A chemical-genetic strategy reveals distinct temporal requirements for SAD-1 kinase in neuronal polarization and synapse formation
© Kim et al; licensee BioMed Central Ltd. 2008
Received: 09 July 2008
Accepted: 22 September 2008
Published: 22 September 2008
Neurons assemble into a functional network through a sequence of developmental processes including neuronal polarization and synapse formation. In Caenorhabditis elegans, the serine/threonine SAD-1 kinase is essential for proper neuronal polarity and synaptic organization. To determine if SAD-1 activity regulates the establishment or maintenance of these neuronal structures, we examined its temporal requirements using a chemical-genetic method that allows for selective and reversible inactivation of its kinase activity in vivo.
We generated a PP1 analog-sensitive variant of SAD-1. Through temporal inhibition of SAD-1 kinase activity we show that its activity is required for the establishment of both neuronal polarity and synaptic organization. However, while SAD-1 activity is needed strictly when neurons are polarizing, the temporal requirement for SAD-1 is less stringent in synaptic organization, which can also be re-established during maintenance.
This study reports the first temporal analysis of a neural kinase activity using the chemical-genetic system. It reveals that neuronal polarity and synaptic organization have distinct temporal requirements for SAD-1.
An emerging theme from recent studies of neural development is that many genes are employed repeatedly by different developmental processes. For example, morphogens such as WNTs are important not only for neural patterning but also for synaptogenesis [1, 2]. Similarly, secreted factors such as UNC-6/netrin and semaphorins are required for both axon guidance and synapse formation [3–7], and gamma-protocadherins promote both neuronal survival and synapse formation [8, 9]. These molecules may serve multiple functions through a single or multiple genetic pathways during the different developmental processes.
Distinguishing the multiple roles of a gene in neuronal development is often challenging because the developmental processes are highly interdependent: neurons that fail to polarize cannot form functional synapses, and conversely, failure in establishing synapses may lead to axon retraction and subsequent abnormalities in axon growth. Therefore, the involvement of a gene in later differentiation stages could be masked by an early-stage arrest. Addressing this issue with conventional genetic methods is not always possible. Gene knock-outs lead to an irreversible, often complete loss of gene function, and their mutant phenotypes are likely to reveal only the earliest roles during development. Temperature sensitive (ts) or partial loss-of-function (LOF) genetic alleles are a commonly used alternative. However, ts alleles are available for only a very small number of genes, and partial LOF alleles may carry sufficient residual activity to obscure the functional identification. The conditional Cre-Lox recombination-induced gene knock-out system allows temporal and tissue-specific gene inactivation; but few Cre lines permit the tight temporal control required to analyze the neuronal differentiation events that transition from one stage to the next within a narrow window of time. Therefore, a complete but reversible inactivation strategy that allows for tight temporal control and tissue specificity would be an ideal approach to addressing this issue .
In this study, we adopted this chemical-genetic approach to study the temporal requirements for a serine/threonine kinase, SAD-1 (Synapses of amphids defective-1). Initially identified in Caenorhabditis elegans, SAD-1 is required for proper neuronal polarity and synaptic organization [20, 21]. In sad-1 LOF mutants, synaptic vesicles and other presynaptic proteins are trafficked to both axons and dendrites, and vesicle clusters are abnormally diffuse at synapses. Its mammalian orthologs, SAD-A and SAD-B, also play multiple roles in neural development. Mouse SAD-A and SAD-B (also known as BRSK2 and BRSK1, respectively) function redundantly to regulate neuronal polarity in vivo [22, 23], and SAD-B regulates the release of synaptic neurotransmitters in cultured rat neurons .
The mechanism of SAD-1-mediated regulation of neuronal polarity and synaptic organization is not well-understood. Our recent study showed that SAD-1 physically interacts with a scaffolding protein, NAB-1 (Neurabin-1), and that this interaction is essential for regulating neuronal polarity but not synaptic organization . These data suggest that SAD-1 regulates neuronal polarity through pathways that are distinct from those for synaptic organization. Whether SAD-1 is required for the establishment or maintenance of the two neuronal structures is unknown. To dissect the temporal requirements for SAD-1 in neuronal polarity and synaptic organization, precise temporal inactivation of SAD-1 activity in vivo is needed. The kinase activity of SAD proteins is essential for their in vivo functions [20, 23], making SAD-1 an ideal candidate for a chemical-genetic system that allows for inducible and reversible inactivation of kinase activity.
The chemical-genetic strategy has been used in yeast, Arabidopsis, Drosophila, mammalian cell lines, and mice [13–19]. Here we report the first successful application of this method in C. elegans. Using this system, we discovered that the kinase activity of SAD-1 is required at developmental stages that coincide with the establishment of neuronal polarity and synaptic organization. However, while inactivating SAD-1 when neurons are establishing polarity and synapses led to irreversible defects in neuronal polarity, defects in synaptic organization were reversible and corrected by SAD-1 activity during maintenance. Therefore, these differentiation events have distinct temporal requirements for SAD-1 activity.
L123A SAD-1 is functional and can be inactivated by a PP1 analog in vitro
To generate a SAD-1 variant that is sensitive to PP1 analogs, its gate-keeper residue, leucine 123, was mutated to an alanine (L123A; Figure 1A). The kinase activity of L123A SAD-1 was compared to that of the wild-type enzyme in an in vitro kinase assay using tau as a substrate  (Figure 1B). Upon activation by the LKB1 complex , both wild-type and L123A mutant enzymes were able to phosphorylate tau at S262. L123A SAD-1 was 30% as active as the wild-type enzyme even at the highest ATP concentrations tested (Figure 1B). An alteration in activity of this magnitude is not uncommon for kinases with mutated gate-keeper residues, and in most cases the decrease in activity does not affect the ability of the kinase to properly function in vivo .
We next tested the sensitivity of wild-type and L123A SAD-1 to a PP1 analog, 1NA-PP1. The wild-type enzyme was unaffected by 1NA-PP1, but L123A SAD-1 was inhibited in a dose-dependent manner (Figure 1B). In summary, L123A SAD-1 retains kinase activity and is selectively sensitive to a PP1 analog.
L123A SAD-1 is functional in vivo
To determine if L123A SAD-1 can substitute for wild-type SAD-1 in vivo, we asked whether it can rescue neuronal polarity and synaptic organization phenotypes of sad-1 null mutants. The L123A mutation was introduced to a sad-1 construct that rescues defects in both neuronal phenotypes of sad-1 mutants [20, 21]. This construct, P sad-1 -sad-1(L123A), was introduced into sad-1 (ky289) null mutants along with juIs1, a fluorescent marker of synaptic vesicles in GABAergic neurons (P unc-25 -snb-1::gfp) . The juIs1 marker has been shown to be a reliable reporter for neuronal polarity  and synaptic organization [20, 21].
We also asked whether L123A SAD-1 could rescue the synaptic organization defect in sad-1 mutants. In wild-type animals, juIs1 puncta in DD axons were round and discrete along the dorsal nerve cord, displaying the characteristic 'beads-on-a-string' morphology (Figure 2Di, Ei). In sad-1 mutants, juIs1 puncta appeared diffuse or smaller (Figure 2Dii, Eii). This defect in the juIs1 morphology correlates with a more diffuse distribution of synaptic vesicles in sad-1 mutants as shown by electron microscopy . For a quantitative analysis of the juIs1 morphology, the width of each punctum was measured, and the population distribution of punctum widths was examined. As shown in Figure 2F, sad-1 mutants displayed a broader distribution of punctum widths, or 'lower kurtosis' of the distribution curve, than wild-type animals, consistent with the appearance of both diffuse and smaller puncta. In the L123A SAD-1-expressing sad-1 mutants, juIs1 puncta appeared more round and discrete (Figure 2E, F). Thus, L123A SAD-1 rescues synaptic organization as well as polarity defects in sad-1 mutants.
L123A SAD-1 can be selectively inactivated by 1NA-PP1 in vivo
These data show that L123A SAD-1 is both functional and selectively inactivated by 1NA-PP1 in vivo. We therefore hereafter refer to L123A SAD-1 as an analog-sensitive version of SAD-1 kinase (SAD-1as) and L123A SAD-1-expressing sad-1 mutants as SAD-1as animals.
SAD-1 kinase activity is required for the establishment of neuronal polarity and synaptic organization
As noted above, life-long exposure of SAD-1as animals to 1NA-PP1 led to defects in neuronal polarity and synaptic organization (Figure 3). To ask whether the kinase activity of SAD-1 is required for the establishment or maintenance of neuronal polarity, we exposed SAD-1as animals to 1NA-PP1 during early larval stages or later (Figure 4B) and counted ectopic juIs1 puncta in adults (Figure 4C). Inhibiting SAD-1as throughout establishment and not maintenance was sufficient to abolish its rescuing effects on the polarity defect (Figure 4Bi, C). Moreover, inhibiting SAD-1as for as few as 30 hours during L1 and L2 was sufficient to elicit the same level of polarity defect (Figure 4Bii, C). Conversely, an exposure to 1NA-PP1 throughout maintenance had little effect on the polarity phenotype of SAD-1as animals (Figure 4Biii, C). These results suggest that SAD-1 activity is required for the establishment, but not the maintenance, of neuronal polarity.
In contrast to the dramatic effects of early larval inhibition on adult neuronal polarity, inactivating SAD-1as for either three days or 30 hours in early larvae had little effect on the juIs1 morphology in adults (Figure 4B, D). Diffuse puncta were observed occasionally after three days of inactivation (Figure 4Di), but this subtle abnormality was not quantitatively significant (Figure 4Div).
Distinct temporal requirements exist for SAD-1 kinase activity during neuronal development
Our study demonstrates the first application of the chemical-genetic system in C. elegans. To further test our findings from using this new methodology, we employed an independent method. Instead of inactivating SAD-1, we utilized a heat-shock (HS) inducible system to express wild-type SAD-1 in sad-1 null mutants. If SAD-1 determines neuronal polarity solely in developing neurons, expression of SAD-1 after the L2 stage would not rescue the neuronal polarity defect in adults. In contrast, if SAD-1 could re-establish synaptic organization in the maintenance stage, expression of SAD-1 after the L2 stage should rescue the synaptic morphology defect in adults.
We report here the first successful application of the chemical-genetic system to analyzing the temporal requirements of a kinase in C. elegans. A PP1 analog-sensitive version of SAD-1 kinase, SAD-1as, was functional and sensitive to 1NA-PP1-mediated inhibition, both in vitro and in vivo. Using this system, we temporally inactivated SAD-1 kinase during different stages of the C. elegans life-cycle and found that SAD-1 activity is necessary in establishing both neuronal polarity and synaptic organization but dispensable for their maintenance (Figure 7). However, while SAD-1 activity is required strictly during a narrow window of time to establish neuronal polarity, its activity is not temporally restricted to the establishment stage and sufficient to re-establish synaptic organization in later stages. Our study thus reveals temporally distinct requirements for SAD-1 activity during neuronal differentiation.
A chemical-genetic approach in analyzing kinase functions in C. elegans
We showed that SAD-1as can be generated by mutating the gate-keeper residue, allowing inducible, fast, and reversible inhibition of its kinase activity with a specific small-molecule inhibitor. In this study, a small-volume liquid culture system was devised that offered several advantages over conventional plate cultures (Additional files 1 and 2; described in Materials and methods). The small volumes of the liquid culture required only microgram quantities of 1NA-PP1, reducing the quantity of inhibitor used 30-fold. The liquid culture also facilitated efficient administration of the membrane-permeable inhibitor through feeding.
PP1 analogs effectively blocked the activity of as-kinases at nanomolar concentrations in yeast and mammalian cell cultures [13, 16]. In our study, micromolar-range 1NA-PP1 was required to achieve complete inhibition of SAD-1as (Figure 3). This is likely caused by the poor permeability of C. elegans cuticles, only allowing entry through feeding. Micromolar concentrations of PP1 analogs were also required to inhibit mammalian as kinase activities in intact animals through oral administration [14, 16]. Therefore, the efficacy of 1NA-PP1 in C. elegans is comparable to that in mice in vivo.
The kinetics of 1NA-PP1 entry in C. elegans is unknown. However, we do not think slow kinetics of entry was a confounding factor in our analyses because the shortest exposure time of 30 hours was sufficient for 1NA-PP1 entry and SAD-1as inactivation (Figure 7Av, vii). Indeed, a similar PP1 analog, 1NM-PP1, has been shown to cross the murine blood-brain barrier within minutes . We also do not believe differential susceptibilities to 1NA-PP1 entry existed during different larval stages of the C. elegans life-cycle because the phenotypic difference in synaptic organization between the lifetime exposure (Figure 7Aii) and early larval exposures (Figure 7Avi, vii) suggests that there was sufficient 1NA-PP1 entry and inhibition of SAD-1as across the larval stages.
Although the chemical-genetic system has proven to be quite a versatile and effective tool for regulating the activity of different kinases in yeast and cell culture studies, its application to intact animal models has been limited. Many kinase families display strong functional conservation across different animal species. The ease of genetic manipulation, simple physiology, and short developmental cycle make C. elegans an attractive target for using chemical genetics to temporally dissect the functional requirements of kinases. Our protocol may be applicable to other kinases or even other proteins containing ATP-binding domains  in C. elegans.
Distinct temporal requirements for SAD-1 activity in neuronal polarization and synapse formation
We previously demonstrated that SAD-1 regulates neuronal polarity and synaptic organization through different genetic pathways . Using inducible modulation of protein activity (as) and of mRNA expression (HS), we have now demonstrated that the neuronal structures also display different temporal requirements for SAD-1 activity.
The molecular basis for the temporal distinction between neuronal polarity and synaptic organization is unknown. The simplest explanation is that SAD-1 targets different substrates during polarization and synapse formation. While the substrates involved in neuronal polarization may only be present or active during the establishment stage, those involved in synapse formation may be present throughout establishment and maintenance. The mammalian orthologs of SAD-1, SAD-A and SAD-B, have been shown to regulate neuronal polarity through phosphorylation of tau, a microtubule associated protein [22, 23], and subsequent promotion of microtubule dynamics during axon extension . As an activator of microtubule dynamics, SAD-1 would define neuronal polarity only during the establishment stage. At synapses, the diffuse vesicle clustering in sad-1 mutants may reflect a failure in regulating vesicle release at the active zone or vesicle retrieval at the periactive zone. SAD-1 might phosphorylate distinct substrates that are required for organizing the endo- and exo-cytosis apparatus or for facilitating the interactions between vesicles and active zone/periactive zone regions.
To further our understanding of SAD-1, it is critical to identify all of its physiological substrates. The chemical-genetic system has been used to identify substrates of kinases in in vitro assays [33, 34]. Exploiting our SAD-1as system with substrate-labelling analogs may facilitate the identification of substrates and provide insights to the mechanism of SAD-1 in the future.
Materials and methods
All C. elegans strains were cultured at 22°C using standard procedures on Nematode Growth Medium  with OP50 Escherichia coli as the food source.
juIs1 (Punc-25-snb-1::gfp) has been described previously . pJH685 (Psad-1-sad-1(L123A)) was co-injected with a Podr-1-gfp marker into sad-1 (ky289); juIs1 animals and integrated into the C. elegans genome by UV irradiation. The integrants were out-crossed four times against wild-type N2 animals to generate hpIs89. From this, hpIs89; sad-1 (ky289); juIs1 was generated and termed 'SAD-1as animals'. pJH73 (Punc-115-sad-1) was co-injected with a Podr-1-gfp marker into sad-1 (ky289); juIs1 animals to generate sad-1 (ky289); juIs1; hpEx1043 animals. pJH1360 (P HS -sad-1) was co-injected with a Podr-1-gfp marker into sad-1 (ky289); juIs1 animals to generate sad-1 (ky289); juIs1; hpEx1431 animals. pJH1368 (P HS -gfp::sad-1) was co-injected with a Plin-15-lin-15 into lin-15 animals to generate lin-15; hpEx1421 animals.
The L123A mutation was engineered by oligonucleotide site-directed mutagenesis in a sad-1 genomic DNA fragment to generate plasmid pBNL32. pJH685 (Psad-1-sad-1(L123A)) was generated by sub-cloning the Nhe I/Xma I fragment of pBNL32 into pJH630, which contains the minimal rescuing region (Sac II/Avr II fragment) from the cosmid F15A2. pJH73 was generated by inserting a sad-1 mini-gene fragment from pJH56 under the unc-115 promoter into the pBSK vector using the Not I and Bam HI sites. pJH1360 was generated by inserting a sad-1 mini-gene fragment from pJH56 into the HS pPD118.26 vector using the Bam HI and Apa I sites. pJH1368 was generated by cloning gfp from pJH21 into the 5' end of sad-1 in pJH1360 using the Bam HI site.
Protein purification and kinase assays
Wild-type and L123A versions of SAD-1 cDNA were sub-cloned into pGEX6p-1. Proteins were expressed in E. coli BL21 by inducing with 0.1 mM isopropyl beta-D-thiogalactoside (IPTG) for 24 hours at room temperature with aeration. Purification was performed as described in  except that elution was performed using PreScission protease (GE Healthcare, Buckinghamshire, England) according to the manufacturer's specifications. Eluted protein was snap-frozen at -80°C until use. Due to low yields of protein, quantification and normalization of the wild-type and L123A proteins was performed by western blot using an anti-SAD-1 antibody previously described .
For kinase assays, an activation reaction was first performed to phosphorylate SAD-1 at the activation loop threonine, a modification required for full activity of AMP-activated protein kinase (AMPK) family members . In this reaction, approximately 5 μg of wild-type or L123A SAD-1 was incubated with 100 ng of LKB1 complex (Upstate, Billerica, MA, USA) at room temperature for 30 minutes in a kinase buffer (KB) containing 25 mM Tris-HCl, 10 mM MgOAc, 1 mM dithiothreitol, 0.1% (v/v) Triton X-100, and 0.1 mg/ml bovine serum albumin, pH 7.4 plus 100 μM ATP in a volume of 50 μL. SAD-1 kinase assays were performed using purified tau as a substrate: 400 ng of purified 4R tau (a kind gift of MA Glicksman, Brigham & Women's Hospital, Cambridge, MA, USA) diluted in TBS was added to each well of a 96-well tissue culture dish (standard tissue culture treated polystyrene; Corning, Lowell, MA, USA) followed by overnight incubation at 4°C. The wells were washed with four changes of TBS with 0.1% Tween-20 (TBS-T) and were then equilibrated with KB prior to the start of the assay. Immediately prior to the start of the assay, the KB was aspirated and 10 μL of KB containing the desired concentrations of DMSO/1NA-PP1 (final DMSO concentration was 3.3%) was added. The activation reaction was diluted in the same buffer, but the ATP concentration was adjusted to 500 μM, and 20 μL aliquots were added to the wells (30 μL final reaction volume). Reactions, performed in triplicate, were incubated at room temperature and were stopped after 30 minutes by aspirating the liquid and adding 50 μL of 10 mM EDTA. To measure phosphorylation of immobilized tau at Ser262, an ELISA was performed using an anti-phosphoTau [S262] antibody (1:1000; Stressgen, Ann Arbor, MI, USA) and a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:25,000; Jackson Immunoresearch, West Grove, PA, USA). Antibodies were diluted in Superblock TBS blocking solution (Pierce, Rockford, IL, USA), and bound antibody was visualized using TMB liquid chromogenic substrate (Sigma-Aldrich, St Louis, MO, USA). After sufficient signal developed, the reactions were stopped by addition of an equal volume of 1 N HCl, and the plates were read in a Spectramax 384 plate reader. Background signal was determined by measurements from multiple wells that did not contain kinase reactions.
Observation of juIs1 puncta in VD neurons
As there is no VD-specific promoter, selective visualization of juIs1 puncta in VD neurons was achieved by inhibiting expression of the juIs1 marker in DD neurons as previously described [21, 27]. Briefly, double-stranded RNA against the unc-30 transcription factor – which activates the unc-25 promoter used to express the juIs1 marker – was synthesized from pJH573 as previously described  and injected at 100 ng/μL into adult animals carrying the juIs1 marker . Progenies of the injected animals that retained the GFP signal in all 13 of the VD neuron cell bodies but in none of the six DD neuron cell bodies were scored.
DMSO and 1NA-PP1 treatments in C. elegans
Animals were exposed to DMSO (Sigma-Aldrich, Oakville, Ontario, Canada) or 1NA-PP1 in liquid culture. Overnight LB cultures of OP50 E. coli were divided into 500 μL aliquots, and pellets were collected and stored at -20°C until use. Each pellet was resuspended in 650 μL of S-medium. Animals were incubated in 150 μL of the OP50 E. coli resuspension containing 0.33% DMSO or 33 μM 1NA-PP1/0.33% DMSO added to the wells of a 96-well tissue culture dish (Falcon, St Louis, MO, USA). Exposures throughout the C. elegans life-cycle for validation studies were achieved by treating fourth larval (L4) stage animals and observing their offspring in an adult stage. The solutions were renewed every three days. Exposures until earlier than the adult stage were performed by treating L4 animals and transferring their offspring onto OP50 E. coli-seeded agar plates at desired stages, assessed by the stage of gonad development. Early first larval (L1) stage-arrested animals were obtained by lysing gravid adult animals in hypochlorite on unseeded agar plates and allowing them to develop at 22°C for 16–18 hours. Late second larval stage (L2) animals were recovered by transferring and keeping early L1-arrested animals on OP50 E. coli-seeded agar plates for 30 hours at 22°C. Detailed procedures on the incubations are described in Additional files 1 and 2.
All HS treatments were performed by incubating animals at 30°C for 7 hours. A gradual decrease in the GFP::SAD-1 protein expression level, reflected by the intensity of GFP fluorescence, was observed following HS over the course of a day. Therefore, for post-L2 HS treatments, late L2 animals obtained as described above were heat-shocked twice, 15 hours apart.
Puncta quantification and statistical analyses
Significance-testing employed paired t-test for the in vitro kinase assay and the Wilcoxon rank-sum test for the VD neuron analyses as implemented in the R statistical environment (v2.6.2) . juIs1 puncta were quantified by an in-house developed program called 'punctaanalyser' using the MatLab software (v6.5; Mathworks, Inc., Natick, MA, USA) and analyzed using kernel density estimation implemented in R (v2.6.2).
- C. elegans :
green fluorescent protein
Synapses of amphids defective
analog-sensitive version of SAD-1 kinase.
We thank MA Glicksman for tau protein; K Shen for the Podr-1-gfp injection marker; P Candido and AZ Fire for the pPD118.26 vector; C Mok and T Kawano for the 'punctaanalyser' program; PC Boutros for statistical assistance and critical comments on the manuscript; and members of MZ's laboratory for critical review of the manuscript. This work was funded by an NIH grant (RO1AI440099) to KMS, a grant from NIH (NINDS) to JRS, and an NSERC discovery grant (112033912) awarded to MZ. JSMK is a recipient of NSERC graduate student fellowships (CGS M and PGS M). BNL is a fellow of the Damon Runyon Cancer Research Foundation (DRG 1914-06).
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