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. 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 Y maze 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. 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-AD mice 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. 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.
Abstract Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a −/− mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a −/− basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a −/− Lyn −/− showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a −/− hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.
Vascular inflammation and a dysfunctional blood-brain-barrier have been implicated in the pathogenesis of Alzheimer's disease. However, the role of leukocyte trafficking mechanisms in Alzheimer's disease in the induction of neuropathological changes and memory deficit is unclear. We carried out two-photon laser-scanning microscopy (TPLSM) experiments in the brain of mice with five familial Alzheimer's disease (5xFAD) mutations presenting amyloid pathology, and 3xTgAD mice with both amyloid and tau pathology and observed that fluorescently labeled neutrophils and activated lymphocytes adhere to the vascular endothelium and migrate into the brain parenchyma in these animals. Blocking monoclonal antibodies to LFA-1 (alphaLbeta2) and VLA-4 (alpha4beta1) integrins significantly inhibited neutrophil and lymphocyte adhesion in brain vessels, suggesting that leukocyte integrins play a role in leukocyte extravasation in Alzheimer's disease models. In addition, our TPLSM data showed that leukocytes from LFA-1 deficient mice are completely unable to adhere or crawl in the blood vessels and thus to transmigrate in the brain parenchyma of Alzheimer's disease mice. LFA1 or VLA-4 inhibition using monoclonal antibodies clearly improved memory function compared to mice treated with a control antibody, restoring performance to levels comparable with wild-type age-matched littermates in Y maze and contextual fear conditioning tests. The role of LFA-1 integrin was confirmed by crossing 3xTg-AD animals with the LFA-1-deficient Itgal-/- strain. We found that the 3xTg-AD mice lacking LFA-1 integrin showed improved memory in cognitive tests compared to wild-type animals. These findings were supported by neuropathological data showing a lower density and activation state of microglia in 3xTg-AD mice deficient of LFA-1 integrin compared to 3xTg-AD aged-matched controls. We also found that the temporary LFA-1 blockade with an anti-LFA-1 antibody during the early stages of disease preserved the cognitive functions of the mice at later time points suggesting that therapeutic blockade of leukocyte adhesion during the early stages of disease provides a long-term beneficial effect on cognition in older mice. Current Alzheimer's disease therapies provide only temporary improvement and marginally reduce the rate of cognitive decline. Therefore, we propose that targeting integrins controlling leukocyte trafficking may represent a new therapeutic strategy in Alzheimer's disease.
Calcium dobesilate (calcium 2,5-dihydroxybenzenesulfonate, CaD) is an oral drug currently used for the treatment of chronic venous insufficiency and microangiopathies such as diabetic retinopathy. Treatment with CaD prevents reactive gliosis and neuronal loss in animal models of diabetic retinopathy. Furthermore, CaD reduces reactive oxygen species production by neutrophils in vitro and lowers the production of pro-inflammatory cytokines in disease models. However, its effect on neuroinflammation and Alzheimer's disease (AD) is unknown and our goal was to determine the potential therapeutic effect of CaD in AD models. In vitro adhesion assays on integrin ligands were performed to analyze the effect of CaD on rapid adhesion under static conditions. Flow adhesion assays using Bioflux technology were performed to assess the effect of CaD under physiopathological conditions. Chemotaxis assays were performed using 3μm pore transwells and flow cytometry. 3xTg-AD mice, which present both amyloid and tau pathology were treated with CaD to determine the effect of this drug on memory decline and neuropathological changes. We recently demonstrated that neutrophils migrate into the brain and contribute to the induction of cognitive deficits in animal models of AD. Treatment of neutrophils with CaD reduced integrin-dependent rapid adhesion triggered by chemokines and TNF under static conditions. CaD also significantly inhibited neutrophil adhesion under flow conditions suggesting that this drug may interfere with neutrophil trafficking in vivo. Interestingly, CaD did not reduce chemotaxis and integrin expression, suggesting that it selectively interferes with signal transduction machinery controlling rapid adhesion and integrin activation. Notably, oral treatment for one month with CaD of 3xTg-AD mice led to a significant restoration of memory in Y maze and contextual fear conditioning behavioral tests when compared to control mice. Neuropathological studies showed that treatment with CaD reduced amyloid deposition mainly in the cortex and led to a drastic reduction of microglial activation in the hippocampus and cortex when compared to control animals. CaD has anti-inflammatory properties and can improve cognition in animal models of AD, suggesting that this drug may offer a new therapeutic strategy in AD with rapid translation into the clinic.
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