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The role of immune cells in Alzheimer's disease

Periodic Reporting for period 3 - IMMUNOALZHEIMER (The role of immune cells in Alzheimer's disease)

Reporting period: 2019-09-01 to 2021-02-28

"Alzheimer’s disease is the most common form of dementia and it affects more than 35 million people worldwide. Based on statistical studies on the aging population, it is estimated that by 2030 nearly 66 million people will have Alzheimer’s disease and by 2050 Alzheimer's will be a global epidemic with rates exceeding 115 million individuals. Alzheimer’s disease drives to death within 3 to 9 years after diagnosis and THERE IS NO CURE FOR THE DISEASE. Current drugs provide symptomatic benefit for up to 1 year, but there are no disease-modifying therapies.
Alzheimer's disease is characterized by a progressive deterioration of cognitive functions, and the neuropathological features include amyloid beta deposition, aggregates of hyperphosphorylated tau protein, and the loss of neurons in the central nervous system (CNS). Research efforts in the past decades have been focused on neurons and other CNS resident cells, but this ""neurocentric"" view has not resulted in disease-modifying therapies.
Growing evidence suggests that inflammation mechanisms are involved in Alzheimer's disease and the project team has recently shown an unexpected role for neutrophils in Alzheimer's disease, supporting the innovative idea that circulating leukocytes contribute to disease pathogenesis.
The MAIN GOAL of IMMUNOALZHEIMER is to study the role of circulating immune cells in animal models of Alzheimer's disease, focusing on neutrophils and T cells. We will study leukocyte-endothelial interactions in CNS microcirculation in intravital microscopy experiments. Leukocyte trafficking will be also studied inside the brain parenchyma by using two-photon microscopy, which will allow us to characterize leukocyte dynamic behaviour and the crosstalk between migrating leukocytes and CNS cells. The effect of therapeutic blockade of leukocyte-dependent inflammation mechanisms will be determined in animal models of Alzheimer's disease. Finally, the presence of neutrophils and T cells will be studied on brain samples from Alzheimer's disease patients and we will correlate leukocyte accumulation and microenvironmental positioning to disease severity.
Overall, IMMUNOALZHEIMER will generate fundamental knowledge to the understanding of the role of immune cells in neurodegeneration and will unveil novel therapeutic strategies to address Alzheimer’s disease.
The MAIN GOAL of our project is to study the role of immune cells in the pathogenesis of animal models of Alzheimer’s disease and to identify novel potential therapeutic targets for this disease.
During the first 30 months, at OBJECTIVE I we characterized neutrophil-driven inflammation and cognitive decline in Alzheimer’s disease. Particularly, we investigated neutrophil accumulation in models of Alzheimer’s disease and characterized the molecular mechanisms controlling neutrophil-endothelial interactions in intravital microscopy studies in brain microcirculation. We also characterized neutrophil intraparenchymal dynamics including the interactions with neural cells and identified the previously unknown existence of cell-cell interactions in animals with Alzheimer’s-like disease. We demonstrated the impact of blockade of trafficking mechanisms on disease and our data showed that therapeutic inhibition of key migration mechanisms improves memory and reduces microglial activation, amyloid deposition and tau hyperphosphorylation (numerous dissemination activities derived from these results). Finally, at Objective I we obtained exciting data correlating neutrophil accumulation to correlate disease severity in patients with Alzheimer’s disease.
At OBJECTIVE II we determined the impact of T cells on cognitive decline and neuropatological changes in mice with Alzheimer’s-like disease. Notably, we found a previously unknown role for specific T cell subpopulations in disease pathogenesis. Moreover, we found that early inhibition of T cell function blocks disease development and improves memory in animal models of Alzheimer’s disease. We also characterized the molecular mechanisms controlling the interactions between inflamed brain endothelium and T cells and studied the motility behavior and interactions between T cells and neural cells. We found a role for specific integrins in activated T cell motility inside inflamed central nervous system, suggesting these molecules may represent novel therapeutic targets in Alzheimer’s disease. We also characterized the molecular mechanisms controlling T cell trafficking mechanisms in the Alzheimer’s disease brain and the contacts between migrating leukocytes and neural cells. All these cutting-edge microscopy studies, which represent a large part of the project, were initially planned to be performed exclusively in vivo. Indeed, animal models are invaluable to address critical questions regarding the pathogenesis of human diseases and are essential for testing new therapeutic strategies. However, animal models are also very costly, may lack sufficient sensitivity in imaging experiments, are time consuming and present several ethical issues.
In recent years new powerful and groundbreaking approaches have emerged in the context of tissue and organ 3D cultures, coupling co-culture of many cells of different origins to microfluidic devices, allowing the building of increasingly complex in vitro micro environments recapitulating, at least partially, the complexity of in vivo conditions. These 3D cutting-edge approaches, globally called organoids, organ chips or lab on chips, hold the promise to revolutionize bio-medical research having many unquestionable advantages, including a level of complexity reasonably resembling the in vivo counterpart, better standardized and reproducible experimental contexts, higher sensitivity of imaging experiments, possibility to study fine molecular mechanisms under dynamic conditions, opportunity of technological scale up, containment of costs and less ethical issues to address compared to in vivo studies
In the context of IMMUNOALZHEIMER, to complement imaging studies in vivo, and to better characterize the molecular mechanisms controlling neutrophil and T cell trafficking in Alzheimer’s disease and interactions with neural cells, we set up an innovative 3D tissue model of Alzheimer’s brain parenchyma using primary microglia cells and primary neurons and astrocytes producing amyloid beta and hyperphosphorylated tau. Also, we are currently implementing this pioneering in vitro model of Alzheimer’s cerebral parenchyma with the addition of microfluidics and an underflow model simulating the complexity of blood-brain barrier (BBB) in a fluidic lab on chip.
During the first scientific reporting period, we also characterized the accumulation of T cell subpopulations in brains from subjects with AD to further complete the innovative picture on immune cell-dependent damage in AD pathogenesis.
Overall, the results obtained during the first scientific reporting period point to circulating leukocytes as central players in the induction of chronic neuroinflammation and neurodegeneration and show novel disease mechanisms and potential therapeutic targets in Alzheimer’s disease.
During the reporting period, the team published eight manuscripts with the ERC support (one is in press) and, additionally, has: two manuscripts under revision, three manuscripts very close to submission and five manuscripts in preparation.

1) Rossi B, Constantin G. Live Imaging of Immune Responses in Experimental Models of Multiple Sclerosis.
Front Immunol. 2016 Nov 21;7:506.

2) Zenaro E, Piacentino G, Constantin G. The blood-brain barrier in Alzheimer's disease.
Neurobiol Dis. 2016 Jul 15. pii: S0969-9961(16)30165-6.

3) Pietronigro E, Della Bianca V, Zenaro E, Constantin G. NETosis in Alzheimer's disease
Front Immunol. 2017 Mar 2;8:211. doi: 10.3389/fimmu.2017.00211.

4) Zenaro E, Constantin G. Targeting neuroinflamamtion in the treatment and prevention of Alzheimer’s disease. Drugs of the future, 2017; 42(1), pp. 21-42.

5) Liebner S, Dijkhuizen RM, Reiss Y, Plate KH, Agalliu D and Constantin G. Functional morphology of the blood-brain barrier in health and disease.
Acta Neuropathol. 2018 Mar;135(3):311-336.

6) Zhang X, Wang Y, Yuan J, Li N, Pei S, Xu J, Luo X, Mao C, Liu J, Yu T, Gan S, Zheng Q, Liang Y, Guo W, Qiu J, Constantin G, Jin J, Qin J, Xiao Y.
Macrophage/microglial Ezh2 facilitates autoimmune inflammation through inhibition of Socs3.
J Exp Med. 2018 Apr 6. pii: jem.20171417. doi: 10.1084/jem.2017141.

7) Farinazzo A, Angiari S, Turano E, Bistaffa E, Dusi S, Ruggieri S, Bonafede R, Mariotti R, Constantin G, and Bonetti B. Nanovesicles from adipose-derived mesenchymal stem cells inhibit T lymphocyte trafficking and ameliorate chronic experimental autoimmune encephalomyelitis.
Sci Rep. 2018 May 10;8(1):7473. doi: 10.1038/s41598-018-25676-2.

8) Zenaro E, Rossi B, Constantin G. A role for neutrophils in neurodegeneration. Immunobiology, in press.
Alzheimer’s disease is the most common form of dementia affecting more than 35 million people worldwide and its prevalence is projected to nearly double every 20 years with a tremendous social and economic impact on the society. There is no cure for Alzheimer's disease and current drugs only temporarily improve disease symptoms.
The research activities proposed in IMMUNOALZHEIMER are therefore in the context an important challenge for the society: the Alzheimer's disease global epidemic. Our project goes beyond the state-of-the-art of the amyloid hypothesis and “neurocentric” views in Alzheimer’s disease proposing a role for circulating immune cells in disease pathogenesis.
IMMUNOALZHEIMER is an INTERDISCIPLINARY project based on the innovative idea of a role for circulating leukocytes in AD pathogenesis. Our expertise in immunology, leukocyte trafficking and neuroinflammation, as well as our skills in advanced microscopy and animal models of Alzheimer’s disease place us at the leading edge for the study of the circulating immune cells in this disease. We will also use a cutting-edge 3D system, to provide more insight into the molecular mechanisms responsible for leukocyte-mediated damage in Alzheimer’s disease, facilitating the achievement of project objectives and reducing the number of laboratory animals.
THE IMMUNOALZHEIMER’s team is therefore in the unique position to bring innovative ideas in the field leading to a more comprehensive picture of disease pathogenesis in which the role of immune cells has the potential to take the stage.
Overall the studies proposed in IMMUNOALZHEIMER are pioneering and we expect to identify novel disease mechanisms and potential therapeutic targets in Alzheimer’s disease.