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LeVine Lab

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PROJECTS

Projects in the lab apply biophysical, immunological, structural, molecular biological, and biochemical techniques to understand protein misfolding and its effects at the molecular level. Currently the focus is on the Ab peptide and AD.  The biological relevance of the concepts developed at the molecular level are investigated in cellular and animal systems as well as in human disease tissue through collaborations with other labs both in the Center on Aging and elsewhere which provide the breadth of expertise required to understand disease mechanisms.

Finally, as the result of the PI’s long career in the pharmaceutical industry, an eye is kept on the possibilities for applications of our findings in translational research to assist in the development of therapies for chronic neurodegenerative diseases. 

Specific areas of effort include:

Mechanism of Ab42 Oligomer Formation

            Soluble multimeric forms of the Alzheimer’s b-peptide are thought to be the toxic species leading to neuronal dysfunction manifesting as clinical dementia in Alzheimer’s disease.  We are studying the assembly of oligomer structures at physiological concentrations by immunological and sophisticated single molecule fluorescence methods. By understanding the process of their formation and how they relate to cellular effects we hope to catalyze efforts to pharmacologically manipulate their production or characteristics to halt the progression of neurodegeneration in Alzheimer’s disease.

Inhibitors of Ab42 Oligomer Formation

            We are using a Tecan Genesis 2000 Workstation industrial-scale robot in collaboration with Dr Randal Voss (Biology) to screen a LOPAC library of 1280 pharmaceutical compounds and a 16,000 compound Maybridge HitFinder™ pharmacophore library for inhibitors and modulators of Aβ42 oligomer formation in preparation for future large-scale library screening and medicinal chemistry efforts.

Ab42 Oligomers as Biomarkers for Alzheimer’s Disease

            Although Alzheimer’s b-peptide (Ab) oligomers are increasingly believed to be a major etiologic agent for neurodegeneration, their association with disease progression has not been well characterized, mainly due to a lack of analytical tools.  We have developed a highly sensitive immunological approach to detect soluble oligomeric Ab that we are using to correlate their accumulation with the progression of cellular pathology through the brain from tissue donated to the Sanders-Brown Brain Bank collection in collaboration with Drs. Bill Markesbery and Steve Scheff (Sanders-Brown).

Aβ Pathogenesis in Inclusion Body Myositis

            Inclusion Body Myositis is the most common muscle disease of the elderly resulting in both distal and proximal skeletal muscle atrophy leading to profound muscle weakness. IBM that is refractory to antinflammatory treatments is postulated to be driven by intracellular Aβ accumulation and deposition. In collaboration with Dr. M. Paul Murphy’s laboratory we are investigating potential common mechanisms between AD and IBM which could respond similarly to therapies.

Chronic Neurodegenerative Disease and the Chaperone System

            Cells of organisms from bacteria to humans possess quality control mechanisms to prevent the aggregation of misfolded proteins resulting from biochemical stressors or errors during synthesis of proteins. A primary protective system is a network of chaperone/cochaperone proteins that unfold misfolded proteins and then either refold them or target them for degradation.  In humans this system and ancillary processes successfully protect the brain against the accumulation of misfolded proteins for 50+ years.  Then, in an unexplained transition, the situation changes as an increasing proportion of people begin developing brain pathology that eventually manifests as dementia in Alzheimer’s disease, movement disorders in Parkinson’s disease, and other chronic dysfunction. 

            We are using biochemical and biophysical approaches to study the interaction of Ab, which is prone to misfolding into toxic species, with components of the chaperone system in vitro. Our early results suggest that the complex network of factors regulating chaperones interacts with Ab and may account for the multiple effects of this peptide on cells. The Alzheimer’s Association is funding our study of the chaperone capacity and refolding activity in control and AD brain and in cellular model systems.  

POTENTIAL FUTURE PROJECTS  

Seeding

            A major impediment to the understanding and development of effective therapeutics for Alzheimer’s disease is the lack of an animal model that recapitulates the human disease. Transgenic animals that overproduce the precursor (AβPP) for the amyloid plaque-forming peptide develop the amyloid pathology with associated immune response but not the characteristic neurodegeneration or neurofibrillary tangles. Following the paradigm used for prion infection of transgenic animals which produce the normal form of the prion protein, seeding of the brains of young AβPP transgenic animals with dilute soluble extracts of AD brain robustly produces plaques.

1)  Walker, L. C., Callahan, M. J., Bian, F., Durham, R. A., Roher, A. E. and Lipinski, W. J. (2002) Exogenous induction of cerebral beta-amyloidosis in betaAPP-transgenic mice. Peptides 23: 1241-1247.

2)  Walker, L. C., Bian, F., Callahan, M. J., Lipinski, W. J., Durham, R. A. and LeVine, H. (2002) Modeling Alzheimer's disease and other proteopathies in vivo: Is seeding the key? Review Article. Amino Acids 23: 87-93.

3)  Kane, M. D., Lipinski, W. J., Callahan, M. J., Bian, F., Durham, R. A., Schwarz, R. D., Roher, A. E. and Walker, L. C. (2000) Evidence for seeding of beta -amyloid by intracerebral infusion of Alzheimer brain extracts in beta -amyloid precursor protein-transgenic mice. J Neurosci 20: 3606-3611.  

 Neither extracts from control aged human brain nor amyloid fibrils made from the synthetic peptide produce this response.  The identity of the seeding agent and when and where it is produced in the brain is unknown. Collaborators in the early stages of this work are Dr. Steve Scheff (Sanders-Brown) and Dr. Lary C. Walker (Emory University).

Structural and ligand binding studies with Thioflavine T – like benzothiazole compounds being used for Aβ plaque imaging in AD indicate that Aβ plaques and fibrils are polymorphic. These forms may differ in their ability to elicit biological responses and could be related to the seeding phenomena we have observed. This could account for the lack of a strong neurodegeneration in mouse models of AD and other animals with human sequence Aβ that develop plaques but suffer no significant neuronal death.  We are developing methodologies to investigate this possibility.