Isabel Cardoso

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.

Doxycycline disrupts transthyretin amyloid: evidence from studies in a FAP transgenic mice model

Familial amyloidotic polyneuropathy is an autosomal dominant disorder mainly characterized by the extracellular deposition of transthyretin, with special involvement of the peripheral nerve. Several animal models have been generated, including transgenic mice carrying the most prevalent TTR mutation (TTR Val30Met). TTR‐Val30Met mice without endogenous TTR (TTR‐Val30Met X TTR‐KO) were previously analyzed in our laboratory and ∼60% of the animals over 1 year of age were found to have deposition as amyloid, i.e., with Congo red (CR) ‐positive material, constituting a good tool to investigate the effect of drugs on TTR deposition and fibrillogenesis. We recently showed that the drug doxycycline acts in vitro as a TTR fibril disrupter. In the present work we assessed the activity of this drug in vivo in the TTR‐Met30Val X TTR‐KO mice. Doxycycline was administrated in the drinking water to 23‐to 28‐month‐old mice over a period of 3 months. Immunohistochemistry analyses revealed no differences in nonfibrillar TTR deposition between treated (n=11) and untreated mice (n=11). However, CR‐positive material was observed only in the control group (untreated) whereas none of the animals treated with doxycycline was CR‐positive. Immunohistochemistry for several markers associated with amyloid, such as matrix metalloproteinase‐9 (MMP‐9) and serum amyloid P component (SAP), was performed. MMP‐9 was altered with significantly lower levels in treated animals compared with the control group. Mouse SAP was absent in treated animals, being observed only in untreated animals presenting TTR congophilic deposits. These results indicate that doxycycline is capable of disrupting TTR CR‐positive amyloid deposits and decreases standard markers associated with fibrillar deposition, being a potential drug in the treatment of amyloidosis. FASEB J. 20, 234–239 (2005)

4′-iodo-4′-Deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producing noncytotoxic species: screening for TTR fibril disrupters

Transthyretin Leu55Pro is one of the most aggressive mutations in familial amyloidotic polyneuropathy, an autosomal dominant disorder characterized by extracellular deposition of fibrillar amyloid protein. This variant has the ability to form fibrils in vitro under physiological conditions (PBS, pH 7.4). We studied by transmission electron microscopy the effect of the drug 4′‐iodo‐4′‐deoxydoxorubicin (I‐DOX) on the in vitro assembly of TTR Leu55Pro fibrils by following fibril growth over a 15 day period. Our results showed that I‐DOX at a concentration of 10−5 M/100 µg fibrils does not inhibit fibril formation in up to 10 days since fibrils identical to the ones present in the untreated sample were observed. However, after 15 days of treatment, only round particles, resembling soluble native TTR, were observed. We also tested the ability of tetracyclines and nitrophenols to interfere with amyloid fibril formation for 17 days; the group of compounds tested showed fibril disruption activity to different extents: doxycycline and 2,4‐dinitrophenol resulted in complete disaggregation of fibrils. The species generated upon I‐DOX and tetracyclines treatments were nontoxic, as revealed by the lack of significant caspase‐3 activation on a Schwannoma cell line, making them potential therapeutic drugs in TTR‐related and other amyloidosis.—Cardoso, I., Merlini, G., Saraiva, M. J. 4′‐iodo‐4′‐Deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producing noncytotoxic species: screening for TTR fibril disrupters. FASEB J. 17, 803–809 (2003)

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

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.

Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor.

Alzheimers disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid β-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T4 and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1–14) and (15–42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an αAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted αAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

Transthyretin binding to A-Beta peptide – Impact on A-Beta fibrillogenesis and toxicity

It has been suggested that transthyretin (TTR) is involved in preventing A‐Beta fibrillization in Alzheimers disease (AD). Here, we characterized the TTR/A‐Beta interaction by competition binding assays. TTR binds to different A‐Beta peptide species: soluble (Kd, 28 nM), oligomers and fibrils; diverse TTR variants bind differentially to A‐Beta. Transmission electron microscopy (TEM) analysis demonstrated that TTR is capable of interfering with A‐Beta fibrillization by both inhibiting and disrupting fibril formation. Co‐incubation of the two molecules resulted in the abolishment of A‐Beta toxicity. Our results confirmed TTR as an A‐Beta ligand and indicated the inhibition/disruption of A‐Beta fibrils as a possible mechanism underlying the protective role of TTR in AD.

Transthyretin and Alzheimer's disease: where in the brain?

Transthyretin (TTR), a carrier protein for thyroxine and retinol in plasma and cerebrospinal fluid (CSF), has been shown to bind the amyloid beta peptide. Accordingly, TTR has been suggested to protect against amyloid beta deposition, a key pathological feature in Alzheimers disease (AD). Supporting this view are the reduced TTR levels found in CSF of patients with AD, as well as reports of altered TTR expression in the cortex and hippocampus of AD rodent models. Importantly, early characterization of TTR distribution revealed the choroid plexus as the site of TTR synthesis within the brain. To resolve this controversy we used precise laser microdissection technology to assay for TTR mRNA expression. Our results clearly demonstrate that TTR is not produced in the brain parenchyma of wild-type mice nor in two different transgenic mouse models of AD, suggesting that contamination by choroid plexus contributed to the recent results indicating TTR production in various brain regions. The relevance of TTR to AD should now take into consideration TTR production by the choroid plexus and its ability, in the CSF, to sequester the amyloid beta peptide.

Binding of epigallocatechin‐3‐gallate to transthyretin modulates its amyloidogenicity

MINT‐7294529: TTR (uniprotkb:P02766) and TTR (uniprotkb:P02766) bind (MI:0407) by comigration in non‐denaturing gel electrophoresis (MI:0404)

Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative.

In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation.

Activation of ERK1/2 MAP kinases in Familial Amyloidotic Polyneuropathy

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.