Scientific Report 2007

Molecular and Experimental Medicine

Division of Rheumatology Research

W.M. Keck Autoimmune Disease Center

Pathogenesis of Late-Onset Genetic Diseases Related to Abnormalities of Protein Conformation

J.N. Buxbaum, N. Reixach, Z. Ye, L. Friske, T. Coelho,* D. Jacobson,** G. Gallo,*** C. Tagoe,**** A. Roberts,***** E. Masliah^†

* Hospital Geral Santo António, Porto, Portugal
** Boston University School of Medicine, Boston Massachusetts
*** NYU School of Medicine, New York, New York
**** Albert Einstein College of Medicine, Bronx, New York
***** Molecular and Integrative Neuroscienes Department, Scripps Research
^† University of California, San Diego, California

We are studying the pathogenesis of a group of hereditary and sporadic human diseases, the transthyretin amyloidoses, that are the result of age-dependent protein misfolding. The misfolded molecules are deposited in the heart, kidneys, and peripheral nerves, producing organ-specific disease. We use 4 major approaches: biophysical analysis of recombinant and naturally occurring murine and human transthyretin, animals transgenic for human transthyretin, cell cultures to determine how the misfolded proteins injure their cellular targets, and genetic epidemiology to identify potential disease carriers and assess the effects of other hereditary and environmental factors on the disease.

Currently, we are completing 2 studies on the clinical impact of the transthyretin mutation Val122Ile, an allele carried by 3%–4% of African Americans. One study, carried out in collaboration with the Cardiovascular Heart Study, a multiinstitutional cooperative study of risk for cariovascular disease in community-dwelling individuals more than 65 years old, indicates that African American carriers of the allele who are more than 70 years old have a higher frequency of new-onset congestive heart failure than do controls matched for age, sex, and ethnic background and also have more features of heart disease consistent with cardiac amyloidosis. In the second study, a case control analysis done in collaboration with D. Jacobson, Boston University of Medicine; G. Gallo, NYU School of Medicine; and C. Tagoe, Albert Einstein College of Medicine, we compared living carriers of the allele with controls matched for age, sex, and ethnic background. We found an increased frequency of cardiac changes associated with cardiac amyloidosis in the carriers more than 65 years old. These data reinforce the notion that this late-onset genetic disorder is a significant health risk for the elderly African Americans who have this gene.

Our animal models continue to give us insights into the biology of these diseases of protein structure. In our continuing collaboration with D.R. Salomon, Department of Molecular and Experimental Medicine, we have shown that pathologic deposition of transthyretin in the hearts and kidneys of the same animals results in different molecular pathways of injury and response. The role of the molecular chaperoning response of the liver in sparing the heart from exposure to damaging protein aggregates appears to be quite clear, although the details remain to be elucidated. In collaboration with Dr. Salomon, J. Kelly, Department of Chemistry, and T. Coelho, Hospital Geral Santo António, we will be extending our observations to patients with the hereditary forms of transthyretin.

In biophysical investigations related to aging, we have explored the effects of oxidation on the stability of transthyretin, a protein known to be more susceptible to in vivo aggregation as humans become older. In this model, we showed that methionine oxidation actually reduces the tendency of transthyretin to form fibrils similar to those seen in the amyloidoses but makes the protein more toxic to cultured cells. These data challenge theories of aging that invoke generalized severe oxidation as an explanation for increased protein aggregation as people become older.

In the course of our studies with transgenic animals, we have examined the transcriptional profiles of various organs during normal aging of the animals. These analyses have revealed that few transcriptional features of aging are shared by different organs. Some organs show relative increases in transcription; others, decreases; and others, no quantitative change. The organs also differ qualitatively, with different degrees and distribution of expression of genes in various functional categories, particularly genes responsible for inflammatory responses in the absence of any apparent inflammatory stimulus apart from aging. These observations are quite consistent with current ideas that aging may represent a chronic low-grade inflammatory state, although perhaps not involving the same cells as conventional inflammatory disorders of infectious or noninfectious etiologies.

Studies in our murine model of human disease have also revealed, not unexpectedly, that even proteins of similar structure and function in mice and humans may have strikingly different biophysical properties. We have been able to synthesize recombinant mouse transthyretin, which we compared with recombinant human transthyretin. Both types of transthyretin are tetrameric proteins, but mouse transthyretin is more stable than is human transthyretin to chemical denaturants, and it is not amyloidogenic. Furthermore, the incorporation of mouse transthyretin subunits into the human protein results in a stable heterotetramer. These results parallel those obtained in vivo. In animals that have both the human protein and the mouse protein, transthyretin circulates as human-mouse heterotetramers, and this protein is more stable than the human transthyretin circulating in sera from animals in which the gene for mouse transthyretin is inactive. The disease model is apparent only when the human gene is highly expressed in excess of the mouse gene or is minimally or moderately expressed in animals in which the endogenous gene has been molecularly silenced.

A number of studies during the past 10 years have suggested an interaction between transthyretin and the amyloid β-peptide in the pathogenesis of Alzheimer's disease. We have crossed mice that have a human Alzheimer's gene with animals in which human transthyretin was overexpressed or in which the mouse transthyretin gene was silenced. Overexpression of human transthyretin suppressed the behavioral and neuropathologic phenotype usually seen in the mice with the Alzheimer gene, and the absence of the mouse gene was associated with earlier and more severe onset of the neuropathologic changes. We also discovered a specific interaction of purified human and mouse transthyretin with aggregates of the amyloid β-peptide, suggesting that the effects on the phenotype are mediated by an interaction between transthyretin and the peptide in vivo. These data clearly indicate an important effect of transthyretin in the pathogenesis of disease in these animals that may be relevant to the disease in humans. The presence of such a pathway would suggest other potential modes of therapy for the human disease.

Publications

Bartfai, T., Waalen, J., Buxbaum, J.N. Adipose tissue as a modulator of clinical inflammation: does obesity reduce the prevalence of rheumatoid arthritis? J. Rheumatol. 34:488, 2007.

Buxbaum, J.N. The amyloidoses. In: Cecil Textbook of Medicine, 23th ed. Goldman L., Ausiello, D. Saunders, Philadelphia, in press.

Buxbaum, J.N. The amyloidoses. In: Rheumatology, 4th ed. Hochberg, M.C., et al. (Eds.). Mosby, St. Louis, in press.

Buxbaum, J.N. The genetics of the amyloidoises: interactions with immunity and inflammation. Genes Immun. 7:439, 2006.

Maleknia, S.D., Reixach, N., Buxbaum, J.N. Oxidation inhibits amyloid fibril formation by transthyretin. FEBS J. 273:5400, 2006.

Reixach, N., Adamski-Werner, S.L., Kelly, J.W., Koziol, J., Buxbaum, J.N. Cell based screening of inhibitors of transthyretin aggregation. Biochem. Biophys. Res. Commun. 348:889, 2006.

Tagoe, C.E., Reixach, N., Friske, L., Mustra, D., French, D., Gallo, G., Buxbaum, J.N. In vivo stabilization of L55P transthyretin (TTR) by murine wild-type TTR and diflunisal in transgenic mice. Amyloid, in press.