Diesel Exhaust Exposure Alters The Expression Of Networks Implicated in Neurodegeneration in Zebrafish Brains Part 1

Abstract

Neurodegenerative diseases are a major cause of disability in the world, but their etiologies largely remain elusive. Genetic factors can only account for a minority of risk for most of these disorders, suggesting environmental factors play a significant role in the development of these diseases. 

Neurodegenerative diseases refer to a type of disease that causes the degeneration of brain cells due to different reasons. Common ones include Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. These diseases affect neurons in the brain, resulting in impaired brain function and symptoms such as memory, thinking, and behavioral changes.

However, we don't need to lose our positive attitude because of these illnesses. There are many ways to help patients improve memory and relieve symptoms. Let's take a look at some of these methods:

First, get some physical exercise. Research shows that physical activity improves blood circulation in the brain and increases the release of chemicals like dopamine and endorphins, which help protect and improve the function of neurons. At the same time, exercise can also help reduce problems such as anxiety, depression, and insomnia, making patients more pleasant to face life.

Second, maintain social relationships. It's also very important that our social networks are closely connected to related areas of the brain, and social interaction can not only reduce stress, but also improve brain health by increasing happiness, fighting depression, and more.

Finally, eat well and get enough sleep. Both of these aspects can have important effects on brain health. A good diet not only needs to include enough nutrients, but it also needs to avoid repeatedly consuming too much sugar, saturated fat, and cholesterol. In addition, getting enough sleep can help combat memory problems at various stages, especially diseases such as Alzheimer's disease.

In short, neurodegenerative diseases do not mean giving up expectations for life. While maintaining a positive attitude, it is also necessary to adopt scientific and effective methods to improve the quality of life. More laughter and more sunshine will help you stay healthy. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the multiple active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health through a variety of pathways.

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Prolonged exposure to air pollution has recently been identified to increase the risk of Alzheimer's and Parkinson's diseases, but the molecular mechanisms by which it acts are not well understood. 

Zebrafish embryos exposed to diesel exhaust particle extract (DEPe) lead to dysfunctional autophagy and neuronal loss. Here, we exposed zebrafish embryos to DEPe and performed high throughput proteomic and transcriptomic expression analyses from their brains to identify pathogenic pathways induced by air pollution. DEPe treatment altered several biological processes and signaling pathways relevant to neurodegenerative processes, including xenobiotic metabolism, phagosome maturation, and amyloid processing. The biggest induction of gene expression in brains was in Cyp1A (over 30-fold). 

The relevance of this expression change was confirmed by blocking induction using CRISPR/Cas9, which resulted in a dramatic increase in sensitivity to DEPe toxicity, confirming that Cyp1A induction was a compensatory protective mechanism. 

These studies identified disrupted molecular pathways that may contribute to the pathogenesis of neurodegenerative disorders. Ultimately, determining the molecular basis of how air pollution increases the risk of neurodegeneration will help in the development of disease-modifying therapies.

Keywords 

Air pollution · Dementia · Parkinson's disease · Alzheimer's disease · Transcriptomics · Proteomics.

Introduction

Air pollution is a major contributor to mortality and is associated with respiratory disease, heart disease, stroke, lung cancer, and diabetes. Emerging epidemiological evidence supports a link between air pollution exposure and the development of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases (AD and PD) (Fu et al. 2019). There is a paucity of research investigating the mechanisms by which air pollution may increase the risk of AD and PD, but the few that do exist are supportive of a causal relationship (Fu et al. 2019).

 The accumulation of protein inclusions in the brain is a universal feature of neurodegenerative disorders and is likely a common pathway leading to neuronal dysfunction and death. For example, the formation of amyloid-beta (A-beta) and tau structures are the pathological hallmarks of AD and α-synuclein (α-syn) aggregates or Lewy Bodies in PD (Ross and Poirier 2004; Woulfe 2008). 

Alterations in proteostasis appear to at least partially underlie the formation of these aggregates, but the precise mechanisms are still unknown and may vary in individuals depending on genetic and environmental risk factors. We do know that increased expression or decreased degradation of A-beta or α-syn can lead to AD and PD (Bostancıklıoğlu 2019; Johnson et al. 2019). 

Other common features in neurodegenerative disorders are inflammation and oxidative stress. Activated microglia, the inflammatory cells in the CNS, can be readily appreciated in autopsies of AD and PD brains and likely contribute to the pathogenesis of neurodegeneration (Kannarkat et al. 2013). This inflammation and possibly mitochondrial dysfunction are thought to lead to oxidative stress which is also appreciated in autopsied brains (Picca et al. 2020). 

The majority of mechanistic studies on air pollution and neurodegeneration to date have focused on inflammation (Jayaraj et al. 2017). Air pollution appears to increase both CNS inflammation and oxidative stress in animal and human brains (Calderón-Garcidueñas et al. 2004, 2007, 2008a, b  and c; Levesque et al.  2011a, b,  2013; Moulton and Yang 2012; Mumaw et al. 2017; Yokota et al. 2013a, b). 

Of particular interest are the findings that air pollution increases the expression of some inflammatory genes in the olfactory bulb (OB) of mice, a brain region where PD pathology is seen very early in the disease (Levesque et al. 2011a, b; Yokota et al. 2013a). Inflammation was also seen in dogs living in urban areas compared to those living in rural areas, and the authors speculated that these changes were due to high levels of air pollution (Calderón-Garcidueñas et al. 2003). 

The same authors also suggested that α-syn accumulated in the brains of people living in cities due to air pollution (Calderón-Garcidueñas et al. 2004, 2008a, b). In a recent study in a zebrafish model, exposure to DEPe was reported to cause neurotoxicity and a significant decrease in neuron number by disrupting autophagy (Barnhill et al. 2020). 

DEPe contains many of the toxic components of air pollution and is commonly used to model exposure in cell culture systems (Costa et al. 2014; Hesterberg et al. 2010; Levesque et al. 2011b). Autophagy is an essential intracellular mechanism for the removal and eradication of misfolded proteins and damaged organelles. 

Indeed, precise autophagic activity is necessary for cellular homeostasis, and dysfunctional autophagy in neurons leads to altered survival and neurodegeneration (Kesidou et al. 2013). There are clear limitations in many of these studies, but they all suggest that air pollution exposure increases the risk of AD and PD. Our understanding of the mechanisms by which pollution increases this risk is limited, but ultrafine particles and several components of air pollution can enter the brain either directly through the olfactory bulb or through the lungs via the bloodstream (Kilian and Kitazawa 2018). 

These particles are composed of a carbon core and adsorbed compounds such as polycyclic aromatic hydrocarbons, metals, nitrate, sulfate, and other elements and constitute a significant proportion of air pollutants, especially in urban areas. Many of these compounds have been implicated in oxidative stress and cellular toxicity adding plausibility to a causal association of air pollution and neurodegeneration.

Detailed multidisciplinary investigations, including cellular and animal models, can give insight into the causes of disease and direct future therapeutic approaches (Cannon and Greenamyre 2011). Among the model organisms used for in vivo studies, Danio rerio (zebrafish) is a powerful model for studying toxicology, molecular genetics, toxicogenomics, and drug discovery. 

Most biological pathways are highly conserved in vertebrates, and the majority of functional human genes have homologs in zebrafish (Vaz et al. 2018). In addition, zebrafish embryos develop rapidly and independently, allowing for analysis at any desired developmental stage. These features, together with rapid reproduction and low cost of husbandry, make zebrafish a favorable model for studying the toxic effects of environmental exposures. 

To better understand the molecular mechanisms behind the pathogenesis of the disease, a wide range of high-throughput gene expression profiling approaches have been developed. Mass spectrometry–based proteomics and high-throughput RNA sequencing hold particular promise in identifying biological pathways of interest. These techniques have been widely used to understand the response of cellular and animal models to environmental stimuli (Duan et al. 2017; García-Estrada et al. 2013; Jami et al. 2014a, b, 2015; Kosalková et al. 2012). 

In this study, we have pursued expression analyses of the effects of DEPe treatment on both the proteome and transcriptome profiles within the heads of zebrafish embryos. Isolation of samples from the head which is mainly composed of the brain tissue allowed for obtaining more tissue-specific profiles and minimized effects originating from other tissues. We describe here altered gene expression and protein profiles in several pathways implicated in neurodegeneration in the heads of DEPe-exposed zebrafish. These results provide new insights into the possible mechanisms by which air pollution increases the risk of AD and PD.

Material and methods

Fish treatment and CRISPR/Cas9-mediated gene knockdown

All studies were approved by the UCLA Animal Rights Committee. Zebrafish (AB) were bred by light stimulation for 1  h, and a total number of 200 eggs were incubated for 24  h at 28  °C. The resulting embryos were dechorionated in pronase (2  mg/ ml) and treated with either DEPe (Standard Reference Materials, NIST, Gaithersburg, MD) at a final concentration of 20  μg/ml or vehicle until 5  days post-fertilization (DPF). 

This concentration was selected since it resulted in neuron loss as previously reported but without significant mortality (Barnhill et al. 2020). Cyp1A knockdown fish was prepared as follows. Using tools available at http://crispr.mit.edu, the CRISPR RNA (Cyp1A-crRNA) was designed based on the location of suitable PAM sites in the first exon of the Cyp1A gene. 

The best design was purchased from IDT DNA (www.idtdna.com) together with the standard CRISPR-Cas9 tracrRNA. The crRNA for the Cyp1A gene and tracrRNA were separately resuspended in 10 mM Tris–EDTA to yield a 100 μM final concentration. Then, tracrRNA was combined with crRNA in 1×duplex buffer (100  mM potassium acetate; 30 mM HEPES, pH 7.5; IDT DNA) to yield a final concentration of 10  μM. The mixture was heated to 95 °C for 5 min in a boiling water bath and slowly cooled to room temperature to allow for the annealing of the complementary nucleotide sequences in crRNA and tracrRNA and the formation of functional guide RNA (gRNA). 

A volume of 3 μL of annealed gRNA was then combined with the same volume of a donor single-strand DNA (ssDNA; 200 μM) as the template sequence for mutant Cyp1A. An amount of 3  μg of Cas9 Nuclease (IDT) in 1×injection buffer (5  mM KCl; 0.1  M sodium phosphate, pH 6.8) was added to the mixture to form the final CRISPR/Cas9 ribonucleoprotein (Cyp1A-RNP) complexes. 

For the preparation of the Scramble RNP (SC-RNP), the specific Cyp1A-crRNA was substituted by a commercial Scramble-crRNA with no target on the genome. The sequence of all oligonucleotides used in this work is listed in Supplementary Table 1. Eggs were injected under a stereomicroscope with around 3 nL of RNP injection mixture. 

Five biological replicate injections (each replicate included 400–600 eggs injected with Cyp1A-RNP and 100–200 injected with SC-RNP) were performed, and all microinjections were completed within 60 min. Eggs were placed into 100 mm plastic Petri dishes and incubated at 28 °C in egg water. The survival rate of embryos was monitored for up to 7  days. 

Images of surviving fish were reviewed for scoring malformation. Each larva was scored in a blinded manner for the presence of 4 developmental malformations including head malformation, tail malformation, cardiac edema, and yolk edema. Larvae with all 4 malformations would be scored 4, whereas normal developing larvae would score 0. The statistical T-test was used to evaluate the significant differences.

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