What Can the Humble Fruit Fly Teach Us About Alzheimer's Disease?

What Can the Humble Fruit Fly Teach Us About Alzheimer’s Disease?
Seminar presented by Amrit Mudher
Southampton University School of Biological Sciences
In association with The Alzheimer’s Society

Abstract

Alzheimer’s disease, modelled here in Drosophila, is characterised by to types of lesion, identifiable post-mortem in AD patients. These lesions are neuritic plaques, caused by β amyloid oligomer aggregation, and neurofibrillary tangles caused by oligomers of tau protein. Hyperphosphorylation of tau results in microtubule instability, loss of neuronal cytoskeletal integrity and impaired axonal transport. GSK inhibitors are shown to rescue this phenotype by inhibiting phosphorylation of tau, decreasing its solubility. It is possible that some small, insoluble species of tau confer protection against tauopathies. A new therapy, a microtubule stabilising agent known as NAP, is currently in phase 3 clinical trials. This therapy has yet to show ant toxicity.

Introduction

Alois Alzheimer, the namesake of the disease, was first to state that Alzheimer’s disease (AD) has a distinct and recognisable neuropathological substrate (Mudher. 2012). There are two primary lesion types associated with the disease: neurofibrillary tangles (fig. 1a) and neuritic, or senile plaques (fig. 1b) (Mudher. 2012) (Perl. 2010). The result of these lesions is neuronal loss and a visible reduction in brain tissue.

Figure 1a: (Mudher.2012): Neurofibrillary tangles, caused by accumulations of abnormally phosphorylated tau protein within the perikaryal cytoplasm of neurons. Tissue obtained post mortem from an AD patient (Perl. 2010).

Figure 1b: (Mudher. 2012): A neuritic/senile plaque in tissue obtained post-mortem from an AD patient. The plaque is made up of β-amyloid deposits surrounded by abnormal neurites (Perl. 2010).

Definitive diagnosis of AD can only be made at autopsy, because there are no definitive biomarkers to test for the disease. Distinguishing between normal ageing and AD, particularly in early stages of the disease, is also very difficult (Perl. 2010). Studies using autopsy-derived tissue, and animal models, are therefore vital for scientific progress in the understanding of AD, and the search for therapies.
Drosophila melanogaster is a model well suited to such research. Drosophila has a long history as a powerful genetic model; indeed, it has been used as a model organism for some 100 years. Its genome has been sequenced and highly annotated. It has thus provided researchers with an array of powerful and effective genetic tools, which have been used to study the underlying cellular and molecular bases of many key physiological processes and disease pathologies. A summary of factors which make Drosophila an ideal model organism:

• It’s small size

• Inexpensive to maintain (it costs approximately £2000 for a six month study using flies, which would take an equivalent of 3 years in rodents at a cost of approximately £250 000)

• Rapid propagation

• Short life span

• Genetic tractability

• 75% of genes implicated in human disease have Drosophila orthologues.

(Mudher. 2012) (Cowan et al. 2010).

Introduction

The focus of this research is how tau affects neuronal homeostasis.
The normal function of tau is microtubule binding in neuron axons. It conveys stability to the cytoskeleton of these structures. Microtubules are cylindrical in shape. They provide a network, like ‘roads’, via which transport vesicles move within the cells (fig. 2). Hyperphosphorylation of tau (p-tau) results in reduced binding with microtubules, loss of cytoskeletal integrity, and reduced axonal transport (Cowan et al. 2010). This can be viewed in vivo, using live fly larvae. The larval cuticle is
clear wih greatly innervated musculature beneath. This is a well characterised network of motor and sensory neurons, therefore, it is possible to visualise physiological and/or pathological processes that occur within them. This is done by tagging a protein that participates in these processes with a fluorescent marker. Visualisation occurs in real time. An example of this type of experiment was carried out by Cowan et al (2010, Modelling Tauopathies in Drosophila: Insights from the Fruit Fly). UAS/GAL4 was used to drive expression of GFP (green fluorescent protein) tagged neuropeptide Y and a wild-type isoform of human tau (0N3R-, which is constitutively hyperphosphorylated) within motor neurons (see results) (Cowan et al. 2010).
So the question must be, is neuronal dysfunction caused by the hyperphosphorylation of tau alone, or the later formation of tangles?

Figure 2: Microtubules in neuron axons are cylindrical in shape. They are composed of tubulin. Kinesin plays a role in vesicle transport along the microtubule network, which act like ‘roads’ (Stebbings. 2005).

Results

True to the prediction made by the tau microtubule hypothesis, expression of p-tau, which causes dissociation and solubility, leads to loss of microtubule integrity. As a result axonal transport is disrupted, there is dysfunction within synapses, and locomotor impairment (Cowan et al. 2010). This is seen as ‘traffic jams on the roads’; vesicles passage along axons (via microtubules) is blocked. Ultimately, p-tau toxicity results in neuronal death by apoptosis. Together, these problems result in the behavioural phenotype of AD, even prior to the presence of p-tau aggregations or neuronal death (Mudher. 2012). Loss of microtubule integrity can be seen in figure 3a and 3b.
It is possible to rescue the behavioural phenotype, restoring microtubule number and integrity, using GSK-3β inhibitors (Glycogen Synthase Kinase-3β). GSK inhibitors therefore must increase levels of h-tau (human tau). Figure 4 depicts this. However, a side effect of GSK inhibitors is the appearance of electron-dense granular structures within the cytoplasm of neurons. These are aggregations of granular tau oligomers.

Figure 3a: (Top) Healthy tissue. Borders of cells are well-defined by plasma membranes, and the
microtubules are clearly visible as small circular structures (Mudher. 2012.) (Mudher
et al. 2004).

Figure 3b: (Bottom) Tissue affected by hyperphosphorylated tau protein. Cells have lost their well defined shape and cell contact is poor – gaps are present. Microtubules are deformed (see black arrows) (Mudher. 2012) (Mudher et al. 2004).

Figure 4: Levels of human tau are increased with GSK inhibition (Mudher. 2012).

Discussion

The physiological and pathological nature of AD means cognitive function in patients is progressively impaired. Glycogen synthase kinase is responsible for phosphorylation of serine and threonine residues on target substrates, in this case tau. It is this that prompts dissociation of tau from microtubules, creating the instability. It is this (together with formation of neuritic plaques) which results in cognitive impairment. It is possible to reverse damage (prior to tangle formation) and restore phenotype with GSK inhibitors. This therapy increases tau by decreasing its solubility. In its insoluble state, tau binds to microtubules, restoring integrity and normal function of neurons.
Further work is underway to investigate AD therapies which increase microtubule stability, and the possibility that they could prevent and rescue tau-mediated phenotypes. One such agent is NAP (full name NAPVSIPQ) is able to preferentially bind with tubulin in neurons and glial cells. It promotes microtubule assembly and reduces p-tau in vitro. Peripheral administration (intravenous) of NAP shows significant efficacy in various in vivo models (Matsuouka et al. 2008), and is now in phase 3 clinical trials as a therapy for AD (Mudher. 2012).

Conclusion

Certain species of small insoluble Tau proteins are non-toxic and are thought to confer protection against tauopathies such as AD. Hope for future tauopathy therapies lies with microtubule stabilising agents, such as NAP, which unlike current therapies, shows no toxicity at this stage of clinical trials (Mudher. 2012).

Bibliography

Cowan, C.M.; Sealey, M.A.; Quraishe, S.; Targett, M.T.; Marcellus, K.; Allan, D.; Mudher, A. (2011). Modelling Tauopathies in Drosophila: Insights from the Fruit Fly. International Journal of Alzheimer's Disease. 2011 (598157), 1 - 16.
Matsuouin, Y.; Jouroukhin, Y.; Li, H.F.; Feng, Li.; Lecanu, L.; Walker, B.R.; Plantel, E.; Aracanio, O.; Gozes, I.; Aisen, P. (2008). A Neuronal Microtubule-Interacting Agent, NAPVSIPQ, Reduces Tau Pathology and Enhances Conitive Function in a Mouse Model Of Alzheimer's Disease. Journal of Pharmacology and Experimental Therapeutics. 325 (1), 146 - 153.
Mudher, A. (2012). Seminar Powerpoint presentation. 2/11/2012.
Mudher, A.; Shepherd, D.; Newman, T.A.; Mildren, P.; Jukes, J.P.; Squire, A. Mears, A.; Drummond, J.A.; Berg, S.; Mackay, D.; Assuni, A.A.; Bhat, R.; Lovestone, B. (2004). GSK-3beta Inhibition Reverses Axonal Transport Defects and Behavioural Phenotype in Drosophila. Molecular Psychology. 9 (5), 522 – 30.
Perl, D.P. (2010). Neuropathology of Alzheimer's Disease. Mt. Sinai J Med. 77 (1), 32 - 42.
Stebbings, H (2005). Cell Motility. Online: eLS. 1.