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Human Molecular Genetics Advance Access originally published online on June 18, 2007
Human Molecular Genetics 2007 16(16):1959-1971; doi:10.1093/hmg/ddm143
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Published by Oxford University Press 2007

SUT-1 enables tau-induced neurotoxicity in C. elegans

Brian C. Kraemer1,2,* and Gerard D. Schellenberg1,2,3,4

1 Geriatrics Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, WA 98108, USA, 2 Division of Gerontology and Geriatric Medicine, Department of Medicine, 3 Division of Neurogenetics, Department of Neurology and 4 Department of Pharmacology, University of Washington, Seattle, WA 98195, USA

* To whom correspondence should be addressed at:, Seattle Veterans Affairs Puget Sound Health Care System, S182, 1660 South Columbian Way, Seattle, WA 98108, USA. Tel: +1 2062773275; Fax: +1 2067642569; Email: kraemerb{at}u.washington.edu

Received March 29, 2007; Accepted June 3, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We previously reported a transgenic Caenorhabditis elegans model for tauopathies in which expression of human tau in neurons caused insoluble phosphorylated tau accumulation, neurodegeneration and uncoordinated movement (Unc). To identify genes participating in tau neurotoxicity, we conducted a forward genetic screen for mutations that ameliorate tau-induced uncoordination. The recessive mutation sut-1(bk79) partially suppresses the Unc phenotype, tau aggregation and neurodegenerative changes caused by tau. We identified the sut-1 gene and found it encodes a novel protein. We conducted a yeast two hybrid screen to identify SUT-1 binding partners and found UNC-34, the C. elegans homolog of the cytoskeletal regulatory protein Enabled (ENA). In vitro protein binding assays and genetic studies validated the interaction between SUT-1 and UNC-34. The SUT-1/UNC-34 protein–protein interaction plays a role in both the normal function of UNC-34 and in the tau-induced phenotype. Thus, we have found a conserved molecular pathway participating in tau neurotoxicity in C. elegans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The mammalian neuronal cytoskeleton contains tubulin protein polymerized into microtubules (MTs), actin protein polymerized into microfilaments (MFs) and three distinct neurofilament protein subunits polymerized into neurofilament fibrils (NFs). Tau is a microtubule-associated protein found in neuronal axons and is involved in regulating MT dynamics (reviewed in 1). Normally, unphosphorylated tau protein binds to MTs stimulating MT polymerization and promoting MT stabilization (2). In cooperation with other cytoskeletal components, neuronal MTs function to transport cargo from the cell body to synapses along axons, to develop and maintain axonal structure, and to support growth cone and neuronal migration (reviewed in 3). Also, tau may participate in dynamic actin-mediated processes (e.g. neuronal process extension) either by directly binding to actin (4), or by interacting with specific kinases (5) that control these processes, or by binding to proteins that in turn bind actin directly. The Caenorhabditis elegans protein closest to tau is the protein with tau-like repeats-1 (ptl-1). In adult worms, PTL-1 is only expressed in 6 touch neurons, but is not required for the mechanosensory function of these neurons (6,7) or for locomotion.

In a number of neurodegenerative disorders, tau polymerizes into abnormal filaments forming neurofibrillary and glial tangles (2,8). These disorders, collectively called tauopathies, include Alzheimer's disease (AD), Down syndrome, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Pick's disease, Guam amyotrophic lateral sclerosis/Parkinson's dementia complex and frontotemporal dementia with parkinsonism chromosome 17 type with tau pathology (FTDP-17T) (2,9). While the role tau-containing lesions play in most of these disorders remains unclear, in FTDP-17T autosomal dominant mutations in the gene encoding tau (MAPT) cause the disease (10–12). These mutations demonstrate changes in tau can directly cause neurodegeneration. However, the precise mechanism of tau-induced neurodegeneration remains unclear. One hypothesis is that abnormal tau disrupts axonal MT function resulting in synaptic loss and neuronal dysfunction. As neurons accumulate aggregated tau deposits, MT function becomes increasingly impaired until MT axonal transport is disrupted causing neurodegeneration (13,14). Alternatively, tau aggregates could be intrinsically toxic, killing neurons directly. This paradigm holds that FTDP-17T mutations cause disease by promoting tau self-aggregation either by reducing the affinity of tau for MTs resulting in an increase in free tau concentrations (15), or by accelerating the rate of tau self-aggregation (16,17). Furthermore, tau in its pathological state is hyperphosphorylated suggesting tau phosphorylation state could play an essential role in neurodegeneration (18).

To explore the possible causes of tau-induced neurodegeneration, we developed a transgenic C. elegans model of human tauopathy diseases using either normal human tau coding sequences, or tau with FTDP-17 mutations P301L or V337M as transgenes (19). In this model, the pan neuronal aex-3 promoter (20) drives expression of tau in all neurons. The initial phenotype seen in the transgenic lines is uncoordinated locomotion (Unc), a phenotype characteristic of a variety of C. elegans nervous system defects. As the Unc phenotype becomes more severe with age, phosphorylated, insoluble tau begins to accumulate, which is a hallmark of human tauopathies. A progressive neurodegenerative phenotype occurs with gaps and bulges developing in GABAergic nerve cord axons and subsequent neuronal loss. By age 9 days, abnormal tau aggregates are observed by transmission EM in axons that show severe degenerative changes that include axoplasmic clearing, dilated regions and onionskin membranous infolds typical of other C. elegans neurodegenerative phenotypes. For these phenotypes, the FTDP-17 mutant transgenic lines are more severe than lines that express the normal human tau sequence. Thus, the model recapitulates several key features seen in human tauopathy disorders. To identify genes that participate in tau neurotoxicity, we carried out a forward genetic screen to identify mutations that prevent the tau-induced Unc phenotype. We isolated a recessive mutation that suppresses the strong Unc phenotype induced by tau. We call the mutated gene suppressor of tau 1 (sut-1). Animals carrying a loss of function mutation in sut-1(allele bk79) are resistant to the toxic effects of tau indicating that the product of this gene, the SUT-1 protein, is essential for tau neurotoxicity in C. elegans.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The C. elegans tauopathy model used here is transgenic for the human tau protein with the V337M FTDP-17 mutation (strain T337), and the transgene is expressed in all neurons. The result is an uncoordinated worm with reduced motility and a neurodegenerative phenotype. To identify genes required for this phenotype, we mutagenized strain T337 and screened for mutants with normal (non-Unc) locomotion. We isolated 72 putative suppressor mutants with near-normal locomotion, 11 of which bred true. Here we describe the characterization of one mutant line called bk79. The tau transgene was not altered by mutagenesis in bk79 because out-crossed mutant lines had Unc progeny similar to those of the T337 parental strain. When allele bk79 is present in the homozygous state in the parental T337 line, motility is restored to near wild type levels as measured by liquid thrashing assays (Fig. 1A). In the absence of the tau transgene, bk79 has normal motility essentially identical to N2, the wild type C. elegans strain used (Fig. 2E).


Figure 1
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  		Figure 1. sut-1(bk79) suppresses tau induced locomotion defects. (A) Liquid thrashing assays for staged 1, 3 and 5 day old worms. Each data point is the mean (±SEM) thrashing rate for 20 worms. (B) sut-1 genomic region. The minimum sut-1 region as determined by genetic mapping was between SNP markers uCe2-571 and uCe2-725. Predicted genes are shown as boxed arrows. Cosmid coverage is shown below the transcripts. Cosmid T13B5 is sufficient to rescue the sut-1 phenotype. (C) Sequence of sut-1 cDNA and SUT-1 protein. Shown is the early stop found in the sut-1(bk79) mutant.

 


Figure 2
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  		Figure 2. sut-1 allele bk79 is a loss-of-function mutation. sut-1(bk79) are deficient in sut-1 mRNA and SUT-1 Protein. (A) Diagram of the sut-1 locus; bars represent exons. The diagram is proportional to the actual sizes of the exons and introns. (B) Northern blot of polyA-selected mRNA from N2, T337 and sut-1(bk79) probed with sut-1 cDNA sequence (top) or actin (bottom). The prominent 890 nt band corresponds to the predicted transcript. sut-1 mRNA is virtually undetectable in the sut-1(bk79) mutant strain. (C) Immunoblots probed with SUT-1 specific antibodies or E7 anti-tubulin antibody. SUT-1 protein is not detectable in sut-1(bk79) mutant lysates. (D and E) Liquid thrashing measurements for 20 staged 4 day old worms of each genotype. Strains used are: D, T337/+ heterozygotes and T337;sut-1(bk79) homozygotes. Statistical analysis of liquid thrashing data using Students t-test yielded P = 0.00022 for comparison of T337/+ and T337;sut-1(bk79) homozygotes. E, Thrash rates for non-Tg animals (N2) compared to sut-1(bk79) homozygotes in the absence of the tau transgene; there is no significant difference between N2 and sut-1. (F–H) Immunostaining using SUT-1 specific antibodies. (H–J) DAPI nuclear staining of the same animals shown in panels above. (F and I) Head staining in N2 adult. (G and J) Head staining in homozygous T337 adults. H and K, head staining in homozygous T337;sut-1(bk79) adult.

 
We used single nucleotide polymorphism mapping (21) to localize the bk79 mutation to chromosome II between polymorphic markers uCe2-577 and uCe2-725, an interval of ~298 kb (Fig. 1B). To localize the mutated gene more precisely, we injected the bk79 mutant line with pools of cosmid clones spanning the interval between uCe-2-577 and uCe2-725. We found a single cosmid, T13B5 (Fig. 1B), that when present as an extrachromasomal transgene, prevented the bk79 suppression of the tau induced Unc phenotype. T13B5 contains seven complete genes. A genomic PCR fragment containing the gene T13B5.8 relieved bk79 suppression of tau when expressed as a transgene. We sequenced the exons of T13B5.8 in bk79 and found an early stop codon in exon 3 encoding a Y39X change (Fig. 1C). Thus, bk79 contains a mutation in gene T13B5.8, and we renamed this gene sut-1.

The predicted gene product encoded by the sut-1 gene is a novel 234 amino acid long protein (SUT-1). The gene has six predicted exons and covers ~2 kb of genomic sequence (Fig. 2A). We sequenced three cDNAs (yk459d2, yk636d5 and yk657g8) confirming the predicted open reading frame for sut-1. Northern blot analysis shows a single sut-1 transcript consistent in size with the 890 nucleotide transcript (Fig. 2B). sut-1 mRNA is essentially undetectable in sut-1(bk79) homozygotes, presumably because the nonsense mutation early in the coding sequence causes sut-1(bk79) mRNA degradation via nonsense mediated decay (22,23). To study SUT-1 protein, antibodies were raised against full-length recombinant SUT-1. Immunoblotting shows that SUT-1 protein is abundantly expressed in N2 and T337, but is not detected in the sut-1(bk79) mutant (Fig. 2C). The SUT-1 protein migrates at ~26 kD consistent with the predicted size of 26.3 kD. Human tau expression does not appear to dramatically influence the protein expression level or mobility of SUT-1 protein (Fig. 2C).

To determine the expression pattern of SUT-1 protein, we stained worms with SUT-1 specific antibodies. N2 animals showed prominent specific SUT-1 staining within most cells and tissues including neurons (Fig. 2F–K). SUT-1 is present both in the nuclear and cytoplasmic compartments, with nuclear staining being predominant. SUT-1 protein exhibited nucleo-cytoplasmic expression from mid embryogenesis to adulthood. The expression and distribution of SUT-1 protein appears unchanged in non-transgenic when compared with tau transgenic animals. In contrast, the sut-1(bk79) mutant did not show staining with SUT-1 antibodies, consistent with immunoblotting and Northern blotting results (Fig. 2B and C), showing the bk79 allele is a null mutation.

One of the pathological features shared between tau transgenic worms and authentic human tauopathy is aggregation of phosphorylated, insoluble tau. To examine how loss of sut-1 function affects tau, we examined the expression level, phosphorylation and the formation of insoluble tau protein in worms homozygous for the human tau transgene and the sut-1(bk79) mutation. Transgene expression levels were evaluated in four independent worm lysates by immunoblotting using ß-tubulin levels as an internal control (Fig. 3A). Tau protein is reduced by 28% relative to the parental tau transgenic strains, as determined by quantitative western blotting using tau specific primary and 125I labeled secondary antibodies. This decrease is not sufficient to alleviate the tauopathy phenotype, as heterozygous tau transgenic animals that express 50% of the tau found in the homozygous strains still exhibit a tauopathy phenotype (Fig. 2D). To assess tau phosphorylation, we probed total protein worm lysate immunoblots with phosphorylation-specific tau antibodies PHF-1, AT8 and 12E8. These antibodies recognize tau phosphorylated at S396/S404, S199/S202 and S262, respectively (Fig. 3B). We saw no change in tau phosphorylation state in the T337;sut-1(bk79) line relative to the parental T337 line. To determine how the sut-1(bk79) mutant effects tau aggregation, we sequentially extracted worm lysates using buffers of increasing solublizing strength (24). We initially homogenized T337 or T337;sut-1(bk79) worm pellets in RAB, a high salt buffer, yielding the soluble tau fraction. We re-extracted material insoluble in RAB with RIPA, a detergent containing buffer yielding the detergent soluble fraction. Subsequently, we recovered detergent insoluble material by extraction with formic acid (FA) (Fig. 3C). We observed that sut-1(bk79) caused a dramatic decrease in the levels of detergent soluble and detergent insoluble tau, both of which are abnormal aggregates of this protein. Thus, normal sut-1 activity is required for the accumulation of appreciable amounts of insoluble tau in our tauopathy model.


Figure 3
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  		Figure 3. Effects of sut-1 allele bk79 on tau protein. (A) Immunoblot of quadruplicate T337 and T337;sut-1 lysates probed for tau and tubulin. (B) Immunoblot of T337 and T337;sut-1(bk79) lysates probed with tau specific phosphorylation dependent antibodies. (C) Sequential extraction of insoluble tau protein from T337 and T337;sut-1(bk79) mutant lysates. RAB is the soluble fraction; RIPA contains detergent soluble tau, while FA contains the detergent insoluble material formic-acid (FA) solubilized protein.

 
To investigate the effect of loss of sut-1 function on tau-induced neurodegeneration, we explored the structural integrity of GABAergic neurons. To monitor neuronal integrity, we used an unc-25::GFP reporter transgene that expresses green fluorescent protein (GFP) in all 19 GABAergic motor neurons (25). In the parental N2 strain, both the dorsal and ventral nerve cords are continuous and contain the normal complement of 19 inhibitory motor neurons (13 Ventral D type and 6 Dorsal D type GABAergic neurons—Fig. 4A and B). We previously showed tau transgenic animals exhibit age dependent discontinuities in both dorsal and ventral cord axons (see also Fig. 4C and D) (19). Likewise, neurons are lost in an age dependent fashion. The sut-1(bk79) mutation partially ameliorates the tau induced neurodegenerative disruption of axons and loss of neurons (Fig. 4E and F). This partial suppression of neurodegeneration is consistent with the incomplete suppression of the locomotion phenotype in sut-1(bk79) mutant (Fig. 1A).


Figure 4
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  		Figure 4. Loss of sut-1 ameliorates tau-induced neurodegeneration. All strains shown contain a reporter transgene that marks GABAergic neurons with GFP (unc-25::GFP) from strain CZ1200. Left is anterior, down is ventral. (A) Reporter strain CZ1200 in a non-transgenic wild type background at 4 days of age. (B) Higher magnification view of CZ1200. No neurodegenerative changes are evident. (C) Four day old T337 animal exhibits loss of neurons (arrows), and gaps or interruptions of both dorsal and ventral nerve cords (arrowheads) in all animals. Bright head fluorescence (in panels C-F) is myo-2:GFP, the marker transgene used to identify tau transgenic worms. (D) Higher magnification view of T337. (E) Four day old T337;sut-1(bk79) animal showing that loss of sut-1 ameliorates many of the defects seen in C and D. (F) Higher magnification of T337;sut-1(bk79). (G) Measurement of extent of neurodegenerative events for N2, T337 or T337;sut-1(bk79). Neurodegenerative events are broken down into three categories: neuronal loss (neurons), interruption of ventral cord continuity (VC axons), or interruption of dorsal cord continuity (DC axons). Measurements are the average from 50 animals for each strain and error bars are SEM. Using the Students t-test, T337;sut-1(bk79) is significantly different from the parental T337 strain for neuronal loss (P = 1.6 x 10–15), ventral cord gaps (P = 1.1 x 10–11) and dorsal cord gaps (P = 4.5 x 10–23).

 
We conducted a yeast two hybrid screen (26) using a lexA-SUT-1 fusion protein as bait to identify proteins that bind to SUT-1. Briefly, we transformed yeast reporter strain L40ura– containing the lexA-SUT-1 bait construct with the C. elegans cDNA library pRB-1. From 14,600,000 transformants, we recovered 43 colonies that activated both HIS3 and lacZ reporter genes under control of lexA operator sequences. From these, we isolated four cDNA clones encoding the UNC-34 protein, which interact specifically with the SUT-1 bait but not the control bait (MS2 phage coat protein). To test whether SUT-1 and UNC-34 proteins can bind in the absence of yeast proteins, we employed an in vitro protein-binding assay. Briefly, we expressed in Escherichia coli SUT-1 as a fusion protein with glutathione S-transferase (GST) and purified the recombinant protein using glutathione sepharose affinity chromatography. We mixed recombinant GST-SUT-1 fusion protein coated sepharose beads with 35S labeled UNC-34 protein generated by in vitro translation of a full-length unc-34 cDNA. 35S labeled UNC-34 bound specifically to GST-SUT-1 fusion protein coated beads but not GST coated beads (Fig. 5A), demonstrating SUT-1 and UNC-34 proteins can interact in two independent assays. UNC-34 encodes a homolog of Drosophila Enabled (ENA) and is a member of the ENA/vasodilator-stimulated phosphoprotein (ENA/VASP) protein family. The mammalian homolog of unc-34 and ena is encoded by mena. The C. elegans UNC-34 protein shares ~37% amino acid similarity with Mena.


Figure 5
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  		Figure 5. SUT-1 protein binds to UNC-34. (A) In vitro translated full-length UNC-34 binds to recombinant GST-SUT-1, but not recombinant GST alone in a GST-pulldown assay. Multiple bands are likely due to internal initiation at non-start methionines during translation in reticulocyte lysates. (B) UNC-34 domains and single domain constructs. (C) Truncated SUT-1 proteins were labeled with 35S by in vitro translation and assayed for GST-SUT-1 binding. The EVH1 variant bound to SUT-1 but not EVH2 or PRD constructs.

 
UNC-34, Mena and other ENA/VASP proteins characteristically contain three distinct functional domains. These are a proline-rich domain (PRD), Enabled/vasodilator-stimulated phosphoprotein homology domain 1 (EVH1) and EVH domain 2 (EVH2) (Fig. 5B) (27). EVH1 domains are protein–protein interaction sequences that bind via proline-rich motifs (e.g. FPPPP) to a variety of proteins including cytoskeletal-associated proteins. The EVH2 domain promotes multimerization and F-actin binding (28). The PRD domain binds profilin, an actin binding protein, proteins with Src homology 3 and some proteins with WW motifs (e.g. FE65) (27,29). To identify the UNC-34 domain responsible for SUT-1 binding, we generated three UNC-34 deletion constructs each encoding an individual UNC-34 domain. We labeled these truncated proteins with 35S and tested for binding to GST-SUT-1. Only the EVH1 domain bound to SUT-1 (Fig. 5C). Thus, SUT-1 binds to the same domain of UNC-34 that potentially binds to cytoskeletal associated proteins.

Since UNC-34 binds SUT-1, we investigated whether unc-34 activity is required for suppression of the tau phenotype by sut-1(bk79). We constructed double mutant animals using the loss of function mutation sut-1(bk79) and the previously characterized loss-of-function unc-34 allele e566. unc-34(e566) causes defects in axon pathfinding and neuronal migration leading to an Unc phenotype (30,31). We used the liquid thrashing assay to measure the effects of loss-of-function sut-1 and unc-34 alleles, either singularly or as double mutants, in the absence or presence of the tau transgene (Fig. 6). As described above, the sut-1 (bk79) allele suppresses the tau-induced Unc phenotype with the T337;sut-1(bk79) strain having higher thrash rates than the T337 strain alone (Fig. 6). However, when the unc-34 loss-of-function allele is added, the resulting double mutant T337;sut-1(bk79);unc-34(e566) has a much lower thrash rate compared to T337;sut-1(bk79) or T337 alone. Thus, unc-34 activity is required for sut-1(bk79) to suppress the tau-induced Unc phenotype. In addition, unc-34 influences the tau-induced phenotype in the presence of wild type sut-1. The unc-34(e566) allele is an enhancer of the T337 phenotype with T337;unc-34(e566) having lower thrash rates than T337 alone. This result indicates that wild type unc-34 gene partially protects worms from the tau transgene-induced Unc phenotype. Finally, the sut-1 and unc-34 genes also interact in the absence of the tau transgene. While sut-1(bk79) has wild type thrash rates (Fig. 2E), sut-1 acts as an enhancer of the unc-34 mutant as shown by the fact that the double mutant sut-1(bk79);unc-34 (e566) has lower thrash rates than the single mutant unc-34(e566) strain. These genetic interactions between sut-1 and unc-34 are consistent with the SUT-1/UNC-34 protein–protein interactions described above and suggest a scheme where both SUT-1 and UNC-34 act in a common pathway to control tau neurotoxicity (Fig. 7).


Figure 6
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  		Figure 6. UNC-34 function is required for sut-1 suppression of the tau-induced Unc phenotype. (A) Liquid thrashing measurements for staged 4-day-old worms. Strains used are: N2, T337, T337;sut-1(bk79), T337;unc-34(e566), T337;unc-34(e566); sut-1(bk79), unc-34(e566),T337;unc-34(e566), sut-1(bk79) and unc-34(e566); sut-1(bk79). (B) Statistical analysis of liquid thrashing data using Students t-test.

 


Figure 7
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  		Figure 7. Model for the relationship between SUT-1 and tau pathology. (A) Genetic pathway relating SUT-1 to pathological tau. (B) Potential SUT-1 Interaction network. Dashed lines are genetic interactions or indirect interactions and solid lines are physical interactions. Blue arrows represent data from mammalian systems. Black arrows represent data from this worm model. References for human protein–protein interactions: APP-FE6544;68; FE65-Mena29;44; Mena-Profilin69; Diaphanous-Profilin70; Diaphanous-Rho70; DNMBP-Mena39.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Aggregated tau is a part of the neuropathology of several neurodegenerative diseases. In some cases, mutations in MAPT cause the disease (10), clearly establishing abnormal tau as the cause of the disorder. In other tauopathies, different mechanisms lead to tau aggregation and neuronal dysfunction. In all types of tau-related disorders, the process by which tau contributes to cellular dysfunction is not fully understood. To identify genes essential for tau pathogenesis, we generated a C. elegans model for tauopathy disorders where a human mutant tau cDNA is expressed in all neurons (19). The resulting model system exhibits many of the biochemical and cellular features of authentic human tau-related disorders. The phenotype exhibited by this model is sufficiently robust to allow genetic screens for genes that are essential to tau-induced toxicity and degeneration. Here we identified sut-1, a gene essential for tau-induced degeneration. Loss of sut-1 function restores tau transgenic worms to a near-normal phenotype. The Unc phenotype is markedly reduced as is neurodegeneration. Insoluble tau, a characteristic of this model and of human tauopathies, is also essentially eliminated. Thus, loss of a single gene restores worms to a near normal phenotype in the presence of pathologic levels of mutant human tau.

The predicted amino acid sequence of SUT-1 provides limited information concerning the function of this protein. SUT-1 shares sequence similarity with proteins from other nematode species including Ascaris suum, C. briggsae and C. remanei (Supplementary Material, Figure S1). The A. suum gene SL30p is part of the splice leader (SL) trans splicing apparatus (32). While the sequence of SL30p is similar to SUT-1, in C. elegans W02F12.6, is a much closer homologue of SL30p than SUT-1. The relatively low homology between SL30p and SUT-1 leaves open the question of whether the function of SUT-1 is closely related to the function of SL30p. With respect to higher organisms, there is a very limited homology between sut-1 and the human gene PAXIP1, a locus previously implicated in AD (33). However, the homology is weak and the sequence complexity of the aligning regions limited by a high glutamine content. Also, SUT-1 lacks the primary functional domains (BRCT domains) found in PAXIP1. Thus, the relationship between PAXIP1 and SUT-1 function is unclear.

The key finding concerning SUT-1 function is that it physically binds to the UNC-34 protein (Fig. 6). The genetic interaction between the sut-1 and unc-34 genes provides in vivo support (Fig. 7) for the observed physical interaction. UNC-34 is a member of the Ena/VASP protein family that includes the Drosophila melanogaster ortholog Ena, the Dictyostelium discoideum ortholog DdVASP (vasodilator-stimulated phosphoprotein) and vertebrate proteins Mena and VASP in mammals (34). All members of this family have EVH1, PRD and EVH2 domains. The EVH1 and PRD domains bind other proteins and the EVH2 domain may be involved in oligermerization (27,28). In addition to SUT-1, other EVH1 and PRD binding proteins include profilin, FE65, dynamin binding protein (DMNBP), diaphanous, vinculin and zyxin (35).

Functionally, Ena/VASP proteins link signaling pathways to actin dynamics, and are involved in cell migration, adhesion, polarity, endocytosis and other processes. On a molecular level, Ena/VASP antagonizes actin fibril capping thereby promoting actin fiber extension and also inhibiting branching (34,36). On the cellular level, in worms, UNC-34 participates in axonal pathfinding and in neuronal migration during development (31). Both of these processes exhibit dynamic, actin-based plasma membrane protrusions (lamellipodia and filopodia) and UNC-34 is required for filopodia formation (37). In higher organisms, Ena/VASP proteins localize to the leading edge of lamellipodia and modulate the protrusion step of motility. Env/VASP proteins, in conjunction with other leading edge protein binding partners such as profilin and lamellipodin (38), control the dynamic actin structures involved in motility.

One UNC-34 interacting protein of particular interest in our tauopathy model is the DNMBP, also known as TUBA (39). We previously demonstrated RNAi knockdown of the worm DNMBP ortholog exacerbates the tau-induced phenotype in our C. elegans model (40). Dynamin mediates vesicle fission in endocytosis, and DNMBP which binds both dynamin and Mena, connects this process to regulation of the actin cytoskeleton (41,42). In rat brain, DNMBP is located in synapses where it is presumed to participate with dynamin in synaptic vesicle endocytosis (39). In addition, DNMBP was implicated in AD where a SNP allele has been associated with an increased risk in AD (43). The putative interaction between Mena and DNMBP ties a previously identified genetic enhancer of tauopathy (DMNBP) with the genetic suppressor found in this study (sut-1(bk79)) suggesting they have opposing roles, and likely both act through unc-34. This demonstrates a convergence of the complementary screening approaches described previously (40) and in this study (Fig. 7).

The site of Ena/VASP protein interactions with dynamic actin processes is at discrete cytoplasmic locations often associated with membranes. On the other hand, SUT-1 has primarily a nuclear localization though some cytoplasmic staining is also in evidence (Fig. 2E–J). Another protein that has both a nuclear function and binds to an Ena/VASP protein in the cytoplasm is FE65. FE65 binds Mena (29) and can form a tripartite complex with the amyloid precursor protein (APP) (44). APP is important in AD because endolytic cleavage of this protein by ß- and {gamma}-secretase produces Aß, a toxic peptide and primary component of the amyloid plaques found in this disease. APP and FE65 co-localize with Mena in lamellipodia (44), linking APP/FE65 with the regulation of actin dynamics during cell movement. FE65 and APP are also implicated in actin-based neuronal growth cone regulation (44). Mena appears to physically link FE65 and APP to processes involving the regulation of actin dynamics. The FE65–APP interaction also results in regulation of nuclear transcription. FE65, in an APP-dependent process, binds to TIP60, a histone acetyl-transferase (45) and the transcription factor CP2/LSF/LBP1 (46), and the TIP60-FE65 complex can then move to the nucleus and regulate gene expression (47). Thus, FE65-APP complexes not only participate in dynamic actin-mediated processes in the cytoplasm, but also act in the nucleus to regulate expression of genes related to these processes. Likewise, SUT-1 may also have both a cytoplasmic function when bound to UNC-34 and an as yet unknown nuclear function (Fig. 7).

The mechanism by which sut-1 influences the tau-induced pathology in this model system is unclear though the interaction of sut-1 with UNC-34 provides information concerning the molecular pathways affected. Several lines of evidence suggest sut-1 and unc-34 regulate the same process: first, genetic loss of sut-1 suppresses, while loss of unc-34 enhances tau toxicity; second, loss of sut-1 prevents the tau-induced phenotype only when an unc-34 is present; and third, the loss of sut-1 exacerbates the Unc phenotype of an unc-34 loss-of-function mutant (Fig. 7). The relationship between SUT-1 and UNC-34 may be antagonistic and presumably results from the direct interaction between these two proteins. The observation that most of SUT-1 is located in the nucleus suggests SUT-1 could shuttle between cytoplasmic UNC-34 and the nucleus to regulate transcription in response to UNC-34-dependent processes. Precedent for such a signal transduction mechanism exists for the FE65-Mena-APP interactions described above where FE65 complexes with Mena and APP to regulate actin-mediated processes, yet FE65 also can also shuttle to the nucleus to affect transcription. Other proteins that, like SUT-1, bind to EVH1 domains of Ena/VASP proteins and shuttle between the cytoplasm and nucleus include zyxin (48), Homer (49), Profilin (50,51) and Diaphanous1 (52); the latter three proteins affect gene expression changes (53,54).

Actin and related proteins have recently been implicated in tau-related neurodegenerative disease. In a Drosophila tauopathy model, expression of FTDP-17 mutant human tau causes neurodegeneration as well as inducing excess F-actin formation (55). In this fly model, over-expression of actin exacerbates tau toxicity, while destabilizing actin by over-expressing cofilin suppresses tau toxicity. In the same model, rod-like deposits of actin filaments, profilin and tau are observed. Similar deposits are also seen in a tau transgenic mouse model of FTDP-17 (55). In humans, actin-containing protein deposits called Hirano bodies were initially observed in tauopathies such as Guam ALS/PDC, AD, Pick's disease and PSP (56) providing evidence for alternation of actin-mediated processes in neurodegenerative disease (57). Hirano bodies contain actin filaments (58), cofilin and actin depolymerization factor (ADF) (two actin binding proteins) (59). However, Hirano deposits are also seen in normal aging and the actual relationship between these deposits and disease processes is unclear, though it is not unusual for the proteins deposited in neurodegenerative diseases to be intimately connected to the pathogenetic mechanisms involved.

It is tempting to speculate that the process affected in our SUT-1 mutant animals is some sort of presynaptic actin-dependent process such as DMNBP-dependent vesicle fission. This idea is supported by the results of a genome-wide RNAi screen that identified as enhancers not only DMNBP (see above) but also several other neurotransmission genes including aex-1 (human ortholog is UNC13D) which is involved in synaptic vesicle release and acr-14, a nicotinic acetyl choline receptor (40). Also, previous work showed tau can cause a presynaptic deficit in neurotransmission in this model (19). Thus, we propose UNC-34 may be required for generation or maintenance of normal synapses, and SUT-1, either directly, or through nuclear gene regulation, may negatively modulate this function. Under the stress of tau toxicity, SUT-1 suppression of this UNC-34-dependent process is detrimental.

Here we show loss of a single gene can nearly eliminate the toxic effects of tau in tau transgenic C. elegans. This suggests in human tauopathies, functional inhibition of a single protein could be a treatment for neurodegeneration. Thus, identification of SUT-1 reveals a pathway target for drug development. While there is no human ortholog of SUT-1, we demonstrate via the interaction with UNC-34, that SUT-1 is part of a pathway conserved in higher organisms including man. The possibility exists that in higher organisms, there is a protein that is functionally equivalent to SUT-1 but does not have a similar sequence. This has been demonstrated for a number of worm genes. One recent example is for sys-1, a functional beta-catenin, bearing no sequence homology to mammalian beta-catenins. Based on functional criteria, sys-1 can substitute for the bar-1 beta-catenin and the encoded protein shares similar biochemical and transcriptional regulatory properties (60). The situation with SUT-1 may be comparable, except the relevant mammalian functional counterpart remains unknown. Identification of a mammalian analog of SUT-1 that negatively modulates a Mena-dependent pathway could lead to novel therapeutic targets.


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Strains. Bristol strain N2 is the wild type C. elegans strain used (61). Tau transgenic line T337-1 (CK10) was used for the suppressor screen. Line CK10–bkIs10[Paex-3::Tau–337M), Pmyo-2::GFP] carries a chromasomally integrated transgene encoding the 1N4R isoforms of human tau carrying the 337M FTDP-17 mutation with expression driven by the pan-neuronal promoter aex-3, and has a pronounced Unc phenotype (19). CK15 is the CK10 strain backcrossed to the Hawaiian C. elegans isolate CB4586 12 times. CZ1200 carries an integrated unc-25::GFP transgene expressed in GABAergic neurons. The original source of unc-34(e566) mutation is strain CB566. Double mutants were constructed by standard methods. Genotypes were confirmed by PCR and sequencing or restriction digests for all strains with non-obvious phenotypes.

Mutagenesis. Tau transgenic worms were mutagenized using ethyl nitrosurea (ENU) as described (62). Briefly, tau transgenic worms were exposed to 1 mM ENU in M9 for 4 h with gentle rocking. Approximately 50 000 mutagenized haploid genomes were screened. Mutant F2 progeny that had restored motility relative to the parental tau transgenic progeny were selected. This motility selection consisted of washing F2 mutagenized worms of all bacteria using M9. Washed worms were placed on a 245 x 245 mm square agar plate with food at one end. Worms are placed at the opposite end from the food, and worms with normal locomotion rapidly move to the food, while those that have impaired locomotion do not arrive at the food until much later. The animals that reached food first were picked as candidate mutants. Mutants that bred true were subjected to a secondary screen which consisted of testing mutant line for tau expression levels. Only lines with tau protein levels similar to the parental line were retained for mapping and further analysis. The line containing a sut-1 mutation was tested against other isolated alleles by complementation testing. However, all other alleles isolated complemented sut-1, thus only a single sut-1 allele was isolated.

Positional Cloning of sut-1. Single nucleotide polymorphism mapping was conducted essentially as described (21) except that CK15 was used as the Hawaiian strain. The simplified method of Davis et al. (63) was adopted for some mapping. Using these methods sut-1(bk79) was mapped to the interval between markers uce2-577 and uce2-725. To rescue the sut-1 phenotype, pools of cosmids were injected at 30 ng/µl each with Pmyo-2::dsRED at 10 ng/µl as a co-injection marker. The rescuing pool contained cosmids W09G6, M01A4, K02E7 and T13B5. These cosmids were subsequently injected individually at 30 ng/µl with 90 ng/µl pBluescript II KS(+) as carrier and 10 ng/µl of Pmyo-2::dsRED.

Mutation screen. The exons and flanking sequences for genes on T13B5 were amplified by PCR and sequenced. The sequences for sut-1 exons were compared with the sequence for N2 and CK10. A single point mutation was identified in exon 3 of T13B5.8 in sut-1(bk79).

Behavioral assays. Liquid thrashing assays were performed in 20 µl of M9 media (42 mM Na2HPO4, 22 mM KH2PO4, 86 mM NaCl, 1 mM MgSO4) on Teflon-printed slides as previously described (19). Worms were allowed to settle and thrashes counted for 30 s.

Protein extraction. Tau fractions were obtained as described previously (19). To determine if aggregated tau accumulates or is absent from sut-1 animals strains were sequentially extracted using buffers of increasing solublizing strengths and compared with the parental T337 strain (19,24). First, worms were homogenized in high salt reassembly buffer [RAB-High Salt (0.1 M MES, 1 mM EGTA, 0.5 mM MgSO4, 0.75 M NaCl, 0.02 M NaF, 0.5 mM PMSF, 0.1% protease inhibitor cocktail, pH 7.0)] and ultra-centrifuged at 50 000x gravity yielding the soluble fraction (supernatant) and an insoluble pellet. Next, the RAB insoluble material was re-extracted with an ionic and non-ionic detergent containing RIPA buffer (50 mM Tris,150 mM NaCl,1% NP40,5 mM EDTA,0.5% DOC,0.1% SDS, 0.5 mM PMSF, 0.1% protease inhibitor cocktail, pH 8.0) and centrifuged as above yielding abnormal tau in the supernatant. Finally, the detergent insoluble pellet was re-extracted with 70% FA to solublize detergent insoluble tau. The three fractions were analyzed using quantitative western blotting with tau specific primary and 125I-labeled secondary antibodies.

Immunoblotting. Protein samples were boiled 5 min and loaded onto 10% pre-cast SDS–PAGE gels (Biorad). For quantitative immunoblotting, we detected human tau using antibody 17026 at a dilution of 1:3000 (A gift of Virginia Lee) as described previously (19). We used anti-tubulin antibody at a dilution of 1:1000 (Developmental Studies Hybridoma Bank). SUT-1 antibody was prepared as described below and used at a dilution of 1:1000. 125I-labled goat anti-mouse or goat-anti-rabbit IgG were the secondary antibody reagents used at a dilution of 1:1000 (New England Nuclear). Signals were quantitated using a Packard Cyclone phosphorimager.

Immunocytochemistry. Worms were fixed in paraformaldehyde and permeablized by freeze cracking as described (64). Fixed whole animals were stained with SUT-1 specific affinity purified rabbit antibodies at a dilution of 1:500. AlexA 568 conjugated anti-rabbit antibody (Molecular Probes) was used as the secondary antibody at a dilution of 1:500.

Measurement of neuronal degeneration. The unc-25::GFP transgene was crossed into the background of the tau transgenic strains assayed. Fifty worms of each genotype were developmentally staged and analyzed for neuronal structure defects as previously described (19).

Yeast two hybrid screening. Yeast two hybrid screening was carried out as described (65), except that yeast strain L40ura- carrying pLexA-SUT-1 plasmid was transformed with the LamdaACT RB-2 library and plated on SD-trp-leu-his, 100 mM 1,2,4 3-amino triazole. Colonies were picked after 5 days and cDNA-containing plasmids were rescued into E. coli. Recovered cDNA plasmids were reintroduced into L40 containing either pLexA–SUT1 or pLexA–MS2 coat protein. Those cDNAs that activated expression with SUT-1, but not MS2 coat protein, were analyzed further.

Recombinant protein purification. The SUT-1 protein expression construct was prepared by inserting the sut-1 cDNA into the pGEX 6P-1 expression vector (Pharmacia) to generate a construct encoding a GST-SUT-1 fusion protein. The GST moiety allows one-step affinity purification of recombinant protein on Glutathione coupled sepharose beads. A log-phase culture of BL21(DE3) cells carrying the pGEX-SUT-1 vector was induced for 3 h at 37ºC with shaking. Glutathione sepharose (Pharmacia) was used as the affinity resin; cells were harvested, lysed and recombinant protein was purified as previously described (66).

SUT-1 antibody preparation. SUT-1 antibodies were prepared commercially using the Invitrogen Zymed antibody service. Purified recombinant GST-SUT-1 protein was used as the immunogen. Antisera were affinity purified using pure SUT-1 protein cleaved from the GST moiety using the Zymed antibody affinity purification service.

GST pulldown assays. In vitro protein-binding assays were performed essentially as described (65) except the binding buffer contained 0.5% Bovine Serum Albumin (BSA), 0.1% Tween-20, 100 mM NaCl, 5 mM DTT, 20 mM HEPES, pH 7.4. GST or GST-SUT-1 fusion protein was bound to glutathione-sepharose as described (65). The amount of GST or GST-SUT-1 bound was determined by eluting the bound protein and analyzing them by SDS–PAGE followed by coomasie blue staining (67). Radiolabeled (35S) UNC-34 was produced using the TNT reticulocyte lysate (Promega) according to the manufacture's methods. Labeled protein was then added to equivalent amounts of glutathione beads to which either GST alone or GST-SUT-1 fusion protein was bound and incubated with beads at 4°C with gentle rotation for 60 min. Beads were pelleted, washed five times in binding buffer and eluted by boiling in SDS–PAGE sample buffer. Eluted, labeled proteins were analyzed by SDS–PAGE. In the figures, the amount of material shown in lanes marked ‘input’ is 10% of the amount used in the GST-pulldown experiments.


   SUPPLEMENTARY MATERIAL
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 ABSTRACT
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 RESULTS
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Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
This work was supported by a Department of Veterans Affairs including a Merit Review Entry Program Grant (BCK). The work was also supported by NIA grant PO1 AG17586 (GDS, V.-M. Lee, PI). We thank Dr. James H. Thomas for advice regarding C. elegans genetics and strains. We thank the C. elegans Genetics Center for providing strains. We thank Leo Anderson, Harmony Danner, Elaine Loomis and Lindsey Foley for outstanding technical assistance. We thank Yuji Kohara for the sut-1 cDNAs yk459d2, yk636d5, yk657g8, Andrew Fire for providing C. elegans expression vectors, Yishi Jin for strain CZ1200, Virginia Lee for antibody 17026, Peter Davies for antibody PHF1, Peter Seubert for antibody12E8 and the ß-tubulin antibody E7 is from the Developmental Studies Hybridoma Bank (NICHD). We also thank Virginia Lee, Joeseph Buxbaum, Tom Blumenthal and Peg MacMorris for critical reading of the manuscript.

Conflict of Interest statement. The authors state that they have no conflict of interest.


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