- Email: [email protected]
Lead exposure and tau hyperphosphorylation: An in vitro study
Accepted Manuscript Title: Lead exposure and tau hyperphosphorylation: An in-Vitro study Authors: Syed Waseem Bihaqi, Aseel Eid, Nasser H. Zawia PII: DOI: Reference:
S0161-813X(17)30160-2 http://dx.doi.org/doi:10.1016/j.neuro.2017.07.029 NEUTOX 2222
To appear in:
NEUTOX
Received date: Revised date: Accepted date:
6-4-2017 20-7-2017 22-7-2017
Please cite this article as: Bihaqi Syed Waseem, Eid Aseel, Zawia Nasser H.Lead exposure and tau hyperphosphorylation: An in-Vitro study.Neurotoxicology http://dx.doi.org/10.1016/j.neuro.2017.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Lead Exposure and tau Hyperphosphorylation: An in-Vitro Study Syed Waseem Bihaqi3, Aseel Eid2 and Nasser H. Zawia1, 2, 3*
1
Department of Biomedical & Pharmaceutical Sciences, 2Interdisciplinary Neuroscience Program,
3
George and Anne Ryan Institute for Neuroscience; University of Rhode Island, Kingston, RI,
USA
*
Corresponding Author:
Nasser H. Zawia, Ph.D. University of Rhode Island 7 Green House road College of Pharmacy, Room 380 Kingston, RI 02881 Office Phone: (401) 874-5909 Lab Phone: (401) 874-7648 Fax: (401) 874-2516 Email: [email protected]
Highlights
Exposure to the environmental toxicant Pb results in an increase of protein levels of total tau as well as site specific hyperphosphorylation of tau in an in-vitro model.
Exposure to Pb also elevated the levels of CDK5 as well as its activator p25.
These changes in protein expression were detected a week later (144 h) after Pb exposure was ceased.
Abstract The presence of fibrillary lesions, which are mainly composed of the microtubule associated protein tau (MAPT) in neurons, has gained immense recognition due to their presence in numerous neurodegenerative diseases, including Alzheimer's disease (AD). Dysregulation of tau is related with its altered site-specific phosphorylation which is followed by tau polymerization, neuronal dysfunction and death. Previous reports by us suggest that molecular alterations favor abundant tau phosphorylation and immunoreactivity in the frontal cortex of aged primates and rodents with past exposure to lead (Pb). Here we report the involvement of Pb-induced alterations in tau and hyperphosphorylation of tau in differentiated Human Neuroblastoma SH-SY5Y cells exposed to a series of Pb concentrations (5-100 µM) for 48 h. These cells were analyzed for the protein expression of total tau, site-specific tau hyperphosphorylation, cyclin dependent kinase 5 (CDK5) and p35/p25 at selected time points (24-144 h), after Pb exposure had ceased. Western blot analysis revealed aberrant tau levels as well as site-specific tau hyperphosphorylation accompanied by elevated CDK5 levels and altered protein ratio of p35/p25 particularly at 72 and 144 h. These changes provide additional evidence that neurodegenerative events are subject to environmental influences.
Key words: CDK5; Hyperphosphorylated Tau; Lead (Pb); p35/p25; SH-SY5Y; Tau protein.
1. Introduction The presence of a proteinaceous aggregate is the hallmark of many neurological disorders characterized by neuronal dysfunction and eventually cell death. Tauopathies refer to the group of neurodegenerative diseases associated with the aggregates of neurofibrillary tangles (NFT) composed of the tau protein (Gendron and Petrucelli, 2009). An intact microtubule structure is
essential for normal neuronal functioning, synaptic plasticity and transmission, such as neurite growth, synaptogenesis and axonal transport (Garcia and Cleveland, 2001). Neurofibrillary degeneration in the absence of β-amyloid is seen in several tauopathies such as Guam parkinsonism dementia complex, dementia pugilistica, corticobasal degeneration, Pick's disease, frontotemporal dementia with parkinsonism linked with chromosome 17 (FTDP-17), and progressive supranuclear palsy (Komori, 1999). These tauopathies are all accompanied by neocortical lesions that are clinically characterized by dementia (Iqbal et al., 2010). Lead (Pb) has been recognized as a potent environmental toxin due to its worldwide distribution. While epidemiological studies have not been able to provide substantial evidence that can correlate Pb exposure with onset of neurodegenerative diseases, correlation between exposure to Pb and cognitive decline in humans has been established in several longitudinal and crosssectional studies in the elderly (Nordberg et al., 2000; Weisskopf et al., 2007). High concentrations of Pb have also been found in the brains of patients with AD (Wu et al., 2008) along with diffuse NFT and calcification (Haraguchi et al., 2001). Recent reports from our lab on rodents have demonstrated that rodents with developmental exposure to Pb performed poorly on cognitive tests as aged adults (Bihaqi et al., 2014b). In addition, studies by us on primate and rodent brains have shown that developmental exposure to Pb induces a latent overexpression of tau accompanied by site specific hyperphosphorylation of tau as well as increased tau immunoreactivity in the frontal cortex (Bihaqi et al., 2014a; Bihaqi and Zawia, 2013). In the present study, we exposed differentiated SH-SY5Y cells to a series of Pb concentrations and monitored the protein expressions of tau, site specific hyperphosphorylation of Tau, and enzymes associated with its regulation such as CDK5 and p35/25, at different time points
(24 h, 48 h, 72 h, 144h), after Pb exposure had ceased. This cell culture model was used to recapitulate whether exposure to Pb had an impact on human cells and to decipher further the mechanisms involved in mediating such neurotoxicity
2. Material and Methods 2.1. Cell culture SH-SY5Y cells were procured from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in T-75 tissue culture flasks (Cyto-one, USA Scientific) containing Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium (Invitrogen, MD) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μ g/ml streptomycin, and 2mM Lglutamine then incubated in a CO2 incubator maintained at 5% CO2 and 37°C. Cells at the density of 105 cells /ml were sub-cultured in T-25 tissue culture flasks (Cyto-one, USA Scientific) containing 5ml each of growth media. After 24 h, cells were differentiated with 10 μM all-trans retinoic acid (Sigma-Aldrich, MO) dissolved in the media containing 1% FBS and observed for neurite out-growth. Cells were examined for neurite outgrowth at 48 h, 72 h, and on day 6, with media being changed after every 48 h (Huang et al., 2011; Jamsa et al., 2004). A 20X objective lens on a Nikon ECLIPSE camera (TE2000-E) adapted to the microscope was used to examine the morphology of cultured cells and to obtain photomicrographs.
2.2. Pb Exposure Differentiated SH-SY5Y cells were exposed to Pb as follows: A 10 mM Pb stock solution was prepared by dissolving the appropriate amount of Pb acetate (Sigma Aldrich, MO) in sterile double distilled H2O. Experimental Pb concentrations were prepared by dilution of a stock solution in DMEM/F12 medium containing 1% FBS, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml
streptomycin, and 2 mM L-glutamine. Furthermore, differentiated cells were incubated with 0, 5, 50, and 100 μM of Pb for 48 h at 37˚C, with 0 μM Pb cells serving as the control group. Following Pb exposure, cells were washed with PBS and switched into normal medium, the cells were harvested at 24 h, 48 h, 72 h, and 144 h with media being changed every three days. 2.3. Sample preparation and Western blot The cells were lysed in Radio-Immunoprecipitation Assay (RIPA) lysis buffer (150 mM NaCl, 25 mM Tris-HCl at pH 8.0, 1% NP-40, 10 mM NaF, 1 mM Na3VO4) containing 1% protease inhibitor, incubated on ice for 30 min. The samples were sonicated and vortexed for 5 min before centrifugation at 10,000 × g for 20 min. The supernatants were collected and used for Western blot analysis. Protein concentration was determined by Pierce Bicinchoninic Assay (BCA) kit (Thermo Scientific, Waltham, MA, USA). For Western blotting a total of 40 µg of protein was electrophoresed on 10-12% polyacrylamide gel at 100 V for 2 h and then transferred to polyvinylidene Difluoride (PVDF) membranes (GE-Healthcare, Piscataway, NJ, USA). Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in Tris Buffer Saline + 0.1% Tween 20 (TBST) at room temperature for 1 h. Immunoblotting was performed after overnight exposure to the following antibodies diluted at 1:1000 with gentle agitation on a shaker at 4C, mouse anti-Tau 46, anti-phospho-Tau [rabbit anti threonine-212 (pThr-212) (Sigma Aldrich, MO), rabbit anti threonine-181 (p-Thr-181), mouse anti pSer-396 (Cell Signaling technology, MA), rabbit anti p-Ser 235 (Abcam, MA)], rabbit anti-CDK5, and rabbit anti p35/25 (C64B10) (Cell Signaling technology, MA). On the following day, membranes were washed and exposed for 1 h to goat anti mouse/goat anti rabbit IRDye® 680LT infrared dye (LI-COR Biotechnology, NE), then diluted at 1: 10,000. The images were developed using Odyssey infrared imaging system (Model- 9120, LI-COR Biotechnology, NE). As a control for
equal protein loading, membranes were stripped and reprobed for 2 h with mouse glyceraldehyde 3 phosphate dehydrogenase (GAPDH) antibody diluted at 1:5000 (Sigma-Aldrich, MO) at room temperature followed by washing and re-exposure to goat anti mouse IRDye®680LT infrared dye. After transferring to a PVDF membrane, the gel was stained with Bio-safe Coomassie blue stain (Bio-Rad, Hercules, CA) to assess the loading of the samples. Tau-related protein biomarkers were analyzed in the present study and normalized against GAPDH. 2.4. Densitometry and statistical analysis The intensities of Western blot bands were quantified by Image-Quant™ 5.2 software (GE, NJ). All measurements were made in triplicate and all values were represented as mean ± S.E.M. The significance of difference between means between groups was obtained with one-way ANOVA, Tukey- Kramer multiple comparison post-test and student Newman-Keuls comparison post-test, using Graph-pad Prism 3.0 computer software. The level of significance was set at P < 0.05.
3. Results The Pb concentrations used in the present study have been used in our previous in-vitro studies that have established these concentrations as non-cytotoxic (Huang et al., 2011). The pictorial representation shows the range of Pb concentrations. Changes in biomarkers of tauopathy were changing at ranges when there was no overt cytotoxicity (Figure 1).
3.1. Latent effect of Pb on total tau protein expression In the present study, an antibody directed against total tau was used to determine the effect of Pb exposure on differentiated SH-SY5Y cells at various time points, after Pb exposure had
ceased. Western blot analysis revealed a significant (P<0.05) increase in the protein levels of total tau at 144 h in cells which had been exposed to 50 µM Pb, significant (P<0.05) increase in the total tau protein expression was also observed at 72 h and 144h in cell previously exposed to 100 µM Pb (Figure 2B).
3.2. Effect of Pb on site-specific phosphorylation: Protein expression of Thr-212, Thr-181, Ser-396, and Ser-235 in differentiated SH-SY5Y cells, which were exposed to 5-100 µM Pb for 48 h and analyzed for changes in protein expression, was undertaken at different time points after Pb exposure was ended (Figure 3A). Our results revealed significant increases in the expression of Thr-181 at 144h (P<0.01) in SH-SY5Y cells previously exposed to 100 µM Pb (Figure 3B). Also, a similar increase was observed in the expression of Thr-212 which was significant at 72 h (P<0.05) and 144 h (P<0.01) post-exposure (Figure 3C). Antibodies directed against select serine residues were used to evaluate the effect on Ser396, and Ser-235 in differentiated SH-SY5Y cells which were exposed to 5-100 µM Pb for 48 h (Figure 4A). Normalized values revealed significant increase in the levels of Ser-235 at 144 h (P<0.05) in cells previously exposed to 100 µM Pb (Figure 4 B); increases in the levels was also observed on Ser-396 at 72 h and 144 h in cells formerly exposed to 50 µM Pb (P<0.05) and 100 µM Pb (P<0.05, P<0.01) (Figure 4 C).
3.3. Effect of Pb exposure on CDK5 and p35/25 Kinases and their respective activators direct the degree of tau phosphorylation. Differentiated SH-SY5Y cells exposed to 5-100 µM Pb for 48 h and were analyzed at different
time points post-exposure to determine changes in the CDK5 (Figure 5A) and its activators p35/p25 (Figure 6A). Western blot analysis revealed an increase in the expression of CDK5, which was significant at 144 h (P<0.05) in cells which had been exposed to 100 µM of Pb (Figure 5 B). These cells also revealed a decrease in the p35 levels at 72 h and 144 h in cells with prior exposure to 100 µM Pb. However, significance (P<0.05) decreases were only observed at 144 h post exposure (Figure 6B). Western blot analysis also revealed the effect of Pb on p25, a proteolytic cleavage product of p35. Significant (P<0.05) increased levels of p25 were observed at 144 h in cells exposed to 100 µM Pb (Figure 6 C).
4. Discussion The heavy metal Pb is a potent neurotoxin that poses a widespread public health hazard especially in developing children due to its casual correlation between low doses of Pb and a decrease in the Intelligence Quotient (IQ) (Needleman et al., 1990; Schwartz, 1994). Published reports from our lab on in-vivo and in-vitro models have provided convincing evidence that indicated early life exposure to this toxicant has a latent impact on the onset of neurodegenerative processes. These latent changes occurring in the aging brain were associated with epigenetic reprogramming (Bihaqi et al., 2011; Bihaqi and Zawia, 2012; Eid et al., 2016), thus rendering an individual susceptible to latent onset of diseases (Needleman and Gatsonis, 1990). The hyperphosphorylation of tau at serine and threonine residues is the primary hallmark feature for various tauopathies, including AD, resulting in the formation of NFTs. Abnormal hyperphosphorylation has been associated with the loss of microtubule stability and function, resulting in a loss of axonal or dendritic transport (Alonso et al., 1996). In the present study, we investigated the effect of Pb concentration (0-100 µM) on both total tau and hyperphosphorylated
tau levels in differentiated SH-SY5Y cells. These cells were exposed to Pb for 48 h and were analyzed at various time points after Pb exposure had ceased. We have previously established that these concentrations have no significant cytotoxicity on differentiated SH-SY5Y cells at the concentration range used (Bihaqi and Zawia, 2012). Utilizing the human SH-SY5Y cells provides a unique opportunity to understand the influence of environmental exposure on the human tau gene, and its potential role in tauopathies. Published reports from our lab on primates and rodents with developmental exposure to Pb exhibited a latent increase in phosphorylation at Thr 181, Thr 212, Ser 396, and Ser 235 (Bihaqi et al., 2014a; Bihaqi and Zawia, 2013). Furthermore, in-vitro studies carried out by us on differentiated SH-SY5Y have revealed that exposure to a series of Pb concentrations for 48 h result in a latent increase in the expression of Amyloid Precursor Protein (APP), a week following the exposure (Bihaqi and Zawia, 2012). Consistent with our earlier finding, our data revealed a gradual increase in the expression of human total tau as well as site specific hyperphosphorylation levels of tau that persisted for one week after Pb exposure had ceased. Although a significant increase in the site-specific phosphorylation was observed at time points; particularly 72 h and 144h, no significant difference was observed in the levels of phosphorylation when compared with total tau. The presence of various proline directed phosphorylated sites in the AD brain suggests the involvement of kinases such as CDK5 as its highest expression and associated kinase activity are detected only in the nervous systems (Hellmich et al., 1992; Imahori and Uchida, 1997; Tsai et al., 1993). Association of CDK5 with its activators p35 and p39 which are found in abundance in the post mitotic neurons is pivotal for its activity. The activator p35 is vastly studied and can be enzymatically cleaved to p25. Aberrant activation of p25 can delocalize and deregulate CDK5,
and over-activation of CDK5 due to p25 has been associated with NFT formation (Cruz et al., 2003; Dhavan and Tsai, 2001). Seminal studies by Bihaqi and coworkers have revealed increases in CDK5 and p25 levels and decreases in p35 levels in aged primate and rodent brain samples exposed to Pb as infants (Bihaqi et al., 2014a; Bihaqi and Zawia, 2013). Data of the in-vitro investigation revealed a latent increase in the levels of CDK5 and p25, whereas p35 was subsequently lowered in differentiated SHSY5Y cells which were previous exposure to Pb for 48 h. In conclusion, this study demonstrates that exposure to the environmental toxicant Pb results in an increase of protein levels of tau as well as site specific hyperphosphorylation of tau in an in-vitro model. These changes in the protein expression were significantly detected a week after Pb exposure had ceased. This study is significant in that it provides evidence for the ability of this environmental agent as a potential inducer of both tau protein levels, and hyperphosphorylated tau.
Acknowledgement This research was supported by NRF Fund No. 31M091, the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (grant no. 1R15AG023604-01), and by grant NIH-5RO1ES015867-03. The research core facility was funded (P20RR016457) by the National Center for Research Resources, a component of NIH.
References Alonso, A.C., Grundke-Iqbal, I., Iqbal, K., 1996. Alzheimer's disease hyperphosphorylated tau sequesters normal tau into tangles of filaments and disassembles microtubules. Nat Med 2(7), 783787. Bihaqi, S.W., Bahmani, A., Adem, A., Zawia, N.H., 2014a. Infantile postnatal exposure to lead (Pb) enhances tau expression in the cerebral cortex of aged mice: relevance to AD. Neurotoxicology 44, 114-120. Bihaqi, S.W., Bahmani, A., Subaiea, G.M., Zawia, N.H., 2014b. Infantile exposure to lead and late-age cognitive decline: relevance to AD. Alzheimers Dement 10(2), 187-195. Bihaqi, S.W., Huang, H., Wu, J., Zawia, N.H., 2011. Infant exposure to lead (Pb) and epigenetic modifications in the aging primate brain: implications for Alzheimer's disease. J Alzheimers Dis 27(4), 819-833. Bihaqi, S.W., Zawia, N.H., 2012. Alzheimer's disease biomarkers and epigenetic intermediates following exposure to Pb in vitro. Curr Alzheimer Res 9(5), 555-562. Bihaqi, S.W., Zawia, N.H., 2013. Enhanced taupathy and AD-like pathology in aged primate brains decades after infantile exposure to lead (Pb). Neurotoxicology 39, 95-101. Cruz, J.C., Tseng, H.C., Goldman, J.A., Shih, H., Tsai, L.H., 2003. Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles. Neuron 40(3), 471-483. Dhavan, R., Tsai, L.H., 2001. A decade of CDK5. Nat Rev Mol Cell Biol 2(10), 749-759. Eid, A., Bihaqi, S.W., Renehan, W.E., Zawia, N.H., 2016. Developmental lead exposure and lifespan alterations in epigenetic regulators and their correspondence to biomarkers of Alzheimer's disease. Alzheimers Dement (Amst) 2, 123-131.
Garcia, M.L., Cleveland, D.W., 2001. Going new places using an old MAP: tau, microtubules and human neurodegenerative disease. Curr Opin Cell Biol 13(1), 41-48. Gendron, T.F., Petrucelli, L., 2009. The role of tau in neurodegeneration. Mol Neurodegener 4, 13. Haraguchi, T., Ishizu, H., Takehisa, Y., Kawai, K., Yokota, O., Terada, S., Tsuchiya, K., Ikeda, K., Morita, K., Horike, T., Kira, S., Kuroda, S., 2001. Lead content of brain tissue in diffuse neurofibrillary tangles with calcification (DNTC): the possibility of lead neurotoxicity. Neuroreport 12(18), 3887-3890. Hellmich, M.R., Pant, H.C., Wada, E., Battey, J.F., 1992. Neuronal cdc2-like kinase: a cdc2related protein kinase with predominantly neuronal expression. Proc Natl Acad Sci U S A 89(22), 10867-10871. Huang, H., Bihaqi, S.W., Cui, L., Zawia, N.H., 2011. In vitro Pb exposure disturbs the balance between Abeta production and elimination: the role of AbetaPP and neprilysin. Neurotoxicology 32(3), 300-306. Imahori, K., Uchida, T., 1997. Physiology and pathology of tau protein kinases in relation to Alzheimer's disease. J Biochem 121(2), 179-188. Iqbal, K., Liu, F., Gong, C.X., Grundke-Iqbal, I., 2010. Tau in Alzheimer disease and related tauopathies. Curr Alzheimer Res 7(8), 656-664. Jamsa, A., Hasslund, K., Cowburn, R.F., Backstrom, A., Vasange, M., 2004. The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation. Biochem Biophys Res Commun 319(3), 993-1000. Komori, T., 1999. Tau-positive glial inclusions in progressive supranuclear palsy, corticobasal degeneration and Pick's disease. Brain Pathol 9(4), 663-679.
Needleman, H.L., Gatsonis, C.A., 1990. Low-level lead exposure and the IQ of children. A metaanalysis of modern studies. JAMA 263(5), 673-678. Needleman, H.L., Schell, A., Bellinger, D., Leviton, A., Allred, E.N., 1990. The long-term effects of exposure to low doses of lead in childhood. An 11-year follow-up report. N Engl J Med 322(2), 83-88. Nordberg, M., Winblad, B., Fratiglioni, L., Basun, H., 2000. Lead concentrations in elderly urban people related to blood pressure and mental performance: results from a population-based study. Am J Ind Med 38(3), 290-294. Schwartz, J., 1994. Low-level lead exposure and children's IQ: a meta-analysis and search for a threshold. Environ Res 65(1), 42-55. Tsai, L.H., Takahashi, T., Caviness, V.S., Jr., Harlow, E., 1993. Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system. Development 119(4), 10291040. Weisskopf, M.G., Proctor, S.P., Wright, R.O., Schwartz, J., Spiro, A., 3rd, Sparrow, D., Nie, H., Hu, H., 2007. Cumulative lead exposure and cognitive performance among elderly men. Epidemiology 18(1), 59-66. Wu, J., Basha, M.R., Zawia, N.H., 2008. The environment, epigenetics and amyloidogenesis. J Mol Neurosci 34(1), 1-7.
Figure Legends Figure 1. Pb exposure in differentiated SH-SY5Y cells. Cells were exposed for 48 h to a series of Pb concentrations (A) 0 μM (control), (B) 5μM, (C) 50 μM, (D) 100 μM. Photomicrographs were obtained with a 20 X objective lens on a Nikon ECLIPSE camera (TE2000-E) adapted to the microscope.
Figure 2. Total tau protein expression in differentiated SH-SY5Y cells after exposure to graded concentrations of Pb. Differentiated cells were exposed to Pb (0-100 μM) for 48 h and were analyzed at specific time points after Pb exposure was ceased. Above are representative Western blot bands of tau protein at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.
Figure 3. Protein expression of phosphorylated tau protein Thr-181 and Thr-212 in differentiated SH-SY5Y cells after exposure to increasing concentrations of Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after the Pb exposure was ceased. Above are representative Western blot bands of Thr-181 and Thr-212 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B and C). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; **P<0.01, *P<0.05 compared to control.
Figure 4. Protein expression of phosphorylated tau protein Ser-235 and Ser-396 in differentiated SH-SY5Y cells after exposure to various concentrations of Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after the Pb exposure was ceased. Above are representative Western blot bands of Ser-235 and Ser-396 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B and C). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.
Figure 5. CDK5 protein expression in differentiated SH-SY5Y cells after exposure to Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after Pb exposure had ceased. Above are representative Western blot bands of CDK5 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.
Figure 6. Protein expression of p35 and p25 in differentiated SH-SY5Y cells after exposure to various concentrations of Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after Pb exposure was ended. Above are representative Western blot bands of p35 and p25 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (Band C). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.
S0161-813X(17)30160-2 http://dx.doi.org/doi:10.1016/j.neuro.2017.07.029 NEUTOX 2222
To appear in:
NEUTOX
Received date: Revised date: Accepted date:
6-4-2017 20-7-2017 22-7-2017
Please cite this article as: Bihaqi Syed Waseem, Eid Aseel, Zawia Nasser H.Lead exposure and tau hyperphosphorylation: An in-Vitro study.Neurotoxicology http://dx.doi.org/10.1016/j.neuro.2017.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Lead Exposure and tau Hyperphosphorylation: An in-Vitro Study Syed Waseem Bihaqi3, Aseel Eid2 and Nasser H. Zawia1, 2, 3*
1
Department of Biomedical & Pharmaceutical Sciences, 2Interdisciplinary Neuroscience Program,
3
George and Anne Ryan Institute for Neuroscience; University of Rhode Island, Kingston, RI,
USA
*
Corresponding Author:
Nasser H. Zawia, Ph.D. University of Rhode Island 7 Green House road College of Pharmacy, Room 380 Kingston, RI 02881 Office Phone: (401) 874-5909 Lab Phone: (401) 874-7648 Fax: (401) 874-2516 Email: [email protected]
Highlights
Exposure to the environmental toxicant Pb results in an increase of protein levels of total tau as well as site specific hyperphosphorylation of tau in an in-vitro model.
Exposure to Pb also elevated the levels of CDK5 as well as its activator p25.
These changes in protein expression were detected a week later (144 h) after Pb exposure was ceased.
Abstract The presence of fibrillary lesions, which are mainly composed of the microtubule associated protein tau (MAPT) in neurons, has gained immense recognition due to their presence in numerous neurodegenerative diseases, including Alzheimer's disease (AD). Dysregulation of tau is related with its altered site-specific phosphorylation which is followed by tau polymerization, neuronal dysfunction and death. Previous reports by us suggest that molecular alterations favor abundant tau phosphorylation and immunoreactivity in the frontal cortex of aged primates and rodents with past exposure to lead (Pb). Here we report the involvement of Pb-induced alterations in tau and hyperphosphorylation of tau in differentiated Human Neuroblastoma SH-SY5Y cells exposed to a series of Pb concentrations (5-100 µM) for 48 h. These cells were analyzed for the protein expression of total tau, site-specific tau hyperphosphorylation, cyclin dependent kinase 5 (CDK5) and p35/p25 at selected time points (24-144 h), after Pb exposure had ceased. Western blot analysis revealed aberrant tau levels as well as site-specific tau hyperphosphorylation accompanied by elevated CDK5 levels and altered protein ratio of p35/p25 particularly at 72 and 144 h. These changes provide additional evidence that neurodegenerative events are subject to environmental influences.
Key words: CDK5; Hyperphosphorylated Tau; Lead (Pb); p35/p25; SH-SY5Y; Tau protein.
1. Introduction The presence of a proteinaceous aggregate is the hallmark of many neurological disorders characterized by neuronal dysfunction and eventually cell death. Tauopathies refer to the group of neurodegenerative diseases associated with the aggregates of neurofibrillary tangles (NFT) composed of the tau protein (Gendron and Petrucelli, 2009). An intact microtubule structure is
essential for normal neuronal functioning, synaptic plasticity and transmission, such as neurite growth, synaptogenesis and axonal transport (Garcia and Cleveland, 2001). Neurofibrillary degeneration in the absence of β-amyloid is seen in several tauopathies such as Guam parkinsonism dementia complex, dementia pugilistica, corticobasal degeneration, Pick's disease, frontotemporal dementia with parkinsonism linked with chromosome 17 (FTDP-17), and progressive supranuclear palsy (Komori, 1999). These tauopathies are all accompanied by neocortical lesions that are clinically characterized by dementia (Iqbal et al., 2010). Lead (Pb) has been recognized as a potent environmental toxin due to its worldwide distribution. While epidemiological studies have not been able to provide substantial evidence that can correlate Pb exposure with onset of neurodegenerative diseases, correlation between exposure to Pb and cognitive decline in humans has been established in several longitudinal and crosssectional studies in the elderly (Nordberg et al., 2000; Weisskopf et al., 2007). High concentrations of Pb have also been found in the brains of patients with AD (Wu et al., 2008) along with diffuse NFT and calcification (Haraguchi et al., 2001). Recent reports from our lab on rodents have demonstrated that rodents with developmental exposure to Pb performed poorly on cognitive tests as aged adults (Bihaqi et al., 2014b). In addition, studies by us on primate and rodent brains have shown that developmental exposure to Pb induces a latent overexpression of tau accompanied by site specific hyperphosphorylation of tau as well as increased tau immunoreactivity in the frontal cortex (Bihaqi et al., 2014a; Bihaqi and Zawia, 2013). In the present study, we exposed differentiated SH-SY5Y cells to a series of Pb concentrations and monitored the protein expressions of tau, site specific hyperphosphorylation of Tau, and enzymes associated with its regulation such as CDK5 and p35/25, at different time points
(24 h, 48 h, 72 h, 144h), after Pb exposure had ceased. This cell culture model was used to recapitulate whether exposure to Pb had an impact on human cells and to decipher further the mechanisms involved in mediating such neurotoxicity
2. Material and Methods 2.1. Cell culture SH-SY5Y cells were procured from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in T-75 tissue culture flasks (Cyto-one, USA Scientific) containing Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium (Invitrogen, MD) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μ g/ml streptomycin, and 2mM Lglutamine then incubated in a CO2 incubator maintained at 5% CO2 and 37°C. Cells at the density of 105 cells /ml were sub-cultured in T-25 tissue culture flasks (Cyto-one, USA Scientific) containing 5ml each of growth media. After 24 h, cells were differentiated with 10 μM all-trans retinoic acid (Sigma-Aldrich, MO) dissolved in the media containing 1% FBS and observed for neurite out-growth. Cells were examined for neurite outgrowth at 48 h, 72 h, and on day 6, with media being changed after every 48 h (Huang et al., 2011; Jamsa et al., 2004). A 20X objective lens on a Nikon ECLIPSE camera (TE2000-E) adapted to the microscope was used to examine the morphology of cultured cells and to obtain photomicrographs.
2.2. Pb Exposure Differentiated SH-SY5Y cells were exposed to Pb as follows: A 10 mM Pb stock solution was prepared by dissolving the appropriate amount of Pb acetate (Sigma Aldrich, MO) in sterile double distilled H2O. Experimental Pb concentrations were prepared by dilution of a stock solution in DMEM/F12 medium containing 1% FBS, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml
streptomycin, and 2 mM L-glutamine. Furthermore, differentiated cells were incubated with 0, 5, 50, and 100 μM of Pb for 48 h at 37˚C, with 0 μM Pb cells serving as the control group. Following Pb exposure, cells were washed with PBS and switched into normal medium, the cells were harvested at 24 h, 48 h, 72 h, and 144 h with media being changed every three days. 2.3. Sample preparation and Western blot The cells were lysed in Radio-Immunoprecipitation Assay (RIPA) lysis buffer (150 mM NaCl, 25 mM Tris-HCl at pH 8.0, 1% NP-40, 10 mM NaF, 1 mM Na3VO4) containing 1% protease inhibitor, incubated on ice for 30 min. The samples were sonicated and vortexed for 5 min before centrifugation at 10,000 × g for 20 min. The supernatants were collected and used for Western blot analysis. Protein concentration was determined by Pierce Bicinchoninic Assay (BCA) kit (Thermo Scientific, Waltham, MA, USA). For Western blotting a total of 40 µg of protein was electrophoresed on 10-12% polyacrylamide gel at 100 V for 2 h and then transferred to polyvinylidene Difluoride (PVDF) membranes (GE-Healthcare, Piscataway, NJ, USA). Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in Tris Buffer Saline + 0.1% Tween 20 (TBST) at room temperature for 1 h. Immunoblotting was performed after overnight exposure to the following antibodies diluted at 1:1000 with gentle agitation on a shaker at 4C, mouse anti-Tau 46, anti-phospho-Tau [rabbit anti threonine-212 (pThr-212) (Sigma Aldrich, MO), rabbit anti threonine-181 (p-Thr-181), mouse anti pSer-396 (Cell Signaling technology, MA), rabbit anti p-Ser 235 (Abcam, MA)], rabbit anti-CDK5, and rabbit anti p35/25 (C64B10) (Cell Signaling technology, MA). On the following day, membranes were washed and exposed for 1 h to goat anti mouse/goat anti rabbit IRDye® 680LT infrared dye (LI-COR Biotechnology, NE), then diluted at 1: 10,000. The images were developed using Odyssey infrared imaging system (Model- 9120, LI-COR Biotechnology, NE). As a control for
equal protein loading, membranes were stripped and reprobed for 2 h with mouse glyceraldehyde 3 phosphate dehydrogenase (GAPDH) antibody diluted at 1:5000 (Sigma-Aldrich, MO) at room temperature followed by washing and re-exposure to goat anti mouse IRDye®680LT infrared dye. After transferring to a PVDF membrane, the gel was stained with Bio-safe Coomassie blue stain (Bio-Rad, Hercules, CA) to assess the loading of the samples. Tau-related protein biomarkers were analyzed in the present study and normalized against GAPDH. 2.4. Densitometry and statistical analysis The intensities of Western blot bands were quantified by Image-Quant™ 5.2 software (GE, NJ). All measurements were made in triplicate and all values were represented as mean ± S.E.M. The significance of difference between means between groups was obtained with one-way ANOVA, Tukey- Kramer multiple comparison post-test and student Newman-Keuls comparison post-test, using Graph-pad Prism 3.0 computer software. The level of significance was set at P < 0.05.
3. Results The Pb concentrations used in the present study have been used in our previous in-vitro studies that have established these concentrations as non-cytotoxic (Huang et al., 2011). The pictorial representation shows the range of Pb concentrations. Changes in biomarkers of tauopathy were changing at ranges when there was no overt cytotoxicity (Figure 1).
3.1. Latent effect of Pb on total tau protein expression In the present study, an antibody directed against total tau was used to determine the effect of Pb exposure on differentiated SH-SY5Y cells at various time points, after Pb exposure had
ceased. Western blot analysis revealed a significant (P<0.05) increase in the protein levels of total tau at 144 h in cells which had been exposed to 50 µM Pb, significant (P<0.05) increase in the total tau protein expression was also observed at 72 h and 144h in cell previously exposed to 100 µM Pb (Figure 2B).
3.2. Effect of Pb on site-specific phosphorylation: Protein expression of Thr-212, Thr-181, Ser-396, and Ser-235 in differentiated SH-SY5Y cells, which were exposed to 5-100 µM Pb for 48 h and analyzed for changes in protein expression, was undertaken at different time points after Pb exposure was ended (Figure 3A). Our results revealed significant increases in the expression of Thr-181 at 144h (P<0.01) in SH-SY5Y cells previously exposed to 100 µM Pb (Figure 3B). Also, a similar increase was observed in the expression of Thr-212 which was significant at 72 h (P<0.05) and 144 h (P<0.01) post-exposure (Figure 3C). Antibodies directed against select serine residues were used to evaluate the effect on Ser396, and Ser-235 in differentiated SH-SY5Y cells which were exposed to 5-100 µM Pb for 48 h (Figure 4A). Normalized values revealed significant increase in the levels of Ser-235 at 144 h (P<0.05) in cells previously exposed to 100 µM Pb (Figure 4 B); increases in the levels was also observed on Ser-396 at 72 h and 144 h in cells formerly exposed to 50 µM Pb (P<0.05) and 100 µM Pb (P<0.05, P<0.01) (Figure 4 C).
3.3. Effect of Pb exposure on CDK5 and p35/25 Kinases and their respective activators direct the degree of tau phosphorylation. Differentiated SH-SY5Y cells exposed to 5-100 µM Pb for 48 h and were analyzed at different
time points post-exposure to determine changes in the CDK5 (Figure 5A) and its activators p35/p25 (Figure 6A). Western blot analysis revealed an increase in the expression of CDK5, which was significant at 144 h (P<0.05) in cells which had been exposed to 100 µM of Pb (Figure 5 B). These cells also revealed a decrease in the p35 levels at 72 h and 144 h in cells with prior exposure to 100 µM Pb. However, significance (P<0.05) decreases were only observed at 144 h post exposure (Figure 6B). Western blot analysis also revealed the effect of Pb on p25, a proteolytic cleavage product of p35. Significant (P<0.05) increased levels of p25 were observed at 144 h in cells exposed to 100 µM Pb (Figure 6 C).
4. Discussion The heavy metal Pb is a potent neurotoxin that poses a widespread public health hazard especially in developing children due to its casual correlation between low doses of Pb and a decrease in the Intelligence Quotient (IQ) (Needleman et al., 1990; Schwartz, 1994). Published reports from our lab on in-vivo and in-vitro models have provided convincing evidence that indicated early life exposure to this toxicant has a latent impact on the onset of neurodegenerative processes. These latent changes occurring in the aging brain were associated with epigenetic reprogramming (Bihaqi et al., 2011; Bihaqi and Zawia, 2012; Eid et al., 2016), thus rendering an individual susceptible to latent onset of diseases (Needleman and Gatsonis, 1990). The hyperphosphorylation of tau at serine and threonine residues is the primary hallmark feature for various tauopathies, including AD, resulting in the formation of NFTs. Abnormal hyperphosphorylation has been associated with the loss of microtubule stability and function, resulting in a loss of axonal or dendritic transport (Alonso et al., 1996). In the present study, we investigated the effect of Pb concentration (0-100 µM) on both total tau and hyperphosphorylated
tau levels in differentiated SH-SY5Y cells. These cells were exposed to Pb for 48 h and were analyzed at various time points after Pb exposure had ceased. We have previously established that these concentrations have no significant cytotoxicity on differentiated SH-SY5Y cells at the concentration range used (Bihaqi and Zawia, 2012). Utilizing the human SH-SY5Y cells provides a unique opportunity to understand the influence of environmental exposure on the human tau gene, and its potential role in tauopathies. Published reports from our lab on primates and rodents with developmental exposure to Pb exhibited a latent increase in phosphorylation at Thr 181, Thr 212, Ser 396, and Ser 235 (Bihaqi et al., 2014a; Bihaqi and Zawia, 2013). Furthermore, in-vitro studies carried out by us on differentiated SH-SY5Y have revealed that exposure to a series of Pb concentrations for 48 h result in a latent increase in the expression of Amyloid Precursor Protein (APP), a week following the exposure (Bihaqi and Zawia, 2012). Consistent with our earlier finding, our data revealed a gradual increase in the expression of human total tau as well as site specific hyperphosphorylation levels of tau that persisted for one week after Pb exposure had ceased. Although a significant increase in the site-specific phosphorylation was observed at time points; particularly 72 h and 144h, no significant difference was observed in the levels of phosphorylation when compared with total tau. The presence of various proline directed phosphorylated sites in the AD brain suggests the involvement of kinases such as CDK5 as its highest expression and associated kinase activity are detected only in the nervous systems (Hellmich et al., 1992; Imahori and Uchida, 1997; Tsai et al., 1993). Association of CDK5 with its activators p35 and p39 which are found in abundance in the post mitotic neurons is pivotal for its activity. The activator p35 is vastly studied and can be enzymatically cleaved to p25. Aberrant activation of p25 can delocalize and deregulate CDK5,
and over-activation of CDK5 due to p25 has been associated with NFT formation (Cruz et al., 2003; Dhavan and Tsai, 2001). Seminal studies by Bihaqi and coworkers have revealed increases in CDK5 and p25 levels and decreases in p35 levels in aged primate and rodent brain samples exposed to Pb as infants (Bihaqi et al., 2014a; Bihaqi and Zawia, 2013). Data of the in-vitro investigation revealed a latent increase in the levels of CDK5 and p25, whereas p35 was subsequently lowered in differentiated SHSY5Y cells which were previous exposure to Pb for 48 h. In conclusion, this study demonstrates that exposure to the environmental toxicant Pb results in an increase of protein levels of tau as well as site specific hyperphosphorylation of tau in an in-vitro model. These changes in the protein expression were significantly detected a week after Pb exposure had ceased. This study is significant in that it provides evidence for the ability of this environmental agent as a potential inducer of both tau protein levels, and hyperphosphorylated tau.
Acknowledgement This research was supported by NRF Fund No. 31M091, the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (grant no. 1R15AG023604-01), and by grant NIH-5RO1ES015867-03. The research core facility was funded (P20RR016457) by the National Center for Research Resources, a component of NIH.
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Figure Legends Figure 1. Pb exposure in differentiated SH-SY5Y cells. Cells were exposed for 48 h to a series of Pb concentrations (A) 0 μM (control), (B) 5μM, (C) 50 μM, (D) 100 μM. Photomicrographs were obtained with a 20 X objective lens on a Nikon ECLIPSE camera (TE2000-E) adapted to the microscope.
Figure 2. Total tau protein expression in differentiated SH-SY5Y cells after exposure to graded concentrations of Pb. Differentiated cells were exposed to Pb (0-100 μM) for 48 h and were analyzed at specific time points after Pb exposure was ceased. Above are representative Western blot bands of tau protein at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.
Figure 3. Protein expression of phosphorylated tau protein Thr-181 and Thr-212 in differentiated SH-SY5Y cells after exposure to increasing concentrations of Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after the Pb exposure was ceased. Above are representative Western blot bands of Thr-181 and Thr-212 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B and C). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; **P<0.01, *P<0.05 compared to control.
Figure 4. Protein expression of phosphorylated tau protein Ser-235 and Ser-396 in differentiated SH-SY5Y cells after exposure to various concentrations of Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after the Pb exposure was ceased. Above are representative Western blot bands of Ser-235 and Ser-396 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B and C). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.
Figure 5. CDK5 protein expression in differentiated SH-SY5Y cells after exposure to Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after Pb exposure had ceased. Above are representative Western blot bands of CDK5 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (B). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.
Figure 6. Protein expression of p35 and p25 in differentiated SH-SY5Y cells after exposure to various concentrations of Pb. Differentiated cells were exposed to Pb (0-100μM) for 48 h and were analyzed at specific time points after Pb exposure was ended. Above are representative Western blot bands of p35 and p25 at 24 h, 48 h, 72 h and 144 h (A) and below is the quantification after normalization to GAPDH (Band C). Each data point of the bar diagrams is the mean ± SEM. Three independent experiments were performed in triplicate; *P<0.05 compared to control.