Alzheimer’s disease (AD) is a serious neurodegenerative disease leading to dementia. With globally increasing life expectancy, a number of patients suffering from AD is expected to rise. It is estimated that approximately 74.7 million people worldwide will live with AD or a related form of dementia by 2030, and this number will continue increasing to 131.5 million in 2050. AD is categorized as a protein-misfolding disease. Accumulation of misfolded amyloid-β (Aβ) peptides in the brain is thought to be an early driver of AD, triggering a cascade of pathogenic processes and causing neuronal cell death. Currently, there are only six drugs on the market for the treatment of AD, none of which can stop or reverse the disease progression. This further justifies the urgency for developing alternative strategies against misfolded protein aggregates in AD patients’ brains.
Recently graphene oxide (GO) nanoflakes have attracted significant attention in biomedical areas due to their high carrier mobility, unparalleled thermal conductivity, and excellent biocompatibility. GO nanoflakes are two-dimensional sp2-hybridized carbon nanomaterials that have been largely explored in cancer therapies, drug delivery and biosensing applications. The GO nanoflakes are hydrophilic and interact well with biomolecules such as proteins. Several in vitro studies showed that GO sheets can inhibit the Aβ aggregation process and promote the clearance of Aβ via cutting mature amyloid fibrils into pieces. A recent in vivo study demonstrated that GO nanoflakes could ameliorate cognitive impairment in the AD mouse model by clearing of Aβ aggregates. However, there still persists a knowledge gap on the underlying mechanisms of GO penetration into cells, clearance of amyloid aggregates and improvement of cell survival in vivo.
Due to the strong conservation of the cellular protein quality control system among eukaryotic cells, which are cells of organism with a membrane-bound nucleus, the yeast Saccharomyces cerevisiae has become a powerful model organism for studying misfolded proteins and their implication in human pathologies. We are one of the few groups in the world that have established a yeast AD model with Aβ42 expression, which is the form of Aβ most prone to form aggregates and enriched in AD patients’ brains. This AD model mimics the chronic cytotoxicity due to accumulated Aβ42 proteins, such as shorter life span, stronger stress in the endoplasmic reticulum (major site for protein synthesis and transport), dysfunctional protein quality control system, and reduced mitochondrial function. Therefore, it offers an attractive platform for potential AD therapeutic development.
Our study showed that GO nanoflakes could penetrate Aβ42-expression yeast cells without affecting their morphological integrity. Internalized GO nanoflakes significantly increased the viability of the Aβ42 strain throughout the chronological life span (CLS). We further examined whether the size of GO nanoflakes impacted its effect on the Aβ42 strain. Even though both small flakes (mean diameter of 90 nm) and larger flakes (mean diameter of 250 nm) showed beneficial effects on cell survival, the larger flakes aggregated into insoluble clumps after 7 day’s cultivation, while the small flakes remained uniformly dispersed.
AD is a complicated disease including the interplay of many cellular networks. Therefore, we combined an in-depth proteome analysis and follow-up experiments to investigate how GO nanoflakes helped to reduce Aβ42 cytotoxicity. The proteome analysis showed that GO nanoflake treatment could activate the unfolded protein response (UPR)-related processes, which help cells to control protein homeostasis in the endoplasmic reticulum. Induction of UPR tends to reduce endoplasmic reticulum stress by enhancing protein folding and misfolded protein degradation, and repressing protein biosynthesis. It is suggested that the GO nanoflakes treated Aβ42-expressing cells are more capable of reducing the misfolded protein burden generated by Aβ42 aggregates to increase the cellular viability. To verify this hypothesis, we stressed GO-treated cells with L-azetidine-2-carboxylic acid (AZE), which is an analogue of L-proline. When AZE substitutes proline during protein biosynthesis, it results in protein misfolding, aggregation, and endoplasmic reticulum stress. The AZE treatment strongly decreased cellular viability, while cells treated with GO nanoflakes showed improved viability. Furthermore, we discovered that internalized GO nanoflakes directly interacted with Aβ42 and reduced the amount of Aβ42 aggregates.
Brain cells are highly susceptible to oxidative stress with their high oxygen consumption and insufficient antioxidant activity. This can explain why oxidative stress has been implicated in the pathogenesis of AD and the crucial role of redox homeostasis. The nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) and nicotinamide adenine dinucleotide phosphate (NADP+)/reduced NADP+ (NADPH) redox couples are essential for maintaining redox homeostasis. They are mainly generated in central carbohydrate metabolism including glycolysis and pentose phosphate pathway (PPP). Proteome data showed that most of the proteins on glycolysis and PPP pathways were upregulated upon GO nanoflakes treatment, compared to Aβ42 strain without GO treatment. We measured the ratio of NADPH/(NADPH + NADP+) and NADH/(NADH + NAD+) in the Aβ42 strain, GO nanoflakes treatment significantly increased these ratios compared to untreated strain. Measurement of cellular levels of reactive oxygen species (ROS) showed that the GO nanoflakes treatment significantly reduced the ROS levels following the aging process. Hence, we speculated that GO nanoflakes act through two independent pathways to mitigate Aβ42 toxicity in yeast. In one pathway, GO nanoflakes act directly to suppress Aβ42 accumulation. In another pathway, GO nanoflakes act indirectly to increase cellular capacity to handle misfolded proteins and oxidative stress, which is a presently unknown mechanism.
How to apply GO nanoflakes to AD patients is still a question for the future. In addition to the potential application in AD treatment, our studies also showed that treatment with GO nanoflakes could reduce the toxicity of accumulated Huntington’s (HTT) protein, which develops when the CAG trinucleotide repeats happen in the HTT gene and cause the polyglutamine (polyQ) expansion. The accumulation of HTT protein contributes to the pathogenesis of Huntington’s disease (HD), which is an inherited neurodegenerative disease. Consequently, GO nanoflakes hold great potential for further application in the field of other neurodegenerative diseases, which share a common hallmark of progressive accumulation of misfolded proteins inside neurons. The next step we would like to test the beneficial effects of GO nanoflakes in other AD model organisms, such as mammalian cell lines, and investigate the possibility of developing a GO based drug delivery system for AD therapeutics.
Graphene Oxide Attenuates Toxicity of Amyloid‐β Aggregates in Yeast by Promoting Disassembly and Boosting Cellular Stress Response
Graphene oxide reduces the toxicity of Alzheimer’s proteins
Xin Chen is a senior research at the department of Life Sciences, Chalmers University of Technology, Sweden. She obtained an MD degree from University of South China and PhD degree in Genetics from Shanghai Jiao Tong University, China. She is an expert in applying human cell biology with omics data to study human diseases and look for potential therapies. Her research also focuses on application of metabolic engineering strategies including gene deletion, gene overexpression, CRISPR technology to construct yeast cell factories for pharmaceutical protein production.
Ivan Mijakovic is a Chaired Professor of Bacterial Systems Biology at the Chalmers University of Technology, Sweden. He is also a Professor and group leader at the Technical University of Denmark. Prof. Mijakovic obtained his Ph.D. degree from the University Paris XI. He is an expert on bacterial protein phosphorylation and signalling, and his group investigates the physiology of bacterial model organisms and pathogens. The Mijakovic group also develops metabolic engineering strategies, and new approaches to fight bacterial infections: novel antibacterial agents, coatings, and graphene-based sensors for infection.
Santosh Pandit is a senior researcher at the department of Life Sciences, Chalmers University of Technology, Sweden. He obtained his Ph.D. degree from Jeonbuk National University, Republic of Korea. His area of expertise includes bacterial biofilms, microbial resistant biomedical surfaces, and antimicrobial agents. His current research focuses on the antimicrobial coatings of graphene and other 2D materials on biomedical devices to prevent the device associated infections. His research also focuses on finding novel antimicrobial/anti-biofilm agents for the treatment of biofilm associated infection as well as to reduce the possibility of antimicrobial resistance development.
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