chondroitin-sulfates and Amyloid-Neuropathies--Familial

Familial Amyloidotic Polyneuropathy (FAP) is a disorder characterized by the extracellular deposition of fibrillar Transthyretin (TTR) amyloid, with a special involvement of the peripheral nerve. We had previously shown that doxycycline administered for 3 months at 40 mg/Kg/ml in the drinking water, was capable of removing TTR amyloid deposits present in stomachs of old TTR-V30M transgenic mice; the removal was accompanied by a decrease in extracellular matrix remodeling proteins that accompany fibrillar deposition, but not of non-fibrillar TTR deposition and/or markers associated with pre-fibrillar deposits. On the other hand, Tauroursodeoxycholic acid (TUDCA), a biliary acid, administrated to the same mouse model was shown to be effective at lowering deposited non-fibrillar TTR, as well as the levels of markers associated with pre-fibrillar TTR, but only at young ages. In the present work we evaluated different doxycycline administration schemes, including different periods of treatment, different dosages and different FAP TTR V30M animal models. Evaluation included CR staining, immunohistochemistry for TTR, metalloproteinase 9 (MMP-9) and serum amyloid P component (SAP). We determined that a minimum period of 15 days of treatment with a 8 mg/Kg/day dosage resulted in fibril removal. The possibility of intermittent treatments was also assessed and a maximum period of 15 days of suspension was determined to maintain tissues amyloid-free. Combined cycled doxycycline and TUDCA administration to mice with amyloid deposition, using two different concentrations of both drugs, was more effective than either individual doxycycline or TUDCA, in significantly lowering TTR deposition and associated tissue markers. The observed synergistic effect of doxycycline/TUDCA in the range of human tolerable quantities, in the transgenic TTR mice models prompts their application in FAP, particularly in the early stages of disease.

Topics: Amyloid Neuropathies, Familial; Animals; Biglycan; Biomarkers; Blotting, Western; Chondroitin Sulfates; Disease Models, Animal; Dose-Response Relationship, Drug; Doxycycline; Drug Administration Schedule; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Extracellular Matrix Proteins; Heat-Shock Proteins; Immunohistochemistry; Matrix Metalloproteinase 9; Mice; Prealbumin; Proteoglycans; Stomach; Taurochenodeoxycholic Acid

Familial Amyloidotic Polyneuropathy (FAP) is a disorder characterized by the extracellular deposition of fibrillar Transthyretin (TTR) amyloid, with a special involvement of the peripheral nerve. Several extracellular matrix proteins have been found elevated in tissues from FAP patients, namely metalloproteinase-9 (MMP-9), neutrophil gelatinase associated lipocalin (NGAL) and biglycan. In this work we assessed the levels of MMP-9, tissue inhibitor of metalloproteinase 1 (TIMP-1), NGAL, biglycan and chondroitin sulphate (CSPG) in an FAP V30M TTR-related transgenic mouse model at different stages of TTR deposition and after two different treatment approaches to remove fibrillar deposits. Immunohistochemistry or RT-PCR analysis showed that biglycan was already increased in animals presenting TTR deposited in a non-fibrillar state, whereas MMP-9, TIMP-1, NGAL and CSPG were elevated only in mice with TTR amyloid deposits. Mice treated with doxycycline, a TTR fibril disrupter, presented lower levels of MMP-9, TIMP-1 and NGAL, suggestive of matrix recovery. Mice immunized with TTR Y78F to remove TTR deposition showed significantly lower levels of all the five tested markers, suggesting removal of fibrillar and non-fibrillar deposits. Cellular studies using oligomeric TTR showed induction of MMP-9 when compared to soluble TTR, large aggregates or fibrils. Furthermore, this induction was neutralized by an anti-receptor for advanced glycation end products (RAGE) antibody, indicating RAGE engagement in this process. Further studies in a larger number of tissue samples will indicate the application of these ECM markers in parallel with Congo Red staining in tissue characterization of pre-clinical and clinical stages in FAP and other amyloidoses.

Topics: Amyloid Neuropathies, Familial; Animals; Biglycan; Cell Line, Tumor; Chondroitin Sulfates; Disease Progression; Extracellular Matrix; Extracellular Matrix Proteins; Follow-Up Studies; Inflammation; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Models, Biological; Proteoglycans; Rats; Tissue Inhibitor of Metalloproteinase-1