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*LIOTHYRONINE
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*Neuroblastoma
Endocrinology Vol. 139, No. 6 2692-2698
Copyright © 1998 by The Endocrine Society

ARTICLES

Thyroid Hormones Regulate ß-Amyloid Gene Splicing and Protein Secretion in Neuroblastoma Cells1

Maria Jesús Latasa2,3, Borja Belandia3 and Angel Pascual

Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, 28029 Madrid, Spain

Address all correspondence and requests for reprints to: Dr. Angel Pascual, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: apascual{at}iib.uam.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ß-amyloid protein (Aß), the major component of the senile plaques found in Alzheimer brains, derives from a larger ß-amyloid precursor protein (APP). Alternative splicing of the APP gene yields three major APP messenger RNAs (mRNAs), which, in turn, give rise to the APP770, APP751, and APP695 protein isoforms. In this study we examined the effects of thyroid hormone on APP expression in N2a-ß neuroblastoma cells. T3 caused a significant increase in the APP770 mRNA band, in detriment of the APP695 mRNA, which was proportionately reduced. In agreement with these results, T3 markedly altered the relative ratio of intracellular APP isoforms, increasing the amount of APP770 and causing an equivalent reduction of the immature APP695 isoform. In accordance with these results, the soluble APP695-derived form was specifically reduced in the culture medium obtained from T3-treated cells. In contrast, the increase in intracellular APP770 was not followed by an enhanced release of soluble derivatives of this isoform. These results suggest that T3 regulates not only APP gene splicing, but also the processing and secretion of the APP peptides. According to our results, thyroid hormone might play a role in the development of Alzheimer’s disease by modulating the intracellular and extracellular contents of APP isoforms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALZHEIMER’S disease is a degenerative disorder of the central nervous system that causes mental deterioration and progressive dementia and is accompanied by neuropathological lesions, including the presence of senile plaques, of which the ß-amyloid protein (Aß), a hydrophobic 39- to 43-residue amino acid peptide, is the major component (1, 2). As illustrated in Fig. 1Go, the ß-amyloid protein is proteolytically derived from a set of alternatively spliced ß-amyloid precursor proteins (APP) that are encoded by a single gene located on human chromosome 21 (3). Expression of APP plays a central role in Alzheimer’s disease, and it has been suggested that an increase in the production of this protein might actively contribute to the development of this pathology (4, 5, 6). The APP gene is expressed in virtually all mammalian tissues and gives rise to three major APP messenger RNAs (mRNAs) that encode for the isoforms APP695, APP751, and APP770, all of them containing the ß-amyloid peptide sequence. In addition, the 751 and 770 isoforms contain a 56-amino acid Kunitz-type protease inhibitor domain. The APP isoforms are membrane glycoproteins that are proteolytically cleaved by the action of a set of enzymes referred to generically as secretases (Fig. 1Go). The {alpha}-secretase cleaves the protein between the residues Lys16 and Leu17 of the Aß domain, precluding the generation of amyloidogenic fragments (7, 8). Proteolytic cleavage of APP by {alpha}-secretase results in the release of soluble full-length amino-terminal fragments (sAPP), which appear to be involved in neurotropic events, such as neuronal development or neurite extension (9). In contrast, the synthesis and release of amyloidogenic fragments require the successive action of the ß-secretase, which cleaves the precursor at the amino-terminus of the Aß sequence, and the {gamma}-secretase, which, in turn, cuts at positions 39–43 of this domain (10, 11, 12).



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  		Figure 1. Schematic diagram of the APP, showing its location in the membrane and the position of the alternatively spliced exons 7 (Kunitz domain) and 8 (Spl.8). The APP770 isoform contains both sequences, whereas APP751 only contains the Kunitz region. Both domains are absent in the APP695 isoform. The diagram also shows the epitopes of the various antibodies used. Cleavage sites for {alpha}, ß, and {gamma} secretases have been identified with arrows.

 
APP processing can be regulated by a variety of compounds in a cell-dependent manner. Activation of the protein kinase C or protein kinase A signal transduction pathways specifically stimulates secretory nonamyloidogenic processing, thus increasing the release of soluble fragments of APP (13, 14, 15, 16). A similar effect is induced by 17ß-estradiol, a ligand of the steroid/thyroid hormone superfamily of nuclear receptors, in a human breast carcinoma cell line (17). In contrast, overexpression of APP has been shown to induce the activity of the alternative processing that generates the ß-amyloid peptide (18).

The antiprotease activity associated with Kunitz-containing APP peptides might locally affect its own processing. Therefore, the relative ratio of Kunitz (770 and 751) to non-Kunitz (695) isoforms may also play a role in Alzheimer’s disease. In this respect, different factors, including retinoic acid, another ligand of the nuclear receptor superfamily, can specifically alter APP pre-mRNA splicing (19, 20, 21, 22) and APP processing (16, 23, 24) in a variety of cell types.

An apparent relationship between thyroid status and Alzheimer’s disease has been suggested. Hypothyroidism is accompanied by neurological symptoms that might, in a way, resemble those observed in Alzheimer’s disease. Moreover, and although a strong link between thyroid hormones and Alzheimer’s disease has not been yet established, it has been suggested that a history of thyroid dysfunction might represents a risk factor for that pathology (25, 26). In addition, thyroid hormones normally exert their action by binding to nuclear receptors, and a reduction of thyroid hormone receptor mRNA levels in Alzheimer hippocampal cells has been described (27).

In the present study, we examined the effects of T3 on the splicing and secretion of APP in N2a-ß neuroblastoma cells, a stably transfected subline of N2a cells that expresses the ß1 isoform of thyroid hormone receptor. Our results show that T3 affects not only the splicing but also the secretion of APP in neuroblastoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and antibodies
DMEM and FCS were purchased from BioWhittaker (Verviers, Belgium). Resin AG1X8 was obtained from Bio-Rad (Richmond, CA). Polyclonal antibody 369A against the cytoplasmic domain of APP (23) was a gift from Dr. Samuel E. Gandy (Cornell University Medical Center, New York, NY). The polyclonal antibody R7, recognizing the Kunitz APP region (28), was a gift from Dr. N. Robakis (Mount Sinai School of Medicine, New York, NY). The monoclonal antibody 22C11, which recognizes the amino-terminus of APP, was purchased from Boehringer Mannheim (Mannheim, Germany), and the 6E10 and 4G8 monoclonal antibodies raised against the ß-amyloid peptide were obtained from Senetek (Maryland Heights, MO). The epitopes recognized by the different antibodies are depicted at the top of Fig. 1Go. The second biotinylated antirabbit antibody and the peroxidase-conjugated streptavidin used in Western blot analysis were purchased from Amersham International (Aylesbury, UK). Reverse transcriptase and other reagents used for the PCR analysis of DNA were obtained from Promega (Madison, WI).

Cell culture
Murine N2a-ß neuroblastoma cells, a gift from Dr. Dussault (29), were cultured in DMEM supplemented with 10% FCS depleted of thyroid hormones by treatment with resin AG1X8, as previously described (30). Previous to the experiments, cells were plated in 90-mm diameter culture tissue dishes (Falcon, Becton Dickinson, Plymouth, UK), and 24–48 h later were incubated in the absence or presence of T3. At the times indicated in the text, media and cells were collected and stored frozen until posterior analysis. Parental N2a cells were grown in medium containing 10% FCS as described by Ortiz-Caro et al. (31), but the experiments were carried out in the medium containing thyroid hormone-depleted serum.

Western blot analysis
Cellular proteins were extracted by lysis with a buffer [150 mM NaCl, 50 mM Tris (pH 8), 2 mM EDTA, 1% Triton, and 0.1% SDS] containing the protease inhibitors phenylmethylsulfonylfluoride (1 mM) and leupeptin (10 µg/ml). The protein content of cells was determined by using the BCA assay (Pierce, Rockford, IL), according to the manufacturer’s instructions. Equal amounts (40 µg) of cell extracts were then electrophoresed in an 8% SDS-polyacrylamide gel and transferred to an Immobilon polyvinylidine difluoride membrane. Nonspecific binding was blocked with 5% nonfat dried milk in Tris-buffered saline and 0.1% Tween-20 for 2–3 h at room temperature, and cellular APP was detected with a 1:1500 dilution of the rabbit polyclonal antibody 369A, raised against the carboxyl-terminal domain of human APP, or with the same dilution of the polyclonal antibody R7, which specifically recognizes the Kunitz-containing isoforms of APP. After 1-h incubation at room temperature, the membrane was washed and incubated with a second biotinylated antirabbit antibody (1:2000) for an additional hour, washed again, and finally incubated for 1 h with 1:2000 peroxidase-conjugated streptavidin. All incubations took place at room temperature, and detection by enhanced chemiluminiscence (ECL, Amersham International) was carried out according to manufacturer’s indications. The monoclonal antibody 6E10, which specifically recognizes the Aß-containing APP isoforms, was used at a final concentration of 1.5 µl/ml.

Secreted full-length APP isoforms were detected by the same method from 50 µl (1:100 from total) of conditioned medium using monoclonal antibody 22C11 at a final concentration of 10 µg/ml. A minimal variation of the method was introduced to detect the 4-kDa ß-amyloid peptide in the culture medium. In this case, 50- to 100-µl aliquots of medium were electrophoresed in a 16.5% Tris-tricine gel, and the peptide was analyzed with different dilutions (0.1–1.5 µl/ml) of anti-Aß monoclonal antibodies 6E10 and 4G8.

The apparent molecular mass (kilodaltons) of the detected bands was always determined using a wide range protein standard (Mark 12 from NOVEX, San Diego. CA). The intensity of the bands was quantified by densitometric scan of the autoradiograms, and the results for a particular isoform are expressed as a percentage of the total APP, either cellular or soluble.

RT-PCR amplification of mRNA
RT-PCR was used to analyze the differential expression of APP mRNA isoforms. Two oligonucleotides that flanked exons 6 and 9 of the APP gene were used. The forward primer was 5'-TGAAGACAAAGTAGTAGAAGT-3', and the reverse primer was 5'-ACCTGGGACATTCTCTCTCGGTGCTTGGC-3'. Total RNA was extracted from cell cultures by the guanidine thiocyanate method (32), and first strand complementary DNA (cDNA) synthesis was performed on 5–6 µg RNA using 20 U AMV reverse transcriptase and 100 ng reverse primer. The PCR reaction was set up using the cDNA previously obtained in a standard 100-µl buffer containing 5 U Taq polymerase and 100 ng of the appropriate primers. These primers were chosen to generate fragments of 562, 505, and 337 bp corresponding to the three major mRNA isoforms. Samples were subjected to 25–30 cycles of amplification (denaturation at 94 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 2 min). PCR products were analyzed on 3% NuSieve-GTG agarose and visualized by ethidium bromide staining.

Statistics
Unless indicated otherwise, all data points are the mean of at least duplicate cultures that did not normally vary by more than 10–15% or are the mean ± SEM of the results obtained in at least three separate experiments performed in duplicate. The significance of differences was calculated with Student’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of T3 on the cell-associated APP protein
Cellular APP isoforms were detected from cell lysates by Western blot using antibody 369A. As illustrated in Fig. 2AGo, 5Go nM T3 induced a significant change in the pattern of intracellular APP species, mainly affecting the 95- and 125-kDa bands. Based upon earlier reports (23, 24, 33), the smallest 95-kDa band could represent the immature APP695 isoform, whereas the lowest mobility bands more likely represent the mature Kunitz-containing APP770 and APP751 species. According to this, T3 consistently induces a reduction of immature APP695 as well as an increase in the mature APP770 form after 24–48 h of treatment. In addition, the intermediate bands do not appear to be affected by the thyroid hormone. However, a complete definition of those intermediate bands has not been possible, and a specific modification of one of those species cannot be definitely discarded. Figure 2BGo illustrates the densitometric quantitation of the different bands at 48 h of treatment. The intensity of the 95-kDa band, which represents the immature APP695 isoform, was reduced to less than 50% of the control value, whereas the 125-kDa band (APP770) was increased by more than 2-fold in the cells after T3 treatment. As indicated above, the bands from 105–118 kDa, which were considered a unique species in the densitometric analysis, were not affected by T3. No effects were detected either in N2a-ß cells before 12 h of T3 treatment or in parental N2a cells, in which the nuclear T3 receptor is expressed at very low concentrations, at any of the time periods analyzed (data not shown).



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  		Figure 2. Western blot analysis of cell-associated isoforms of APP. Cellular proteins were extracted by lysis from cells previously incubated in the absence or presence of 5 nM T3. At the indicated times, samples containing 40 µg protein were electrophoresed in an 8% polyacrylamide gel and transferred to an Immobilon membrane. The immunoreactive bands were analyzed with carboxyl-terminal antibody 369A and visualized by ECL. A, Autoradiogram of a representative experiment. The apparent sizes (kilodaltons) of bands are indicated at the left. B, Densitometric analysis of bands obtained from cells incubated with T3 for 48 h. The intermediate bands containing the 105- to 118-kDa isoforms have been considered as a single species. Data are the average ± SE from three separate experiments performed in duplicate and are expressed as a percentage of the total content of immunoreactive bands. Controls are untreated N2a-ß cells. In this and subsequent figures: *, P < 0.05; **, P < 0.01.

 


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  		Figure 5. Regulation of APP secretion by T3. After 24 or 48 h of incubation of N2a-ß cells in the absence or presence of 5 nM T3, the secreted APP isoforms were analyzed by Western blot with monoclonal antibody 22C11 in 50-µl aliquots (1% of total) of conditioned medium. A, Autoradiogram for a representative experiment. The exposure time was longer for the 24-h culture groups. B, Densitometric quantification of the Kunitz-containing band and the 93-kDa band (APP695 derivative) in the culture medium after 48 h of incubation. Data are the average ± SE from three separate experiments performed in duplicate and are expressed as a percentage of the total APP secreted forms.

 
The effect of T3 on the APP770 isoform was confirmed in two separate experiments performed with the R-7 antibody, which specifically recognizes the Kunitz-containing APP isoforms (28). Identical results were obtained in both experiments. As illustrated in Fig. 3Go, thyroid hormone induces a specific increase in the slowest mobility band, which corresponds to the APP770 isoform. The effect was observed after 24–48 h of treatment with 0.1 or 5 nM T3, although the increase was less marked with the lowest hormone concentration. As expected, the results were essentially identical to those observed using the 369A antibody, and the Kunitz-containing APP770 was increased by approximately 2-fold in N2a-ß cells incubated in the presence of 5 nM T3 for 48 h.



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  		Figure 3. Western blot analysis of Kunitz-containing isoforms in N2a-ß cells. Proteins obtained from N2a-ß cells incubated in the absence (control) or presence of 0.1 or 5 nM T3 for 24–48 h were analyzed with R7 antibody. A, A characteristic autoradiogram; B, densitometric quantitation of the slowest mobility band (APP770) in the group of cells incubated for 48 h. Each bar in B is representative of four datasets (two separate experiments performed in duplicate) in control and 5 nM T3-treated cells and is representative of only two datasets (duplicates of one experiment) in the 0.1 nM T3-treated cells. The cellular content of APP770 has been expressed as a percentage of the total APP detected.

 
Antibody 369A can also recognize different isoforms of APLP, an amyloid precursor-like protein that is identical to APP except for the lack of the Aß sequence. One of these isoforms, which contains 695 residues (APLP695), might run together with APP695. Cultured neuronal cells normally express very low levels of APLP695 (34), and it is unlikely that this isoform could interfere and mask the effect of T3 on APP in N2a-ß cells. However, to definitely establish the identity and regulation of APP695, we performed additional experiments using monoclonal antibody 6E10, which specifically recognizes APP, but not APLP, isoforms. As shown in Fig. 4AGo, antibody 6E10 detects a band identical to the 95-kDa species observed with the 369A antibody. As illustrated in Fig. 4BGo, the regulation of this band is also identical to that obtained with antibody 369A. These results confirm those presented in Fig. 2Go, and although we cannot discard a possible contribution of APLP forms to the species detected with the antibody 369A, it can be concluded that T3 specifically decreases the cellular content of APP695.



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  		Figure 4. Specific detection of APP isoforms with monoclonal antibody 6E10. Cellular proteins were extracted by lysis from N2a-ß cells incubated in the absence (control) or presence of 5 nM T3 for 24–48 h, and aliquots of 40 µg protein were analyzed by Western blot with monoclonal antibody 6E10. Results were essentially identical in two separate experiments. A, Autoradiogram of a representative experiment. The apparent size (kilodaltons) of the highest mobility band is indicated at the right by an arrow. B, Densitometric analysis of the 95-kDa band obtained from cells after 48 h of incubation.

 
Influence of T3 on the accumulation of APP secreted forms into the culture medium
The full-length soluble derivatives of APP released into the culture medium were analyzed with monoclonal antibody 22C11. A similar pattern of bands was observed at the two time points studied, 24 and 48 h. As shown in Fig. 5Go, incubation of cells with 5 nM T3 resulted in a significant and specific reduction of the 93-kDa band, which represents the sAPP695 species accumulated in the medium. As observed in Fig. 5BGo, the intensity of the 93-kDa band was reduced to approximately 50% of the control value after 48 h of incubation with T3. As occurred with the cell-associated APPs, these effects were not observed in the parental N2a cell line. However, and contrary to the effect observed on the intracellular pattern of proteins, no significant differences in the intensity of the soluble Kunitz-containing bands were observed at 24 or 48 h between the T3 and control groups. Taken together, these results suggest that thyroid hormones may selectively affect not only the processing but also the release of APP.

Effects of T3 on APP mRNA isoforms
To examine whether the specific changes induced by T3 on the cellular APP isoforms were the result of changes in the alternative splicing of the APP mRNA precursor, we analyzed the relative ratios of the different mRNA isoforms by RT-PCR. The alternative splicing pattern of exons 7 and 8 was studied using primers that amplify sequences between exons 6 and 9. Three major bands were detected at the expected positions of 562 bp (APP770), 505 bp (APP751), and 337 bp (APP695). Figure 6Go illustrates the results obtained in a representative experiment in which the cDNA was amplified through 30 cycles. Similar results were obtained in an additional experiment performed under the same conditions and in two separate experiments performed with only 25 cycles. Although under our conditions the RT-PCR analysis is not quantitative, the relative abundance of bands was essentially identical after 25 or 30 cycles, thus indicating that in that phase the intensity of bands is proportional to the initial concentration of the corresponding isoforms.



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  		Figure 6. Amplification and analysis of RNA by RT-PCR. The presence of the transcripts that encode the different APP isoforms was analyzed by RT-PCR in cells incubated in the absence (0 h) or presence of 5 nM T3 for 48 and 72 h. The figure illustrates duplicates of a representative experiment. Three bands of 562, 505, and 337 bp, which correspond to the major mRNA isoforms (770, 751, and 695), were observed.

 
As shown in Fig. 6Go, incubation of cells for 48 h with 5 nM T3 significantly altered the splicing pattern, decreasing the content of APP695 in favor of the APP770 form. Thus, T3 treatment led to a pattern of isoforms that, to some extent, resembled that in nonneuronal cells. The effect was not yet evident at 24 h of incubation (data not shown), but it was maintained after 72 h of treatment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Expression and metabolism of APP play a central role in the development of Alzheimer’s disease. It has been previously described that ligands of nuclear receptors, such as 17ß-estradiol or retinoic acid, the active metabolite of vitamin A, are involved in the splicing and/or processing of APP (17, 20, 21, 22). In this study, we have demonstrated that T3, another ligand of the nuclear receptor superfamily, could also play an important role in the regulation of these processes.

T3, the more active form of the thyroid hormones, specifically alters APP gene splicing in N2a-ß neuroblastoma cells, decreasing the relative amount of APP695 mRNA in favor of APP770 mRNA, which encodes a Kunitz-containing form. The changes induced by T3 give rise to a pattern of APP mRNA that, to some extent, resembles that of peripheral and nonneuronal cells.

The precise mechanism(s) involved in the regulation of APP by this hormone remains unclear. However, the lack of effect of T3 on APP expression in the parental N2a cell line, which expresses very low levels of hormone receptor (31), strongly suggests a receptor-mediated mechanism. The effects induced by the thyroid hormone on the pattern of APP mRNA isoforms could be secondary to the regulation of specific factors involved in the alternative splicing of the gene. In this respect, thyroid hormone as well as retinoic acid can regulate the expression of SmN, a specific splicing protein that is only expressed in adult heart and brain (35). Although the precise function of this protein remains unclear, this finding suggests a potential role of this or another yet unidentified splicing factor in the response of neuronal cells to thyroid hormones.

T3 alters the intracellular pattern of the APP isoforms, leading to a significant reduction of the immature non-Kunitz APP695 isoform and an increase in the Kunitz-containing mature forms. This pattern of intracellular proteins shows a good correlation with the T3-induced distribution of APP mRNA isoforms and is probably the direct result of the alternative mRNA splicing. In contrast, the parallelism between the mRNA and cellular holoprotein patterns could not be extended to the T3-induced changes in the sAPP isoforms released to the culture medium. Treatment of N2a-ß cells with T3 leads to a diminished accumulation of sAPP695, but not to an increase in other soluble isoforms.

Additionally, efforts have been made to detect the amyloid peptide in the medium. Unfortunately, the levels of Aß produced from endogenous APP in N2a-ß cells are extremely low, and this precludes a reliable observation of the peptide.

As the pattern of released forms in the T3-treated cells did not correlate with the pattern of cellular APP, it might be speculated that T3 can regulate the synthesis and/or activity of some protease(s) involved in the metabolism of this precursor. In this respect, it has been reported that tacrine, a drug extensively used for the treatment of Alzheimer’s disease, effectively reduces the secretion of soluble APP derivatives by a mechanism that probably involves inhibition of an acetylcholinesterase-associated proteolytic activity (36). As the effects of tacrine in human neuroblastoma IMR-32 cells in part resemble those elicited by T3 in N2a-ß cells, a similar mechanism might be proposed. However, the differential effect exerted by T3 on the secretion of soluble APP695 and APP770/751 into the culture medium suggests a more complex mechanism involving additional effects of the thyroid hormones in the regulation of sAPP secretion. Further experiments will be necessary to clarify the mechanisms by which thyroid hormones regulate the processing and release of APP isoforms.

The relevance of our results for the management of Alzheimer’s patients is still unclear at present. On the one hand, it has been reported that isoforms containing the Kunitz protease inhibitor domain are preferentially expressed in the Alzheimer brain (37), whereas the 695 form is reduced (4). According to these descriptions, the thyroid hormones should be considered a risk factor because they induce a pattern of APP in which the Kunitz-containing forms are preferentially expressed. On the other hand, it has been suggested that deposition of ß-amyloid in senile plaques may be mainly derived from APP695. In agreement with these reports, thyroid hormones might protect against the amyloid deposition by decreasing the presence of APP695 and its soluble derivative sAPP isoform into the cells and the extracellular space, respectively.

It is evident that new experiments should be performed to fully understand the role of thyroid hormone in Alzheimer’s disease. However, the results described in the present work support the idea that thyroid hormones might play a role in this pathology by modulating the cellular and extracellular content of APP transcripts through a mechanism that probably involves binding to the nuclear receptor and expression of genes that encode different splicing factors that must be identified in future studies. In addition, and as T3 receptor mRNA levels are decreased in Alzheimer hippocampal cells (27), the cellular sensitivity to T3 might be affected in Alzheimer’s disease.


   Acknowledgments
 
We thank S. E. Gandy for providing the polyclonal antibody 369A, and Drs. J. Puymirat and J. H. Dussault for the N2a-ß cells.


   Footnotes
 
1 This work was supported by grants from the Dirección General de Investigación Científica y Técnica (PB93–0135), Comisión Interministerial de Ciencia y Tecnología (SAF 97–0183), and Fondo de Investigación Sanitaria (94/0272). Back

2 Recipient of a fellowship from the Departamento de Educación y Cultura del Gobierno de Navarra. Back

3 M.J.L. and B.B. contributed equally to this work, and each of them should be considered as primary author. Back

Received November 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Glenner GG, Wong CW 1984 Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120:885–890[CrossRef][Medline]
  2. Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K 1985 Amyloid plaque core protein in Alzheimer’s disease and Down syndrome. Proc Natl Acad Sci USA 82:4245–4249[Abstract/Free Full Text]
  3. Selkoe DJ 1994 Cell biology of the amyloid ß-protein precursor and the mechanism of Alzheimer’s disease. Annu Rev Cell Biol 10:373–403[CrossRef]
  4. Neve RL, Finch EA, Dawes LR 1988 Expression of the Alzheimer amyloid precursor gene transcripts in the human brain. Neuron 1:669–677[CrossRef][Medline]
  5. Yoshikawa K, Aizawa T, Hayashi Y 1992 Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor. Nature 359:64–67[CrossRef][Medline]
  6. Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, Guido T, Hagopian S, Johnson-Wood K, Khan K, Lee M, Leibowith P, Lieberburg I, Little S, Masliah E, McConlogue L, Montoya-Zavala M, Mucke L, Paganini L, Penniman E, Power M, Schenk D, Seubert P, Snyder B, Soriano F, Tan H, Vitale J, Wadsworth S, Wolozin B, Zhao J 1995 Alzheimer-type neuropathology in transgenic mice overexpressing V717F ß-amyloid precursor protein. Nature 373:523–527[CrossRef][Medline]
  7. Esch FS, Keim PS, Beattie EC, Blacher RW, Culwell AR, Oltertsdorf T, McClure D, Ward PG 1990 Cleavage of amyloid ß peptide during constitutive processing of its precursor. Science 248:1122–1124[Abstract/Free Full Text]
  8. Sisodia SS, Koo EH, Beyreuther K, Unterbeck A, Price DL 1990 Evidence that ß-amyloid protein in Alzheimer’ disease is not derived by normal processing. Science 248:492–495[Abstract/Free Full Text]
  9. Furukawa K, Barger SW, Blalock EM, Mattson MP 1996 Activation of K+ channels and suppression of neuronal activity by secreted beta-amyloid-precursor protein. Nature 379:74–78[CrossRef][Medline]
  10. Seubert P, Oltertsdorf T, Lee MG, Barbour R, Blonquist C, Davis DL, Bryant K, Fritz LC, Galasko D, Thal LJ, Lieberburg L, Shenk DB 1993 Secretion of ß-amyloid precursor protein cleaved at the amino-terminus of the ß-amyloid peptide. Nature 361:260–263[CrossRef][Medline]
  11. Shoji M, Golde TE, Ghiso J, Cheung TT, Estus S, Shaffer LM, Cai XD, McKay DM, Tintner R, Frangione B, Younkin SG 1992 Production of the Alzheimer amyloid ß protein by normal proteolytic processing. Science 258:126–129[Abstract/Free Full Text]
  12. Haass C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A, Ostaszewski BL, Lieberburg I, Koo EH, Schenk D, Teplow DB, Selkoe DJ 1992 Amyloid ß-peptide is produced by cultured cells during normal metabolism. Nature 359:322–325[CrossRef][Medline]
  13. Efthimiopoulos S, Punj S, Manolopoulos V, Pangalos M, Wang GP, Refolo LM, Robakis NK 1996 Intracellular cyclic AMP inhibits constitutive and phorbol ester-stimulated secretory cleavage of amyloid precursor protein. J Neurochem 67:872–875[Medline]
  14. Xu H, Sweeney D, Greengard P, Gandy S 1996 Metabolism of Alzheimer ß-amyloid precursor protein: regulation by protein kinase A in intact cells and in a cell-free system. Proc Natl Acad Sci USA 93:4081–4084[Abstract/Free Full Text]
  15. Hung AY, Haass C, Nitsch RM, Qiu WQ, Citron M, Wurtman RJ, Growdon JH, Selkoe DJ 1993 Activation of protein kinase C inhibits cellular production of the amyloid ß-protein. J Biol Chem 268:22959–22962[Abstract/Free Full Text]
  16. Gabuzda D, Busciglio J, Yankner BA 1993 Inhibition of ß-amyloid production by activation of protein kinase C. J Neurochem 61:2326–2329[Medline]
  17. Jaffe AB, Toran-Allerand CD, Greengard P, Gandy S 1994 Estrogen regulates metabolism of Alzheimer amyloid ß-precursor protein. J Biol Chem 269:13065–13068[Abstract/Free Full Text]
  18. Fucuchi K, Kamino K, Deeb SS, Smith AC, Dang T, Martin GM 1992 Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity. Biochem Biophys Res Commun 182:165–173[CrossRef][Medline]
  19. Fukuyama R, Chandrasekaran K, Rapoport SI 1993 Nerve growth factor-induced neuronal differentiation is accompanied by differential induction and localization of the amyloid precursor protein APP in PC12 cells and variant PC12S cells. Mol Brain Res 17:17–22[Medline]
  20. König G, Masters CL, Beyreuther, K 1990 Retinoic acid induced differentiated neuroblastoma cells show increased expression of the ßA4 amyloid gene of Alzheimer’s disease and an altered splicing pattern. FEBS Lett 269:305–310[CrossRef][Medline]
  21. Hung AY, Koo EH, Haass C, Selkoe DJ 1992 Increased expression of ß-amyloid precursor protein during neuronal differentiation is not accompanied by secretory cleavage. Proc Natl Acad Sci USA 89:9439–9443[Abstract/Free Full Text]
  22. Pan JB, Monteggia LM, Giordano T 1993 Altered levels and splicing of the amyloid precursor protein in the adult rat hyppocampus after treatment with DMSO or retinoic acid. Mol Brain Res 18:259–266[Medline]
  23. Buxbaum JD, Gandy SE, Cicchetti P, Ehrlich ME, Czernik AJ, Fracasso RP, Ramabhadran TV, Unterbeck AJ, Greengard P 1990 Processing of Alzheimer ß/A4 amyloid precursor protein: modulation by agents that regulate protein phosphorylation. Proc Natl Acad Sci USA 87:6003–6006[Abstract/Free Full Text]
  24. Buxbaum JD, Oishi M, Chen HI, Pinkas-Kramarski R, Jaffe EA, Gandy S, Greengard P 1992 Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer ß/A4 amyloid protein. Proc Natl Acad Sci USA 89:10075–10078[Abstract/Free Full Text]
  25. Heyman A, Wilkinson WE, Hurwith BJ, Schmechel D, Sigmon AH, Weinberg T, Helms MJ, Swift M 1983 Alzheimer’s disease: genetic aspects and associated clinical disorders. Ann Neurol 14:507–515[CrossRef][Medline]
  26. Mortimer JA 1989 Genetic and environmental risk factors for Alzheimer’s disease: key questions and new approaches. In: Mortimer JA, Altman HJ, Altomar BN (eds) Alzheimer’s and Parkinson Diseases. Plenum Press, New York, pp 85–100
  27. Sutherland MK, Wong L, Somerville MJ, Handley P, Yoong L, Bergeron C, Crapper McLachlan DR 1992 Reduction of thyroid hormone receptor c-erbA{alpha} mRNA levels in the hippocampus of Alzheimer as compared to Huntington brain. Neurobiol Aging 13:301–312[CrossRef][Medline]
  28. Refolo LM, Salton SRJ, Anderson PM, Robakis NK 1989 Nerve and epidermal growth factors induce the release of the Alzheimer amyloid precursor from PC12 cell cultures. Biochem Biophys Res Commun 164:664–670[CrossRef][Medline]
  29. Lebel JM, Dussault JH, Puymirat J 1994 Overexpression of the ß1 thyroid receptor induces differentiation in neuro-2a cells. Proc Natl Acad Sci USA 91:2644–2648[Abstract/Free Full Text]
  30. Samuels HH, Stanley F, Casanova J 1979 Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105:80–85[Abstract]
  31. Ortiz-Caro J, Yusta B, Montiel F, Aranda A, Pascual A 1986 Identification and characterization of L-triiodothyronine receptors in cells of glial and neuronal origin. Endocrinology 119:2163–2167[Abstract]
  32. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  33. Weidemann A, König G, Bunke, D, Fisher P, Salbaum JM, Masters CL, Beyreuther K 1989 Identification, biogenesis and localization of precursors of Alzheimer’s disease A4 amyloid protein. Cell 57:115–126[CrossRef][Medline]
  34. Sandbrink R, Master CL, Beyreuther K 1994 Similar alternative splicing of a non-homologous domain in ßA4-amyloid protein precursor-like proteins. J Biol Chem 269:14227–14234[Abstract/Free Full Text]
  35. Gerrelli D, Huntriss JD, Latchman DS 1994 Antagonistic effects of retinoic acid and thyroid hormone on the expression of the tissue-specific splicing protein SmN in a clonal cell line derived from rat heart. J Mol Cell Cardiol 26:713–719[CrossRef][Medline]
  36. Lahiri DK, Lewis S, Farlow MR 1994 Tacrine alters the secretion of the beta-amyloid precursor protein in cell lines. J Neurosci Res 37:777–787[CrossRef][Medline]
  37. Johnson SA, McNeill T, Cordell B, Finch CE 1990 Relation of neuronal APP-751/APP-695 mRNA ratio and neuritic plaque density in Alzheimer’s disease. Science 248:854–857[Abstract/Free Full Text]



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