Relationship between amyloid deposition and intracellular structural changes in familial amyloidotic polyneuropathy

Transthyretin-related familial amyloidotic polyneuropathy is a systemic amyloidosis caused by mutations in the transthyretin gene. Extracellular deposition of amyloid is the common pathologic hallmark of amyloidoses including Alzheimer disease, AL amyloidosis, AA amyloidosis, and familial amyloidotic polyneuropathy. However, the exact relationship between amyloid deposition and cell death has not yet been clarified. To elucidate this relationship, we studied the effect of transthyretin amyloid fibrils and prefibrillar aggregates on cells by using autopsy tissues obtained from 8 patients with familial amyloidotic polyneuropathy, as well as cultured cell lines. Ultrastructural studies of amyloid-laden cardiomyocytes showed that intracellular structural changes correlated with the degree of amyloid deposition and may reflect metabolic disturbances caused by physical limitations imposed by the amyloid deposits. Amyloid-laden vascular endothelial cells, mesangial cells, smooth muscle cells, Schwann cells, and cardiomyocytes, however, had well-preserved cell nuclei and showed no apoptotic changes, even when cells were completely surrounded by prefibrillar transthyretin aggregates and amyloid fibrils. Synthesized prefibrillar transthyretin aggregates, transthyretin fibrils, and amyloid fibrils obtained from patients with familial amyloidotic polyneuropathy evidenced no cytotoxicity in cell culture experiments. Our data thus indicate that neither transthyretin amyloid fibrils nor prefibrillar transthyretin aggregates directly induced apoptosis. However, cellular metabolic disturbances caused by cells' being physically confined by amyloid deposits may induce cell degeneration.     

The potassium permanganate method. A reliable method for differentiating amyloid AA from other forms of amyloid in routine laboratory practice.

Alterations in affinity of amyloid for Congo red after incubation of tissue sections with potassium permanganate, as described by Wright el al, were studied. The affinity of amyloid for Congo red after incubation with potassium permanganate did not change in patients with myeloma-associated amyloidosis, familial amyloidotic polyneuropathy, medullary carcinoma of the thyroid, pancreatic island amyloid, and cerebral amyloidosis. Affinity for Congo red was lost after incubation with potassium permanganate in tissue sections from patients with secondary amyloidosis and amyloidosis complicating familial Mediterranean fever (consisting of amyloid AA). Patients with primary amyloidosis could be divided into two groups, one with potassium-permanganate--sensitive and one with potassium-permanganate--resistant amyloid deposits. These two groups correlated with the clinical classification in typical organ distribution (presenting with nephropathy) and atypical organ distribution (presenting with cardiomyopathy, nephropathy, and glossopathy) and the expected presence of amyloid AA or amyloid AL. Potassium permanganate sensitivity seems to be restricted to amyloid AA. The potassium permanganate method can be important in dividing the major forms of generalized amyloidosis in AA amyloid and non-AA amyloid. This can be used for differentiating early stages of the disease and cases otherwise difficult to classify. It is important to define patient groups properly, especially in evaluating the effect of therapeutic measures. (Am J Pathol 97:43--58, 1979).     

Prealbumin variants in the amyloid fibrils of Swedish familial amyloidotic polyneuropathy.

Amino-acid sequence analysis of an amyloid fibril protein from a patient with Swedish familial amyloidotic polyneuropathy showed homology with prealbumin but with heterogeneous N-terminal deletions. One-third of the molecules had the same amino acid substitution, methionine for valine in position 30, as in familial amyloidosis of Portuguese, Japanese and Swedish-American type. A protein with the same antigenic properties and size was found in the fibrils of two other patients with Swedish FAP while the amyloid fibrils in two further patients predominantly contained a smaller prealbumin-derived protein. Cyanogen bromide cleavage of this protein revealed no evidence for a methionine residue in position 30.     

4'-Iodo-4'-deoxydoxorubicin disrupts the fibrillar structure of transthyretin amyloid

Transthyretin (TTR) is a tetrameric protein synthesized mainly by the liver and the choroid plexus, from where it is secreted into the plasma and the cerebrospinal fluid, respectively. Some forms of polyneuropathy, vitreopathy, and cardiomyopathy are caused by the deposition of normal and/or mutant TTR molecules in the form of amyloid fibrils. Familial amyloidotic polyneuropathy is the most common form of TTR amyloidosis related to the V30M variant. It is still unclear the process by which soluble proteins deposit as amyloid. The treatment of amyloid-related disorders might attempt the stabilization of the soluble protein precursor to retard or inhibit its deposition as amyloid; or aim at the resorption of the deposited amyloid. The anthracycline 4'-iodo-4'-deoxydoxorubicin (I-DOX) has been shown to reduce the amyloid load in immunoglobulin light-chain amyloidosis. We investigated 1) whether I-DOX has affinity for TTR amyloid in tissues, 2) determined the I-DOX binding constants to TTR synthetic fibrils, and 3) determined the nature of the effect of I-DOX on TTR fibrils. We report that 1) I-DOX co-localizes with amyloid deposits in tissue sections of patients with familial amyloidotic polyneuropathy; 2) I-DOX strongly interacts with TTR amyloid fibrils and presents two binding sites with k(d) of 1.5 x 10(-11) mol/L and 5.6 x 10(-10) mol/L, respectively; and 3) I-DOX disrupts the fibrillar structure of TTR amyloid into amorphous material, as assessed by electron microscopy but does not solubilize the fibrils as confirmed by filter assays. These data support the hypothesis that I-DOX and less toxic derivatives can prove efficient in the treatment of TTR-related amyloidosis.     

Analyses of pathogenesis and therapeutic approaches for hereditary amyloidosis

Amyloidosis is a disorder of protein metabolism in which normally soluble autologous proteins are deposited in tissues as abnormal insoluble fibrils, causing structural and functional disruptions. We have recently identified the novel localized amyloidosis accompanied by trichiasis. The precursor protein of amyloid deposits was mutated lactoferrin and all the patients had lactoferrin Glu561Asp. The disease was classified hereditary amyloidosis whose risk factor is trichiasis. We examined the therapeutic possibilities for mutated transthyretin(ATTR) related familial amyloidotic polyneurpathy(FAP), one of the systemic amyloidoses. Cr3+ suppressed amyloid formation by stabilizing ATTR structure in vitro. BSB is a useful new diagnostic tool to detect amyloid deposits both in in vitro and in vivo and may have therapeutic potential for preventing amyloid deposition. Gene therapy using single-stranded oligonuclotides(SSOs) may become a promising tool for therapy instead of liver transplantation. SSOs with athrocollagen effectively replaced the TTR gene both in vitro and in vivo.     

Amyloidosis and oxidative stress

Recent studies on oxidative stress have revealed that free radical injury appears to be involved in either the amyloid formation process or in post-fibrillar modification in several types of amyloidosis. Here, we report the role of oxidative stress in the pathogenesis of dialysis-related amyloidosis(DRA) and familial amyloidotic polyneuropathy(FAP), and propose radical scavenger treatment for such amyloidosis. For patients under maintenance hemodialysis, EC-SOD Arg213Gly was a risk factor for the progression of DRA, atherosclerosis, and renal failure causing hemodialysis. In FAP patients who had EC-SOD Arg213Gly, massive amyloid deposition which may be related to increased oxidative stress in loco was found especially prominently around blood vessels in the interstitial tissues. Histological and biochemical examinations revealed that oxidative stress is deeply connected with amyloid formation mechanisms in FAP. We started radical scavenger therapy, such as N-acetyl cysteine, vitamin E and vitamin C in 20 Swedish FAP patients for 6 months. Although no improvement was found in the amount of amyloid deposition in biopsy specimens, modified body mass index(mBMI), an index of nutritional status, tended to be increased, suggesting the therapeutic possibility of radical scavenger treatment for amyloidosis.     

Amyloid protein of vessels in leptomeninges, cortices, choroid plexuses, and pituitary glands from patients with systemic amyloidosis

The cerebrum, cerebellum, and choroid plexuses from 16 patients with systemic amyloidosis, and the pituitary glands from 14 of these patients, were investigated histologically and immunohistochemically. Cerebrovascular amyloid (CVA) was found in the leptomeninges and cortices of six patients with systemic amyloidosis, including two patients with amyloid A protein (AA) amyloidosis related to serum amyloid A protein, one with AL amyloidosis related to immunoglobulin light chain (AL), two with familial type I amyloidotic polyneuropathy (FAP), and one with senile systemic amyloidosis (SSA). CVA protein from two patients with FAP reacted with anti-human prealbumin antibody similar to that of the visceral organs of these two patients. CVA in SSA reacted with anti-human prealbumin antibody and anti-beta protein antibody. Vascular amyloid was frequently noted in the pituitary glands and choroid plexuses of patients with systemic amyloidosis, and was found to be identical to that in the visceral organs (heart, kidney, and intestine) of these patients. CVA in the leptomeninges and cortices from two patients with AA amyloidosis and one with AL amyloidosis reacted with anti-beta protein monoclonal antibody but not with anti-human AA monoclonal antibody, anti-human A lambda antisera, and anti-human A kappa antisera. We suggest that amyloid proteins of AA and AL amyloidosis do not readily accumulate in the vessels in the leptomeninges and cortices even though the proteins circulate, and that beta protein is not derived from a serum precursor.     

Wild-type transthyretin-derived amyloidosis in various ligaments and tendons

Transthyretin-derived amyloid deposition is commonly found in intercarpal ligaments of patients with senile systemic amyloidosis. However, the frequency of transthyretin-derived amyloid deposits in ligaments of other tissues remains to be elucidated. This study aimed to determine the frequency of amyloid deposition and the precursor proteins of amyloid found in orthopedic disorders. We studied 111 specimens from patients with carpal tunnel syndrome (flexor tenosynovium specimens), rotator cuff tears (rotator cuff tendon specimens), and lumbar canal stenosis (yellow ligament specimens). To identify amyloid precursor proteins, we used immunohistochemical staining with antibodies that react with transthyretin, immunoglobulin light chain, amyloid A protein, and β(2)-microglobulin. By means of Congo red staining, we identified 47 (42.3%) amyloid-positive samples, 39 of which contained transthyretin-derived amyloid (18 flexor tenosynovium specimens, 5 rotator cuff tendon specimens, and 16 yellow ligament specimens). Genetic testing and/or clinical findings suggested that all patients with transthyretin amyloid deposits did not have familial amyloidotic polyneuropathy. The occurrence of amyloid deposition in those tissues depended on age. These results suggest that transthyretin-derived amyloid deposits may occur more frequently in various ligaments and tendons than originally expected. In the future, such amyloid deposits may aid determination of the pathogenesis of ligament and tendon disorders in older patients.     

Liver transplantation and new therapeutic approaches for familial amyloidotic polyneuropathy (FAP)

Liver transplantation has been considered as a promising therapy to halt the progression of clinical symptoms in familial amyloidotic polyneuropathy (FAP) because most transthyretin (TTR) is produced by the liver. In addition, domino liver transplantation using an FAP patient’s liver has been performed because of a shortage of donor livers. However, because the use of liver transplantation as therapy for FAP has given rise to several problems, an alternative treatment is needed. We have tried several other approaches. Recent studies suggested that certain metal ions affect amyloidogenesis. Among metal ions tested in an in vitro amyloid formation study, Cr³⁺ increased stability of both normal and mutant TTR tetramers and suppressed TTR amyloidogenesis induced by low pH. Our findings indicate that Cr³⁺ acts to suppress TTR amyloidogenesis. BSB, a Congo red derivative that binds to amyloid fibrils in FAP as well as to those in senile plaques in Alzheimer’s disease, effectively suppressed TTR amyloid formation in vitro. BSB may thus be useful for preventing amyloid formation. Free radical scavenger therapy was also tried in FAP patients but yielded no conclusive results. Immunization for transgenic mice having the ATTR V30M gene using ATTR Y78P resulted in suppression of amyloid deposits. Finally, an RNA/DNA chimera and single-stranded oligonucleotides (SSOs) were tested in vitro and in vivo in an attempt to repair the amyloidogenic TTR gene in the liver and retina. On the basis of results achieved so far, SSO is a promising tool for gene therapy.     

Transthyretin-its function and pathogenesis

Transthyretin (TTR) is a transport protein for retinol-binding protein and thyroxin, and works as a rapid turnover protein. Recently, it has been used as a nutrition assessment protein in the assessment of the acute phase nutritional status in various diseases because it contains four tryptophans in the tetramer of the protein and its plasma half life is 1.9 days. However, the wild-type protein and its mutated form become a precursor protein of amyloid fibrils in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy(FAP), respectively. Recent biochemical and pathological studies revealed that instability of the terameric form of TTR by mutation and post-translational modifications leads to amyloid formation in the tissues of SSA and FAP. In the process of TTR amyloid formation, misfolding of TTR, the trigger of amyloid formation, is also induced. For these amyloid formation mechanisms, Cr3+ administration, BSB(FSB) therapy, gene therapy, and antibody therapy are now on-going therapeutic projects for FAP and SSA.     

Abdominal fat tissue aspirate in human amyloidosis: light, electron, and immunofluorescence microscopic studies

Abdominal fat tissue aspirates from 12 patients with biopsy-proved amyloidosis were investigated by different morphologic techniques. By light microscopy, after staining of the fat tissue aspirates with Congo red and examination with a polarizing microscope, positive results were obtained in nine patients with amyloidosis, two of the three with primary (AL) amyloidosis and seven of the nine with secondary (AA) amyloidosis. By indirect immunofluorescence, using AA antiserum, positive results were obtained in five of the nine cases of AA amyloidosis (aspirates from these five patients were positive on Congo red staining). By electron microscopy, amyloid fibrils were observed in five cases of amyloidosis (two of the AL and three of the AA type, all positive on Congo red staining). Although amyloid was demonstrated less frequently by immunofluorescence and electron microscopy, perhaps because of the small numbers of fat particles examined, it seems that, with Congo red staining, abdominal fat tissue aspiration is a simple and sensitive method for the diagnosis of amyloidosis. Immunofluorescence studies allow discrimination between the different types of amyloidosis. The method could be used in patients in whom other types of tissue biopsy are not recommended because of risks of bleeding or other problems.     

Amyloid fibril composition is related to the phenotype of hereditary transthyretin V30M amyloidosis

Swedish familial systemic amyloidosis with polyneuropathy (FAP) depends on a mutation leading to a methionine-for-valine substitution in transthyretin. The disease appears with different clinical manifestations, including age of onset and involvement of the heart. Liver transplantation is currently the only curative treatment, but progressive cardiomyopathy may occur post-transplant. Two amyloid deposition patterns have previously been described in the heart. In one, the amyloid consists partially of transthyretin fragments and is weakly stainable by Congo red, while in the other, only full-length molecules are found and the fibrils have a strong affinity for Congo red. The present study aimed to see whether these morphological and biochemical variations have clinical implications. Subcutaneous adipose tissue biopsies were taken from 33 patients with Val30Met FAP and examined by microscopy, electrophoresis and western blot. Clinical data included age, sex, duration of disease and echocardiographic determination of the interventricular septum (IVS) thickness. It was found that fibrils composed of only full-length transthyretin were associated with early age of onset (44.8 +/- 12.9 years), no clinical cardiac involvement and a strong affinity for Congo red. In contrast, presence of transthyretin fragments in the amyloid was associated with late age of onset (67.3 +/- 7.0 years), signs of cardiac involvement and weak Congo red staining. For each individual, the same molecular type of amyloid was found in different organs. This is the first report showing that variations in clinical appearance of familial ATTR amyloidosis are associated with specific structural differences in the amyloid fibrils, and therefore may have a molecular cause. The molecular type of amyloid can be determined from a subcutaneous fat tissue biopsy. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pulmonary vascular amyloidosis in aged dogs. A new form of spontaneously occurring amyloidosis derived from apolipoprotein AI.

The N-terminus of a mutant form of apolipoprotein AI [apoAI] has previously been shown to be the subunit protein of amyloid fibrils in a human kindred with a form of familial amyloid polyneuropathy (FAP, type III) and in a recently reported kindred with a form of non-neuropathic hereditary amyloidosis. In this study, we demonstrate by amino-acid sequence analysis, that a form of vascular amyloidosis occurring in the lungs of aged dogs is derived from a N-terminal fragment of apoAI and that no amino acid substitution is present in this confirmed sequence. This represents the first documentation of apoAI as a precursor for a form of amyloidosis in animals, and provides the first documentation of apoAI as a precursor for amyloid fibrils in a form of age-associated ("senile") amyloidosis. Secondary structure prediction analysis of the N-terminal regions of normal human and dog apoAI indicated a propensity for beta-pleated sheet conformation, and thus amyloidogenesis, in 40 and 45% of the respective sequences. These results suggest that apoAI (like transthyretin) may serve as an amyloid precursor protein for both familial and senile forms of amyloidosis. ApoAI should, therefore, be considered as a potential amyloid precursor when forms of human senile amyloidosis of unknown origin are evaluated.     

An Experimental Model in Mink for Studying the Relation Between Amyloid Fibril Protein AA and the Related Serum Protein, SAA

Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amyloids. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.     

An Experimental Model in Mink for Studying the Relation Between Amyloid Fibril Protein AA and the Related Serum Protein, SAA

Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amy-loidosis. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.     

Analyses of amyloid formation mechanism in familial amyloidotic polyneuropathy and therapeutic trial

Familial amyloidotic polyneuropathy (FAP) is characterized by systemic accumulation of amyloid fibrils in the peripheral nerves and other organs. FAP ATTR Val30Met is the most common of the familial forms of amyloidosis. In the Kumamoto district, 5 different points of mutation in transthyretin (TTR) have been discovered. To make a diagnosis of FAP, histochemical analysis using ATTR Val30Met monoclonal antibody and FAP patients' hair, and mass spectrometry which can analyze TTR post-translational modifications in the blood circulation and cerebrospinal fluid. From our examinations, oxidative stress and beta protein metabolism is deeply connected with amyloid formation mechanism. Liver transplantation for FAP is only the therapy to save the life of FAP patients. By 1999, we had 17 FAP patients who underwent liver transplantation. They are all alive and showed some improvement predominantly in autonomic dysfunction after the surgery. Liver transplantation revealed that FAP does not progress if the TTR gene in the liver is normalized, suggesting the therapeutic possibility of gene therapy to the liver in FAP patients.     

Prealbumin in Swedish Patients with Senile Systemic Amyloidosis and Familial Amyloidotic Polyneuropathy

A prealbumin (PA)-like protein was demonstrated in amyloid fibrils both from a patient with senile systemic amyloidosis (SSA) and from a patient in northern Sweden with familial amyloidotic polyneuropathy (FAP). The investigated properties of this protein were similar in the two types of fibrils. The protein had molecular weight, antigenic determinants, and at least one cysteinyl residue in common with the subunit of normal PA. In contrast to normal PA, it contained disulphide-linked subunits and was to some extent bound to the fibril via the cysteinyl residues. The noncovalent forces between its subunits were weaker than in normal PA. It constituted part of the previously described AScl protein of the SSA fibrils. Proteins with lower molecular weight than the PA monomer were major proteins in both SSA and FAP fibrils. These proteins had similar properties in the two kinds of fibril and may be derived from PA. PA in serum from Swedish patients with FAP and SSA was normal with regard to the isoelectric pH of the monomers and tetramers.     

Amyloid and nonfibrillar deposits in mice transgenic for wild-type human transthyretin: a possible model for senile systemic amyloidosis

The human serum protein transthyretin (TTR) is highly fibrillogenic in vitro and is the fibril precursor in both autosomal dominant (familial amyloidotic polyneuropathy [FAP] and familial amyloidotic cardiomyopathy [FAC]) and sporadic (senile systemic amyloidosis [SSA]) forms of human cardiac amyloidosis. We have produced mouse strains transgenic for either wild-type or mutant (TTRLeu55Pro) human TTR genes. Eighty-four percent of C57BI/6xDBA/2 mice older than 18 months, transgenic for the wild-type human TTR gene, develop TTR deposits that occur primarily in heart and kidney. In most of the animals, the deposits are nonfibrillar and non-Congophilic, but 20% of animals older than 18 months that bear the transgene have human TTR cardiac amyloid deposits identical to the lesions seen in SSA. Amino terminal amino acid sequence analysis and mass spectrometry of the major component extracted from amyloid and nonamyloid deposits revealed that both were intact human TTR monomers with no evidence of proteolysis or codeposition of murine TTR. This is the first instance in which the proteins from amyloid and nonfibrillar deposits in the same or syngeneic animals have been shown to be identical by sequence analysis. It is also the first time in any form of amyloidosis that nonfibrillar deposits have been shown to systematically occur temporally before the appearance of fibrils derived from the same precursor in the same tissues. These findings suggest, but do not prove, that the nonamyloid deposits represent a precursor of the fibril. The differences in the ultrastructure and binding properties of the deposits, despite the identical sizes and amino terminal amino acid sequences of the TTR and the dissociation of deposition and fibril formation, provide evidence that in vivo factors, perhaps associated with aging, impact on both systemic precursor deposition and amyloid fibril formation.     

Deposition of Transthyretin in Early Stages of Familial Amyloidotic Polyneuropathy : Evidence for Toxicity of Nonfibrillar Aggregates

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.