Diesel Exhaust Exposure Alters The Expression Of Networks Implicated in Neurodegeneration in Zebrafish Brains Part 3
Mar 04, 2024
The results of proteomics comparison revealed the upregulation (overrepresentation) of ACTG2 (actin, gamma 2), CAT (catalase), GSR (glutathione-disulfide reductase), GST (glutathione S-transferase), HMOX1 (heme oxygenase 1), and PRDX1 (peroxiredoxin 1), as well as the downregulation of ubiquitin-conjugating enzyme ubcM2, RAP1A (RAP1A, member of RAS oncogene family), UBE2E3 (ubiquitin-conjugating enzyme E2 E3), and ACTA1 (actin, alpha 1).
Proteomics is a large-scale scientific technology that studies the types, structures, quantities, and functions of proteins in organisms. In recent years, more and more studies have shown that proteomics is closely related to memory.
Researchers have discovered that proteins in the brain can affect signaling between neurons, thereby affecting cognitive activities such as learning and memory. For example, most protein molecules required for memory encoding and storage processes are synthesized at synapses. These protein molecules are not only directly involved in the conduction and transduction of nerve signals, but can also affect the synaptic connections and their stability between neurons, thereby affecting the generation and consolidation of memory.
Research has also found that certain proteins play a crucial role in the memory process. For example, CaMKII protein kinase in neurons regulates the strength and stability of synaptic connections and plays an important role in learning and memory. Another important protein is BDNF, which can promote the growth of neurons, synaptic strengthening, and neuronal survival, thereby enhancing the consolidation and maintenance of memory.
In addition, some studies have found that physical health and diet also have significant effects on proteomics and memory. Adequate sleep, appropriate exercise, and a low-fat, high-protein diet can promote protein synthesis and repair, which is beneficial for enhancing memory and cognitive abilities.
In summary, proteomics and memory are closely linked. Scientists studying the life sciences are increasingly discovering how to modulate the proteome to enhance memory and prevent treatments for cognitive disorders such as Alzheimer's disease. Therefore, we should pay attention to the results of scientific research and maintain good eating, living, and exercise habits to maintain a healthy, strong brain and have a better memory. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors. These substances are very important for memory and learning. In addition, Cistanche deserticola can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrients and energy, thereby improving brain vitality and endurance.
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Among them, the downregulation of ubcM2, an inhibitor of Nrf2, can lead to the higher activity of this pathway. Since different components of the cytoskeleton (ACTG2 and ACTA1) behave differentially and considering the lack of significant differential expression in Nrf2 or Keap1, it can be concluded that the exposure to DEPe may induce the rearrangement of the cytoskeleton (in favor of Nrf2 translocation to the nucleus) and activation of the Nrf2 pathway indirectly via oxidative stress stimuli (Fig. 4).
These findings are well supported by our transcriptomic analysis, which shows the upregulation of PRDX, GSR, and GST (downstream genes). However, the transcriptome analysis further detailed that c-Fos and FRA1 (two inhibitors of nuclear Nrf2) are significantly downregulated and therefore allow for higher Nrf2 activity during the DEPe treatment.
Phagosome maturation
Phagosome maturation facilitates the trafficking of internalized particles toward a series of increasingly acidifed, membrane-bound subcellular structures, resulting in particle degradation. After sealing or scission from the surface membrane, the phagosome is fused with early endosomes, late endosomes, and lysosomes accompanied by significant acidifying changes in its composition, converting it to an oxidative and degradative milieu.
The phagosome movement occurs based on cytoskeletal protein binding (microtubules), leading to the fusion of endosomes and lysosomes sequentially in a dynamic manner (Gotthardt et al. 2002). During the maturation, the GTPase Rab5 drives the fusion of early endosomes via interaction with several molecules, including the early endosome antigen 1 (EEA1), VPSS34 complex, and SNARE proteins.
Moreover, acidification occurs simultaneously via vATPase (a vacuolar proton pump) that translocates H+ across the membrane and further acidifies the lumen (Desjardins et al. 1994).
Although no significant involvement of this pathway was found at the transcriptomic level, our proteomic analysis shows downregulation of ATP6V1A (ATPase H+transporting V1 subunit A), ATP6V1E1, ATP1B2B, ATP1B3A, ATP6V1F, DYNLRB1 (dynein light chain roadblock-type 1), TUBA1C (tubulin alpha 1c), TUBB2A, TUBB4A, and VAMP2 (vesicle-associated membrane protein 2) which suggest that the treatment with DEPe may lead to lower phagosome maturation activity or altered lysosomal pH (Fig. 4). This is consistent with our recent report that DEPe exposure results in lower neuronal autophagic fux and suggests a mechanism underlying neuronal toxicity of air pollution (Barnhill et al. 2020).
Amyloid processing
Amyloid plaques are hallmarks of neuropathological brain lesions in AD and PD with dementia. These structures are composed of A-beta peptide, which is central to the pathophysiology of AD (SadighEteghad et al. 2015).
Amyloid beta is processed by gamma-secretase complex (Presenilin1/2) and beta-secretase (BACE1) from the Type 1 transmembrane protein-Amyloid protein (APP). It is known that abnormal Ca2+infux leads to the aberrant activation of Calpain, which plays a role in the phosphorylation of the microtubule-associated protein Tau via the CDK5 pathway.
Furthermore, oxidative stress associated with the accumulation of A-beta contributes to the activation of several kinases, including MAP kinases, resulting in the phosphorylation of tau. The pathological hyperphosphorylation of tau has an essential role in the destabilization of microtubules (Sadigh-Eteghad et al. 2015). These changes are particularly relevant to the pathogenesis of AD and PD and could be a contributing factor in how air pollution increases risk.
Our expression analyses suggest a destabilizing impact of DEPe on the regulators of amyloid processing as three regulators including CAPNS1 (calpain small subunit 1), MARK1 (microtubule affinity regulating kinase 1), and PRKAR1B (protein kinase cAMP-dependent type I regulatory subunit beta) were downregulated at the protein level (Fig. 4).
Mitochondrial dysfunction
As the main consumers of oxygen in a cell, mitochondria contain a multitude of redox carriers to transfer electrons to oxygen, which results in the formation of reactive oxygen species (ROS).
These organelles contain an antioxidant defense system to detoxify ROS and avoid oxidative damage to other cellular components. DEPe exposure led to changes in the expression of key proteins that likely resulted in mitochondrial dysfunction. As shown in Fig. 4, there is a significant decline in the protein level of ATP synthase (ATP5F1D, ATP5mea), Cytochrome c oxidase (COX7A2), NADH dehydrogenase (NDUFA10, NDUFS8), 2-oxoglutarate dehydrogenase (OGDH), and Cytochrome b-c1 complex subunit 2 (UQCRC2) that play pivotal roles in electron transportation.
This could alter ATP production and also directly result in elevated ROS production and toxicity. Mitochondrial dysfunction has been proposed to contribute to the pathogenesis of AD and PD and, as with some of the aforementioned pathways, air pollution might alter risk by disrupting the function of this critical organelle.
Axonal guidance signaling
The formation of neuronal connections requires the extension of axons that migrate toward their synaptic targets. At the axon leading edge, the axonal growth cone contains receptors with the ability to sense attractive and repulsive guidance cues that are required for the navigation of the axon.
In addition to BMP, Shh, and Wnt (that have been recently implicated in axonal guidance), there are at least four major groups of guidance cues: (i) Netrins and DCC/ UNC-5 receptors; (ii) Plexin, semaphorins, and neuropilin receptors; (iii) Slits and Robo receptors; and (iv) Ephrins and Eph receptors. These proteins may either be secreted and associated with the extracellular matrix (Slits, Netrins, and some Semaphorins) or be anchored to the cell surface (Ephrins and some other Semaphorins) (Stoeckli 2018). Treatment with DEPe impacts several aspects of axonal guidance.
At the protein level, several important factors and regulators were altered upon the treatment. The overrepresentation of PLXNB2 (plexin B2) and underrepresentation of several proteins including ARHGEF11 (Rho guanine nucleotide exchange factor 11), CHMP1A (charged multivesicular body protein 1A), COPS5 (COP9 signalosome subunit 5), GNAT1 (G protein subunit alpha transducin 1), GNAT2 (G protein subunit alpha transducin 2), GNB5 (G protein subunit beta 5), MYL1 (myosin light chain 1), MYL2 (myosin light chain 2), NCK1 (adaptor protein 1), PLXNA1 (plexin A1), PRKAR1B (protein kinase cAMP-dependent type I regulatory subunit beta), RAC3 (Rac family small GTPase 3), RAP1A (a member of RAS oncogene family), SHANK2 (SH3 and multiple ankyrin repeat domains 2), TUBA1C (tubulin alpha 1c), TUBB2A (tubulin beta 2A class IIa), and TUBB4A (tubulin beta 4A class Iva) suggest a significant impairment of axon guidance in the developing embryo (Supplementary Table 5).
Interestingly, the axonal guidance pathway was not significantly affected at the transcriptomic level, which suggests that the involvement of this pathway may be a downstream outcome through which some of the effector/regulator proteins have been degraded.
Changes in axonal guidance pathways described here might reflect neurotoxic pathways relevant to AD and PD or simply reflect interference of neural development in developing embryos. Analysis of expression profiles in DEPe-exposed adults would help resolve this question.
Other pathways
Our proteomic and transcriptomic analyses also demonstrated the involvement of several pathways and metabolic routes including melatonin degradation, circadian rhythm, Sertoli cell-Sertoli cell junction signaling, epithelial adherens junction signaling, thyroid hormone metabolism II (via conjugation and/or degradation), RhoA signaling, inosine-5′- phosphate biosynthesis II, GP6 signaling pathway, RhoGDI signaling, extrinsic prothrombin activation pathway, and the coagulation system. Many of these changes could reflect alterations due to the interruption of development.
For example, both proteomic and transcriptomic analyses revealed that the phototransduction pathway has the largest changes in expression upon DEPe treatment. As shown in Supplementary Table 5, several genes, including Opsin, GRK, S-arrestin, Transducin-α, Transducin-γ, PDC, and GUCA have an attenuated level of transcription due to the exposure to DEPe. Due to the lack of evidence of induction of any element in this pathway, DEPe treatment appears to hurt the development of the visual system. This is consistent with our observation that the eyes never fully developed and are small following the treatment.
Functional assessment
In this report, the significant upregulation of cytochrome P450 (Cyp1A) was the most prominent effect of DEPe exposure. Cyp1A metabolizes many endogenous and exogenous compounds and increased expression in the brain may be protective against, or contribute to, toxicity. To better understand the role of Cyp1A in DEPe toxicity, we utilized CRISPR/ Cas9 to generate a Cyp1A knockdown model. Since the gene-editing procedure in zebrafish is likely to be a partial process resulting in mosaic embryos, we use the term "knockdown" (KD) instead of "knock out." Using a sequence-specific guide-RNA, we converted the first CCA PAM site to a TGA (stop) codon in the first exon of Cyp1A. A non-target guide-RNA was alternatively used as scramble control (SC).
Total RNA was then isolated from the heads of up to 20 KD and SC embryos (5 pdf), and the knockdown efficiency was validated by qPCR analysis. Similar to what was observed in the high throughput RNA-Seq study, this experiment demonstrated a sharp induction (30.59-fold from the baseline) of Cyp1A upon DEPe treatment in the SC embryos, while the KD embryos lacked such an increase in expression (Fig. 5a).
The survival of six groups of embryos during a 7-day time course (n≥40) is shown in Fig. 5b. Briefly, at 7 pdf, the average survival was 89.1%, 74.4%, 79.4%, 68.5%, 48.8%, and 7.6% for the WT/DMSO, WT/ DEPe, SC/DMSO, SC/DEPe, KD/DMSO, and KD/ DEPe, respectively. Thus, KD of Cyp1A resulted in reduced survival but was dramatically reduced in the DEPe-treated fish compared to controls.
Furthermore, KD also resulted in a significantly higher rate of morphological malformations (Fig. 5c). Thus, the induction of Cyp1A expression by DEPe appears to be a protective compensatory mechanism. A significant limitation of our findings is that we measured Cyp1A induction of expression in the zebrafish brains but KD experiments reduced expression in the entire embryo.
Importance and implications for neurodegenerative diseases
Air pollution is known to negatively impact the quality of life and play an important role in the development of human and animal diseases. Using high throughput expression analyses, we showed that DEPe treatment altered several biological processes and signaling pathways in the heads of zebrafish embryos heads. Alterations in phagosome formation, amyloid processing, and mitochondrial function are particularly relevant to neurodegeneration.
Surprisingly, our results did not indicate any clear involvement of inflammation, which has been hypothesized to play an important role in air pollution toxicity. This is likely because we measured expression only in the heads of 5-day-old zebrafish and not the entire body. These studies intended to determine how the brain is impacted by exposure to DEPe.
The brain does contain microglia, which likely are activated by DEPe, but they compose such a small fraction of the cells in the brain that any changes may be undetectable. In addition, microglia are not present and functioning in the brain of developing larvae until 3–5 pdf. Because of this, early exposure may not be the most relevant for studying inflammatory responses. The induction of xenobiotic metabolism is also likely relevant as a compensatory or protective response to air pollution.
Our Cyp1A KD model validated the protective role of this pathway and represents an excellent example of how expression studies can provide important clues to determine mechanisms of toxicants such as air pollution. It is not known if polymorphisms in the Cyp1A gene alter the risk of AD or PD in humans exposed to air pollution, but these results suggest a potential gene-environment interaction and deserve further investigation.
One important question to address is the relevance of DEPe exposures to air pollution. DEPe is the most common mixture used in air pollution research for cell culture and other models that are not amenable to inhalation chambers. The dichloromethane extracts of DEP contain hydrophobic moieties such as PAHs that are most likely to enter the bloodstream and bioaccumulate in the brain. A recent study determined the concentration of several PAHs in human autopsy brains and found that they contained concentrations of PAHs very similar to those used in the studies presented here (Pastor-Belda et al. 2019). Thus, the exposures to zebrafish brains are very relevant, as they contain PAHs and other moieties that have been observed to bioaccumulate in the human brain.
Acknowledgments We thank the following agencies for funding this work: The Levine Foundation (JMB), grants from the National Institute of Environmental Health Sciences, NIEHS T32ES015457 (LMB, SK), and The Parkinson's Alliance (JMB).
Author contribution Conception and design of the study (MSJ, LMB, JMB); Acquisition of data (MSJ, HM, LMB, SL): Analysis and interpretation of data (MSJ, LMB, JMB); Drafting the article or revising it critically for important intellectual content (MSJ, HM, LMB, JMB).
Funding These studies were supported by The Levine Foundation (JMB), grants from the National Institute of Environmental Health Sciences, NIEHS T32ES015457 (LMB, SK), and The Parkinson's Alliance (JMB).
Data Availability All data are available in our supplemental files.
Declarations
Ethics approval All animal experiments were approved by the UCLA Animal Research Committee.
Consent for publication All authors approve of this submission for publication.
Competing interests The authors declare no competing interests.
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