Can you provide a brief history of your academic career? What led you to research neuroinflammation and neurodegeneration?

My journey started with a neuroscience-focused biochemistry degree at Imperial College London, UK. After taking a year out to work in a neuroscience lab, I joined the laboratory of Professors Chris Miller and Chris Shaw at the Institute of Psychiatry, King’s College London to study for a PhD in Molecular Neuroscience. There, I was exposed to neurodevelopment and the mechanisms of neurodegeneration found in Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). I then moved to Dr Harish Pant’s laboratory at the National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), USA, where I studied the role and inhibition of aberrant cyclin-dependent kinase (Cdk5) activity in neurodegeneration. Subsequently, I joined the National University of Singapore, extending the Cdk5 work and discovering novel mechanisms of neuroinflammation as an early trigger for subsequent degeneration. I then joined the GlaxoSmithKline Neural Pathways discovery unit to work on drug discovery in ALS, Huntington’s disease and spinal muscular atrophy.

What is the principal goal of your work on Cdk5?

My aim is to understand its regulation during normal development, and the transformation that occurs during its shift to the aberrant hyperactive state. There are many top-quality labs studying the role of Cdk5 in neurodevelopment, but I want to study its role in neurodegeneration. Leading on from this, there are more specific

research avenues I would like to pursue, especially those relating to therapeutic applications based on our results with particular reference to neuroinflammation.

Why is research into Cdk5 activity – specifically its conversion from truncated activator p35 to p25 – important for advancing understanding of neurodegenerative diseases?

We have to be clear that the conversion of p35 to p25 is not necessarily the primary event in a neurodegenerative cascade, though it should remain a focus for ongoing research. Our ambition is to cure diseases; until then, we are endeavouring to develop effective treatments to alleviate the associated symptoms. The p35 to p25 conversion is a consequence of calcium entry into the neuron as a result of neurotoxic stress. If you wanted to go upstream, then calcium influx and protease inhibition might be steps forward – but these approaches have been unsuccessful for a number of reasons. Our data have shown that it is possible to target p25/Cdk5 activity and almost completely eliminate the neurodegenerative changes reminiscent of Alzheimer’s disease in a number of systems; these need to be extended into more translational approaches.

What role does p25/Cdk5 play in the pathogenesis of numerous neurodegenerative diseases, including ALS, Parkinson’s and Alzheimer’s?

A number of research groups around the world have reported the formation of p25 in ALS, Parkinson’s and Alzheimer’s. It is not particularly surprising that when neurons die there is a cascade of reactions and various

proteases are activated. It should be noted, however, that there has been some debate regarding the formation of p25, especially in the Alzheimer’s field; this questioning of existing knowledge should continue as it’s healthy for research progression.

I want to stick to a mechanistic hypothesis; neuronal death is initiated by an unknown toxic event, there is calcium entry and subsequent protease activation to cleave p35 to form p25. When associated with Cdk5, p25 causes aberrant hyperphosphorylation of tau, neurofilaments and amyloid, which then results in accumulations in the neuronal cell body, ‘choking’ the neuron and causing cell death. Deregulation of the normal behaviour of these proteins can be found in a number of neurodegenerative diseases.

How are your endeavours advancing the field of neuroscience?

I would like to think that we are making significant progress towards a better understanding of neurodegeneration – in particular, in diseases that display aberrant phosphorylation and accumulations of key neuronal proteins such as neurofilaments, tau and amyloid. Our critics will say that we are only focusing on instances where p25 is involved and are therefore not investigating pan neurodegeneration or even Alzheimer’s. However, we are using models where the mechanistic progress of neurodegeneration can be modelled, including neuroinflammation, tau and amyloid phosphorylation, as well as accumulation and neuronal death. Our research is also paving the way for, and working towards, new therapeutic approaches.

Building on his work on the mechanisms of neurodegeneration, Dr Sashi Kesavapany is researching possible future treatments for a variety of diseases, including Alzheimer’s and amyotrophic lateral sclerosis

Defying neuronal death

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DR SASHI KESAVAPANY

THERE ARE CURRENTLY an estimated 47.5 million people worldwide suffering from Alzheimer’s disease or other dementias, with this number forecast to increase to 75.6 million in 2030 and 135.5 million in 2050. Age is, of course, a very important risk factor and, with developed and developing countries showing longer life expectancies and radically ageing populations, neurodegenerative diseases are set to become ever more common.

The effects of these diseases are not confined to patients; the social, financial and, indeed, emotional costs are high, especially because of the need for substantial care, whether through families, the state or other providers. In 2010, the estimated global cost of dementia was US $604 billion, which would make it the world’s 18th largest economy if it were a country. It has been estimated that this figure will have increased by 85 per cent by 2030. While some progress has been made in the treatment of conditions such as multiple sclerosis, Alzheimer’s remains a disease for which there is no substantially effective therapy

to halt or reverse its effects. Additionally, the nature of its symptoms, and a lack of understanding of them, makes the disease particularly distressing for all those involved.

Clearly, research addressing neurodegeneration and its specific mechanisms is not only an important facet of neuroscience as an academic discipline, it is also highly significant from a broader perspective. There are key implications for treating the symptoms of these chronic conditions and, ultimately, developing a fuller understanding of the pathologies and curative therapeutic approaches.

Dr Sashi Kesavapany from the Department of Biochemistry at the National University of Singapore is striving to advance knowledge in the field through experimental exploration. The suggestion that there is a relationship between neuroinflammation and neurodegeneration is not new, but the specific mechanisms are incompletely understood. His group has already gained some insight into the process: “An early initiation of neuroinflammation triggers

Researchers in the Department of Biochemistry at the National University of Singapore are elucidating the role of cyclin dependent kinase 5 in the pathogenesis of neurodegenerative diseases

Inhibiting inflammationand reducing degeneration

FROM IN VITRO TO IN VIVO

While carrying out research at the National Institute of Neurological Disorders and Stroke (NINDS), Dr Harish Pant’s laboratory – in which Kesavapany worked – discovered that peptides from truncations of p35 (the Cdk5 activator) could both hyperactivate and inhibit the kinase, hence potentially promoting or reducing neurodegeneration. This work was initially performed in vitro, in mammalian cell lines before extending these data into neuronal cell lines and then primary neurons. In every instance, inhibition of deregulated Cdk5 activity reduced phosphorylation of the tau protein, an accepted signature of Alzheimer’s.

More recently, at the National University of Singapore, Kesavapany and co-workers have extended their work to an in vivo model, using a specially produced transgenic mouse. It was this research that led to the identification of lysophosphatidylcholine as the soluble factor that the neurons were releasing. The data showed that when this early trigger was inhibited, there was complete rescue of the pathology in the central nervous system – a striking and encouraging result.

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PROJECT OR NAME

NEURODEGENERATION INHIBITION

OBJECTIVETo gain further insight into the role of cyclin dependent kinase 5 (Cdk5) deregulation in the pathogenesis of neurodegenerative diseases through in vivo and in vitro investigations.

KEY COLLABORATORSDr Harish C Pant, Chief Cytoskeletal Protein Regulation Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), USA • Dr Tej K Pareek, Department of Pediatrics, Case Western Reserve University, USA • Professor Sally Frautschy, Department of Neurology, University of California, Los Angeles, USA • Associate Professor Markus Wenk, Department of Biochemistry, National University of Singapore • Associate Professor Chian Ming Low, Department of Pharmacology, National University of Singapore • Dr Kai-Hsiang Chuang, Singapore Bioimaging Consortium, A*STAR, Singapore

PARTNERSVerdure Sciences (Dr Blake Ebersole)

FUNDINGAcademic Research Fund, National University of Singapore

National Medical Research Council, Ministry of Health

CONTACTAdjunct Associate Professor Sashi Kesavapany Neurobiology Discovery Team Leader,GSK Neural Pathways DPU

Department of BiochemistryNational University of Singapore Block MD 78 Medical Drive02-06117597 Singapore

T +65 6398 3302E sashi.s.kesavapany@gsk.com

www.med.nus.edu.sg/bch/kess.htm

SASHI KESAVAPANY obtained his BSc with honours at Imperial College London, UK, in 1996, and his PhD at the Institute of Psychiatry, UK, in 2000. He

is currently Team Leader at GlaxoSmithKline and Adjunct Associate Professor in the Department of Biochemistry at the National University of Singapore. Kesavapany’s main research interests are: the role of p25/Cdk5 in neurodegeneration and rescue afforded by specific p25/Cdk5 inhibition; investigating the early neuroinflammatory trigger in neurodegeneration, with implications in Alzheimer’s disease and amyotrophic lateral sclerosis; the role of Cdk5 in mitotic cells; and Huntington’s disease discovery research.

INTELLIGENCE a cycle of neurodegeneration,” Kesavapany explains. “In our system, astrogliosis precedes microgliosis, while there is also a pronounced recruitment of peripheral inflammatory cells, likely through a compromised blood-brain barrier.” If uncontrolled, the inflammation results in cell death, which then propagates the inflammatory cycle.

AN INFLAMMATORY CYCLEMore specifically, one focus of Kesavapany’s research is elucidating the role of cyclin-dependent kinase (Cdk5) hyperactivation in this process, with related attention to its conversion from truncated activator p35 to p25. “It is a mechanism that underlies the neurodegenerative process, and we believe that the neuroinflammation caused by p25/Cdk5 contributes to a cycle of neuronal death, which in turn causes even greater neuroinflammation and neuronal death – it is a vicious cycle,” he elaborates. “We have found that when we inhibited the early inflammation, we could both reduce the pathological hallmark formation and neuronal death in the p25Tg mouse we studied.”

TARGETING TRIGGERSKesavapany and colleagues have, to date, identified one of the p25/Cdk5-mediated neuroinflammation pathways: lysophosphatidylcholine (LPC). Striking a note of caution, he warns: “I want to be clear that we do not think we have identified the only pathway; there will be other mediators involved, perhaps with different aspects of neuroinflammation and subsequent neurodegeneration”. However, their data suggested that this pathway could be highly significant. Firstly, they indicated that when p25 is present, a neuron-based enzyme called cPLA2 is activated to produce LPC; this initiates an astroglial activation cascade resulting in cytokine production and microglial activation. Secondly, they showed that inhibiting cPLA2 and consequently LPC production results in robust decreases of astrogliosis – implying that this could be an important pathway to target for inhibition of neuroinflammation.

SELECTIVE INHIBITION OF ABERRANT CDK5 HYPERACTIVATIONIn view of the team’s discovery, inhibiting p25/Cdk5 would clearly be desirable. However, p35/Cdk5 is essential for the central nervous system (CNS), and thus a treatment that also inhibits p35/Cdk5 activity in the CNS would be unusable. Therefore, it is a significant finding for Kesavapany’s group that the Cdk5 inhibitory peptide (CIP) works selectively, inhibiting p25/Cdk5 and not p35/Cdk5. “It is very important for the field to be moving forward in this way,” Kesavapany reveals. “The process has been challenging; the in vivo system used was by far the most complex approach we have been faced with.” Similar studies are now in the pipeline.

Future research will concentrate on selectivity/specificity and means of delivery, looking at both small molecule and peptide approaches. The small molecule approach requires allosteric inhibitors that are not active site or ATP pocket binders, as there are selectivity and specificity problems with this approach, whereas with peptides, the means of delivery is the challenge.

Kesavapany notes that while these approaches reduce the activation of cPLA2, and consequently reduce LPC production, there is another possible (closely related) angle of attack: “This takes the form of an inhibitor programme for cPLA2 with similar effects downstream”. There are different stages of the process and it is possible that the problem could be addressed more easily or advantageously at a different one, using another aspect of what is essentially the same end result. The data from Kesavapany’s work also support a possible multifaceted approach for Alzheimer’s therapeutics, namely, specific inhibition of deregulated Cdk5 activity and targeting of an anti-inflammatory signalling pathway, in isolation or in combination. There is a great deal of work in store, but there are a number of potential research pathways, and the outlook is promising.

Neurobiology Discovery Team

From left to right: Dr Jeyapriya Sundaram, Research Fellow; Dr Sashi Kesavapany, Team Leader; Dr Charlene Poore, Research Fellow; Mr Noor Hazim, Laboratory Manager.

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