Increased Cerebrospinal Fluid Levels of 3,3',5'-Triiodothyronine in Patients with Alzheimer’s Disease
http://www.100md.com
《临床内分泌与代谢杂志》
Abstract
Cerebrospinal fluid (CSF) levels of rT3 were evaluated in 21 euthyroid patients with overt Alzheimer’s disease (AD) and 18 matched healthy controls. The assessment also included trans-thyretin and total T3 and T4 CSF concentrations. Despite normal circulating thyroid hormone levels, AD subjects showed significantly increased rT3 levels and an increased rT3 to T4 ratio in the face of unchanged CSF total T4 and transthyretin levels. These results suggest an abnormal intracerebral thyroid hormone metabolism and possibly the occurrence of brain hypothyroidism, either as a secondary consequence of the ongoing process or as a cofactor in the progression of the disease.
Introduction
ALZHEIMER’S DISEASE (AD) is a degenerative disorder of the central nervous system (CNS) characterized by loss of neurons in the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaques, neurofibrillary tangles, and neuropil thread formation. Several laboratory and clinical data suggest that thyroid dysfunction might be relevant to the pathogenesis of AD. Evidence exists in both embryonic and adult laboratory mammals showing that thyroid hormones (TH) play a key role in the development and maintenance of basal forebrain cholinergic neurons typically involved in AD (1, 2). A reduction of c-erbA TH receptor mRNA has been found in AD brains at the level of the hippocampus, another brain structure primarily affected by the disease (3). Furthermore, in vitro studies have shown that TH may regulate the gene expression of the amyloid precursor protein from which the A? peptide, the main component of ?-amyloid that accumulates in the neuritic plaques of AD patients, is derived (4, 5).
Population-based studies also suggest that either hypo- or hyperthyroidism increases the risk of developing AD in the elderly (6, 7, 8). However, to date, no evidence of thyroid dysfunction has been found in AD patients. Indeed, available data only relate to the measurement of TH in the serum, where no major alterations have been detected during AD diagnosis (9). Furthermore, circulating TH levels do not properly reflect TH metabolism in the CNS (10, 11, 12, 13). Thus, the question of whether cerebral metabolism of these hormones is modified in AD remains to be clarified.
Because most of the solutes from the cerebral extracellular spaces directly flow into the cerebrospinal fluid (CSF), analysis of CSF TH levels might provide better clues for investigating the status of TH metabolism and the role of TH in the brain of AD patients (14). In the CNS, TH metabolism is finely regulated by the activity of two different deiodinases, type 2 (D2) and type 3 (D3) iodothyronine deiodinases (11, 12, 13). In fact, D2 and D3 activity balance has been shown to be critical for allowing adequate intraneuronal concentration of the most biologically active form, T3 (11, 13). D2 is responsible for the conversion of T4 to T3 and therefore is considered an activating enzyme. Its localization seems to be mainly glial (26). In contrast to the peripheral tissues, where most of the nuclear-bound T3 is imported from the plasma pool, the supply of the physiologically active hormone T3 in the brain depends mainly on the cellular uptake and intracellular deiodination of T4 by D2. Thus, D2 is believed to play a critical role in the maintenance of TH function in the CNS. D3 catalyzes the conversion of T4 and T3 into the inactive metabolites rT3 and 3,3'-diiodothyronine (T2), respectively, by inner ring deiodination. D3 is considered an inactivating enzyme, because rT3 and T2 do not activate the TH receptor. D3 localization is mainly neuronal. Both D2 and D3 activities have been previously demonstrated in the human brain (15). The topographical pattern of rT3 distribution within the human CNS is coincidental with that of D3, suggesting that most of the rT3 present in the CNS is locally generated from T4 via the intraneuronal 5-deiodination pathway catalyzed by D3 (11, 12, 13, 15).
In this study we assessed TH levels in CSF and serum samples from patients with AD and healthy subjects. We focused on the rT3 concentration as a potential indicator of changes in the cerebral metabolism of TH (16, 17, 18).
Subjects and Methods
Subjects
Twenty-one subjects (14 men and seven women) with diagnosis of probable AD were included in this study. This cohort was a subset from 68 consecutive subjects admitted to the Department of Neurological Sciences, Second University of Naples, between January 1995 and December 2000. All included patients were within 24–36 months after the onset of dementia symptoms. Each subject underwent neurological examination, standard neuropsychological evaluation (Mini-Mental State Examination, Mental Deterioration Battery) and laboratory investigations (brain magnetic resonance imaging or computed tomography, electroencephalogram, and serum and CSF analyses). The 21 individuals included in this study matched the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association Work Group diagnostic criteria for probable AD (9). All subjects were revised 6–12 months after the first assessment to confirm the diagnosis of AD. At reassessment, none of the subjects showed signs of concomitant complicating neurological disease. Furthermore, family history data were negative for familiar AD or other degenerative CNS diseases in all subjects. None of the patients had thyroid autoimmunity or dysfunction.
At admission, each subject showed normal nutritional state (body mass index, 20–27) and normal liver and renal functions and was not taking any medication. The median Mini-Mental State Examination score in our patients was 8.0, indicating overt CNS involvement at the time of lumbar puncture.
Eighteen consecutive nondemented subjects (10 men and eight women) admitted to the Department of Neurological Sciences, Second University of Naples, during the same period served as controls (Table 1). They presented with symptoms requiring CSF analysis. This, however, as well as other laboratory investigations did not disclose any neurological or systemic disorders, so no diagnosis of neurological disease was made at the time of discharge. These control subjects matched the AD group for age, sex (Table 1), and level of education. Moreover, none of the control subjects was taking any medication at the time of lumbar puncture.
CSF and serum analysis
After obtaining informed consent, approximately 8 ml CSF were taken by lumbar puncture between 0800–1000 h together with 10 ml venous blood. An aliquot of each serum and CSF sample was used for routine analyses, and the remaining (3–6 ml) was frozen within 20 min from sampling and stored at –80 C until hormone determinations. Fresh serum routine analyses included glucose, albumin, and Igs (Nephelometry, Dade Behring, Deerfield, IL). Fresh CSF routine analyses included cell count, evaluation of glucose, total protein, albumin and Igs (Nephelometry; Dade Behring). The CSF/blood albumin quotient (AbQ) as well as the serum/CSF IgG index were calculated. All CSF hormone measurements were conducted at the same time within the same assay.
For assessment of thyroid function, TSH, free T4, free T3, rT3, and transthyretin (TTR) levels were evaluated in serum samples by standard procedures. Total T4 (TT4) and T3 (TT3), rT3, and TTR levels were determined in CSF samples by highly sensitive RIAs, following the procedure described by Campos-Barros et al. (15). The rT3 assay was validated by recovery tests using two different rT3 amounts (5 and 10 ng) added to CSF aliquots. The recovered amounts were 101 ± 3.1% and 98.3 ± 0.8%, respectively. The sensitivity limits of the assays were 3.0 ng/dl (0.03 nmol/liter), 2.0 (0.03 nmol/liter), and 1.5 ng/dl (0.05 nmol/liter) for TT4, TT3, and rT3, respectively.
Because TT3 concentrations in individual CSF samples were close to [AD patients, 0.5–1.5 ng/dl (0.0076–0.023 nmol/liter); controls, 1.8–4.6 ng/dl (0.027–0.070 nmol/liter)] or even below the limit of determination (2.0 ng/dl) of the assay, TT3 determinations were carried out in concentrated pools of CSF samples (16).
Each pool consisted of four or five CSF samples (AD patients) or three CSF samples (controls). The pooling of samples was performed blindly without knowledge of the results of previous hormone assays. According to the procedure described by Nishikawa et al. (16), 1.6-ml CSF aliquots were lyophilized and reconstituted with 125 μl charcoal-treated, iodothyronine-free CSF (concentration factor, 1 x 12.8). One hundred microliters of the concentrated samples were used for the TT3 RIA. Each assay was carried out in duplicate. The intraassay variations were 5.4%, 5.6%, and 6.7% for TT4, TT3, and rT3, respectively. CSF TTR levels were evaluated by nephelometry using commercially available kits (Dade Behring).
Statistical analysis
Data are expressed as the mean ± SEM. Statistical comparisons were performed using the t test for unpaired data. Frequencies were compared using the 2 test, with Fisher’s correction, when appropriate. The relationship between variables was analyzed using Pearson’s correlation coefficient. Statistical significance was assumed at P 0.05.
Results
In AD patients, serum concentrations of TSH, free T4, free T3, and rT3 were not significantly different from those in the control group (Table 1). Moreover, no significant differences were found in the CSF levels of TT4 [107.9 ± 7.3 ng/dl (1.4 ± 0.09 nmol/liter) vs.98.9 ± 5.2 ng/dl (1.3 ± 0.07 nmol/liter); t = –0.98; P = 0.34], TTR [151.0 ± 1.4 mg/liter (27.5 ± 0.25 mmol/liter) vs. 145.0 ± 0.81 mg/liter (26.4 ± 0.15 mg/liter); t = 1.33; P = 0.43], AbQ (0.49 ± 0.02 vs. 0.47 ± 0.01; t = 0.61; P = 0.81), and the IgG index (0.45 ± 0.02 vs. 0.44 ± 0.02; t = 0.43; P = 0.67) between AD patients and healthy subjects.
In contrast, TT3 levels in the concentrated CSF pools were significantly and markedly lower in AD patients (n = 5) than in controls [n = 6; 57.7 ± 3.2 ng/dl (0.89 ± 0.05 nmol/liter) vs. 148.9 ± 13.9 ng/dl (2.3 ± 0.21 nmol/liter); t = 5.87; P < 0.001], whereas CSF rT3 levels were significantly enhanced (Fig. 1A) in AD patients compared with controls [12.6 ± 0.9 ng/dl (0.19 ± 0.02 nmol/liter) vs. 6.1 ± 0.29 ng/dl (0.10 ± 0.004 nmol/liter); t = –4.5; P < 0.001]. Moreover, as shown in Fig. 1, B and C, TT3/TT4 molar ratios (0.009 ± 0.001 vs. 0.026 ± 0.003; t = 5.6; P < 0.001) were significantly lower in AD patients than in healthy subjects, whereas a significant increase in the rT3/TT4 molar ratio (0.14 ± 0.008 vs. 0.09 ± 0.008; t = –4.0; P < 0.001) was observed in AD patients compared with controls.
CSF rT3 concentrations were significantly correlated with CSF TT4 levels (r = 0.68; P < 0.001) in normal controls as well as in AD patients (r = 0.62; P < 0.003), thus indicating that in both cases the amount of rT3 in CSF was linked to T4 metabolism.
Discussion
In this study, patients with overt AD were found to have normal thyroid function, as assessed by standard evaluation of serum TH levels. CSF rT3 concentrations were, however, significantly enhanced compared with those in healthy subjects, suggesting an impairment of intracerebral TH metabolism during development of the disease.
Animal studies have shown that in the euthyroid brain, approximately 70–80% of the nuclear-bound T3 originates from intracerebral T4 to T3 conversion, whereas only a modest 20–30% is obtained from the plasma pool (11, 13, 19). The amount of nuclear-bound T3 in the brain is regulated by the fine-tuned and well coordinated activities of D2 and D3 deiodinases (11, 13, 15). A critical role is played by the D3 enzyme, which transforms T4 and T3 into the inactive metabolites rT3 and T2, respectively, protecting neuronal cells from excessive T3 exposure. In fact, D3 activity increases in hyperthyroidism, whereas it decreases in the hypothyroid status (10). The increased CSF rT3 concentration as well as the reduced CSF T3 levels found in AD patients may reflect an enhancement of D3 activity in this disease. This hypothesis is supported by the 2-fold increase in the rT3/T4 molar ratio we found in the CSF of AD patients, which suggests that a considerable amount of T4 is transformed into rT3 in the CNS of AD patients. This might implicate the incidence of local hypothyroidism at the level of brain tissue, given the known inverse relationship between rT3 and T3 production (13).
An increase in the CSF rT3 concentration has been found in other degenerative, vascular, and inflammatory diseases of the CNS (16, 17, 18, 20). This finding could be linked to a relatively unspecific passage of rT3 from the serum into the CSF across an eventually damaged blood-CSF barrier, or it could reflect an enhancement of D3 activity in the CNS. In previous studies, markers of blood-CSF barrier integrity (i.e. AbQ) and indexes of intracerebral inflammation (i.e. IgG index) were not reported (16, 17, 18, 20). Moreover, the small size and clinical heterogeneity of the patient groups investigated in those studies (16, 17, 18, 20) did not allow us to draw any conclusion about the specificity of TH changes observed in the examined disorders. Therefore, in our study we investigated the status of brain TH metabolism in a cohort of AD patients that was homogenous with respect to clinical diagnostic criteria, disease duration, and various physiological parameters (i.e. TTR, IgG index, AbQ, and total T3) that may affect intracerebral TH metabolism.
The CSF rT3 concentrations determined in our study were lower than those found in previous reports (16, 17, 18), but compare well with values reported more recently by Labudova et al. (20). Indeed, different RIA protocols for iodothyronine determinations may not always generate comparable results, especially if they are conducted with different antisera, which may display variable antigen selectivities.
We cannot conclude whether the increased production of rT3 in the CNS of AD patients is an adaptive phenomena to the disease or is a relevant physiopathological event (21). Cytokines are involved in the pathological process underlying AD (22), and they may enhance the activity of D3 with a consequential increase in rT3 production, as observed in the systemic euthyroid sick syndrome (13, 23). A decrease in the T4 to T3 conversion rate may favor the development of senile plaques due to the lack of T3 inhibitory effects on ?-amyloid gene expression (4) or may promote the formation of the amyloid precursor protein isoforms preferentially expressed in the brain of patients with AD (5). Moreover, rT3 itself is known to be a very effective competitive inhibitor of D2-mediated deiodination of T4 to T3 and has also been reported to be a physiological regulator of D2 activity during development (24, 25). This suggests that an increase in local rT3 production could hypothetically contribute to induce a local hypothyroid status. In addition to this, D2 activity is known to be predominantly expressed in astrocytes (26), which appear to be the primary affected targets of early pathophysiological events in AD (27). Thus, an impairment of astrocytic D2 activity might be the primary event affecting TH metabolism in AD brain. This could, in turn, explain the lower T4 to T3 conversion rate, as indicated by the lower T3/T4 ratio, and the increase in CSF rT3 levels, as observed in our AD patients.
In the CSF, TTR is the main iodothyronine-binding protein transferring T4 from the blood into the brain across the blood-choroid plexus barrier (28, 29). Although, an adequate concentration of TTR in the CSF does not seem to be required for preservation of the euthyroid status of the brain (30), it might be important for maintaining the intracerebral proteins as amyloid fibrils in soluble form (31). Indeed, previous studies reported decreased CSF TTR concentrations in late-onset AD patients, possibly due to epithelial atrophy of the choroid plexus (31, 32). However, we did not observe any significant difference in CSF TTR levels between AD patients and healthy subjects. Such a discordant result is probably due to the different age and post-disease onset stage of our patients compared with those previously studied by others (31).
In this regard, the maintenance of normal CSF TTR values in the subjects included in the present study support the hypothesis that the increase in CSF rT3 levels was not secondary to an advanced stage of AD disease, because at the time of lumbar puncture, the choroid plexus epithelium still produced normal amounts of TTR. Note that our AD group included subjects who were within the range of 24–36 months post-disease onset, because our aim was to investigate whether alterations in brain TH metabolism could play a role in the development of overt AD symptoms.
rT3 has been previously reported to cross the choroid plexus-CSF barrier (33). Because our AD patients had serum rT3 levels comparable to those in control subjects, and the choroid plexus-CSF barrier was likely to be intact, we infer that the increase in CSF rT3 levels was caused by abnormal intracerebral TH metabolism. This interpretation is supported by the findings of, respectively, decreased and increased TT3/TT4 and rT3/TT4 ratios in AD patients compared with control subjects.
In conclusion, our study reveals an increased CSF concentration of rT3 and a higher rT3 to TT4 molar ratio in the face of decreased CSF TT3 levels and a lower TT3/TT4 ratio in patients with overt AD. These findings suggest that even with an apparent systemic euthyroid status, local or tissue hypothyroidism may occur during the progress of the disease. Follow-up studies should clarify whether this condition is also present during early AD and how it develops during the advanced stages of the disease.
Footnotes
First Published Online October 13, 2004
Abbreviations: AbQ, Albumin quotient; AD, Alzheimer’s disease; CNS, central nervous system; CSF, cerebrospinal fluid; D2, type 2 deiodinase; D3, type 3 deiodinase; T2, 3,3'-diiodothyronine; TH, thyroid hormone; TT3, total T3; TT4, total T4; TTR, transthyretin.
Received June 8, 2004.
Accepted September 28, 2004.
References
Calza L, Giardino L, Aloe L 1997 Thyroid hormone regulates NGF content and p75LNGFR expression in the basal forebrain of adult rats. Exp Neurol 143:196–206
Patel AJ, Hayashi M, Hunt A 1987 Selective persistent reduction in choline acetyltransferase activity in basal forebrain of the rat after thyroid deficiency during early life. Brain Res 422:182–185
Sutherland MK, Wong L, Somerville MJ, Handley P, Yoong L, Bergeron C, McLachlan DR 1992 Reduction of thyroid hormone receptor c-erbA mRNA levels in the hippocampus of Alzheimer as compared to Huntington brain. Neurobiol Aging 13:301–312
Belandia B, Latasa MJ, Villa A, Pascual A 1998 Thyroid hormone negatively regulates the transcriptional activity of the ?-amyloid precursor protein gene. J Biol Chem 46:30366–30371
Latasa MJ, Belandia B, Pascual A 1998 Thyroid hormones regulate ?-amyloid gene splicing and protein secretion in neuroblastoma cells. Endocrinology 139:2692–2698
Yoshimasu F, Kokmen E, Hay ID, Beard CM, Offord KP, Kurland LT 1991 The association between Alzheimer’s disease and thyroid disease in Rochester, Minnesota. Neurology 41:1745–1747
Ganguli M, Burmeister LA, Seaberg EC, Belle S, DeKosky ST 1996 Association between dementia and elevated TSH: a community-based study. Biol Psychiatry 40:714–725
Kalmijn S, Mehta KM, Pols HA, Hofman A, Drexhage HA, Breteler MM 2000 Subclinical hyperthyroidism and the risk of dementia. The Rotterdam study. Clin Endocrinol (Oxf) 53:733–737
McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM 1984 Clinical diagnosis of Alzheimer’s disease: report of the NINCDS ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s disease. Neurology 34:939–944
Dratman MB, Crutchfield FL, Gordon JT, Jennings AS 1983 Iodothyronine homeostasis in rat brain during hypo and hyperthyroidism. Am J Physiol 245:E185–E193
Leonard JL 1992 Regulation of T3 production in the brain. Acta Med Austriaca 19(Suppl 1):5–8
Silva JE, Leonard JL, Crantz FR, Larsen PR 1982 Evidence for two tissue-specific pathways for in vivo thyroxine 5'-deiodination in the rat. J Clin Invest 69:1176–1184
Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23:38–89
Galasko D 1999 Cerebrospinal fluid opens a window on Alzheimer disease. Arch Neurol 56:655–656
Campos-Barros A, Hoell T, Musa A, Sampaolo S, Stoltenburg G, Pinna G, Eravci M, Meinhold H, Baumgartner A 1996 Phenolic and thyrosyl ring iodothyronine deiodination and thyroid hormone concentrations in the human central nervous system. J Clin Endocrinol Metab 81:2179–2185
Nishikawa M, Inada M, Naito K, Ishii H, Tanaka K, Mashio Y, Nakao K, Nakai Y, Udaka F, Imura H 1981 3,3',5'-Triiodothyronine (reverse T3) in human cerebrospinal fluid. J Clin Endocrinol Metab 53:1030–1035
Thompson Jr P, Burman KD, Wright FD, Potter MW, Wartofsky L 1982 Iodothyronine levels in human cerebrospinal fluid. J Clin Endocrinol Metab 54:653–655
Kirkegaard C, Faber J 1991 Free thyroxine and 3,3',5'-triiodothyronine levels in cerebrospinal fluid in patients with endogenous depression. Acta Endocrinol (Copenh) 124:166–172
Malin JP, Kodding R, Fuhrmann H, von zur Muhlen A 1989 T4, T3 and rT3 levels in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis. J Neurol 236:57–59
Labudova O, Cairns N, Koeck T, Kitzmueller E, Rink H, Lubec G 1999 Thyroid stimulating hormone-receptor overexpression in brain of patients with Down syndrome and Alzheimer’s disease. Life Sci 64:1037–1044
van Doorn J, van der Heide D, Roelfsema F 1983 Sources and quantity of 3,5,3'-triiodothyronine in several tissues of the rat. J Clin Invest 72:1778–1792
Eikelenboom P, Van Gool WA 2004 Neuroinflammatory perspectives on the two faces of Alzheimer’s disease. J Neural Transm 111:281–294
Wartofsky L, Burman KD 1982 Alterations in thyroid function in patients with systemic illness: the "euthyroid sick syndrome." Endocr Rev 3:164–217
Kaplan MM 1986 Regulatory influences on iodothyronine deiodination in animal tissues. In: Henneman G, ed. Thyroid hormone metabolism. New York: Marcel Dekker; 231–253
St Germain DL, Galton VA 1997 The deiodinase family of selenoproteins. Thyroid 7:655–668
Guadano-Ferraz A, Obregon MJ, St Germain DL, Bernal J 1997 The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc Natl Acad Sci USA 94:10391–10396
Abramov AY, Canevari L, Duchen MR 2003 Changes in intracellular calcium and glutathione in astrocytes as the primary mechanism of amyloid neurotoxicity. J Neurosci 23:5088–5095
Chanoine JP, Alex S, Fang SL, Stone S, Leonard JL, Korhle J, Braverman LE 1992 Role of transthyretin in the transport of thyroxine from the blood to the choroid plexus, the cerebrospinal fluid, and the brain. Endocrinology 130:933–938
Dratman MB, Crutchfield FL, Schoenhoff MB 1991 Transport of iodothyronines from the bloodstream to brain: contributions by blood:brain and choroid:cerebrospinal fluid barriers. Brain Res 554:229–236
Palha JA, Fernandes R, de Escobar GM, Episkopou V, Gottesman M, Saraiva MJ 2000 Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin-null mouse model. Endocrinology 141:3267–3272
Serot JM, Christmann D, Dubost T, Couturier M 1997 Cerebrospinal fluid transthyretin: aging and late onset Alzheimer’s disease. J Neurol Neurosurg Psychiatry 63:506–508
Riisoen H 1988 Reduced prealbumin (transthyretin) in CSF of severely demented patients with Alzheimer’s disease. Acta Neurol Scand 78:455–459
Hennemann G, Docter R, Friesema ECH, De Jong M, Krenning EP, Visser TJ 2001 Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev 22:451–476(Simone Sampaolo, Angel Ca)
Cerebrospinal fluid (CSF) levels of rT3 were evaluated in 21 euthyroid patients with overt Alzheimer’s disease (AD) and 18 matched healthy controls. The assessment also included trans-thyretin and total T3 and T4 CSF concentrations. Despite normal circulating thyroid hormone levels, AD subjects showed significantly increased rT3 levels and an increased rT3 to T4 ratio in the face of unchanged CSF total T4 and transthyretin levels. These results suggest an abnormal intracerebral thyroid hormone metabolism and possibly the occurrence of brain hypothyroidism, either as a secondary consequence of the ongoing process or as a cofactor in the progression of the disease.
Introduction
ALZHEIMER’S DISEASE (AD) is a degenerative disorder of the central nervous system (CNS) characterized by loss of neurons in the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaques, neurofibrillary tangles, and neuropil thread formation. Several laboratory and clinical data suggest that thyroid dysfunction might be relevant to the pathogenesis of AD. Evidence exists in both embryonic and adult laboratory mammals showing that thyroid hormones (TH) play a key role in the development and maintenance of basal forebrain cholinergic neurons typically involved in AD (1, 2). A reduction of c-erbA TH receptor mRNA has been found in AD brains at the level of the hippocampus, another brain structure primarily affected by the disease (3). Furthermore, in vitro studies have shown that TH may regulate the gene expression of the amyloid precursor protein from which the A? peptide, the main component of ?-amyloid that accumulates in the neuritic plaques of AD patients, is derived (4, 5).
Population-based studies also suggest that either hypo- or hyperthyroidism increases the risk of developing AD in the elderly (6, 7, 8). However, to date, no evidence of thyroid dysfunction has been found in AD patients. Indeed, available data only relate to the measurement of TH in the serum, where no major alterations have been detected during AD diagnosis (9). Furthermore, circulating TH levels do not properly reflect TH metabolism in the CNS (10, 11, 12, 13). Thus, the question of whether cerebral metabolism of these hormones is modified in AD remains to be clarified.
Because most of the solutes from the cerebral extracellular spaces directly flow into the cerebrospinal fluid (CSF), analysis of CSF TH levels might provide better clues for investigating the status of TH metabolism and the role of TH in the brain of AD patients (14). In the CNS, TH metabolism is finely regulated by the activity of two different deiodinases, type 2 (D2) and type 3 (D3) iodothyronine deiodinases (11, 12, 13). In fact, D2 and D3 activity balance has been shown to be critical for allowing adequate intraneuronal concentration of the most biologically active form, T3 (11, 13). D2 is responsible for the conversion of T4 to T3 and therefore is considered an activating enzyme. Its localization seems to be mainly glial (26). In contrast to the peripheral tissues, where most of the nuclear-bound T3 is imported from the plasma pool, the supply of the physiologically active hormone T3 in the brain depends mainly on the cellular uptake and intracellular deiodination of T4 by D2. Thus, D2 is believed to play a critical role in the maintenance of TH function in the CNS. D3 catalyzes the conversion of T4 and T3 into the inactive metabolites rT3 and 3,3'-diiodothyronine (T2), respectively, by inner ring deiodination. D3 is considered an inactivating enzyme, because rT3 and T2 do not activate the TH receptor. D3 localization is mainly neuronal. Both D2 and D3 activities have been previously demonstrated in the human brain (15). The topographical pattern of rT3 distribution within the human CNS is coincidental with that of D3, suggesting that most of the rT3 present in the CNS is locally generated from T4 via the intraneuronal 5-deiodination pathway catalyzed by D3 (11, 12, 13, 15).
In this study we assessed TH levels in CSF and serum samples from patients with AD and healthy subjects. We focused on the rT3 concentration as a potential indicator of changes in the cerebral metabolism of TH (16, 17, 18).
Subjects and Methods
Subjects
Twenty-one subjects (14 men and seven women) with diagnosis of probable AD were included in this study. This cohort was a subset from 68 consecutive subjects admitted to the Department of Neurological Sciences, Second University of Naples, between January 1995 and December 2000. All included patients were within 24–36 months after the onset of dementia symptoms. Each subject underwent neurological examination, standard neuropsychological evaluation (Mini-Mental State Examination, Mental Deterioration Battery) and laboratory investigations (brain magnetic resonance imaging or computed tomography, electroencephalogram, and serum and CSF analyses). The 21 individuals included in this study matched the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association Work Group diagnostic criteria for probable AD (9). All subjects were revised 6–12 months after the first assessment to confirm the diagnosis of AD. At reassessment, none of the subjects showed signs of concomitant complicating neurological disease. Furthermore, family history data were negative for familiar AD or other degenerative CNS diseases in all subjects. None of the patients had thyroid autoimmunity or dysfunction.
At admission, each subject showed normal nutritional state (body mass index, 20–27) and normal liver and renal functions and was not taking any medication. The median Mini-Mental State Examination score in our patients was 8.0, indicating overt CNS involvement at the time of lumbar puncture.
Eighteen consecutive nondemented subjects (10 men and eight women) admitted to the Department of Neurological Sciences, Second University of Naples, during the same period served as controls (Table 1). They presented with symptoms requiring CSF analysis. This, however, as well as other laboratory investigations did not disclose any neurological or systemic disorders, so no diagnosis of neurological disease was made at the time of discharge. These control subjects matched the AD group for age, sex (Table 1), and level of education. Moreover, none of the control subjects was taking any medication at the time of lumbar puncture.
CSF and serum analysis
After obtaining informed consent, approximately 8 ml CSF were taken by lumbar puncture between 0800–1000 h together with 10 ml venous blood. An aliquot of each serum and CSF sample was used for routine analyses, and the remaining (3–6 ml) was frozen within 20 min from sampling and stored at –80 C until hormone determinations. Fresh serum routine analyses included glucose, albumin, and Igs (Nephelometry, Dade Behring, Deerfield, IL). Fresh CSF routine analyses included cell count, evaluation of glucose, total protein, albumin and Igs (Nephelometry; Dade Behring). The CSF/blood albumin quotient (AbQ) as well as the serum/CSF IgG index were calculated. All CSF hormone measurements were conducted at the same time within the same assay.
For assessment of thyroid function, TSH, free T4, free T3, rT3, and transthyretin (TTR) levels were evaluated in serum samples by standard procedures. Total T4 (TT4) and T3 (TT3), rT3, and TTR levels were determined in CSF samples by highly sensitive RIAs, following the procedure described by Campos-Barros et al. (15). The rT3 assay was validated by recovery tests using two different rT3 amounts (5 and 10 ng) added to CSF aliquots. The recovered amounts were 101 ± 3.1% and 98.3 ± 0.8%, respectively. The sensitivity limits of the assays were 3.0 ng/dl (0.03 nmol/liter), 2.0 (0.03 nmol/liter), and 1.5 ng/dl (0.05 nmol/liter) for TT4, TT3, and rT3, respectively.
Because TT3 concentrations in individual CSF samples were close to [AD patients, 0.5–1.5 ng/dl (0.0076–0.023 nmol/liter); controls, 1.8–4.6 ng/dl (0.027–0.070 nmol/liter)] or even below the limit of determination (2.0 ng/dl) of the assay, TT3 determinations were carried out in concentrated pools of CSF samples (16).
Each pool consisted of four or five CSF samples (AD patients) or three CSF samples (controls). The pooling of samples was performed blindly without knowledge of the results of previous hormone assays. According to the procedure described by Nishikawa et al. (16), 1.6-ml CSF aliquots were lyophilized and reconstituted with 125 μl charcoal-treated, iodothyronine-free CSF (concentration factor, 1 x 12.8). One hundred microliters of the concentrated samples were used for the TT3 RIA. Each assay was carried out in duplicate. The intraassay variations were 5.4%, 5.6%, and 6.7% for TT4, TT3, and rT3, respectively. CSF TTR levels were evaluated by nephelometry using commercially available kits (Dade Behring).
Statistical analysis
Data are expressed as the mean ± SEM. Statistical comparisons were performed using the t test for unpaired data. Frequencies were compared using the 2 test, with Fisher’s correction, when appropriate. The relationship between variables was analyzed using Pearson’s correlation coefficient. Statistical significance was assumed at P 0.05.
Results
In AD patients, serum concentrations of TSH, free T4, free T3, and rT3 were not significantly different from those in the control group (Table 1). Moreover, no significant differences were found in the CSF levels of TT4 [107.9 ± 7.3 ng/dl (1.4 ± 0.09 nmol/liter) vs.98.9 ± 5.2 ng/dl (1.3 ± 0.07 nmol/liter); t = –0.98; P = 0.34], TTR [151.0 ± 1.4 mg/liter (27.5 ± 0.25 mmol/liter) vs. 145.0 ± 0.81 mg/liter (26.4 ± 0.15 mg/liter); t = 1.33; P = 0.43], AbQ (0.49 ± 0.02 vs. 0.47 ± 0.01; t = 0.61; P = 0.81), and the IgG index (0.45 ± 0.02 vs. 0.44 ± 0.02; t = 0.43; P = 0.67) between AD patients and healthy subjects.
In contrast, TT3 levels in the concentrated CSF pools were significantly and markedly lower in AD patients (n = 5) than in controls [n = 6; 57.7 ± 3.2 ng/dl (0.89 ± 0.05 nmol/liter) vs. 148.9 ± 13.9 ng/dl (2.3 ± 0.21 nmol/liter); t = 5.87; P < 0.001], whereas CSF rT3 levels were significantly enhanced (Fig. 1A) in AD patients compared with controls [12.6 ± 0.9 ng/dl (0.19 ± 0.02 nmol/liter) vs. 6.1 ± 0.29 ng/dl (0.10 ± 0.004 nmol/liter); t = –4.5; P < 0.001]. Moreover, as shown in Fig. 1, B and C, TT3/TT4 molar ratios (0.009 ± 0.001 vs. 0.026 ± 0.003; t = 5.6; P < 0.001) were significantly lower in AD patients than in healthy subjects, whereas a significant increase in the rT3/TT4 molar ratio (0.14 ± 0.008 vs. 0.09 ± 0.008; t = –4.0; P < 0.001) was observed in AD patients compared with controls.
CSF rT3 concentrations were significantly correlated with CSF TT4 levels (r = 0.68; P < 0.001) in normal controls as well as in AD patients (r = 0.62; P < 0.003), thus indicating that in both cases the amount of rT3 in CSF was linked to T4 metabolism.
Discussion
In this study, patients with overt AD were found to have normal thyroid function, as assessed by standard evaluation of serum TH levels. CSF rT3 concentrations were, however, significantly enhanced compared with those in healthy subjects, suggesting an impairment of intracerebral TH metabolism during development of the disease.
Animal studies have shown that in the euthyroid brain, approximately 70–80% of the nuclear-bound T3 originates from intracerebral T4 to T3 conversion, whereas only a modest 20–30% is obtained from the plasma pool (11, 13, 19). The amount of nuclear-bound T3 in the brain is regulated by the fine-tuned and well coordinated activities of D2 and D3 deiodinases (11, 13, 15). A critical role is played by the D3 enzyme, which transforms T4 and T3 into the inactive metabolites rT3 and T2, respectively, protecting neuronal cells from excessive T3 exposure. In fact, D3 activity increases in hyperthyroidism, whereas it decreases in the hypothyroid status (10). The increased CSF rT3 concentration as well as the reduced CSF T3 levels found in AD patients may reflect an enhancement of D3 activity in this disease. This hypothesis is supported by the 2-fold increase in the rT3/T4 molar ratio we found in the CSF of AD patients, which suggests that a considerable amount of T4 is transformed into rT3 in the CNS of AD patients. This might implicate the incidence of local hypothyroidism at the level of brain tissue, given the known inverse relationship between rT3 and T3 production (13).
An increase in the CSF rT3 concentration has been found in other degenerative, vascular, and inflammatory diseases of the CNS (16, 17, 18, 20). This finding could be linked to a relatively unspecific passage of rT3 from the serum into the CSF across an eventually damaged blood-CSF barrier, or it could reflect an enhancement of D3 activity in the CNS. In previous studies, markers of blood-CSF barrier integrity (i.e. AbQ) and indexes of intracerebral inflammation (i.e. IgG index) were not reported (16, 17, 18, 20). Moreover, the small size and clinical heterogeneity of the patient groups investigated in those studies (16, 17, 18, 20) did not allow us to draw any conclusion about the specificity of TH changes observed in the examined disorders. Therefore, in our study we investigated the status of brain TH metabolism in a cohort of AD patients that was homogenous with respect to clinical diagnostic criteria, disease duration, and various physiological parameters (i.e. TTR, IgG index, AbQ, and total T3) that may affect intracerebral TH metabolism.
The CSF rT3 concentrations determined in our study were lower than those found in previous reports (16, 17, 18), but compare well with values reported more recently by Labudova et al. (20). Indeed, different RIA protocols for iodothyronine determinations may not always generate comparable results, especially if they are conducted with different antisera, which may display variable antigen selectivities.
We cannot conclude whether the increased production of rT3 in the CNS of AD patients is an adaptive phenomena to the disease or is a relevant physiopathological event (21). Cytokines are involved in the pathological process underlying AD (22), and they may enhance the activity of D3 with a consequential increase in rT3 production, as observed in the systemic euthyroid sick syndrome (13, 23). A decrease in the T4 to T3 conversion rate may favor the development of senile plaques due to the lack of T3 inhibitory effects on ?-amyloid gene expression (4) or may promote the formation of the amyloid precursor protein isoforms preferentially expressed in the brain of patients with AD (5). Moreover, rT3 itself is known to be a very effective competitive inhibitor of D2-mediated deiodination of T4 to T3 and has also been reported to be a physiological regulator of D2 activity during development (24, 25). This suggests that an increase in local rT3 production could hypothetically contribute to induce a local hypothyroid status. In addition to this, D2 activity is known to be predominantly expressed in astrocytes (26), which appear to be the primary affected targets of early pathophysiological events in AD (27). Thus, an impairment of astrocytic D2 activity might be the primary event affecting TH metabolism in AD brain. This could, in turn, explain the lower T4 to T3 conversion rate, as indicated by the lower T3/T4 ratio, and the increase in CSF rT3 levels, as observed in our AD patients.
In the CSF, TTR is the main iodothyronine-binding protein transferring T4 from the blood into the brain across the blood-choroid plexus barrier (28, 29). Although, an adequate concentration of TTR in the CSF does not seem to be required for preservation of the euthyroid status of the brain (30), it might be important for maintaining the intracerebral proteins as amyloid fibrils in soluble form (31). Indeed, previous studies reported decreased CSF TTR concentrations in late-onset AD patients, possibly due to epithelial atrophy of the choroid plexus (31, 32). However, we did not observe any significant difference in CSF TTR levels between AD patients and healthy subjects. Such a discordant result is probably due to the different age and post-disease onset stage of our patients compared with those previously studied by others (31).
In this regard, the maintenance of normal CSF TTR values in the subjects included in the present study support the hypothesis that the increase in CSF rT3 levels was not secondary to an advanced stage of AD disease, because at the time of lumbar puncture, the choroid plexus epithelium still produced normal amounts of TTR. Note that our AD group included subjects who were within the range of 24–36 months post-disease onset, because our aim was to investigate whether alterations in brain TH metabolism could play a role in the development of overt AD symptoms.
rT3 has been previously reported to cross the choroid plexus-CSF barrier (33). Because our AD patients had serum rT3 levels comparable to those in control subjects, and the choroid plexus-CSF barrier was likely to be intact, we infer that the increase in CSF rT3 levels was caused by abnormal intracerebral TH metabolism. This interpretation is supported by the findings of, respectively, decreased and increased TT3/TT4 and rT3/TT4 ratios in AD patients compared with control subjects.
In conclusion, our study reveals an increased CSF concentration of rT3 and a higher rT3 to TT4 molar ratio in the face of decreased CSF TT3 levels and a lower TT3/TT4 ratio in patients with overt AD. These findings suggest that even with an apparent systemic euthyroid status, local or tissue hypothyroidism may occur during the progress of the disease. Follow-up studies should clarify whether this condition is also present during early AD and how it develops during the advanced stages of the disease.
Footnotes
First Published Online October 13, 2004
Abbreviations: AbQ, Albumin quotient; AD, Alzheimer’s disease; CNS, central nervous system; CSF, cerebrospinal fluid; D2, type 2 deiodinase; D3, type 3 deiodinase; T2, 3,3'-diiodothyronine; TH, thyroid hormone; TT3, total T3; TT4, total T4; TTR, transthyretin.
Received June 8, 2004.
Accepted September 28, 2004.
References
Calza L, Giardino L, Aloe L 1997 Thyroid hormone regulates NGF content and p75LNGFR expression in the basal forebrain of adult rats. Exp Neurol 143:196–206
Patel AJ, Hayashi M, Hunt A 1987 Selective persistent reduction in choline acetyltransferase activity in basal forebrain of the rat after thyroid deficiency during early life. Brain Res 422:182–185
Sutherland MK, Wong L, Somerville MJ, Handley P, Yoong L, Bergeron C, McLachlan DR 1992 Reduction of thyroid hormone receptor c-erbA mRNA levels in the hippocampus of Alzheimer as compared to Huntington brain. Neurobiol Aging 13:301–312
Belandia B, Latasa MJ, Villa A, Pascual A 1998 Thyroid hormone negatively regulates the transcriptional activity of the ?-amyloid precursor protein gene. J Biol Chem 46:30366–30371
Latasa MJ, Belandia B, Pascual A 1998 Thyroid hormones regulate ?-amyloid gene splicing and protein secretion in neuroblastoma cells. Endocrinology 139:2692–2698
Yoshimasu F, Kokmen E, Hay ID, Beard CM, Offord KP, Kurland LT 1991 The association between Alzheimer’s disease and thyroid disease in Rochester, Minnesota. Neurology 41:1745–1747
Ganguli M, Burmeister LA, Seaberg EC, Belle S, DeKosky ST 1996 Association between dementia and elevated TSH: a community-based study. Biol Psychiatry 40:714–725
Kalmijn S, Mehta KM, Pols HA, Hofman A, Drexhage HA, Breteler MM 2000 Subclinical hyperthyroidism and the risk of dementia. The Rotterdam study. Clin Endocrinol (Oxf) 53:733–737
McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM 1984 Clinical diagnosis of Alzheimer’s disease: report of the NINCDS ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s disease. Neurology 34:939–944
Dratman MB, Crutchfield FL, Gordon JT, Jennings AS 1983 Iodothyronine homeostasis in rat brain during hypo and hyperthyroidism. Am J Physiol 245:E185–E193
Leonard JL 1992 Regulation of T3 production in the brain. Acta Med Austriaca 19(Suppl 1):5–8
Silva JE, Leonard JL, Crantz FR, Larsen PR 1982 Evidence for two tissue-specific pathways for in vivo thyroxine 5'-deiodination in the rat. J Clin Invest 69:1176–1184
Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23:38–89
Galasko D 1999 Cerebrospinal fluid opens a window on Alzheimer disease. Arch Neurol 56:655–656
Campos-Barros A, Hoell T, Musa A, Sampaolo S, Stoltenburg G, Pinna G, Eravci M, Meinhold H, Baumgartner A 1996 Phenolic and thyrosyl ring iodothyronine deiodination and thyroid hormone concentrations in the human central nervous system. J Clin Endocrinol Metab 81:2179–2185
Nishikawa M, Inada M, Naito K, Ishii H, Tanaka K, Mashio Y, Nakao K, Nakai Y, Udaka F, Imura H 1981 3,3',5'-Triiodothyronine (reverse T3) in human cerebrospinal fluid. J Clin Endocrinol Metab 53:1030–1035
Thompson Jr P, Burman KD, Wright FD, Potter MW, Wartofsky L 1982 Iodothyronine levels in human cerebrospinal fluid. J Clin Endocrinol Metab 54:653–655
Kirkegaard C, Faber J 1991 Free thyroxine and 3,3',5'-triiodothyronine levels in cerebrospinal fluid in patients with endogenous depression. Acta Endocrinol (Copenh) 124:166–172
Malin JP, Kodding R, Fuhrmann H, von zur Muhlen A 1989 T4, T3 and rT3 levels in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis. J Neurol 236:57–59
Labudova O, Cairns N, Koeck T, Kitzmueller E, Rink H, Lubec G 1999 Thyroid stimulating hormone-receptor overexpression in brain of patients with Down syndrome and Alzheimer’s disease. Life Sci 64:1037–1044
van Doorn J, van der Heide D, Roelfsema F 1983 Sources and quantity of 3,5,3'-triiodothyronine in several tissues of the rat. J Clin Invest 72:1778–1792
Eikelenboom P, Van Gool WA 2004 Neuroinflammatory perspectives on the two faces of Alzheimer’s disease. J Neural Transm 111:281–294
Wartofsky L, Burman KD 1982 Alterations in thyroid function in patients with systemic illness: the "euthyroid sick syndrome." Endocr Rev 3:164–217
Kaplan MM 1986 Regulatory influences on iodothyronine deiodination in animal tissues. In: Henneman G, ed. Thyroid hormone metabolism. New York: Marcel Dekker; 231–253
St Germain DL, Galton VA 1997 The deiodinase family of selenoproteins. Thyroid 7:655–668
Guadano-Ferraz A, Obregon MJ, St Germain DL, Bernal J 1997 The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc Natl Acad Sci USA 94:10391–10396
Abramov AY, Canevari L, Duchen MR 2003 Changes in intracellular calcium and glutathione in astrocytes as the primary mechanism of amyloid neurotoxicity. J Neurosci 23:5088–5095
Chanoine JP, Alex S, Fang SL, Stone S, Leonard JL, Korhle J, Braverman LE 1992 Role of transthyretin in the transport of thyroxine from the blood to the choroid plexus, the cerebrospinal fluid, and the brain. Endocrinology 130:933–938
Dratman MB, Crutchfield FL, Schoenhoff MB 1991 Transport of iodothyronines from the bloodstream to brain: contributions by blood:brain and choroid:cerebrospinal fluid barriers. Brain Res 554:229–236
Palha JA, Fernandes R, de Escobar GM, Episkopou V, Gottesman M, Saraiva MJ 2000 Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin-null mouse model. Endocrinology 141:3267–3272
Serot JM, Christmann D, Dubost T, Couturier M 1997 Cerebrospinal fluid transthyretin: aging and late onset Alzheimer’s disease. J Neurol Neurosurg Psychiatry 63:506–508
Riisoen H 1988 Reduced prealbumin (transthyretin) in CSF of severely demented patients with Alzheimer’s disease. Acta Neurol Scand 78:455–459
Hennemann G, Docter R, Friesema ECH, De Jong M, Krenning EP, Visser TJ 2001 Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev 22:451–476(Simone Sampaolo, Angel Ca)