LRRK2 and RAB8A regulate cell death after lysosomal damage in macrophages through cholesterol-related pathways

IF 5.1 2区医学 Q1 NEUROSCIENCES Neurobiology of Disease Pub Date : 2024-11-01 DOI:10.1016/j.nbd.2024.106728

Josefine Fussing Tengberg , Francesco Russo , Tau Benned-Jensen , Jacob Nielsen

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Abstract

Activating mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are among the most common genetic causes of Parkinson's disease (PD). The mechanistic path from LRRK2 mutations to PD is not established, but several lines of data suggest that LRRK2 modulation of lysosomal function is involved. It has previously been shown that LRRK2 is recruited to lysosomes upon lysosomal damage leading to increased phosphorylation of its RAB GTPase substrates in macrophage-derived RAW 264.7 cells. Here, we find that LRRK2 kinase inhibition reduces cell death induced by the lysosomotropic compound LLOMe in RAW 264.7 cells showing that lysosomal damage and LRRK2 functionally interacts in both directions: lysosomal damage can lead to activation of LRRK2 signaling and LRRK2 inhibition can attenuate LLOMe-induced cell death. The effect is lysosome specific, as only lysosomal stressors and not a variety of other cell death inducers could be modulated by LRRK2 kinase inhibition. We show with timing and Lysotracker experiments that LRRK2 inhibition does not affect the immediate lysosomal permeabilization induced by LLOMe, but rather modulates the subsequent cellular response to lysosomal damage. siRNA-mediated knockdown of LRRK2 and its main substrates, the RAB GTPases, showed that LRRK2 and RAB8A knockdown could attenuate LLOMe-induced cell death, but not other RAB GTPases tested. An RNA sequencing study was done to identify downstream pathways modulated by LLOMe and LRRK2 inhibition. The most striking finding was that almost all cholesterol biosynthesis genes were strongly downregulated by LLOMe and upregulated with LRRK2 inhibition in combination with LLOMe treatment. To explore the functional relevance of the transcriptional changes, we pretreated cells with the NPC1 inhibitor U18666A that can lead to accumulation of lysosomal cholesterol. U18666A-treated cells were less sensitive to LLOMe-induced cell death, but the attenuation of cell death by LRRK2 inhibition was strongly reduced suggesting that LRRK2 inhibition and lysosomal cholesterol reduces cell death by overlapping mechanisms. Thus, our data demonstrates a LRRK2- and RAB8A-mediated attenuation of RAW 264.7 cell death induced by lysosomal damage that is modulated by lysosomal cholesterol.

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LRRK2 和 RAB8A 通过胆固醇相关途径调控巨噬细胞溶酶体损伤后的细胞死亡。

富亮氨酸重复激酶 2（LRRK2）的激活突变是帕金森病（PD）最常见的遗传病因之一。从 LRRK2 基因突变到帕金森病的机理尚未确定，但一些数据表明 LRRK2 对溶酶体功能的调节与此有关。以前的研究表明，溶酶体受损时，LRRK2 会被招募到溶酶体，导致巨噬细胞衍生的 RAW 264.7 细胞中其 RAB GTPase 底物的磷酸化增加。在这里，我们发现 LRRK2 激酶抑制剂可减少溶酶体趋向性化合物 LLOMe 在 RAW 264.7 细胞中诱导的细胞死亡，这表明溶酶体损伤和 LRRK2 在功能上是双向互动的：溶酶体损伤可导致 LRRK2 信号的激活，而 LRRK2 抑制剂可减轻 LLOMe 诱导的细胞死亡。这种效应是溶酶体特异性的，因为只有溶酶体应激物而不是其他各种细胞死亡诱导物能被 LRRK2 激酶抑制所调节。siRNA 介导的 LRRK2 及其主要底物 RAB GTPases 的敲除表明，LRRK2 和 RAB8A 的敲除能减轻 LLOMe 诱导的细胞死亡，但不能减轻其他 RAB GTPases 的测试。为了确定受 LLOMe 和 LRRK2 抑制作用调节的下游通路，进行了一项 RNA 测序研究。最引人注目的发现是，几乎所有胆固醇生物合成基因都被LLOMe强烈下调，而在LRRK2抑制与LLOMe处理相结合的情况下则被上调。为了探索转录变化的功能相关性，我们用NPC1抑制剂U18666A预处理细胞，这种抑制剂可导致溶酶体胆固醇的积累。U18666A 处理的细胞对 LLOMe 诱导的细胞死亡不那么敏感，但 LRRK2 抑制对细胞死亡的减弱作用却大大降低，这表明 LRRK2 抑制和溶酶体胆固醇通过重叠的机制减少细胞死亡。因此，我们的数据证明了 LRRK2 和 RAB8A 介导的溶酶体损伤诱导的 RAW 264.7 细胞死亡的减弱作用受到溶酶体胆固醇的调节。

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来源期刊

Neurobiology of Disease 医学-神经科学

CiteScore

11.20

自引率

3.30%

发文量

270

审稿时长

76 days

期刊介绍： Neurobiology of Disease is a major international journal at the interface between basic and clinical neuroscience. The journal provides a forum for the publication of top quality research papers on: molecular and cellular definitions of disease mechanisms, the neural systems and underpinning behavioral disorders, the genetics of inherited neurological and psychiatric diseases, nervous system aging, and findings relevant to the development of new therapies.