Research Interests

(Ia) Role of caspases in neuronal programmed cell death.
Members of the caspase family of aspartate proteases have been shown to be a requisite part of the pathway of programmed cell death in a number of acute and chronic forms of neural injury in vivo. Consistent with this, inhibition of caspase function has been shown to confer protection from both developmental and injury-induced PCD in a number of cell types including neurons. In order to assess the therapeutic potential of caspase inhibition with respect to mammalian CNS injury, we are examining the injury response of CNS neurons in genetically modified caspase null murine mutants in vivo to several forms of traumatic neural injury. In addition, these null mutants allow the relative contribution of caspase family members to developmental and neuronal death to be established.

Past projects:

(Ib) Targeted deletion of the bcl-2 gene in mice.
The proto-oncogene bcl-2 has been shown to be a potent negative regulator of programmed cell death.   While mice containing a targeted deletion of the bcl-2 locus show significant immune and nephrologic defects, they show no overt effects within the nervous system; perhaps due to co-expression of functionally redundant bcl-2 family members.   To examine the effects of bcl-2 within the nervous system in greater detail, we have performed a detailed examination of bcl-2 -/- mice, using animals provide by the laboratory of Dr. D.Y. Loh.   The results of these studies demonstrate that bcl-2 -/- mice exhibit a specific and preferential loss of small caliber (gamma) motor axons; suggesting a defect in the latter phase of motor neurogenesis. At present, we are examining the functional interactions of bcl-2 family members with several PCD effectors
in vivo .

(Ic) CNTF transgenic mice.
It has come to be appreciated in recent years that neurotrophic factors can prevent both developmental and injury-induced programmed cell death.   In order to determine the ability of ciliary neurotrophic factor (CNTF) to prevent these forms of injury, we have generated linesof transgenic mice which over-express CNTF under the control of either glial (GFAP), neuronal (NSE), or ubiquitous (h-Actin) promoters.   Analysis of these mice demonstrate that CNTF can prevent both developmental and injury-induced forms of programmed cell death in motor neurons.  Results also suggest that the presence of CNTF outside the central nervous system can result in detrimental metabolic consequences (CNTF-induced cachexia).   As a result of these studies, we have also produced the first immunohistochemical map of CNTF expression within the murine central nervous system.

(Id) Analysis of neuronal survival in CNTF transgenic mice.

(Ie) Sensitivity of GlurB and mGluR5 null mice to calcium-mediated PCD.

Role of EphB-family receptors in regulating neural innervation.

Meaningful functional recovery within the adult central nervous system following injury requires both the survival of injured neurons and proper re-innervation of these neurons to their topographically appropriate targets. To gain a greater understanding of the process of neural innervation during development, and re-innervation following adult CNS injury, we are examining the Eph family of axon guidance molecules. Eph receptors represent a large family of receptor tyrosine kinases whose expression is predominantly confined to the neural tissue. This family can be sub-divided into two groups (A and B), based upon the type of ligand with which they (predominantly) interact (GPI-linked or transmembrane respectively).

During developmental target innervation, eph receptors and their ligands are expressed on the surface of growing axons. Interaction of axons with neurons bearing a cognate partner results in a modification of axonal growth (typically axon repulsion). The interaction of eph receptors with their ligands is thought to control aspects of neural specificity within the CNS. Murine mutants which containing specific genetic modifications of eph receptor family members, can thus be utilized to examine the functional consequences of Eph-mediated axon guidance within the developing and adult CNS. Our goal is to gain a more complete understanding of the regulatory abilities of these molecules with the aim of modifying neural growth response following injury, to enhance functional recovery within the damaged CNS.

At present, our examination of EphB2 (-/-) mice has revealed that these animals exhibit a specific loss of axonal projections within the anterior commissure of the CNS, suggesting that the eph B2 receptor is capable of providing extremely specific instructive cues for the guidance of cortical neurons which are independent of cell survival effects.   We are presently producing additional alleles of the eph B2 receptor in order to further define its function.   In addition, we have recently demonstrated that the eph B2 receptor becomes up-regulated following injury in some CNS neurons.   We are presently examining the mechanism of this up-regulation and determine its relavance to postnatal CNS injury.

(IIIa) Genetic modification and transplantation of neural stem cells.
A variety of neurotrophic factors have been shown to rescue an array of different neural cells following neural injury. However, problems such as short biological half-lives, systemic side effects, low neural penetrance, the inability to target discreet neural groups, and problems associate with prolonged intrathecal administration of these factors; severely limit their current therapeutic use. These effects also limit our understanding of their true therapeutic potential, as much current knowledge is based upon fairly acute studies. While retro-, adeno-, and herpes virus vectors have been used (in vivo and ex vivo) as delivery vehicles to some of the problems indicated above, these approaches also possess significant drawbacks including, neural toxicity, host immune reactions, low efficiency in constitutive expression; as well as the potential for recombination with endogenous viral targets.

An alternate approach is to modify endogenous stem cells to express a given neurotrophic factor. Stem cells modified through the use of viral vectors exhibit several of the problems indicated above, and are generally subject to a substantial reductions in transgene expression over time. However stem cells modified through the introduction of mammalian DNA transgenes exhibit a far more consistent pattern of long-term expression. Cell lines can thus be engineered to produce a given neurotrophic agent, as well as possess selective drug sensitivities. Stem cells present within adult ecto- and mesodermal derivatives are currently being modified using this approach. The aim of these studies is to allow the production of significant quantities of genetically modified (isogenic) graft material for functional repair studies.

(IIIb) Bio-engineered guidance cues to enhance target-specifc neural re-innervation following injury.

(IIIc) Genetically engineered bio-implants which regulate programmed cell death following CNS injury.

(IIId) Pharmacologic regulators of programmed cell death.

Past projects:

In vivo effects of N-acetylcysteine.
In vivodelivery of neurotrophic proteins still pose significant problems with respect to the therapeutic use of these factors, particularly for conditions of chronic neural injury.  Many of these problems could be ameliorated if small molecules could be developed which mimic the cell survival properties of neurotrophins.   Recently, work by Dr. M. Noble and others have demonstrated that the glutathione precursor N-acetylcysteine, which reduces the levels of reactive oxygen species within the cell, can promote the survival of neurons in vitro; in a manner similar to that observed for neurotrophins.

To examine the ability of N-acetylcysteine (NAC) to protect motor neurons in vivo, wobbler mice were given a daily oral dose of NAC for nine weeks (wobbler is a model of naturally-occurring lower motor neuron degeneration similar in some respects to Werdnig-Hoffman disease and amyotrophic lateral sclerosis in man).   The results demonstrate that NAC significantly reduces motoneuron loss and axonal atrophy in wobbler mice, and substantially improves forelimb function.   We are currently examining the ability of NAC to promote neuronal survival in several paradigms of acute brain and spinal cord injury.

CNTF induced cachexia.
Given the diverse distribution of many neurotrophic receptors, it is important to thoroughly understand the effects of these agents in different organ systems; in order to maximize their therapeutic potential.  Two components of the tripartite CNTF receptor, LIFR beta and gp 130 are widely distributed throughout the body.  The CNTF-specifying component CNTFr alpha is primarily restricted to neural tissue, but has also been localized to skeletal muscle and liver hepatocytes.  Because of the embryonic lethality observed in animals which express a secreted form of CNTF under the control of the beta actin promoter, we examined the effects of systemic application of CNTF.   Our results demonstrate that when present in the systemic circulation at concentrations of > 100 ng/ml, CNTF induced a potent syndrome of body wasting resulting in death in 7-10 days (adult rate/mouse).   This wasting involved the sequential depletion of metabolic energy stores and could be completely reversed upon the removal of CNTF from the circulation.