Microglia are the primary immune cells of the central nervous system (CNS); they have dynamic roles in brain development, maintaining homeostasis, and responding to insult and injury, thereby contributing to CNS health. However, increasing evidence highlights a role for microglial dysfunction in many brain pathologies, like Alzheimer’s disease (AD) and Parkinson’s disease (PD). Understanding how and when microglia are beneficial, detrimental or both, is an active area of research and, elucidating the underlying mechanisms involved in neurodegeneration could uncover novel therapeutic avenues. In this work, I investigated how microglial functions and neuronal loss were affected by calreticulin and β-galactosidase, two proteins associated with ageing, chronic inflammation, and neurodegeneration.

The aggregation of amyloid-β to form oligomers and insoluble amyloid plaques in the brain is a hallmark of AD. Amyloid-β can be directly neurotoxic and induce pro-inflammatory activation of microglia, which may contribute to neurodegeneration. Molecular chaperone proteins are commonly found intracellularly, where they interact with proteins to prevent their aggregation and facilitate proper folding. The endoplasmic reticulum-resident chaperone protein, calreticulin, can be released from microglia and has been found to bind amyloid-β. I investigated whether exogenous calreticulin affects amyloid-β aggregation and amyloid-β induced neurotoxicity. In vitro assays revealed inhibition of amyloid-β fibrillisation by calreticulin, and transmission electron microscopy showed that calreticulin promoted formation of larger amyloid-β oligomers. Furthermore, exogenous calreticulin was protective in the context of amyloid-β-induced neuronal loss in primary mixed neuronal-glial cultures. Together this data suggests that calreticulin might act as an extracellular chaperone for amyloid-β and be neuroprotective, hence treatments increasing extracellular calreticulin in the brain might be beneficial for AD.

Glycohydrolase enzymes, including neuraminidase 1 (Neu1) and β-galactosidase, play a fundamental role in the degradation of glycoproteins and glycolipids, to maintain cellular constituent turnover and glycosylation homeostasis. Neu1 hydrolyses terminal sialic acid residues to expose galactose residues, which can then be hydrolysed by β-galactosidase. Previously it was thought that these enzymes were exclusively located in lysosomes, but recent evidence has found Neu1 activity also associated with the external surface of the plasma membrane. As Neu1 can be structurally and functionally coupled to β-galactosidase in a lysosomal multienzyme complex, I investigated whether BV-2 microglia and primary rat microglia have increased extracellular β-galactosidase activity when activated by a variety of inflammatory stimuli, including lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Inflammatory activation of microglia increased β-galactosidase activity at the cell surface and increased β-galactosidase protein levels extracellularly. Extracellular β-galactosidase might remove galactose residues from the surface of microglia and neurons, potentially disrupting homeostasis. I found that addition of β-galactosidase to primary mixed neuronal-glial cultures caused a significant loss of neurons and promoted microglial activation. Whereas inhibition of β-galactosidase in LPS-stimulated cultures reduced LPS-induced neuronal loss and microglial activation, suggesting that β-galactosidase may activate microglia in a way that promotes neuronal loss.

Together, this work elucidates novel effects of calreticulin and β-galactosidase on microglial function and neuronal loss, which may contribute to understanding the roles of these proteins in neurodegeneration and disease.