The complement cascade is phylogenically ancient pathogen recognition and response system that forms the initial frontier of mammalian innate immunity. Complement forms the bridge between humoural and cellular immunity through the generation of the anaphylatoxins, C3a and C5a, to recruit leukocytes to a pathogenic insult. However, in recent years, novel roles for the complement system outside of innate immunity have been demonstrated, especially in the developing embryo. To add to these novel roles, this thesis explores the role of the complement anaphylatoxin receptors in neural development.
Previous studies in our laboratory have indicated a novel role for C5aR1 in the prevention of neural tube defects in folate-deficient dams. In these studies, C5aR1 was localised to the neuroepithelium throughout the period of neural tube closure and the precursor to C5a, C5, was also shown to be present. It has previously been unclear, both in this study and others, what other components of the complement system are present in the developing embryo that may lead to the generation of anaphylatoxins. Here we demonstrate that the classical and alternative pathways, at the time of murine neural tube closure, lack expression of key propagators. Our data demonstrate the C2/4- independent and extrinsic pathways remain patent as a plausible means of C5a generation.
In order to trace C5aR1-expressing cells within the developing embryo, a transgenic mouse was created using a modified bacterial artificial chromosome approach. A CreErt2-IRES-EGFP transgene was inserted at the second exon of C5ar1 in order to induce expression from the C5aR1 promoter region. Resulting litters demonstrated transgene expression that was responsive to LPS stimulation and similar to previously reported C5aR1 expression within the CNS. However, crossing with a cre- reporter line demonstrated that the protein produced from the transgene was non-functional.
In separate studies, both C3aR and C5aR1 were shown to be expressed by neural progenitor cells from neural tube closure to birth, and both receptors localised to the apical attachment of progenitor cells. This thesis demonstrated C5a is a constituent of embryonic cerebrospinal fluid and that C5a- C5aR1 interaction activates aPKC to promote neural progenitor proliferation. Blockade of C5aR1 during development leads to behavioural abnormalities in the resulting litters and microstructural brain abnormalities on MRI. C3aR, conversely, promotes differentiation of neural progenitors.
The functions of C5aR1 and C3aR seem to be somewhat redundant during development as anaphylatoxin knockout animals are not known to exhibit any gross neurological abnormalities. However, here we demonstrate subtle deficits in memory and anxiety in these animals – perhaps resulting from impaired neuroplasticity or development.
In summary, this thesis demonstrates the presence and function of the complement anaphylatoxin receptors in neural progenitor cells. As complement becomes a more viable target for the treatment of inflammatory disorders of pregnancy, this work provides a caution for clinicians by elucidating an important neurodevelopmental role for C3aR and C5aR1.