1. Introduction
Cultured SGC neurons have been regularly used as an in vitro model for sympathetic neurons and in studying axon growth and trafficking, synaptogenesis, microtubule dynamics, axon regeneration and degeneration and neuronal survival [
1]. They can be grown as dissociated cells or explants and as a co-culture system with glial cells [
2]. Sympathetic neurons can be prepared from embryonic or postnatal rats or mice using dissected SCGs and grown as pure neuronal cultures in the presence of nerve growth factor (NGF, [
3,
4]). These homogenous neuronal cultures can be processed for immunocytochemical staining, biochemical studies and physiological analysis [
5,
6]. SCG neurons extend axon-like neurites that rapidly grow to several millimeters making them an ideal system to study the properties of growing and degenerating axons growth in the context of the neuronal cytoskeleton [
7]. What has not always been considered in these studies is the rapid changes in the expression of critical cytoskeletal protein, which can affect the properties of the neurons and interpretation of the results. In particular, there is a dramatic switch in the expression of tau from the low molecular weight (LMW) isoforms in early embryonic stages to Big tau at late postnatal stages which directly affects the dynamics of the microtubules as well as the properties of the axons with respect to growth, transport and possible degeneration [
8,
9].
The Big tau Isoform has been discovered 30 years ago [
10,
11,
12] composed of the additional 4a exon which dramatically increased the size and likely the 3D structure of tau. Big tau was initially shown to be expressed in the peripheral nervous system (PNS) and later it has also been mapped to specific areas of the central nervous system that project to the periphery such as cranial nerve motor nuclei as well as retinal ganglion cells and the cerebellum [
13]. There remains however a significant gap of knowledge about the developmental changes in tau expression with respect to the switch from the LMW isoforms to Big tau, the differential effects that it has on microtubule dynamics and the properties of axons that express this isoform. So far, it is known that although the 4a exon defining Big tau has a stable size of about 254-aa in a wide range on vertebrates, its sequence is not conserved among species; for example, showing almost no homology between mammals and non-mammals vertebrates [
14,
15].
The present study is designed to be a roadmap for the expression of the tau isoforms during development and in culture following the timeline of the switch to Big tau. This could serve as a reference for future studies taking into account the critical forms of tau expression particularly when studying microtubule dynamic and the properties of axons in growth, regeneration and degeneration. Using antibodies for all tau isoforms and those specific for Big tau we have presented structural, immunocytochemical and developmental data of SCG in vivo and in vitro including dissociated neuronal cultures and explants response to injury.
2. Experimental Procedures
2.1. Tissue Preparation
For morphological analysis, adult Sprague Dawley rats (4 months-old) were perfused transcardially by 0.9% normal saline followed by ice-cold 4% paraformaldehyde (PFA) fixative in phosphate buffer. All tissues needed for this study (SCG and DRGs, dorsal root ganglia) were dissected out and kept in same fixative overnight, and then transferred into 30% sucrose for 3–5 days. For SCG dissection, SCG was identified on the underside of a bifurcation of the carotid artery. Under dissecting microspore, the skin at the throat was cut from the chin to the upper chest. The fat pad around the sides of neck was cut away first. To visualize the carotid artery, three muscles needed to clip away by first removing the superior-most muscle (closer to head), then the second one (close to the low chest) and finally the muscle lying parallel to the trachea. Using forceps to hold the artery at the point of the bifurcation, SCG was underneath the bifurcation and could be completely removed. Tissues were embedded in M1 and cut either cross or sagittal sections at 20 µm. Sections were collected on Superfrost Plus (Fisher Scientific, Hampton, NH, USA) slides and kept at −20 °C.
For Polymerase chain reaction (PCR) and Western blots, fresh tissue was dissected from adult rat brain, DRGs, and SCGs at 1–7 weeks-old postnatally, adult (4 months-old), as well as aged (over 12 months-old), then transferred quickly into 1.5 mL tube with rNase, dNase Pyrogen-free (Capital Scientific, Inc., Austin, TX, USA) on dry ice. All samples were stored at −80 °C. For analysis of SCG explant, samples were collected following culturing for 8 days and 4 h or 5 days after lesion in the same 1.5 mL tubes and stored at −80 °C.
2.2. Primary SCG Culture
SCGs were dissected from rats at different ages: postnatal days 3, 14, 21, 28. To dissociate the tissue into single cells, SCGs were cut into small pieces and treated enzymatically with incubation in collagenase for 15 min at 37 °C followed by trypsin for 45 min also at 37 °C. Reaction was stopped by adding blocking medium (L-15 base medium, 1.2% D-glucose, 2% Glutamax, 2% Pen/strep, penicillin-streptomycin, 10% FBS, fetal bovine serum). SCGs were transferred into a 1.5 mL tube after removal of blocking medium, added 1 mL of plating medium (blocking medium with NGF 0.1 ng/mL), triturating the ganglia 10–15 times with P1000 pipette (Eppendorf North America, Enfiled, CT, USA) and then plated in 120 μL as suspension in a 24-well plate with coverslips coated with poly-D-lysine, (PDL, 1 mg/mL) and laminin (10 μg/mL) using 6–8 coverslips in a plating density of 4000 cells/coverslip. Cells were cultured for 3–10 days, and then fixed with 4% PFA for 30 min at room temperature. Coverslips were then washed 3 times with phosphate-buffered saline (PBS), and then stored at 4 °C for immunocytochemical staining.
2.3. SCG Explant Culture
SCGs were dissected from postnatal day 3 (P3) rats. Ganglia were cut into two pieces and placed on a 35 mm dish with 14 mm glass bottom pre-coated with PDL and laminin same as described above with limited culture medium for 2 h. More culture medium was added 2 h after incubation. Medium was changed every other day. After 8 days of culturing, neurites from SCG explant were cut using a 30-gauge needle and allowed to continue to grow. SCG explants were fixed with 4% PFA at different times after the lesion (4 h and 5 days) and washed with PBS 3 times. SCG explants in PBS were stored at 4 °C for staining with a Big tau specific antibody 1:1000 (Fischer lab, Philadelphia, PA, USA) described in Boyne et al., 1995 and a Tuj antibody (Covance, Biolegend, San Diego, CA, USA), 1:500).
To analyze the ratio of Big tau/Tuj staining in different regions of the explants, images were processed using Image J (NIH, Bethesda, MD, USA) as described in [
16]. Briefly, we defined “proximal” areas inside 150 µm distance from the cell body and “distal” outside this area outlining 3 regions of 100 × 100 pixels for each area where the densities of Big tau and Tuj staining were measured and expressed as a ratio of Big Tau to Tuj. Values of the 3 regions of each image were averaged and Student’s
t-test (SigmaPlot 13, Palo Alto, CA, USA) was performed to determine the significance of the differences between the proximal and the distal ratio in both time points (4 h and 5 days post lesion).
2.4. Western Blots
Tissue homogenates were disrupted and homogenized in cold lysis buffer (RIPA: 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS) in the presence of protease inhibitor cocktail using a Fisher brand Sonic Dismembrator. Protein determination was done by Pierce™ BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). The tissue homogenates were stored at −80 °C. Equal amounts of protein from each sample were denatured in 4×protein sample loading buffer (LI-COR) containing 2-mercaptoethanol and boiled at 95 °C for 5 min. Samples and 2 μL of the Chameleon duo pre-stained protein ladder (LI-COR, Lincoln, NE, USA) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4%–12% polyacrylamide bis-tris gels (Invitrogen, Waltham, MA, USA). After distilled water washing and dry transfer (Thermo Fischer iBlot system, Waltham, MA, USA), PVDF membranes were washed in Tris-buffered saline pH 7.4 (TBS) and then blocked in Intercept TBS blocking buffer (LI-COR) for 1 h at room temperature, followed by incubation with primary antibodies: Big tau ([
13] diluted 1:1000), 3′ Tau [
9] diluted 1:5000, actin (Millipore, Burlington, MA, USA, 1:10,000) at 4 °C overnight. Membranes were washed with TBS and then incubated with fluorescently labeled IRDye secondary antibodies (LI-COR) for 1 h at room temperature. After several TBS washes, membranes were washed in distilled water and digital fluorescent visualization of signals were detected at 700 and/or 800 nm channels using the Odyssey
® CLx Imaging System (LI-COR). All primary and secondary antibodies were diluted in Intercept Antibody Diluent (TBS). To assess the levels of Big tau and LMW tau during development, the average densities of the corresponding bands from the original raw data of early (weeks 1–3) and late postnatal times (weeks 5–7) were calculated and plotted.
2.5. Polymerase Chain Reaction (PCR)
RNA extraction and purification was done using a RNeasy Mini Kit (Qiagen, Germantown, MD, USA). cDNA was obtained using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, ref. 4368814, Thermo Fisher Scientifics, Waltham, MA, USA). Primers were designed with the BLAST program to include sequences of exon 2 (5′—TCA GAA CCA GGG TCG GA—3′) to exon 4A (5′—GCA GGT TGC TTG TCA GTT GG—3′), exon 4A (5′—GGG TTC CAT CCC ACT TCC TG—3′) to exon 6 (5′—GGT GGT TCA CCT GAT CCT GG—3′); and exon 9 (5′—CCG TCT GCC AGT AAG CGC—3′) to exon 12 (5′ –CTA CCT GGC CAC CTC CTG GC—3′).
All primers were purchased from IDT (Integrated DNA Technologies, Inc., Coralville, IA, USA). PCR were run using a Taq DNA polymerases Kit (ABM, Richmond, BC, Canada) on a thermal cycler (Bio-Rad, Hercules, CA, USA).
2.6. Immunocytochemical/Histochemical Staining
Immunocytochemical tissue analysis was performed with the following antibodies used at single or double staining: Big tau 1:1000 (Fischer lab), 3′-Tau 1:1000 (Fischer lab), Tuj 1:500 (Covance), IB4 1:2000 (Invitrogen, Waltham, MA, USA), CGRP 1:2000 (Peninsula, Rheinstrasse, Switzerland), Parvalbumin 1:500 (Abcam, Waltham, MA, USA). Sections were washed 3 times in PBS and incubated with blocking buffer (PBS with 0.3% triton X-100, 0.5 mg/mL BSA, 0.01% thimerosal, 5–10% goat serum) for 1 h at room temperature, then incubated overnight with the primary antibodies for either single or double staining. On the following day, sections were washed 3 times in PBS, 10 min each, incubated with goat anti-mouse or goat anti-rabbit antibodies conjugated to FITC or rhodamine red (Jackson ImmunoResearch, West Gove, PA, USA) for 2 h, and then cover-slipped with anti-fade fluoromount-G (SouthernBiotech). All the staining experiments were repeated to assure the rigor of our data using a representative figure to illustrate the results.
For analysis of dishes or coverslips cultured with cells, cells were washed with PBS 3 times, incubated with blocking buffer for 15 min, and then incubated with the primary antibodies: Big tau 1:1000, Tuj 1:500 (Covance), and MAP1B 1:1000 (Fischer lab) for 1–2 h. Coverslips were washed with PBS, and then incubated with secondary antibodies: goat anti-mouse or goat anti-rabbit conjugated to FITC or rhodamine red (Jackson ImmunoResearch, West Grove, PA, USA) for 30 min, and then with anti-fade fluoromount-G (SouthernBiotech, Birmingham, AL, USA).
Slides or coverslips were visualized using a Leica DM5500B fluorescent microscope (Leica Microsystems, Wetzlar, Germany) with a Retiga-SRV camera (QImaging, Tucson, AZ, USA) and selected images were captured using Slidebook software (Olympus, Tokyo, Japan).
2.7. Statistical Analysis
SigmaPlot software package (Inpixon HQ, Palo Alto, CA, USA) was used to perform statistical analysis (one-way Anova, Student t-test) with a power of 0.05.
4. Discussion
The studies presented here analyzed the expression of Big tau in SCG tissue and cultures setting a time frame for the transition of the tau isoforms during development and accordingly the properties of the neuronal cultures derived from different stages of development. The transition from LMW tau to Big tau starts during embryonic stages and is completed at about week 4–5 postnatally setting the SCG axons with an isoform of tau characteristic of PNS neurons (e.g., DRG). It also defined a postnatal stage where both LMW tau and Big tau a co-expressed. Why is this dramatic transition necessary remains a matter of speculation [
8], but what is clear and can be informative is that the 4a exon defining Big tau is very large with little homology across the different vertebrate species. Indeed, our recent study [
15] which analyzed the exon structure of MAPT (microtubule-associated protein tau
, the tau gene) in vertebrates revealed that Big tau containing the 4a exon was present early in vertebrate evolution with low sequence conservation despite a stable size range of about 250aa. It suggested that the appearance of the significantly larger isoform of tau may have repeated during evolution, underscoring the importance of Big tau in supporting novel physiological functions required for a complex nervous system. For example, it is possible that the expression of Big tau is an adaptation of neurons with long axons that project to the periphery (e.g., PNS) to achieve robust and efficient axonal transport by acting as a much larger spacer between neighboring microtubules (the projection domain is increased from about 200 aa to >500 aa). Accordingly, the cross-sections of axonal microtubules have shown a spacing of ~35 nm when induced by Big tau compared to ~20 nm when induced by LMW tau [
17]. Another provocative idea is that that 4a exon of Big tau changes the folding of the protein in a way that avoids the generation of aggregates and thus provides a protective mechanism to otherwise vulnerable neurons This idea is consistent with the metabolic stress of long projecting axons and the reduced or late appearance of tauopathy symptoms related to aggregation observed in these neurons. Indeed, one can also point out to areas of the CNS such as the cerebellum, which express Big tau and appear to be less vulnerable to tauopathies. The PCR analysis of the Big tau transcript of SCG have not only confirmed the presence of the 4a exon but have also revealed the presence of exons 2/3 and 10. The latter, which is one of the microtubule binding domains (MTBD) indicates that Big tau has the 4R isoform, which is typical of the adult tau.
Besides the general characterization of tau expression in SCG we expect that this work will also align the results and interpretation of cell biological studies using SCG cultures with the expression of the specific isoforms of tau. For example, a standard protocol of SCG cultures is using neurons derived from early postnatal time points when they express both LMW tau and Big tau [
9,
18,
19,
20,
21,
22,
23,
24]. In these and many other studies few have considered the unusual properties of SCG cultures where microtubules are associated with the unique isoform of Big tau even when studying axonal properties closely related to cytoskeleton structure and function. In practice, antibodies specific to Big tau have not been commercially available and the story of Big tau that originated in the 90′s has faded away. In the few cases that Big tau antibodies were available or when the analysis of tau expression included biochemical or molecular methods using Western blots and PCR, respectively [
9] the presence of Big tau was noted. The challenge then and now remains how to compare and interpret the results of neurons expressing different tau isoforms. There has certainly been impressive progress studying the 6 variants of the LMW tau particularly when dealing with the alternative splicing of exon 10, which is one of the MTBD, as well as the relationship between the expression of tau variants and tauopathies [
25,
26,
27,
28,
29]. Big tau however remains in the twilight of speculation about its physiological function and relationship to tauopathies.
In contrast to the uniform staining of SCG, representing autonomic system neurons, DRG neurons, representing sensory neurons, express Big tau selectively reflecting the known heterogeneity of the sensory neurons and their diverse function [
30]. Indeed, recent molecular analysis of DRG neurons [
31,
32] expanded the unique profile of many subtypes. It is therefore interesting that SCG neuron which provide sympathetic innervation to different areas of the head and neck (e.g., pineal gland, the choroid plexus, the eye, carotid body and the salivary and thyroid glands) and may have different phenotypes [
6] all express Big tau. Both sensory and autonomic system neuron originate from the neural crest, both start expressing Big tau after E16 [
13] but they differentiate and specialize in distinct pathways. We believe that elucidating the function of Big will shed light on the similarity and difference among these neurons in the context of the neuronal cytoskeleton in general and tau function in particular.
In the last part of this study, we used SCG explants to analyze the expression of Big tau during growth and regrowth following lesion. Although our lesion/regeneration experiments with the explants shown in
Figure 8,
Figure 9 and
Figure 10 are preliminary and will need confirmation in both in vitro and in vivo systems, the results show that the explants are capable of rapid and robust regrowth of their neurites following a lesion. While the regrowing neurites contain microtubule, Big tau expression seems to be lagging and does not show at the distal neurite at 4 h but is there at the later timepoint 5 days after the lesion. These results are consistent with previous studies showing the expression of Big tau only in mature peripheral neurons (sensory and autonomic) and that the sequestration of Big tau in SCG cultures does not impair their growth [
9]. Indeed, in regeneration experiments of sciatic nerve crushing the levels of Big tau have been reduced during the regeneration phase [
33]. What then drive the growth/regeneration of the explants shown in
Figure 9 and
Figure 10? Most likely MAP1B, which is expressed in the SCG cultures (
Figure 6), and which has been shown to be associated with axon growth in both the CNS and PNS neurons [
34]. It seems therefore that mature SCG and other neurons that express Big tau retain endogenous growth associated proteins as well as an environment of Schwann cell conducive for regeneration. In contrast CNS neurons have a reduced capacity for regeneration but a better capacity for plasticity associated with the complex structure and function of the brain. The story of the selective expression of Big tau can therefore be an instructive example of the tradeoff in the balancing works of evolution.