The leucine-rich repeat kinase LRRK2/Park8 controls regulated exocytosis in neurons and endocrine cells of the pancreas

Endocrine cells of the pancreas share many physiological features with neurons, although they have different embryological origins. Regulated membrane fusion resulting in hormone exocytosis in endocrine cells is controlled by the same core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) fusion machinery that mediates neurotransmitter release at synapses. The leucine-rich repeat kinase 2 (LRRK2), a protein expressed in neurons and associated with inherited Parkinson\u2019s disease, has been recently shown to interact with specific synaptic proteins and influence synaptic transmission. Given the functional similarities between neurons and endocrine cells of the pancreas, here we first verified LRRK2 expression in islet of Langerhans, and then we investigated its possible involvement in hormone release. By means of RT-PCR and western blotting experiments, we detected LRRK2 expression in beta and alpha cell lines and in human islets of Langerhans. Using the pH-sensitive dye acridine orange as a tool to monitor exocytosis/endocytosis and elisa assays in the presence and the absence of specific LRRK2 inhibitors, we demonstrated LRRK2 involvement in the control of vesicles trafficking and hormone release in both alpha and beta cells. Our data for the first time identified LRRK2 as a novel component of the secretory machinery of endocrine cells of the pancreas and further highlight the similarity between neurons and endocrine cells

Threonine 67 is a key component in the coupling of the NSS amino acid transporter KAAT1

The crystallizations of the prokaryotic LeuT and of the eukaryotic DAT and SERT transporters represent important steps forward in the comprehension of the molecular physiology of Neurotransmitter: Sodium Symporters, although the molecular determinants of the coupling mechanism and of ion selectivity still remain to be fully elucidated. The insect NSS homologue KAAT1 exhibits unusual physiological features, such as the ability to use K+ as the driver ion, weak chloride dependence, and the ability of the driver ion to influence the substrate selectivity; these characteristics can help to define the molecular determinants of NSS function. Two non-conserved residues are present in the putative sodium binding sites of KAAT1: Ala 66, corresponding to Gly 20 in the Na2 site of LeuT, and Ser 68, corresponding to Ala 22 in the Nal site. Thr 67 appears also to be significant since it is not conserved among NSS members, is present as threonine only in KAAT1 and in the paralogue CAATCH1 and, according to LeuT structure, is close to the amino acid binding site. Mutants of these residues were functionally characterized in Xenopus oocytes. The T67Y mutant exhibited uptake activity comparable to that of the wild type, but fully chloride-independent and with enhanced stereoselectivity. Interestingly, although dependent on the presence of sodium, the mutant showed reduced transport-associated currents, indicating uncoupling of the driver ion and amino acid fluxes. Thr 67 therefore appears to be a key component in the coupling mechanism, participating in a network that influences the cotransport of Na+ and the amino acid

The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.

Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely \u3b23-Cav2.1 at N-terminus part and \u3b2-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause diseas

PCSK9 deficiency reduces insulin secretion and promotes glucose intolerance: the role of the low-density lipoprotein receptor

Aims PCSK9 loss of function genetic variants are associated with lower low-density lipoprotein cholesterol but also with higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Here, we investigated the molecular mechanisms underlying this association. Methods and results Pcsk9 KO, WT, Pcsk9/Ldlr double KO (DKO), Ldlr KO, albumin AlbCre+/Pcsk9LoxP/LoxP (liver-selective Pcsk9 knock-out mice), and AlbCre-/Pcsk9LoxP/LoxP mice were used. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology, and cholesterol accumulation in pancreatic islets were studied in the different animal models. Glucose clearance was significantly impaired in Pcsk9 KO mice fed with a standard or a high-fat diet for 20\u2009weeks compared with WT animals; insulin sensitivity, however, was not affected. A detailed analysis of pancreas morphology of Pcsk9 KO mice vs. controls revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels and decreased plasma insulin, and C-peptide levels. This phenotype was completely reverted in Pcsk9/Ldlr DKO mice implying the low-density lipoprotein receptor (LDLR) as the proprotein convertase subtilisin/kexin Type 9 (PCSK9) target responsible for the phenotype observed. Further studies in albumin AlbCre+/Pcsk9LoxP/LoxP mice, which lack detectable circulating PCSK9, also showed a complete recovery of the phenotype, thus indicating that circulating, liver-derived PCSK9, the principal target of monoclonal antibodies, does not impact beta-cell function and insulin secretion. Conclusion PCSK9 critically controls LDLR expression in pancreas perhaps contributing to the maintenance of a proper physiological balance to limit cholesterol overload in beta cells. This effect is independent of circulating PCSK9 and is probably related to locally produced PCSK9

Evolutionary-game-based dynamical tuning for multi-objective model predictive control

Model predictive control (MPC) is one of the most used optimization-based control strategies for large-scale systems, since this strategy allows to consider a large number of states and multi-objective cost functions in a straightforward way. One of the main issues in the design of multi-objective MPC controllers, which is the tuning of the weights associated to each objective in the cost function, is treated in this work. All the possible combinations of weights within the cost function affect the optimal result in a given Pareto front. Furthermore, when the system has time-varying parameters, e.g., periodic disturbances, the appropriate weight tuning might also vary over time. Moreover, taking into account the computational burden and the selected sampling time in the MPC controller design, the computation time to find a suitable tuning is limited. In this regard, the development of strategies to perform a dynamical tuning in function of the system conditions potentially improves the closed-loop performance. In order to adapt in a dynamical way the weights in the MPC multi-objective cost function, an evolutionary-game approach is proposed. This approach allows to vary the prioritization weights in the proper direction taking as a reference a desired region within the Pareto front. The proper direction for the prioritization is computed by only using the current system values, i.e., the current optimal control action and the measurement of the current states, which establish the system cost function over a certain point in the Pareto front. Finally, some simulations of a multi-objective MPC for a real multi-variable case study show a comparison between the system performance obtained with static and dynamical tuning.Peer ReviewedPostprint (author's final draft

Drive-by-wire vehicle stabilization and yaw regulation: A hybrid model predictive control design

Electronic Stability Control (ESC) and Active Front Steering (AFS) have been introduced in production vehicles in recent years, due to improved vehicle maneuverability and the effects in reducing single vehicle accident. We propose a hybrid Model Predictive Control (MPC) design for coordinated control of AFS and ESC. By formulating the vehicle dynamics with respect to the front and rear tire slip angles and by approximating the tire-force characteristics by piecewise affine functions, the vehicle dynamics are formulated as a linear hybrid dynamical system. This model is used to design a hybrid model predictive controller. The proposed model formulation allows one to visually analyze the stability region of the closed-loop system and to assess the stabilizing capability of the hybrid MPC controller. Simulations of the controller in closed-loop with an accurate nonlinear model are presented

Methods and compositions for the diagnosis and treatment of diabetes

Both type 1 (DMT1) and type 2 (DMT2) diabetes mellitus are characterized by the slow loss of the insulin-producing beta cells in the pancreas and by a long preclinical stage when diagnosis and preventive therapies are already possible. Our inventors identified a novel DMT1 auto-antigen which is selectively expressed by beta cells and likely involved in the control of their progressive death. The identified molecules represent novel targets or tools for the development of diagnostic tests and cytoprotective therapies for diabetes mellitus

Vehicle yaw stability control by coordinated active front steering and differential braking in the tire sideslip angles domain

Vehicle active safety receives ever increasing attention in the attempt to achieve zero accidents on the road. In this paper, we investigate a control architecture that has the potential of improving yaw stability control by achieving faster convergence and reduced impact on the longitudinal dynamics. We consider a system where active front steering and differential braking are available and propose a model predictive control (MPC) strategy to coordinate the actuators. We formulate the vehicle dynamics with respect to the tire slip angles and use a piecewise affine (PWA) approximation of the tire force characteristics. The resulting PWA system is used as prediction model in a hybrid MPC strategy. After assessing the benefits of the proposed approach, we synthesize the controller by using a switched MPC strategy, where the tire conditions (linear/saturated) are assumed not to change during the prediction horizon. The assessment of the controller computational load and memory requirements indicates that it is capable of real-time execution in automotive-grade electronic control units. Experimental tests in different maneuvers executed on low-friction surfaces demonstrate the high performance of the controller