Gennj Piacentino

Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin

Inflammation is a pathological hallmark of Alzheimers disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimers disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-β (Aβ) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aβ42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimers disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimers disease–like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimers disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimers disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimers disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimers disease.

The blood-brain barrier in Alzheimer's disease.

Alzheimers disease (AD) is a chronic neurodegenerative disorder characterized by the pathological accumulation of amyloid beta (Aβ) peptides and neurofibrillary tangles containing hyperphosphorylated neuronal tau protein. AD pathology is also characterized by chronic brain inflammation, which promotes disease pathogenesis. In this context, the blood-brain barrier (BBB), a highly specialized endothelial cell membrane that lines cerebral microvessels, represents the interface between neural cells and circulating cells of the immune system. The BBB thus plays a key role in the generation and maintenance of chronic inflammation during AD. The BBB operates within the neurovascular unit (NVU), which includes clusters of glial cells, neurons and pericytes. The NVU becomes dysfunctional during AD, and each of its components may undergo functional changes that contribute to neuronal injury and cognitive deficit. In transgenic animals with AD-like pathology, recent studies have shown that circulating leukocytes migrate through the activated brain endothelium when certain adhesion molecules are expressed, penetrating into the brain parenchyma, interacting with the NVU components and potentially affecting their structural integrity and functionality. Therefore, migrating immune system cells in cerebral vessels act in concert with the modified BBB and may be integrated into the dysfunctional NVU. Notably, blocking the adhesion mechanisms controlling leukocyte–endothelial interactions inhibits both Aβ deposition and tau hyperphosphorylation, and reduces memory loss in AD models. The characterization of molecular mechanisms controlling vascular inflammation and leukocyte trafficking could therefore help to determine the basis of BBB dysfunction during AD and may lead to the development of new therapeutic approaches.

TIM-1 Glycoprotein Binds the Adhesion Receptor P-Selectin and Mediates T Cell Trafficking during Inflammation and Autoimmunity

Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease.

Regulatory T Cells Suppress the Late Phase of the Immune Response in Lymph Nodes through P-Selectin Glycoprotein Ligand-1

Regulatory T cells (Tregs) maintain tolerance toward self-antigens and suppress autoimmune diseases, although the underlying molecular mechanisms are unclear. In this study, we show that mice deficient for P-selectin glycoprotein ligand-1 (PSGL-1) develop a more severe form of experimental autoimmune encephalomyelitis than wild type animals do, suggesting that PSGL-1 has a role in the negative regulation of autoimmunity. We found that Tregs lacking PSGL-1 were unable to suppress experimental autoimmune encephalomyelitis and failed to inhibit T cell proliferation in vivo in the lymph nodes. Using two-photon laser-scanning microscopy in the lymph node, we found that PSGL-1 expression on Tregs had no role in the suppression of early T cell priming after immunization with Ag. Instead, PSGL-1-deficient Tregs lost the ability to modulate T cell movement and failed to inhibit the T cell–dendritic cell contacts and T cell clustering essential for sustained T cell activation during the late phase of the immune response. Notably, PSGL-1 expression on myelin-specific effector T cells had no role in T cell locomotion in the lymph node. Our data show that PSGL-1 represents a previously unknown, phase-specific mechanism for Treg-mediated suppression of the persistence of immune responses and autoimmunity induction.

TREATMENT WITH CALCIUM DOBESILATE REDUCES NEUROINFLAMMATION AND IMPROVES COGNITION IN A MOUSE MODEL OF ALZHEIMER’S DISEASE

activated in brains following TBI. We show here that PERK activation following injury is time dependent and cell specific. We also show inhibition of PERK following traumatic brain injury in two month old mice affected tau pathology, overall protein synthesis, broad neuronal function, cognition, and gliosis. Conclusions: Our data provide novel insight into the physiological mechanisms of TBI, and suggest that PERK plays an important role in injury progression and cellular response. These data also suggest that the timing of PERK inhibition is critical during recovery. Overall, we show that the exploration of PERK inhibition as a therapeutic treatment for traumatic brain injury is an attractive option.

BLOCKADE OF ALPHA4 INTEGRINS AMELIORATES COGNITIVE DYSFUNCTION AND NEUROPATHOLOGICAL CHANGES IN TRANSGENIC ANIMALS WITH ALZHEIMER'S-LIKE DISEASE

Background: Blood-derived leukocyte subpopulations, including lymphocytes, monocytes and neutrophils, have been identified in the brains of patients with Alzheimer’s disease (AD) and in corresponding animal models, but their role in disease pathogenesis is unclear. We have recently demonstrated that neutrophils infiltrate the AD brain and that neutrophil depletion has therapeutic effect in AD transgenic mouse models. However, leukocyte trafficking mechanisms in the central nervous system (CNS) during AD are largely unknown and our goal was to determine the role of alpha4 integrins in disease pathogenesis. Methods: Intravital microscopy studies were performed to visualize and analyze leukocyte-vascular interactions in 3xTg-AD mice, which present both amyloid and tau pathology. Contextual fear conditioning and Ymaze tests were performed to analyze the cognitive deficit. Neuropathological studies were performed to evaluate amyloid beta deposition, tau hyperphosphorylation, microglial activation and expression of synaptic proteins. Results: Intravital microscopy studies performed in the CNS microcirculation of 3xTg-AD mice showed that blockade of alpha4 integrins prevents activated T cell rolling and firm adhesion on brain endothelium. Treating 3xTg-AD mice with an anti-alpha4 integrin antibody starting during early disease phases in mice already presenting cognitive deficits clearly improved memory function compared to mice treated with a control antibody in Y maze and contextual fear conditioning tests. These findings were supported by neuropathological data showing a reduction in Abeta deposition and lower density and activation state of microglia in the cortex and hippocampus of 3xTg-ADmice treated with anti-alpha4 antibody compared to animals treated with an isotype control antibody. Moreover, anti-apha4 treatment reduced tau hyperphosphorylation and restored synaptic protein expression compared to control animals. Notably, restoration of cognitive function in mice with temporary anti-alpha4 treatment during early disease was maintained also at later time points in aged animals, suggesting that therapeutic blockade of leukocyte adhesion during the early stages of disease provides a long-term beneficial effect on cognition in older mice. Conclusions: Antibodies anti-alpha4 integrin are currently used for the treatment of patients with autoimmune diseases and our data suggest that blockade of alpha4 integrins may represent a novel therapeutic approach in AD that has the potential for rapid translation into the clinic.

Targeting leukocyte integrins has therapeutic effect in Alzheimer's-like disease

demonstrated that the classic TSPO ligands, Ro5-4864 and PK11195, increase brain steroid levels, reduce Ab accumulation, and improve cognition in a mouse model of AD. Here we evaluated the steroidogenic efficacy and acute behavioral effects of three TSPO imidazopyridine ligands, CLINDE, PBR175, and PBR162, which we have previously shown to be non-toxic in vitro. CLINDE, which exhibited the most favorable behavioral and pharmacokinetic profile, was investigated further for its specificity and binding affinity in human brain. Methods: The acute behavioural effects of the three novel TSPO ligands were compared with Ro5-4864, in 3 month old castrated C57BL/6J mice. Two hours after ligand injection (3 mg/kg i.p.), anxiety-, depression-, and memory-related behaviors were assessed in the elevated-plus maze, open field maze, tail-suspension test, and object recognition tests respectively. Brain steroids levels were measured by LC-MS/MS. To determine if the behavioral effects of TSPO ligands were mediated via increased neurosteroidogenesis, mice were pretreated with a steroidogenesis inhibitor, aminogluthetimide (10mg/ kg). The pharmacokinetic properties of the novel ligands were assessed by competitive PET imaging [11C]PK-11195. The specificity of action of CLINDE was determined in TSPO knockout mice, and the affinity for TSPO (Ki value) in human brain homogenate was investigated in vitro by [11C] PK-11195 binding assay. Results: CLINDE and PBR175 improved learning and memory performance equally well as Ro5-4864. These improvements were completely ablated by aminogluthetimide, confirming that the beneficial effects were mediated through an increase in neurosteroidogenesis. Anxietyand depression-related behaviors were unaffected by the TSPO ligands. Competitive PET studies indicated that CLINDE showed the most long lasting binding to TSPO. The specificity of behavioral effects of CLINDE was confirmed. The affinity of CLINDE in high-affinity binder (HAB) and low-affinity binder (LAB) subjects were 5.5 and 29.7 respectively. Conclusions:These findings indicate that CLINDE and PBR175 rapidly promote neurosteroidogenesis, thereby enhancing learning and memory in vivo. These second generation TSPO ligands are promising therapeutic candidates with improved pharmacokinetic properties compared to classic TSPO ligands.

Krebs cycle potentiation has immunosuppressive effects and inhibits experimental autoimmune encephalomyelitis development

Background: Immunometabolism represents a novel field of investigation that links immune cell functions with the biochemical changes correlated to their intracellular metabolic state. Recent studies suggest that immune responses can be regulated by modulating immune cell metabolism, and that metabolic intervention can affect autoimmune disease development in animalmodels. Pantethine (PTTH) is ametabolic precursor of coenzyme A and a Krebs cycle stimulator, andwas successfully used in cardiovascular riskpatients. The aimof our studywas to investigate the effect ofmetabolic interventionwith pantethine on the effector functions of encephalitogenic T cells and on the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the murine model of human multiple sclerosis (MS). Results: Metabolomic and phosphoproteomic studies followed by bioinformatics computational analysis shown that, compared to naïve T cells, PLP139–151-specific encephalitogenic T cells undergo a metabolic reorganization known as Warburg effect, with reduced fatty acid oxidation (FAO) and Krebs cycle rate, strongly increased aerobic glycolysis and shunt of glycolysis intermediates to the pentose phosphate pathway (PPP). This metabolic shift led to increased synthesis of nucleotide, lipid andamino acid building blocks necessary for cell growthandproliferation. In vitro treatment with PTTH rapidly induced Krebs cycle potentiation in encephalitogenic T cells, followed by increased FAO, reduced glycolysis, block of PPP and inhibition of macromolecule biosynthesis. This metabolic tuning strongly affected antigen-specific proliferation and cytokine production by encephalitogenic T cells and inhibited integrin-mediated T cell adhesion in vitro. Accordingly, treatment with PTTH during the preclinical phase of disease prevented the development of both chronic (C-EAE) and relapsing–remitting (RR-EAE) EAE models, by blocking T cell activation and leukocyte adhesion in pial venules, leading to reduced pathogenic cell trafficking to the central nervous system. Notably, therapeutic treatment with PTTH significantly reduced the severity of established EAE in both C-EAE and RR-EAE models. Conclusions: Our data demonstrate that metabolic regulation of encephalitogenic T cell effector functions with pantethine has therapeutic effect on EAE and may represent a novel therapeutic strategy for MS. This work was supported by the European Research Council (ERC) grant Neurotrafficking (to G.C.).

NEUTROPHILS INDUCE ALZHEIMER'S-LIKE DISEASE VIA LFA-1-INTEGRIN AND NEUTROPHIL EXTRACELLULAR TRAPS

Methods: Confocal microscopy studies were performed to evaluate inflammation mechanisms. Intra-vital microscopy studies using two-photon microscopy were performed to visualize and analyze the movement of neutrophils inside brain vessels and in the parenchyma. In vitro rapid adhesion assays were performed on integrin ligands whereas and integrin affinity was measured using Image Stream technology. Neuropathological studies, fear conditioning and Y maze tests were performed to analyze the effect of inflammation mechanism inhibition on disease.