Deposition of transthyretin in early stages of familial amyloidotic polyneuropathy: evidence for toxicity of nonfibrillar aggregates

Abstract

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of transthyretin (TTR) amyloid fibrils, particularly in the peripheral nervous system. No systematic immunohistochemical data exists relating TTR deposition with FAP progression. We assessed nerves from FAP patients in different stages of disease progression (FAP 0 to FAP 3) for TTR deposition by immunohistochemistry, and for the presence of amyloid fibrils by Congo Red staining. The nature of the deposited material was further studied by electron microscopy. We observed that early in FAP (FAP 0), TTR is already deposited in an aggregated nonfibrillar form, negative by Congo Red staining. This suggested that in vivo, preamyloidogenic forms of TTR exist in the nerve, in a stage before fibril formation. Cytotoxicity of nonfibrillar TTR was assessed in nerves of different FAP stages by immunohistochemistry for macrophage colony-stimulating factor. FAP 0 patients already presented increased axonal expression of macrophage colony-stimulating factor that was maintained in all other stages, in sites related to TTR deposition. Toxicity of synthetic TTR fibrils formed in vitro at physiological pH was studied on a Schwannoma cell line by caspase-3 activation assays and showed that early aggregates but not mature fibrils are toxic to cells. Taken together, these results show that nonfibrillar cytotoxic deposits occur in early stages of FAP.

Figure 1.

A: TTR immunohistochemistry (left) and Congo Red staining (right) of normal nerves (top), FAP 0 patients (middle), and FAP 3 patients (bottom). Original magnifications, ×40. B: TTR immunogold labeling and electron microscopy of nerves from a FAP 0 patient (left; original magnification, ×84,000), a FAP 3 patient (middle; original magnification, ×84,000), and a FAP 3 patient using preadsorbed anti-TTR (C, right; original magnification, ×66,000). Scale bar, 100 nm.

Figure 2.

MCSF immunohistochemistry of nerves of FAP 0 patients (n = 4) (top left; original magnification, ×60); FAP 3 patients (n = 3) (top middle; original magnification, ×40), and from normal individuals (top right; original magnification, ×40). Co-localization of MCSF antigens in FAP 0 with the neuronal marker neurofilament 200 (bottom left; original magnification, ×60). Quantitation of immunohistochemical images corresponding to M-CSF staining in control individuals and FAP patients (bottom right). Data are represented as percent occupied area ± SD (*, P < 0.03; ^‡, P < 0.005).

Figure 3.

A: TEM of negatively stained TTR Leu55Pro: soluble, initial aggregates, 6- and 15-day fibrils. Scale bar, 100 nm. B: Activation of caspase-3 in RN22 cells exposed for 48 hours to 2 mmol/L of the different species shown in A: soluble TTR (sol. TTR), initial aggregates (I. aggreg.), 6-day fibrils (6d fibrils), and 15-day fibrils (15d fibrils). *, P < 0.03; ^‡, P < 0.003).