Modulation of the neuroinflammatory response following genetic and environmental manipulations in the zebrafish (Danio Rerio)

Abstract

Aging is an inevitable process through which organisms experience functional and physical decline. Cellular changes such as mitochondrial dysfunction, telomere attrition, and loss of proteostasis constitute the main components of this process. One of the hallmarks of brain aging is the increased inflammatory status of the brain. This process is named neuroinflammation and is seen both in the development of neurodegenerative disorders and in healthy aging. The over-activation of microglia and astrocytes, increased secretion of pro-inflammatory cytokines and reactive oxygen species, NLRP3/NALP3 inflammasome activation, and the upregulation of NF-κB signaling pathway are among the markers of neuroinflammation. Deregulated nutrient sensing through mammalian target of rapamycin pathway(mTOR), impaired neurogenesis, and synaptic integrity over time are among the common outcomes of this process. Genetic susceptibility is also another factor to contribute the vulnerability to inflammation. Therefore, further studies are necessary to investigate the effects of the inflammation-stimulating agents and the genetic susceptibility. Thus, this study aimed to develop copper sulfate as an inflammatory stimulating agent for both zebrafish embryos and adults. For this objective, we conducted long-term and short-term copper sulfate embryo treatment studies. The gene expression results showed that the inflammatory response was quite predictable in embryos by increasing pro-inflammatory markers early on and later increasing anti-inflammatory cytokines. Secondly, we conducted copper sulfate and rapamycin treatment in very old (38 months) zebrafish animals to investigate the impact of the inflammation on mTOR signaling and synaptic integrity. The protein expression results indicated that rapamycin was an effective for mTOR suppression in very old animals but copper sulfate was not able to stimulate a robust inflammatory response. Also, synaptic integrity markers were mostly stable among treatment groups in very old animals. Finally, we used tsc2+/- adult animals and applied rapamycin treatment to very old (33 months) tsc2+/- adults to assess the effect of overactive mTOR signaling and the possibility of reversing this in the progression of inflammation. Comparatively, we wanted to understand the effect of copper sulfate exposure in very old (43 months) ztor+/- animals that have a downregulated mTOR pathway. The results showed that the rapamycin effect was not significant between wild-type and tsc2+/- animals in terms of pro-inflammatory cytokines and autophagy markers. Similarly, the copper sulfate effect was not different between wild type and ztor+/- animals for pro-inflammatory markers. However, autophagy markers decreased significantly in mutants. In conclusion, this study showed that aging affects the regulation of the inflammatory response within the brain. Also, genetic manipulations on the mTOR pathway would provide crucial insights to investigate the neuroinflammatory profile of the brain in the course of aging.