An Insight Into Alzheimer’s Disease
AT first sight, the humble fruitfly seems an unlikely candidate to open up the secrets of human disease. Yet it is proving a remarkably useful aid in fight against severe illnesses like Alzheimer's disease.Human neurodegenerative diseases share a number of common pathological features: a) an evidence of extensive neuronal dysfunction and loss; b) the clinical symptoms are usually a reflection in the brain region where the neuronal dysfunction/ loss occurs; c) the pathological hallmarks are often composed of aggregations of normal neuronal proteins that appear to acquire abnormal characteristics in disease states; and d) the incidence of most neurodegenerative diseases increases as we grow.The pathological mechanisms by which normal neuronal proteins become abnormal in disease lead to aggregate formation, of neuronal dysfunction and neuronal loss which is poorly understood. This is partly due to lack of adequate model organisms within which these pathological features can be simulated. The common fruitfly known as Drosophila melanogaster, is one model organism which holds huge promise for facilitating the understanding of pathogenic mechanisms that underlie human neurodegenerative disease.It may seem unremarkable as it hovers around old fruit in domestic settings but, in actual, the fruitfly has played a significant role in elucidating many biological processes in both normal and abnormal conditions. Much of the credit for its contribution in this field goes to the repertoire of powerful genetic tools which makes it an experimental tracking model. Drosophila models have helped unravel pathological mechanisms that underlie complex human ailments such as Alzheimer's disease.
Studying human disease with fruitflies
At first glance the fruitfly appears to be too simple and too different from more complex species in relevance to the studies conducted on flies. However, upon closer examination one finds a remarkable conservation of fundamental cellular pathways across the species which implies the structure and function of basic cellular machinery which is likely to be very similar in flies and humans. This has been found out that over 50 per cent of genes implicated in human disease have counterparts (called orthologues) in Drosophila.
Some of these human orthologues have been shown to rescue phenotypes created by knocking out the Drosophila orthologue, showing that they are functionally interchangeable. Moreover many important biological pathways (such as developmental pathways, circadian rhythms, etc) were first delineated in Drosophila and later when their human counterparts were identified it was clear that such pathways are largely conserved across the species.
Similarly pathological hallmarks such as Alzheimer's, Parkinson's, and Huntington's disease have been successfully replicated in Drosophila. When the genes implicated in these human diseases, it was expressed in the fly nervous system. This shows that the basic cellular machinery for responding to cellular insults is conserved and it may be possible to gain a better understanding of normal and abnormal human responses by studying them in Drosophila first.
Genetic manipulation of flies is easier due to the existence of a number of powerful and sophisticated genetic tools. Firstly p element transposons also called "jumping genes" enable the insertion of foreign genes into the chromosomes of Drosophila making it relatively easy to create transgenic fly to express genes associated with human disease. This approach has been used to create transgenic flies expressing human genes implicated in Alzheimer's, Huntington's and Parkinson's disease.
When a transgenic fly is created it is possible to use the UAS-GAL4 expression system to exert spatial and temporal control over the expression of the transgene. This is advantageous because neurodegenerative events have a predilection for specific neuronal populations in different neurodegenerative diseases.
Once a Drosophila model of human disease is created and a phenotype (a physiological or behavioural change is seen after expression of the disease-specific gene) emerges, it is possible to carry out high throughput enhancer/suppressor screens. This is the process of crossing the human disease- gene-expressing transgenic fly with a fly from another stock, to see if the phenotype is exacerbated or rescued. This enables the identification of other key players in the neurodegenerative cascade. There are two types of enhancer/suppressor screens that are usually undertaken: biased hypothesis driven screens and unbiased screens.
In biased hypothesis the human-disease-gene-expressing transgenic fly is crossed with another stock expressing a gene which has also been implicated in that disease. These screens rely on prior knowledge and hypotheses about individual diseases. Such approaches have been undertaken using Drosophila models of Alzheimer's disease, Parkinson's disease and Huntington's disease and they have also helped to test and the validity of current hypotheses.
Alzheimer's Disease (AD) is the commonest cause of dementia in the elderly. It is characterised by extensive loss of neurones and the appearance of two striking pathological hallmarks: neurofibrillary tangles (NFTs) and neurofibrillary plaques (NPs). NFTs are rope-like aggregates that grow within an affected nerve cell and they are made up of a building block protein called tau. NPs are also aggregates but they are made up of another building block protein called amyloid unlike NFTs, they deposit outside and around affected nerve cells. Both tau and amyloid are normal proteins but they appear to acquire some abnormal characteristics in AD.
The "tau and tangle" hypothesis was put forward to explain the pathogenic role played by abnormal tau proteins in AD. The first consequence was the inability to perform its normal job in nerve cell. Tau is a microtubule associated protein - this means that it is normally found in axons of the neurons, where it binds to unstable structural proteins called microtubules. When microtubules are stabilised, they form long cytoskeletal tracks within the axon over which transport of material can take place. This axonal transport is vital for the survival of the neurone and also its communication with targets. Tau therefore plays a very important role in enabling the maintenance of axonal transport.
The "tau and tangle" hypothesis proposes that when tau becomes abnormal, its ability to bind and to stabilise the microtubules diminishes and then microtubular cytoskeletal tracks collapse. The axonal transport, which relies on intact tracks, becomes compromised and the neurone is then unable to communicate effectively with its target. As a result, it is likely the neuronal networks in the specific part of brain which becomes dysfunctional and will be reflected in a clinical symptom. But in the case of AD, the tau proteins first become abnormal, invariably leading to loss of memory.
A Drosophila stock existed in which a green-fluorescent tag had been attached to cargo is transported within axons. It is therefore possible to study axonal transport using this Drosophila stock.
"Tau and tangle" hypothesis
We used this stock of flies to test the ‘tau and tangle' hypothesis by expressing human tau in transgenic flies that were already expressing the green-fluorescent cargo. We found that normally the fluorescent cargo is rapidly transported along with axon and therefore can be seen evenly distributed throughout the axon. However, when we express human tau in the same axons, there is a striking change in the distribution of the fluorescent cargo - instead of being evenly distributed throughout the axons, it is now "stuck" in "pile-ups" at various points in the axon. This is analogous to road traffic accidents. The experiment clearly shows that the "tau and tangle" hypothesis is correct - excessive expression of human tau can lead to a disruption of axonal transport. Furthermore, the disruption of axonal transport is clearly severe enough to compromise the ability of the neurons to communicate with their targets (which are muscle cells), because the larvae exhibit crawling defects and electrophysiological abnormalities at their neuromuscular junctions. Although defects have also been reported in another Drosophila model in which human tau was over-expressed in the fly's brain regions which regulate learning and memory.
Despite the emergence of these behavioural defects, there was no evidence of any cell death or tangle formation in either of these models. The result implies that when tau protein becomes abnormal and makes the nerve cells "sick" by disrupting their axonal transport. This suggests in AD the earliest symptoms arise not because of tangle formation or death of neurones but possibly because of tau-mediated neuronal dysfunction.
Phosphorylation is the process of adding inorganic phosphate groups to proteins to alter their physiological properties. Tau, like many other cellular proteins, also undergoes phosphorylation. In AD, tau has been shown phosphorylated to a greater extent than normal and can be said "hyperphosphorylated".
Studies on phosphorylation state of tau, plays a role in the mechanism by which it disrupts axonal transport in our transgenic model. To test this hypothesis, enhancement the phosphorylation state of tau by co-expressing it with a tau kinase (an enzyme which adds phosphate groups to tau) called glycogen synthase kinase-3 beta (GSK-3b) in our transgenic flies. These double transgenic flies exhibited greater axonal transport and locomotor defects.
In a converse approach, tau transgenic flies were fed with lithium chloride (LiCl), a drug which reduces the phosphorylation state of tau, and found its mediated-axonal transport and locomotor defects were significantly ameliorated. These results lead to a drug trial in which the therapeutic value of LiCl as a treatment of AD should have to be investigated. Similar findings have emerged from other Drosophila models in which human tau has been over-expressed - in all cases, enhancing the phosphorylation state of tau exacerbates the tau-induced phenotypes.
These results collectively imply that the phosphorylation state of tau plays a critical role in the process by which tau disrupts neuronal function and that drugs which can reduce "hyperphosphorylation" of tau may have therapeutic potential in AD.
Drosophila models faithfully replicate aspects of human disease. And because of their repertoire of elegant genetic tools, they are highly experimental. The studies undertaken using such models enable scientists to dissect the pathological mechanisms which underlie neurodegenerative events in human disease. It is evident that neither neuronal death nor pathological hallmarks are necessary for the manifestation of clinical symptoms - neuronal dysfunction caused early on in the disease process may be a culprit. This is a significant finding because it suggests therapeutic interventions which should focus on rescuing neuronal dysfunction but not on preventing the formation of pathological hallmarks.
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