LRRK2 Kinase Inhibition Attenuates Astrocytic Activation in Response to Amyloid β1-42 Fibrils

Intracerebral accumulation of amyloid-β in the extracellular plaques of Alzheimer’s disease (AD) brains represents the main cause of reactive astrogliosis and neuroinflammatory response. Of relevance, leucine-rich repeat kinase 2 (LRRK2), a kinase linked to genetic and sporadic Parkinson’s disease (PD), has been identified as a positive mediator of neuroinflammation upon different inflammatory stimuli, however its pathogenicity in AD remains mainly unexplored. In this study, by using pharmacological inhibition of LRRK2 and murine primary astrocytes, we explored whether LRRK2 regulates astrocytic activation in response to amyloid-β1-42 (Aβ1-42). Our results showed that murine primary astrocytes become reactive and recruit serine 935 phosphorylated LRRK2 upon Aβ1-42 fibril exposure. Moreover, we found that pharmacological inhibition of LRRK2, with two different kinase inhibitors, can attenuate Aβ1-42-mediated inflammation and favor the clearance of Aβ1-42 fibrils in astrocytes. Overall, our findings report that LRRK2 kinase activity modulates astrocytic reactivity and functions in the presence of Aβ1-42 deposits and indicate that PD-linked LRRK2 might contribute to AD-related neuroinflammation and pathogenesis.


Introduction
Alzheimer's disease (AD) is the most devastating neurodegenerative disease in the adult population and represents the most frequent cause of dementia worldwide [1]. AD is neuropathologically characterized by progressive neurodegeneration, together with deposition of aggregated proteins, extracellular amyloid-β and intracellular hyperphosphorylated Tau [1,2]. Although the mechanisms underlying AD onset and progression are not yet fully elucidated, it is well known that neuroinflammation plays a critical role in the pathology [3,4]. Neuroinflammation, driven by activation of microglia and astrocytes, is a defense mechanism that protects the brain against pathogens or other inflammatory stimuli, with beneficial effects on brain tissue repair [5]. However, a sustained inflammatory response can turn cytotoxic and lead to cellular and tissue damage, thus promoting neurodegeneration and disease progression [4][5][6]. In this regard, several studies have reported the involvement of chronic neuroinflammation and glia activation in AD [7][8][9]. Reactive microglia and astrocytes have been detected in proximity to amyloid-β plaques in post-mortem brains of AD patients and animal models [10][11][12]. In addition, elevated levels

Primary Astrocytes
C57BL/6J wild-type mice were maintained under a 12 h light-dark cycle at room temperature (RT, 22 • C) with ad libitum food and water. Animal procedures were performed in accordance with European Community Directive 2010/63/UE and approved by the Ethics Committee of the University of Brescia (Project ID: 800-2017). Moreover, the research protocol has been approved by the Ethics Committee of IRCCS San Giovanni di Dio-Fatebenefratelli (n • 90-2021 and Prot. 290/2021).
Primary astrocytes were derived from pups at post-natal days 2-4 (P2-P4). Briefly, cerebral cortices were dissociated in cold PBS; the cell suspension was maintained at RT for 5 min and the top fraction was centrifuged at 1000 rpm 5 min. The cells were then resuspended in astrocytes medium containing high-glucose DMEM (Immunological Sciences, Rome, Italy), 10% fetal bovine serum (FBS, ThermoFisher Scientific, Waltham, MA, USA), 2mM L-Glutamine (ThermoFisher Scientific) and penicillin/streptomycin (Ther-moFisher Scientific). Cells obtained from five brains were then seeded in 175 cm 2 flasks and maintained at 37 • C with 5% CO 2 . After 4 days, the medium was changed and then the cells were maintained in culture until confluence (DIV7-9), when the cells were processed for experimental applications. The purity of primary astrocytic culture was verified by immunostaining with CD11b and glial fibrillar acidic protein (GFAP) antibodies, microglia and astrocyte markers, respectively. In our cultures, the amount of microglia contaminants was~13%.
The fibrillization of Aβ 1-42 fibrils was verified by ThioflavinT (ThioT, Merck/Sigma Aldrich) assays and transmission electron microscopy (TEM). In brief, 7 µg of fibrils were incubated for 1 min at RT with 5 µM of ThioT. Control measurement was performed with 5 µM ThioT in HCl for detection of background fluorescence intensity. Fluorescence emission was recorded at 482 nm with excitation at 450 nm by using the PerkinElmer ® EnSight-Multimode Plate Reader. For TEM, Aβ 1-42 1 µM samples were incubated on a 400 mesh formvar-coated grid (TAAB Ltd., Berks, UK) for 2 min at RT. After removing the excess solution from the grid, samples were negatively stained with Uranyless (Electron Microscopy Sciences) for 2 min at RT and examined with a transmission electron microscope (Tecnai G2 Spirit; FEI Company, Eindhoven, The Netherlands) at 80 kV.

Reactive Oxygen Species (ROS) Detection
ROS generation was measured using the CellROX ® Green Reagent (C10444, Ther-moFisher Scientific) following manufacturer's instructions. Briefly, astrocytes were treated with 10 µM Aβ 1-42 fibrils, or 10 mM-DMSO HCl as a control, for 16 h. Successively, astrocytes were washed once with 1X PBS and treated with 5 µM CellROX ® Green Reagent in cell medium for 30 min at 37 • C. After treatment, the cells were washed three times with 1X PBS and the ROS fluorescence was detected using PerkinElmer ® EnSight-Multimode Plate Reader, setting the excitation at 485 nm and the emission at 520 nm.
Chloroquine (CQ, Merck/Sigma Aldrich), an inhibitor of lysosomal activity, was dissolved in ultrapure distilled water and used on primary astrocytes at 25 µM for 16 h.
During all the treatments, primary astrocytes were cultured in medium containing 1% FBS.

Cell Immunofluorescence and Imaging
Cells were washed once with PBS 1X and then fixed with paraformaldehyde (PFA) 4% pH 7.4 15 min at RT. After three washes with PBS 1X, permeabilization with PBS/Triton-X-100 0.3% was performed for 5 min at RT. Next, cells were saturated with blocking solution (FBS 5% in PBS/Triton-X-100 0.3%) for 1 h at RT and incubated with primary antibody anti-β amyloid clone 6E10 (Biolegend 803004, 1:50), anti-GFAP (Invitrogen 13-0300, 1:500) and anti-CD11b (BD Biosciences 550282, 1:100) diluted in blocking solution. After three washes in PBS 1X, cells were incubated with secondary antibody AlexaFluor 488 or 594 (ThermoFisher Scientific, 1:500) for 1 h at RT. After several washes in PBS 1X, cells were mounted using Prolong Gold Antifade reagent containing DAPI (ThermoFisher Scientific). Images were acquired with a Zeiss Axioplan2 fluorescence microscope with a 63× oil immersion objective (Carl Zeiss AG, Oberkocken, Germany). For the experiments related to lysosomal activity inhibition, primary astrocytes were exposed to 25 µM CQ and to 2 µM LRRK2 GSK inhibitor for 120 min and 90 min, respectively, before the treatment with Aβ 1-42 fibrils. The cells were analyzed 16 h after the treatment with Aβ 1-42 fibrils. After treatment, the cells were fixed and immunostained for GFAP and Aβ 1-42 as described above. Quantification of intracellular Aβ 1-42 was performed using ImageJ software, calculated as fluorescence intensity divided by cellular area (detected by GFAP) and expressed as fluorescence intensity/µm 2 . At least thirty cells were randomly chosen in a minimum of three independent experiments. Images were acquired using a Zeiss Axioplan2 microscope with a 63× oil-immersion objective (Carl Zeiss AG).

Statistical Analysis
All data are expressed as mean ± SEM and represent at least three sets of experiments. Statistical significance of differences between two groups was assessed by unpaired t-test, while multiple comparison was measured using one-Way ANOVA followed by Tukey's post hoc test. Cumulative frequency distributions were compared with a Kolmogorov-Smirnov test. Data were analyzed using Prism software (GraphPad) and statistical significance was taken at p < 0.05.

Astrocytic Activation in Response to Aβ 1-42 Fibrils Priming
In order to investigate a potential role of LRRK2 in astrocytic activation upon ADrelated aggregates, we first generated and validated Aβ 1-42 fibrils ( Figure 1). Aβ 1-42 fibrils were prepared from Aβ 1-42 monomeric protein incubated for 48 h to induce aggregation. The formation of fibrils was verified using several approaches. Specifically, Western blot confirmed the presence of a smear with large Aβ aggregates with high molecular weight (Figure 1a), ThioT assay detected a greater amount of fluorescence signal in fibrils preparation compared to control solvent ( Figure 1b) and TEM examination reported thread-like fibrillar structures (Figure 1c). Taken together, these results indicate the high quality of our Aβ 1-42 fibril-enriched preparation.
Aβ1-42 fibrils, or with DMSO-HCl as a control, and we assessed th cytokine, one of the most crucial pro-inflammatory cytokines in AD a stress-related protein [45]; and the generation of ROS. As show observed a significant increase of all the inflammatory markers ana Aβ1-42 fibrils triggered astrocytes toward a pro-inflammatory and re Next, we asked whether astrocytes were able to internalize A performed a time-course experiment in which cells were exposed to 1 h, 2 h and 4 h and we evaluated fibril uptake through flow cyto FACS analysis of MFI (Figure 2c), the uptake of Aβ1-42 fibrils was cle h of treatment and further increased at longer time points. Moreo ability of astrocytes to internalize Aβ1-42 fibrils, even on fixed cells, via in the Figure 2d. Taken together, these results indicate that the treatm triggered astrocytes toward a reactive phenotype with the ability to  Given that multiple lines of evidence have indicated that amyloid-β deposits trigger glia activation and inflammation [3,40], we investigated whether Aβ 1-42 fibrils were able to activate primary astrocytes. To this purpose, we treated murine primary astrocytes with Aβ 1-42 fibrils, or with DMSO-HCl as a control, and we assessed the induction of IL-1β cytokine, one of the most crucial pro-inflammatory cytokines in AD [41][42][43][44]; of clusterin, a stress-related protein [45]; and the generation of ROS. As shown in Figure 2a,b, we observed a significant increase of all the inflammatory markers analyzed, indicating that Aβ 1-42 fibrils triggered astrocytes toward a pro-inflammatory and reactive phenotype. in fibril-enriched preparation compared to control solvent DMSO-HCl. Data are representative of three independent preparations and are expressed as mean ± SEM. Data were analyzed using unpaired t-test, * p = 0.0120. (c) TEM performed on Aβ1-42 preparation reveals thread-like fibril structure. Scale bar 500 nm and 200 nm. Representative images of astrocytes exposed to Aβ1-42 fibrils for 2 h, fixed and immunostained for Aβ1-42 (green) and GFAP (red). Nuclei were stained with DAPI (blue). Scale bar 10 μm.

LRRK2 Kinase Inhibition Attenuates Aβ1-42 Fibril-Mediated Astrocytic Activation
Several studies, including some from our group, have revealed that LRRK2 kinase activity is crucial to mediate glia activation in response to different stimuli, such as LPS [23][24][25], HIV-1 Tat protein [46], manganese [47] and α-synuclein fibrils [22,23,27]. However, evidence linking LRRK2 to AD-related inflammation is still missing. Thus, we explored the possibility that LRRK2 is involved in astrocytic inflammation mediated by Aβ1-42 fibrils. We first investigated whether LRRK2 was activated upon Aβ1-42 fibril exposure. To this aim, we analyzed the phosphorylation of LRRK2 at serine 935 (pS935), which has been reported to be increased in microglia upon exposure to inflammatory stimuli [22,46,48,49]. Of interest, we observed that Aβ1-42 fibrils triggered an increment of pSer935 -LRRK2 compared to control astrocytes (Figure 3a), suggesting that LRRK2 is involved in the cellular pathways activated by Aβ1-42 fibrils and that are phosphorylated/activated upon being challenged, even in astrocytes, as observed in microglia.
Then, we assessed whether LRRK2 kinase activity controls the induction of astrocytic inflammation. Thus, we treated cells with Aβ1-42 fibrils for 16 h in the presence of LRRK2 kinase inhibition. To this aim, we used two different compounds, GSK [50] and IN-1 [51]. Of note, both LRRK2 inhibitors attenuate the inflammatory response mediated by Aβ1-42 fibrils priming, as revealed by the strong reduction of IL-1β (Figure 3b,c). Taken together, Next, we asked whether astrocytes were able to internalize Aβ 1-42 fibrils. We first performed a time-course experiment in which cells were exposed to FAM-Aβ 1-42 fibrils for 1 h, 2 h and 4 h and we evaluated fibril uptake through flow cytometry. As shown by FACS analysis of MFI (Figure 2c), the uptake of Aβ 1-42 fibrils was clearly detectable after 1 h of treatment and further increased at longer time points. Moreover, we observed the ability of astrocytes to internalize Aβ 1-42 fibrils, even on fixed cells, via imaging, as reported in the Figure 2d. Taken together, these results indicate that the treatment with Aβ 1-42 fibrils triggered astrocytes toward a reactive phenotype with the ability to internalize fibrils.

LRRK2 Kinase Inhibition Attenuates Aβ 1-42 Fibril-Mediated Astrocytic Activation
Several studies, including some from our group, have revealed that LRRK2 kinase activity is crucial to mediate glia activation in response to different stimuli, such as LPS [23][24][25], HIV-1 Tat protein [46], manganese [47] and α-synuclein fibrils [22,23,27]. However, evidence linking LRRK2 to AD-related inflammation is still missing. Thus, we explored the possibility that LRRK2 is involved in astrocytic inflammation mediated by Aβ 1-42 fibrils. We first investigated whether LRRK2 was activated upon Aβ 1-42 fibril exposure. To this aim, we analyzed the phosphorylation of LRRK2 at serine 935 (pS935), which has been reported to be increased in microglia upon exposure to inflammatory stimuli [22,46,48,49]. Of interest, we observed that Aβ 1-42 fibrils triggered an increment of pSer935 -LRRK2 compared to control astrocytes (Figure 3a), suggesting that LRRK2 is involved in the cellular pathways activated by Aβ 1-42 fibrils and that is phosphorylated/activated upon being challenged, even in astrocytes, as observed in microglia. these results indicate that LRRK2 kinase activity is a regulator of Aβ1-42 fibril-mediated inflammation in astrocytes.

Astrocytes with LRRK2 Kinase Inhibition Exhibited Increased Clearance of Aβ1-42 Fibrils
Previous studies have shown that LRRK2 affects the phagocytic functions of glia upon inflammatory priming [46,52,53]. Starting from these observations, we explored whether LRRK2 kinase activity was involved in the uptake of Aβ1-42 fibrils by astrocytes through FAM-Aβ1-42 fibrils and flow cytometry. To this aim, we treated astrocytes with LRRK2 inhibitors (GSK or IN-1) or, as control, DMSO-containing medium, for 90 min before exposing cells to FAM-Aβ1-42 fibrils for 2 h, after which we performed FACS analysis. By analyzing the intracellular FAM-Aβ1-42 signal of cells as MFI, we found that astrocytes treated with LRRK2 inhibitors exhibited a strong reduction of the MFI compared to cells treated with FAM-Aβ1-42 fibrils alone (Figure 4a,b), indicating that astrocytes with LRRK2 inhibition exhibited a decreased amount of intracellular Aβ1-42 fibrils. To investigate if the reduced MFI was mediated by the effect of LRRK2 kinase activity on the degradation rather than the uptake of FAM-Aβ1-42 fibrils, we quantified the intracellular Aβ1-42 after blocking lysosomal degradation with CQ. To this aim, we treated cells with GSK alone or in combination with CQ before the treatment with Aβ1-42 fibrils for 16 h and we quantified the intracellular Aβ1-42 fluorescence signal after fixation. Of interest, the cumulative frequency distribution analysis revealed that astrocytes treated with CQ display Aβ1-42 fluorescence signal consistently shifted toward higher intensity values with respect to cells treated with LRRK2 GSK inhibitor alone (Figure 4c,d), showing a homogenous increase of intracellular Aβ1-42 in the presence of CQ. Taken together, these results indicate that LRRK2 might be involved in the degradation process of Aβ1-42 fibrils and, importantly, suggest that the kinase inhibition of LRRK2 may favor the clearance of Aβ1-42 fibrils in astrocytes. Then, we assessed whether LRRK2 kinase activity controls the induction of astrocytic inflammation. Thus, we treated cells with Aβ 1-42 fibrils for 16 h in the presence of LRRK2 kinase inhibition. To this aim, we used two different compounds, GSK [50] and IN-1 [51]. Of note, both LRRK2 inhibitors attenuate the inflammatory response mediated by Aβ 1-42 fibrils priming, as revealed by the strong reduction of IL-1β (Figure 3b,c). Taken together, these results indicate that LRRK2 kinase activity is a regulator of Aβ 1-42 fibril-mediated inflammation in astrocytes.

Astrocytes with LRRK2 Kinase Inhibition Exhibited Increased Clearance of Aβ 1-42 Fibrils
Previous studies have shown that LRRK2 affects the phagocytic functions of glia upon inflammatory priming [46,52,53]. Starting from these observations, we explored whether LRRK2 kinase activity was involved in the uptake of Aβ 1-42 fibrils by astrocytes through FAM-Aβ 1-42 fibrils and flow cytometry. To this aim, we treated astrocytes with LRRK2 inhibitors (GSK or IN-1) or, as control, DMSO-containing medium, for 90 min before exposing cells to FAM-Aβ 1-42 fibrils for 2 h, after which we performed FACS analysis. By analyzing the intracellular FAM-Aβ 1-42 signal of cells as MFI, we found that astrocytes treated with LRRK2 inhibitors exhibited a strong reduction of the MFI compared to cells treated with FAM-Aβ 1-42 fibrils alone (Figure 4a,b), indicating that astrocytes with LRRK2 inhibition exhibited a decreased amount of intracellular Aβ 1-42 fibrils. To investigate if the reduced MFI was mediated by the effect of LRRK2 kinase activity on the degradation rather than the uptake of FAM-Aβ 1-42 fibrils, we quantified the intracellular Aβ 1-42 after blocking lysosomal degradation with CQ. To this aim, we treated cells with GSK alone or in combination with CQ before the treatment with Aβ 1-42 fibrils for 16 h and we quantified the intracellular Aβ 1-42 fluorescence signal after fixation. Of interest, the cumulative frequency distribution analysis revealed that astrocytes treated with CQ display Aβ 1-42 fluorescence signal consistently shifted toward higher intensity values with respect to cells treated with LRRK2 GSK inhibitor alone (Figure 4c,d), showing a homogenous increase of intracellular Aβ  in the presence of CQ. Taken together, these results indicate that LRRK2 might be involved in the degradation process of Aβ 1-42 fibrils and, importantly, suggest that the kinase inhibition of LRRK2 may favor the clearance of Aβ 1-42 fibrils in astrocytes. Representative fluorescence microscopy images of astrocytes exposed to CQ and LRRK2 inhibitor, or LRRK2 GSK inhibitor alone, before treatment with Aβ1-42 fibrils for 16 h, fixed immunostained for Aβ1-42 (green) and GFAP (red). Nuclei were stained with DAPI (blue). bar 1

Discussion
Chronic neuroinflammation widely contributes to neurodegeneration progression of several neurodegenerative diseases, including AD [13]. In this reg amyloid-β accumulates in the form of amyloid plaques in the CNS and represents main cause of neuroinflammation [3,5,54,55]. Of interest, in the last decade LRRK2 been identified as a key regulator of the neuroinflammatory response in microglia astrocytes upon inflammatory priming [21,26,56]. However, studies exploring the ro LRRK2 in amyloid-β-related inflammation are still missing. In this work, by using LR kinase inhibition, we found that LRRK2 modulates astrocyte activation upon exposu AD-related amyloid deposits. Specifically, we established for the first time that inhibition of LRRK2 kinase activity attenuates the induction of IL-1β cytokine and fa the clearance of Aβ1-42 fibrils, suggesting that LRRK2-related astrocytic functions m contribute to AD pathology.
Neuroinflammation represents a well-described event in AD and it is now emer

Discussion
Chronic neuroinflammation widely contributes to neurodegeneration and progression of several neurodegenerative diseases, including AD [13]. In this regard, amyloid-β accumulates in the form of amyloid plaques in the CNS and represents the main cause of neuroinflammation [3,5,54,55]. Of interest, in the last decade LRRK2 has been identified as a key regulator of the neuroinflammatory response in microglia and astrocytes upon inflammatory priming [21,26,56]. However, studies exploring the role of LRRK2 in amyloidβ-related inflammation are still missing. In this work, by using LRRK2 kinase inhibition, we found that LRRK2 modulates astrocyte activation upon exposure to AD-related amyloid deposits. Specifically, we established for the first time that the inhibition of LRRK2 kinase activity attenuates the induction of IL-1β cytokine and favors the clearance of Aβ 1-42 fibrils, suggesting that LRRK2-related astrocytic functions might contribute to AD pathology.
Neuroinflammation represents a well-described event in AD and it is now emerging as one of the leading causes of the pathology [6,13,[57][58][59]. Astrogliosis has been detected in the brains of AD patients [60] and of animal models [61,62]. In addition, several studies have shown that extracellular amyloid-β deposits trigger glial activation and inflammatory response [3,63]. Furthermore, glia reactivity and neuroinflammation have been reported to cause amyloid-β and tau protein aggregation, which are detrimental to neuronal and synaptic health [14]. Overall, these observations indicate that neuroinflammation is involved in different aspects of AD pathology and plays a crucial role in the progression of the disease. In this scenario, LRRK2, which is a positive mediator of neuroinflammation, could mediate and contribute to AD pathogenesis. To test this hypothesis, we asked whether LRRK2 can be activated and modulate astrocytic reactivity upon Aβ 1-42 fibrils exposure. To assess inflammatory response in our experiments, we analyzed IL-1β induction, which is one of the most relevant pro-inflammatory cytokines, reported to be crucial for AD neuroinflammation and pathogenesis [3,4,64]. As expected, we found that Aβ 1-42 fibrils mediate astrocytic activation, as detected by the strong generation of IL-1β, clusterin and ROS generation. Interestingly, for the first time, we observed that LRRK2 is activated upon Aβ 1-42 fibril priming, as shown by the increased phosphorylation levels of LRRK2 Ser935, thus indicating that LRRK2 is actively involved in astrocytic pathways triggered by amyloid-β. In accord with these findings, our research and that of other groups have shown increased levels of phosphorylated LRRK2 in microglia upon different inflammatory stimuli, which correlated to the production of pro-inflammatory cytokines [22,46,48,49,65].
Of note, given that LRRK2 has been robustly associated to the modulation of inflammatory response [21,26,66], we then investigated whether LRRK2 kinase activity was implicated in the amyloid-β -mediated astrocytic inflammation. Our results showed that astrocytes with LRRK2 kinase inhibition exhibit a strong reduction of IL-1β generation after Aβ 1-42 fibril exposure. These observations indicate that LRRK2 kinase activity regulates inflammation mediated by amyloid-β fibrils and that, as well as in microglia, LRRK2 might be a common modulator of the astrocytic inflammatory response. In support to this hypothesis, Munoz and colleagues reported that LRRK2 IN-1 inhibitor attenuated IL-6 cytokine secretion upon IL-1β priming in human primary astrocytes [28]. This is in accord with what we found in microglial cells, which exhibited reduced levels of IL-1β and cyclooxygenase-2 upon LPS priming when treated with LRRK2 inhibitors [23]. LRRK2 was found to control the induction of inflammation through protein kinase A (PKA)-mediated phosphorylation of nuclear factor kappa-B (NF-κB) inhibitory subunit p50 [23]. Our findings, that kinase inhibition of LRRK2 also exhibits a strong attenuation of IL-1β production in astrocytes upon exposure to Aβ 1-42 fibrils, suggest that, also in astrocytes, the modulatory effects of LRRK2 on inflammatory response to amyloid-β fibrils may involve the PKA-NFκB pathway.
Microglia and astrocytes, efficient scavengers of the brain [67], have been found to be closely associated with amyloid plaques in AD brains and their involvement in amyloidβ clearance has gained increasing interest over the years [38,68,69]. In this context, AD pathology is hypothesized to be caused by an imbalance between amyloid-β production and clearance that leads to amyloid-β accumulation in the brain [70], thus supporting the importance of glia functions in the disease. Interestingly, LRRK2, through its kinase activity, has been shown to affect also phagocytosis and clearance of amyloid proteins by glial cells [46,52,71]. Specifically, it has been demonstrated that microglia with LRRK2 genetic deletion exhibited increased clearance of a-synuclein [53] and astrocytes with an enhanced LRRK2 kinase activity (LRRK2 pathological mutations) displayed reduced degradative capacity [52,72,73]. Starting from these premises, we then investigated whether LRRK2 was involved in the uptake/clearance of amyloid-β fibrils. Interestingly, our results revealed that the inhibition of LRRK2 kinase activity increases the ability of astrocytes to degrade Aβ 1-42 fibrils. This is an important observation as it has been shown that astrocytes can engulf large amounts of amyloid-β but fail to properly digest the amyloid-β, which then accumulates and eventually leads to severe lysosomal and cellular dysfunctions [74].
Engulfment of Aβ 1-42 by astrocytes may initially be a protective clearance mechanism, but an overburden can clearly be detrimental for astrocytic functions, leading, with time, to cell dysfunction, Aβ 1-42 secretion and progression of AD [74]. Taken together, our findings indicate that LRRK2 is involved in the endo-lysosome pathway that leads to Aβ 1-42 fibril degradation and, importantly, suggest that LRRK2 kinase inhibition might enhance the ability of astrocytes to clear amyloid-β and to preserve cell physiology.

Conclusions
Neuroinflammation strongly contributes to AD pathophysiology. Using LRRK2 kinase inhibition, we found that LRRK2 modulates the activation of astrocytes upon exposure to Aβ 1-42 fibrils in vitro. Whereas the inhibition of LRRK2 kinase activity strongly attenuated the induction of IL-1β, it favored the clearance of Aβ 1-42 fibrils by astrocytes. Overall, our study showed that LRRK2 kinase activity controls astrocyte reactivity induced by Aβ 1-42 fibrils and indicates that LRRK2 might be involved in AD-related neuroinflammation and pathogenesis. However, future studies performed on transgenic mouse models of AD and on the post-mortem brain of AD patients might shed more light in the implication of PD-linked LRRK2 to AD pathogenesis and could contribute to dissecting novel pathological mechanisms for pharmacological intervention to limit AD progression.

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets supporting the conclusion of this article are available in the ZENODO repository (10.5281/zenodo.7463238) from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare has no competing interest.