Introduction: Recent genome wide association studies have identified the C4 locus, specifically C4A in Schizophrenia (SCZ) risk¹. in vivo and in vitro models of SCZ have highlighted that disease onset is at least in part due to dysfunctional complement mediated synaptic pruning². Astrocytes are the predominant cellular source of C4 in the brain and influence neuronal C4 secretion. Further work is needed to clarify how astrocytes contribute to excessive neuronal synaptic pruning in SCZ and whether this can be modulated. Emerging evidence suggests individuals with SCZ carrying certain C4 haplotypes have dysbiosis of the gut microbiome³. Futhermore, dietary probiotic supplementation has shown promising protective effects in clinical trials and in models of SCZ^4,5. Such revelations highlight the potential of probiotic use as a tool to modulate complement driven synaptic pruning and as an adjunctive treatment for SCZ.

Objectives: The key aims of this project are to investigate the effects of probiotic conditioned media on modulating C4 expression first in relevant immortalised cells lines. This investigation will be furthered by utilising an IPSC model of neurons and astrocytes that models this SCZ specific genetic risk. We will probe the efficacy of probiotic metabolites on ameliorating this aberrant C4 production in these cells and thereby, aberrant synaptic pruning, and investigate the underlying mechanism.

Methods: qPCR, ELISA and immunocytochemistry were carried out to confirm C4 expression and production in immortalised human derived neural and glial cell lines. Cell lines used were differentiated SH-SY5Y (neuronal), 1321N1(astrocytic), HMC3 (microglial). Immortalised cell lines were exposed to either lipopolysaccharide (LPS), interferon gamma (IFN-y) or interleukin-6 (IL-6), inflammatory cytokines shown to be increased in SCZ patients, for 48 hours. RNA was extracted and RT-qPCR performed to assess levels of C4 expression. Three consortia of probiotic metabolite containing conditioned media were added to cell lines for 48 hours with and without inflammatory stimuli to assess the potential anit-inflammatory impact on C4 expression.

Results: Astrocytic and neuronal cell lines were shown to produce and secrete C4. Exposure to all inflammatory stimuli significantly increased C4 expression in these cell types. Exposure to probiotic conditioned media, resulted in a down regulation of basal C4 levels compared to untreated cells. Additionally, the increased expression of C4 seen upon exposure to inflammatory stimuli was blocked by supplementing conditioned media. C4 expression was significantly reduced across all conditioned when cells were exposed to conditioned media, with both SH-SY5Y and 1321N1 cells showing consistently lower expression levels of C4 compared to stimulated cells alone. The same protective effects were seen across almost three consortia of conditioned media. All experiments were carried out with an n=3 and Two way ANOVA was used for statistical analysis in all experiments.

 

 

Discussion: Confirming that astrocytes are the source of C4 in the brain highlights their importance as a target for mediating C4 production through probiotic supplementation. These results demonstrate the potential neuromodulatory effects of probiotic metabolites have when challenged with multiple inflammatory stimuli at reducing C4 expression and production; further strengthening their potential use as supplementary treatments for SCZ.