act as a neuroprotective mechanism and prevent neuronal damage. However, in pathological conditions, excessive and long-term microglial activation leads to releasing the proinflammatory cytokines, called chronic neuroinflammation (Kim and Joh 2006), which interfere with insulin signaling elements, such as IRSs (Copps and White 2012, Kim and Feldman 2012). Proinflammatory cytokines decrease IRS interactions with insulin receptors through serine phosphorylation IRSs, reduce insulin sensitivity and cause insulin resistance (Copps and White 2012, Kim and Feldman 2012).
IRS1 and 2 are highly expressed in different regions of the brain, and regulate GSK3β activity which is involved in regulating many cellular processes such as protein synthesis, cell survival, and metabolism (Speed, Saunders et al. 2011). GSK3β overactivation initiates several intracellular signaling cascades which promote apoptotic cell death (Ghasemi, Haeri et al. 2013). GSK3β plays an important role in the pathogenesis of neurodegenerative diseases, and inhibition of its activity can be considered as a therapeutic strategy in reducing the pathology and severity of PD (Duka, Duka et al. 2009, Li, Liu et al. 2014). Insulin exerts some of its neuroprotective effects through inactivating GSK3β by phosphorylation on Ser9, which is mediated by IRS/PI3K/Akt signaling pathway. Therefore, in pathological conditions correlated with chronic neuroinflammation, insulin resistance occurs, which is characterized by decrease in insulin receptor and IRSs, concomitant with increased activity of GSK3β.
Several studies have indicated a link between insulin resistance and brain dysfunction (Ma, Wang et al. 2015, Maciejczyk, Żebrowska et al. 2019). Dopaminergic neurons, in particular, have a high energy demand, which may partly explain their increased sensitivity to hyperglycemia (Lv, Yuan et al. 2021). Insulin receptors are expressed in substantia nigra, and development of insulin resistance reduces the insulin-dependent release of dopamine (Akhtar and Sah 2020). It has been shown that chronic insulin resistance due to a high-fat diet disrupts nigrostriatal function by reducing the release and clearance of dopamine (Vijiaratnam, Girges et al. 2021). There is evidence that insulin signaling pathways are disrupted in PD. Insulin receptors are reduced in the striatum of PD patients, accompanied by decrease of the release and clearance of dopamine (Morris, Bomhoff et al. 2011). It has been also indicated that insulin resistance due to a high-fat diet in mice, leads to more severe motor deficits induced by 6-OHDA than control mice, suggesting that insulin resistance increases the risk of PD pathology (Sharma and Taliyan 2018). Clinical studies have shown that uncontrolled diabetes acts as a risk factor to develop PD (Ou, Wei et al. 2021). Insulin resistance can also induce iron deposition in dopaminergic neurons, which lead to production of highly reactive radicals and neuronal dysfunction (Pignalosa, Desiderio et al. 2021). Consistent with these evidences, our findings revealed that LPS impaired motor function of the animals via, at least in part, induction of insulin resistance. Moreover, there was also overexpression of TLR4 following LPS injection, proposing the interaction of TLR4 and insulin signaling pathway and the role of TLR4 in LPS-induced insulin resistance.
α-Synuclein aggregation and accumulation in the brain, especially in dopaminergic neurons, is the main pathological hallmark of PD. This presynaptic protein is involved in many physiological processes including synaptic transmission, neurotransmitter release and mitochondrial function (Bendor, Logan et al. 2013). However, when aggregating in the oligomeric form, it becomes toxic and causes mitochondrial dysfunction via interaction with the respiratory chain complexes (Chinta, Mallajosyula et al. 2010). Besides, α-synuclein is also involved in neuroinflammatory processes through activation of TLR4 on microglia and astrocytes (Codolo, Plotegher et al. 2013, Fellner, Irschick et al. 2013, Rannikko, Weber et al. 2015, Hughes, Choi et al. 2019, Gorecki, Anyaegbu et al. 2021). Previous studies have shown that LPS induces overexpression of α-synuclein via overactivation of microglial cells (Niu, Wang et al. 2020). It has been shown that chronic microglial overactivation due to overexpression of α-synuclein lead to motor impairments in mice (Drouin-Ouellet, St-Amour et al. 2015). Here, we observed overexpression of α-synuclein in the striatum of animals that received LPS concomitant with overexpression of TLR4. Administration of insulin and TLR4 blocker (TAK242) could decrease them. Insulin has been indicated to activate autophagy through PI3K/Akt/mTOR pathway, and promote degradation of accumulated toxic proteins like α-synuclein (Heras-Sandoval, Pérez-Rojas et al. 2014). In the present study, α-synuclein protein was attenuated following insulin treatment, while S961 could not reduce α-synuclein. This proposes that α-synuclein clearance might be mediated by mTOR inhibition via IRS/ PI3K/Akt pathway. In parallel with these findings, during an in vitro study insulin could reduce α-synuclein in PC12 cells due to treatment with MPP+ (Ramalingam and Kim 2017). Furthermore, other studies have previously reported that rapamycin, an inhibitor of mTORC1, decreased α-synuclein aggregation and prevented dopaminergic neuron loss (Sarkar, Davies et al. 2007, Tain, Mortiboys et al. 2009).