Roskamp Institute Articles

As I mentioned before, most of the PhD students and research assistants from our Institute participated in poster session  that was organized as a part of VA Research Week event series. Here you will find the abstract of our posters, enjoy

Development of the Barnes Maze as an Alternative to the Morris Water Maze Following TBI in Mice.

Scott Ferguson^1,2, Benoit Mouzon^1,2, John Phillips¹, Vani Ganapapthi¹, Alex Bishop¹, Gogce Kayihan^1,2, Venkatarajan Mathura^1,2, Michael Mullan^1,2 , Fiona Crawford^1,2

¹The Roskamp Institute, Sarasota, FL, USA; ² James A Haley Veterans Administration, Tampa, FL, USA

Traumatic brain injury (TBI) is suffered by approximately 1.4 million people in the United States each year. TBI is the leading cause of death and disability in the most active population (under 45 years of age) in industrialized countries. Within the military, TBI is associated with 59% of blast-associated injuries seen at Walter Reed Army Medical Center, and between January 2003 and May 2005, 31% of all admissions to WRAMC had a brain injury. Apolipoprotein E (APOE) polymorphisms are known to impact the outcome after TBI, with the APOE4 allele (and concomitant ApoE4 expressed protein) associated with worse outcome than ApoE3 following TBI.   As part of our TBI research program we are exploring the molecular, neurobehavioral and neuropathological outcome after TBI in mouse models of injury, including APOE transgenic mice.

In order to evaluate differential outcomes of injury and its effects on motor skills and memory, we have optimized a series of neurobehavioral tests in mice.  The Rotarod test measures motor skill and learning via a programmable rotating bar.  Rotarod has shown the ability to distinguish between injured and uninjured mice, and has shown appropriate trends between differing levels of injury.  Morris water maze is a test of spatial memory and learning originally designed for rats but later adapted for mice.  APOE3 mice performed better than APOE4 mice in our Rotarod results, demonstrating an APOE genotype-dependent effect on motor function following TBI.  However, in the cognitive paradigm not only did we fail to detect any APOE genotype-dependent effects, we observed no significant differences in performance between injured and uninjured mice.  We therefore explored other cognitive paradigms for their ability to discriminate between injured and uninjured mice.

The Barnes maze is analogous to the Morris water maze in that it is also a test of spatial memory and learning, but because swimming is not involved it is associated with less stress than MWM typically induces in mice.  Others have shown that there is a strain-dependent effect on the ability of mice to learn the water maze task as well as the Barnes maze task.  Given that C57BL/6J mice are reported to perform better on the Barnes maze task, and this is the background strain utilized in our research, we optimized a paradigm of the Barnes maze for use in our TBI studies.  Our results show a statistically significant effect of injury on the spatial memory and learning of C57BL/6J wild type mice.  Future studies will re-examine the effect of APOE genotype on spatial memory following TBI using this test.

This research was funded by a Department of Defense award (W81XWH-07-1-0700) to Dr. Fiona Crawford and by the Roskamp Foundation

APP is Internalized After CD40 Ligation Which Increases Aβ Production

Ghania Ait-Ghezala, Jeremy Frieling, Myles Mullan, Claude-Henry Volmar, and Michael J. Mullan.

CD40, a member of the tumor necrosis factor receptor superfamily, and its cognate ligand CD40L are both elevated in the brains of Alzheimer’s disease (AD) patients compared to controls. We have shown that pharmacological or genetic interruption of CD40/CD40L interaction results in mitigation of AD-like pathology in vivo in transgenic AD mouse models, and in vitro. Recently, we showed that CD40L stimulation could increase Aβ levels, but the mechanism causing this phenomenon is not known. Here we show that CD40 ligation triggers internalization of APP and that internalization by endocytosis is associated with increased Aβ production. Furthermore, anthocyanins, which are known to impact trafficking to and from lipid rafts, impair the production of Aβ by CD40L stimulated CD40. However, anthocyanins have no effect on CD40L treatment of a neuroblastoma cell line over-expressing the C-99 APP fragment suggesting that CD40L internalization has no effect on γ-secretase. This finding is consistent with previous data suggesting that endocytosis increases BACE activity. In summary, these data suggest that a general mechanism of increased Aβ generation may be lipid raft mediated internalization of APP allowing increased BACE activity on its substrate.

Neurobehavioral profiles of two mouse models of Gulf War Illness

Laila Abdullah¹, Alex Bishop¹, John Phillips¹, Benoit Mouzon^1,2, Scott Ferguson^1,2, Vani Ganapathi¹, Myles Mullan^1,2, Ghania Ait-Ghezala PhD^1,2, Michael Mullan MD, PhD^1,2 and Fiona Crawford PhD^1,2

Affiliations: ¹Roskamp Institute, 2040 Whitfield Ave, Sarasota, FL, 34243, ²James A. Haley VA Hospital, 13000 Bruce B. Downs Blvd, Tampa, FL, 33612.

Background: Gulf War Illness (GWI) is a multisymptom condition associated with service in the 1990-1991 Persian Gulf War conflict and affects around 250,000 US veterans. It is largely attributed to combined exposure to pyridostigmine bromide (PB) and overuse of pesticides and insect repellants.  After nearly two decades, there is still no treatment for GWI and the underlying pathologic factors associated with the observed central nervous system (CNS)-based symptoms in veterans remain unclear.  Current GWI animal models do not demonstrate the full spectrum of neurobehavioral features reported to be associated with GWI, which makes it particularly difficult to explore the efficacy of possible therapeutic options.  Therefore, we tested two different treatment paradigms in order to establish a mouse model of GWI, which exhibits motor, cognitive and anxiety-related symptoms that are observed in veterans with this illness.  Methods: For model A, a previously established treatment paradigm was used which showed pathological changes suggestive of neurodegeneration, however extensive neurobehavioral profiling was not performed. Treated C57BL6 mice received oral administration of 1.3mg/kg of PB in water, dermal application of 0.13mg/kg of permethrin (PER) and 40 mg/kg of N-N-diethyl m-toluamide 2 (DEET) in 70% ethanol and 5 minutes of restrained stress daily for 28 days, whereas control mice received vehicle for the same duration.  For model B, a new treatment paradigm was developed where CD1 mice in the treatment group were administered 2mg/kg of PB and 200 mg/kg of PER via i.p. in DMSO daily for 10 days and the control group received DMSO only.  Following treatment, neurobehavioral profiles were examined using the Rotarod test to assess motor deficits, the Open Field test for anxiety-related changes and the Morris Water Maze test to assess spatial memory.  Results: In model A, treatment was associated with significant impairment in sensorimotor function and presence of anxiety-related behavior, but there was no deficit in spatial memory.  In model B, a delayed adverse effect of treatment was observed on the outcome measures of anxiety and spatial memory, but there was no evidence of sensorimotor impairment.  Conclusion: These findings suggest that combined exposure to PB and pesticides/insect repellents may lead to CNS-based effects in mice that mimic some of the clinical symptoms observed in veterans with GWI.  Additional studies are required to determine whether all three neurobehavioral features can be produced in one mouse model and whether these changes correlate with pathological features associated with neurodegeneration.

Acknowledgment: Funding for this research is provided by a Congressionally Directed Medical Research award (GW080094) to Dr. Fiona Crawford.

Proteomic identification of plasma TBI biomarkers

Benoit Mouzon^1,2, Alex Bishop¹, Gogce Kayihan^1,2, Ben Katz¹, Scott Ferguson^1,2, Jon Reed¹, Venkatarajan Mathura¹, Michael Mullan^1,2 and Fiona Crawford^1,2

¹Roskamp Institute, Sarasota, Florida

²James A. Haley Veterans’ Hospital, Tampa, Florida

Traumatic Brain Injury (TBI) is a major cause of mortality and morbidity in both military and civilian populations. The current lack of prognostic biomarkers for TBI confounds treatment and management of patients and is of increasing concern as the TBI population grows.  As part of our TBI research program we are generating brain and plasma proteomic profiles from APOE3 and APOE4 transgenic mice which demonstrate relatively favorable and unfavorable outcomes respectively, following TBI.  In this study we used proteomic approaches to identify the changes in plasma protein profiles in APOE3 and APOE4 mice following severe TBI, in order to determine peripheral biomarkers associated with a poor outcome after TBI.

Using a quantitative proteomics approach (isobaric tagging for relative and absolute quantitation – iTRAQ) we have identified proteins that are significantly modulated as a function of APOE genotype, injury and the interactive term of “genotype*injury”. Analysis of modulated plasma proteins revealed significant differences in proteomic response at 24 hours, 1 month and 3 months post injury across genotypes.  From these proteomic datasets we have identified 83 proteins at the 24 hour timepoint, 170 at 1 month and 129 at 3 months post TBI. For each timepoint, the identified proteins included those whose response was dependent on injury or the injury*genotype interaction, suggesting them as potential biomarkers of injury or outcome following injury.

In pilot validation studies, using antibody-based approaches in the original plasma from these mice, we have demonstrated the validity of our approach.  These preliminary data clearly demonstrate plasma protein changes that are not only injury-dependent but also interaction-dependent. The identified proteins include biomarkers that have been previously implicated in human TBI, and their time course and relationship to neurobehavior and pathology are now to be examined in these mouse models.  Importantly these results demonstrate the presence of TBI-dependent and interaction-dependent plasma proteins at a 3 months time point, which is a considerable time post-injury in the mouse model and will potentially be of significance for combat veterans receiving assessment at extended periods post-injury.  Furthermore, our identification of clusters of related proteins indicates disturbance of particular biological modules which increases their value beyond that of solitary biomarkers.  Clinical assays for many of these proteins are already established, which will facilitate translation of our findings from mouse to human.

The biomarker panels developed from this work will aid clinicians in the determination of diagnosis, prognosis, appropriate treatment and monitoring response to treatment, all of which are urgently needed in TBI management. The next step will be to investigate these potential biomarkers in human TBI patients and those studies will begin this year with the VA patient population and our clinical collaborators.

Acknowledgement:  This research was funded by a Department of Defense award (W81XWH-07-1-0700) to Dr. Fiona Crawford.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949

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