Calreticulin and other candidate Alarmins and Opsonins from Microglia
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
PhD project strategic theme: Biosciences for an integrated understanding of health
Alarmins are soluble molecules released from damaged cells that promote the non-infectious inflammatory activation of immune cells. This activation is important for initiating the early stage innate immune response to tissue damage or disease. Calreticulin, an intracellular chaperone of the endoplasmic reticulum, is a candidate alarmin to microglia - the resident macrophage of the brain. During the rotation project, it was found that calreticulin was released from damaged cells, chemoattracted microglia in vitro, and stimulated their release of tumour necrosis factor-alpha and interleukin-6; cytokines associated with an inflammatory phenotype. Thus, calreticulin may act as an intermediate between tissue damage in the brain and the microglial immune response to that damage.
The PhD project would build on these findings. Firstly, by understanding the mechanistic pathways involved, including the mechanism of cellular release, and the receptor (or receptors) on microglia for calreticulin and downstream signalling. Secondly, the consequences for microglia in terms of the inflammatory profile and neurotoxicity. Thirdly, it will be tested whether calreticulin acts as an opsonin for microglial phagocytosis of synapses, myelin and neurons. Fourthly, if calreticulin acts as an opsonin, then whether this promotes antigen presentation to T cells will be determined. To determine whether calreticulin release occurs in vivo, calreticulin and cytokine levels will be measured in the cerebrospinal fluid of human patients that have either suffered from traumatic brain injury or not. Similarly, calreticulin and cytokine levels will be measured in the cerebrospinal fluid of rats and mice from various disease models, including brain trauma. Samples would be acquired across multiple acquisition timepoints post-injury to better elucidate the physiological pattern of calreticulin release and relate this to the inflammatory profile.
As the microglial immune response is a multi-layered process involving numerous complexes and molecules, further to calreticulin other soluble mediators will be investigated for their potential as microglial alarmins and/or opsonins. The secretome of primary microglia under various conditions will be acquired, purified, and then fragmented into identifiable proteins by mass spectrometry. Candidate proteins will then be assessed for their ability to act as alarmins or opsonins for microglia as above.
Concurrently with this project, the microglial receptor P2Y6 will be assessed to test whether it has a role in developmental synaptic phagocytosis (pruning). Too little synaptic pruning is implicated in autism, and too much pruning in schizophrenia. Preliminary research in the Brown lab indicates that the P2Y6 receptor may be required for microglial phagocytosis of synapses; and behavioural testing of 3-4 month old P2Y6-/- mice indicates they have memory deficits. This project will further test whether one-month-old P2Y6-/- mice have social deficits and/or increased synaptic density, and whether this is due to reduced microglial phagocytosis of synapses.
Alarmins are soluble molecules released from damaged cells that promote the non-infectious inflammatory activation of immune cells. This activation is important for initiating the early stage innate immune response to tissue damage or disease. Calreticulin, an intracellular chaperone of the endoplasmic reticulum, is a candidate alarmin to microglia - the resident macrophage of the brain. During the rotation project, it was found that calreticulin was released from damaged cells, chemoattracted microglia in vitro, and stimulated their release of tumour necrosis factor-alpha and interleukin-6; cytokines associated with an inflammatory phenotype. Thus, calreticulin may act as an intermediate between tissue damage in the brain and the microglial immune response to that damage.
The PhD project would build on these findings. Firstly, by understanding the mechanistic pathways involved, including the mechanism of cellular release, and the receptor (or receptors) on microglia for calreticulin and downstream signalling. Secondly, the consequences for microglia in terms of the inflammatory profile and neurotoxicity. Thirdly, it will be tested whether calreticulin acts as an opsonin for microglial phagocytosis of synapses, myelin and neurons. Fourthly, if calreticulin acts as an opsonin, then whether this promotes antigen presentation to T cells will be determined. To determine whether calreticulin release occurs in vivo, calreticulin and cytokine levels will be measured in the cerebrospinal fluid of human patients that have either suffered from traumatic brain injury or not. Similarly, calreticulin and cytokine levels will be measured in the cerebrospinal fluid of rats and mice from various disease models, including brain trauma. Samples would be acquired across multiple acquisition timepoints post-injury to better elucidate the physiological pattern of calreticulin release and relate this to the inflammatory profile.
As the microglial immune response is a multi-layered process involving numerous complexes and molecules, further to calreticulin other soluble mediators will be investigated for their potential as microglial alarmins and/or opsonins. The secretome of primary microglia under various conditions will be acquired, purified, and then fragmented into identifiable proteins by mass spectrometry. Candidate proteins will then be assessed for their ability to act as alarmins or opsonins for microglia as above.
Concurrently with this project, the microglial receptor P2Y6 will be assessed to test whether it has a role in developmental synaptic phagocytosis (pruning). Too little synaptic pruning is implicated in autism, and too much pruning in schizophrenia. Preliminary research in the Brown lab indicates that the P2Y6 receptor may be required for microglial phagocytosis of synapses; and behavioural testing of 3-4 month old P2Y6-/- mice indicates they have memory deficits. This project will further test whether one-month-old P2Y6-/- mice have social deficits and/or increased synaptic density, and whether this is due to reduced microglial phagocytosis of synapses.
