Neuroprotection Trials in Mouse Models of Neurodegeneration

Bethesda, Maryland
June 29, 2005

The lack of effective treatment for devastating neurodegenerative diseases has stimulated great interest in the development of neuroprotective drugs that can prevent or treat progressive loss of neural function leading to serious impairment and death. The use of mouse models for testing promising therapeutic compounds as an intermediate step between cell culture assays and large animal and human studies was the focus of a meeting of investigators using such models. The goal is to stimulate progress by maximizing the number of different mouse trials with promising neuroprotective agents and then looking across multiple interventions and mechanisms of action to see how the results might prove mutually informative. The meeting was sponsored jointly by the National Institute of Neurologic Diseases and Stroke (NINDS), The Michael J. Fox Foundation and the Amyotrophic Lateral Sclerosis Association. It was held in Bethesda, Maryland on June 29, 2005. The NINDS was represented by Dr. Story Landis, Director, NINDS, and Program Directors, Dr. Jill Heemskerk and Dr. Diane Murphy; The Michael J. Fox Foundation was represented by its Associate Director of Research Programs, Dr. Todd Sherer, and the Amyotrophic Lateral Sclerosis Association by its Vice President and Science Director, Dr. Lucie Bruijn.

The ongoing research program that brought these investigators together supports a broad survey of available compounds to identify the most promising ones for further development and translation into clinical treatments. Many of the studies presented were based on prior work that employed a consortium of 26 academic laboratories. This group experimentally screened 1040 FDA approved compounds compiled by the NINDS (the NIH Custom Collection) for activity on mechanisms of action that play a part in neurodegeneration. Based on assay results, compounds considered most promising were selected for studies in animal models of human neurodegenerative diseases. Some studies presented at the meeting utilized genetic models of disease and others used animals pre-treated with neurotoxins that produce lesions of the nervous system and behavioral deficits similar to those found in human diseases.

Animal models employed included those for: Amyotrophic Lateral Sclerosis (G93A SOD-1 mouse, and mice pre-treated with a neurotoxin: paclitaxel), Huntington's Disease (N171082Q mouse), Spinocerebellar Ataxia Type 1 (SCA-1 mouse) and Parkinson's Disease (A53T Hu alpha-synuclein mouse, and also mice and rats pre-treated with one of two neurotoxins: 1-methyl-4-phenyl-1, 2, 3 6-tetrahydropyridine (MPTP), or rotenone). A summary of the tested interventions presented is included as Appendix 1.

Compounds from NINDS FDA Approved Drug Screening Program

Dr. Lucie Bruijn of the Amyotrophic Lateral Sclerosis Association presented results of a study of Ceftriaxone in the ALS mouse. Ceftriaxone, a drug selected for its assessment on a battery of ALS-relevant assays, was administered to a mouse model of ALS at disease onset. Mice treated with Ceftriaxone showed an increased survival as compared to placebo treated mice. Additionally, in pre-clinical models it crosses the blood brain barrier and is well tolerated during long-term use. Clinical trials of the neuroprotective effects of Ceftriaxone in ALS patients are planned to begin soon.

Dr. Robert Friedlander of Harvard Medical School pointed to evidence of mitochondrial dysfunction in multiple neurodegenerative diseases. He presented data on mitochondrial permeability transition (PT) and its inhibition by the drugs nortryptilene, a tricyclic anti-depressant, and promethazine, an antihistamine, using mouse models of Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS) and ischemia. PT represents the opening of pores in the inner mitochondrial membrane causing a loss of membrane potential, generation of reactive oxygen species and probably the release of apoptogenic factors leading to cell death. By inhibiting PT with drugs, mitochondrial function is preserved and neurodegeneration prevented. He suggested that co-administration of multiple PT inhibitors may provide valuable therapeutic strategies for neuroprotection. The focus on a mechanism of action (PT) found in multiple diseases suggests that a common therapeutic regimen may offer neuroprotection in conditions of diverse aetiologies.

Dr. Carine Cleren from Weill Medical College of Cornell University demonstrated that both promethazine and celastrol protect mice against MPTP, a neurotoxin, that produces a model of Parkinson's Disease (PD). Promethazine (PMZ) accumulates in brain mitochondria in vivo and inhibits Ca2+-induced mitochondrial PT in rat liver mitochondria in vitro. Mice treated with MPTP sustained a significant loss of dopaminergic neurons within the substantia nigra pars compacta that was strongly attenuated by PMZ treatment. This protection is thought to result from reducing mitochondrial membrane depolarization and delaying mitochondrial PT. In a second set of experiments, mice were treated with celastrol before and after injections of MPTP. The loss of dopaminergic neurons induced by MPTP in the substantia nigra pars compacta was significantly attenuated by celastrol treatment. Moreover, celastrol treatment significantly reduced the depletion in dopamine concentration induced by MPTP. Celastrol induced heat shock protein 70 (HSP70) within dopaminergic neurons, decreased tumor necrosis factor-alpha and nuclear factor k B immunostaining. These results indicate that Celastrol and PMZ are strong neuroprotective agents capable of protecting dopaminergic neurons against MPTP toxicity in vivo and are promising neuroprotective agents for the treatment of PD.

Dr. Patrick Weydt from the University of Washington Medical Center studied the effects of cannabinol (CBN) on disease progression and survival in a mouse model of Huntington's Disease (HD). He used the N171082Q mouse model of HD with CBN administered to the mice with osmotic minipumps. His data showed that while CBN (5mg/kg/day) can be detected in mouse urine with the THC test, it did not improve survival in a mouse model of HD. There was no beneficial effect detectable in this study. CBN was not toxic and did not affect motor function, wasting or thermoregulation in the HD mice studied. Future plans include the use of higher doses of CBN (100mg/kg/day) and alternate drug delivery routes.

Dr. Mary Abood of the California Pacific Medical Center Research Institute chose cannabinoids for study in a mouse model of ALS. This was based on previous studies showing neuroprotection with these compounds in multiple sclerosis and excitotoxicity. Two major theories for motor neuron vulnerability are susceptibility to excitotoxicity and oxidative damage; cannabinoids are thought to reduce both. Dronabinol (delta 9 tetrahydrocannabinol) was administered to ALS mice (G93 SOD-1). Motor impairment and survival were the measured outcomes. A 6% increase in motor performance and an extension in mean survival of 5.1% in the mice receiving dronabinol as compared to controls wese found. A study of dronabinol together with another cannabinoid failed to increase survival time, showing it to be less beneficial than dronabinol alone.

Antioxidants

Dr. Todd Sherer of the Michael J. Fox Foundation presented data showing that chronic melatonin administration protects rats against the neurodegenerative effects of chronic rotenone infusion, including oxidative damage and alpha-synuclein upregulation. This is of interest because chronic rotenone infusion reproduces features of PD that include nigrostriatal dopaminergic degeneration and motor abnormalities. He also demonstrated that this protection was not achieved by altering rotenone levels or rotenone-induced complex 1 dysfunction in brain.

Dr. Gail Zeevalk of the UMDNJ Robert Wood Johnston Medical School has studied the use of the ethyl ester of glutathione (GEE) as a neuroprotectant. Glutathione is one of the major antioxidants found in cells and approaches to increase its concentration might decrease oxidative damage to neurons in PD. Experiments were conducted to determine if GEE would protect midbrain dopamine neurons from oxidative stress or mitochondrial impairment. Sprague Dawley rats received chronic delivery of MPP+ into the left lateral ventricle via osmotic minipump with co-infusion of glutathione ethyl ester. Brain levels of glutathione were elevated and it appeared that intracellular glutathione provided neuroprotection. Glutathione elevation provided neuroprotection in vitro and in vivo in models of acute and chronic mitochondrial impairment. Translation to clinical situations is severely limited by toxicity of the drug, the need for central delivery and rapid clearance from cells requiring daily administration.

Neuroprotective Strategies in PD Models

Dr. Gloria Meredith of the Chicago Medical School studied the neuroprotection of dopamine neurons by antioxidants in a chronic MPTP mouse model of PD. She utilized a variety of methods including, stereology, immunohistochemistry, measurement of dopamine levels in the striatum and midbrain, and functional tests of motor agility. She advocated the use of the forepaw reach test as compared to the widely used rotarotor test, stating that testing of motor performance in different laboratories can produce discordant data based on whether the test used measures the activity of the forepaws or the hind limbs. Compounds tested were sodium salicylate, cystamine and minocycline. It was concluded that administration of cystamine or salicylate after MPTP rescues dopamine neurons, striatal dopamine levels and behavior, and that both compounds appear to work as anti-oxidants in rescuing dopamine cells and restoring motor function. In these studies, minocycline had no effects on dopamine cells or behavior but did reduce microglial density.

Dr. Jay Schneider of Thomas Jefferson University has shown partial neuroprotective effects of nicotinamide and L-PDMP in acute and sub-acute MPTP models of PD in mice. Beta-Nicotinamide adenine dinucleotide (NAD) generates ATP in mitochondria and NAD depletion is considered a critical factor in cell death following oxidative stress. Animal injury models have shown NAD to be neuroprotective against oxidative stress. It also may protect against apoptotic as well as necrotic destruction of neurons. (L) 1-phenyl1-2decanoylamino-3-morpholino-1-propanol (L-PDMP) is an analogue of ceramide that enhances the activities glycosyltransferases leading to higher concentrations of gangliosides including GM1. It also protects against ischemic injury to brain tissue. While GM1 penetrates the blood brain barrier poorly, L-PDMP crosses into brain and can be taken orally. Experiments in C57/BL6J mice showed that nicotinamide pre-treatment spared striatal dopamine levels in the acute MPTP model with minor effects in the sub-acute model. There was greater cell survival compared to striatal dopamine levels. The cause of this finding is unclear. L-PDMP increased GM1 levels but had no significant effects on striatal dopamine levels. L-PDMP showed different effects depending on the lesion model.

Dr. Moussa Youdim of the Technion Institute, Israel administered a brain permeable, multifunctional iron chelator-brain selective MAO A-B inhibitor (M30) in mouse models of neurodegenerative disease. In a MPTP treated mouse model of PD it attenuates the dopamine depleting action of the neurotoxin and increases striatal levels of dopamine, serotonin and noradrenaline, while decreasing their metabolites. Since dopamine is equally well metabolized by MAO-A and -B, it is expected that M30 would have a greater dopamine potentiation in PD than selective MAO-B inhibitors, since MAO-B inhibitors do not alter brain dopamine. He posits that drugs such as M-30, a multi-functional anti-PD agent, will be superior to mono-functional anti-PD drugs currently in clinical use. This suggests that the treatment of neurodegenerative diseases where there are multiple pathologies will be best approached using polypharmacology, or "dirty" drugs" (single agents with mutiple mechanisms of action).

Dr. Mike Lee of the Johns Hopkins University did a preliminary study using an alpha-synuclein transgenic mouse model of PD. Expression of A53T Hu-alpha-synuclein leads to fatal neurological disease in these mice. The onset of disease features ataxia, slowness, freezing, dystonia and slight muscle atrophy, with death in 14-21 days. Nerotoxicity may be due to an intracellular decrease in soluble oligomers and an increase in insoluble fibrils. Baicalein, a flavonoid, has been shown to dissolve fibrils and stabilize oligomers that are increased only in the areas of brain exhibiting pathology in these mice. Administration of bacalein to these mice led to death in all mice while those in the control group all survived. The meaning of this study is unclear. There was discussion of whether the combination of fibrils and oligomers could be overloading the lysosomes leading to a change in cell pH and cell death. It might be worth exploring whether treatment with lower doses of baicalein could prevent fibril formation, allowing the lysosomes the chance to clear the oligomers.

Neuroprotective Strategies in Genetic Models

Dr. Robert Ferrante of Boston University studied transcriptional dysregulation in ALS. He described experiments using HDAC inhibitors with the goal of prolonging survival and regulating expression of anti-apoptotic genes in transgenic ALS mice. The administration of sodium phenylbutyrate (PBA) to ALS mice resulted in amelioration of cytosolic accumulation of cytochrome c as compared to controls. It also reduced caspase-9 and -3 activation and enhanced histone acetylation in treated ALS mice causing them, in these examples, to more closely resemble normal mice. He considers agents selected for their ability to counteract abnormal molecular mechanisms in neurodegenerative disease such as PBA to be attractive for further investigation.

Dr. Jonathan D. Glass of Emory University School of Medicine studied the potential of calpain inhibitors for neurodegenerative diseases. Calpains are calcium activated cysteine proteases that are active in models of neurodegeneration including peripheral neuropathy, spinal cord and head injury, and stroke. Taxol (paclitaxel) causes a sensory neuropathy in humans and mice with axonal degeneration as the major pathological feature. A possible mechanism of action by which neuropathy is induced includes activation of calpains by taxol. Dr Glass believes that ALS is a distal axonaopathy and presents evidence for this in mice and man. He has shown that in a mouse model, chronic administration of a calpain inhibitor prevents axonal degeneration and preserves function. Further development of calpain inhibitors is needed along with studies of their pharmakokinetics, drug delivery and combination with other protease inhibitors.

Dr. Parminder Vig of the University of Mississippi Medical Center studied the effects of intranasal administration of Insulin-Like Growth Factor-1 (IGF-1) in SCA-1 transgenic mice. IGF-1 and its receptor are expressed in cerebellar Purkinje cells and the goal of this work was to see if intranasal IGF-1 would have beneficial effects on the pathogenesis of disease in this mouse model. Mice receiving IGF-I and control mice were assessed on an accelerating rotating rod. Motor performance was improved in the SCA-1 mice receiving IGF-1. Additionally, double mutant mice created by mating SCA-1 transgenic mice with IGF-1 overexpressing transgenic mice showed that the double mutants have improved motor coordination on the rotating rod with decreased Purkinje cell pathology. These data suggest that IGF-I may be a good candidate for treating SCA-1.

Need for Standardized Methods of Mouse Model Experimentation

Concern was expressed about the difficulties encountered in reproducing the results achieved in some studies of neuroprotection. An example given was the disparate results reported in studies of minocyline. Differences in experimental methods might be the explanation. It was thought that greater specification of the animals employed, including their genetic and phenotypic characterization, source, age, weight, and conditions of housing and diet need to be reported. Additionally, details related to the putative neuroprotective agent including the dose, and route and time of day of administration are needed. Descriptions of any equipment used for behavioral testing as well as time of day of testing and procedures for scoring should likewise be routinely reported. A discussion of the use of rotarod to assess motor performance revealed apparent differences in scoring rules among investigators (also different design of rotarod equipment used in Europe versus the United States was noted). The use of a newly developed, automated gait analysis system seemed an attractive alternative. Some investigators were wary that standardization, if overly restrictive, could inhibit inquiry and discovery. One approach might be the establishment of a common minimum data set to be used in studies of animal models of neuroprotection.

Combinatorial Therapeutics

The use of polypharmacology or "dirty drugs" was thought to be a promising approach for neuroprotective therapy. Current treatment of many diseases, e.g., hypertension, cancers and infectious diseases often utilizes multiple medications. Additionally, using lower doses of each of several drugs may decrease unwanted side effects. The importance of dose finding studies was emphasized and there was general agreement about the difficulty of receiving funding for such studies in the absence of programs dedicated to this goal.

Current State of Neuroprotection

Patients suffering from neurodegenerative diseases currently have inadequate therapies available to them. The use of mouse models for the development of treatments for human neurodegenerative diseases holds promise but has not yet lead to the initiation of human clinical trials. Clearly, more work needs to be done as we pursue effective pharmaceutical treatments for these devastating diseases. Greater investments in translational research are aimed at fast-tracking potentially valuable treatments.

Mary Abood, Ph.D., Scientist
California Pacific Medical Center Research Institute
Forbes Norris MDA/ALS Center

Lucie Bruijn, Ph.D., Science Dir. & Vice President
The ALS Association

Carine Cleren, Ph.D., Professor and Chairman
Weill Medical College of Cornell University
Department of Neurology and Neuroscience

Robert Ferrante, Ph.D., M.Sc., Director
Bedford VA Medical Center
Translational Therapeutics Laboratory

Robert Friedlander, M.D.
Harvard Medical School
Brigham and Women's Hospital
Department of Neurosurgery

Jennifer Gatchel, Student
Baylor College of Medicine - Zoghbi Laboratory
Neuroscience Graduate Program

Jonathan Glass, M.D., Professor
Emory University School of Medicine
Neurology Center for Neurodegnerative Disease

Jill Heemskerk, Ph.D., Program Director
National Inst. of Neurological Disorders and Stroke
National Institutes of Health

Thomas Jeitner, Ph.D., Assistant Professor
Medical College of Wisconsin
Department of Biochemistry

Story Landis, Ph.D., Director
National Inst. of Neurological Disorders and Stroke
National Institutes of Health

Gloria E. Meredith, Ph.D., Professor and Chair
Chicago Medical School
Rosalind Franklin Univ. of Medicine and Science
Departments of Cellular and Molecular Pharmacology

Lauren Murphree, Ph.D.
National Inst. of Neurological Disorders and Stroke
National Institutes of Health
Anticonvulsant Screening Program

Diane Murphy, Ph.D., Program Director
National Inst. of Neurological Disorders and Stroke
National Institutes of Health

Eugene Oliver, Ph.D.
National Inst. of Neurological Disorders and Stroke
National Institutes of Health

Jay Schneider, Ph.D., Professor
Thomas Jefferson University
Departments of Pathology, Anatomy and Cell Biology

Todd Sherer, Ph.D.
Associate Director of Research Programs
Michael J Fox Foundation for Parkinson's Research

Parminder Vig, Ph.D., Professor
University of Mississippi Medical Center
Department of Neurology

Patrick Weydt, M.D., Senior Fellow
University of Washington Medical Center
Department of Laboratory Medicine (La Spada Lab)

Moussa Youdim, Ph.D.,
Professor and Director of Pharmacology
Technion-Faculty of Medicine
Eve Topf and NPF Centers

Gail Zeevalk, Ph.D.
Associate Professor of Neurology
UMDNJ - Robert Wood Johnston Medical School
Department of Neurology