The Synergistic Effects of LRRK1 Inhibition and TLR4 Activation on Apoptotic, Inflammatory Parameters and α-Synuclein Expression in HMC-3 and SH-SY5Y Cell Lines

Abstract

LRRK1 protein is sole paralog of LRRK2 protein, which plays a role in autosomal dominant Parkinson’s disease inheritance in humans. Both proteins share similar domain organization characteristics. Like LRRK2, LRRK1 also possesses kinase and GTPase activities. However, role of LRRK1 in pathogenesis of Parkinson’s disease remains poorly understood. In this study, we aimed to evaluate cytokine expression levels and expression of apoptotic and inflammatory proteins in commonly used HMC-3 microglial and SH-SY5Y neuroblastoma cell lines following inhibition of LRRK1 by IN04 inhibitor. Cytokine expression was assessed through IL-6, apoptosis via caspase-3 activation, and proliferation and inflammation through p38 phosphorylation. Additionally, effect of IN04 on α-synuclein expression was evaluated. Furthermore, the synergistic effect of IN04 with LPS was investigated, as LPS induces inflammation through TLR4 activation. To this end, four experimental groups were established: control, IN04, LPS, and IN04+LPS. According to our results, IN04 treatment increased α-synuclein expression in both HMC-3 and SH-SY5Y cells. IN04 treatment elevated caspase-3 activation and p38 phosphorylation in HMC-3 cells, whereas interestingly, it decreased both parameters in SH-SY5Y cells. Moreover, IN04 treatment increased IL-6 expression in HMC-3 cells, while in SH-SY5Y cells, combination of IN04 with LPS led to an increase in IL-6 expression.

Keywords

• Parkinson
• Inflammation
• LRRK1
• Microglia
• Apoptosis

HMC-3 ve SH-SY5Y Hücre Hatlarında LRRK1 İnhibisyonunun ve TLR4 Aktivasyonunun, Apoptotik, İnflamatuar Parametreler ve α-Sinüklein Ekspresyonu Üzerindeki Sinerjistik Etkileri

Abstract

LRRK1 proteini insanda otozomal dominant Parkinson kalıtımında rol oynayan LRRK2 proteinin tek paralogudur. Her iki protein domain organizasyonu açısından benzer özelliklere sahiptir. LRRK1 proteini de LRRK2 proteini gibi kinaz ve GTPaz aktivitesine sahiptir. LRRK1 proteinin Parkinson patogenezindeki rolü tam anlaşılamamıştır. Bu sebeple yapmış olduğumuz çalışmada LRRK1 proteinin IN04 inhibitörü ile inhibe edilmesi sonucu Parkinson araştırmalarında yaygın olarak kullanılan HMC-3 mikroglia hattı ve SH-SY5Y nöroblastoma hücre hatları üzerindeki sitokin ekspresyon düzeylerinin değerlendirilmesi ve apoptotik, inflamatuvar proteinlerin ekspresyon düzeylerinin belirlenmesi hedeflenmiştir. Sitokin ekspreyonu IL-6 üzerinden, apoptoz kaspaz-3 aktivasyonu üzerinden, proliferasyon ve inflamasyon p38 fosforilasyonu üzerinden değerlendirilmiştir. Ayrıca IN04’un α-sinüklein ekspresyonu üzerinden etkisi değerlendirilmiştir. Bunun yanında LPS uygulanması sonucu IN04’ un LPS ile sinerjistik etkisi tespit edilmiştir. LPS, TLR4 aktivasyonuna yol açarak inflamasyon sürecini başlatmaktadır. Bu amaçla yapmış olduğumuz çalışmada kontrol, IN04, LPS, IN04+LPS olmak üzere 4 adet deney grubu oluşturulmuştur. Elde ettiğimiz verilere göre IN04 uygulaması sonucu HMC-3 ve SH-SY5Y hücrelerinde α-sinüklein ekspresyonu artmaktadır. IN04 uygulaması HMC-3 hücrelerinde kaspaz-3 aktivasyonunu ve p38 fosforilasyonunu artırırken, ilginç bir şekilde SH-SY5Y hücrelerinde kaspaz-3 aktivasyonu ve p38 fosforilasyonunu azaltmaktadır. IN04 uygulamasına bağlı olarak HMC-3 hücrelerinde IL-6 ekspreyonu artarken, SH-SY5Y hücrelerinde IN04’un LPS ile kombinasyonu sonucu IL-6 ekspresyonu artmaktadır.

Keywords

• Parkinson
• İnflamasyon
• LRRK1
• Mikroglia
• Apoptoz

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