Molecular Neuropharmacology Laboratory

Prof Philip Beart
Prof Philip Beart

Laboratory Head

Professor Philip Beart BSc (Hons) (Adel) PhD (ANU) DSc (Melb)

Contact Details

Email:

philip.beart@florey.edu.au

Phone:

+61 (0)3 8344 1955

Fax:

+61 (0)3 9347 0446

Number of

Staff:        3
Students: 7

Research Interests

The Molecular Neuropharmacology team focuses its activities on understanding the injury processes that contribute to acute and chronic degeneration within in the brain. Various mechanisms, including excitotoxicity, oxidative stress and inflammation appear to underlie the patholologic chain of events occurring in ischemic-hypoxic conditions, Parkinson’s disease and amyotrophic lateral sclerosis (a form of motor neuron disease). Glial cells are also intimately involved in the maintenance of normal synaptic transmission and during brain injury become activated, changing their phenotype and energetics, and synthesising inflammatory and trophic factors.

Progress towards the big picture of understanding neuronal injury and glial activation pertinent to brain injury is achieved by examining the regulation of cell surface receptors, transporters, second messengers and genes. Our laboratory interfaces studies in cultured cells and brain slices, with whole animal studies to understand degenerative and reparative processes. Additionally, we apply multidisciplinary strategies that allow us to attack questions from various directions.

Astrocyte biology has become a topic of key focus of our recent efforts. Glia outnumber neurons 10:1 in mammals, but surprisingly have been called the “forgotten half of the brain”. We have increasing evidence that astrocytes function as sensors for brain injury and that they may respond to changes in the extracellular milieu by altering their phenotype and the cellular localisation of glutamate transporters. Astrocytes by virtue of their physical interfaces with neurons, vasculature and ventricular space are well positioned for this role and have already been proposed to adapt their energy metabolism to neuronal injury. Moreover, exciting new findings from our laboratory suggest that astrocytes respond to hypoxic stress by releasing groups of proteins that act in concert to produce neuroprotection. These findings are providing evidence for a novel cascade of glial-neuronal signalling. Our current efforts address the time-course of adaptative changes in glia and the switching on of these signalling cascades.

Current Projects

Tolerance in brain: studies of endogenous mechanisms which can minimize brain cell death in models of stroke and perinatal asphyxia

Nicole Jones, Percy Chu, Tim Brown, Luba Kardashyan, Jenny Callaway & Philip Beart

Hypoxia is an important feature of many neurodegenerative conditions, including stroke and perinatal asphyxia. Depending upon the degree of hypoxia, the outcomes can range from mild neurological damage to death. Thus, there is a great need to develop novel drugs that can reduce neurological damage following hypoxia. Neurologists have long known that transient ischaemic attacks can reduce the severity of a subsequent major stroke. Previous research has shown that pretreatment (or preconditioning) with a mild, non-damaging exposure to hypoxia can protect against a subsequent brain injury. We have been analysing the contributions of hypoxia-inducible factor (HIF-1) to this form of tolerance. HIF-1 is a key regulator of adaptative responses to hypoxia and is involved in modulating expression of cytoprotective genes central to angiogenesis, erythropoiesis and energetics. The activity of HIF-1 is inversely regulated by a group of prolyl hydroxylases (PHD) enzymes.

Our studies examine how mild hypoxia and PHD inhibition can protect the brain against injury in models of hypoxic-ischaemic (HI) brain insults. We employ a model of hypoxia-induced tolerance to ischaemic injury in neonatal rats including use of PHD inhibitors desferrioxamine (DFO) and cobalt chloride (CoCl2). Using behavioural tests in concert with morphological indices, we find that preconditioning with hypoxia, DFO or CoCl2 24h prior to HI insult can prevent functional deficits observed at 5 weeks after HI and have long-term protective against injury. Furthermore hypoxic preconditioning increases expression of HIF-1α and PHD-2 in neonatal rat cortex indicating their likely involvement in this neuroprotective effect. We are also investigating preconditioning in primary glial and neuronal culture systems. These studies provide novel evidence in cortical astrocytes for a PHD/HIF system whereby preconditioning with PHD inhibitors is cytoprotective against oxidative stress. This effect involves increased levels of HIF-1 and VEGF suggesting glial-neuronal signalling mediating beneficial effects in brain injury.

Roles and regulation of glutamate transporters in central nervous system

Ross O’Shea, Linda Lau, Chrissandra Zagami, Mark Farso, Nicole Wallis, Nicole Jones & Philip Beart

A family of glutamate transporters (EAAT1-5) mediate the high-affinity uptake of extracellular glutamate, and permit normal excitatory synaptic transmission, as well as protecting against excitotoxicity. Dysregulation of glutamate transport contributes to the pathology of ischaemia and motor neuron disease, and altered expression or function of EAATs has been identified in these and other neurological conditions. Thus up-regulation of EAAT activity may be an in-built neuroprotective mechanism since levels of potentially toxic L-glutamate rise in may forms of brain injury.

Our primary objectives involve characterising brain EAAT function and its regulation in cultured preparations (cells and brain slices) and in vivo during brain injury. The relationship between various stressors, and the expression, localisation and activity of EAATs is being explored using a variety of cytochemical and neurochemical techniques. Interestingly, the shape of astrocytes in culture changes rapidly in response to injury and to treatments that alter cellular signalling or the cytoskeleton. In pure astrocyte cultures, we have succeeded in dissecting the temporal sequence of changes in EAAT abundance and function, and are currently relating them to astrocytic phenotype. These studies reveal that many treatments that alter astrocyte shape also increase EAAT function and expression, suggesting that EAAT activity is intimately linked to astrocyte activation. By monitoring actin stress fibres using fluorescent dyes and confocal microscopy, we have found that astrocyte stellation leads to the redistribution of EAAT1/2 on the cellular surface, as well as inducing the production of EAAT protein. Other work in spinal cells, maintained in primary culture, has demonstrated that EAATs localised to astrocytes act to minimise the effects of cellular stressors by retaining transporter molecules at the cell surface in manner whereby they become redistributed on the astrocytic processes. Moreover, EAATs respond differentially to oxidative stressors and excitotoxicity maintaining function by increasing affinity or capacity, respectively. This homeostatic process is accompanied by astrocyte stellation and represents a new endogenous mechanism for maintaining brain function in the face of potentially destructive insults. Thus EAATs in glia may act as sensors being activated to respond to potentially toxic stimuli that would injure neurons.

Roles for the intrinsic mitochondrial pathway in neuronal apoptosis

Philip Beart, Shanti Diwakarla, Linda Mercer & Liubov Kardashyan

Mitochondrial dysfunction has been described in various neuropathologies, including Parkinson’s, Huntington’s and motor neuron diseases, and in HI brain injury. The subsequent energy deficits and generation of toxic free radicals contributes to cellular injury by activating mitochondrial death pathways, which involve the redistribution of mitochondrial proteins to activate proteases. Classically, these events are considered to involve caspases, but we have shown that in neuronal apoptosis many cytodestructive events involve caspase-independent pathways, especially involving apoptosis-inducing factor (AIF) and calpain. This program is a continuing collaboration with Professor Phillip Nagley, Department of Biochemistry and Molecular Biology, Monash University.

We have found and continue to reveal many paradoxes as we document how mitochondrial-induced signalling controls neuronal “death” signalling. As we attempt to disentangle the amazing complexity of the permutations and combinations of likely events, our findings reveal that in cortical neurons slow calcium-dependent excitotoxicity is hierarchical involving depolarisation of mitochondria and delayed release of most pro-apoptotic proteins, but with little evidence of caspase activation. Other insults produce injury which follows closely the classical recruitment of the intrinsic mitochondrial pathway. However, in virtually all studies we see evidence of crosstalk between caspase-dependent and -independent signalling, although the extent of crosstalk depends upon the neuron phenotype and the nature of the insult.

For example, when studying dopamine neurons using drugs that produce models of parkinsonism, we see the balance of this crosstalk between mitochondrial signalling cascades tipped in favour of caspase-independent apoptosis involving AIF. These finding imply that caspase-independent injury needs further attention in Parkinson’s disease. In other studies, we have developed a primary culture model of striatal GABAergic neurons that die slowly in Huntington’s disease - our goal here is to understand the patterns of recruitment of mitochondrial signalling, which has been implicated in the neuropathology of this crippling disorder. 3-Nitropropionic acid (3NP), a mitochondrial complex II inhibitor, produces an animal model of Huntington’s disease and when we studied this neurotoxin in vitro it produced very slow induction of apoptosis. 3NP, unlike the oxidative stressor SIN-1, only resulted in late activation of caspase-3 when producing apoptosis. However, we were able to demonstrate that the caspase-independent marker AIF was mobilised for both insults. Our intention is to continue our analyses of the chronological involvement of markers up- and down-stream of mitochondrial recruitment so as to determine how the balance between caspase-dependent and -independent apoptosis is affected by neuronal phenotype and the type of insult. These studies are directly relevant to cellular injury in many brain pathologies and the targeting of drug strategies.

White matter injury and long-term regenerative mechanisms in hypoxic-ischemic brain insult: opportunities to explore novel therapeutics

Philip Beart, Bevyn Jarrott, Nicole Jones & Jenny Callaway

Axonal injury is recognised as a feature of early multiple sclerosis, axonal loss is considerable in amyotrophic lateral sclerosis and also important in acute disorders such as head injury and stroke. These types of white matter injury can be conveniently studied in rat models of HI which allow the evaluation of functional outcomes after therapeutic inventions by analyses of behavioural and histopathological endpoints. In particular, models employed allow (a) therapeutic agents normally impermeable to the brain to be tested, (b) long-term evaluation of therapeutics to be undertaken in conscious animal models of multiple functional parameters, and (c) correlation of behavioural improvements with morphological indices. Ongoing work focuses on the abilities of trophic factors to minimise injury and where, for example, BDNF produces functional improvements in both models of adult and neonatal white matter injury.

Laboratory Techniques

Funding

International Collaborators

Research