Ellerby Lab

Overview

The Lisa Ellerby laboratory studies cell death mechanisms and polyglutamine expansion diseases, with an emphasis on the relationship and interplay between proteases and cell death.

Molecular and Cellular Mechanisms of Polyglutamine Expansion Diseases

Huntington's disease

Huntington's disease (HD), one of the most extensively studied of the CAG/polyglutamine diseases, is an autosomal dominant neurodegenerative disease resulting in neuronal loss and dysfunction in the striatum and cortex, and characterized by involuntary movements, personality changes, dementia, and early death. A primary focus of our research is the characterization of the transgenic animal and cellular models that delineate the abnormal functions of the HD protein. We have shown that seven of the eight polyglutamine expansion disease proteins are cleaved by cell death proteases-the caspases.

Huntingtin protein

The cDNA for Htt codes for a protein of 348 kDa composed of over 3100 amino acids. The protein product of the Htt gene has widespread expression both in the brain and the periphery. The function of Htt is unknown. Co-localization of Htt with vesicles, microtubules, and synaptic elements suggests that it may play a role in vesicle and organelle transport (DiFiglia et al. 1995; Gutekunst et al. 1995; Sharp et al. 1995; Velier et al. 1998). Structural analysis of the Htt protein using threading alignment programs suggests it is composed of HEAT repeats (Andrade and Bork, 1995). HEAT repeats are tandemly repeated sequences of about 40 amino acids that form two helices. The HEAT repeats make up four predicted domains of huntingtin (HH). Numerous studies have demonstrated that Htt is a substrate for several different types of proteases. The amino terminal 600 amino acids (aa) of Htt contains cleavage sites for caspases, calpains, and aspartyl proteases, and the polyglutamine tract (Q) resides at the extreme amino terminus. Many studies have demonstrated that Htt fragments are toxic to cells, particularly in the presence of expanded polyglutamine tracts. These observations show that cleaved Htt has the potential to initiate neuronal death; for this reason, characterization of the proteases that can cleave Htt is the focus of our research. Despite significant progress in identifying the proteases that cleave Htt in vivo, and despite delineation of many of the sites of cleavage in Htt, little is known about the interrelationships among the different cleavage events, particularly in vivo. Currently, we do not know if cleavage occurs sequentially or simultaneously by distinct proteolytic pathways. However, we can postulate based on structural analysis of Htt a mechanism of proteolytic cleavage events. The caspase-calpain sites do not lie in the HEAT domain structures of Htt but rather in an area of low complexity where proteins are often disordered. The smaller N-terminal aspartic endopeptidase site characterized by Lunkes et al. (2002) lies in the structured HH1 domain. From this structural analysis, we hypothesize that proteolytic cleavage of Htt occurs in a series of sequential steps, the initial cleavage occurring in the calpain-caspase protease susceptibility domain of Htt, which triggers a conformational change, and that subsequent cleavage generates smaller N-terminal Htt fragments resulting in further toxicity to the cell.

Summary of hypothesis and analogy to APP processing and Alzheimer's disease

We hypothesize that caspase-calpain cleavage is a critical step in a sequential cleavage sequence generating toxic N-terminal Htt fragments. This hypothesis comes from (1) previous studies suggesting the existence of cleavage fragments in HD postmortem brain tissue and transgenic models; (2) previous studies indicating that fragments with expanded repeats are considerably more toxic than full-length Htt with an expanded repeat; (3) predictions about the structure of the Htt protein, indicating that the calpain- caspase region is embedded in a proteolytically susceptible (i.e., relatively unstructured) region of the protein, and thus that caspase-calpain cleavage might be an initial proteolytic event in a sequence; and (4) our published studies derived from both cell culture and transgenic mouse models. Our model is similar to the cleavage of APP in Alzheimer's disease (AD), which is increasingly well understood, and in which several proteolytic steps are key to pathogenesis. Careful analysis of proteolytic events in AD has greatly clarified its pathogenesis. Investigations of APP, PS1, and PS2 proteolysis have furthered our understanding of proteolysis at the membrane interface, led to the discovery of new proteases, and provided an understanding of how proteolysis regulates transcriptional activities of APP in the nucleus. We believe it is important to study the proteolytic cleavage events in HD to identify therapeutic targets for it.

Therapeutic strategies to attenuate HD

We have found that crossing transgenic caspaseresistant mice with transgenic HD mouse models improves their performance on a rotor rod. These observations suggest that specific caspases attenuate the progression of HD, and that blocking these caspases may yield benefit in the treatment of HD. Further studies will use these newly designed transgenic mice to determine if particular cell death proteases are involved in Huntington's-related striatal cell death. We have initiated a collaboration with Kunlin Jin, MD, PhD, and David Greenberg, MD, PhD, both at the Buck Institute, to replace the cells lost in HD by stimulating neurogenesis (new neurons in the brain).

References

Andrade, M.A. and Bork, P. 1995. HEAT repeats in the Huntington's disease protein. Nat Genet 11:115-116.

DiFiglia, M., Sapp, E., Chase, K., Schwarz, C., Meloni, A., Young, C., Martin, E., Vonsattel, J. P., Carraway, R., and Reeves, S. A. 1995. Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14:1075-1081.

Gutekunst, C. A., Levey, A. I., Heilman, C. J., Whaley, W. L., Yi, H., Nash, N. R., Rees, H. D., Madden, J. J., and Hersch, S. M. 1995. Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies. Proc Natl Acad Sci U S A 92:8710-8714.

Sharp, A. H., Loev, S. J., Schilling, G., Li, S. H., Li, X. J., Bao, J., Wagster, M. V., Kotzuk, J. A., Steiner, J. P., Lo, A., et al. 1995. Widespread expression of Huntington's disease gene (IT15) protein product. Neuron 14:1065-1074.

Velier, J., Kim, M., Schwarz, C., Kim, T. W., Sapp, E., Chase, K., Aronin, N., and DiFiglia, M. 1998. Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways. Exp Neurol 152:34-40.

Lunkes, A., Lindenberg, K. S., Ben-Haiem, L., Weber, C., Devys, D., Landwehrmeyer, G. B., Mandel, J. L., and Trottier, Y. 2002. Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol Cell 10:259-269.

Xia, J., Lee, D. H., Taylor, J., Vandelft, M., and Truant, R. 2003. Huntingtin contains a highly conserved nuclear export signal. Hum Mol Genet 12:1393-1403.