Familial amyloid polyneuropathy (FAP) is a neurodegenerative disorder associated with extracellular deposition of mutant transthyretin (TTR) amyloid fibrils, particularly in the peripheral nervous system. We have hypothesized that binding of TTR fibrils to the receptor for advanced glycation end products (RAGE) on critical cellular targets is associated with a destructive stress response underlying peripheral nerve dysfunction. Analysis of nerve biopsy samples from patients with FAP (n ϭ 16) at different stages of disease (0-3), compared with agematched controls (n ϭ 4), by semiquantitative immunohistology and in situ hybridization showed increased levels of RAGE, beginning at the earliest stages of the disease (FAP 0; p Ͻ 0.02) and especially localized in axons. Upregulation of proinflammatory cytokines (tumor necrosis factor-␣ and interleukin-1␤) (approximately threefold; p Ͻ 0.02) and the inducible form of nitric oxide synthase (iNOS) (ϳ2.5-fold; p Ͻ 0.04) was also observed in a distribution overlapping RAGE expression. Tyrosine nitration and increased activated caspase-3 in axons from FAP patients ( p Ͻ 0.03) were apparent. Although these data suggest the presence of ongoing neuronal stress, there was no upregulation of neurotrophins (nerve growth factor and neurotrophin-3) in FAP nerves. Studies on cultured neuronallike, Schwann, and endothelial cells incubated with TTR fibrils displayed RAGE-dependent expression of cytokines and iNOS at early times (6 and 12 hr, respectively), followed by later (24 hr) activation of caspase-3 and DNA fragmentation. We propose that the interaction of TTR fibrils with RAGE may contribute to cellular stress and toxicity in FAP. Furthermore, there is an apparent lack of responsiveness of Schwann cells in FAP nerve to provide neurotrophic factors.

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.

Familial amyloid polyneuropathy (FAP) is characterized by extracellular deposition of transthyretin (TTR) aggregates and amyloid fibrils, particularly in the peripheral nervous system (PNS) and is accompanied with changes in connective tissue. Given the invasiveness of nerve biopsy, FAP salivary glands (SGs) were used in microarray analysis; biglycan and neutrophil gelatinase-associated lipocalin (NGAL), two genes related to extracellular matrix (ECM) remodeling were overexpressed in FAP. Results were validated by RT-PCR and immunohistochemistry both in SG and in nerve biopsies of different stages of disease progression. Matrix metalloproteinase-9 (MMP-9), which exists as a complex with NGAL, was also increased in FAP and in vitro degraded TTR aggregates and fibrils; however in the presence of serum amyloid P, a universal amyloid component, TTR fibrils became resistant to MMP-9 proteolysis. Biglycan, NGAL, and MMP-9 are transcriptionally up-regulated by NF-kappaB, a transcription factor that is activated in FAP nerves and SG. Given the relationship between inflammation and ECM remodeling, and the increase of proinflammatory cytokines in FAP, IL-10 expression in FAP nerves was investigated; IL-10 increased after fibril deposition, suggesting a balance between proinflammatory and anti-inflammatory mechanisms. Changes in ECM-related proteins and inflammatory events may be relevant for therapy in FAP and other neurodegenerative disorders.

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Familial amyloid polyneuropathy (FAP) is characterized by deposition of mutated transthyretin (TTR) in the peripheral nervous system. Prior to amyloid fibrils, nonfibrillar TTR aggregates are deposited inducing oxidative stress with increased nitration (3-NT). As the major source of TTR is the liver, liver transplantation (LT) is used to halt FAP. Given the shortage of liver donors, domino LT (DLT) using FAP livers is performed. The correlation between TTR deposition in the skin and nerve was tested in biopsies from normal individuals, asymptomatic carriers (FAP 0) and FAP patients; in FAP 0, nonfibrillar TTR was observed both in the skin and nerve in the same individuals; in patients, amyloid was detected in both tissues. The occurrence of amyloidosis in recipients of FAP livers was evaluated 1-7 years after DLT: TTR deposition occurred in the skin 3 years after transplantation either as amyloid or aggregates; in one of the recipients, fibrillar TTR was present in the epineurium 6 years after DLT. Deposits were scarce and 3-NT immunostaining was irrelevant. Nerve biopsies from DLT recipients had no FAP-related neuropathy. Our findings suggest that TTR amyloid formation occurs faster than predicted and that TTR of liver origin can cross the blood-nerve barrier. Recipients of FAP livers should be under surveillance for TTR deposition and tissue damage.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by the extracellular deposition of transthyretin (TTR), especially in the PNS. Given the invasiveness of nerve biopsy, salivary glands (SG) from FAP patients were used previously in microarray analysis; mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) was down-regulated in FAP. Results were validated by RT-PCR and immunohistochemistry both in SG and in nerve biopsies of different stages of disease progression. MKP-3 was also down-regulated in FAP SG biopsies. Given the relationship between MKPs and MAPKs, the latter were investigated. Only extracellular signal-regulated kinases 1/2 (ERK1/2) displayed increased activation in FAP SG and nerves. ERK1/2 kinase (MEK1/2) activation was also up-regulated in FAP nerves. In addition, an FAP transgenic mouse model revealed increased ERK1/2 activation in peripheral nerve affected with TTR deposition when compared to control animals. Cultured rat Schwannoma cell line treatment with TTR aggregates stimulated ERK1/2 activation, which was partially mediated by the receptor for advanced glycation end-products (RAGE). Moreover, caspase-3 activation triggered by TTR aggregates was abrogated by U0126, a MEK1/2 inhibitor, indicating that ERK1/2 activation is essential for TTR aggregates-induced cytotoxicity. Taken together, these data suggest that abnormally sustained activation of ERK in FAP may represent an early signaling cascade leading to neurodegeneration. Keywords: amyloid, extracellular signal-regulated kinases 1/ 2, familial amyloidotic polyneuropathy, mitogen-activated protein kinase phosphatase 1, transthyretin.

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No significant side effects occurred after the surgical procedure, and adequate material for pathological analysis was always obtained. Amyloid deposition was found in 91% of the patients. Patients with negative biopsies (N = 7) were all in the earlier stage of the disease. Two asymptomatic carriers had biopsies with amyloid deposition. We conclude that LSG biopsy is a useful, sensitive and minimal invasive method to detect amyloid deposition.