Summary

Alzheimer’s disease is a progressive, debilitating disease that accounts for 60-80% of all dementia cases, however, it remains untreatable. In this blog, we provide an overview of many commonly used organisms for modeling AD, and compare their advantages and limitations.

Introduction

Currently, over 5 million people in the USA are living with Alzheimer’s disease (AD), and due to the aging population, this number is expected to nearly triple to 14 million people by 2060 (CDC, 2020). AD is a neurodegenerative disorder that affects memory, behavioural and social skills, and motor abilities, among other functions (Mayo Clinic, 2020).  AD is associated with many risk factors including age, family history/genetics, and lifestyle and is estimated to be the third leading cause of deaths in Americans (NIH, 2019a). Despite the prevalence, severity, and considerable research on AD, there is no known cure.

The discovery of successful Alzheimer’s therapeutics, and understanding the mechanisms behind the disorder, is reliant on animal models which are able to predict the progression of the disease. Pathologically, AD has two main features: the accumulation of extracellular beta-amyloid (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) (Janowsky & Zheng, 2017). Traditionally, research has focused on the formation, and prevention, of these plaques and tangles. More and more, however, researchers are seeing mixed pathologies in patients with AD, indicating that beta-amyloid plaques and tau tangles might only be partially responsible for the disease while vascular and immune system contributions might be much more important than previously recognized (Neff, 2019). 

Understanding the mechanisms behind Alzheimer’s Disease, and identifying effective drugs and treatment options,  relies on animal models to predict the progression of the disease. The future in AD looks promising as researchers are combating this heterogeneous disorder by adopting a multi-model approach in their research, and new technological advances such as CRISPR. 

In this article, we will discuss the advantages and limitations of commonly used organisms for modeling AD: the Drosophila melanogaster (fruit fly), Caenorhabditis elegans, Danio rerio (zebrafish), and mouse.

Drosophila melanogaster (fruit fly)

The Drosophila melanogaster, more commonly known as the fruit fly, has been used in genetic research for over a century thanks to its short lifespan, low cost,  and large brood size ( Prüßing, Voigt, & Schulz, 2013). Since it is a well established model, Drosophila research has the advantages of a wide variety of genetic tools, publicly available fly stocks, and databases (Allocca, Zola & Bellosta, 2018). 

For  Alzheimer’s research specifically, the Drosophila‘s fully sequenced genome and RNAilibrary allow for genetic mutants to be screened for the toxicity of beta-amyloid and tau (Moloney et al, 2010). The Drosophila also has biological similarities with humans, such as the function and structure of its brain, which make it a valuable model for AD as well as neurodegenerative disease in general (Moloney et al, 2010). 

Furthermore, the Drosophila is an excellent model for diseases which are characterized by their protein aggregates, such as Alzheimer’s Disease. This is because the Drosophila can process human amyloid precursor protein (APP) and has an endogenous orthologue, and thus, can provide insight into protein folding/misfolding (Moloney et al, 2010).

While the Drosophila is well-suited for genetic studies, behavioral studies using this model organism are time-consuming and result in limited conclusions (Prüßing, Voigt, & Schulz, 2013). What makes it good for genetics, its simplicity, is also its limitation, making it have relatively low relevance to humans compared with mice. Because the Drosophila has functional and structural similarities with humans but relatively uncomplex behavior, they are an excellent model to use in tandem with other models, specifically mice models as mice have the complex behavior that Drosophila lack.

Figure 1. Image via Moloney et al., 2010.

Caenorhabditis elegans (worm) 

It might be surprising that a microscopic worm without a brain is a viable system for studying a neurological disorder, however, C. elegans have proven themselves to be valuable models for modeling learning and memory impairments, as seen in Alzheimer’s Disease. C. elegans have been used in the field of disease-specific research since 1995 when Dr. Christopher Link introduced a transgenic model of AD (Link, 1995). Link chose the invertebrate model, in part, because C. elegans age quickly, cost significantly less than other animal models, and have their cell lineages and neural connectomes mapped. C. elegans are also highly amenable to genetic manipulation, especially with the advent of CRISPR (Neff, 2019). In Link’s model, the transgenic worm expressed human beta-amyloid peptide in the body wall and progressively became paralyzed, enabling the study of cell-external neurotoxicity (Link, 1995). While C. elegans do not naturally have the amyloid beta peptide, it can be introduced via  human genes through a process known as ‘humanization’. Thus, thanks to humanizations, researchers can study beta amyloid peptide toxicity using C. elegans. However, these humanized models may still act differently in a mammalian/human system (Neff, 2019). 

C. elegans do not have brains or adaptive immune systems which make them structurally less translational than other, more complex, models. That being said, humanized C. elegans have displayed some of the neurodegeneration of AD, making them a promising model for the future of Alzheimer’s research. C. elegans can be a particularly useful tool by complementing other models, acting as a precursor model before testing on more expensive and time-consuming models. 

Danio rerio (zebrafish)

Zebrafish are a commonly used, and highly validated, model for human neurodegenerative diseases such as Alzheimer’s Disease (Saleem & Kannan, 2018). Zebrafish have been successful models for human neurological disorders in part, due to their short lifespans, large broods, and transparent embryos, which enables researchers to visualize individual genes and aids in genetic manipulation. In addition, the zebrafish’s ability to act as an in vivo model using real-time imaging techniques, such as Ca2+ imaging, makes them an especially useful model in disease-specific research as  this allows for observation of the disease throughout the zebrafish’s life without interfering in the disease’s pathogenesis (Saleem & Kannan, 2018). 

Although zebrafish are small fish, there is a high resemblance between the neuroanatomic and neurochemical pathways of the zebrafish brain and the human brain (Saleem & Kannan, 2018). Notably for Alzheimer’s research, zebrafish also possess the main excitatory glutamatergic and inhibitory GABAergic neurotransmitter circuits – serotonin, dopamine, histamine, and acetylcholine neurotransmitters – making them an ideal model for neurodegenerative diseases which are known to affect these circuits. Furthermore, the zebrafish’s genome has several gene orthologs similar to those mutated in human Familial Alzheimer’s disease (FAD). 

Zebrafish are growing in popularity as they offer a lower maintenance alternative to traditional mouse models. Zebrafish and mice have comparable brain function and organizations, and similar to mice, zebrafish also show similar behavioral patterns to humans, which allows them to act as behavioral models (Takai et al., 2020). 

Caution should be taken when using zebrafish in pharmacological studies, however, as these studies often use chemicals in the fish’s water, and since  fish absorb the chemicals through their skin and gills, it is not possible to quantify the precise amount the fish are processing  (Bhattarai et al., 2017). There are also limitations to using zebrafish for Aβ1-42 peptide research though, since zebrafish have the unique ability to regenerate neurons along their rostrocaudal brain axis. The neurodegeneration caused by Aβ has been shown to activate this neuroregeneration, and so, this ability may undermine zebrafish’s ability to act as an Alzheimer’s model. (Bhattarai et al., 2017). While this unique ability may deter some researchers from using zebrafish in their studies, it does present the opportunity to research the regeneration of neurons. 

Thus, with the zebrafish’s structural and behavioral complexity, it can be considered a good model to bridge the gap between cellular models and preclinical assays.

Figure 2. Advantages of Zebrafish for modeling Alzheimer’s Disease (Image via: Saleem & Kannan, 2018).

Mus musculus (Mouse)

Mouse models have been used for Alzheimer’s Disease research for decades without yielding much success. There are different opinions as to why that is the case: some researchers attribute this to incomplete models (modeling just plaques, or just tangles), some researchers say it is the over-interpretation of the models, and some researchers think that the inability for mice to accurately translate to the clinic is due to the strain of the mouse model being used (Neff, 2019). The attention to the strain of mouse being used is a relatively new concern, but an important one for Alzheimer’s research, as most AD mouse models come from a strain which has shown to be overly resistant to Alzheimer’s neuropathologies. This concern has led to an effort to diversify the strains being used, and including wild mice in their breeding programs to develop a model with greater validity (Onos et al., 2019). 

One of the main advantages of using a mouse model of Alzheimer’s Disease, however, is its long history in the field: over 100 genetically engineered mouse lines have been developed to address AD, and there are established techniques for genetic manipulation and humanization in mice (Neff, 2019). Overall, mice are also considered to have a high translational value to humans, even with the difficulty the AD field has had with this. 

Although there are numerous mouse models for Alzheimer’s, they are predominantly models of Early Onset, familial, Alzheimer’s Disease. This severely restricts the research being conducted, as this type of AD only represents approximately 5% of the AD population. Thus, the field is still restricted in its knowledge of AD pathogenesis for its different types. 

Mice also do not naturally create amyloid beta, or tau (but can be genetically modified to), and can differ to humans in their structure, neurobiology, and immune systems, raising the question as to whether these differences inhibit their ability to act as a successful, translational model for AD (Neff, 2019). In 2016, to address these concerns, the MODEL-AD program was created; the program focuses on late-set Alzheimer’s Disease, and developing more useful models for this type of Alzheimer’s.

Conclusion

As previously discussed, researchers are beginning to expand the study of Alzheimer’s Disease beyond beta-amyloid to other variables which have been overlooked. The models discussed in this article can help in the effort to approach Alzheimer’s differently, studying this complex and varied disease through many different organisms which all have their own advantages and limitations. 

The future of Alzheimer’s Disease research will combine model systems, using certain, more simplistic models (such as Drosophila and C. elegans) as an original test, helping to narrow down genes which are involved in AD, before targets are studied in more complex models (such as Zebrafish and mice). In this way, models work in tandem to help researchers understand Alzheimer’s Disease, and identify potential therapeutic targets in a faster, more cost-effective way.

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