Abstract
Familial amyloidotic polyneuropathy (FAP) is a type of systemic amyloidosis characterized by peripheral and autonomic neuropathy. Although FAP is a typical autosomal dominant disorder caused by a point mutation in the TTR gene, the average age at onset varies significantly among different countries. This discrepancy clearly suggests that a combination of intrinsic factors as well as extrinsic (environmental) factors shapes the development of FAP. However, these factors are difficult to analyze in humans, because detailed pathologic tissue analysis is only possible at autopsy. Thus, mouse models have been produced and used to disentangle these factors. This review covers the mouse models produced thus far and how these models are applied to analyze intrinsic and extrinsic factors involved in disease development and to test drug efficacy.
1 Introduction
Familial amyloidotic polyneuropathy (FAP) or transthyretin (TTR)-associated amyloidosis (ATTR) is an autosomal dominant disorder caused by a point mutation in the TTR gene. Since the first description of Portuguese patients with FAP by Andrade (1952)[1], many similar cases have been reported in various countries. According to the online registry for hereditary amyloidosis mutations (http://www.amyloidosismutations.com), 146 TTR mutations are associated with human amyloidosis. The major sites of TTR synthesis are known to be the liver and the choroid plexus of the brain[2,3,4]. TTR is a 127-amino acid, 55-kDa protein composed of four identical, noncovalently associated subunits[5–6]. TTR serves as a transport molecule for thyroxine (T4) and retinol-binding protein 4 (RBP4) [7].
The process of TTR amyloidogenesis involves rate-limiting dissociation of the TTR tetramer, followed by partial unfolding of monomers to yield nonfibrillar aggregates, protofibrils and mature amyloid fibrils[8,9,10]. We initiated our studies on FAP in 1985. At that time, the pathologic processes were almost unknown. This is mainly because detailed pathologic tissue analysis is only possible at autopsy. By that time, a large amount of amyloid has usually accumulated in many tissues.
Although FAP is a typical dominantly inherited disease, the average age at onset varies significantly among different countries with a median or mean of 33.5 years in Portugal, 51.3 years in France, 67.5 years in Great Britain, 56.7 years in Sweden and 33.4 years in Japan[11,12,13,14,15]. The reason for the different ages of onset is unknown. However, these results suggest that a combination of intrinsic factors as well as extrinsic (environmental) factors are involved in the development of FAP and that onset occurs earlier in warm climates, but later in cold climates.
To gain insight into the pathogenesis of FAP, we first produced a transgenic mouse by introducing the human mutant TTR gene into fertilized mouse eggs[16]. Since then, several groups, including ours, have generated transgenic mice that carry human TTR genes with various mutations, such as Met30, Ser10/Met30, Pro55, or Ser84[15,16,17,18,19,20,21,22,23]. In this review, we will discuss intrinsic and extrinsic factors involved in the development of FAP revealed by the use of transgenic models and the application of these models to test drug efficacy.
2 Intrinsic factors
2.1 Mutant TTR gene as a genetic factor
A significant component of amyloid fibrils isolated from FAP patients was found to be TTR[24]. Moreover, amyloid fibrils were found to be composed of mutant TTR[25,26,27]. To prove that the presence of mutant TTR is the cause of disease, we produced a transgenic mouse by introducing a mutant TTR (hM30) gene with a metallothionein (MT) promoter into fertilized eggs from C57BL/6 mice. Pathologic analyses revealed that amyloid deposition occurred in various tissues including the heart, kidney, intestinal tract and skin, but not in the peripheral nervous system[23], suggesting that the presence of the mutant TTR gene was the cause of FAP.
2.2 Unknown factors related to the late-onset nature of amyloid deposition
Clinical studies on FAP patients clearly showed that the disease is late-onset, as described in the introduction section. Then, the question is whether amyloid deposition itself or clinical symptoms are late-onset in nature? Harats et al.[28] demonstrated that the accumulation of amyloid itself did not start until late in life. To examine when amyloid deposition starts in a mouse model, we produced a transgenic mouse by introducing an MT-hTTRV30M (MT-hM30) gene, which carried a human mutant TTR gene with a metallothionein promoter. We found that amyloid deposition started at 6 months of age and that the amount and extent of amyloid deposition increased with aging, suggesting the late-onset nature of FAP.
It has been thought that this late-onset amyloid deposition might be correlated with a low level of serum hMet30 before puberty. To examine this possibility, we produced a transgenic mouse by introducing a 0.6-hTTRVal30Met (0.6-hM30) or 6.0-hTTRVal30Met (6.0-hM30) gene that contains an approximately 0.6-kb or 6.0-kb upstream region of the human TTR (hTTR) gene, respectively[17]. Then, we examined the temporal expression pattern. We found that both the 0.6-hM30 and 6.0-hM30 gene expression started from the fetal stage. The serum levels of hTTR increased up to 7-fold after birth and reached an adult level at 4 weeks of age. Thus, there is a time gap between increased serum hTTR levels and amyloid deposition, suggesting the involvement of other factor(s) in amyloid deposition. The reason for the time gap is not yet known.
2.3 Correlation between the serum hTTR level and amount of amyloid deposition
Generally speaking, the amount of amyloid deposition is not correlated with the serum level of TTR (Table 1). The amounts of amyloid deposition varied greatly depending on breeding conditions, type of mutation, etc. For example, Sousa et al.[29] reported that amyloid deposition was observed in only one out of 25 mice, while we[30,31,32] reported that amyloid deposition was observed in most of the mice even though both groups used the same Tg(6.0-hTTRM30) mice. In the case of the TTRL55P mutation, the amounts of amyloid deposition were much less than those in the TTRV30M mutation.
Serum TTR concentrations and amounts of amyloid deposition
| Author | Mouse | Frequency of amyloid deposition | |||||
|---|---|---|---|---|---|---|---|
| Construct | Ttr | Serum conc. | up tp 5 months | 6 – 11 months | 12 – 17 months | 18 – 24 months | |
| Kohno 1997[31] | 6.0-hTTRV30M | +/+ | 30 – 65 μg/mL | not done | 1/6 | 2/6 | 4/6 |
| 6.0-hTTRV30M | −/− | 22 – 79 μg/mL | not done | 3/6 | 2/6 | 3/6 | |
| Takaoka 2004[19] | 7.2-hTTRV30 | +/+ | 38 – 53 μg/mL | not done | 0/1 | 0/8 | 0/27 |
| 7.2-hTTRC10:V30M | +/+ | 48 – 57 μg/mL | not done | 0/4 | 0/19 | 14/27 | |
| 7.2-hTTRC10S:V30M | +/+ | 4 – 78 μg/mL | not done | 0/4 | 0/11 | 0/40 | |
| Inoue 2008[30] | 0.6-hTTRV30M | +/+ | 15 – 30 μg/mL | not done | 0/2 | 4/8 | 4/6 |
| 6.0-hTTRV30M | +/+ | 137 – 145 μg/mL | not done | 0/12 | 7/11 | 6/6 | |
| MT-hTTRV30M | +/+ | 10 – 48 μg/mL | not done | 1/8 | 10/13 | 7/7 | |
| Teng 2001[21] | 19.2kb hTTRV30 | +/+ | 1000 – 3500 μg/mL | not done | not done | 0/15 | 13/83 |
| 19.2kb hTTRL55P | +/+ | 10 – 30 μg/mL | not done | not done | 0 | 0 | |
| Sousa 2002[29] | MT-hTTRL55P | −/− | 50 – 200 μg/mL | 0/21 | 0/12 | 0/11 | 0/11 |
| 6.0-hTTRV30M | +/+ | not done | 0/24 | 0/25 | 0/25 | 1/25 | |
| Ttr−/− :6.0-hTTRV30M | −/− | not done | 1/1 | 0/2 | 13/13 | 16/16 | |
| Li 2018[80] | Ttr+/+ :6.0-hTTRV30M | +/+ | 142 – 149 μg/mL | not done | 8/10 | 6/7 | 12/12 |
| Ttr−/− :6.0-hTTRV30M | −/− | 131 – 143 μg/mL | not done | 5/10 | 5/7 | 12/12 | |
| TtrV30/V30:Rbp4RBP4 | −/− | 6.0 – 6.6 μg/mL | not done | 10/10 | 7/8 | 12/12 | |
| TtrV30/M30:Rbp4RBP4 | −/− | 5.5 – 5.9 μg/mL | not done | 0/10 | 6/6 | 8/8 | |
The amounts of amyloid deposition varied greatly depending on housing conditions, type of mutation, etc. For example, Sousa et al. reported that amyloid deposition was observed only one out of 25 mice, while we reported that amyloid deposition was observed most of mice even though both groups used the same 6.0-hTTRV30M.
The relationship between amounts of amyloid deposition and serum TTR concentrations can be analyzed only when breeding conditions are the same. Initially, we used two strains of mice, Tg(0.6-hM30) and Tg(6.0-hM30). We found that amounts of amyloid deposition were related to serum TTR concentrations to some extent only when we kept transgenic mice under the same conventional (CV) conditions[20]. Then, we used 3 strains of mice, namely, Tg(MT-hM30), Tg(0.6-hM30) and Tg(6.0-hM30). The amount of amyloid deposition was highest in Tg(MT-hM30), followed by Tg(6.0-hM30), and lowest in Tg(0.6-hM30) (Table 2). This order was surprising because the serum hTTR level determined by Western blot assay was approximately 15 mg/dL, 3 mg/dL and 5 mg/dL in Tg(6.0-hM30), Tg(0.6-hM30) and Tg(MT-hM30), respectively. To examine the cause of this discrepancy, we performed Northern blot analysis. As shown in Fig. 1, the 0.6-hM30 gene or the 6.0-hM30 gene was expressed only in the liver or in the liver and brain, respectively. Serum TTR is mainly derived from the liver, and its expression level in this tissue is important for amyloid deposition. In contrast, the MT-hM30 gene is expressed not only in tissues where the mouse Ttr (mTtr) gene is expressed but also in other tissues lacking expression of the mTtr gene. We previously showed that hTTR protein was associated with mouse TTR (mTTR) protein to form hybrid tetramers in transgenic mice[16]; therefore, hTTR expressed in tissues other than liver would form homotetramers composed of hTTR. Based on the expression level in each tissue, the serum concentration of hTTR homotetramers was estimated to be 3 μg/dL, 900 μg/dL, and 2 500 μg/dL in the Tg(0.6-hM30), Tg(6.0-hM30), and Tg(MT-hM30) lines, respectively (Fig. 1). This result suggests that the concentration of the hM30 homotetramer is related to the amount of amyloid deposition.
Amyloid deposition in various Tg Lines
| Tg Lines | Tissue | 6 months | 12 months | 18 months | 24 months |
|---|---|---|---|---|---|
| 0.6-hMet30 | heart | − | − | − ~ + | − |
| small intestine | − | − | + ~ + + | − ~ + | |
| kidney | − | − | − | − | |
| skin | − | − | − ~ + | − | |
| sciatic nerve | − | − | − | − | |
| 6.0-hMet30 | heart | − | − ~ + | − | + ~ + + + |
| small intestine | − | − ~ + + | − ~ + | + + | |
| kidney | − | − ~ ± | − | + + + | |
| skin | − | − ~ + | − | − ~ + | |
| sciatic nerve | − | − | − | − | |
| MT-hMet30 | heart | − | − ~ + | + ~ + + | + + + |
| small intestine | − ~ + | − ~ + + + | + + | + + + | |
| kidney | − | − ~ + | + + ~ + + + | + + + | |
| skin | − | + | + | + + | |
| sciatic nerve | − | − | − | − |
Amyloid deposition in 6.0-h Met30 was earlier than that in 0.6-h Met30. This was expected from the data that serum TTR level was higher in 6.0-h Met30 than in 0.6-h Met30 line. However, amyloid deposition in MT-h Met30 was earlier and larger than those in other lines.
Relationship between the concentration of hTTR homotetramer and the amount of amyloid deposition
Concentrations of hTTR homotetramer were estimated from the amounts of TTR mRNA and the serum hTTR concentrations. Amounts of amyloid deposition were correlated with the concentration of the hTTR homotetramer.
2.4 TTR proteolysis
Several studies reported the existence of different TTR molecules in amyloid deposits, one is full-length TTR and the other cleaved TTR[33,34,35,36]. Thus, TTR proteolysis was recognized as one of the mechanisms driving TTR amyloid formation. In the highly amyloidogenic variant, TTRS52P, the 49–127 TTR fragment was the most frequent fragment detected in fibrils[37–38]. The specific fragmentation at Lys48 in the TTR polypeptide suggested that a trypsin-like serine protease was involved in proteolysis[39].
Plasmin is structurally similar to trypsin and in silico studies suggested plasmin as a plausible pathophysiological candidate protease involved in the process of TTR amyloid formation[40]. To prove the involvement of plasmin in amyloid deposition, Slamova et al.[41] produced a mouse model of cardiac TTR amyloidosis with transgenic expression of human TTRS52P. This model showed substantial TTR amyloid deposits in the heart and tongue. The amyloid fibrils contained both full-length human TTR and the residue 49–127 cleavage fragment similar to TTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin were abundant within the cardiac and lingual amyloid deposits, which contained marked serine protease activity. Furthermore, the knockout of the physiological inhibitor of plasmin, α2-antiplasmin, enhanced amyloid formation. These findings indicate that the production of plasmin by local uPA induced cleavage of TTR and cardiac TTR amyloid deposition.
On the other hand, serine protease inhibitors (Serpins), particularly SERPINA1 (alpha-1-antitrypsin [AAT]), were shown to be related to other amyloid diseases, such as Alzheimer's disease[42,43,44]. SERPINA1 is best known as an inhibitor of serine proteases[45]. Like TTR, SERPINA1, one of the extracellular chaperons, is mostly expressed in the liver and represents a major protein of human serum. Recently, Niemietz et al.[46] demonstrated upregulation of SERPINA1 after TTR knockdown in hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells of FAP patients. They also showed that SERPINA1 inhibited in vitro aggregation of TTR, suggesting that SERPINA1 may have a role in the pathogenesis of TTR amyloidosis. Furthermore, SERPINA1 downregulation resulted in specific elevated TTR expression in hepatoma cells, and serum TTR was elevated after mSERPINA1 knockdown by antisense treatment in Ttr−/−:TTRM30 mice, leading to increased TTR deposition in several tissues, including dorsal root ganglia and intestine[47]. Similar results were reported by Bezerra et al.[48]. They found that SerpinA1 partially inhibited plasmin-mediated TTR proteolysis and aggregation in vitro. In addition, downregulation of SerpinA1 increased serum TTR levels and deposition in the cardiac tissue of older Ttr−/−:Tg(V30M) mice. Following SerpinA1 knockdown, the TTR fragments were observed in the heart of young and old mice but not in other tissues. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. These results indicate that SerpinA1 inhibits TTR proteolysis and aggregation in vitro and in vivo.
2.5 Amyloid deposition in peripheral nerves
In FAP patients, amyloid deposition usually occurs in peripheral nerves. However, such amyloid deposition was not observed in any of the transgenic models, although nonfibrillar deposits were reported in the sciatic nerve in Tg(6.0-hTTRV30M):Hsf1−/− (6.0-hM30/Hsf1−/−) mice[49]. This finding could be due to a low level of hTTR expression or the absence of hTTR expression in the choroid plexus. However, in the 6.0-hM30 line, the serum TTR level was equivalent to that in humans. Thus, this explanation is unlikely valid.
The choroid plexus is one of the major sites of TTR production. As the perineurium of the peripheral nerve is open to the subarachnoid space, it is possible that hMet30 present in cerebrospinal fluid is transported to the peripheral nerve through the perineurium. As shown before, the 6.0-hMet30 gene was expressed in the choroid plexus at a level similar to that of the endogenous mouse TTR gene. However, no amyloid deposition in the peripheral nerve was observed in 6.0-hM30 mice.
Another possibility is as follows. Tagoe et al.[50] showed that the TTR tetramer was much more stable in the presence of murine protein because the TTR circulates as hybrid human/murine heterotetramers in Tg(hTTRLeu5Pro). Reixach et al.[51] demonstrated that heterotetramers comprising mouse and human subunits are kinetically more stable than human homotetramers in vitro and are considered to inhibit dissociation and subsequent amyloid formation. Thus, stabilization of TTR tetramers may be responsible for the absence of amyloid deposition in peripheral nerves. However, Kohno et al.[31] produced a Ttr−/−:Tg(6.0-hTTRMet30) (−/−:6.0-hM30) mouse strain by mating Ttr knockout mice with Tg(6.0-hM30) mice. As this strain lacks the mouse Ttr gene, only hTTR homotetramers are present in serum. No amyloid deposition was observed in the peripheral nervous system of these mice.
Another factor that can stabilize the TTR tetramer is RBP4. In hepatocytes, newly synthesized RBP4 associates with retinol and TTR in the endoplasmic reticulum and is then secreted into the blood[52]. In transgenic lines, TTR is associated with mouse RBP4. To examine the role of the TTR-RBP4 complex in amyloid deposition, we produced TtrhTTRVal30orf/hTTRVal30orf:Rbp4hRBP4orf/hRBP4orf (TtrhV30o/hV30o:Rbp4hR/hR) and TtrhTTRVal30orf/hTTRMet30orf:Rbp4hRBP4orf/hRBP4orf (TtrhV30o/hM30o:Rbp4hR/hR) mice[32]. Amyloid deposition in the perineurium of the sciatic nerve was observed in TtrhV30o/hM30o:Rbp4hR/hR but not in TtrhV30o/hV30o:Rbp4hR/hR. Thus, the physicochemical properties of the TTR–RBP4 complex in the microenvironment surrounding the peripheral nerve or autonomic nerve may be important for amyloid deposition.
2.6 Cause of neuropathy
Amyloid deposits due to mutant TTR are pathognomonic in FAP[53–54]. Apart from fibrillar amyloid deposits, Sousa et al.[55] demonstrated that TTR was already deposited in an nonfibrillar TTR aggregate before amyloid deposition. Nonfibrillar TTR deposits were also demonstrated in Tg(TTRV30)[21] and in Ttr−/−: Tg(TTRLeu55Pro) transgenics[29]. Then, toxicity of nonfibrillar TTR aggregates was demonstrated in nerves of FAP patient by showing increased axonal expression of inflammation and oxidative stress-associated molecules[55–56]. Similarly, approximately twofold increases in the levels of nitrotyrosine in tissues with TTR deposition were observed in Ttr−/−: Tg(TTRLeu55Pro) mice[29]. Furthermore, TTR deposits were observed in the dorsal root ganglia of 6.0-hTTRV30M transgenic mice, and the mutant TTR secreted from the Schwann cells was inhibitory to neurite outgrowth in vitro[57].
Amyloid deposition was observed in the peripheral nervous systems of our double humanized mice, TtrhV30o/hM30o:Rbp4hR/hR. However, the amount of amyloid deposition in the sciatic nerve was too little to assess neurotoxicity. Because neuropathy has not been reported in any of transgenic mouse models until recently, evidence for relationship between toxicity of nonfibrillar TTR aggregates or amyloid deposition and neuropathy has been lacking until 2018. A TTR-A97S mutation is the most prevalent TTR mutation in Taiwan and is responsible for late-onset polyneuropathy in the sixth to seventh decades with marked axonal degeneration[58–59]. Kan et al.[60] produced knock-in mice carrying wild-type and A97S human TTR by replacing mTtr with the hTTR without altering the promoter and enhancer sequences of mTtr. These mice were backcrossed to C57BL/6 female to produce the heterozygotes with the genotypes hTTRV30/mTtr and hTTRA97S/mTtr. These aged TTR-A97S knock-in mice (> 104 weeks) showed small fiber neuropathy with decreased intraepidermal nerve fiber density and decreased mechanical thresholds. These mice also showed large fiber neuropathy with reduced myelinated nerve fiber density in sural nerves and reduced sural sensory nerve action potential amplitudes. Amyloid deposits were observed in sensory nerves of these mice, suggesting that the neurotoxicity of TTR-A97S was caused by amyloid deposits. However, amyloid deposition was only detected in a portion of tissues, mainly in sensory nerves, nonfibrillar TTR aggregates may also exert neurotoxic effect on the peripheral nerve.
2.7 Role of phagocytosis for elimination of amyloid deposits
Immunohistochemical examination of sural nerve biopsies in patients with amyloidotic neuropathy shows co-aggregation of TTR with several proteins, including apolipoprotein E, serum amyloid P and components of the complement cascade[61].
Complement component 1q (C1q) is the first subcomponent of the C1 complex of the classical pathway of complement activation and was suggested to be a modifier gene in onset of FAP by a Cypriot cohort of ATTR V30M patients[62]. Panayiotou et al.[63] demonstrated that C1q deficient ATTR V30M mice exhibited a 60% increase in amyloid deposition compared to their wild-type mice. C1q is one of early components of complement and has a protective role. In contrast, late components such as C3a and C5a exacerbate neuroinflammation[64]. The C5 complement system factor is cleaved by C5 convertases producing the C5a and C5b molecules. C5a acts as an anaphylactic molecule by attracting C5a receptor bearing cells such as macrophages and neutrophils, leading to a pro-inflammatory response[65]. Activation of the C5a receptor in the CNS could have a detrimental role leading to neurotoxicity or a neuroprotective role through phagocytosis[66]. To elucidate the role of the complement in the pathogenesis of FAP amyloidosis, Fella et al.[67] administered a C5a receptor agonist or a C5a receptor antagonist (PMX53) and subsequently analyzed disease phenotype in Ttr−/−: Tg(TTRV30M) mice 1 week after treatment. They showed that treatment with the C5a receptor agonists significantly ameliorated amyloid deposition while C5a receptor antagonist PMX53 exacerbated amyloid deposits. They also performed mass spectrometry-based proteomic analysis and showed substantial phagocytic cell activation, as well as the increased expression of proteolytic peptidases accompanying the reduction in amyloid deposition. These results suggested that administration of the C5a receptor agonists increased recruitment of phagocytic cells resulting in clearance of amyloid deposits.
The above results suggest that an antibody to one of amyloid components may be used to enhance phagocytosis by macrophages and to reduce amyloid deposits. In fact, Michalon et al.[68] developed a recombinant human monoclonal immunoglobulin G1 against the disease-associated TTR aggregates. This antibody removed TTR deposits ex vivo from patient-derived myocardium by macrophages, as well as in vivo from mice grafted with patient-derived TTR fibrils in a dose-and time-dependent fashion. The biological activity of ATTR removal involved antibody-mediated activation of phagocytic immune cells including macrophages. These data suggest that this antibody can be used to treat patients with TTR-associated cardiomyopathy.
2.8 Role of SAP in amyloid deposition
Amyloid fibrils are composed of two proteins. The main component is different and specific to each type of amyloidosis. A minor component that is common among amyloidosis is the amyloid P component which is derived from and identical to serum amyloid P component (SAP). SAP is synthesized by hepatocytes and is composed of two pentameric disk-like rings formed by five identical glycosylated subunits[69]. SAP has the property of Ca2+-dependent binding to specific ligands such as amyloid fibrils[70]. We investigated whether the presence of human SAP can affect amyloid deposition in MT-hM30: hSAP double-transgenic mice. We used a Tg(hSAP) line whose serum hSAP level is 42 μg/mL because this level is equal to that in humans. However, the onset, progression and tissue distribution of amyloid deposition were the same as those in single-transgenic mice carrying only the hMet30 gene. These results suggest that SAP does not play an important role in amyloid deposition; instead, SAP is just an additive component to preexisting amyloid protein.
3 Extrinsic factors
As mentioned before, it is obvious that extrinsic factors affect the development of FAP because the average age at onset differs significantly among different countries. Transgenic mouse models can be used to examine such extrinsic factors. Extrinsic factors are usually defined as factors that are outside the body. However, in addition to the breeding environment, we will discuss intestinal flora, inflammation and oxidative stress as extrinsic factors because these factors can be affected by environmental conditions.
3.1 Breeding environment
Experimental animals can be kept under specific pathogen-free (SPF) conditions or CV conditions. Using these conditions, we can examine the effect of the breeding environment on amyloid deposition. We used 3 mouse strains, 0.6-hM30, 6.0-hM30 and MT-hM30. Each line carries a transgene containing the human genomic Met30 gene, but not cDNA. Thus, we can examine the effect of housing conditions on different promoters. These mice were kept under either CV or SPF conditions until 24 months of age and were examined for amyloid deposition[30]. Under CV conditions, amyloid deposition was observed in all strains, although the onset and degree of amyloid deposition were different among these strains. However, no amyloid deposition was observed in any of these strains under SPF conditions. We also examined whether the duration that the strains were kept under CV conditions is related to the extent of amyloid deposition. The extent of amyloid deposition is indeed correlated with the duration under CV conditions. The serum levels of hM30 were similar in mice housed under CV and SPF conditions. Thus, the lack of amyloid deposition under SPF conditions was not due to alterations in serum TTR levels.
3.2 Intestinal flora
One major consideration regarding differences between CV and SPF conditions may be intestinal flora. Over the past decade, the influence of microbial dysbiosis has been shown for a variety of chronic diseases including neurological disorders[71]. We examined the effect of intestinal flora on amyloid deposition using the 6.0-hM30 line.
The 6.0-hM30 mice were kept under SPF conditions for three months after birth[72]. Then, these mice were transferred to CV conditions or remained under SPF conditions. Amyloid deposition was examined at 6, 12, 18, 24 and 28 months of age. Amyloid deposition was observed only in mice kept under CV conditions. Then, we examined the intestinal flora (Fig. 2)[72]. Under SPF conditions, the intestinal flora remained fairly constant, and the major flora was the group I symbiotes, such as gram-positive anaerobic cocci. In contrast, under CV conditions, the group III weak pathogens such as yeast, staphylococci, and Pseudomonas, increased, while the group I symbiotes decreased. In association with this difference, we observed infiltration of numerous neutrophils in the digestive tract in mice maintained under CV conditions. Neutrophil infiltration may lower the pH in the intestine, leading to dissociation of the TTR tetramer and amyloid deposition. In any case, these results suggest that the intestinal flora plays an important role in amyloid deposition.
Effect of intestinal flora on amyloid deposition
The 6.0-hM30 mice were kept under SPF or CV conditions. Amyloid deposition was observed only in mice maintained under CV conditions. Under SPF conditions, the intestinal flora remained fairly constant, and the major flora was group I symbiotes, such as gram-positive anaerobic cocci. In contrast, under CV conditions, the group III weak pathogens such as yeast, staphylococci, and Pseudomonas, increased, while that of group I symbiotes decreased. GPAC, gram positive anaerobic cocci; GPAR, gram positive anaerobic rods.
3.3 Inflammation
Inflammation has long been a well-known symptom of many infectious diseases, but molecular and epidemiological research increasingly suggests that it is also intimately linked to a broad range of noninfectious diseases. To examine whether inflammation accelerates amyloid deposition, MT-hM30 mice were injected with lipopolysaccharide (LPS) every 5 or 6 days[73]. Inflammation was confirmed by monitoring the serum SAP levels, as SAP is an acute-phase protein in mice. As expected, the serum level of SAP in these transgenic mice increased up to approximately 16-fold within 10 h after the injection of LPS and remained at high levels until 96 h later. Thus, we injected LPS every five days. However, LPS did not enhance amyloid deposition in MT-hM30 mice, although amyloid A amyloidosis was observed in the liver and spleen, suggesting that inflammation itself was enhanced by LPS injection. These results suggest that repeated acute inflammation and the sustained high serum level of SAP did not affect the onset and extent of TTR-associated amyloid deposition.
3.4 Oxidative stress
Using X-ray crystallography, Terry et al.[74] showed that the SH side chain of cysteine at position 10 (Cys10), the only thiol group in the TTR subunit, forms a hydrogen bond with Gly57 in normal TTR. However, the substitution of valine at position 30 with methionine causes separation of the sulfur of Cys10 and NH of Gly57, resulting in a free SH residue in Cys10. This result suggests a crucial role for the free Cys10 residue and possible involvement of physiological factors affecting Cys residue reactivity in TTR amyloidogenesis.
To examine the importance of the Cys10 residue, we produced 3 lines of transgenic mice, namely, Tg(7.2-hTTRCys10:Val30cDNA) (7.2-hC10:V30c), Tg(7.2-hTTRCys10:Val30MetcDNA) (7.2-hC10:M30c), and Tg(7.2-hTTRCys10SercDNA:hVal30Met) (7.2-hS10:M30c) (Fig. 3)[19]. Serum TTR levels ranged from 38 to 78 μg/mL among these transgenic mice, and there was no statistically significant difference in the hTTR concentrations. Histological analyses were performed at 9 months, 15 to 19 months, 21 to 24 months of age. Amyloid deposition was found only in the 7.2-hC10:M30c lines, but not in 7.2-hC10:V30c or 7.2-hS10:M30c. Free SH can form disulfide bridges with other TTR monomers or other molecules, such as hemopexin or transferrin[75], resulting in amyloid deposition. Disulfide bridges may be inhibited by reducing agents, such as glutathione. Oral intake of such agents may prevent amyloid deposition. In any case, the SH residue in Cys10 plays a crucial role in TTR Val30Met amyloidogenesis in vivo.
Role of Cys10 in amyloid deposition
Three recombinant cDNAs encoding normal hTTRC10:V30, hTTRC10:M30, and hTTRSer10:Met30 were introduced into the cDNA cloning site of the plasmid pTG.27; the resultant plasmids were designated 7.2-hC10:V30c, 7.2-hC10:M30c, and 7.2-hS10:M30c, respectively. Amyloid deposition was observed only in 7.2-hC10:M30c mice.
4 Application for evaluation of drug efficacy
To assess the in vivo efficacy of drug candidates, animal models that reproduce the pathology of FAP are required. TTR circulates as a 1:1 molar complex with RBP4. The biding affinity of hTTR with hRBP4 is different from that with mRBP4; thus, it is necessary to produce double-humanized mice for both TTR and RBP4 genes. By using a Cre-mediated recombination system, we produced humanized mice carrying either human normal (Val30) or mutant (Met30) TTR cDNA at the mouse Ttr locus[76]. By using the same strategy, we also created a humanized mouse carrying the human RBP4 gene at the mouse Rbp4 locus[77]. Then, we crossed these mice to produce three strains of double humanized mice, namely TtrhTTRVal30cDNA/hTTRVal30cDNA:Rbp4hRBP4cDNA/hRBP4cDNA (TtrhV30c/hV30c:Rbp4hRc/hRc), TtrhTTRVal30cDNA/hTTRMet30cDNA:Rbp4hRBP4cDNA/hRBP4cDNA (TtrhV30c/hM30c:Rbp4hRc/hRc) and TtrhTTRval30MetcDNA/hTTRval30MetcDNA:Rbp4hRBP4cDNA/hRBP4cDNA (TtrhM30c/hM30c:Rbp4hRc).
CHF5074 (CSP-1103) [1-(30,40-dichloro-2-fluoro[1,10-biphenyl]-4-yl)cyclopropane carboxylic acid] is a nonsteroidal anti-inflammatory derivative that lacks cyclooxygenase inhibitory activity. Recent X-ray analysis of the TTR crystal structure revealed that CHF5074 binds to the T4 sites of the TTR tetramer and prevents acidic pH-induced TTR dissociation in vitro[78].
Using these double-humanized mice, we examined the short-term effect of oral CHF5074 administration on hTTR tetramer stabilization[79]. The data showed that CHF5074 stabilized both normal and mutant TTR tetramers against denaturing stresses due to both acid and urea, thereby increasing the serum TTR levels of mice fed a CHF5074-containing diet. Therefore, our double-humanized mice may be an effective model to evaluate the stabilizing effects of small molecules for hTTR tetramers.
In our double-humanized mice, the serum levels of human TTR and human RBP4 were 4% and 25% of those of human serum TTR and RBP4, respectively[32]. These levels might be too low to test drug efficacy. To produce an ideal mouse model, we created a TTR exon-humanized mouse in which only mouse exons, but not introns, were replaced with the corresponding human exons using genome editing technology[80]. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial-specificity. Thus, these TTR exonhumanized mice will be useful for verifying novel treatments for FAP, including gene therapies.
Acknowledgements
This work was partly supported by Grants-in-Aid for Scientific Research (61480439, 63440082, 10470506, 13470509, 17200028 to K.Y.; 21590361, 24590404, 19K07354 to Z.L.) from the Japan Society for the Promotion of Science. The manuscript has been carefully edited by Nature Research Editing Service (http://authorservices.springernature.com).
Conflict of interests
Part of this study was done in collaboration with TransGenic, Inc.
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