Tau Is Hyperphosphorylated in the Insulin-Like Growth Factor-I Null Brain
http://www.100md.com
《内分泌学杂志》
Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Abstract
IGF action has been implicated in the promotion of oxidative stress and aging in invertebrate and murine models. However, some in vitro models suggest that IGF-I specifically prevents neuronal oxidative damage. To investigate whether IGF-I promotes or retards brain aging, we evaluated signs of oxidative stress and neuropathological aging in brains from 400-d-old Igf1–/– and wild-type (WT) mice. Lipofuscin pigment accumulation reflects oxidative stress and aging, but we found no difference in lipofuscin deposition in Igf1–/– and WT brains. Likewise, there was no apparent difference in accumulation of nitrotyrosine residues in Igf1–/– and WT brains, except for layer IV/V of the cerebral cortex, where these proteins were about 20% higher in the Igf1–/– brain (P = 0.03). We found no difference in the levels of oxidative stress-related enzymes, neuronal nitric oxide synthase, inducible nitric oxide synthase, and superoxide dismutase in Igf1–/– and WT brains. Tau is a microtubule-associated protein that causes the formation of neurofibrillary tangles and senile plaques as it becomes hyperphosphorylated in the aging brain. Tau phosphorylation was dramatically increased on two specific residues, Ser-396 and Ser-202, both glycogen synthase kinases target sites implicated in neurodegeneration. These observations indicate that IGF-I has a major role in regulating tau phosphorylation in the aging brain, whereas its role in promoting or preventing oxidative stress remains uncertain.
Introduction
RECENT STUDIES IN Caenorhabditis elegans and mice have implicated the insulin/IGF system in reduced longevity and oxidative stress resistance (1, 2). Reduced expression of insulin, IGF-I, or downstream signaling molecules extend the life span significantly (1, 3, 4, 5). Also, mice heterozygous for IGF-I receptor gene deletion display increased resistance to oxidative stress and increased lifespan (6). However, IGF-I appears to protect neurons from oxidative stress in vitro (7, 8, 9). In addition, reduction in circulating and brain IGF-I levels have been associated with aging and neurodegenerative conditions, and IGF-I has been suggested as a therapeutic agent in amyotrophic lateral sclerosis and Alzheimer’s disease (10, 11, 12). Thus, it has been suggested that increasing IGF-I may ameliorate the deterioration of brain aging and neurodegenerative disorders.
In this study, we used IGF-I gene-targeted mice (Igf1–/–) to investigate whether IGF-I promotes or retards brain aging. Age-related neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases are strongly correlated with oxidative stress. Hence, we examined biomarkers of oxidative damage, such as lipofuscin and nitrotyrosine residues in aged Igf1–/– and WT brains. Lipofuscin consists of intracellular autofluorescent pigments that are deposited in neuronal soma and processes during the aging process (13). The accumulation of nitrotyrosine residues is an indicator of the generation of reactive nitrogen species during aging progression. We also examined enzymes that regulate oxidative activity, such as superoxide dismutase (SOD) and nitric oxide synthase (NOS). SOD catalyzes the conversion of superoxide radicals to H2O2, which is converted to H2O by peroxidase, protecting brain cells against oxidative stress (14). NOS, on the other hand, catalyzes synthesis of nitric oxide (NO), which then reacts with superoxide to form toxic peroxinitrite, causing cell damage during aging (15). In addition, brain aging is associated with increased phosphorylation of tau, a microtubule-associated protein involved in microtubule assembly and stabilization. Tau hyperphosphorylation is believed to lead to its disengagement from microtubule, destabilizing microtubule and disrupting normal microtubule-dependent processes (16). The unbound tau is thought to be more resistant to degradation and more prone to aggregation, culminating in the formation of neurofibrillary tangles (NFTs) (16, 17). Hyperphosphorylated, insoluble tau in the form of NFTs is associated with cognitive dysfunction in normal aging as well as a variety of late onset neurodegenerative disorders termed "tauopathies" (16, 18). We therefore evaluated tau protein levels and phosphorylation status in the aged Igf1–/– and WT control brains to elucidate IGF-I’s role in regulation of this important neuropathological factor.
Materials and Methods
Animals
The mice used in this study were from an Igf1 deletion line derived and genotyped as previously described (19). Animals were studied under protocols approved by the NICHD Animal Use and Care Committee. Male Igf1–/– mice and age-sex-matched littermates were euthanized at postnatal d 400 in CO2 chamber followed by decapitation. Brains were rapidly dissected, cut along the sagittal midline, and snap frozen. One brain half was used for protein preparation, and the other half was sectioned for histochemistry. Sections 10-μm thick were cut sagittally at –20 C, thaw-mounted onto poly-L-lysine-coated slides, and stored at –70 C until use.
Lipofuscin detection
Frozen sections were thawed and directly mounted with DAPI media (Vector Laboratories, Burlingame, CA). The sections were then examined directly under fluorescent microscope. The intracellular autofluorescent pigments known as lipofuscin gave a bright yellow fluorescent signal, whereas DAPI-stained nuclei gave a blue fluorescent signal. For the Sudan Black B staining, frozen sections were first fixed in 4% formaldehyde for 5 min, rinsed in water, and immersed in Sudan Black B reagent (Sigma Diagnostics, St. Louis, MO) overnight. After quick differentiation in 70% EtOH, the slides were washed in 50, 30% EtOH, water and then counterstained in Nuclear fast red (Sigma Diagnostics) for 3 min.
Immunohistochemistry
Immunohistochemistry was performed by the avidin-biotin-immunoperoxidase method (20). Briefly, frozen sections were fixed in 4% formaldehyde for 10 min then washed in 1x Tris/NaCl buffer [50 mM Tris/150 mM NaCl/0.1% Triton (pH 7.5)]. To remove endogenous peroxidase activity, slides were placed in 3% H2O2 solution for 10 min and then washed in the Tris/NaCl buffer. Nonspecific signal was blocked by incubating slides with 10% normal goat serum in blocking solution [1% BSA/0.02% Na-Azide/0.05% Triton X-100 in Tris/NaCl buffer (pH 7.5)] for 45 min. The sections were then incubated at 4 C with antinitrotyrosine antibody (1:500; Chemicon, Temecula, CA) overnight. After washing, the sections were incubated with biotinylated secondary antibodies (1:400; New England Biolabs, Beverly, MA) at room temperature for 40 min. The signal was amplified using the ABC peroxidase method (Vector Laboratories) and visualized with 3,3'-diaminibenzidine (Vector Laboratories). The semiquantitation of nitrotyrosine immunosignals was carried out in a blinded fashion. The signals were captured at x200 using a video camera and analyzed using NIH image software (Image 1.57, NIH) in several brain structures, including frontal cortex layer II/III, layer VI/V, thalamus (ventrolateral thalamic nuclei and ventramedial thalamic nuclei), hypothalamus (lateral hypothalamus area) and striatum (accumbens nuclei). Background signal from each slide was subtracted before further analysis. Two sections from each brain were scored and n = 4 and 5 for Igf1–/– and WT controls, respectively. Differences between groups were compared by ANOVA followed by Fisher’s least significant different tests.
Immunoblotting
Immunoblotting was performed according to Cheng et al. (21). Mouse brains (0.4 g) were homogenized in a boiling solution containing 10 mM Tris (pH 7.4), 1 mM sodium orthovanadate, and 1% sodium dodecyl sulfate at a ratio of 1 g tissue to 17.5 ml solution. Ten microliters of each sample were loaded and resolved on NuPAGE 10% Bis-Tris Gels (Invitrogen Life Technologies, Carlsbad, CA) and transferred to nitrocellulose membranes using electrophoretic transfer cells (Bio-Rad, Hercules, CA). To ensure equal loading and transfer efficiency, the membranes were prestained with Ponceau S solution (Sigma) before immunodetection. Primary antibodies were purchased and diluted to use as follows: antiphospho-tau-396 (1:500; Zymed Laboratories, South San Francisco, CA), neuronal NOS (nNOS) and inducible NOS (iNOS) (1:1000; Chemicon International, Temecula, CA), SOD1 (1:500; Chemicon), tau-1 (1:500; Chemicon), AT-8 (1:100; Pierce Endogen, Rockford, IL), 12E8 (1:5000; provided by Dr. Peter Seubert at Elan Pharmaceuticals, Inc., San Francisco, CA) and tau-5 (1:500; Biosource International Inc., Camarillo, CA). After incubation with primary antibodies for 90 min and washed, the membranes were incubated with horseradish peroxidase-linked secondary antibodies for 60 min. Protein bands were then visualized using SuperSignal West Pico detection reagents (Pierce, Rockford, IL) on a Kodak image machine (Kodak, Rochester, NY). Digital image of the results from each blot was obtained, and the intensity of protein bands revealed by each specific antibody was compared and analyzed using Kodak 1D image-analysis software. Differences between groups were compared by ANOVA followed by Fisher’s least significant different tests (n = 3–4 for Igf1–/– brains and n = 5 for WT controls). All the immunoblottings were repeated at least once, and the data were confirmed.
In situ hybridization
The tau clone (Image clone ID no. 746947, ATCC no. 1015357) was obtained from American Type Culture Collection (ATCC, Manassas, VA). This tau cDNA clone was sequenced and confirmed to contain the complete mouse tau coding region and partial 5' and 3' untranslated sequences. The cDNA clone was linearized with EcoRI for use as a template to synthesize antisense cRNA probe. The cRNA probe synthesis and in situ hybridization protocol were detailed previously (22). After hybridization, slides were exposed to Kodak Bio-Max MR film (Eastman Kodak, Rochester, NY) for 1 d and later dipped in Kodak NTB2 emulsion for 2 d. Parallel sections were hybridized to sense probes and processed together with antisense hybridization sections to serve as hybridization background control. Background signal from a sense probe was subtracted from raw data before further analysis. Film image was analyzed by NIH image program (Image 1.57, NIH). The quantitation of hybridization signal was carried out in a blinded fashion. Four measurements in each brain structure, including frontal cortex, thalamus and granule cell layer of cerebellum were taken for each animal to obtain group means. Hybridization signals on CA2 and CA3 of hippocampus were captured using a monochrome video camera under x400 magnification and silver grains were counted with assistance of NIH image version 1.57 software. Statistical significance between two groups was compared by ANOVA followed by Fisher’s least significant different tests (n = 4 for Igf1–/– brains and n = 5 for WT control).
Results
IGF-I null mice
The Igf1–/– mice have a very high perinatal mortality, largely due to hypoventilation (19). The mice that survive are dwarfs, reaching approximately 30–40% of WT size, depending on the strain (23). Whereas behavior is difficult to measure precisely in dwarfs, our study did not find any evidence for major neurological defects (20).
Lipofuscin
Accumulation of autofluorescent lipoprotein pigments (lipofuscin) in nerve cells is a typical feature of brain aging. Thus, we examined whether IGF-I deletion affected the lipofuscin deposition in aged brains by comparing sections from Igf1 gene-deleted and age-matched WT mice. There were no apparent differences in the distribution or levels of autofluorescent signals in 400-d-old Igf1–/– brains compared with WT control brains (Fig. 1). This result was confirmed by lipid staining using Sudan Black B (data not shown).
Nitrotyrosine residues
To address whether IGF-I modulates brain oxidative stress, we investigated the deposition of nitrotyrosine residues (NT), the end-products of oxidative reactions. We found that NT distribution was similar in Igf1–/– and WT brains (Fig. 2). For example, NT immunostaining was apparent in Purkinje cells (Fig. 2, A and B) and in pyramidal neurons of the cerebral cortex (Fig. 2, C and D). The intensity of the immunostaining appeared similar in Purkinje cells, although they were markedly smaller in the Igf1–/– brain. Semiquantitation of immunostaining in cortical pyramids suggested a modest but significant increase in NT accumulation (P = 0.03) in cortical layer IV/V of Igf1–/– brains (Fig. 2E). However, NT levels were similar in Igf1–/– and WT brains in the thalamus, hypothalamus, striatum, and cerebellum (not shown).
Oxidation regulatory enzymes
We also evaluated enzymes involved in production of nitric oxide, nitric oxide synthetases (NOS) in these brains by immunoblotting (Fig. 3). We found similar levels of nNOS and iNOS in Igf1–/– and WT brains. There was a trend toward higher iNOS levels in Igf1–/– brains, but the difference between groups did not reach statistical significant (Fig. 3). SOD1 levels were also similar in Igf1–/– and WT brains (Fig. 3).
Tau
NFTs accumulate during brain aging as a result of increased phosphorylation of the microtubule-associated protein tau (17). To examine the effect of IGF-I deletion on tau phosphorylation in aged brains, we used Western blotting to evaluate the phosphorylation status of tau in Igf1–/– and WT brains. Anti-tau-396 antibodies recognize tau phosphorylated at serine 396, a site predominantly phosphorylated by GSK-3 (glycogen synthase kinase 3) and prominent in pathological paired helical filaments (PHF) of neurofibrillary tangles (24). Tau phosphorylation at this specific epitope was increased by approximately 7-fold in the Igf1–/– brain compared with WT (P = 0.004) (Fig. 4). Another phospho-tau-specific antibody, AT-8, which recognizes PHF-associated tau, phosphorylated at serine 202, also detected a significant approximately 10-fold increased in Igf1–/– brain homogenate compared with WTs (P = 0.02). However, tau phosphorylation at serine 262 detected by 12E8 antibody showed no difference between Igf1–/– and WT brains. Total tau levels detected by the phospho-independent antibody, tau-5, were similar in Igf1–/– and WT brains (Fig. 4). Consistent with the above observations, the nonphosphorylated tau population, as detected by anti-tau-1 antibody (25, 26), was reduced in the Igf1–/– aged brains (P < 0.01). This result shows that tau is heavily phosphorylated on two serine sites in the brains of animals with IGF-I deficiency, suggesting that IGF-I normally prevents tau hyperphosphorylation at these specific sites.
To further investigate tau expression in Igf1–/– and WT mice, we performed in situ hybridization on anatomically matched brain sections. The anatomical distribution of tau mRNA was similar in Igf1–/– and WT mice (Fig. 5). Tau mRNA was concentrated in the dentate gyrus and Ammon’s horn (Fig. 5, A and B) and in the granule cell layer as well as Purkinje cells of cerebellum (Fig. 5, C and D). Quantitative analyses in temporal cortex, thalamus, granule cell layer of cerebellum and CA2 and CA3 of hippocampus revealed that tau mRNA levels were similar in Igf1–/– and WT brains (data not shown).
Discussion
This study investigated the effects of IGF-I deletion on concomitants of brain aging, namely accumulation of oxidation by-products and tau phosphorylation. We found that aged Igf1–/– brains have similar levels of nitrotyrosine residues and lipofuscin deposition compared with WT brains. We found no difference in levels of enzymes involved in oxidative stress, such as NOS and SOD1, in Igf1–/– and WT control brains. These observations suggest that IGF-I normally has little effect on oxidative damage during brain aging. However, we found that the microtubule-associated protein tau is much more highly phosphorylated in the Igf1 null brain, suggesting that IGF-I normally prevents tau hyperphosphorylation in the brain.
In healthy neurons, tau proteins regulate microtubule function in the nerve processes. Hyperphosphorylation dislodges tau from the microtubule surface, resulting in the accumulation of insoluble, toxic tau peptides and compromised axonal integrity (16, 17). In the brains of individuals with Alzheimer’s disease (AD) and other neurodegenerative diseases, hyperphosphorylated tau is aggregated into intraneuronal deposits or NFTs (17). Analysis of tau protein from AD brains by mass spectrometry and sequencing revealed several persistently phosphorylated sites that are recognized by tau-directed antibodies, including tau-1 site (residues 191–225), carboxyl-terminal portion of the protein (residues 386–438) and Ser 262 (the numbering referred to the longest human tau isoform) (27, 28). In this report, we demonstrated the Ser-396, adjacent to microtubule-binding domain, and Ser-202, inside the tau-1 antibody binding site, are highly phosphorylated in the IGF-I null brain. Both sites are implicated in NFT formation. However, Ser-262 phosphorylation site was not altered by IGF-I deletion. Moreover, IGF-I deletion is not associated with reduced tau mRNA or protein levels. These observations suggest that IGF-I regulates tau phosphorylation on specific phosphorylation sites in the brain, but does not have a general effect on tau synthesis.
Tau-directed protein kinases may be divided into three groups: 1) second-messenger-activated kinases, including protein kinase C, protein kinase A, and Ca2+/calmodulin-dependent kinase; 2) Ser/Pro-directed kinases, including MAPK, cyclin-dependent kinase 5, cell division cycle 2, GSK3, GSK3, and PAR-1 (29); and 3) other kinases, including p110mapk and casein kinase (for review see Ref.30). These different kinases phosphorylate tau at various specific as well as overlapping sites (30), as for which kinases are most critical for AD-type NFT formation still under active debate. The present study illuminates the role of GSK3 in tau phosphorylation in vivo because IGF-I inhibits this kinase activity and specific GSK3 targets sites, Ser202 and Ser-396, are hyperphosphorylated in the IGF-I null brain. These GSK3 phosphoryated sites were well documented in in vitro studies (30, 31, 32, 33). It is also well known that activation of IGF-I/insulin receptors triggers activation of the serine/threonine protein kinase Akt (34), which phosphorylates GSK-3 ser9, resulting in its inhibition (35). We have previously shown that both GSK-3 and Akt phosphorylation are significantly reduced in the Igf1 null brain compared with WT (36), suggesting that overactive GSK-3 may cause tau hyperphosphorylation in the Igf1 null brain. All of these data indicate that disturbance in IGF-I signaling cascade leads to aberrant tau hyperphosphorylation, potentiating the formation of NFT.
GSK3’s contribution to tau-mediated neurodegeneration is unsettled; GSK3 overexpression was associated with tau hyperphosphorylation and neurodegeneration in some studies (37, 38, 39) but was associated with reduced neurodegeneration in a different murine model (40). This latter study in a complex, multiple transgenic model, found that although more hyperphosphorylated tau was present, neither an increase in insoluble tau aggregates nor the presence of paired helical filaments or tangles was observed. However, in human brains NFTs invariably contain tau phosphorylated on GSK3 target sites and GSK3 is consistently found colocalized with pretangle and tangle-bearing neurons (41). The mouse brain does not normally exhibit the neuropathological hallmarks of human brain aging and neurodegenerative disorders such as amyloid plaques or NFTs (42), and we did not detect such abnormal markers in either Igf1 null or WT brain in this study. However, the present study provides an important in vivo loss-of-function model illustrating a role for IGF-I in regulation of tau phosphorylation by inhibition of GSK3, potentially impacting tau-mediated neurodegeneration in the mature/aging murine brain. This may be a somewhat more physiological model than previous studies based on GSK3 overexpression in cell culture or transgenic animals for its link to tau hyperphosphorylation (33, 37, 43).
This study found similar levels of SOD and NOS enzymes, and similar markers of oxidative damage in Igf1–/– and WT brains, except for a small increase in nitrotyrosine residues detected in cortical layer IV/V in the Igf1null mouse. This could mean that IGF-I does not normally have a major role in mature brain oxidative activity or antioxidant defenses. However, lipofuscin and NT residues are relatively nonspecific markers of neuronal oxidative activity, and it remains possible that IGF-I is active in specific metabolic pathways we have not detected with this methodology.
IGF-I and/or insulin have been implicated in other aspects of neurodegenerative disease (12, 44). The human brain affected by Alzheimer-type deterioration is consistently found to exhibit reduced glucose use (45, 46). We have previously shown a highly significant positive correlation between local IGF-I expression and brain glucose use in the maturing mouse brain (36), suggesting that reduction in local or perhaps circulating IGF-I levels may contribute to reduced glucose metabolism and cerebral dysfunction in the adult. Recently, a potential relation between reduced insulin/IGF-I signaling and -amyloid accumulation has also been suggested (46, 47). Therefore, reduced IGF-I/insulin signaling is implicated in several major pathological features of AD, including amyloidosis, NFT, cell demise and aberrant brain glucose use. These observations suggest that preventing the age-related decline in circulating (48) and brain IGF-I levels (49, 50) may help reduce the risk of AD.
Acknowledgments
We thank Dr. Shu-Hui Yen at Mayo Clinic for discussion in tau pathologies and Dr. Peter Seubert at Elan Pharmaceuticals, Inc. (San Francisco, CA) for providing the 12E8 antibody.
Footnotes
First Published Online August 25, 2005
Abbreviations: AD, Alzheimer’s disease; GSK, glycogen synthase kinase; iNOS, inducible NOS; NFTs, neurofibrillary tangles; NOS, nitric oxide synthase; nNOS, neuronal NOS; PHF, paired helical filaments; SOD, superoxide dismutase; WT, wild type.
Accepted for publication August 15, 2005.
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Abstract
IGF action has been implicated in the promotion of oxidative stress and aging in invertebrate and murine models. However, some in vitro models suggest that IGF-I specifically prevents neuronal oxidative damage. To investigate whether IGF-I promotes or retards brain aging, we evaluated signs of oxidative stress and neuropathological aging in brains from 400-d-old Igf1–/– and wild-type (WT) mice. Lipofuscin pigment accumulation reflects oxidative stress and aging, but we found no difference in lipofuscin deposition in Igf1–/– and WT brains. Likewise, there was no apparent difference in accumulation of nitrotyrosine residues in Igf1–/– and WT brains, except for layer IV/V of the cerebral cortex, where these proteins were about 20% higher in the Igf1–/– brain (P = 0.03). We found no difference in the levels of oxidative stress-related enzymes, neuronal nitric oxide synthase, inducible nitric oxide synthase, and superoxide dismutase in Igf1–/– and WT brains. Tau is a microtubule-associated protein that causes the formation of neurofibrillary tangles and senile plaques as it becomes hyperphosphorylated in the aging brain. Tau phosphorylation was dramatically increased on two specific residues, Ser-396 and Ser-202, both glycogen synthase kinases target sites implicated in neurodegeneration. These observations indicate that IGF-I has a major role in regulating tau phosphorylation in the aging brain, whereas its role in promoting or preventing oxidative stress remains uncertain.
Introduction
RECENT STUDIES IN Caenorhabditis elegans and mice have implicated the insulin/IGF system in reduced longevity and oxidative stress resistance (1, 2). Reduced expression of insulin, IGF-I, or downstream signaling molecules extend the life span significantly (1, 3, 4, 5). Also, mice heterozygous for IGF-I receptor gene deletion display increased resistance to oxidative stress and increased lifespan (6). However, IGF-I appears to protect neurons from oxidative stress in vitro (7, 8, 9). In addition, reduction in circulating and brain IGF-I levels have been associated with aging and neurodegenerative conditions, and IGF-I has been suggested as a therapeutic agent in amyotrophic lateral sclerosis and Alzheimer’s disease (10, 11, 12). Thus, it has been suggested that increasing IGF-I may ameliorate the deterioration of brain aging and neurodegenerative disorders.
In this study, we used IGF-I gene-targeted mice (Igf1–/–) to investigate whether IGF-I promotes or retards brain aging. Age-related neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases are strongly correlated with oxidative stress. Hence, we examined biomarkers of oxidative damage, such as lipofuscin and nitrotyrosine residues in aged Igf1–/– and WT brains. Lipofuscin consists of intracellular autofluorescent pigments that are deposited in neuronal soma and processes during the aging process (13). The accumulation of nitrotyrosine residues is an indicator of the generation of reactive nitrogen species during aging progression. We also examined enzymes that regulate oxidative activity, such as superoxide dismutase (SOD) and nitric oxide synthase (NOS). SOD catalyzes the conversion of superoxide radicals to H2O2, which is converted to H2O by peroxidase, protecting brain cells against oxidative stress (14). NOS, on the other hand, catalyzes synthesis of nitric oxide (NO), which then reacts with superoxide to form toxic peroxinitrite, causing cell damage during aging (15). In addition, brain aging is associated with increased phosphorylation of tau, a microtubule-associated protein involved in microtubule assembly and stabilization. Tau hyperphosphorylation is believed to lead to its disengagement from microtubule, destabilizing microtubule and disrupting normal microtubule-dependent processes (16). The unbound tau is thought to be more resistant to degradation and more prone to aggregation, culminating in the formation of neurofibrillary tangles (NFTs) (16, 17). Hyperphosphorylated, insoluble tau in the form of NFTs is associated with cognitive dysfunction in normal aging as well as a variety of late onset neurodegenerative disorders termed "tauopathies" (16, 18). We therefore evaluated tau protein levels and phosphorylation status in the aged Igf1–/– and WT control brains to elucidate IGF-I’s role in regulation of this important neuropathological factor.
Materials and Methods
Animals
The mice used in this study were from an Igf1 deletion line derived and genotyped as previously described (19). Animals were studied under protocols approved by the NICHD Animal Use and Care Committee. Male Igf1–/– mice and age-sex-matched littermates were euthanized at postnatal d 400 in CO2 chamber followed by decapitation. Brains were rapidly dissected, cut along the sagittal midline, and snap frozen. One brain half was used for protein preparation, and the other half was sectioned for histochemistry. Sections 10-μm thick were cut sagittally at –20 C, thaw-mounted onto poly-L-lysine-coated slides, and stored at –70 C until use.
Lipofuscin detection
Frozen sections were thawed and directly mounted with DAPI media (Vector Laboratories, Burlingame, CA). The sections were then examined directly under fluorescent microscope. The intracellular autofluorescent pigments known as lipofuscin gave a bright yellow fluorescent signal, whereas DAPI-stained nuclei gave a blue fluorescent signal. For the Sudan Black B staining, frozen sections were first fixed in 4% formaldehyde for 5 min, rinsed in water, and immersed in Sudan Black B reagent (Sigma Diagnostics, St. Louis, MO) overnight. After quick differentiation in 70% EtOH, the slides were washed in 50, 30% EtOH, water and then counterstained in Nuclear fast red (Sigma Diagnostics) for 3 min.
Immunohistochemistry
Immunohistochemistry was performed by the avidin-biotin-immunoperoxidase method (20). Briefly, frozen sections were fixed in 4% formaldehyde for 10 min then washed in 1x Tris/NaCl buffer [50 mM Tris/150 mM NaCl/0.1% Triton (pH 7.5)]. To remove endogenous peroxidase activity, slides were placed in 3% H2O2 solution for 10 min and then washed in the Tris/NaCl buffer. Nonspecific signal was blocked by incubating slides with 10% normal goat serum in blocking solution [1% BSA/0.02% Na-Azide/0.05% Triton X-100 in Tris/NaCl buffer (pH 7.5)] for 45 min. The sections were then incubated at 4 C with antinitrotyrosine antibody (1:500; Chemicon, Temecula, CA) overnight. After washing, the sections were incubated with biotinylated secondary antibodies (1:400; New England Biolabs, Beverly, MA) at room temperature for 40 min. The signal was amplified using the ABC peroxidase method (Vector Laboratories) and visualized with 3,3'-diaminibenzidine (Vector Laboratories). The semiquantitation of nitrotyrosine immunosignals was carried out in a blinded fashion. The signals were captured at x200 using a video camera and analyzed using NIH image software (Image 1.57, NIH) in several brain structures, including frontal cortex layer II/III, layer VI/V, thalamus (ventrolateral thalamic nuclei and ventramedial thalamic nuclei), hypothalamus (lateral hypothalamus area) and striatum (accumbens nuclei). Background signal from each slide was subtracted before further analysis. Two sections from each brain were scored and n = 4 and 5 for Igf1–/– and WT controls, respectively. Differences between groups were compared by ANOVA followed by Fisher’s least significant different tests.
Immunoblotting
Immunoblotting was performed according to Cheng et al. (21). Mouse brains (0.4 g) were homogenized in a boiling solution containing 10 mM Tris (pH 7.4), 1 mM sodium orthovanadate, and 1% sodium dodecyl sulfate at a ratio of 1 g tissue to 17.5 ml solution. Ten microliters of each sample were loaded and resolved on NuPAGE 10% Bis-Tris Gels (Invitrogen Life Technologies, Carlsbad, CA) and transferred to nitrocellulose membranes using electrophoretic transfer cells (Bio-Rad, Hercules, CA). To ensure equal loading and transfer efficiency, the membranes were prestained with Ponceau S solution (Sigma) before immunodetection. Primary antibodies were purchased and diluted to use as follows: antiphospho-tau-396 (1:500; Zymed Laboratories, South San Francisco, CA), neuronal NOS (nNOS) and inducible NOS (iNOS) (1:1000; Chemicon International, Temecula, CA), SOD1 (1:500; Chemicon), tau-1 (1:500; Chemicon), AT-8 (1:100; Pierce Endogen, Rockford, IL), 12E8 (1:5000; provided by Dr. Peter Seubert at Elan Pharmaceuticals, Inc., San Francisco, CA) and tau-5 (1:500; Biosource International Inc., Camarillo, CA). After incubation with primary antibodies for 90 min and washed, the membranes were incubated with horseradish peroxidase-linked secondary antibodies for 60 min. Protein bands were then visualized using SuperSignal West Pico detection reagents (Pierce, Rockford, IL) on a Kodak image machine (Kodak, Rochester, NY). Digital image of the results from each blot was obtained, and the intensity of protein bands revealed by each specific antibody was compared and analyzed using Kodak 1D image-analysis software. Differences between groups were compared by ANOVA followed by Fisher’s least significant different tests (n = 3–4 for Igf1–/– brains and n = 5 for WT controls). All the immunoblottings were repeated at least once, and the data were confirmed.
In situ hybridization
The tau clone (Image clone ID no. 746947, ATCC no. 1015357) was obtained from American Type Culture Collection (ATCC, Manassas, VA). This tau cDNA clone was sequenced and confirmed to contain the complete mouse tau coding region and partial 5' and 3' untranslated sequences. The cDNA clone was linearized with EcoRI for use as a template to synthesize antisense cRNA probe. The cRNA probe synthesis and in situ hybridization protocol were detailed previously (22). After hybridization, slides were exposed to Kodak Bio-Max MR film (Eastman Kodak, Rochester, NY) for 1 d and later dipped in Kodak NTB2 emulsion for 2 d. Parallel sections were hybridized to sense probes and processed together with antisense hybridization sections to serve as hybridization background control. Background signal from a sense probe was subtracted from raw data before further analysis. Film image was analyzed by NIH image program (Image 1.57, NIH). The quantitation of hybridization signal was carried out in a blinded fashion. Four measurements in each brain structure, including frontal cortex, thalamus and granule cell layer of cerebellum were taken for each animal to obtain group means. Hybridization signals on CA2 and CA3 of hippocampus were captured using a monochrome video camera under x400 magnification and silver grains were counted with assistance of NIH image version 1.57 software. Statistical significance between two groups was compared by ANOVA followed by Fisher’s least significant different tests (n = 4 for Igf1–/– brains and n = 5 for WT control).
Results
IGF-I null mice
The Igf1–/– mice have a very high perinatal mortality, largely due to hypoventilation (19). The mice that survive are dwarfs, reaching approximately 30–40% of WT size, depending on the strain (23). Whereas behavior is difficult to measure precisely in dwarfs, our study did not find any evidence for major neurological defects (20).
Lipofuscin
Accumulation of autofluorescent lipoprotein pigments (lipofuscin) in nerve cells is a typical feature of brain aging. Thus, we examined whether IGF-I deletion affected the lipofuscin deposition in aged brains by comparing sections from Igf1 gene-deleted and age-matched WT mice. There were no apparent differences in the distribution or levels of autofluorescent signals in 400-d-old Igf1–/– brains compared with WT control brains (Fig. 1). This result was confirmed by lipid staining using Sudan Black B (data not shown).
Nitrotyrosine residues
To address whether IGF-I modulates brain oxidative stress, we investigated the deposition of nitrotyrosine residues (NT), the end-products of oxidative reactions. We found that NT distribution was similar in Igf1–/– and WT brains (Fig. 2). For example, NT immunostaining was apparent in Purkinje cells (Fig. 2, A and B) and in pyramidal neurons of the cerebral cortex (Fig. 2, C and D). The intensity of the immunostaining appeared similar in Purkinje cells, although they were markedly smaller in the Igf1–/– brain. Semiquantitation of immunostaining in cortical pyramids suggested a modest but significant increase in NT accumulation (P = 0.03) in cortical layer IV/V of Igf1–/– brains (Fig. 2E). However, NT levels were similar in Igf1–/– and WT brains in the thalamus, hypothalamus, striatum, and cerebellum (not shown).
Oxidation regulatory enzymes
We also evaluated enzymes involved in production of nitric oxide, nitric oxide synthetases (NOS) in these brains by immunoblotting (Fig. 3). We found similar levels of nNOS and iNOS in Igf1–/– and WT brains. There was a trend toward higher iNOS levels in Igf1–/– brains, but the difference between groups did not reach statistical significant (Fig. 3). SOD1 levels were also similar in Igf1–/– and WT brains (Fig. 3).
Tau
NFTs accumulate during brain aging as a result of increased phosphorylation of the microtubule-associated protein tau (17). To examine the effect of IGF-I deletion on tau phosphorylation in aged brains, we used Western blotting to evaluate the phosphorylation status of tau in Igf1–/– and WT brains. Anti-tau-396 antibodies recognize tau phosphorylated at serine 396, a site predominantly phosphorylated by GSK-3 (glycogen synthase kinase 3) and prominent in pathological paired helical filaments (PHF) of neurofibrillary tangles (24). Tau phosphorylation at this specific epitope was increased by approximately 7-fold in the Igf1–/– brain compared with WT (P = 0.004) (Fig. 4). Another phospho-tau-specific antibody, AT-8, which recognizes PHF-associated tau, phosphorylated at serine 202, also detected a significant approximately 10-fold increased in Igf1–/– brain homogenate compared with WTs (P = 0.02). However, tau phosphorylation at serine 262 detected by 12E8 antibody showed no difference between Igf1–/– and WT brains. Total tau levels detected by the phospho-independent antibody, tau-5, were similar in Igf1–/– and WT brains (Fig. 4). Consistent with the above observations, the nonphosphorylated tau population, as detected by anti-tau-1 antibody (25, 26), was reduced in the Igf1–/– aged brains (P < 0.01). This result shows that tau is heavily phosphorylated on two serine sites in the brains of animals with IGF-I deficiency, suggesting that IGF-I normally prevents tau hyperphosphorylation at these specific sites.
To further investigate tau expression in Igf1–/– and WT mice, we performed in situ hybridization on anatomically matched brain sections. The anatomical distribution of tau mRNA was similar in Igf1–/– and WT mice (Fig. 5). Tau mRNA was concentrated in the dentate gyrus and Ammon’s horn (Fig. 5, A and B) and in the granule cell layer as well as Purkinje cells of cerebellum (Fig. 5, C and D). Quantitative analyses in temporal cortex, thalamus, granule cell layer of cerebellum and CA2 and CA3 of hippocampus revealed that tau mRNA levels were similar in Igf1–/– and WT brains (data not shown).
Discussion
This study investigated the effects of IGF-I deletion on concomitants of brain aging, namely accumulation of oxidation by-products and tau phosphorylation. We found that aged Igf1–/– brains have similar levels of nitrotyrosine residues and lipofuscin deposition compared with WT brains. We found no difference in levels of enzymes involved in oxidative stress, such as NOS and SOD1, in Igf1–/– and WT control brains. These observations suggest that IGF-I normally has little effect on oxidative damage during brain aging. However, we found that the microtubule-associated protein tau is much more highly phosphorylated in the Igf1 null brain, suggesting that IGF-I normally prevents tau hyperphosphorylation in the brain.
In healthy neurons, tau proteins regulate microtubule function in the nerve processes. Hyperphosphorylation dislodges tau from the microtubule surface, resulting in the accumulation of insoluble, toxic tau peptides and compromised axonal integrity (16, 17). In the brains of individuals with Alzheimer’s disease (AD) and other neurodegenerative diseases, hyperphosphorylated tau is aggregated into intraneuronal deposits or NFTs (17). Analysis of tau protein from AD brains by mass spectrometry and sequencing revealed several persistently phosphorylated sites that are recognized by tau-directed antibodies, including tau-1 site (residues 191–225), carboxyl-terminal portion of the protein (residues 386–438) and Ser 262 (the numbering referred to the longest human tau isoform) (27, 28). In this report, we demonstrated the Ser-396, adjacent to microtubule-binding domain, and Ser-202, inside the tau-1 antibody binding site, are highly phosphorylated in the IGF-I null brain. Both sites are implicated in NFT formation. However, Ser-262 phosphorylation site was not altered by IGF-I deletion. Moreover, IGF-I deletion is not associated with reduced tau mRNA or protein levels. These observations suggest that IGF-I regulates tau phosphorylation on specific phosphorylation sites in the brain, but does not have a general effect on tau synthesis.
Tau-directed protein kinases may be divided into three groups: 1) second-messenger-activated kinases, including protein kinase C, protein kinase A, and Ca2+/calmodulin-dependent kinase; 2) Ser/Pro-directed kinases, including MAPK, cyclin-dependent kinase 5, cell division cycle 2, GSK3, GSK3, and PAR-1 (29); and 3) other kinases, including p110mapk and casein kinase (for review see Ref.30). These different kinases phosphorylate tau at various specific as well as overlapping sites (30), as for which kinases are most critical for AD-type NFT formation still under active debate. The present study illuminates the role of GSK3 in tau phosphorylation in vivo because IGF-I inhibits this kinase activity and specific GSK3 targets sites, Ser202 and Ser-396, are hyperphosphorylated in the IGF-I null brain. These GSK3 phosphoryated sites were well documented in in vitro studies (30, 31, 32, 33). It is also well known that activation of IGF-I/insulin receptors triggers activation of the serine/threonine protein kinase Akt (34), which phosphorylates GSK-3 ser9, resulting in its inhibition (35). We have previously shown that both GSK-3 and Akt phosphorylation are significantly reduced in the Igf1 null brain compared with WT (36), suggesting that overactive GSK-3 may cause tau hyperphosphorylation in the Igf1 null brain. All of these data indicate that disturbance in IGF-I signaling cascade leads to aberrant tau hyperphosphorylation, potentiating the formation of NFT.
GSK3’s contribution to tau-mediated neurodegeneration is unsettled; GSK3 overexpression was associated with tau hyperphosphorylation and neurodegeneration in some studies (37, 38, 39) but was associated with reduced neurodegeneration in a different murine model (40). This latter study in a complex, multiple transgenic model, found that although more hyperphosphorylated tau was present, neither an increase in insoluble tau aggregates nor the presence of paired helical filaments or tangles was observed. However, in human brains NFTs invariably contain tau phosphorylated on GSK3 target sites and GSK3 is consistently found colocalized with pretangle and tangle-bearing neurons (41). The mouse brain does not normally exhibit the neuropathological hallmarks of human brain aging and neurodegenerative disorders such as amyloid plaques or NFTs (42), and we did not detect such abnormal markers in either Igf1 null or WT brain in this study. However, the present study provides an important in vivo loss-of-function model illustrating a role for IGF-I in regulation of tau phosphorylation by inhibition of GSK3, potentially impacting tau-mediated neurodegeneration in the mature/aging murine brain. This may be a somewhat more physiological model than previous studies based on GSK3 overexpression in cell culture or transgenic animals for its link to tau hyperphosphorylation (33, 37, 43).
This study found similar levels of SOD and NOS enzymes, and similar markers of oxidative damage in Igf1–/– and WT brains, except for a small increase in nitrotyrosine residues detected in cortical layer IV/V in the Igf1null mouse. This could mean that IGF-I does not normally have a major role in mature brain oxidative activity or antioxidant defenses. However, lipofuscin and NT residues are relatively nonspecific markers of neuronal oxidative activity, and it remains possible that IGF-I is active in specific metabolic pathways we have not detected with this methodology.
IGF-I and/or insulin have been implicated in other aspects of neurodegenerative disease (12, 44). The human brain affected by Alzheimer-type deterioration is consistently found to exhibit reduced glucose use (45, 46). We have previously shown a highly significant positive correlation between local IGF-I expression and brain glucose use in the maturing mouse brain (36), suggesting that reduction in local or perhaps circulating IGF-I levels may contribute to reduced glucose metabolism and cerebral dysfunction in the adult. Recently, a potential relation between reduced insulin/IGF-I signaling and -amyloid accumulation has also been suggested (46, 47). Therefore, reduced IGF-I/insulin signaling is implicated in several major pathological features of AD, including amyloidosis, NFT, cell demise and aberrant brain glucose use. These observations suggest that preventing the age-related decline in circulating (48) and brain IGF-I levels (49, 50) may help reduce the risk of AD.
Acknowledgments
We thank Dr. Shu-Hui Yen at Mayo Clinic for discussion in tau pathologies and Dr. Peter Seubert at Elan Pharmaceuticals, Inc. (San Francisco, CA) for providing the 12E8 antibody.
Footnotes
First Published Online August 25, 2005
Abbreviations: AD, Alzheimer’s disease; GSK, glycogen synthase kinase; iNOS, inducible NOS; NFTs, neurofibrillary tangles; NOS, nitric oxide synthase; nNOS, neuronal NOS; PHF, paired helical filaments; SOD, superoxide dismutase; WT, wild type.
Accepted for publication August 15, 2005.
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