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Human Molecular Genetics Advance Access originally published online on April 6, 2006
Human Molecular Genetics 2006 15(9):1483-1496; doi:10.1093/hmg/ddl067
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Published by Oxford University Press 2006

Molecular pathways that influence human tau-induced pathology in Caenorhabditis elegans

Brian C. Kraemer1,2, Jack K. Burgess1,2, Jin H. Chen1,2, James H. Thomas3 and Gerard D. Schellenberg1,2,4,5,*

1Geriatrics Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA and 2Division of Gerontology and Geriatric Medicine, Department of Medicine, 3Department of Genome Sciences, 4Division of Neurogenetics, Department of Neurology and 5Department of Pharmacology, University of Washington, Seattle, WA, USA

* To whom correspondence should be addressed at: Geriatric Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle Division, 1660 S. Columbian Avenue, Seattle, WA 98108-1597, USA. Tel: +1 2067642701; Fax: +1 2067642569; Email: zachdad{at}u.washington.edu

Received January 7, 2006; Accepted March 16, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Mutations in the gene encoding tau cause frontotemporal dementia with parkinsonism—chromosome 17 type (FTDP-17). In FTDP-17, Alzheimer's disease, and other tauopathies, aggregated hyper-phosphorylated tau forms the neurofibrillary tangles characteristic of these disorders. We previously reported a Caenorhabditis elegans model for tauopathies using human normal and FTDP-17 mutant tau as transgenes. Neuronal transgene expression caused insoluble phosphorylated tau accumulation, neurodegeneration and uncoordinated (Unc) movement. Here we describe a genome-wide RNA-mediated interference (RNAi) screen for genes that modify the tau-induced Unc phenotype. We tested RNAi sequences for 16 757 genes and found 75 that enhanced the transgene-induced Unc phenotype. Forty-six of these genes have sequence similarity to known human genes and fall into a number of broad classes including kinases, chaperones, proteases and phosphatases. The remaining 29 modifiers have sequence similarity only with other nematode genes. To determine if the enhancers are specific for the tau-induced Unc behavior, we exposed several non-tau Unc mutants to tau RNAi enhancer clones. Fifteen enhancers modified phenotypes in multiple Unc mutants, whereas 60 modified only the Unc phenotype in the tau transgenic lines. We also introduced the tau transgene into the background of genetic loss-of-function mutations for a subset of the enhancer genes. Tau transgenic animals homozygous for loss of these enhancer genes exhibited increased impaired motility relative to the tau transgene line alone. This work uncovers novel candidate genes that prevent tau toxicity, as well as genes previously implicated in tau-mediated neurodegeneration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Tau is a microtubule-associated protein (MAP) found primarily in neuronal axons. Tau protein binds to microtubules (MTs) stimulating MT polymerization and promoting stabilization. In a number of neurodegenerative diseases, hyperphosphorylated tau forms filaments that aggregate in neurons as neurofibrillary tangles (NFTs) (1Go,2Go). In some diseases, aggregated tau also appears in glial cells. Disorders sharing similar pathological insoluble tau deposits, collectively called tauopathies, include Alzheimer's disease (AD), Down syndrome, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, Guam amyotrophic lateral sclerosis/Parkinson's dementia complex and frontotemporal dementia with parkinsonism chromosome 17 type (FTDP-17) (3Go). Although the role aggregated tau plays in most of these disorders is not understood, for FTDP-17, autosomal-dominant mutations in MAPT, the gene encoding tau, cause the disease (4Go–6Go). These mutations demonstrate that altered tau can be the primary cause of neurodegeneration. However, the precise mechanism by which tau causes FTDP-17 and how tau contributes to other tauopathies not caused by MAPT mutations remains unclear. One hypothesis is that abnormal tau disrupts MT-mediated axonal transport resulting in dysfunctional neurons that accumulate tau aggregates and subsequently degenerate (7Go,8Go). Another hypothesis posits tau aggregates are intrinsically toxic, killing neurons directly. In this scenario, FTDP-17 mutations cause tau aggregation either by reducing the affinity of tau for MTs resulting in an increase in free tau concentrations (9Go) or by accelerating the rate of tau self-aggregation (10Go,11Go). Furthermore, pathologic tau is hyperphosphorylated, suggesting the phosphorylation state of tau may play a prominent role in neurodegeneration (12Go).

To explore the possible causes of tau-induced neurodegeneration, we developed a transgenic Caenorhabditis elegans model of human tauopathy diseases using either normal human tau coding sequences or tau with FTDP-17 mutants 301L or 337M (13Go). We expressed tau in all neurons by using the pan neuronal aex-3 promoter (14Go). Both the normal and the FTDP-17 mutant tau transgenic lines showed progressively worsening uncoordinated (Unc) locomotion, a phenotype characteristic of a variety of C. elegans nervous system defects. The Unc phenotype was accompanied by progressive accumulation of phosphorylated insoluble human tau, reduced cholinergic neurotransmission, substantial neurodegeneration seen as bulges and gaps in nerve cords and subsequent neuronal loss. The observed neurodegenerative phenotype was significantly worse in the mutant tau lines (337M-1 and 301L-1) relative to the normal tau lines. Thus, the model recapitulates many of the features seen in human tauopathy disorders. To identify specific worm genes participating in this tau-induced phenotype, we conducted a genome-wide screen using RNA-mediated interference (RNAi) to reduce or eliminate gene function for most C. elegans genes. We used a library of RNAi clones that targets ~85% of all C. elegans genes (15Go). When 16 757 RNAi sequences were screened for alteration of the tau-induced Unc phenotype, 75 enhancer genes were identified. These enhancers were screened for effects on other Unc-producing mutations to determine which modifiers were specific for the tau-induced Unc phenotype. In addition, for a subset of enhancer genes in which loss-of-function mutants were available, we generated double mutants with the tau transgene expressed on a homozygous loss-of-function background to confirm the results observed with RNAi. The modifier genes identified include kinases, chaperones, proteases, phosphatases, enzymes and genes of unknown function. Some of these genes have previously been implicated in tau-induced pathology in humans but others have not. This latter group provides novel insight into tau-induced neurodegenerative mechanisms.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 REFERENCES
 
To identify potential modifiers of the tau-induced pathologic phenotype in our model, we used RNAi to find genes that enhanced the transgene-induced Unc phenotype. We used line 337M-1 because it exhibits consistent strong Unc behavior and has the most severe overall phenotype relative to the 301L FTDP-17 mutant and the normal tau lines. RNAi sequences were introduced into the model by feeding transgenic worms 16 757 Escherichia coli clones harboring dsRNA-expressing plasmids corresponding to ~86% of all C. elegans predicted genes (15Go). We fed the transgenic worms to each E. coli clone individually, and scored for alterations in motility relative to untreated 337M-1 worms.

In the initial screen, we identified 1217 clones that enhanced (worsened) the 337M-1 Unc phenotype. To eliminate false-positive clones (Fig. 1), we first tested positive RNAi clones in parallel on the 337M-1 and the non-transgenic (N2) lines to ensure that the Unc phenotype was more severe in the transgenic line than in the N2 line. Clones that failed to recapitulate the initial enhancer phenotype and those giving comparable phenotypes in the N2 and 337M-1 were eliminated leaving 75 clones (Fig. 1, Tables 1 and 2). No RNAi clone suppressed the tau-induced Unc phenotype.


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  		Figure 1. Flowchart of RNAi screening for modifiers of tau-induced phenotype.

 

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  		Table 1. RNAi enhancers of the tau-induced Unc phenotype
 

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  		Table 2. The effect of RNAi clones on non-tau Unc mutants
 
The RNAi clones in Table 1 may target genes that specifically protect against toxic tau or may alternatively be genes that generally protect against multiple causes of neuronal dysfunction. To differentiate between these two possibilities, we screened the 75 clones in Table 1 for the ability to enhance the locomotion phenotype in four other non-tau Unc strains (unc-3, unc-76, unc-78 and unc-87; Table 2). The unc-3 gene encodes an early B-cell factor (EBF) family transcription factor required for normal ventral cord axonal outgrowth, and mutations in unc-3 cause disorganization of the ventral nerve cord (16Go). unc-76 encodes a novel protein implicated in cell adhesion and unc-76 defects cause abnormal axonal fasciculation and growth cone morphology (17Go). Because an Unc phenotype can be caused by either a neuronal or a muscle defect, we also tested the clones against strains with mutations in genes encoding muscle proteins (unc-78 and unc-87; Table 2). unc-78 encodes an actin-binding protein that acts as a regulator of actin polymerization. Defects in unc-78 cause disorganization of actin filaments and muscular dysfunction (18Go). unc-87 encodes a calponin family member and unc-87 mutations cause paralysis due to muscle fiber abnormalities (19Go). When the 75 RNAi clones were screened against these four Unc mutants, two classes of responses resulted.

The largest group identified consisted of 60 clones that only enhanced the tau-induced Unc phenotype and not the phenotype of the other four Unc lines tested (e.g. C09D4.3; Table 2). These RNAi clones target genes for knockdown that potentially are specific for tau-induced neurodegeneration. For most of these, RNAi feeding did not visibly affect the non-transgenic control (N2). The exception was LLC1.2 (DLD) which caused partially penetrant embryonic lethality. Of these 60 tau-specific modifiers, 38 have homologous human genes. Of these homologues, six were previously implicated as participants in tau pathology either in human disease or in other animal models. These are: WNT2, a wingless signaling pathway ligand (20Go); TTBK2, a kinase shown to phosphorylate tau and tubulin in vitro (21Go); GSK-3ß, a tau kinase potentially involved in hyperphosphorylation of tau (22Go–27Go); TAOK1 (MARKK), a kinase that activates MARK by phosphorylation (28Go,29Go) which in turn phosphorylates tau (30Go); CSTE, a protease expressed in the degenerating neurons of AD (31Go); and CHRNA7, a nicotinic acetylcholine receptor up-regulated in AD brain (32Go–34Go).

A second group consisting of 15 RNAi clones affected the Unc phenotype in both tau and in some or all other Unc strains tested (e.g. T09B4.10). In this class, nine C. elegans genes had human homologs, of which several have been previously implicated in tauopathies. The tauopathy-related homologs include protein phosphatase PP2A that dephosphorylates tau (35Go,36Go) and binds directly to tau (37Go), and CHIP (Carboxyl terminus of Hsc70 Interacting Protein), an Hsp70 co-chaperone and E3 ubiquitin ligase that induces tau ubiquitination and degradation and also increases tau aggregation (38Go,39Go). Although most of the RNAi clones that affected non-tau Unc lines affected both neuronal (unc-3 and unc-76) and muscle (unc-78 and unc-87) Unc strains (e.g. F38H4.9, PP2A subunit C), RNAi clones B0304.3 and R13A1.8 enhanced only the neuronal Unc strains, and clone R106.1 only affected the muscle Unc strains. A number of these clones [F48E8.5 (PP2A-A), F38H4.9 (PP2A-C), K09C4.3 (HSPA2) and F59G1.2] also affected the non-transgenic N2 line.

Given the variability in strength of RNAi knockdown from gene to gene, we obtained true genetic loss-of-function allele lines for candidate genes in which the enhancing effect was specific for the tau-induced Unc phenotype. We examined eight of the tau-specific RNAi candidates that have pre-existing loss-of-function mutations (Table 3). Note that none of these loss-of-function mutations cause a significant Unc phenotype. These loss-of-function mutation lines were crossed with tau transgenic lines to produce doubly homozygous null/tau transgenic lines. The Unc phenotype of these double homozygote strains was compared with 337M-1 single homozygotes by scoring for Unc phenotype severity (Fig. 2). All the aex-1, acr-14, lin-44, sir-2.3, pxn-1, vap-1 and mut-14 mutants clearly increased the severity of the tau Unc phenotype (Fig. 2), consistent with the RNAi screening results. Six other genes (coq-1, kin-18, hsf-1, let-92, tag-177 and hgrs-1) were not tested due to recessive lethality in the absence of tau.


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  		Table 3. Tau-specific modifiers genes with existing loss-of-function alleles
 

Figure 0672
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  		Figure 2. Enhancers of pathological tau exacerbate the Unc phenotype of tau transgenic worms. Animals doubly homozygous for the Tau 337M-1 transgene and loss-of-function mutations for the tau enhancer gene indicated were assessed for the severity of the Unc phenotype using the liquid thrashing assay (13Go). Each bar is the mean for 10 animals with thrash rates depicted as a percentage of the parental 337M-1 line. (A) Staged 2-day-old 337M-1, lin-44/337M-1, aex-1/337M-1, acr-14/337M-1, sir-2.3/337M-1, pxn-1/337M-1, vap-1/337M-1 and mut-14/337M-1 animals were scored for liquid thrashing. Statistical comparisons were made using a two-tailed t-test. Significance levels for comparisons of 337M-1 to mutant lines are P=3.8x10–4 (lin-44/337M-1), P=6.4x10–4 (aex-1/337M-1), P=3.8x10–2 (acr-14/337M-1), P=2.8x10–5 (sir-2.3/337M-1), P=5.9x10–6 (pxn-1/337M-1), P=1.3x10–2 (vap-1/337M-1) and P=2.9x10–6 (mut-14/337M-1). The thrash rates for non-transgenic N2 and 337M-1 at day 2 are 101 and 26 thrashes/minute, respectively. (B) Staged 5-day-old animals were scored for liquid thrashing as above. Significance levels for comparisons of 337M-1 to mutant lines are P=0.069 (lin-44/337M-1), P=0.366 (aex-1/337M-1), P=0.24 (acr-14/337M-1), P=8.6x10–2 (sir-2.3/337M-1), P=0.32 (pxn-1/337M-1), P=0.23 (vap-1/337M-1) and P=0.69 (mut-14/337M-1). The thrash rates for non-transgenic N2 and 337M-1 at day 5 are 116 and 13 thrashes/minute, respectively.

 
We evaluated the effects of genetic loss-of-function mutations on insoluble tau accumulation in transgenic lines. Transgenic lines N-1 and 337M-1 progressively accumulate insoluble tau, with the latter accumulating more insoluble tau and at an earlier time than the N-1 line (13Go). Soluble and insoluble tau proteins were extracted as previously described (13Go) from worms doubly homozygous for 337M-1 and loss-of-function mutations aex-1, acr-14, lin-44, sir-2.3, pxn-1, vap-1 or mut-14. We quantitated tau by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) immunoblotting using antibodies specific for human tau (Fig. 3, Table 4). All but one double-mutant strain had levels of soluble and insoluble tau comparable to the starting 337M-1 single-mutant line. The exception was mut-14/337M-1, which had reduced levels of both soluble and insoluble tau. Thus for all mutations tested, the enhanced impairment conferred by the loss-of-function mutations is not an increase in insoluble tau but rather is the result of the cellular response to the mutant tau.


Figure 0673
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  		Figure 3. Measurement of insoluble tau levels in enhancer strains. Total tau protein and insoluble tau were extracted from equivalent amounts of packed mixed stage worms. Lines used were 337M-1 line and loss-of-function mutant strains crossed with 337M-1 to generate double homozygotes. SDS–PAGE immunoblots of (A) tubulin, (B) total tau and (C) insoluble tau were performed as described (13Go) using tau antibody 17026 and tubulin antibody E7.

 

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  		Table 4. Soluble and insoluble tau in enhancer null strains
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 REFERENCES
 
The RNAi screen described above identified genes that when knocked down enhance the Unc phenotype produced by human tau. There are multiple mechanisms by which these enhancers could act. An RNAi clone could enhance the tau phenotype by increasing tau protein levels, either by affecting transcription or translation, or by altering degradation of either the message or the protein. Another type of enhancer could directly affect processes by which tau becomes toxic. For example, aggregated tau may be the toxic entity causing neurodegeneration. In vitro, tau aggregation occurs through a biphasic mechanism with a slow nucleation step followed by a more rapid elongation phase (40Go). Post-translational modifications such as phosphorylation may affect initiation, elongation or dissociation of tau filaments. Also, conformational changes in tau induced by protein chaperones or small molecules may control aggregation. Enhancers could also affect tau aggregation by decreasing the affinity of tau for MTs and increasing the amount of free tau available to form filaments. Another mechanism by which genes could influence the tau-induced phenotype is through processes involved in neuronal responses to injury. Such would include cellular systems that target mis-folded proteins for degradation and mechanisms that degrade damaged macromolecules and organelles. Finally, enhancers could be genes involved in regenerative responses to injury. These genes could be those that are involved in neurogenesis and synaptogenesis during development that also are invoked when neurons are damaged. The genes in Table 1 provide candidates for most of these mechanisms although it is difficult to unambiguously assign a given gene to a specific enhancer process. The different classes of enhancers as grouped in Table 1 are discussed below with respect to the potential mechanism for influencing tau-induced pathogenesis.

Kinases and phosphatases
In tauopathies, when compared to soluble tau from normal brain, aggregated tau is hyperphosphorylated at a number of serines and threonines. Here we identified six enhancers that are kinases and two that are subunits of a protein phosphatase. The kinases correspond to four human homologs, three of which were previously implicated in tau hyperphosphorylation (Table 1). Likewise the phosphatase identified, PP2A, is implicated in dephosphorylation of tau (41Go). Also PP2A binds tau possibly regulating PP2A activity and/or transport of PP2A along axons (37Go). One kinase enhancer, sgg-1, corresponds to human GSK-3ß, a proline-directed serine/threonine kinase. This kinase phosphorylates tau strongly at S404 and S396, and is less active at T50 and S46, and weaker still at S199, S202, T205 and T212. Two other C. elegans kinases correspond to the human homolog TTBK2, a non-proline-directed kinase that phosphorylates tau at S208 and S210 (21Go). Phosphorylation at these sites primes tau for subsequent phosphorylation by GSK-3ß. Another kinase enhancer, kin-18, corresponds to MARKK, which does not phosphorylate tau directly but does phosphorylate another kinase, MARK. MARK, when activated by MARKK-directed phosphorylation, in turn phosphorylates tau at S214 and S264 (29Go), sites that are in the MT-binding region of tau and at S356 (30Go). PAR-1, the MARK homolog in Drosophilia, stimulates GSK-3ß phosphorylation of tau, presumably by acting as a priming kinase (28Go). In the model used here, transgenic human tau is phosphorylated at a number of these same disease-related sites phosphorylated by MARK and GSK-3ß (e.g. T181, S199, S202, T205, S262, S396, S404 and S422) (13Go).

Interpretation of the role of these kinase and phosphatase enhancers in terms of disease mechanisms is difficult because the role of tau phosphorylation in tauopathies is controversial. Some evidence suggests that phosphorylation contributes to tau aggregation and thus to disease initiation and progression. Likewise, phosphorylation generally decreases tau affinity for MTs (42Go–44Go), thus increasing the amount of free tau available for aggregation. For example, MARK, a non-proline-directed serine/threonine kinase, phosphorylates tau at S262 and S214, which strongly inhibits MT binding (45Go). In this model, higher levels of unbound tau would promote aggregation and lead to disease. However, other work suggests that tau phosphorylation is actually protective against tau aggregation and disease progression. For example, although phosphorylation at sites S262 and S214 does reduce the affinity of tau for MTs, it also inhibits tau self-aggregation (44Go) thus potentially preventing NFT formation and disease progression (46Go,47Go). Thus kinases such as MARK could potentially regulate tau-MT interactions and protect the cell from the toxic effects of excess free tau which can potentially aggregate. Proline-directed serine/threonine kinases such as GSK-3ß phosphorylate sites that inhibit MT binding more weakly. Although some studies suggest that tau phosphorylation protects from aggregation, other work suggests that tau phosphorylation inhibits filament dissociation (48Go) thereby actually promoting filament formation or at least promoting the elongation step (49Go,50Go). Complicating the interpretation of tau phosphorylation and disease is that at least in AD, different sites are phosphorylated at different stages of tangle formation and disease progression. Sites phosphorylated in early stages where abnormal tau is in a pre-tangle state may be different from sites observed in mature NFTs and in terminal extra-cellular ‘ghost’ tangles (51Go). Thus tau phosphorylation appears to continue after filament formation occurs. Also, although tau from disease brains is phosphorylated at 19 or more distinct sites, the average number of phosphates per tau molecule is 6–8 in AD brains (52Go,53Go), indicating that phosphorylated tau is a complex mixture and may not be the product of an ordered phosphorylation pathway.

Another issue confounding the interpretation of specific enhancers in disease is that kinases such as MARK and GSK-3ß are part of regulatory networks where other non-tau proteins are phosphorylated. Thus it is difficult to determine whether the enhancer effects seen here are directly caused by reduced tau phosphorylation or by some other effect on another pathway that indirectly influences tau phosphorylation and/or the tau-mediated Unc phenotype. For example, MARKK regulates MARK activity, which in turn can phosphorylate MAP2, MAP4 and doublecortin, all involved in neuronal homeostasis. MARKK can also phosphorylate dishevelled, affecting Wnt signaling (54Go) and can phosphorylate kinases in the stress-responsive MAP kinase pathways. Likewise, GSK-3ß is a component of the canonical Wnt signaling pathway and regulates MT stability (55Go,56Go).

The role of specific kinases in tauopathy pathogenesis has been experimentally explored using transgenic animal models, and not all the results are consistent. In mice, conditional overexpression of GSK-3ß in adult hippocampal and cortical neurons is detrimental, resulting in increased endogenous tau phosphorylation at GSK-3ß-directed sites, increased somatic-dendritic tau localization, abnormal neuronal morphologies, neuronal death, reactive gliosis and spatial learning deficits (57Go,58Go; see also 59Go–61Go). In contrast, another study showed GSK-3ß overexpression protected tau transgenic mice from human tau-induced motor deficits, axonal swelling, reduced fast-axonal transport and tau hyperphosphorylation (61Go,62Go). Co-overexpression of constitutively active GSK-3ß along with tau resulted in increased tau phosphorylation, and amelioration of the motor impairment, axonal swelling and axonal transport deficit (62Go,63Go). Thus GSK-3ß was protective and restored the function lost by tau overexpression. In other conflicting work using a different tau transgenic mouse, GSK-3ß inhibition reduced tau phosphorylation, tau aggregation and reduced spinal cord neurodegeneration (64Go). However, the motor deficits observed in these animals was not influenced when GSK-3ß was inhibited, suggesting GSK-3ß is detrimental, not protective.

In a Drosophila tauopathy model generated by human tau overexpression in eye, simultaneous overexpression of GSK-3ß (shaggy) and tau enhanced the tau-induced phenotype that included abnormal eyes, neurodegeneration and filamentous aggregated tau (28Go,65Go). The same phenotypes were suppressed by crossing the tau transgenic model with a loss of GSK-3ß function mutant (65Go). Similar results were obtained in another Drosophila model where the tau transgene was expressed in motor neurons. GSK-3ß overexpression enhanced tau-induced abnormal larval locomotion, axonal transport and increased tau phosphorylation (66Go). Loss-of-function GSK-3ß mutants and GSK-3ß inhibitors suppressed the tau-induced phenotype. The Drosophila results are consistent with tau phosphorylation and GSK-3ß promoting tau-induced neurodegeneration.

Drosophilia has also been used as a model to study the interactions of tau and PAR-1, the fly homolog of MARK. In transgenic flies overexpressing tau in the eye, PAR-1 co-overexpression increased tau phosphorylation and enhanced tau-induced eye abnormalities. Conversely, reduction of PAR-1 activity leads to reduced tau phosphorylation and reduced tau toxicity (28Go). However, in another Drosophila study, the opposite results were obtained with PAR-1 overexpression suppressing tau-induced eye abnormalities (67Go).

In the work presented here, knock-down of kinases enhanced the tau-induced phenotype demonstrating at least in this system that the kinases identified are protective. Knock-down of PP2A, which should have the opposite affect at least on tau phosphorylation, also enhanced the tau-induced UNC phenotype. These apparently conflicting findings may indicate that either over- or under-phosphorylation of tau is detrimental. Another explanation is that these kinases and this phosphatase enhance the phenotype by acting on systems downstream from the initial tau-induced event. Simply measuring tau phosphorylation in RNAi-treated worms for these enhancers cannot resolve what phosphorylated proteins are directly responsible for the RNAi-induced tau enhancer phenotype.

Protein folding, stress response and protein degradation
A number of enhancers identified fall into the broad category of cellular response to unfolded proteins and protein degradation. These enhancers could potentially be involved in degrading abnormal tau, either as small aggregates or as more extended filaments. An alternate hypothesis is that these systems attempt to protect cells from mis-folded proteins generated in response to tau-induced neurodegeneration. The enhancer corresponding to human XBP-1 fits this latter hypothesis. This gene encodes a transcription factor, which in response to excess unfolded proteins in the endoplasmic reticulum (ER) activates transcription of grp78 and other ER stress genes. The products from some of these stress-response genes increase the ER protein-folding ability (68Go). Because tau is not a secreted protein or a membrane protein, it is unlikely that mis-folded tau will accumulate in the ER. Rather, tau-induced degeneration presumably interferes with ER protein-folding functions including proper export of receptors involved in neurotransmission. When XBP-1 expression is reduced by RNAi, the accumulation of mis-folded proteins in the ER may become toxic.

Several enhancers implicate the ubiquitination/proteosome pathway in tau-induced neurodegeneration. These include homologs of CHIP, HSC70, HSF1 and possibly FKBP4. CHIP (69Go), an E3 ubiquitin ligase, acts in a complex with HSC70 to target mis-folded proteins for degradation by the proteosome system. Others showed that tau is one such protein that is ubiquitinated by the CHIP/HSC70 complex mechanism (38Go,39Go,70Go). In our C. elegans model, both CHIP and HSC70 homologs are enhancers, consistent with tau, or tau aggregates being toxic, and with CHIP and HSC70 protecting neurons by promoting tau degradation. However, we cannot exclude the possibility that CHIP/HSC70 is degrading some other toxic protein induced by tau or by the degenerative process. Interestingly, the CHIP homolog is also an enhancer of the other muscle and neuronal Unc strains tested, whereas the HSC70 homolog is only an enhancer in the tau transgenic line (Table 2). In another C. elegans transgenic model, where human tau is expressed only in mechanosensory neurons, expression of human HSP70 protects against the transgenic tau-induced neuronal dysfunction, a result consistent with our finding that loss of hsp-1 is an enhancer (Table 1) (71Go). HSF1 is a stress-response transcription factor induced by the accumulation of damaged proteins that controls expression of the ubiquitination/proteosome pathway for protein degradation (72Go). FKBP4 is a multifunctional protein that binds HSP90, has a peptidyl-propyl cis–trans isomerase domain and may be involved in protein re-folding pathways (73Go).

Transcription
We identified a number of putative transcriptional regulators that specifically modify the tau phenotype. These likely play a role in alleviating tau pathology indirectly through their transcriptional targets. These targets are at present unknown, but may include some of the genes we have already identified. None of these genes have been previously implicated in tauopathy, but this does not preclude their involvement in tau pathology.

Proteolysis
Many potential protease genes were also identified in our screen. One hypothesis for how such proteases might protect neurons from tau is that these proteases normally degrade toxic tau. Thus loss of protease function could lead to increased tau toxicity. None of the identified proteases are known to degrade tau. However, tau can be degraded by a proteasome-independent process, suggesting that tau can be a substrate for specific proteases (74Go). Perhaps, one of these identified proteases acts directly to degrade tau in worms.

Neurotransmission and signaling
Another class of genes identified includes those involved in neurotransmission and cell signaling. One such enhancer is acr-14, which encodes an acetylcholine receptor that is a homolog of human CHRNA7. acr-14 is expressed primarily in the ventral nerve cord motor neurons and nerve ring motor neurons (75Go,76Go). In man, CHRNA7 is upregulated in AD brain (33Go) and potentially mediates Aß-directed phosphorylation of tau through ERK and JNK-1 kinases (34Go). Another enhancer that influences neurotransmission is aex-1, which is expressed in muscle, and is part of a retrograde-signaling mechanism that regulates synaptic neurotransmitter vesicle release (77Go). Identification of both acr-14 and aex-1 as enhancers is consistent with our previous work showing that the tau-induced Unc phenotype is the result of pre-synaptic dysfunction as opposed to a post-synaptic defect (13Go). dyb-1, a worm homolog of human dystrophin, also influences cholinergic transmission (78Go). Other signaling enhancers not directly connected to neurotransmission but possibly involved in nervous system development include lin-44, a homolog of human WNT2 homolog, and B0511.12, a homolog of the Drosophilia pecanex gene, which regulates neurogenesis.

Neuronal regeneration
At least one enhancer (F56H1.5) is potentially involved in neuronal regeneration after injury. The mammalian homolog of F56H1.5 is AGTPBP1, also called nna1, a gene that is up-regulated during axonal regeneration in response to axonal injury. Also, mice with mutations in this gene undergo neurodegeneration in Purkinje neurons, retinal photoreceptors, olfactory bulb mitral neurons and selected thalamic neurons (79Go).

Modifiers in other models
Other genetic modifier screens have been performed using invertebrate models of polyglutamine diseases, Parkinson disease and tauopathies. Comparison of the results from the different systems could reveal mechanisms common to multiple disorders. For polyglutamine diseases, Nollen et al. (80Go) used a C. elegans model transgenic for a polyglutamine protein to perform a genome wide RNAi screen to find genes that enhance aggregation of the transgene. Their screen identified 186 enhancers that fell into five functional classes: RNA synthesis and processing, protein synthesis, protein folding, protein degradation and protein trafficking. Comparison of the genes in these classes to our results shows that although there was only one gene in common (hsp-1), both screens found heat shock genes involved in unfolded protein response, and genes for ubiquitin-mediated protein degradation. Also, hsf-1, identified in our screen, acted as an enhancer of polyglutamine aggregation model though this gene was not identified in the RNAi screen. Notably the Nollen et al. polyglutamine screen differed from our work in that we did not find large numbers of RNA synthesis/processing, protein synthesis or protein transport genes. In other screens using Drosophila transgenic for polyglutamine proteins, genes involved in ubiquitin-mediated processes, and unfolded protein response genes were identified (81Go,82Go). Willingham et al. (83Go) used yeast to screen for modifiers of both polyglutamine and {alpha}-synuclein toxicity. Even though both polyglutamine disease and Parkinson's disease ({alpha}-synuclein model) are protein aggregation diseases and ubiquitin-mediated processes have been implicated in both disorders, there was little overlap between the modifiers identified for each protein. This led to the conclusion that different disease mechanisms are involved for these two disorders. The polyglutamine screen did identify a number of stress response, protein folding and ubiquitin-related genes, a finding consistent with the C. elegans and Drosophila polyglutamine screens discussed above. For tauopathy modifiers, one other screen has been performed in a Drosophila line transgenic for human tau (67Go). Common modifiers identified in both the Drosophila screen and our work include the serine-threonine kinase corresponding to MARKK and the phosphatase PP2A. In the Drosophila work, up-regulation of MARKK and PP2A resulted in an enhanced phenotype, whereas in our study, down-regulation of these two genes led to an enhanced phenotype. In contrast, up-regulation of MARK in Drosophila suppressed the tau-induced phenotype.

In summarizing the results from multiple protein aggregation disease models, most screens yield genes for protein folding, stress response and ubiquitin/proteasome-mediated protein degradation.

Summary
Although our RNAi screen identified enhancers with a wide variety of function, several classes of related genes stand out. Of particular interest are kinases and phosphatases that potentially affect tau phosphorylation, and stress-response systems that handle mis-folded proteins. In part, these two groups are notable because they contain genes that encode proteins previously implicated in tau-mediated disease pathways. Although identification of genes previously implicated in tau pathogenesis (e.g. GSK-3ß, PP2A and CHIP) validates the C. elegans model used here, the goal of this work is to identify new pathways involved in tau-driven neurodegeneration. However, additional work is needed to understand how these new candidates for tau modifiers might fit into human tauopathies. The group of enhancers unique to nematodes will be particularly difficult to understand.

The RNAi methodology employed permits rapid screening of gene function in C. elegans, but this approach has notable drawbacks. In worm neurons, RNAi is less effective as compared to other tissue types (84Go,85Go). Also, some worm genes are resistant to RNAi knockdown (15Go). These limitations prevent us from ruling out any gene as acting to prevent tau toxicity on the basis of a negative result from our RNAi screen. Thus, although we did identify many genes that normally function to prevent tau toxicity, many other genes that prevent tau toxicity most likely remain unidentified by the RNAi approach.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
REFERENCES
 
Strains
Bristol strain N2 was the wild-type C. elegans (86Go) and line 337M-1 was used for the primary RNAi screen (13Go). Line 337M-1 is transgenic for 1N4R tau with the 337M FTDP-17 mutation with expression driven by the pan-neuronal promoter aex-3 (13Go). 1N4R tau is one of the more abundant of the six isoforms found in human brain and contains sequences from the alternatively spliced exons 2 and 10. For RNAi secondary screening, mutant strains unc-3(cb151), unc-76(cb911), unc-78(cb1217) and unc-87(cb1216) were used (16Go–19Go,87Go).

RNAi treatment
Screening of RNAi clones was carried out using the RNAi bacterial feeding procedure essentially as described (88Go). Briefly, each E. coli clone (strain HT115) from the C. elegans genome RNAi library (from the United Kingdom Molecular Research Council Recourse Center) was grown overnight on NGM growth media supplemented with 25 µg/ml carbenecillin and 1 mM isopropyl-ß-D-thiolgalactopyranoside to induce double-stranded RNA expression. Hypochlorite-purified embryos were seeded onto induced bacterial lawns and both adults and any progeny were scored after 4–6 days for enhancement or suppression of the tau-induced Unc phenotype. The identity of library clones were confirmed by automated DNA sequencing.

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 (13Go). Worms were allowed to settle and thrashes counted for 30 s.

Protein extraction
Tau fractions were obtained as described previously (13Go). Pelleted worms were resuspended in an equal amount (w/v) of high salt RAB buffer [100 mM 2-(N-morpholino) ethanesulfonic acid (MES), 1 mM ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 0.5 mM MgSO4 and 20 mM NaF] and lysed by sonication. This lysate was used for total tau measurements. This lysate was then centrifuged at 40 000g for 40 min to obtain an RAB-insoluble pellet which was resuspended by sonication in 0.2 g/ml sucrose, 18.5 mM Tris (pH 6.8), 2 mM EDTA, 80 mM DTT and 2% SDS. This material was used directly for the insoluble tau faction measurements (13Go,89Go).

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. 125I-labeled goat anti-rabbit IgG was the secondary antibody (New England Nuclear) and signals were quantitated using a Packard Cyclone phosphorimager.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH grant AG017586 (G.D.S.), the Virtual Research Institute, Nippon Boehringer Ingelheim Co., Ltd (G.D.S.), a Department of Veterans Affairs Merit Review Entry Program Grant (B.C.K.) and the Alzheimer's Association (NIRG-02-3586 to B.C.K.). The ß-tubulin antibody E7 is from the Developmental Studies Hybridoma Bank (NICHD). We thank Leo Anderson, Harmony Danner and Susan Danner for excellent technical assistance.

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


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