Báo cáo khoa học: Evolutionary changes to transthyretin: evolution of transthyretin biosynthesis doc

Thus, normal growth and development requires tightly regulated levels of THs to reach the nucleus of cells throughout the body and brain, and a strong network of buffering and regulatory

Evolutionary changes to transthyretin: evolution of

transthyretin biosynthesis

Samantha J Richardson

School of Medical Sciences, RMIT University, Bundoora, Vic., Australia

Introduction

Thyroid hormones (THs) are essential for normal

growth and development, and for regulation of the

basal metabolic rate The two major thyroid hormones

are 5¢,3¢,5,3-tetraiodo-[L]-thyronine (thyroxine, T4)

and 3¢,5,3-triiodo-[L]-thyronine (T3) THs are

synthe-sized by the thyroid gland and then secreted into the

bloodstream (see Fig 1) In mammals, most of the TH

produced by the thyroid gland is in the form of T4,

which has higher affinity than T3 for the TH

distribu-tor proteins (THDPs) in the blood [1] However, T3

has higher affinity than T4 for the thyroid hormone

receptors (TRs) [2] More than 99% of TH in blood is

bound to THDPs, which prevent avid nonspecific

par-titioning of THs into membranes THs dissociate from

THDPs and can enter cells via TH transporters or by

passive diffusion as a result of their lipophilicity THs can be deiodinated by a family of deiodinases to either activate (T4–T3) or deactivate [T4–rT3 (reverse T3), T3–T2, etc.] THs [3] Within cells, THs bind to specific cytosolic TH-binding proteins before being

translocat-ed into the nucleus THs elicit their effects by binding

to TR⁄ RXR dimers in the nucleus, and together with co-activator or co-repressor proteins, directly modulate the expression of specific genes (see Fig 1)

Many genes regulated by THs are involved in growth and development, particularly of the brain [4] Thus, normal growth and development requires tightly regulated levels of THs to reach the nucleus of cells throughout the body and brain, and a strong network

of buffering and regulatory feedback systems in order

Keywords

amphibians; birds; brain; choroid plexus;

eutherians; evolution; fish; gene regulation;

liver; marsupials; monotremes; reptiles;

thyroid hormones; transthyretin; vertebrates

Correspondence

S J Richardson, School of Medical

Sciences, RMIT University, PO Box 71,

Bundoora, Vic 3083, Australia

Fax: +61 3 9925 7063

Tel: +61 3 9925 7897

E-mail: samantha.richardson@rmit.edu.au

(Received 2 February 2009, revised 11 June

2009, accepted 12 June 2009)

doi:10.1111/j.1742-4658.2009.07244.x

Thyroid hormones are involved in growth and development, particularly of the brain Thus, it is imperative that these hormones get from their site of synthesis to their sites of action throughout the body and the brain This role is fulfilled by thyroid hormone distributor proteins Of particular inter-est is transthyretin, which in mammals is synthesized in the liver, choroid plexus, meninges, retinal and ciliary pigment epithelia, visceral yolk sac, placenta, pancreas and intestines, whereas the other thyroid hormone distributor proteins are synthesized only in the liver Transthyretin is syn-thesized by all classes of vertebrates; however, the tissue specificity of trans-thyretin gene expression varies widely between classes This review summarizes what is currently known about the evolution of transthyretin synthesis in vertebrates and presents hypotheses regarding tissue-specific synthesis of transthyretin in each vertebrate class

Abbreviations

ApoAI, apolipoprotein AI; CSF, cerebrospinal fluid; LAMP-1, lysosome-associated membrane protein; RBP, retinol-binding protein; T3, 3¢,3,5-triiodo-[L]-thyronine; T4, 3¢,5¢,3,5,-tetraiodo-[L]-thyronine; TBG, thyroxine-binding globulin; TBPA, thyroxine-binding prealbumin; TH, thyroid hormone; THDP, thyroid hormone distributor protein; TLP, transthyretin-like protein; TRE, thyroid hormone response elements.

to maintain euthyroid homeostasis For example,

insufficient TH during gestation in humans leads to

irreversible brain damage and mental retardation

Many hormones affect neurogenesis in the adult brain

[5] In rodents, THs are required for normal cycling of

adult neural stem cells in the subventricular zone [6]

A dramatic example of the effect of THs on

develop-ment is the metamorphosis of tadpoles into frogs: the

animal changes from an aquatic herbivore (with a long

intestine) with gills and a tail, to a terrestrial

carnivo-rous (with a short intestine) tetrapod with lungs This

remarkable transition requires a finely regulated

co-ordination of gene-transcription events directing

apoptosis, resorption and tissue remodelling, which is

driven by THs [7] This illustrates the importance of

the quantitative, temporal and spatial requirements of

TH distribution during development

Often, the focus of TH-regulated events is on the

interaction of the THs with their receptors,

co-modula-tors and the thyroid hormone response elements

(TREs) in the target genes However, this is just the

final step in a long chain of events that have been

quantitatively regulated at each step The movement of

THs from the thyroid gland to a target cell is governed

by the THDPs in the blood and cerebrospinal fluid

(CSF) In humans (but not in all vertebrates or even in

all mammals), the THDPs in blood are albumin,

trans-thyretin and thyroxine-binding globulin (TBG) These

three proteins are synthesized by the liver and secreted

into the bloodstream Transthyretin has intermediate

affinity for THs, between those for albumin (lower

affinity) and TBG (higher affinity) Together, they

form a buffering network system for TH distribution

in the blood [8] The brain is separated from the rest

of the body by a set of interfaces often referred to as

‘the blood–brain barrier’, which actually consists of four barrier interfaces [9] Only one THDP is made in the brain, namely transthyretin Transthyretin is syn-thesized by the epithelial cells of the choroid plexus [10], which is the blood–CSF barrier and produces most of the CSF This transthyretin is secreted exclu-sively into the CSF and is involved in the transport of THs from the blood into the brain and throughout the CSF [11] This review will address the evolution of transthyretin synthesis in vertebrates, specifically: the sites of transthyretin synthesis; the evolution of tissue-specific transthyretin synthesis in fish, amphibians, rep-tiles, birds, monotremes, marsupials and eutherians; the regulation of transthyretin gene expression; and the change of transthyretin ligand in mammals

Transthyretin

Transthyretin was discovered in 1942 in both human CSF [12,13] and human serum [14] It was originally named ‘prealbumin’ because it was the only plasma protein that migrated anodal to albumin during elec-trophoresis Transthyretin has a molecular mass of about 55 kDa and is composed of four identical subunits of about 14 kDa It was not until Ingbar used

a Tris–malate buffer (rather than the then standard barbital buffer) for the electrophoretic analysis of serum that prealbumin was identified as a thyroid hor-mone-binding protein [15] (barbital inhibits binding of THs to transthyretin) Thus, the name was changed to

‘thyroxine-binding prealbumin’ (TBPA) A decade

Fig 1 Five classes of TH-binding proteins.

The thyroid gland secreted TH

(predomi-nantly T4 in mammals) into the blood,

where it binds THDPs (1) TH can dissociate

from THDPs and enter cells by passive

diffusion, or via TH transporter proteins (2).

Within the cell, THs can be deiodinated by

deiodinases (3) and bind cytosolic

TH-binding proteins (4) Within the nucleus,

T3 binds TH receptors (TRs) (5) NB:

deiodinases D1, D2 and D3 have different

locations with a cell; TRs change their

conformation upon binding to DNA ,

albumin; , transthyretin (TTR); , TBG;

, TH transporter; , deiodinase; ,

cytosolic TH-binding protein; , TR; , TR

bound to DNA ([18] Used with permission.)

later, Raz and Goodman [16] discovered that TBPA

also bound retinol-binding protein (RBP) In 1981 the

name was finally changed again to ‘transthyretin’,

which describes its roles in the TRANSport of

THY-roid hormones and RETINol-binding protein [17] For

details of the structure of transthyretin, see the review

in this series by Dr Hennebry

Transthyretin synthesis has been identified in the

liver, in the choroid plexus of the brain, and in the

meninges, retinal and ciliary pigment epithelia, visceral

yolk sac, placenta, pancreas and intestine (see below),

whereas albumin synthesis and TBG synthesis have

only been identified in the liver

Ligands of transthyretin

To assess the selection pressures governing the

regulation of tissue-specific transthyretin synthesis,

the functions of transthyretin must be considered

Transthyretin has multiple ligands that can be divided

into two categories: ‘natural’ and ‘synthetic’ The

natu-ral ligands of transthyretin include: thyroid hormones

(T3 and T4) and RBP, which itself binds retinol, metal

ions, plant flavonoids, apolipoprotein AI (ApoAI) and

lysosome-associated membrane protein (LAMP-1) The

synthetic ligands include nonsteroidal

anti-inflamma-tory drugs, polychlorinated biphenols, industrial

pollu-tants and flame retardants [18] As these synthetic

compounds can displace THs from transthyretin, they

can act as potent endocrine disruptors Furthermore,

these endocrine disruptors can be transported into the

brain via binding to transthyretin synthesized by the

choroid plexus and have the potential to accumulate in

the brain However, as this review is focused on the

evolution of transthyretin synthesis, only the natural

ligands of transthyretin will be discussed For reviews

on non-TH ligands of transthyretin, readers are

direc-ted to excellent reviews published previously [19–24]

TH

In human blood, 99.97% of T4 and 99.70% of T3 is

bound to the THDPs albumin, transthyretin and TBG

[25] Of these, TBG has the highest affinity for T4 and

T3 (1.0· 1010 and 4.6· 108m)1, respectively),

trans-thyretin has intermediate affinity (7.0· 107 and

1.4· 107m)1, respectively) and albumin has the lowest

affinity (7.0· 105 and 1.0· 105m)1, respectively)

Together, these three THDPs form a buffering

net-work for free T4 in blood (24 pm), which could assist

in protection against hypothyroidism (abnormally low

levels of free TH in blood) or hyperthyroidism

(abnor-mally high levels of free TH in blood) [8]

The function of THDPs is to ensure an even dis-tribution of TH throughout tissues and to maintain

a circulating TH pool of sufficient size in the blood and CSF [26] To determine which of the three THDPs contributes most effectively to the delivery

of THs to tissues, the dissociation rates and the cap-illary transit times have to be considered In brief, the dissociation rates for T4 and T3 from TBG are 0.018 and 0.16 s)1, respectively; from transthyretin are 0.094 and 0.69 s)1, respectively; and from albu-min are 1.3 and 2.2 s)1, respectively [27] Thus, given the capillary transit times for various tissues [28], transthyretin is responsible for much of the immedi-ate delivery of THs to tissues [29] An analogy by Ingbar describes it quite nicely: ‘TBG is the savings account for thyroxine and TBPA is the checking account’ [30]

In mammals, transthyretin, albumin and TBG have higher affinity for T4 than for T3 (see above), and, as the concentrations of both free and total T4 are higher than those of T3, T4 is often referred to as the ‘trans-port form’ of TH As T3 has higher affinity than T4 for the TH nuclear receptors [2], T3 is often referred

to as the ‘active form’ of TH However, in birds, rep-tiles, amphibians and fish, transthyretin has a higher affinity for T3 than for T4 (see review in this series by

Dr Prapunpoj) and these animals do not have TBG in their blood Therefore, these animals could have a potentially greater ratio of T3 to T4 in their blood than mammals By contrast, in mammals, transthyretin and TBG distribute T4 (the precursor form) around the blood rather than T3 (the ‘active’ form), which binds to the nuclear receptors This allows for tissue-specific activation of T4–T3 by deiodinases, at the pre-cise sites where T3 is required, giving a greater level of control of TH action in mammals This could be a selection pressure for the change in ligand binding of transthyretin from T3 (in fish, amphibians, reptiles and birds) to T4 (in mammals)

RBP RBP was first described by Kanai et al., in 1968 [31], and was found to be bound to transthyretin in serum

It was suggested that the transthyretin–RBP⁄ retinol complex (80 kDa) or the retinol ⁄ RBP–transthyretin– RBP⁄ retinol complex (100 kDa) prevented loss of RBP–retinol (21 kDa) via glomerular filtration in the kidneys [16] The RBP–retinol complex has higher affinity for transthyretin than apoRBP [32] The X-ray crystal structures of RBP–transthyretin complexes have demonstrated that up to two molecules of RBP can bind one tetramer of transthyretin [33]

The hypothesis that RBP binds to transthyretin to

prevent loss of RBP and retinol by filtration in the

kid-neys may hold true for eutherians (‘placental

mam-mals’), but it is not immediately convincing when

considering other animals For example, there are two

Orders of marsupials: the Diprotodonta (e.g

kanga-roos, koalas and wombats) and the Polyprotodonta

(e.g Tasmanian devil, dunnarts and Antechinus) Adult

Diprotodonta have transthyretin in their blood, whereas

adult Polyprotodonta do not have transthyretin in their

blood [34] This raises the question as to whether there

is a difference in the glomerular filtration size cut-off in

diprotodont marsupials compared with that of

polypro-todont marsupials Similarly, all of the species of

sexu-ally mature fish, amphibians, reptiles and monotremes

studied have RBP in their blood, but not transthyretin

This raises questions as to whether the glomerular

filtra-tion cut-off is significantly smaller in noneutherians, or

if a plasma protein other than transthyretin fulfills the

role of binding RBP to prevent its loss via the kidneys

If the function of transthyretin was to prevent loss of

RBP–retinol through the kidneys, one might speculate

that hepatic transthyretin synthesis would have

co-evolved with hepatic RBP synthesis and that genes

for both transthyretin and RBP would have similar

developmental and evolutionary expression patterns

Metal ions, plant flavonoids, ApoAI and LAMP-I

The vast majority of data on transthyretin binding to

metal ions [35], plant flavonoids [20], ApoAI [36] and

LAMP-I [37] pertain to eutherian transthyretins

Therefore, this data set is not broad enough to build

hypotheses regarding selection pressures leading to the

binding of these compounds by transthyretins during

evolution Thus, it is not yet possible to produce a

sec-tion on the influence of these ligands on the evolusec-tion

of transthyretin synthesis

Sites of transthyretin synthesis

Liver

Transthyretin is synthesized by the liver and secreted

into the blood [38], where it binds THs and RBP⁄

reti-nol However, transthyretin–RBP⁄ retinol can also be

secreted from the liver as a complex [39] Thus, hepatic

transthyretin is involved in the distribution of THs

and retinol throughout the body via the blood The

protein-bound pool of THs is believed to counteract

the avid partitioning of the lipophilic THs into the

lipid membranes and to maintain a circulating pool of

THs in the bloodstream [26] Very recently, it has been

revealed that transthyretin is also involved in periph-eral nerve regeneration [40]

Choroid plexus Transthyretin is synthesized by the choroid plexus epi-thelial cells and secreted into the CSF [10] At least in rodents, this transthyretin is involved in the movement

of T4 (but not of T3) from the blood into and within the brain, as previously reviewed [18] In addition, transthy-retin synthesized by the choroid plexus and secreted into the CSF and interstitial fluid is involved in the delivery

of TH to stem cells and progenitor cells within the sub-ventricular zone of the brain [41], which requires TH for cell cycle regulation [6] The absence of transthyretin synthesized by the choroid plexus results in reduced apoptosis of progenitor cells in the subventricular zone

of the adult mouse brain [41], spatial reference memory impairment [42], increased exploratory activity and reduced depressive behaviour [43], and overexpression

of the neuropeptide Y phenotype [44] Reduced levels of transthyretin have been reported in the CSF of patients suffering from depression, Alzheimer’s disease and Down’s syndrome [18] In the light of reports of decreased transthyretin synthesis and secretion in the brains of ageing mammals [45], the role of transthyretin

in the ageing brain requires further investigation

Visceral yolk sac Transthyretin and RBP synthesized in the visceral yolk sac of rodents has been suggested to be involved in the transport of THs and retinol from the maternal circu-lation to the developing fetus [46,47] Further support for this came from a previous publication [48] in which

it was demonstrated that both transthyretin and RBP are secreted across the basolateral membrane towards the fetal circulation; the report also suggested that the visceral yolk sac could be the source of plasma pro-teins for the fetus before the fetal liver is functional

Placenta Transthyretin synthesis by the eutherian placenta has been suggested indirectly [49] and more recently dem-onstrated directly [50], where it has been proposed to

be involved with the transfer of THs from the mother

to the fetus

Retinal and ciliary pigment epithelia of the eye Transthyretin is synthesized by the retinal pigment epithelium of the eye in several eutherian species [51]

and is secreted across the apical membrane into the

extracellular matrix, together with RBP that is also

synthesized by the retinal pigment epithelium [52]

Transthyretin and RBP synthesized by the retinal

pig-ment epithelium have been proposed to be involved in

the delivery of retinol to Mu¨ller and amacrine cells

[52], where it is converted to retinal, which is required

for photoreceptor function More recently,

transthyre-tin synthesis by the ciliary pigment epithelium was

identified, at about one-third of the levels found in the

retinal pigment epithelium [53]

Intestine

Transthyretin synthesis has been identified in human

intestines during fetal development [54], but not in the

intestine of adult rats [55] A function for transthyretin

synthesized by the intestine has not yet been defined

However, as the intestines are extrahepatic tissue with

the highest concentration of THs [56], a role for TH

distribution or transport seems likely

Pancreas

Transthyretin synthesis in the islets of Langerhans of

rat pancreas has previously been described [57]

Recently, a role for transthyretin in promoting

glu-cose-induced increases in cytoplasmic calcium ion

concentration and insulin release in pancreatic beta

cells has been proposed [58] A role for the

transthy-retin tetramer in protection against beta cell apoptosis

was also proposed, having implications for type 1

diabetes in humans

Other tissues

A single observation of extremely low levels of

trans-thyretin synthesis by the meninges in rat brain has

been reported [59] Transthyretin synthesis (detected by

PCR) has also been identified in the skin, heart,

skele-tal muscle, kidney, testis, gills and pituitary in a species

of adult fish (sea bream, Sparus aurata) [60] Functions

for transthyretin synthesized in these tissues have not

yet been identified

Sites of transthyretin synthesis

throughout vertebrate evolution

Fish

Among teleost fish, transthyretin synthesis in the whole

animal has been reported during early embryogenesis

in sea bream (S aurata) [60] Masu salmon

(Oncorhyn-chus massou) synthesize transthyretin in their liver only during smoltification (a process driven by THs) [61], and subsequently Atlantic salmon (Salmo salar) and Chinook salmon (Oncorhynchus tshawytscha) were reported to undergo hepatic transthyretin synthesis only during smoltification [62] Hepatic transthyretin synthesis was also detected in 3-year-old tuna (Thun-nus orientalis) [63]

A comprehensive survey of tissues in adult sea bream revealed a wide distribution of transthyretin transcripts after PCR analysis (which is a more sensi-tive method than those used in other studies refer-enced) in liver, intestine, whole brain, kidney, testis, gills and pituitary However, only the signal in the liver could be confirmed by northern blotting analysis [60] Until now, there have been no published data on transthyretin synthesis by the choroid plexus of teleost fish There is an unpublished report that fish choroid plexus does not synthesize transthyretin (G Schreiber, personal communication); however, using PCR, Santos and Power [60] amplified transthyretin transcript from the whole brain of adult sea bream, which presumably contains the choroid plexus Whether this transthyretin was synthesized by the choroid plexus remains to be investigated

Of the agnathan fish, two species (from two different genera) of lamprey have been studied [64] Transthyre-tin cDNAs were cloned and sequenced from Petromy-zon marinus and Lampetra appendix These are the first transthyretin sequences from vertebrates basal to tele-ost fish The N-terminal regions of transthyretin subunits from both species were longer than those from other vertebrates Transthyretin was found to be syn-thesized in the liver of lampreys throughout their life cycles and the synthesis of transthyretin was elevated during metamorphosis In other vertebrates, a transient increase in transthyretin⁄ THDP coincides with the increase in TH levels during development (mammals), metamorphosis (amphibians) or smoltification (fish) [62] These processes are (at least in part) driven by THs However, in these two species of lampreys, the increasein transthyretin gene expression coincides with

a decrease in plasma TH levels [64] The Agnatha are

at least 530 Myr old, and the function of THs in lam-preys appears to be different from that in most other vertebrates, as a decrease in TH triggers metamorpho-sis, rather than an increase in TH concentrations [65] Accordingly, lamprey metamorphosis can also be induced by goitrogens [66] It is intriguing that in amphibians an increase in hepatic transthyretin gene expression coincides with an increase in TH concentra-tion in the blood, which drives metamorphosis, whereas

in lampreys an increase in hepatic transthyretin gene

expression is concurrent with a decrease in plasma TH

concentration, which drives metamorphosis

It appears that fish have a wider variety of patterns

of hepatic transthyretin synthesis compared with other

classes of vertebrates These patterns include: hepatic

transthyretin synthesis only during times of increased

TH levels in serum; hepatic transthyretin synthesis

throughout the life cycle; or hepatic transthyretin

syn-thesis throughout the life cycle but an increase during

times of decreased TH levels in serum Fish comprise

an extremely diverse class of vertebrates, including

sev-eral highly derived lineages, which could explain the

diversity of hepatic transthyretin synthesis patterns

The evolutionary structural precursor to

transthyre-tin is the transthyretransthyre-tin-like protein (TLP) (see the

review in this series by Dr Hennebry) TLPs have been

identified in all Kingdoms, but transthyretins have

only been identified in the Phylum Chordata [67] The

transthyretin gene probably arose as a duplication of

the TLP gene around the stage of divergence of the

echinoderms (see the review by Dr Hennebry) TLPs

do not bind THs, and at least some are involved in

uric acid degradation [68] Of the vertebrate

trans-thyretins, lamprey transthyretins are most closely

related to TLPs

Amphibians

There are three Orders within the Class Amphibia:

Anura (frogs and toads), Urodela (newts and

salaman-ders) and Gymnophiona (caecilians) Of these, only a

few species of Anura have been investigated regarding

transthyretin synthesis

For the amphibian species studied thus far, hepatic

transthyretin synthesis occurs around the time of

meta-morphosis, which is driven by increased TH levels in

plasma In Rana catesbieana, hepatic transthyretin

syn-thesis was only detected just before the climax of

meta-morphosis [69,70], whereas in Xenopus laevis, hepatic

transthyretin gene expression occurs only during

meta-morphosis [71]

Transthyretin is not synthesized in the choroid

plexus of adult or metamorphosing frogs

(Limno-dynastes dumerili), cane toads (Bufo marinus) [72],

in X laevis tadpole brain [71], or in R catesbeiana

tadpole choroid plexus [70]

Reptiles

There are four Orders of extant reptiles: Squamata

(liz-ards and snakes), Chelonia (turtles and tortoises),

Crocodilia (crocodiles, alligators and caimans) and

Rhynchocephalia (the tuatara)

Transthyretin was not detected in the blood of adult tuatara (Sphenodon punctatus), Kreft’s tortoise (Emy-dura kreftii), saltwater crocodile (Crocodylus porosus), stumpy-tailed lizard (Tiliqua rugosa), garden skink (Lampropholis guichenoti), or bearded dragon (Amphib-olurus barbatus) [34] Transthyretin synthesis has only been detected in reptilian liver during development [62] (see the review by Dr Yamauchi in this miniseries) All four species of reptiles that were investigated – stumpy-tailed lizards (T rugosa) [73], the red-eared sli-der turtle (Trachemys scripta), the common snapping turtle (Chelydra serpentine) [74] and the salt-water crocodile (C porosus) [75] – were found to synthesize transthyretin in their choroid plexus

Transthyretin mRNA was detected in the eyes of 1-year-old salt-water crocodiles (C porosus), but not in the liver or heart [75] Transthyretin mRNA was not detected in the liver, eye, brain (excluding choroid plexus), heart or kidney of adult stumpy-tailed lizards (T rugosa) [73]

Birds Transthyretin synthesis was detected in both the cho-roid plexus and the liver of chickens (Gallus gallus), pigeons (Columba livia), quails (Coturnix japonica) and ducks (Anas platyrhynchos) at all ages investigated from hatching until adult [76] Transthyretin is also synthesized in the liver of adult geese (Anser anser) [34], zebra finch (Taeniopygia guttata), budgerigar (Melopsittacus undulatus), peafowl (Pavo cristatus) and penguin (Eudyptula minor novaehollandiae) (S Rich-ardson, unpublished observations)

Adult chickens (G gallus) were studied in further detail, with transthyretin mRNA detected in RNA extracts from liver, choroid plexus and eye, but not detected in lung, brain (without choroid plexus), heart, spleen, intestine, kidney or skeletal muscle [77]

The group of extant birds that are believed to have branched earliest from the common lineage with reptiles are the ratites These include the emu, cassowary, ostrich and rhea Transthyretin was detected in the serum from adult emu (Dromaius novahollandiae), ostrich (Stru-thio camelus) [78] and rhea (Rhea americana), and also from ostrich chicks (S Richardson, unpublished obser-vations) This suggests that as soon as the avian lineage diverged from the reptilian lineage, the transthyretin gene was expressed in the liver of adult animals

Monotremes Unfortunately, there are no large breeding colonies of monotremes, which renders animals available for

investigation as scarce Hence, only adult animals have

been investigated thus far Transthyretin was not

detected in the serum of adult echidnas

(Tachyglos-sus aculeatus), either during hibernation or during

arousal, or from platypus (Ornithorhynchus anatinus)

[34] or zaglossus (Zaglossus bruijni) (S Richardson,

unpublished observation) However, transthyretin was

found to be synthesized by the choroid plexus of the

only monotreme investigated: the echidna [34]

Marsupials

Australian marsupials can be divided into two Orders:

the evolutionarily older Polyprotodonta (e.g

Tasma-nian devil, dunnart) and the younger Diprotodonta

(koala and kangaroo) Polyprotodonta are carnivores

and have many teeth on their upper and lower jaws

that are suitable for tearing and chewing flesh, whereas

Diprotodonta are herbivores and have two large teeth

on their upper and lower jaws that are suitable for

grazing Concordant with their diets, Polyprotodonta

have relatively short digestive tracts, whereas

Dip-rotodonta have longer digestive tracts (These points

will be referred to later in the review.)

Australian polyprotodont marsupials synthesize

transthyretin in their livers only during development

[62] (see the review by Dr Yamauchi in this miniseries)

and not as adults [34,79,80], whereas transthyretin was

synthesized by the choroid plexus of all ages of

marsu-pials investigated [34,79] By contrast, diprotodont

marsupials synthesize transthyretin in their liver and

choroid plexus throughout life [34,79]

All American marsupials are polyprotodont and are

believed to be closer to the ancestral marsupial than

the Australian marsupials Synthesis of transthyretin

by the choroid plexus has only been studied in one

American marsupial species: the short-tailed grey

opos-sum (Monodelphis domestica) Synthesis of

transthyre-tin by the choroid plexus was detected during

development from the day of birth [81] and in the

adult [79]

In 1973, Davis and Jurgelski reported that 177

Virginia opossums (Didelphis virginiana) did not have

transthyretin in their serum [82] However, a more

recent study has shown that M domestica, D

virgini-ana, Caluromys lanatus (woolly opossum) and

Dromici-ops australis (monito del monte) do have transthyretin

in their blood [83] By contrast, transthyretin was not

detected in serum from Marmosa sp., Metachirus sp.,

Chironectes sp or Philander sp However, positive

con-trols were not available for these latter species, so these

results are inconclusive as they could be false negatives

[83] Similarly to the situation in eutherians,

transthy-retin is a negative acute-phase plasma protein in mar-supials (see below), and thus individuals that were not healthy or were stressed at the time of blood or liver collection could have yielded negative results because

of the acute-phase response This could also explain the absence of transthyretin in D virginiana serum, investigated by David and Jurgelski

For a summary of the evolutionary history and phylogenetic relationships of marsupials, see below

Eutherians Eutherians are the group of vertebrates in which trans-thyretin biology has been most intensively studied, in particular rodents and humans Rats were used for the bulk of basic research carried out on transthyretin, whereas humans have been investigated in detail for normal transthyretin physiology and in particular for transthyretin-related diseases, namely the transthyretin amyloidoses Furthermore, in the past 15 years a pleth-ora of genetically engineered mouse models for human transthyretin-related diseases have been created and investigated Perhaps some of the tissues synthesizing transthyretin in eutherians also synthesize transthyretin

in other species, but this has yet to be investigated Tissues in adult eutherians known to synthesize transthyretin include: liver, choroid plexus, visceral yolk sac, placenta, retinal and ciliary pigment epithelia, pancreas and meninges Functions for transthyretin synthesized by these tissues (where known) are described above

From the evolutionary perspective, a study investi-gating hepatic transthyretin synthesis in the eutherian Order Insectivora was carried out, as these animals are believed to be most similar to the common ancestors

of eutherians and marsupials Hepatic transthyretin synthesis was detected in each species studied: shrews (Sorex ornatus californicus and Sorex araneus), hedge-hogs (Erinaceus europaeus) and moles (Talpa euro-paea) This indicates that hepatic transthyretin gene expression in eutherians probably appeared before the diversification of eutherian lineages [84]

Negative acute-phase regulation of the transthyretin gene in the liver but not

in the choroid plexus

Transthyretin is a typical ‘negative acute-phase plasma protein’ (i.e following trauma, surgery, inflammation

or malnutrition, the transthyretin gene in the liver is down-regulated and consequently the transthyretin concentration in the blood decreases) [85] This is also the case for the albumin gene As there is only one

transthyretin gene per haploid genome in rats (and

now known to be the situation for several other

spe-cies), the question arose as to whether the transthyretin

gene was also under negative acute-phase regulation in

the choroid plexus Intriguingly, the transthyretin gene

in the choroid plexus was not under negative

acute-phase regulation (i.e the transthyretin gene is

regu-lated independently in the liver and in the choroid

plexus) [86]

As transthyretin synthesis is involved in

transport-ing THs into the brain, as the brain is dependent on

THs for normal development, as the developing and

adult brain are sensitive to the effects of THs and as

hepatic albumin and transthyretin are negative

acute-phase plasma proteins (resulting in a reduction of

total circulating TH in blood during the acute phase),

it was proposed that when the body is experiencing

trauma or inflammation, normal rates of transthyretin

gene transcription in the choroid plexus would ensure

that the brain would be protected against

hypothy-roidism [86]

As some marsupials synthesize transthyretin in

their liver, an investigation into whether hepatic

transthyretin gene regulation was also under negative

acute-phase regulation in marsupials was carried out

Following either brain surgery or injection of

lipo-polysaccharide, hepatic transthyretin synthesis was

down-regulated in M domestica, a South American

opossum [87] As the common ancestor of eutherians

and marsupials is presumably more closely related to

American marsupials than to Australian marsupials

or to eutherians, this suggests that (at least in

mam-mals) as soon as transthyretin is synthesized in the

liver, its gene is under negative acute-phase

regula-tion [87]

A summary of the data from Costa and colleagues

on the transcription factors governing tissue-specific

regulation of transthyretin gene transcription in rats has been previously published [18]

Transthyretin gene regulation during evolution

In this section, only adult animals are considered (for regulation of the transthyretin gene during develop-ment in various classes of vertebrates, see the minire-view in this series by Dr Yamauchi) The choroid plexus and liver have been investigated for transthyre-tin synthesis in all classes of adult vertebrates Trans-thyretin synthesis by other tissues has not been studied

as thoroughly (usually only in eutherians or fish), hence there are insufficient data to make generaliza-tions about the evolution of transthyretin synthesis in tissues other than the choroid plexus and the liver

Liver For a comprehensive analysis, serum from adult indi-viduals from about 150 species was analysed for the presence of THDPs All species studied were found to have albumin, and in some species (e.g fish, amphibi-ans, reptiles and some mammals) albumin was the only THDP [34] Therefore, it was concluded that albumin is the phylogenetically oldest THDP in adult vertebrates Birds and eutherians had transthyretin in addition

to albumin, and an interesting situation became appar-ent amongst the Australian marsupials: some had albu-min as their only THDP, and others had transthyretin

in addition to albumin Those that had transthyretin

in serum belonged to the Order Diprotodonta, whereas those that did not have transthyretin in their serum belonged to the Order Polyprotodonta [34,80] (for an evolutionary tree based on the fossil record, see Fig 2) TBG-like proteins were detected in serum from

Fig 2 Evolutionary ⁄ developmental tree for

transthyretin synthesis in the choroid plexus

and liver of vertebrates Evolutionary tree

showing approximate divergence times for

vertebrate groups, based on the fossil

record Superimposed are symbols

indicat-ing the onset of transthyretin synthesis in

vertebrates ++, onset of transthyretin

syn-thesis in the choroid plexus, in juveniles and

in adults of extant species; LD, hepatic

transthyretin synthesis during development

only; ?LD, possible onset of hepatic

trans-thyretin synthesis during development only;

+, hepatic transthyretin synthesis during

development and in adult MYA, million

years ago ([62] Used with permission.)

various mammalian species, but no clear phylogeny

was apparent

Diprotodont marsupials have two large teeth on

their upper and lower jaws and are herbivores (e.g

kangaroos and wombats), whereas polyprotodont

marsupials have many teeth on their upper and lower

jaws and are carnivores (e.g Tasmanian devils and

dunnarts)

According to the fossil record, marsupials originated

in the region of Laurasia, which is now North America,

and were polyprotodont [88] From there, they

migrated to what is now South America (for a

sche-matic diagram of the positions of these continents

about 150 Ma, see Fig 3) and those in the northern

region died out From South America, some marsupials

migrated back to (what is now) North America and

others migrated across Gondwanaland About 45 Ma,

Gondwanaland began to break up into South America,

Antarctica and Australia [89] There are fossils of

mar-supials in Antarctica (e.g Seymour island) [90], and

many marsupials were isolated on the Australian

conti-nent Shortly after the separation of Gondwanaland,

there was a radiation of marsupials in Australia, which

included the divergence of diprotodont marsupials from

polyprotodont marsupials [88] (See Figs 2 and 3.) It

was previously suggested that in marsupials, the

trans-thyretin gene was turned on in the liver when the

‘younger’ Diprotodonta had diverged from the ‘older’

Polyprotodonta [34,83], whereas transthyretin was syn-thesized in the liver as soon as the avian and eutherian lineages evolved [34,78,84]

The digestive tracts of herbivorous marsupials (diprotodont) are longer than those of carnivorous marsupials (polyprotodont) [91] The intestines are the extrathyroidal tissue with the highest TH content [56], and it has been suggested that the THDPs may be responsible for the regulation of delivery of THs into the intestines [92] It was previously proposed that the increase in lipid pool (e.g length of intestine) was a selection pressure for ‘turning on’ adult hepatic trans-thyretin gene expression It was argued that as the transthyretin gene was already being expressed in the choroid plexus of all reptiles, birds and mammals, the onset of adult hepatic transthyretin gene expression would have simply required a change in distribution of transcription factors [8,34,93]

However, more recent data on hepatic transthyretin synthesis during development [61,62,69–71], revealed that all species studied had hepatic transthyretin syn-thesis at some stage during development, often coincid-ing with an increase in serum TH concentrations In some species, hepatic transthyretin synthesis continued into adult life, whereas in other species the gene was turned off during late stages of development This led

to a re-evaluation of the data and hypotheses regard-ing selection pressures for what was previously described as the ‘onset of adult hepatic transthyretin synthesis’, which should now be viewed as selection pressure for ‘maintaining hepatic transthyretin synthe-sis throughout life’ In light of this, the revised hypoth-eses for selection pressures for maintaining hepatic transthyretin synthesis throughout life are as follows Hypothesis 1 Maintaining hepatic transthyretin gene expression in adulthood is related to the increase in lipid pool to body mass ratio A study by Hulbert and Else [94] compared many physiological parameters of rep-tiles (which do not have transthyretin in their blood) and eutherians (which do have transthyretin in their blood) of similar body mass Amongst other data, they showed that internal organs were larger in adult euthe-rians, which therefore had larger lipid pools and conse-quently a greater lipid volume to body mass ratio, than reptiles of a similar body weight As THs are lipophilic and preferentially partition into the lipid phase rather than the aqueous phase [95,96], the increase in the relative size of the lipid pool could have been a selection pressure for maintaining hepatic trans-thyretin synthesis during adult life As transtrans-thyretin has higher affinity than albumin for THs, the presence

of transthyretin in the blood would contribute to ensuring a circulating pool of THs, thereby

counteract-Fig 3 Marsupial migration in relation to the movement of tectonic

plates The positions of the land masses currently known as North

America (N.A.), South America (S.A.), Africa (Afr), Antarctica (Ant),

India (Ind) and Australia (Aus) about 150 Ma Arrows indicate the

directions of three major marsupial migrations over about 100 Myr:

1., from North America to South America; 2., from South America

to North America and via Antarctica to Australia; 3., extensive

radia-tion of marsupials within Australia (Data from [88–90] Figure from

[18], used with permission.)

ing the increased sink (lipid pool) for TH to

poten-tially partition into Another example is comparison of

adult Australian marsupials Diprotodont marsupials

(with longer intestines) have hepatic transthyretin

syn-thesis, whereas adult polyprotodont marsupials (with

shorter intestines) do not (intestines are the

extrahe-patic tissue with the highest concentration of TH) [56]

Hypothesis 2 Maintaining hepatic transthyretin

syn-thesis in adulthood is related to homeothermy

Transthy-retin was found in serum from all studied species of

birds and eutherians, which are known homeotherms

(i.e they maintain their body temperature at or near

37 C by metabolic means) However, transthyretin

was not detected in serum from adult fish, amphibians,

or reptiles (including members from all four extant

Orders: Crocodilia, Squamata, Chelonia and

Rhyncho-cephalia), which are ectotherms (and in whom body

temperature is determined by a combination of

behav-iour and the environment) [8] Marsupials and

mono-tremes are ‘poor endotherms’ (i.e their body

temperatures are 25–32 C, but when placed in cold

environments, cannot maintain their body

tempera-tures as well as ‘true endotherms’) [97] THs are

intri-cately involved with the control of basal metabolic

rate, oxygen consumption and homeothermy The

basal metabolic rates for monotremes, marsupials

and eutherians are approximately 140, 200 and

290 kJÆkg)0.75, respectively [97] A selection pressure

for maintaining hepatic transthyretin synthesis

through-out life could have been to enable the appropriate

distribution of THs throughout the body to maintain

homeothermy

Choroid plexus

Transthyretin is the major protein synthesized and

secreted by the choroid plexus of reptiles, birds,

mono-tremes, marsupials and eutherians, but is not

synthe-sized by the choroid plexus of amphibians [8] or fish

(G Schreiber, unpublished observations) [However,

more recently, transthyretin mRNA has been detected

in whole-brain homogenates of some fish (see above)

It remains to be elucidated if this transthyretin gene

expression is in the choroid plexus] It appears that the

transthyretin gene in the choroid plexus was turned on

once, at the stage of the stem-reptiles (the closest

com-mon ancestor to reptiles, birds and mammals), but not

of amphibians and fish (see Fig 2) The early reptiles

were the first to develop traces of a cerebral neocortex

[98], thereby increasing their brain volume As THs are

lipophilic and readily partition into cell membranes,

the increase in brain size may have been the selection

pressure for ‘turning on’ the transthyretin gene in the

choroid plexus This resulted in transthyretin assisting movement of THs from the blood across the blood– CSF barrier into the brain, and also acting as a THDP

in the CSF [8]

Because hepatic transthyretin synthesis is present in all extant classes of vertebrates (including fish, amphib-ians and reptiles) during development, it is possible that the stem-reptiles had the transthyretin gene in their genomes, which may have been expressed in the liver during development, then a change in specificity

of transcription factors could have been all that was required to activate transthyretin synthesis in the cho-roid plexus

The major protein synthesized and secreted by the choroid plexus of juvenile and adult amphibians is the lipocalin prostaglandin D synthetase [72], also known

as beta-trace [99] and Cpl1 [100] Prostaglandin D syn-thetase is a monomeric 20 kDa protein that belongs to the lipocalin superfamily of proteins Lipocalins have a calyx (cup) structure and are specialized in binding small molecules This raises the question of whether this lipocalin was the evolutionary functional precursor

to transthyretin in the choroid plexus [This should not

be confused with TLP, which is probably the evolu-tionary structural precursor of transthyretin (see the review in this miniseries by Dr Hennebry)]

Implications of transthyretin evolving from distributing T3 to T4

It has been demonstrated that 100% of transthyretin synthesized by the choroid plexus is secreted into the CSF, and that none is secreted into the blood [11] In rats, this transthyretin was shown to transport 125I-T4 but not 125I-T3 from the blood across the blood–CSF barrier into the brain [96] However, if the transthyre-tin synthesized by the choroid plexus binds T3 with higher affinity than T4, as is presumably the case for birds and reptiles [78] (fish and amphibians do not syn-thesize transthyretin in the choroid plexus), the ques-tion then arises as to whether in birds and reptiles, T3 (rather than T4) is transported across the blood–CSF barrier into the brain This also raises questions about the evolution of deiodinases in the body, and in partic-ular in specific regions of the brain

The selection pressure leading to the change from transthyretin preferentially binding T3 to T4 could be from transporting the ‘active’ form of the hormone, to transporting a ‘precursor’ form of the hormone This would allow greater flexibility and specificity at the local tissue level to either activate the T4 by deiodinating it to T3, or to inactivate the T4 by deiodinating it to rT3 This could be especially true in the brain, as in the rat