Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

Characterization of Lipopolysaccharide Effects on LRRK2 Signaling in RAW Macrophages.

Dysfunction of the immune system and mitochondrial metabolism has been associated with Parkinson's disease (PD) pathology. Mutations and increased kinase activity of leucine-rich repeat kinase 2 (LRRK2) are linked to both idiopathic and familial PD. However, the function of LRRK2 in the immune cells under inflammatory conditions is contradictory. Our results showed that lipopolysaccharide (LPS) stimulation increased the kinase activity of LRRK2 in parental RAW 264.7 (WT) cells. In addition to this, LRRK2 deletion in LRRK2 KO RAW 264.7 (KO) cells altered cell morphology following LPS stimulation compared to the WT cells, as shown by an increase in the cell impedance as observed by the xCELLigence measurements. LPS stimulation caused an increase in the cellular reactive oxygen species (ROS) levels in both WT and KO cells. However, WT cells displayed a higher ROS level compared to the KO cells. Moreover, LRRK2 deletion led to a reduction in interleukin-6 (IL-6) inflammatory cytokine and cyclooxygenase-2 (COX-2) expression and an increase in lactate production after LPS stimulation compared to the WT cells. These data illustrate that LRRK2 has an effect on inflammatory processes in RAW macrophages upon LPS stimulation.

LRRK2 protects immune cells against erastin-induced ferroptosis.

Ferroptosis is an iron-dependent regulated cell death pathway characterized by excessive lipid peroxidation. It is implicated in many neurodegenerative diseases, including Parkinson's Disease (PD). Mutations and increased leucine-rich repeat kinase 2 (LRRK2) kinase activity are associated with both familial and idiopathic PD pathology. Increased iron deposition was observed in the substantia nigra of LRRK2 mutation-carrying PD patients compared to healthy individuals, suggesting a potential link between LRRK2 and ferroptosis. However, the role of LRRK2 in the immune cells is still not well-understood. This study aims to investigate the effect of LRRK2 on ferroptosis-induced cell death in immune cells. We used LRRK2 parental (WT) and LRRK2 KO (KO) RAW 264.7 murine macrophages. Cells were challenged with the ferroptosis inducer, erastin, and the kinase activity was investigated using the LRRK2 kinase inhibitor, MLi2. Cell metabolism and viability analysis showed that WT cells were more resistant to ferroptosis than the KO cells. Lipid peroxidation and cellular reactive oxygen species (ROS) generation were significantly elevated in the KO cells. Furthermore, mitochondrial membrane potential and mitochondrial respiration were decreased in the KO cells after erastin treatment compared to the WT cells. Inhibition of the LRRK2 kinase function resulted in increased cell sensitivity to erastin. Cell and mitochondrial substrates utilization were altered in the KO and kinase inhibited WT cells compared to WT cells. These results indicate a protective role of LRRK2 against erastin-induced ferroptosis in RAW macrophages and point towards the importance of LRRK2 kinase function in the protective mechanism.

The multifaceted role of LRRK2 in Parkinson's disease: From human iPSC to organoids.

Parkinson's disease (PD) is the second most common neurodegenerative disease affecting elderly people. Pathogenic mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the most common cause of autosomal dominant PD. LRRK2 activity is enhanced in both familial and idiopathic PD, thereby studies on LRRK2-related PD research are essential for understanding PD pathology. Finding an appropriate model to mimic PD pathology is crucial for revealing the molecular mechanisms underlying disease progression, and aiding drug discovery. In the last few years, the use of human-induced pluripotent stem cells (hiPSCs) grew exponentially, especially in studying neurodegenerative diseases like PD, where working with brain neurons and glial cells was mainly possible using postmortem samples. In this review, we will discuss the use of hiPSCs as a model for PD pathology and research on the LRRK2 function in both neuronal and immune cells, together with reviewing the recent advances in 3D organoid models and microfluidics.

Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes: A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors.

Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism, including the classic protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). The aim of this study was to investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Rat hepatocytes were exposed to 400 µmol/l diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining, respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The cAMP-elevating agent 7ß-acetoxy-8,13-epoxy-1α,6ß,9α-trihydroxylabd-14-en-11-one (forskolin), the pan-phosphodiesterase inhibitor IBMX, and EPAC inhibitors 5,7-dibromo-6-fluoro-3,4-dihydro-2-methyl-1(2H)-quinoline carboxaldehyde (CE3F4) and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP, whereas both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. SIGNIFICANCE STATEMENT: This study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, this study proposes the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in drug-induced liver injury (DILI). Both EPAC1 and EPAC2, but not protein kinase A, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of DILI and liver injury involving mitochondrial dysfunction.

Collagen type I alters the proteomic signature of macrophages in a collagen morphology-dependent manner.

Idiopathic pulmonary fibrosis is a progressive lung disease that causes scarring and loss of lung function. Macrophages play a key role in fibrosis, but their responses to altered morphological and mechanical properties of the extracellular matrix in fibrosis is relatively unexplored. Our previous work showed functional changes in murine fetal liver-derived alveolar macrophages on fibrous or globular collagen morphologies. In this study, we applied differential proteomics to further investigate molecular mechanisms underlying the observed functional changes. Macrophages cultured on uncoated, fibrous, or globular collagen-coated plastic were analyzed by liquid chromatography-mass spectrometry. The presence of collagen affected expression of 77 proteins, while 142 were differentially expressed between macrophages grown on fibrous or globular collagen. Biological process and pathway enrichment analysis revealed that culturing on any type of collagen induced higher expression of enzymes involved in glycolysis. However, this did not lead to a higher rate of glycolysis, probably because of a concomitant decrease in activity of these enzymes. Our data suggest that macrophages sense collagen morphologies and can respond with changes in expression and activity of metabolism-related proteins. These findings suggest intimate interactions between macrophages and their surroundings that may be important in repair or fibrosis of lung tissue.

Species-specific metabolic reprogramming in human and mouse microglia during inflammatory pathway induction.

Metabolic reprogramming is a hallmark of the immune cells in response to inflammatory stimuli. This metabolic process involves a switch from oxidative phosphorylation (OXPHOS) to glycolysis or alterations in other metabolic pathways. However, most of the experimental findings have been acquired in murine immune cells, and little is known about the metabolic reprogramming of human microglia. In this study, we investigate the transcriptomic, proteomic, and metabolic profiles of mouse and iPSC-derived human microglia challenged with the TLR4 agonist LPS. We demonstrate that both species display a metabolic shift and an overall increased glycolytic gene signature in response to LPS treatment. The metabolic reprogramming is characterized by the upregulation of hexokinases in mouse microglia and phosphofructokinases in human microglia. This study provides a direct comparison of metabolism between mouse and human microglia, highlighting the species-specific pathways involved in immunometabolism and the importance of considering these differences in translational research.

Pharmacological Inhibition of Epac1 Averts Ferroptosis Cell Death by Preserving Mitochondrial Integrity.

Exchange proteins directly activated by cAMP (Epac) proteins are implicated in a wide range of cellular functions including oxidative stress and cell survival. Mitochondrial-dependent oxidative stress has been associated with progressive neuronal death underlying the pathology of many neurodegenerative diseases. The role of Epac modulation in neuronal cells in relation to cell survival and death, as well as its potential effect on mitochondrial function, is not well established. In immortalized hippocampal (HT-22) neuronal cells, we examined mitochondria function in the presence of various Epac pharmacological modulators in response to oxidative stress due to ferroptosis. Our study revealed that selective pharmacological modulation of Epac1 or Epac2 isoforms, exerted differential effects in erastin-induced ferroptosis conditions in HT-22 cells. Epac1 inhibition prevented cell death and loss of mitochondrial integrity induced by ferroptosis, while Epac2 inhibition had limited effects. Our data suggest Epac1 as a plausible therapeutic target for preventing ferroptosis cell death associated with neurodegenerative diseases.

The tale of proteolysis targeting chimeras (PROTACs) for Leucine-Rich Repeat Kinase 2 (LRRK2).

Here we present the rational design and synthetic methodologies towards proteolysis-targeting chimeras (PROTACs) for the recently-emerged target leucine-rich repeat kinase 2 (LRRK2). Two highly potent, selective, brain-penetrating kinase inhibitors were selected, and their structure was appropriately modified to assemble a cereblon-targeting PROTAC. Biological data show strong kinase inhibition and the ability of the synthesized compounds to enter the cells. However, data regarding the degradation of the target protein are inconclusive. The reasons for the inefficient degradation of the target are further discussed.

A Conserved Role for LRRK2 and Roco Proteins in the Regulation of Mitochondrial Activity.

Parkinson's Disease (PD) is the second most common neurodegenerative disease world-wide. Mutations in the multidomain protein Leucine Rich Repeat Kinase 2 (LRRK2) are the most frequent cause of hereditary PD. Furthermore, recent data suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in PD pathogenesis. Isolated mitochondria of tissue samples from PD patients carrying LRRK2 mutations display a significant impairment of mitochondrial function. However, due to the complexity of the mitochondrial signaling network, the role of LRRK2 in mitochondrial metabolism is still not well understood. Previously we have shown that D. discoideum Roco4 is a suitable model to study the activation mechanism of LRRK2 in vivo. To get more insight in the LRRK2 pathways regulating mitochondrial activity we used this Roco4 model system in combination with murine RAW macrophages. Here we show that both Dictyostelium roco4 knockout and cells expressing PD-mutants show behavioral and developmental phenotypes that are characteristic for mitochondrial impairment. Mitochondrial activity measured by Seahorse technology revealed that the basal respiration of D. discoideum roco4- cells is significantly increased compared to the WT strain, while the basal and maximal respiration values of cells overexpressing Roco4 are reduced compared to the WT strain. Consistently, LRRK2 KO RAW 264.7 cells exhibit higher maximal mitochondrial respiration activity compared to the LRRK2 parental RAW264.7 cells. Measurement on isolated mitochondria from LRRK2 KO and parental RAW 264.7 cells revealed no difference in activity compared to the parental cells. Furthermore, neither D. discoideum roco4- nor LRRK2 KO RAW 264.7 showed a difference in either the number or the morphology of mitochondria compared to their respective parental strains. This suggests that the observed effects on the mitochondrial respiratory in cells are indirect and that LRRK2/Roco proteins most likely require other cytosolic cofactors to elicit mitochondrial effects.

Protective effect of metformin against palmitate-induced hepatic cell death.

Lipotoxicity causes hepatic cell death and therefore plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metformin, a first-line anti-diabetic drug, has shown a potential protective effect against NAFLD. However, the underlying mechanism is still not clear. In this study, we aim to understand the molecular mechanism of the protective effect of metformin in NAFLD, focusing on lipotoxicity. Cell death was studied in HepG2 cells and primary rat hepatocytes exposed to palmitate and metformin. Metformin ameliorated palmitate-induced necrosis and apoptosis (decreased caspase-3/7 activity by 52% and 57% respectively) in HepG2 cells. Metformin also reduced palmitate-induced necrosis in primary rat hepatocytes (P < 0.05). The protective effect of metformin is not due to reducing intracellular lipid content or activation of AMPK signaling pathways. Metformin and a low concentration (0.1 µmol/L) of rotenone showed moderate inhibition on mitochondrial respiration indicated by reduced basal and maximal mitochondrial respiration and proton leak in HepG2 cells. Moreover, metformin and rotenone (0.1 µmol/L) preserved mitochondrial membrane potential in both HepG2 cells and primary rat hepatocytes. In addition, metformin and rotenone (0.1 µmol/L) also reduces reactive oxygen species (ROS) production and increase superoxide dismutase 2 (SOD2) expression. Our results establish that metformin AMPK-independently protects against palmitate-induced hepatic cell death by moderate inhibition of the mitochondrial respiratory chain, recovering mitochondrial function, decreasing cellular ROS production, and inducing SOD2 expression, indicating that metformin may have beneficial actions beyond its glucose-lowering effect and also suggests that mitochondrial complex І may be a therapeutic target in NAFLD.

Linalool attenuates oxidative stress and mitochondrial dysfunction mediated by glutamate and NMDA toxicity.

Mitochondrial dysfunction and inflammation contribute to the initiation and development of several brain pathological conditions, including Alzheimer's disease and cerebral ischemia. Linalool is an aromatic plant-derived monoterpene alcohol with reported anti-inflammatory, and anti-oxidant properties. We investigated the role of linalool on glutamate-induced mitochondrial oxidative stress in immortalized neuronal HT-22 cells. Glutamate induced oxidative stress in neuronal cells, as detected by real-time cell impedance measurements, MTT assay, and analysis of Annexin V/PI. Administration of linalool 100 µM reduced cell death mediated by glutamate. Staining of glutamate-stimulated mitochondria by MitoTracker revealed improved morphology in the presence of linalool. Furthermore, we demonstrated a potential neuroprotective effect of linalool in conditions of oxidative stress by a reduction of mitochondrial ROS and mitochondrial calcium levels, and by preserving mitochondrial membrane potential. Experiments using both high-resolution respirometry and Seahorse Extracellular flux analyzer showed that linalool was able to promote an increase in uncoupled respiration that could contribute to its neuroprotective capacity. Linalool protection was validated using organotypic hippocampal slices as ex vivo model with NMDA as a stimulus to induce excitotoxity cell damage. These results demonstrate that linalool is protective in an in vitro model of glutamate-induced oxidative stress and in an ex-vivo model for excitotoxity, proposing linalool as a potential therapeutic agent against neurodegenerative brain diseases where oxidative stress contributes to the pathology of the disease.