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Alzheimer’s disease and gender differences in C. elegans

Jitendra Kumar1 and Kyungchae Park1
1The Buck Institute for Research on Aging, Novato CA

Correspondence to: Jitendra Kumar (jkumar@buckinstitute.org)

The prevalence and severity of Alzheimer’s disease (AD) are influenced by gender such that two thirds of AD patients are women. However, the biological mechanisms underlying these sex differences are not fully understood. AD begins with slight memory loss and confusion, and eventually leads to severe mental impairment (Mc Khann et al., 1984). It has been previously shown by Guillozet et al. that the formation of amyloid plaques and neurofibrillary tangles in the brain are responsible for memory and other cognitive functions (Guillozet et al., 2003). A recent human trial of AD treatments has proven to be largely unsuccessful. A probable reason is that multiple factors such as food, social relationships, physical activity, gender, climate and stress all play a role in its pathology. One of the most important factors is gender (Viña and Lloret, 2010). Lack of biological gender model systems for AD limits our understanding of the role that gender play in AD pathogenesis. Elucidating the role of gender in an AD model is crucial to prevention, diagnosis, and treatment of AD in different sexes.

In order to examine the role of gender on the etiology of AD, a C. elegans model was used to emulate the pathological processes of AD. The worm apl-1 gene encodes two identical isoforms, orthologous to the human amyloid precursor protein (APP) involved in AD (Wentzell and Kretzschmar, 2010). Recently, a transgenic animal (CL4176) expressing Aβ42 in muscle tissues (Drake et al., 2003) has been established as a model. Interestingly, the Aβ protein expressed in C. elegans self-aggregates like Aβ1–42, and forms fibrillar structures (Link, 1995).

We used male and hermaphrodite CL4176 transgenic animals and performed a paralysis assay (Drake et al., 2003). Synchronized eggs were maintained at 16 ºC and males and hermaphrodites were sorted at late L4 stage. Sorted worms were subjected to 25 ºC to induce the transgene expression and the paralysis was scored successively at 2 hour intervals. The hermaphrodite animals paralyzed earlier in comparison to the males. The mean time duration at which 50% worms were paralyzed was 34.6 hours and 27.3 hours, for males and hermaphrodites, respectively (p< 0.001) (Figure 1A, 1B). The data shows that the male CL4176 takes significantly more time to undergo paralysis compared to the hermaphrodites.

In this paper, we provide a way to study the mechanism and treatment strategy of AD disease based on gender. The observed phenotype is consistent with human beings (Viña and Lloret, 2010). Further studies will allow us to investigate the molecular pathways involved in AD in relation to sex and significantly impact our understanding to identify novel therapeutics in the future.

Figures



Figure 1: (A) Paralysis assays in the CL4176 C. elegans in male and hermaphrodite. (A) Time course of paralysis in the CL4176 male and hermaphrodite animals. Data are expressed as percentage of non-paralyzed worms from the three independent trials (n > 100 worms). (B) The paralysis assays was scored for mean time duration at which 50% worms were paralyzed after up shift temperature to 25 oC. ***, p<0.001.

References

Drake J, Link CD, and Butterfield DA. (2003). Oxidative stress precedes fibrillar deposition of Alzheimer’s disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model. Neurobiol. Aging 24, 415-420. PubMed

Guillozet AL, Weintraub S, Mash DC, and Mesulam MM. (2003). Neurofibrillary tangles, amyloid, and memory in aging and mild cognitive impairment. Arch. Neurol. 60, 729-736. PubMed

Link, CD. (1995). Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 92, 9368–9372. PubMed

McKhann G, Drachman D, Folstein M, Katzman R, Price D, and Stadlan EM. (1984). Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease, Neurology 34, 939-944. PubMed

Viña J, and Lloret A. (2010). Why women have more Alzheimer’s disease than men: gender and mitochondrial toxicity of amyloid-beta peptide. J. Alzheimers Dis. S527- 533. PubMed

Wentzell J, and Kretzschmar D. (2010). Alzheimer’s disease and tauopathy studies in flies and worms. Neurobiol. Dis. 40, 21–28. PubMed

L1 arrest affects dauer decision in C. elegans

Alexander B. Artyukhin1,2 and Leon Avery1
1Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond VA, 2Boyce Thompson Institute for Plant Research, Cornell University, Ithaca NY

Correspondence to: Alexander B. Artyukhin (alex_artyukhin@yahoo.com)

During larval development C. elegans has two distinct time points where it can arrest development until environmental conditions become more favorable. The first one is L1 arrest, which occurs when worms hatch in the absence of food. The second decision point is later, around L1-L2 molt, and if conditions are poor at this time (little food, crowding, and high temperature) worms gain the potential to become dauers. L1 arrest and dauer formation are not mutually exclusive and, in principle, a worm can go through both of them sequentially. However, it is not known whether L1 arrest and dauer formation are coupled, i.e., if experiencing L1 arrest affects a worm’s decision to become a dauer. A naïve prediction would be that worms that experienced L1 starvation should be more susceptible to become dauers since they were already exposed to a harsh environment.

We studied dauer formation of N2 worms in liquid and found that in reality worms that experienced L1 arrest and starvation were less likely to become dauers. In a typical experiment, eggs obtained from an egg prep were incubated at 20 0C in S-complete with a small amount of bacterial food (HB101) and dauer formation was assessed 6 days later based on survival in 1% SDS (Pungaliya et al., 2009). When we let eggs hatch without food and added it 1-4 days later, keeping other conditions the same, significantly fewer worms became dauers (Figure 1). This was not due to slower development after starvation and incomplete dauer formation. We also found that this effect reflected an internal state of the worm rather than exposure to L1 exometabolome during L1 arrest since worms starved as L1s at low and high density later formed dauers with the same probability (Figure 1, during the dauer formation worms were at the same low density of 1 worm/µl to minimize the effect of released chemicals, see below).

Starved L1 worms release numerous metabolites, including several ascarosides, and we anticipated that they might promote dauer formation. Indeed, when S-complete was substituted with conditioned medium from high-density L1 larvae starved for 24 h, more worms became dauers (Fig. 2), consistent with the known dauer-inducing effect of crowding mediated by ascarosides (Ludewig and Schroeder, 2013). However, this result apparently contradicts the starvation effect described above. On one hand, when a worm itself experienced L1 arrest but received no signal from other worms (low density condition), it was less likely to become a dauer and preferred to try to grow to adult. On the other hand, a worm that had not experienced starvation itself but was exposed to chemical signals released by other starved L1 worms, was more likely to become a dauer. The next obvious experiment is to combine these two opposite effects and see which one is stronger. The result of this test (Fig. 2) is that the external starvation signal (mediated by L1 conditioned medium) overrides the internal one (L1 arrest). When making a decision whether to become a dauer, apparently C. elegans worms trust the communal voice more than their own.

While the dauer-inducing effect of L1-conditioned medium is intuitively easy to accept, the negative effect of L1 starvation on subsequent dauer formation is less clear. A recently published model of contrast effects (McNamara et al., 2013) may provide a theoretical framework for understanding this phenomenon. But to keep us from being too comfortable with our simplistic views nature presented us with another puzzle: L1 arrest does not negatively affect dauer formation in C. briggsae.

Figures



Figure 1: Percentage of dauers decreased when N2 worms experienced L1 arrest for 2 days. The outcome did not depend on whether worms were L1 arrested at low density (LD, 1 worm/µl) or high density (HD, 36.5 worms/µl). Washing L1 arrested worms before adding bacterial food did not make a difference either.


Figure 2: Exposure to conditioned medium from starved N2 L1s increased percentage of dauers in both non-starved and L1-starved N2 worms. Results for two independent preparations of L1 medium are shown (L1 medium 1: 21.5 h conditioning with 125 worms/µl at 22 oC; L1 medium 2: 22 h conditioning with 72.5 worms/µl at 22 oC). L1 starvation inhibited dauer formation in S-complete control sample but had no significant effect in L1 medium samples.

References

Ludewig AH and Schroeder FC. (2013). Ascaroside signaling in C. elegans (January 18, 2013), WormBook, ed. The C. elegans Research Community. PubMed

McNamara JM, Fawcett TW, and Houston AI. (2013). An adaptive response to uncertainty generates positive and negative contrast effects. Science 340, 1084-1086. PubMed

Pungaliya C, Srinivasan J, Fox BW, Malik RU, Ludewig AH, Sternberg PW, and Schroeder FC. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc. Natl Acad. Sci. U. S. A. 106, 7708-7713. PubMed

Insulin-like peptide expression in response to L3 stage starvation

Tom Janssen1, Isabel Beets1 and Lisbet Temmerman1
1Functional Genomics and Proteomics Unit, Department of Biology, KU Leuven, Leuven, Belgium

Correspondence to: Tom Janssen (Tom.Janssen@bio.kuleuven.be)

The C. elegans genome encodes a large family of putative insulin-like peptides comprising 40 genes (Pierce et al., 2001; Li et al. 2003). In spite of great interest in the insulin-like pathway, given its function in dauer formation, aging, and L1 arrest, the function of most insulin-like peptides remains uncharacterized. There is evidence, however, that they function as either agonists or antagonists of the insulin-like receptor DAF-2, promoting development or arrest, respectively (Pierce et al., 2001). The lack of loss-of-function phenotypes for most insulin-like peptides points towards redundancy, although to what extent remains unknown. Recent findings of Ritter et al. indicate that no single insulin is the sole agonist or antagonist for coordinating dauer formation through the DAF-2 receptor and that there may be complex patterns of redundancies between multiple insulins (Ritter et al., 2013).

We performed a microarray analysis comparing the gene expression between 24h starved L3 stage N2 and fully fed animals of the same age (Figure 1). The starved animals displayed a delay in growth and were still the size of L3 worms, whereas the fully fed animals developed normally to late L4 stage at the time of sampling. 37 of the insulin-like peptide genes (ins) were represented on the Agilent C. elegans Gene Expression Microarrays (design ID 020186).

25 of the 40 known ins genes were differentially expressed more than twofold in the starved animals (Figure 2). Remarkably, 21 insulins are upregulated under L3 starvation while only 4 are downregulated. The strongest increase in expression was observed for ins-35 (39.7 fold), ins-20 (11.4 fold), ins-16 (6.4 fold) and ins-33 (5.7 fold); the strongest decrease in ins-34 (35.4 fold), ins-37 (10.4 fold), ins-31 (4.6 fold) and ins-7 (3.9 fold). The high number of differentially regulated insulins seems to favor of a possible functional redundancy between these peptides, given that DAF-2 is the likely receptor for most of them. The strong change in transcript level of many of these peptides is indicative of their importance with regard to starvation survival.

INS-4, INS-6, INS-7 and DAF-28 have been suggested to be DAF-2 agonists (Pierce et al., 2001; Hua et al., 2003; Murphy et al. 2003), while INS-1, INS-17, INS-18, INS-33 and INS-35 represent antagonists (Pierce et al., 2001; Liu et al., 2004). Our data is consistent with a possible agonistic role for INS-7 (downregulated) and antagonistic role for INS-1, INS-17, INS-18, INS-33 and INS-35 (upregulated) with regard to L3 starvation and its resulting developmental effects. INS-4 and DAF-28 transcript levels, however, were not differentially regulated, and INS-6 was upregulated, indicating it might act as an antagonist under the condition of L3 starvation. The data also suggests possible new agonistic and antagonistic roles for many other insulin-like peptides, for example: INS-31, INS-34 and INS-37 as agonists, inhibiting developmental arrest; and INS-16, INS-20, INS-24, INS-25, INS-30 and INS-36 as antagonists, stimulating developmental arrest. A functional role for these insulins was not described before and might be specific to the condition of L3 starvation.

Different groups of insulins might therefore act differently depending on the physiological and environmental conditions, a concept that was also recently described in the ‘‘block design’’ by Ritter et al. (2013).

Figures



Figure 1: Sampling setup for the microarray analysis. Green arrow = fed; red arrow = starved; performed on NGM plates.


Figure 2: Differential expression of insulin-like peptide genes in 24h starved N2 animals (from L3 stage) compared to fed animals of the same age. green: upregulated; red: downregulated; boldface: > 2 fold difference (p<0.001)

References

Hua QX, Nakagawa SH, Wilken J, Ramos RR, Jia W, Bass J, and Weiss MA. (2003). A divergent INS protein in Caenorhabditis elegans structurally resembles human insulin and activates the human insulin receptor. Genes Dev. 17, 826-831. PubMed

Li W, Kennedy SG, and Ruvkun G. (2003). daf-28 encodes a C elegans insulin superfamily member that is regulated by environmental cues and acts in the DAF-2 signaling pathway. Genes Dev. 17, 844-858. PubMed

Liu T, Zimmerman KK, and Patterson GI. (2004). Regulation of signaling genes by TGFbeta during entry into dauer diapause in C. elegans. BMC Dev. Biol. 4, 11. PubMed

Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. (2003). Genes that act downstream of DAF16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277-283. PubMed

Pierce SB, Costa M, Wisotzkey R, et al. (2001). Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Dev. 15, 672-686. PubMed

Ritter AD, Shen Y, Fuxman Bass J, et al. (2013). Complex expression dynamics and robustness in C. elegans insulin networks. Genome Res. 23, 954-965. PubMed

Should apparent correspondences in worm and human brain/mind forms interest the worm research community? A case for ‘yes’

Graham P. Rabey
1Late of Anatomy Department, The University of Manchester and Stoke Mandeville Hospital, Manchester and Aylesbury, UK

Correspondence to: Graham P. Rabey (camrabeys@yahoo.co.uk)

The formal inversion theory of brain and mind suggests that there are structural similarities between certain basic brain forms characterised by left-right bicyclic formal inversion (arguably occurring in all bilaterian animals including the worm and humans) and certain basic mind forms characterised by formally inverted duality (most obviously occurring in humans) and that the former provide credible explanations for the latter.

White et al. (1986) data provide a strongly arguable two-perspective or two-truth ground-plan for all bilaterian brains that credibly explains the consistent formally inverted dualities of human mind forms across the arts and sciences, in subjects such as philosophy, logic, mathematics, physics, politics and ideology.

Formal Inversion in Minimal Model Bilaterian Brains. The middle part of Figure 1 is an ASE taste circuit. Signals flow in the direction of the arrows. ASE neurons connect with interneurons AIB and the signals change directions. Previously clockwise left signals travel anticlockwise and the inverse occurs on the other side. ASEL–AIBR connections are one-way and ASER–AIBL links are two-way or circular (indicated by a dotted box). AIB cells connect with motor neurons RIM and the signals change direction again − clockwise to anticlockwise on one side and the inverse on the other side. There are further one-way and two-way or circular connections. The latter are again indicated by a dotted box, but here represent one of four such cycles for graphic clarity. At the last stage, motor neurons connect with muscles so that the worm responds to the stimulus.

The lowermost part of Figure 1 summarises the circuit. Overall, signals in left brain cycles travel in inverse directions to signals in right brain cycles and there are further mutually inverted cyclic paths for the exchange of signals between the two sides. Broken-line sections in the summary are reminders that the signals travel via multiple neurons.

The uppermost part of Figure 1 summarises the probably associated formally inverted nature of gene expression in the ASE sensory neurons (Ortiz et al., 2006) which suggests one key direction for research.

Formal Inversion in Maximal Model Bilaterian Brains. Left-right bicyclic formal inversion seems conserved in humans, as in Figure 2, a severely simplified version of just one of the many ways in which the principle might be realised in the human brain. Numbered layers represent the human cerebral cortex. Layers 3 and 4 are main receivers of cortical and subcortical input and are here represented by cells in layer 4. Layers 2 and 3 provide main outputs to cortical destinations and are here represented by cells in layer 3. Layers 5 and 6 provide main outputs to subcortical destinations and are here represented by cells in layer 5.

Interested readers might wish to visit Google Books and insert the book title Suitable Heresies. This will enable them to read a 3 page introduction, a 3 page brief outline and a 27 page expanded outline of the theory. The 2009 book is the most complete description of the theory available but is not complete on worm connectivity in the amphid complex, referring as it does to all 13 sensory neuron pairs and to all 7 related interneuron pairs but to only 6 illustrative motor neuron pairs for simplicity. This deficiency has been corrected in the 2012 book, which now refers to 12 related motor neuron pairs and has had one neuron pair (previously wrongly included by me) deleted. If any interested worm scientist wishes to contact me by email, I shall be pleased to provide a complimentary copy of the 2012 book, which gives the most up-to-date overview of the perceived two-truth ground plan.

Figures



Figure 1: A minimal model ‘thought’.


Figure 2: Left-right bicyclic formal inversion in minimal (below) and maximal (above) model brains.

References

Ortiz CO, Etchberger JF, Posy SL, Frokjaer-Jensen C, Lockery S, Honog B, and Hobert O. (2006). Searching for neuronal left-right asymmetry: genomewide analysis of nematode receptor-type guanylyl cyclases. Genetics 173, 131-149. PubMed

Rabey GP. (2009). Suitable heresies: a new scientific concept of brain and mind and its startling implications for our times. Troubador Publishing Ltd, Leicester, UK.

Rabey GP. (2012). The formal inversion theory of brain and mind: a short overview. Troubador Publishing Ltd, Leicester, UK.

White JG, Southgate E, Thomson JN, and Brenner S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. London. B 314: 1-340. PubMed

Workshop review: Engineered nucleases for genome editing in nematodes

John Calarco1, Hui Chiu2, Daniel J. Dickinson3, Ari E. Friedland4, Junho Lee5, Te-Wen Lo6, Hillel Schwartz2, Yonatan Tzur4 and Jordan D. Ward7
1FAS Center for Systems Biology, Harvard University, Cambridge MA, 2Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena CA, 3Department of Biology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill NC, 4Department of Genetics, Harvard Medical School, Boston MA, 5Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-742, Korea, 6Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley CA, 8Department of Cellular and Molecular Pharmacology, University of California, San Francisco CA

Correspondence to: Hillel Schwartz,Jordan D. Ward (hillels@caltech.edu)

Introduction

The recent development of genome modification technologies such as TAL effector nucleases (TALENs) and the CRISPR-Cas9 system has allowed unprecedented modification of eukaryotic genomes. TALENs are a sequence-specific nuclease comprised of TALE DNA binding domains fused to the FokI nuclease. CRISPR-Cas9 consists of the Cas9 nuclease plus two small RNAs: one that base-pairs with a 19 bp target (crRNA) and another that activates Cas9 (trcRNA). This system can be simplified by making a synthetic guide RNA (sgRNA), a hybrid of the two small RNAs. Both systems are used to make DNA double-strand breaks at desired genomic locations. At the recent International C. elegans meeting, we presented in a workshop about harnessing these technologies to edit the nematode genome. We have summarized the workshop and describe the reagents and protocols we anticipate distributing to the community. The presentations covered a broad range of successful approaches:

DNA-based (CRISPRs)

A.E.F. (Church lab), J.C., and Y.T. (Colaiàcovo lab) presented their system for targeted mutagenesis, which involved injecting a cocktail of three vectors: a worm codon optimized Cas9 driven by the eft-3 promoter, an sgRNA driven by a U6 promoter, and an mCherry marker driven by the myo-3 promoter (Friedland et al., 2013). Targeting four different genes with this system, they recovered mutant progeny with random inserts and deletions at the expected loci. Progeny of these F1 animals were screened and also carried these mutant alleles, indicating that the targeted disruptions are heritable. Reagents are available on Addgene at http://www.addgene.org/crispr/calarco/.

By co-injecting an engineered homologous recombination template and a single Cas9+sgRNA expression plasmid, D.J.D. (Goldstein lab) and J.D.W. (Yamamoto lab) inserted gfp into endogenous genes, resulting in GFP “knock-in” fusion proteins expressed under the control of all native regulatory elements. They also made multiple targeted point mutations in endogenous genes. The unc-119(+) marker used to select for recombinants can be excised with Cre recombinase, allowing complicated genome edits to be made with minimal “scarring.” Knock-in strains took less than 1 month to produce (about 2 days total hands-on time) and cost about $200 (mainly the cost of PCR primers). Plasmids will be distributed via Addgene after acceptance of the manuscript.

Protein-based (CRISPRs)

J.L. reported work from Jihyun Lee (his lab) and S.W. Cho (J.S. Kim lab); they generated gene-specific heritable mutations by germline injection of Cas9 protein complexed with sgRNA. X-linked genes dpy-3 and unc-1 were selected for targeting to facilitate identifying mutations through their visible phenotypes in homozygotes and hemizygotes. Indels at target sites were successfully confirmed in F1 animals by T7E1 assay and sequencing in both cases. Surprisingly, visible F1 mutants were often observed, and one Dpy mutant turned out to be a trans-heterozygote of two independent mutations in dpy-3, demonstrating the high efficiency of the method.

RNA-based (CRISPRs and TALENs)

H.C. and H.S. (Sternberg lab) injected in vitro-synthesized RNAs into the C. elegans germline: a capped and polyadenylated mRNA for humanized Cas9 and an sgRNA. F2 progeny were inspected for phenotypic homozygous mutants. Mutants were recovered at varying frequencies, up to one allele for every five P0s. A majority of mutations were large deletions (>1 kbp). Analysis of high-throughput sequencing of two closely related but independent dpy-11 mutants did not identify off-target changes to the genome, suggesting CRISPR mutagenesis was highly specific for targeted gene disruption.

T.W.L. (Meyer lab) reported on highly effective strategies using TALENs and CRISPR-Cas9 nucleases to create heritable, precise insertion, deletion, or substitution mutations at specific DNA sequences at targeted endogenous loci. This was achieved by germline injection of nuclease mRNAs and single-strand DNA templates. They created nucleotide changes both close to and far from double-strand breaks to gain and lose genetic function, to tag proteins made from an endogenous gene, and to excise entire loci through targeted FLP-FRT recombination. These methodologies were effective across nematode species divergent by 300 million years: hermaphroditic and gonochoristic species within Caenorhabditis (elegans and species 9) and P. pacificus. Thus, genome-editing tools now exist to transform non-model nematode species into genetically tractable model organisms.

Perspectives

The adoption of these modification technologies promises to transform nematode genetics. Going forward, the rules of CRISPR targeting must be better elucidated, the kinetics of insertion/deletion and homologous recombination events can be optimized, and high-throughput screening strategies must be developed. The workshop highlighted the diversity of techniques successfully developed for nematode genome modification, with the best technique depending on the desired experimental outcome.

References

Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, and Calarco JA. (2013). Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10, 741-743.  PubMed

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