Synergy of combined doxycycline/TUDCA treatment in lowering Transthyretin deposition and associated biomarkers: studies in FAP mouse models

Abstract

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.

Figures

Figure 1

Immunohistochemistry and Congo Red analysis of stomachs from TTR mice treated with doxycycline. Doxycycline was administered in the drinking water (8 mg/Kg/day) for 15 days followed by a suspension period of 15 days (water alone) after which animals were sacrificed, (n = 15); control mice were given water alone (n = 11). The top 2 panels represent CR analyses (arrow pointing at CR birefringence in a non-treated mouse) and MMP-9 IHC. The bottom 2 panels are representative of SAP (arrow pointing at staining which co-localizes with amyloid in a non-treated mouse) and TTR IHC. Scale bar (CR and SAP = 100 μm; MMP-9 and TTR = 200 μm).

Figure 2

Immunohistochemistry analyses for CSPG and biglycan in stomachs from TTR mice treated with doxycycline and TUDCA. Groups of animals analyzed: control mice (control, upper row) (n = 7); then in the descending rows: mice receiving doxycycline alone (40 mg/Kg/day; n = 9); mice receiving TUDCA alone (500 mg/Kg/day; n = 7); mice receiving either high dosages of doxycycline/TUDCA (40 mg/Kg/day and 500 mg/Kg/day, respectively; n = 7). The combined scheme involved the administration of the referred dosage of doxycycline for 15 days, followed by the administration of the referred dosage of TUDCA for 30 days. The cycle was repeated twice; for the individual drug administration, during the interruption periods, mice were receiving water alone. Scale bar = 200 μm; Histogram: quantification of the levels of the referred markers, as described in material and methods.*P < 0.05.

Figure 3

Immunohistochemistry analyses for TTR in stomachs from TTR mice treated with doxycycline and TUDCA. Groups of animals analyzed: in the upper row: control mice (n = 7); mice receiving doxycycline alone (40 mg/Kg/day; n = 9); mice receiving TUDCA alone (500 mg/Kg/day; n = 7); in the lower row: mice receiving either high dosages of doxycycline/TUDCA (40 mg/Kg/day and 500 mg/Kg/day, respectively; n = 7) or receiving low dosages of doxycycline/TUDCA (8 mg/Kg/day and 50 mg/Kg/day, respectively; n = 8). The combined scheme involved the administration of the referred dosage of doxycycline for 15 days, followed by the administration of the referred dosage of TUDCA for 30 days. The cycle was repeated twice; for the individual drug administration, during the interruption periods, mice were receiving water alone. Scale bar = 200 μm; Histogram: quantification of the levels of the referred marker, as described in material and methods.*P < 0.05.

Figure 4

Immunohistochemistry analyses for nitro, Fas and BiP in stomachs from TTR mice treated with doxycycline and TUDCA. Groups of animals analyzed: control mice (control, upper row) (n = 7); then in the descending rows: mice receiving doxycycline alone (40 mg/Kg/day; n = 9); mice receiving TUDCA alone (500 mg/Kg/day; n = 7); mice receiving either high dosages of doxycycline/TUDCA (40 mg/Kg/day and 500 mg/Kg/day, respectively; n = 7) or receiving low dosages of doxycycline/TUDCA (8 mg/Kg/day and 50 mg/Kg/day, respectively; n = 8). The combined scheme involved the administration of the referred dosage of doxycycline for 15 days, followed by the administration of the referred dosage of TUDCA for 30 days. The cycle was repeated twice; for the individual drug administration, during the interruption periods, mice were receiving water alone. Scale bar = 200 μm; Histogram: quantification of the levels of the referred markers, as described in material and methods.*P < 0.05.

Figure 5

Western blot analysis for BiP. Stomachs from mice receiving combined cycled administration of doxycycline/TUDCA (40 mg/Kg/day and 500 mg/Kg/day, respectively; n = 3), mice receiving doxycycline (40 mg/Kg/day; n = 3), mice receiving TUDCA (500 mg/Kg/day; n = 3), and control mice (n = 3). Histogram: densitometry of BiP/actin band intensity, *P < 0.05.