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Published as doi: 10.1096/fj.07-096909.
 (The FASEB Journal. 2008;22:2357-2367.)
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Tau isoform expression and regulation in human cortical neurons

Atul Deshpande, Khin May Win and Jorge Busciglio1

Department of Neurobiology and Behavior and Institute for Brain Aging and Dementia, University of California, Irvine, California, USA

1Correspondence: Department of Neurobiology and Behavior, MH 2205, University of California, Irvine, CA 92697-4550, USA. E-mail: jbuscigl{at}uci.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differential expression, activity, phosphorylation, and oligomerization of tau play a critical role during neuronal development and in a number of age-related neurodegenerative diseases. An experimental system that accurately models the molecular changes involved in tauopathies, particularly changes in tau isoform activity, requires the expression at physiological levels of the full complement of tau isoforms present in the adult human brain. To this end, we analyzed tau expression in human cortical neurons (HCNs) in culture. Here, we show that the isoform profile of tau in HCNs is similar to that in the adult human brain and that isoform expression is regulated during neuronal development and by cellular substrates. Interestingly, 4R tau exhibited a distinct pattern of expression and subcellular localization, suggesting the presence of specific functional roles for tau isoforms in HCNs. Tau phosphorylation, microtubule binding, and subcellular localization were markedly altered by pharmacological manipulation of tau-directed phosphatase activities, which also induced the appearance of tau oligomeric forms associated with memory loss in animal models of tauopathy. Thus, experimentally induced changes in tau activity and function in HCNs recapitulate critical features of tauopathies that may lead to neuronal dysfunction and degeneration in the human brain.—Deshpande, A., Win, K. M., Busciglio, J. Tau isoform expression and regulation in human cortical neurons.


Key Words: tauopathy • Alzheimer • phosphorylation • phosphatase • cytoskeleton


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALZHEIMER’S DISEASE (AD) is characterized by the presence of two main pathological features in the brain of affected individuals: amyloid plaques in the extracellular space, and neurofibrillary tangles (NFTs) inside neuronal cells (1 2 3) . NFTs are composed of bundles of highly phosphorylated tau proteins paired into helical or coiled filaments (PHFs) (1 , 4 5 6 7 8 9) . Hyperphosphorylated tau is also found in aberrant neuronal extensions called dystrophic neurites, which have been associated with synaptic loss and the cognitive decline observed in AD patients (10) . Tau is a neuron-specific microtubule-associated protein preferentially localized in axons (9 , 11 12 13) . Tau pathology involves the release of tau from microtubules, mislocalization in the somatodendritic compartment, and its hyperphosphorylation and self-association into PHFs, which further aggregate into tangles, but the molecular mechanisms and precise sequence of events leading to tangle formation remain poorly understood (1 , 14) . Perturbations in tau are not only present in AD, but also in a number of other neurodegenerative diseases such as Down syndrome, Parkinson’s disease, prion diseases, frontotemporal dementias, and Niemann-Pick disease type C, suggesting the existence of multiple genetic and epigenetic factors that induce and/or facilitate tau pathology (1 , 4 , 15) . Human tau is encoded by a single gene consisting of 16 exons present on chromosome 17q21. Alternate splicing of 11 of these exons results in six tau isoforms found in the central nervous system (1 , 16 17 18) . These splice variants consist of either or all of the E2, E3, and E10 exons and range from 352 amino acids (aa) (fetal form) to 441 aa (full length form) (15) . They differ from each other by the presence of three (3R) or four (4R) C-terminal tandem repeats of 31–32 aa microtubule binding domains encoded by E9, E10, E11, and E12 (11 , 18 , 19) . In addition, the triplets of 3R and 4R isoforms differ from one another by the presence or absence of E2 and E3 to generate tau isoforms with either 0 (form 0N), 29 (form 1N), or 58 (form 2N) aa inserts at the N terminus (Fig. 1 ) (11) . In the adult human brain, the 3R/4R ratio is ~1, but the ratio of 0N, 1N, and 2N isoforms is ~37:54:9 (20 , 21) . The expression of tau isoforms is differentially and developmentally regulated. The fetal brain only expresses the shortest (3R-0N) isoform, whereas the adult brain expresses all six isoforms (19 , 22) . Structural differences among tau isoforms may underlie pathological significance. For instance, 4R isoforms have higher microtubule-binding affinity than 3R isoforms (23) , and the ratio of 4R/3R expression and/or accumulation is altered in certain neurodegenerative diseases, such as progressive supranuclear palsy, in which 4R tau accumulates in the brain lesions (1 , 24 , 25) . Current experimental models for the study of tau pathology include transgenic animals and cultured neurons or neuronal cell lines, which do not fully recapitulate tau pattern of expression in the adult human brain. Only three 4R tau isoforms are expressed in rat, four tau isoforms (three 3R plus one 4R) are found in mice (22 , 26 , 27) , and isoforms expressed in immature neurons appear to predominate in human neuroblastoma cell lines (28) . Modeling the precise cellular and molecular mechanisms associated with tau pathology may require all tau isoforms present in the adult human brain. Consequently, we analyzed the expression of tau in primary human cortical neurons (HCNs) in culture. We found that differentiated HCNs express similar tau isoforms compared to the adult human brain. Isoform-specific expression was regulated during development and by cellular substrates, while pharmacological modulation of phosphatase activities markedly affected tau phosphorylation, microtubule binding, subcellular localization, and oligomerization.


Figure 1
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  		Figure 1. Schematic representation of the six human tau isoforms. E2 and E3 represent exons 2 and 3, respectively. R1–R4 represent the microtubule binding regions, including R2 encoded by exon 10.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal cultures
Neuronal cultures were established from 16- to 21-wk-old human fetal brain tissue as described (29 , 30) . Human fetal cortical tissue samples were procured at the Department of Pathology, Albert Einstein School of Medicine, NY. The protocols for tissue procurement complied with federal and institutional guidelines. Briefly, cortical tissue was dissociated into a single-cell suspension by incubation with 0.25% trypsin/PBS at 37°C for 30 min, and mechanically dissociated by using a fire-polished glass Pasteur pipette. Cells were plated at a density of 20,000 cells/cm2 on glass coverslips in 35- and 100-mm culture dishes. Two hours after plating, the medium was changed to Neurobasal plus N2 and B27 supplements (Invitrogen, Carlsbad, CA, USA).

Polylysine and laminin treatments
Coverslips and culture dishes were initially coated overnight with 20 µg/ml of poly-L-lysine (PL) (Sigma, St. Louis, MO, USA), and washed 3x with sterile water before plating. After PL coating, some dishes were incubated with 20 µg/ml laminin (Invitrogen) for 12 h. Neurons were maintained in Neurobasal medium supplemented with N2 and B27. Laminin (20 µg/ml) was also added to the medium of the cultures growing on laminin substrate. Partial medium changes (50%) were carried out every 5 days.

Immunofluorescence
Neurons were fixed in 4% paraformaldehyde/0.12 M sucrose in PBS for 30 min at 37°C, permeabilized with 0.2% Triton X-100/PBS, and blocked for 1 h with 5% BSA/PBS. The following primary antibodies were used: rabbit anti-tau (1:2000; Dako, Carpinteria, CA, USA); mouse anti-phospho-tau (Ser-396/404) (1:10; clone PHF-1), mouse anti-tubulin III (1:2000; Sigma); mouse anti-MAP2 (1:1000; Chemicon, Temicula, CA, USA); mouse anti-4R tau (clone 4RT; 1:50; gift from Dr. Martin Ingelsson, Uppsala University, Uppsala, Sweden) (31) , mouse anti-4R tau (clone ET-2; 1:5; gift from Dr. Peter Davies, Albert Einstein College of Medicine, New York, NY, USA) (32) , mouse anti-4R tau (clone RD4; 1:20; gift from Dr. Rohan de Silva, University College, London, UK) (33) , mouse anti-3R tau (clone RD3; 1:20) (33) , mouse anti-synaptophysin (1:500; Calbiochem, La Jolla, CA, USA), mouse anti-PSD-95 (1:1000; University of California-Davis Hybridoma Bank, Davis, CA, USA). Primary antibodies were incubated for 12 h at 4°C, followed by a 1 h incubation with fluorescent-conjugated secondary antibodies (Alexa, Invitrogen Molecular Probes). Triple labeling was performed by sequential incubation with the appropriate antibodies. Competition with antigenic peptide, use of nonimmune IgG instead of primary antibody, or omission of primary antibody resulted in complete elimination of specific labeling. An Axiovert 200 inverted microscope (Zeiss, Jena, Germany) was used for specimen examination and imaging. Fluorescent images were captured with a digital camera (Zeiss) at x630 and processed using AxioVision software (Zeiss). To assess colocalization of synaptic markers, we used the apotome device (Zeiss) for z-sectioning. To quantify the frequency of colocalization of fluorescent signals, at least 10 fields were captured at x630 and quantified using an automated AxioVision module.

Image analysis
Neurite length
For quantitative image analysis, three independent sets of experiments were performed using three cultures derived from three different brain specimens. For each experiment, 30 random neurons were measured per time point. Average neuritic lengths were obtained using Image J software [U.S. National Institutes of Health (NIH), Bethesda, MD, USA]. The longest neurite (putative axon) in each cell, the average neurite length, and the average number of neurites per cell was compared for laminin-treated and nontreated groups. The results were expressed as the average ± SEM values for each group.

Synaptic density
To quantify synaptic density, we designed a module in AxioVision, based on the intensity, shape, and size of the fluorescence signal. Images were processed using the colocalizer module to generate signal overlap areas between presysnaptic and postsynaptic fluorescent signals (synaptic areas). A similar module was designed to calculate the area of tubulin class III-positive immunolabeling (total neuronal surface). The results were expressed as the ratio of synaptic area to total neuronal area. To quantify the pixel intensity of total and 4R-tau immunofluorescence along neuronal processes, images were analyzed using the densitometric function of Axiovision software along 10- to 15-µm-long axonal segments. To avoid pixel saturation, the exposure time was adjusted to maintain all fluorescent objects within the linear range.

Western blot analysis
For Western blot analysis, cultures were washed with PBS and harvested in radioimmunoprecipitation assay (RIPA) buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) in PBS] plus protease inhibitors (Complete, Roche Molecular Biochemicals, Indianapolis, IN, USA) and phosphatase inhibitors at 4°C. Cell homogenates were centrifuged at 100,000 g for 30 min. Supernatants were collected, and the protein content was determined using a commercial kit (Bio-Rad, Hercules, CA, USA). Samples were separated on 12% linear SDS gels and electrotransferred to polyvinylidene difluoride (PVDF) (Millipore, Bedford, MA, USA). Membranes were washed, blocked, and incubated overnight with either rabbit anti-tau (1:10,000; Dako) or mouse anti-phospho tau (clone AT-180; 1:500; Pierce Biotechnology, Rockford, IL, USA) or mouse anti-4R tau (clone 4RT; 1:200) antibody, followed by secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h. The reaction was developed using a chemiluminescent substrate kit (Bio-Rad). Protein levels of voltage-dependent anion channel (VDAC) (1:2000, Calbiochem, San Diego, CA, USA) were used as control for protein loading.

Cytoskeletal preparations
Cytoskeletal proteins were obtained in a similar manner; however, before harvesting with RIPA buffer, the cells were placed in microtubule-stabilizing buffer (MSB; 0.13 M HEPES, pH 6.9, 2 mM MgCl2, and 10 mM EGTA) at 37°C for 1 min, and then the cells were incubated with 0.2% Triton X-100 in MSB for 2 min at 37°C (34) . Dot blot analysis was carried out with soluble and cytoskeletal fractions blotted onto a PVDF membrane using a Minifold apparatus (Scheiler & Schuell, Keene, NH, USA) and probed overnight with a polyclonal anti-tau antibody (Dako), followed by an HRP-tagged secondary antibody. The membrane was developed using chemiluminescence, scanned, and quantified using Image J (NIH).

RNA extraction
Total RNA was extracted from frozen tissue or cultured HCNs with the TriZOL reagent according to the manufacturer’s protocol (Invitrogen). Briefly, the tissue samples or cells were homogenized in TriZOL in RNase-free vials, followed by centrifugation at 15,000 g and subsequent incubations in chloroform and isopropanol to obtain the RNA pellet. The pellets were washed with ethanol, resuspended in 50 µl H2O, and immediately placed on dry ice. The suspension was further cleaned and concentrated using RNA Clean-up Kit 5 (Zymo Research, Orange, CA, USA). After measurement of the RNA concentration by spectrophotometry (GeneQuant pro; GE, Cambridge, UK), all samples were adjusted to 1 µg/µl and stored at –20°C until used.

Quantitative PCR
One-step RT-PCR was carried out on total mRNA extracted from tissue or cells using the iScript One-Step RT-PCR SYBR Green kit according to the manufacturer’s protocol (Bio-Rad). Primer pairs were based on the longest human tau mRNA isoform as described (35) . The 3R and 4R primer pairs used were as follows: 3R-forward, 5'-TTGCTCAGGTCAACTGGTTTGTA-3'; 3R-reverse, 5'-ACTGAGAACC TGAAGCACCA-3'; 4R-forward, 5'-TGCAGAT AATTAATAAGAAGCTGGA-3'; and 4R-reverse, 5'-GTGTTTGATATTATCCTTTGAGC-3'. The design and utilization of primer pairs for total tau and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been described previously (36) : tau-forward, 5'-TGACACGGACGCTGGCCTGAA-3'; tau-reverse, 5'-CACTTGGAGGTCACCTTGCTC-3'; GAPDH-forward, 5'-CCATGGCACCGTCAAGGCTGA-3'; GAPDH-reverse, 5'-GCCAGTAGAGGCAGGGATGAT-3'. Total RNA was incubated with iScript one-step RT-PCR master mix reagent, including reverse transcriptase, DNA polymerase, and SYBR Green. The RT-PCR procedure was performed in a series of reactions with an Opticon 2 system (MJ Research/Bio-Rad). The thermal cycle protocol consisted of 10 min at 50°C for reverse transcription and 5 min at 95°C for transcriptase inactivation, followed by 40 10-s cycles at 95°C for denaturation and 1 min for annealing and extension. The annealing temperature for 3R isoforms was 54°C; for 4R isoforms, 51.5°C; for total tau, 58.5°C; and for GAPDH, 58.8°C. The final products were subjected to a melting curve analysis to ensure the specificity of the reaction by holding the sample for 1 min at 95°C followed by 1 min at 55°C and reading the fluorescence every 10 s at increasing temperatures of 0.5°C for 80 cycles. The final step was elongation of the product at 72°C for 10 min. The samples were stored at 4°C until further use. The reaction products were analyzed on 5% agarose gels and visualized using ethidium bromide. Images were captured using the EDAS system (Kodak, Rochester, NY, USA). The level of each tau mRNA was expressed relative to the amount of GAPDH.

Treatments with phosphatase inhibitors
Phosphatase inhibitors were purchased from Calbiochem. All treatments were initiated at day 20 and prolonged for 10 days at the following final concentrations: 5 nM okadaic acid (37) , 10 nM cantharidin (38 39 40) , and 40 pM deltamethrin (41 , 42) . Whole cell homogenates and cytoskeletal and soluble fractions were prepared in RIPA buffer and processed as described above.

Statistical analysis
All experiments were repeated ≥3x in cultures derived from ≥3 different fetal specimens. Each individual experiment was performed in triplicate cultures. Data were analyzed by unpaired Student’s t test and expressed as the mean ± SE. Significance was assessed at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Compartmentalization of tau during HCN differentiation in culture
During morphological differentiation, rodent neurons undergo a series of stereotyped changes first described by Dotti et al. (43) . We found a similar sequence of morphological differentiation in HCNs: 1) formation of lamellipodia, 2) outgrowth of minor processes, 3) development of axon and major processes, and 4) dendritic growth. One of the characteristic features of late-stage neuronal differentiation is the compartmentalization of tau in axonal processes, and its disappearance from dendrites and neuronal bodies. Cortical neurons were fixed at 4, 10, and 20 days in culture for immunofluorescence analysis. A progressive increase in the density of neuronal processes was evident at each time point (Fig. 2 ). At 4 days, tau and MAP2 expression was observed in all neuronal compartments. By 10 days, tau levels in neuronal cell bodies were clearly decreased, and by 20 days, tau was mostly localized in axonal processes. The axonal localization of tau was confirmed by the lack of colocalization with MAP2, which is present in the somatodendritic compartment in differentiated neurons (Fig. 2) . Thus, tau gradually disappeared from HCN cell bodies between days 4 and 10, and by day 20, it was exclusively localized in axonal processes.


Figure 2
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  		Figure 2. Compartmentalization of tau and MAP2 during HCN differentiation in culture. Cells were fixed at 4, 10, and 20 days in vitro (DIV)and immunostained with anti-MAP2 (red) and anti-tau (green) antibodies. Each row represents the same microscopic fields. The first column shows MAP-2 immunoreactivity. The second column illustrates tau immunoreactivity (green) and nuclei stained with Hoechst (blue). The images in the third column correspond to the overlap of red, green, and blue channels. At 4 DIV, neuronal processes initiate a period of fast elongation. At 10 DIV, a dense network of axons and developing dendrites is readily observed. By 20 DIV, tau is completely excluded from cell bodies and MAP-2-positive dendrites and is highly concentrated in the axonal network. Scale bar = 20 µm.

Laminin stimulates neurite growth and synapse formation in HCNs
Tau expression during development was analyzed in HCNs grown on polylysine, a synthetic substrate for neuronal attachment, or laminin, one of the most abundant basement membrane proteins in the mammalian brain. In rodent neurons, laminin activates cell adhesion to the substrate and stimulates axonal growth, possibly by accelerating the expression of specific tau isoforms (44) . Examination of the time course of HCN differentiation indicated that days 4 and 5 constitute a period of significant axonal growth, whereas dendrites develop later. Neuritic length was quantified in HCNs growing on laminin or PL. Cultures were fixed at day 5, and immunofluorescence with anti-tubulin class III antibody was performed to visualize the overall neuronal morphology. Approximately 100 neurons per culture were analyzed in cultures grown on laminin and PL, respectively. The average length of the longest neurites was 109 ± 5.36 and 142 ± 5.11 µm for neurons treated with PL and laminin, respectively, with P = 0.0116 for the difference between PL and laminin conditions (Fig. 3 A). The average neuritic length per neuron was determined and again compared between the two experimental groups. Average length for the PL group was 70 ± 1.77 µm, whereas that for the laminin group was 86 ± 3.01 µm, with P = 0.0112 (Fig. 3B ). The average number of neurites per cell was not significantly different between PL and laminin groups: 2.75 ± 0.23 and 2.66 ± 0.11, respectively (Fig. 3C ). Thus, laminin significantly increased the length of both the longest neurite in each cell and the total neuritic length per cell but had no effect on the number of neurites per cell. We also investigated whether laminin accelerated synapse formation in HCNs. HCNs establish abundant synaptic contacts during wk 2 and 3 in culture (29) . HCNs were triple labeled with the presynaptic marker synaptophysin, the postsynaptic marker PSD-95, and the neuronal marker tubulin class III (Fig. 3D ). Synaptic density was quantified by image analysis by measuring the area of overlap between the presynaptic and postsynaptic markers (see Materials and Methods section), and the results were expressed as a ratio of the areas occupied by synapses (overlap of presynaptic and postsynaptic signals) over total neuronal area (tubulin class III signal). By day 10, HCNs growing on laminin exhibited a 40% increase in synaptic density compared to HCNs growing on PL (Fig. 3E ). However, by day 20, there was minimal difference in synaptic density in HCNs cultured with or without laminin (Fig. 3F ). Thus, laminin accelerates both axonal growth and synapse formation in HCNs.


Figure 3
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  		Figure 3. Laminin enhances neurite outgrowth and accelerates synapse formation in HCNs. Comparison of mean lengths of longest neurites (A), mean total neurite length per cell (B), and total number of neurites per neuron (C) between HCNs grown on PL and laminin substrates. Quantifications were made at days 4 and 5 in culture. Laminin induced an increase of ~30 ± 4.5% in the length of the longest neurites, and ~22 ± 2% increase in mean neuritic length per cell. There were no changes in the number of neurites per cell. Synaptic density was analyzed at 10 and 20 days in vitro (DIV) (D–F). HCNs were fixed and immunolabeled with anti-synaptophysin (green), anti-PSD-95 (red), and anti-tubulin class III (blue) antibodies (D). The extension of the area of overlap (light green/yellow) between presynaptic and postsynaptic markers was quantified using the colocalizer module in AxioVision image analysis software. The synaptic area was normalized to the total neuronal area expressed as anti-tubulin class III-positive immunofluorescence, excluding neuronal cell bodies. By 10 DIV, there was a 68 ± 11% increase in synaptic density in HCNs grown in laminin (E). By 20 DIV, similar synaptic densities were present in PL and laminin substrates (F). Data were analyzed by unpaired Student’s t test and expressed as the mean ± SE. Significance was assessed at P < 0.05.

Developmental and substrate regulation of tau isoform expression
The accelerated development of HCNs growing on laminin provided the opportunity to assess tau expression in neurons growing and extending processes at different rates. RT-PCR and Western blot analysis revealed a progressive increase in both tau mRNA and protein levels, respectively, during HCN development in culture. Interestingly, laminin induced a significant increase in 4R tau expression at day 3 (Fig. 4 A–C), preceding the period of maximal axonal outgrowth at days 4–5. Increased mRNA levels of both 3R and 4R were still observed at days 7, 10, and 15 in laminin-treated cultures. However, by day 20, 3R and 4R mRNA levels were nearly identical in PL- and laminin-treated cultures (Fig. 4A, B ). The 3R and 4R primer pairs yielded 70- and 73-bp products, respectively. When analyzed on a 5% agarose gel, the 3R/4R ratio in HCNs at day 20 was found to be ~1 (Fig. 4C ), similar to the ratio in the normal adult human brain (non-AD; Fig. 4C ) (20 , 21) . Consistent with previous reports, we also found that 4R tau expression was markedly increased in the brain of a Down syndrome subject who developed AD pathology (DS/AD) (45) .


Figure 4
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  		Figure 4. 3R and 4R tau mRNA levels increase progressively with HCN differentiation. Cells were harvested at the indicated time points. Quantitative real-time PCR was performed using four different primer pairs for 3R-tau, 4R-tau, total-tau, and GAPDH, as described in Materials and Methods. Values are expressed as the ratios of Ct values 3R/GAPDH and 4R/GAPDH, respectively, for A and B. A progressive increase in 3R tau expression is observed with time in culture. A relatively small increase in mRNA levels was observed in cultures grown on laminin up to 15 days in vitro (DIV). By 20 DIV, nearly identical levels were observed in laminin- and PL-treated cultures (A). 4R mRNA levels were markedly increased in HCNs growing on laminin at 3 DIV, preceding the period of fast process outgrowth (B). Higher 4R mRNA levels on cells growing on laminin persisted up to 15 DIV, but by 20 DIV, 4R mRNA levels in cultures exposed to laminin or PL were similar. The PCR products of HCNs grown on polylysine for 20 days [HCN (20 DIV)] were analyzed on a 5% agarose gel next to the PCR products generated from the brain of a subject with Down syndrome who developed AD neuropathology (DS/AD), and a nondemented subject (non-AD) (C). Approximately similar levels of 3R and 4R PCR products were present in HCNs and nondemented brain tissue, whereas a clear increase in 4R compared to 3R mRNA level is evident in DS/AD brain. GAPDH levels were comparable in all samples. The -ve lane represents a negative control experiment performed in the absence of sample mRNA, to rule out artifacts due to primer pairs.

To assess tau protein levels, Western blots were probed with a polyclonal antibody that recognizes all isoforms of tau, and the results were expressed as a ratio of tau over the level of the VDAC, whose expression does not change in PL or laminin substrates (Fig. 5 A). The results showed a significant increase in total tau levels in laminin-treated cultures at day 3 (T-tau, Fig. 5A, B ). Tau expression remained higher in laminin-treated cultures up to day 10, but by day 15, total tau levels were similar in PL- and laminin-treated cultures (Fig. 5A, B ). The expression of 4R-tau was assessed using the 4R-specific antibody 4RT (31) . 4R tau protein expression in laminin-treated cultures was higher than in PL-treated cultures at day 3 and remained significantly higher at 10, 15, and 20 days (Fig. 5A, B ), in contrast with total tau levels, which showed no substrate-dependent expression differences at 15 and 20 days (Fig. 5A , B). These results suggest that changes in tau isoform expression associated with the type of substrate and/or growth and maturation rate exist in HCNs well beyond the period of active axonal elongation.


Figure 5
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  		Figure 5. Analysis of tau protein expression in HCNs. A) Cultures growing on polylysine (P) or laminin (L) were harvested at the indicated time points [3, 7, 10, and 15 days in vitro (DIV)], separated on a 12% SDS-polyacrylamide gel, and blotted onto PVDF. The membrane was probed with polyclonal anti-tau (1:10,000, Dako), anti-4R-tau (1:200; 4RT) or anti-VDAC (1:2,000). B) Densitometric quantification was performed as described in Materials and Methods. The results were expressed as a ratio of total tau (T-tau) or 4R-tau over VDAC immunoreactivity. They represent the mean of three independent experiments. The expression level of VDAC increased with neuronal development, but was similar in cells growing on polylysine or laminin at all time points. There was a significant increase in total tau protein levels at 3 DIV in cells growing on laminin (A, B). The differences in the level of tau in polylysine and laminin cultures disappeared as cells progressed in their differentiation. In contrast, higher levels of 4R tau in HCNs growing on laminin were observed at all time points analyzed (A, B). C) HCNs harvested at 40 DIV and normal adult human brain exhibited a similar profile of tau isoforms. The blot was developed with polyclonal anti-human tau antibody (T-tau).

The complexity of the banding pattern of 4R tau isoforms also increased with time in culture, consistent with the notion that expression of 4R isoforms increases with neuronal development (1 , 11 , 15 , 19) . A side-by-side comparison of tau banding patterns in differentiated HCN cultures and adult human brain homogenates after separation in a 12% linear gel showed similar, but not identical, band profiles and stoichiometries after incubation with polyclonal anti-tau (Fig. 5C ), suggesting that the pattern of expression of tau in HCNs resembles the pattern present in the adult human brain.

Differential subcellular localization of 4R tau
Immunofluorescence analysis of differentiated HCNs at 20 days in culture using 4R tau-specific antibody 4RT revealed a distinct pattern of staining characterized by a stippled labeling along axonal processes, which contrasted with the smooth-textured staining obtained with nonisoform-specific tau antibodies (Fig. 6 A) and with the 3R-specific antibody RD3 (data not shown). A similar result was obtained with two other antibodies specific for 4R tau (antibodies ET-2 and RD4, data not shown). The same pattern of 4R tau localization was observed in HCNs grown on PL and laminin substrates. Densitometric analysis of immunofluorescent signals along axonal processes confirmed a relatively even profile of fluorescence intensity in the total tau channel (Fig. 6B , T-tau). In contrast, a highly variable densitometric profile, including multiple peaks and valleys was present in the 4R tau channel (Fig. 6B , 4R-tau), confirming the differential subcellular localization of 4R tau and ruling out the possibility that different signal intensities for total tau and 4R tau might account for their different distribution patterns.


Figure 6
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  		Figure 6. Differential distribution of 3R and 4R tau in HCNs. A) Double immunofluorescence analysis of HCNs at 20 days in culture with anti-4R tau (clone 4RT) and polyclonal anti-total tau antibodies illustrate the stippled appearance of 4R immunoreactivity along axonal processes in sharp contrast with the smooth-textured appearance of total tau immunoreactivity. Insets in the left panels correspond to regions magnified in the right panels. B) Densitometric analysis of immunofluorescence signal intensity along axonal processes revealed significantly larger peaks and valleys in the 4R tau densitometric profile compared to that of total tau. The images correspond to the same microscopic field. Scale bars = 20 µM.

Together, these results show the presence of specific profiles of tau isoform expression and subcellular distribution in HCNs, which are modulated by both substrate cues and neuronal growth rate and differentiation stage.

Reduced phosphatase activity leads to tau pathological changes in HCNs
Several studies indicate that reduced tau dephosphorylation may contribute to tau hyperphosphorylation and tangle pathology (reviewed in refs. 1 , 15 ). These experiments were designed to assess the ability of phosphatase inhibitors to enhance tau pathological changes in HCNs regardless of the signaling mechanisms and/or direct or indirect effect of the inhibitors on the tau molecule. Starting at 20 days, HCNs were treated for 10 days with 40 pM deltamethrin, a phosphatase 2B (PP2B) inhibitor, or 10 nM cantharidin, a specific phosphatase 2A (PP2A) inhibitor. At these concentrations, the effects of deltamethrin and cantharidin are specific for PP2B and PP2A, respectively, and no deleterious effects on neuronal survival, viability, or morphology were observed. Western blot analysis showed no significant changes in tau expression after deltamethrin treatment (Fig. 7 A). However, a clear change in the profile of tau isoform protein bands was evident with cantharidin (Fig. 7A ), suggesting alterations in isoform mobility due to increased phosphate content and a role for PP2A but not PP2B on tau dephosphorylation in HCNs (37 , 45 46 47 48 49) .


Figure 7
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  		Figure 7. Phosphatase inhibitors enhance tau phosphorylation in HCNs. A) Western blot of HCNs treated with 40 pM deltamethrin (Del) and 10 nM cantharidin (Can) for 10 days, homogenized, and probed with polyclonal anti-tau (T-tau), and anti-VDAC antibodies. All treatments were initiated at day 20 in culture. Note the change in the profile of tau isoforms after cantharidin treatment. B) Dot blot quantification of total tau in the soluble fraction of cytoskeletal preparations after treatment with deltamethrin and cantharidin at the concentrations indicated above, and 5 nM okadaic acid (OA). C) There is a significant increase in soluble tau after treatment with cantharidin and okadaic acid, which was accompanied by a marked elevation in phosphorylated tau in the soluble fraction, as shown after blotting with antibody AT-180, which recognizes tau phosphorylated at Thr231. D) Double immunofluorescence analysis with polyclonal anti-tau (T-tau, blue channel) and PHF-1 (phosphorylated tau at Ser-396/404, red channel) illustrates both the increase in phosphorylated tau immunoreactivity and its translocation to the somatodendritic compartment after cantharidin (Can) treatment. PHF-1 immunoreactivity was negative in nontreated cells (Ctrl). E) Western blot of HCNs treated with cantharidin (Can) and okadaic acid (OA) showed induction of ~140- to 170-kDa tau multimers, which labeled positive with an antibody that recognizes all tau isoforms (T-tau). Anti-phosphorylated tau antibody AT-180 preferentially labeled the ~170-kDa bands (P-tau). The multimers were also strongly labeled by anti-4R tau antibody (clone 4RT). Lower MW multimers (~140 kDa) were detected by the conformation-dependent antibody A11, which recognizes oligomeric structures regardless of the protein sequence (A11). Scale bar = 20 µM.

Pathological consequences of tau hyperphosphorylation include loss of microtubule-binding capacity, its release from the microtubular polymer, and its translocation from the cytoskeleton to the soluble cytosolic fraction (37 , 50 , 51) . Quantitative dot blot analysis showed a marked increase on tau levels in the soluble fraction after treatment with both cantharidin and okadaic acid, a strong inhibitor of PP1, PP2A, and PP2B, but not after treatment with deltamethrin (Fig. 7B ). In addition, there was a marked increase in phosphorylated tau in the soluble fraction with cantharidin and okadaic acid, but not with deltamethrin, as evidenced after Western blotting with antibody AT-180 that recognizes tau phosphorylated at Thr-231, considered an early marker of tau phosphorylation (52 53 54) (Fig. 7C ). Immnunofluorescence with antibody PHF-1 against phosphorylated residues Ser-396/404 (54 , 55) showed increased immunoreactivity after cantharidin but not after deltamethrin treatment (Fig. 7D ). In addition, phosphorylated tau was mislocalized in cantharidin-treated neurons. Its immunoreactivity was particularly prominent in cell bodies and initial neuritic segments (red immunofluorescence, Fig. 7D ), in sharp contrast with the pattern of total tau, which is preferentially localized in axonal processes and displayed only faint labeling of neuronal cell bodies and dendrites (blue immunofluorescence, Fig. 7D ).

Recent studies have identified multimeric tau species of ~140–170 kDa that closely correlate with functional memory deficits in two different mouse models of tauopathies (56) , and similar multimeric species have been described in the brain of FTDP-17 and AD patients (56 , 57) . Tau multimers in a very similar range were observed in HCN homogenates after incubation with cantharidin and okadaic acid (Fig. 7E ). A clear increase in bands positioned slightly below and above the 150-kDa marker was observed in blots developed with anti-total tau (Fig. 7E, T -tau). Antibody AT-180 detected the induction in the ~170 kDa band specifically (Fig. 7E , P-tau), consistent with the presence of multimeric hyperphosphorylated tau in the ~170-kDa range (56) . Tau multimers at both 140 and 170 kDa were strongly recognized by anti-4R tau antibody 4RT (Fig. 7E , 4R-tau). Finally, ~140 kDa multimers were also recognized by the conformation-dependent antibody A11, which binds specifically oligomeric assemblies regardless of the protein primary sequence (58) . Thus, phosphatase inhibition induces the appearance of tau oligomeric forms in HCNs, which are composed by both hyperphosphorylated and nonhyperphosphorylated tau.

Collectively, these results indicate that chronic phosphatase inhibition in HCNs leads to a series of pathological changes in tau isoforms, closely resembling tau changes in human tauopathies and transgenic animal models.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The development and characterization of clinically relevant experimental models for the study of neurodegenerative conditions is critical to understand complex disease mechanisms at the molecular level. To this end, we carried out a detailed analysis of tau isoform expression and regulation by developmental, environmental, and pharmacological stimuli in HCNs, the main cell types affected by tau-associated pathologies (reviewed in refs. 1 , 15 ). HCNs developed a complex network of axons and dendrites and numerous synaptic contacts in culture (Fig. 3) (29) . They also recapitulated a similar but not identical expression pattern of tau isoforms found in the adult human brain. After the establishment of neuronal polarity, tau acquired an axonal-specific distribution. Tau levels increased steadily in both PL and laminin substrates as HCNs underwent morphological differentiation. Laminin, one of the most abundant extracellular matrix proteins in the mammalian brain, significantly accelerated neurite growth. The enhancement of axonal elongation by laminin observed at days 4 and 5 was preceded by an up-regulation of tau message and protein levels at day 3 (Figs. 4 and 5) , strongly suggesting a tight relationship between tau expression and the rate of neurite growth in HCNs. Laminin particularly enhanced the expression of 4R tau, suggesting that higher expression of 4R tau may be related to increased microtubule stabilization required during rapid axonal elongation (23 , 59 60 61) .

Synaptic loss is one of the earliest features associated with tau pathology (62) , possibly as a consequence of alterations in microtubule-based transport of organelles and synaptic proteins due to increased tau solubilization (63) , expression (64) , and/or filament formation (27) . The establishment of numerous synapses by HCNs (Fig. 3) (29) will allow us to perform a detailed examination of the time course of tau modifications and their effect on axonal transport and synaptic number and function (unpublished results). Immunofluorescence analysis using three different 4R isoform-specific antibodies resulted in a stippled pattern of staining along axonal processes, which differed from the smooth labeling obtained with antibodies that do not distinguish between tau isoforms (Fig. 6) , or with a 3R tau-specific antibody (data not shown). This finding raises the possibility of tau isoform-specific subcellular localization and function in HCNs, possibly related to distinct interactions of 3R and 4R with different microtubular populations required to regulate dynamic instability in neuronal processes (61) . In mature HCNs, the 4R/3R expression ratio was ~1, similar to the 4R/3R ratio in the adult human brain, and in contrast with the elevated levels of 4R observed in the DS/AD brain (Fig. 4) . In fact, previous studies have reported an excess of 4R tau in several tauopathies and also in AD (21 , 45 , 65 66 67) . This is particularly relevant in light of recent experiments showing that 4R tau suppresses neuronal precursor proliferation and promotes neuronal differentiation and axonal growth in the hippocampus of tau knockin/knockout mice (59) . Thus, alterations in tau isoform levels may lead to pathological changes ranging from unbalanced cytoskeletal modulation to perturbations in neuronal differentiation and process formation. These results underscore the importance of studying tau pathology in an experimental system that recapitulates as close as possible the expression pattern of tau in the human brain and that can replicate the molecular changes present during the disease process. In this regard, HCNs represent a valuable experimental model, which appears to recapitulate important molecular aspects of tau isoform expression in the human brain.

Protein phosphatases are the focus of intense research in the field because they are critical to counterbalance the effect of kinases on tau phosphorylation (1) . Chronic treatment with specific phosphatase inhibitors demonstrated that inhibition of PP2A activity resulted in marked alterations in the band profile of tau isoforms, elevated levels of soluble tau, increased level of phosphorylated tau in the soluble fraction, and translocation of phosphorylated tau to the somatodendritic compartment (Fig. 7) . Consistent with these observations, PP2A is the major phosphatase activity toward phosphorylated tau in the brain (68 , 69) ; it binds to tau, and it mediates its association with microtubules (47 , 70) , indicating an important role for PP2A in tau pathological changes. We have also detected dramatic changes in tau phosphorylation and cystoskeletal association in HCNs after treatment with a number of agents, including proteasomal inhibitors, lysosomal inhibitors, and kinase activators (data not shown), suggesting that tau phosphorylation is regulated at multiple levels, and that alterations in a number of cellular pathways can lead to tau modifications, consistent with the conspicuous presence of tau pathology in neurological conditions with very different etiology.

Finally, we found tau oligomeric forms after phosphatase inhibition in HCNs. Berger and coworkers have recently identified tau multimers in the 140- to 170-kDa range in tauopathy mouse models, as well as in FTDP-17 and AD brain homogenates (56) . Furthermore, the appearance of tau multimers closely correlated with functional memory impairments in tau mutant mice, suggesting the involvement of tau multimeric species in the disease process. Tau multimers in HCNs were clearly recognized by 4R tau-specific antibodies and by the conformation-dependent antibody A11, which recognizes oligomeric species regardless of the protein primary sequence (58) , indicating that 4R tau isoforms are principally involved in tau oligomerization. The intracellular accumulation of tau oligomeric forms appears to impair neuronal function independently of tau phosphorylation state since tau multimers have been shown to derive from both nonhyperphosphorylated (~140 kDa) and hyperphosphorylated tau (~170 kDa) (56) and are induced by multiple cellular insults (unpublished results). Ongoing studies in our laboratory are directed to determine the functional consequences of tau oligomerization in neuronal function, including protein trafficking and synaptic activity. In summary, these studies demonstrate a specific and complex pattern of tau isoform expression in HCNs, which may play a critical role in the development of human tauopathies.


   ACKNOWLEDGMENTS
 
This study was supported by grants from the Alzheimer’s Association, the U.S. National Institutes of Health (HD-38466), and The Institute for Brain Aging and Dementia at University of California, Irvine. We thank Drs. Peter Davies and Charles C. Glabe for reagents and helpful advice, and Drs. Martin Ingelsson and Rohan de Silva for generously providing antibodies.

Received for publication August 30, 2007. Accepted for publication January 10, 2008.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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