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Departments of Physiology (V.A.G., E.T.W., E.A.S.G., C.-A.W., G.A., G.M.S.G., D.L.S.G.) and Medicine (D.L.S.G.), Dartmouth Medical School, Lebanon, New Hampshire 03756; and Department of Psychology and Brain Sciences (A.S.C.), Dartmouth College, Hanover, New Hampshire 03755
Address all correspondence and requests for reprints to: Valerie Anne Galton, Department of Physiology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, New Hampshire 03756. E-mail: Val.galton{at}dartmouth.edu.
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Introduction |
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The formation and degradation of T3 in tissues are dependent primarily on the activities of three selenodeiodinases that catalyze the selective removal of iodine from iodothyronines (2). The type 1 and type 2 deiodinases (D1 and D2) are activating enzymes that catalyze the 5'-deiodination (5'D) of T4 to T3. The type 3 deiodinase (D3) is an inactivating enzyme that catalyzes the 5-deiodination (5D) of both T4 and T3 to their relatively inactive derivatives, rT3 and 3,3'-diiodothyronine, respectively. The D1 can also inactivate TH by catalyzing the 5D of sulfated iodothyronine conjugates (3).
Although there is much indirect evidence that the deiodinases play a major role in determining the level of T3 in serum and extrathyroidal tissues (4, 5, 6), the exact physiological roles of the individual enzymes have not been clearly defined. Nevertheless, it is widely accepted that most of the T3 generated by the D1 in liver is exported to the plasma (7, 8), whereas the D2 generates T3 for local use in specific tissues such as pituitary, brown adipose tissue, and brain (2).
Using a D2-deficient mouse, the D2 knockout (D2KO) mouse, created in this laboratory by genetic techniques (9), the importance of the D2 in regulating the intracellular T3 level in pituitary, brown adipose tissue, and inner ear has been confirmed. Thus, D2KO mice have significant deficits in TSH regulation (9), thermogenesis (10), and auditory function (11), despite a normal circulating level of T3 (9).
Indirect evidence suggests that the D2 also plays an important role in the brain, especially during development; indeed, the level of D2 activity is elevated substantially in the rat brain during the neonatal period when much of the brain development occurs (12, 13). In addition, there are compelling data suggesting that D2 protects the thyroid status of the brain under conditions of TH deficiency. Thus, in adult hypothyroid rats, the T3 content of the brain was normalized by infusing T4 at a dose much lower than that needed to restore a normal serum T4, T3, and TSH profile (14).
In the present study, the D2KO mouse was employed to determine the role of the D2 in regulating the content and action of T3 in the developing mouse brain. As expected, the T3 content in several areas of the brain was significantly reduced. The effect of this reduction on neuronal and neurological function has been assessed.
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Tissue preparation
Neonatal mice were killed on P15. They were euthanized with CO2 and decapitated, and trunk blood was collected. The serum was aspirated after centrifugation and then stored at –20 C for subsequent RIA of T4 and T3. The brain was removed, and 20–50 mg cerebellum (Cb), cerebral cortex (CCx), hypothalamus, and midbrain (the area left when the other three areas have been removed) were weighed and homogenized immediately in 1 ml ice-cold 95% methanol containing 10–4 M propylthiouracil (PTU). The homogenate was transferred to a 2-ml microfuge tube and agitated for 10 min. After centrifuging for 10 min at 12,000 x g, the supernatant was transferred to a fresh microfuge tube. The pellet was extracted twice with 0.25 µl of the methanol solution, and the combined supernatants were evaporated to dryness in a Rotovac apparatus. The residue was stored at –20 C for subsequent determination of the T4 and T3 contents by RIA.
For determination of deiodinase activities, cerebrum (brain minus Cb, pons, and medulla), or pieces of Cb, CCx, and midbrain and hypothalamus were homogenized in 0.25 mM sucrose, 20 mM Tris-HCl (pH 7.6) containing 5 mM dithiothreitol (DTT) as previously described (15) to yield approximately a 1:5 homogenate (wt/vol). The homogenates were centrifuged at 1000 x g for 15 min and the supernatants stored at –20 C for subsequent assay of 5'D and 5D activities.
Total RNA was isolated from pieces of Cb, CCx, and midbrain using a commercial RNA isolation reagent (TRIzol solution; Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.
Assays for T4 and T3 in serum and tissue samples
Total T4 and T3 concentrations in serum were determined using the Coat-A-Count RIA total T4 and total T3 kits (Diagnostic Systems Laboratories, Inc., Webster, TX). Tests with TH-depleted mouse serum (16) indicated that there was no nonspecific effect of mouse serum in the T4 assay, and thus the assays were performed according to the manufacturer’s instructions. The minimal detectable concentration of T4 in the assay was 0.25 µg/100ml.
Because there was a small but significant nonspecific effect of mouse serum in the T3 RIA, the protocol was adjusted as follows. For the standard curve, instead of using 100 µl of the zero calibrator (no T3) and T3 standards as called for in the protocol, the assay tubes contained 50 µl calibrator plus 50 µl TH-depleted mouse serum. The sample tubes contained 50 µl serum sample plus 50 µl zero calibrator. The minimal detectable concentration of T3 in the assay was 10 ng/100 ml.
The contents of total T3 and T4 in brain tissue were determined using the highly sensitive nonequilibrium RIA procedures previously described (17). The T4 and T3 antibodies were obtained from a commercial source (Fitzgerald Industries International, Inc., Concord, MA; T3, catalog no. 20-TR45, cross-reactivity with T4 < 0.38%; T4, catalog no. 20-TR40, cross-reactivity with T3 < 7.5%). Briefly, the RIA buffer consisted of 0.2 M glycine, 0.13 M sodium acetate (pH 8.6) containing 0.02% BSA, and incubation was carried out for 5 d at 4 C. The dried extracts of brain were dissolved in 3 ml RIA buffer. Preliminary tests indicated that up to 50 µl sample could be assayed before linearity with the standard curve was lost. In most experiments, 25 µl sample was used. A combined polyethylene glycol/second antibody separation step was employed. Assay sensitivity was approximately 2 pg/tube for T3 and 4 pg/tube for T4.
Determination of 5'D and 5D activities
The 5'D and 5D activities were assayed according to our published methods (18, 19). Briefly, for determination of 5'D activity, the reaction mixture (total volume, 50 µl) contained approximately 100 µg tissue protein and 1.2 mM EDTA. The substrate was either 1.0 nM [125I]rT3 or [125I]T4, and the cofactor was 20 mM DTT. Incubations were carried out for 1 h at 37 C in the presence and absence of 1 mM PTU, which inhibits the activity of D1 but not that of D2. For determination of 5D activity, the reaction mixture (50 µl) did not contain EDTA, the substrate was 1.0 nM [125I]T3, and the cofactor was 50 mM DTT. Assays were conducted in the presence and absence of 1 mM PTU. In both the 5D and the 5'D assays, protein concentrations were adjusted to ensure that deiodination was less than 20%.
[125I]rT3, [125I]T3, and [125I]T4 (specific activities, 
1000 µCi/µg) were obtained from PerkinElmer Inc. (Norwalk, CT) and were purified by chromatography using Sephadex LH-20 (Sigma Chemical Co., St. Louis, MO) before use. Protein concentrations of all samples were determined according to the method of Comings and Tack (20) using BSA as the standard.
Analysis of mRNA levels by real-time PCR
Aliquots of RNA (90 µg) from each sample were adsorbed on to QIAamp columns included in the QIAGEN (Valencia, CA) RNeasy Mini Kit, and subjected to DNase treatment with the QIAGEN RNase-Free DNase Set. Two micrograms of RNA from the eluate of each column were reverse transcribed to cDNA using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time PCR was carried out using 33 ng of the resulting cDNA samples as template with the SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), according to the manufacturer’s protocol. Samples were run in duplicate. As a standard, cyclophilin cDNA (nine 5-fold dilutions from 1 ng to 2.56 fg) was coamplified in duplicate. The following primers used were: RC3, sense, 5'-CGA TAT TCT TGA CAT CCC GC-3', and antisense, 5'-TCA CAA ACA CAG TAG GGA AG-3'; TrkB, sense, 5'-CAG ATC ACC ACT GGT GCA TTC-3', and antisense, 5'-TGA GCG GAG ATC TGT CTC TC-3; Hairless, sense, 5'-AGC ACT GTG TGG CAT GTG TT-3', and antisense, 5'-ACC CCT GCA TCC AAG TAG CA-3'; and MCT8, sense, 5'-CAT CCA GGC TCT GGG CAA CAG-3', and antisense, 5'-CCT AAT GAG GTG AGC CGT AC-3'. The Bio-Rad (Hercules, CA) I-Cycler 9000 was programmed as follows: 95 C for a 6-min delay, then 40 cycles of 15 sec at 95 C, 15 sec at 60 C, and 30 sec at 72 C. The data generated by the I-Cycler software were expressed in terms of femtograms of DNA amplified per sample, where the amount of cyclophilin DNA was obtained from a standard curve based on the nine known amounts of the standard entered into the cycler program. The experimental values were divided by that of the cyclophilin standard.
Assessment of neurological function
Tests for reflexes.
Posturing, righting, eye blink, ear twitch, whisker-orienting reflex, and constriction and dilation of pupil were determined by standard methods (21).
Test for olfaction.
This test was carried out in a 20 x 16-in. cage. The food was buried under 5 cm of bedding in one corner of the cage. The mouse, which had been fasted for 18 h, was placed in the center of the cage, and the time taken to find the food was recorded. Three trials were conducted with a 2-min break between each. For trials 2 and 3, the food was buried in different corners of the cage.
Tests for coordination and motor system function.
A rotarod apparatus was used to test balance and agility. Two types of experiment were performed. In the first, the mouse was placed on the rotarod apparatus rotating at 4 rpm. The speed was increased by 4 rpm every 30 sec until the mouse fell off. The following functions were determined: latency to fall, maximum rpm at fall, latency to passive rotation, and time in passive rotation. The maximum time of the test was 5 min. A second trial was performed after a 10-min break. The second type of experiment included a learning component. Mice were subjected to two sessions per day, morning and afternoon for 8 d, and a single morning session on d 9. Each session involved three trials. On the first day, the mouse was placed on the rotarod rotating at 10 rpm. Once the mouse had remained on the rotarod for 60 sec in all three trials, the speed was increased by 2.5 rpm for the next session. Otherwise, the speed was held at 10 rpm until the mouse had mastered that speed.
A vertical pole test was used for agility. In this test, mice were placed face upwards near the top of a vertical pole. Normally a mouse will invert itself, wrap its tail round the pole, and run to the base. The time to invert and the time to descend were measured over a maximum period of 60 sec.
Tests for learning and memory.
The Morris water maze 5-d test took place in a small room and employed a tub, approximately 100 cm in diameter, filled with water made opaque with a nontoxic paint powder. A small platform, 12 cm in diameter, was located in one quadrant of the tub just below the water surface. The water was at ambient temperature. A variety of visual cues were placed on the walls of the room. On d 1, each mouse was placed on the platform for 10 sec. The mouse was then placed in the water and the time taken for it to climb on to the platform was recorded. Maximum time allowed per trial was 60 sec. The test was repeated three times, the entry points being from all four quadrants of the tub. The mice were allowed to rest for 5 min between each trial. The entire test was repeated on d 2–5. After the test on d 5, the platform was removed, the mouse placed in the water, and its swimming pattern videotaped for 60 sec. The data were then analyzed to determine the time spent in the quadrant in which the platform had been located (probe test). Finally, the platform was replaced so that it was clearly visible. The mouse was again placed in the water and the time taken for it to climb onto the platform recorded (visible cue test).
The water escape test was also used. To avoid the conditioning that occurs in the water maze test, mice not previously subjected to the water maze test were placed in the tub, which now had a visible escape ladder on the side. The time taken to climb on to the ladder was recorded.
Tests for anxiety level.
The open field test employed a circular field, diameter 36 in., surrounded by a 6-in.-high wall. The test places the desire to explore against the aversion to open spaces. Thus, the time spent in the center of the field relates inversely to the anxiety level of the mouse. The activities of the mouse were recorded on a camera attached to a Videomex data analyzer (Columbus Instruments, Columbus, OH) programmed to record the time spent and distance run in 1) the 3-in.-wide outer perimeter of the field and 2) the inner area of the field (diameter, 30 in.).
The elevated plus maze test (22) employed an apparatus that has two open and two enclosed runways placed in the form of a cross elevated 2 ft above the floor (Columbus Instruments). This test also assesses exploration and aversion with the added dimension of height. The time spent in the open arms relates inversely to the anxiety level of the mouse. Mouse activity was recorded and analyzed as described for the open field test.
Statistical analyses
Data are expressed as mean ± SE. Statistical analyses were carried out using the GB-Stat PPC 6.5.4 computer program (Dynamic Microsystems, Inc., Silver Spring, MD). For comparison of values between two groups of mice, Student’s t test was used. For comparisons among more than two groups, one-way ANOVA was performed, and the differences were assessed using Fisher’s least significant difference (protected t test) test. Statistical significance is defined as P < 0.05.
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TH levels in serum and brain at P15
The serum T4 level was significantly higher in D2KO mice than in WT mice at P15 (Fig. 1
). In contrast, the level of T4 in the hypothyroid mice was less than 0.25 µg/100 ml. The serum T3 level was comparable in the WT and D2KO mice but was reduced by more than 50% in the hypothyroid mice (Fig. 1). Compared with adult WT and D2KO mice, the serum levels of both T4 and T3 were approximately 50% higher at P15 in mice of corresponding genotype (data not shown).
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). The reduction was approximately 50% in the first three brain areas and somewhat less in midbrain. It is notable that for a given brain area, the reduction in T3 content was comparable in D2KO and hypothyroid mice.
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). [In some experiments the increase in T4 content in the Cb did reach statistical significance, (data not shown).] In contrast, the T4 content of these two areas was significantly reduced in the hypothyroid mice.
Deiodinase activity in brain
D2 activity was determined in the cerebrum of WT and hypothyroid WT mice at P15 using [125I]T4, the preferred substrate of the D2. D2 activity was increased almost 4-fold in the hypothyroid brain, in fmol iodide/h·mg protein: WT, 30 ± 5; hypothyroid WT, 115 ± 5; P < 0.001. This activity was not inhibited by PTU, confirming that it was D2.
D1 activity was determined in CCx, Cb, hypothalamus, and midbrain of WT and D2KO mice at P15 using [125I]rT3 as substrate. rT3 is the preferred substrate of the D1. No significant activity was detected in the CCx, hypothalamus, or midbrain. A very low level of 5'D activity, three orders of magnitude less than that present in liver (9), was present in the Cb of both WT and D2KO mice (Table 1). This activity was inhibited by PTU, confirming that it was D1.
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Levels of expression of T3-responsive genes in brain at P15
RC3 and TrkB are TH-responsive neuronal genes that are expressed in many areas of the brain. The transcription of RC3 and TrkB is increased and decreased, respectively, by T3 (23). At P15, the level of RC3 mRNA in the CCx and midbrain was significantly decreased in the hypothyroid mice but not the D2KO mice (Fig. 3). Similarly, the level of TrkB mRNA in the CCx was increased in the hypothyroid mice but not in the D2KO mice. In the midbrain, the TrkB mRNA level was increased in D2KO mice, but the increase was significantly less than that in the hypothyroid mice (Fig. 3). The T3-responsive neuronal genes studied in the cerebellum were Hairless and Srg1. The expression of both is increased by T3 (23). A preliminary study, in which the levels of expression of the two genes were assessed at P5, P10, P15, and P20, indicated that maximal expression occurred at P15 (data not shown). As shown in Fig. 3, the mean level of mRNA for both these genes was reduced in the Cb of the D2KO mice, although only for Hairless was the decrease statistically significant, and significantly greater reductions in the levels occurred in the hypothyroid mice (Fig. 3).
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Locomotion and agility.
In the single-day test on the rotarod apparatus, no differences in performance between the WT and D2KO mice were observed. The test employed 19 WT and 23 D2KO mice. None of the mice resorted to passive rotation. The latency to fall (sec) was as follows: in trial 1, WT, 106 ± 19, and D2KO, 101 ± 17; in trial 2, WT, 130 ± 25, and D2KO, 132 ± 28. Maximum rpm was as follows: in trial 1, WT, 16 ± 3, and D2KO, 15 ± 2; in trial 2, WT, 19 ± 2, and D2KO, 19 ± 2.
In the 8-d test, in which a mouse had to master each rotational speed before the speed could be increased, 11 WT and 12 D2KO mice were tested. Again, there was no difference in performance between the two groups (Fig. 5).
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). The D2KO mice were also significantly slower than WT mice both to invert and to descend.
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). However, in four separate experiments, the D2KO mice consistently took slightly longer than WT mice on any given day, although the differences did not reach statistical significance (Fig. 7A). In addition, in the probe test, the D2KO mice spent less time in the quadrant that had housed the platform than did the WT mice (20 ± 2 vs. 26 ± 1 sec, P < 0.05). That these small differences were not necessarily due to impaired learning and memory skills is strongly suggested by the finding that the D2KO mice took significantly more time than did the WT mice to mount the platform when it was clearly visible (Fig. 7B).
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Anxiety and exploratory levels.
Three sets of WT and D2KO mice were tested in the elevated plus maze (Table 3). The parameters determined were the total distance run, the total time spent running, and the time spent visiting the open and closed arms of the maze. The total distance run and time spent running in the entire maze (both arms) during the 3-min test time by WT and D2KO mice were not significantly different. In experiment 2, the D2KO mice traveled a shorter distance and spent less time moving in the open arms than did the WT mice. However, these differences did not occur in the other two experiments, and the visit time of the D2KO mice in the open arms was comparable to that of the WT mice in all three experiments. Values for the three parameters in the closed arms in all experiments were not significantly different in the WT and D2KO mice.
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Discussion |
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The reduction in brain T3 content in the D2KO mouse was significant (25–50%) but not complete, indicating that the brain is not totally dependent on local production of T3 from T4 by the D2. Clearly, it can obtain a significant amount of T3 from the serum and/or cerebrospinal fluid in the euthyroid state. This observation is consistent with studies of others who have shown that although the majority of the receptor-bound T3 in the CCx and Cb of the euthyroid rat was generated locally from T4 presumably by the D2, as much as 25–50% was obtained directly from the serum (25, 26).
In the four areas of the brain studied, the reduction in T3 content in the D2KO mouse and the hypothyroid mouse was comparable. However, unlike in the D2KO mouse, the level of T4 in both serum and brain of the hypothyroid mouse was greatly reduced. In this setting, the D2 activity in the brain was increased almost 4-fold, and thus, because the serum T3 level was greatly reduced, it is likely the majority of the T3 present in the brain was generated locally from T4 by the D2. Data obtained by others support this conclusion (25).
The impairment of neurological function in the D2KO mouse appears to be minimal. This was not predicted because others have shown that the congenitally hypothyroid (Hyt/hyt) mouse exhibits severe impairments in its locomotion, learning, and memory skills (27), and the neonatal hypothyroid mice used in the present study clearly had abnormalities not evident in the D2KO mice; after P10, they grew poorly and had problems with locomotion and balance, and many died even before weaning. In contrast, survival in the D2KO mouse was not compromised, and locomotion, learning, and memory were seemingly unaffected. The only significant neurological or neurobehavioral abnormalities noted were their reluctance to invert and descend on a vertical pole and to escape from a tub of water. Although these differences could be due to a reduction in the level of anxiety in the D2KO mouse, no evidence of this was obtained when the mice were subjected to the open field and elevated plus maze tests.
Perhaps the most unexpected finding in these studies is the apparent dissociation between T3 content and T3 action in the D2KO brain. Thus, whereas the brain T3 content was decreased to a comparable level in the D2KO and hypothyroid mice, the expression levels of the TH-responsive neuronal genes studied were markedly more abnormal in the hypothyroid mice than in the D2KO mice. In fact, the mRNA levels of the molecular markers were altered in only two of the six determinations made in the D2KO brain, whereas in all six determinations, the levels were markedly altered in the hypothyroid brain. However, there are some caveats to be considered. First, because the expression levels of only four TH-responsive genes were determined, it cannot be assumed that others would be similarly affected. Second, although the four areas of the brain in which the T3 content was determined constituted the entire brain, thus indicating that in the D2KO mouse total brain T3 content is reduced, there is the possibility that T3 content may vary within specific regions. Third, the majority of the determinations of TH content and gene expression reported herein were carried out in brain from neonates at P15, and results might vary at other ages. P15 was selected because this is generally regarded as the time of maximal TH-dependent brain development in mice, and the genes in question are either well or maximally expressed at this time. However, we have observed similar findings at P10. Thus, in the D2KO mouse, the T3 content is reduced 30 and 40% in CCx and Cb, respectively, at P10, whereas the levels of RC3 in CCx and Hairless in Cb are not significantly altered (data not shown). Regardless of these caveats, the correlation of these mild biochemical abnormalities and the mild neurological phenotype in the D2KO mouse is striking.
The current view of TH homeostasis in the brain dictates that the T3 present in neurons is produced predominantly, if not exclusively, from T4 through the activity of the D2 in glial cells, because neurons do not express the D2 (28, 29) (Fig. 8A). Our paradoxical finding that a marked decrease in brain T3 content in the D2KO mouse is accompanied by only relatively mild molecular changes and functional impairment strongly implies that this model of TH homeostasis in brain is inadequate or incomplete and that important compensatory mechanisms must be in play in brain and/or peripheral tissues to minimize functional abnormalities in the face of a deficiency in D2 activity.
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It is also possible that the normal serum T3 level in the D2KO mice helps to maintain peripheral tissues, particularly skeletal muscle, in a euthyroid state that allows for enhanced performance of the D2KO animal relative to the hypothyroid mouse in the functional testing. In fact, this may explain in part the mild overall phenotype of the D2KO mouse.
Three other components of the thyroid system could be compensating for the lack of D2 in the brain: key TH transporters, the elevated T4 content of the brain, and T3 from the serum.
In the last decade, a number of transmembrane proteins capable of transporting TH into cells have been identified (24). Of these, two appear to be specific for TH, organic anion transporting polypeptide 1C1 and MCT8. Organic anion transporting polypeptide 1C1 has been implicated in the transport primarily of T4 across the blood-brain barrier (30), a function that may not be relevant in the D2KO mouse unless significant TH action on gene transcription can be executed by T4 per se (vide infra). However, MCT8 is a transporter of both T3 and T4 and is highly expressed in brain and almost exclusively in neurons (31). The concept that MCT8 is in fact a transporter of T3 into neurons is strongly supported by the finding that humans with mutations in the MCT8 gene have severe X-linked psychomotor retardation, which is associated with low serum T4 and rT3 levels and an elevated serum T3 level (32, 33). These observations are substantiated by the finding that the serum TH profile of the MCT8 knockout mouse is similar to that of humans with the mutated MCT8 gene. In addition, despite the elevated serum T3 level, the T3 content of the cerebrum of MCT8 knockout mice is much lower than that of WT cerebrum (34). In the present study, it was shown that the expression level of MCT8 in both CCx and Cb was comparable in WT, D2KO, and hypothyroid mice. This indicates that an increase in the level of the MCT8 transporter is unlikely to be responsible for any selective preservation of neuronal T3 content in the D2KO mouse. However, the possibility that another T3 transporter might be implicated in the preservation of neuronal T3 content cannot be excluded.
A notable difference between the D2KO mouse and the hypothyroid mouse is their brain T4 content, which is reduced in the latter and increased in the D2KO mouse. Conceivably, in this circumstance, T4 may bind directly to TH receptors and mitigate some of the effects of the reduced level of T3 in the D2KO brain. This assumes that T4 is more than a prohormone, and indeed an earlier study from this laboratory suggests that T4 does have intrinsic activity; in cultures of tadpole red blood cells, which do not express either D1 or D2, the T3 receptor number was increased by T4 as well as T3, both at near physiological concentrations (35). In addition, the possibility that the high T4 level in the brain may afford some protection against the low brain T3 content by means of one or more putative nongenomic actions cannot be excluded (36, 37).
Another interesting possibility arises from a consideration of differences in the source of brain T3 in the D2KO mouse vs. the hypothyroid mouse. As discussed above, the majority of the brain T3 in the rodent is thought to be produced locally from T4 by D2 activity (25, 26) and the fraction is even higher in hypothyroidism when D2 activity is elevated (25, 38). In the absence of the D2, however, the only sources of T3 for the brain are the serum and cerebrospinal fluid. This observation indicates clearly that the current model of TH homeostasis in the brain is incomplete and must be modified to include this latter pathway (Fig. 8B). It also raises the possibility that, at least in some brain regions, the T3 that is produced locally in glial cells may be less accessible to neurons than was previously thought and thus less effective than the T3 obtained from serum in regulating neuronal function. This hypothesis may apply also to the hypothalamic-pituitary-thyroid axis given our observation that the TSH level in D2KO mice is only modestly elevated compared with that in the hypothyroid mouse (9). A recent publication by Cettour-Rose et al. (39) supports this hypothesis; the inhibition of D2 activity in hypothyroid rats by the infusion of rT3 failed to result in an increase in the serum TSH level. Thus, it is possible that although one can readily normalize overall brain T3 content in a hypothyroid animal by providing a relatively modest dose of T4 (14), neuronal function may not return to normal in all brain regions until the serum T3 level is normalized. If correct, the implications of this hypothesis for the treatment of pregnant women and infants with hypothyroidism are obvious.
This hypothesis regarding an important role for the serum T3 in regulating brain function does not negate an important role for the D2 in brain TH homeostasis; as indicated above, some neuronal functions are mildly impaired in the D2KO mouse despite a normal serum T3 level. However, it suggests that the D2 is not as critical for some aspects of brain function as was previously thought, at least in the euthyroid animal.
The D2 is likely to be more important for neuronal function in the hypothyroid mouse. In this setting, the levels of both T4 and T3 in serum are reduced, and the level of D2 activity in the WT brain is substantially increased (25, 38). The increased level of D2 activity allows some measure of compensation for the decrease in serum T4 level, but this is not an option in the brain of the D2KO mouse, which by necessity must obtain its T3 from the serum or cerebrospinal fluid. Thus, in the face of a reduced serum T3 level, the phenotype of the D2KO mouse is likely to be exacerbated. That this is in fact the case is suggested by the finding that D2KO mice born from dams made hypothyroid as described herein rarely survive beyond P6 (Galton, A. V., unpublished data).
In summary, although the brain T3 content in the D2KO mouse is significantly reduced, the molecular and neurobehavioral phenotype of the this animal is unexpectedly mild, suggesting that T3 from the serum, the T4 content in brain, or other homeostatic processes are important for TH action in the brain during development.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online March 1, 2007
Abbreviations: Cb, Cerebellum; CCx, cerebral cortex; 5'D, 5'-deiodination; D1, type 1 deiodinase; D2KO, D2 knockout; DTT, dithiothreitol; P15, postpartum d 15; PTU, propylthiouracil; TH, thyroid hormone; WT, wild type.
Received December 22, 2006.
Accepted for publication February 13, 2007.
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C. Fekete, B. C. G. Freitas, A. Zeold, G. Wittmann, A. Kadar, Z. Liposits, M. A. Christoffolete, P. Singru, R. M. Lechan, A. C. Bianco, et al. Expression Patterns of WSB-1 and USP-33 Underlie Cell-Specific Posttranslational Control of Type 2 Deiodinase in the Rat Brain Endocrinology, October 1, 2007; 148(10): 4865 - 4874. [Abstract] [Full Text] [PDF]
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A. C. Bianco, S. Ribich, and B. W. Kim An Inside Job Endocrinology, July 1, 2007; 148(7): 3077 - 3079. [Full Text] [PDF]
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, P < 0.01 vs. WT and D2KO.
, P < 0.01 vs. WT, P < 0.001 vs. D2KO.
