Chanism of G-Betagamma-Dependent Regulation of Neuronal Differentiation and Neurodegeneration
Neurodegeneration is a pathological condition associated with a progressive loss of neurons, and occurs in many neurological disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD) and Lou Gehrig’s disease (ALS). It is the most common cause of dementia among people 65 and older. With the increasing of life expectancy, the prevalence of neurodegenerative diseases is also increasing rapidly. However, the cause of this disorder is largely unknown, and no effective drugs are available to treat the disease process. Therefore, it is crucial to identify new target(s) and strategies for therapeutic interventions. Cytoskeletal defects followed by dysfunction is a major characteristic of neurodegenerative diseases. During neurodegeneration microtubules (MTs) and proteins associated with MTs are severely altered. Tau is a stabilizing MT-associated protein (MAP) and in normal conditions, tau binds to MT, stabilizing neuron structure and integrity. In Alzheimer’s disease (AD) and in other tauapathies, tau is hyper-phosphorylated leading to tau aggregation and NFT (neurofibriliary tangles) formation, and this pathway emerged as a major feature in the pathogenesis of AD and associated illness. Over activation of GSK3, a downstream effector of PI3K pathway has been shown to play key roles in tau hyperphosphorylation. Previous results from our laboratory have shown that G, an important component of GPCR pathway promotes microtubule (MT) assembly and induces neuronal differentiation of PC12 cells and blocking the interaction between G and tubulin/MTs using G-blocking peptide GRK-CT, disrupted MTs, inhibited neurite outgrowth, and induced axonal damage indicating the possible involvement of G in neurodegeneration. Although G has been implicated in familial AD, and has been connected to APP (Amyloid precursor protein)-dependent toxicity, it has not been investigated as potential pathway for inducing neurodegeneration. In the current investigation we used human neuronal cells SHSY5Y to further understand the mechanism by which G is involved in neuronal differentiation and neurodegeneration. I hypothesize that G is involved in neurodegeneration by disrupting MTs, altering phosphorylation of tau protein, and G-mediated downstream PI3K/pAkt/pGSK3 signaling pathway. This hypothesis was tested in the current study. Gallein, an inhibitor of G, and Tideglusib, a GSK3 inhibitor were used to elucidate the proposed signaling pathway. SHSY5Y cells are a neuroblastoma derived cell line that is commonly used in vitro to understand the process of neurodegeneration (ND) was included in the current thesis. In Specific Aim 1, using biochemical, and immunoconfocal methodologies, I have demonstrated that Gallein (GAL) inhibited neurite formation and disrupted MT assembly. Surprisingly,the immunoblot analysis using p396 TAU (ser at 396 position) specific antibody, I observed the expression of phosphorylated tau was reduced in the presence of GAL, suggesting that MT disruption could also be associated with inhibition of tau phosphorylation (hypo-) similar to that observed in hyper-phosphorylation of Tau protein (Cortés-Gómez, M. Á., et al.,2020). In Specific Aim-2, I have demonstrated the PI3K pathway was affected by GAL as phosphorylation of Akt and GSK3 was inhibited. Similar to GAL, we found tideglusib also disrupted MTs and neurite formation. As found with GAL, tideglusib decreased expression of pAKT, pGSK3, and ps396 Tau in SHSY5Y cells. Tideglusib has been used earlier in phase 2 clinical trial for AD patients. However, the primary outcome from the trial was negative. My results indicating that tideglusib disrupt MTs and neurite formation, could account for this outcome. The current results also suggest that that both G and its downstream effector GSK3 could work in concert to disrupt MTs, inhibit neuronal differentiation, and induce neurodegeneration. The study also indicates that more caution should be taken for developing drugs for the treatment of neurodegeneration, and should first be tested for their effectiveness in vitro cell culture system for MT assembly/ neuronal differentiation.
Olivas, Diana, "Chanism of G-Betagamma-Dependent Regulation of Neuronal Differentiation and Neurodegeneration" (2021). ETD Collection for University of Texas, El Paso. AAI28540436.