Identification and functional characterization of a putative IDE, C28F5.4 (ceIDE-1), in Caenorhabditis elegans: Implications for Alzheimer's disease

    Identification and Functional Characterization of a Putative IDE, C28F5.4 (ceIDE-1), in Caenorhabditis elegans: Implications for Alzheimer’s disease Rizwanul Haque, Aamir Nazir PII: DOI: Reference:

S0304-4165(16)30260-4 doi: 10.1016/j.bbagen.2016.07.013 BBAGEN 28553

To appear in:

BBA - General Subjects

Received date: Revised date: Accepted date:

16 April 2016 30 June 2016 16 July 2016

Please cite this article as: Rizwanul Haque, Aamir Nazir, Identification and Functional Characterization of a Putative IDE, C28F5.4 (ceIDE-1), in Caenorhabditis elegans: Implications for Alzheimer’s disease, BBA - General Subjects (2016), doi: 10.1016/j.bbagen.2016.07.013

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Identification and Functional Characterization of a Putative IDE, C28F5.4 (ceIDE-1), in

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Caenorhabditis elegans: Implications for Alzheimer’s disease

Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology,

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Rizwanul Haquea and Aamir Nazira*

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CSIR-Central Drug Research Institute, Lucknow 226 031, INDIA

To whom correspondence should be addressed:

Dr. Aamir Nazir, Senior Scientist, Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, 226 031, (UP) India, Tel.: +91-522 2612411; Fax: +91-522 2623405; E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract

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Insulin-degrading enzyme (IDE) is a zinc metalloprotease, known to degrade insulin peptide and amyloid-beta (Aβ); the key protein involved in Alzheimer‟s disease (AD). Considering the important role played by IDE in disease progression of AD and T2DM, we endeavored to identify the Caenorhabditis elegans (C. elegans) IDE orthologous genes and test them for their role in AD related outcomes. We employed bioinformatics, reverse genetics and molecular biology approaches towards identification and functional characterization of putative IDE candidates in C. elegans. Using in-silico analysis we have identified seven C. elegans genes that possess HXXEH motif, an identifying marker of IDE. We further carried out functional analysis of the identified genes in Aβ expressing C. elegans strain CL4176 [myo-3/Aβ1–42 long 3′-UTR] via studying effect on Aβ induced toxicity, cholinergic neuroanatomy, content of acetylcholine/acetylcholine-esterase, extent of reactive oxygen species and expression of FOXO transcription factor DAF-16. Our findings reveal that amongst the identified putative IDE orthologs, a functionally uncharacterized gene C28F5.4 had a profound effect on the tested endpoints. Knocking down C28F5.4 aggravated the AD associated conditions by decreasing Aβ induced toxicity, severely compromising cholinergic neuroanatomy, reducing expression of acetylcholine-transporter, decreasing acetylcholine content, elevating ROS, with no effect on DAF-16 stress-response protein. These studies provide crucial insight into the structural/functional orthology of IDEs across human and nematode species and further our understanding of the involvement of these proteins and insulin pathway in AD. Further studies could aid in identifying novel drug-targets and in understanding the common modulating factors between AD and T2DM. Keywords: Alzheimer disease; Insulin Degrading Enzyme; C28F5.4, ceIDE-1; Caenorhabditis elegans (C. elegans); Functional genomics; Amyloid beta. Highlights - C28F5.4 shares 99.2% homology with its human counterpart. - The signature motif HXXEH of IDEs is exhibited by C28F5.4 at its active site. - C28F5.4 may be an interesting target in context of Alzheimer‟s disease.

ACCEPTED MANUSCRIPT 1. Introduction

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Insulin-degrading enzyme (IDE) (EC 3.4.24.56), is a zinc-metalloprotease of the superfamily inverzincin [1]. IDE has retained high level of conservation through the process of evolution [1, 2]. Members of general metalloprotease family that include neprilysin have conserved sequence motif: His-Glu-Xaa-Xaa-His (HEXXH) present at the active site; but IDE is endowed with inversion of this active site motif represented as HXXEH at the active site [3].The active site projects a large crypt (hollow ball which has been cut equatorially) which can fully enclose a wide array of substrates including insulin and amyloid beta (Aβ) [4]. IDE plays a role in the degradation of internalized insulin and also degrades the key protein Aβ, which is involved in the manifestation of Alzheimer‟s disease (AD). AD is a debilitating neurodegenerative disease (ND) characterized by the deposition of misfolded Aβ outside the neuronal surface. The deposition of misfolded Aβ restricts the neurons in getting nutrition from outside and gradually the neurons die. This demise of neurons results in dementia, short-term memory loss, problem in orientation and movement problems, oration issues, mood swings, mismanagement of self and behavioral issues [5]. AD is chronic ND that usually starts slowly but worsens over time. In the later stages bodily functions are lost which ultimately lead to death of the person. Presently there is no cure available for the treatment of AD; the disease has been dealt by symptomatic treatment only. Discovery of IDE as a protease for degrading Aβ has been very encouraging. Genetic linkage and association of AD has been reported on chromosome 10q23-24 in the region harboring the IDE gene [6]. Subsequent genetics studies have shown that genetic variants in a haplotype block spanning IDE, are significantly connected with plasma Aβ levels and are a risk for AD [7]. IDE hypo-functioning has been correlated with decrease in Aβ and insulin degradation; on the other hand IDE deficiency has been reported to cause 50% reduction in Aβ degradation in mice model [8]. IDE has been detected in human CSF, and its activity levels along with mRNA are decreased in brain tissue of AD patients; the cause is attributed to the elevated Aβ species [9]. Although fundamental functions concerning the biological role of IDE have been studied but very less is known about the pathways and regulatory role played by IDE. Studies reported herein were carried out to identify the C. elegans IDE orthologous genes and test them for their role in the AD related outcomes employing Aβ expressing C. elegans strain CL4176 [myo-3/Aβ1–42 long 3′UTR]. The model system C. elegans enables functional genomics studies because of its genetic homology, ease of genetic manipulations via RNAi and other accessible methodologies, further, C. elegans exhibits neurobiology that make it relevant for studying human NDs. The strain CL4176 that has been employed for the studies expresses „human‟ Aβ thus providing a facile platform for exploring role of the putative IDE in the context of AD.

2. Results 2.1. Identification of Orthologs of Human Insulin Degrading Enzyme in C. elegans

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IDE is an evolutionarily conserved zinc-metalloprotease distributed widely across the species ranging from prokaryotes to mammals [2]. It is not the mere sequence similarity, IDE has also retained its active site residues and catalytic property during the course of evolution. IDE is known to degrade a wide array of substrates that include intracellular insulin, glucagon, transforming growth factor-α, insulin-like growth factors I and II, bradykinin and kallidin [10]. It also participates in catabolism of misfolded protein of AD namely Aβ. Deficiencies in IDE function are being coupled with AD and Type-2Diabetes Mellitus (T2DM) but no major role of the mutations in IDE gene have been associated with the disease development so far. Considering the important role played by IDE in the disease progression of AD and T2DM, we endeavored to identify the C. elegans IDE orthologous genes and test them for their role in AD related outcomes. We first analyzed the entire C. elegans genomic sequence with multiple sequences searching tool BLASTp (NCBI) using human IDE as a template. This procedure revealed seventeen C. elegans putative IDE genes, as listed in Table1.The highest homology percentage 99.2% was exhibited by C28F5.4 (henceforth being referred as ceIDE-1), a gene which hasn‟t been categorized yet. In addition to percent homology conservation, another important screen based on the conserved active site residues was carried out. IDE bears highly conserved sequence motif: His-Xaa-Xaa-Glu-His (HXXEH) present at the active site which participates in the catalysis process. The HXXEH motif contains two histidine residues present at 108 and 112 position and one glutamate residue present at 189 position [11]. In this screen, represented in Figure 1, we searched for this HXXEH motif across all the identified seventeen putative IDE candidates predicted by BLAST along with Human, Drosophila and Zebrafish IDE sequences. The HXXEH motif was displayed in Human, Drosophila, Zebrafish, ceIDE-1, C02G6.2, Y70C5C.1, F44E7.4, C02G6.1, C05D11.1 and mppb-1. In our analysis we found that HXXEH motif was conserved in Human, Drosophila, Zebrafish and C. elegans suggesting that IDE has retained its active site residues and catalytic property during the course of evolution. The HXXEH motif based screen led us to identification of seven genes that exhibit both featuresorthology to Human, Drosophila, Zebrafish IDE and possess the marker motif that is specific to IDEs. Next, the identified genes were examined for conserved domain present in their structure using NCBI tools. The seven identified genes were found to possess at least two domains namely Insulinase (Peptidase family M16) and Peptidase_M16_C organization, which is also the characteristic hallmark of the human IDE and members of the IDE superfamily. The employed approach successfully led to identification of seven most closely related orthlogues of Human IDE in C. elegans. These identified genes were advancedfor the subsequent studies and their role in AD related effects was explored. Table 1: The C. elegans IDE orthologous genes identified with „multiple sequences searching tool‟BLASTp (NCBI) using human IDE (sequence was retrieved from Universal Protein Resource, UniProt, as a template.

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Not assigned Not assigned Not assigned Not assigned

3 4 5

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mppb-1

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Not assigned ugt-26

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Y70C5C. 1 C02G6.1

Uncharacterized gene. Predicted to act as IDE/Nardilysin like protein in C. elegnas. Uncharacterized gene. Predicted to act as IDE/Nardilysin like protein in C. elegnas. Uncharacterized gene. Predicted to act as IDE/Nardilysin like protein in C. elegnas. Uncharacterized gene. Predicted to act as IDE/Nardilysin like protein in C. elegnas.

C02G6.2 C28F5.4 (ceIDE1) ZC410.2

C05D11. 1 C10H11. 6

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93.3%

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97.1%

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79.9%

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99.2%

Mitochondrial processing peptidase, beta subunit, and related enzymes (insulinase superfamily) ; Uncharacterized gene. Predicted to act as IDE/Nardilysin like protein in C. elegnas. UDP glycosyltransferase 3 family, polypeptide ; UDP-glucuronosyl and UDPglucosyltransferase ; Glycosyltransferases, related to UDP-glucuronosyltransferase Peroxidase/oxygenase; Protein involved in phenylpropanoid biosynthesis An ortholog of human CTSA (cathepsin A); predicted to have serine-type carboxypeptidase activity.

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F45G2.10 is an ortholog of human FAM96B (family with sequence similarity 96, member B). SMA-9 in C. elegansfunctions in a subset of the TGF-beta-mediated signaling pathways that regulate body size and male tail patterning and morphogenesis. Dnahelicase ; DNA helicase PIF1/RRM3

III

Encodes a homeodomain protein homologous to the Deformed and Sex combs reduced family of homeodomain proteins; lin-39 is required cell autonomously for specification of mid-body region cell fates, including those of the VC neurons and the vulval precursor cells (VPCs), during postembryonic development. Dnahelicase ; DNA helicase PIF1/RRM3

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Dnahelicase ; DNA helicase PIF1/RRM3

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I 96.2%

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Not assigned ctsa-1

F09F3.5 K10B2.2

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T05A10. 1

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Uncharacterized gene. Predicted to act as IDE/Nardilysin like protein in C. elegnas.

% Homolog y 88.1%

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Gene Pair name F44E7.4

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F33H12. 6 C07H6.7

Y16E11 A.2 F59H6.5

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98.1%

96.9% X

23.3%

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29.1%

28.7%

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Y17G7B. 15

Encodes a homolog of centaurin beta, an ArfGTPase activating protein (Arf GAP) that also contains a pleckstrin homology domain and C-terminal ankyrin repeats.

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Figure 1: Multiple sequence alignment of Human, Drosophila, Zebrafish IDE and putative IDE candidates orthologous to C. elegans IDE using Clustal Omega alignment program. (A) Multiple sequence alignment showing highly conserved sequence motif: His-Xaa-Xaa-Glu-His (HXXEH) present at the active site (in red) with two histidine residues present at 108 and 112 positions. (B) Multiple sequence alignment showing conserved glutamate residue present at 189 position that participates in the catalysis process 2.2. Silencingof ceIDE-1 Decreased Aβ Induced Toxicity in Transgenic C. elegans Model of AD Expressing Human Aβ. AD is characterized by progressive demise of neurons in the cerebral tissue, extracellular aggregation/deposition of Aβ, and intracellular formation of fibrils containing hyperphosphorylated tau. The accumulation of Aβ is a major characteristic of this disease. Therefore, we employed

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genetically engineered C. elegans strain, CL4176 [myo-3/Aβ1–42 long 3′-UTR] that has been designed to express temperature-inducible human Aβ peptide in the body wall muscles [12]. These transgenic strains have been previously used to assay the effect of many potential therapeutic agents/genes against Aβ toxicity and aggregation. The protective effects of cocoa peptide, Liuwei Dihuang- a traditional Chinese medicinal formula, Bacopa monnieri- an Indian medicinal plant and other agents against Aβ toxicity have been reported using this transgenic strain. The strain has also been successfully employed for screening synthetic compounds with anti-Aβ aggregation properties [13-17]. An increase in Aβ in the worms leads to early paralysis phenotype, while decrease in Aβ in the worms leads to delayed paralysis. We utilized this transgenic system to assay the protective role (if any) of the selected orthlogous genes of Human IDE in C. elegans. Out of the seven genes evaluated, RNAi induced silencing of only one gene ceIDE-1, significantly decreased Aβ induced toxicity. Worms fed with bacterial strains carrying empty vector (serving as controls) exhibited a 46.43 ± 3.57 % paralysis whereas worms with RNAi of ceIDE-1 showed a paralysis of 18.33 ± 1.67%, RNAi of C02G6.2 showed a paralysis of 32.05 ± 1.28%, RNAi of Y70C5C.1 led to paralysis of 44.39 ± 2.28%, RNAi of C02G6.1 led to a paralysis of 29.30 ± 5.49%, RNAi of C05D11.1 showed a paralysis of 45.30 ± 8.26%, RNAi of mppb-1 showed a paralysis of 36.67 ± 3.33% and RNAi of F44E7.4 led to a paralysis of 71.67 ± 11.67%. Hence the worms with knocked down ceIDE-1 exhibited a reduced paralysis by 2.53 folds (*p<0.05) whereas gene knockdown of C02G6.2, Y70C5C.1, C02G6.1, C05D11.1, mppb-1 and F44E7.4 did not have any significant effect on the paralysis phenotype (Figure 2).

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ns ns OP-50 Vector Control C28F5.4 C02G6.2 Y70C5C.1 C02G6.1 C05D11.1 mppb-1 F44E7.4

Figure 2: Bar graph representation of Aβ-induced toxicity in transgenic CL4176 strain of C. elegans after silencing putative IDE candidate genes. CL4176 expresses temperature-inducible human Aβ peptide in the body wall muscles. An increase in Aβ in the worms leads to early paralysis phenotype, while decrease in Aβ in the worms leads to delayed paralysis. ceIDE-1 RNAi significantly decreased Aβ induced toxicity, leading to a delay in paralysis by 2.53 folds (*p<0.05), whereas gene

ACCEPTED MANUSCRIPT knockdown of C02G6.2, Y70C5C.1, C02G6.1, C05D11.1, mppb-1 and F44E7.4 did not have any significant effect. *p<0.05, ns not significant.

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2.3. Absence of ceIDE-1 Significantly Abolishes the Cholinergic Neuroanatomy and Reduces the Expression of Synaptic Vesicle Acetylcholine Transporter

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The loading of acetylcholine (ACh) into synaptic vesicles is assisted by a synaptic vesicle acetylcholine transporter (VAChT) [18]. In C. elegans this VAChT is encoded by unc-17. Mutations that disrupt complete unc-17 gene function are lethal, indicating the important role of the ACh transporter in proper growth of nematode [19]. In AD patients due to compromised cholinergic physiology, concentrations of ACh tend to decline and are known to be effected as much as 90 percent, a parameter associated with the disease. The only classes of drugs currently licensed for combating AD symptoms are acetylcholinesterase (AChE) inhibitors. AChE breaks down ACh molecule at the neural synapse [20] and inhibiting this molecule ultimately leads to increased availability of neurotransmitter ACh. This prompted us to carry out studies related to cholinergic system. For this, we employed a transgenic C. elegans strain LX929 that expresses unc-17::GFP transgene illustrating the cholinergic neurons. Out of 302 neurons in hermaphrodite C. elegans, only the cholinergic neurons fluoresce green in LX929 making it an ideal strain to study neuronal defects related to AD. The influences of various agents on neuronal development have been tested using this transgenic strain [21, 22]. The candidate genes were knocked down in LX929 and the effect on unc-17::GFP expression level and cholinergic neuroanatomy was assayed. As shown in Figure 3, control worms exhibited intact cholinergic neuroanatomy and a steady GFP expression all along the neurons of the transgenic worms. Gene knockdown of ceIDE-1, C02G6.2, Y70C5C.1, C05D11.1 resulted in reduced GFP expression of UNC-17 while gene knockdown of F44E7.4, C02G6.1 and mppb-1 did not have any effect. There was no obvious effect on gross morphology or mobility of the worms. This study reveals the neuroprotective potential of ceIDE-1, C02G6.2, Y70C5C.1, C05D11.1 genes in maintaining cholinergic neurophysiology.

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Figure 3: Expression of synaptic vesicle acetylcholine transporter (UNC-17) and visualization of neuroanatomy in cholinergic neurons of transgenic LX929 strain. LX929 expresses UNC-17 tagged with GFP in all cholinergic neurons of C. elegans. The worms of the control group exhibit intact cholinergic neuroanatomy and a steady GFP expression all along the cholinergic neurons of the LX929 worms. Gene knockdown of ceIDE-1, C02G6.2, Y70C5C.1, C05D11.1 resulted in complete abolition of the cholinergic neuroanatomy and reduced GFP expression of UNC-17 while gene knockdown of F44E7.4, C02G6.1, mppb-1 did not have any effect. Scalebar, 50 µm.

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2.4. Knockdown of ceIDE-1 Leads to Altered Levels of Acetylcholine but does not Affect Acetylcholine Esterase Activity

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After observing the effect of C. elegans putative IDE candidates on cholinergic neuroanatomy and VAChT, we further went on to check the levels of ACh and the activity of AChE. It has been previously shown that ACh content in the AD model of C. elegans is declined as compared to that of wild type animals and correlates tothat of effects seen in humans [13]. We endeavored to carry out quantification of neurotransmitter ACh and AChE activity after knocking down the candidate genes. We observed that worms of control group exhibited an ACh content of 1.0 ± 0.02 arbitrary units whereas the worms of ceIDE-1, C02G6.2, Y70C5C.1, C02G6.1, C05D11.1, mppb-1, F44E7.4 RNAi group exhibited an ACh content of 0.79 ± 0.02, 0.78 ± 0.02, 1.32 ± 0.05, 1.25 ± 0.06, 0.50 ± 0.02, 0.98 ± 0.01 and 0.59 ± 0.03 arbitrary units respectively (Figure 4A). There was a significant 1.27, 1.29, 1.97, 1.70 fold decrease in ACh content in RNAi of ceIDE-1 (p<0.05), C02G6.2 (p<0.05), C05D11.1 (p<0.01), F44E7.4 (p<0.01) knockdown worms respectively. The worms of the Y70C5C.1 RNAi treatment group exhibited a 1.32 fold increase in the ACh content. Moreover, there was no effect on AChE activity thus exhibiting statistically insignificant differences between control (0.72 ± 0.19) and RNAi of ceIDE-1 (0.61 ± 0.01), C02G6.2 (0.59 ± 0.02), Y70C5C.1 (0.72 ± 0.19), C02G6.1 (1.01 ± 0.02), C05D11.1 (0.38 ± 0.02), mppb-1 (0.57 ± 0.08), F44E7.4 (0.47 ± 0.01) RNAi group (Figure 4B).

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Figure 4: Levels of acetylcholine (ACh; A) and activity of acetylcholine esterase (AChE; B) estimated by amplex red assay in wild-type strain N2 after knocking down C. elegans putative IDE candidates. A significant 1.27, 1.29, 1.97, 1.70 fold decrease in ACh content was observed in ceIDE-1 (p<0.05), C02G6.2 (p<0.05), C05D11.1 (p<0.01) and F44E7.4 (p<0.01) knockdown worms respectively. The worms of the Y70C5C.1 RNAi treatment group exhibited a 1.32 fold increase in the ACh content. C02G6.1 and mppb-1 did not have any significant effect on levels of acetylcholine. The worms of all treatment groups did not show any significant deviation in acetylcholinesterase activity from control. *p<0.05, **p<0.01, ns not significant. 2.5. ROS Levels were Altered upon Knockdown of ceIDE-1

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Multiple studies have proposed that the death of selective neurons in AD is due to the increased oxidative stress and may have a role in the pathogenesis of neuronal death. The evidence supporting the theory is based on the fact that high level of metals like Fe, Al, and Hg, increase lipid peroxidation. Free radicals are generated either by increased protein and DNA oxidation or by decrease in cytochrome c oxidase, advanced glycation end products and SOD-1 in brain [23]. Keeping this in mind we studied the content of alteration of reactive oxygen species (ROS) in worms of control and knockdown groups of C. elegans putative IDE candidate genes. We employed the fluorescent dye 2′,7′-dichlorfluorescein-diacetate (H2DCFDA) to study the ROS levels. We observed a fluorescence intensity of 1415 ± 102.0 relative fluorescence units (RFU) in the control group whereas worms of ceIDE-1, C02G6.2, Y70C5C.1, C02G6.1, C05D11.1, mppb-1, F44E7.4 RNAi groups exhibited 2341 ± 69.61, 1433 ± 31.08, 752.8 ± 79.48, 860.9 ± 66.16, 1001 ± 69.10, 1365 ± 103.2, 1356 ± 135.7 RFU respectively. The results are being presented in Figure 5. Out of the seven candidate genes studied, RNAi of ceIDE-1 exhibited a significant (p<0.01) 65.5% increase in ROS level with respect to that of the control group. RNAi of Y70C5C.1(p<0.01), C02G6.1(p<0.05), C05D11.1(p<0.05) resulted in 46.8 %, 52.7 % and 29.2 % percent decrease respectively, in ROS content, with respect to that of the control group.

Figure 5: Effect of RNAi silencing of C. elegans putative IDE candidates on relative formation of reactive oxygen species (ROS) measured by H2DCFDA assay. H2DCFDA is a chemically reduced, nonfluorescent, acetylated form of fluorescein which is readily converted to a greenfluorescent form by the activity of ROS. An imbalance between the formation and transmission of ROS has been correlated with AD pathogenesis and can exacerbate its progression. ceIDE-1 RNAi exhibited a significant (p<0.01) 65.5 % increase in ROS level with respect to that of the control group. RNAi of Y70C5C.1 (p<0.01), C02G6.1 (p<0.05), C05D11.1 (p<0.05) resulted in 46.8 %, 52.7 % and 29.2 % percent decrease respectively, in ROS content with respect to that of the control group. The RNAi against C02G6.2, mppb-1 and F44E7.4 did not have any significant effect on ROS formation, *p<0.05, **p<0.01, ns not significant.

ACCEPTED MANUSCRIPT 2.6. DAF-16, A Critical Regulator of the Stress Response was not Altered upon Knockdown of ceIDE-1

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DAF-16 is a key transcription factor required for longevity of C .elegans. It codes for the Human forkhead box O (FOXO) homologue and acts in the insulin/IGF-1-mediated signaling pathway. DAF-16 regulates longevity, stress response, dauer formation, fat metabolism, and innate immunity [24]. Since, AD is an age-related disorder and DAF-16 governs insulin like signaling, we endeavored to study the effect of candidate putative IDE genes that are proposed to be a common link between AD and T2DM on DAF-16 expression. For this we employed transgenic C. elegans strain TJ356 expressing DAF-16::GFP. The DAF-16::GFP is expressed in a puncta like structure in the worm across the body length. In our study we examined the expression of puncta cum DAF-16::GFP structure under knock down of C. elegans putative IDE candidates. The mean number of DAF-16-puncta exhibited by worms treated with vector control were 45.25 ± 4.008 whereas mean number of DAF-16-puncta exhibited by worms of groupsceIDE-1, C02G6.2, Y70C5C.1, C02G6.1, C05D11.1, mppb-1, F44E7.4 were 62.50 ± 6.513, 60.00 ± 13.01, 20.25 ± 6.047, 87.00 ± 10.44, 66.00 ± 4.000, 32.75 ± 7.674 and 49.33 ± 1.202 respectively. The result of this study is presented in Figure 6A and quantification of the puncta for every group is plotted graphically in Figure 6B. The worms of C02G6.1 and C05D11.1 RNAi group exhibited 1.92 (p<0.05) and 1.45 folds (p<0.01) increase in the DAF-16 GFP puncta respectively, whereas RNAi of Y70C5C.1 exhibited 2.23 folds (p<0.05) decrease in the DAF-16 GFP puncta. The RNAi against ceIDE-1, C02G6.2, mppb-1 and F44E7.4 did not have any significant effect on DAF-16::GFP.

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Figure 6: (A) The expression of DAF-16::GFP in transgenic TJ356 strain of C. elegans after knocking down C. elegans putative IDE candidates. DAF-16 is a FOXO-family transcription factor of the Insulin like signaling pathway and mediates stress response inside the nematode (B) is the graphical representation for quantification of DAF-16::GFP puncta using DotCount software. The worms of C02G6.1 and C05D11.1 RNAi group exhibited 1.92 (p<0.05) and 1.45 folds (p<0.01) increase in the DAF-16 GFP puncta respectively, whereas Y70C5C.1 exhibited 2.23 folds (p<0.05) decrease in the DAF-16 GFP puncta. The RNAi against ceIDE-1, C02G6.2, mppb-1 and F44E7.4 did not have any significant effect on DAF-16::GFP. Scale bar, 50 µm 3. Materials and Methods 3.1. C. elegans culture and maintenance: C. elegans strains used in this study were propagated on Escherichia coli (E. coli) OP50 strain which served as a food source. OP50 seeding was done as reported previously [25] onto NGM plates to prepare a bacterial lawn excluding gene knockdown (RNAi) experiments. Nematode Growth Medium (NGM; 1L) was prepared by adding 3 g NaCl (sigma), 2.5 g Peptone (sigma), 17 g agar (HIMEDIA) to 975 ml double distilled (dd) H2O.After autoclaving the solution and cooling it down to 55°C, 25 ml KH2PO4(1 M pH 6.0), 1 ml CaCl2(1 M), 1 ml MgSO4(1 M) and 1 ml cholesterol (5 mg/ml) was added. Under sterile conditions the media was poured on petri plates and left to solidify and thereafter stored at 4°C. For preparation of bacterial lawn 500µl of OP50 was spread with the help of

ACCEPTED MANUSCRIPT spreader on NGM plates, dried and incubated overnight at 37oC. C. elegans strains were chunked into this pre seeded OP50 plates and incubated at 22°C for propagation.

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3.2. C. elegans embryo isolation: Gravid (reproductively mature) C. elegans population was collected from previously chunked C. elegans petri plates using 10 ml M9 buffer. The worms were settled down by centrifugation at 1300 rpm for 2 min in a 15 ml conical centrifuge tube. Worms were washed thrice in order to remove any adhering bacteria. To the worm pellet, 5 ml of 1M sodium hydroxide solution and 2mL of sodium hypochlorite (together known as axenizing solution) was added. The solution was subjected to gentle mixing to dissolve the body component following which the embryos were released from the worms into the solution. The isolated embryos were settled down by centrifugation at 1300 rpm for 5 min. The embryo pellets were washed thrice with M9 buffer and were subsequently placed onto experimental plates.

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3.3. C. elegans strains: The C. elegans strains used in this study were: N2 (Bristol): wild-type, CL4176, dvIs27, ([pAF29(myo-3/A-Beta 1-42/let UTR) + pRF4(rol-6(su1006))]): this strain is genetically modified to express a temperature inducible human Aβ protein in the body wall muscle of the worm. This strain is normally propagated at 15oC and upshifting to 25oC induces the expression of human Aβ protein and worms get paralyzed. LX929, vsIs48 ([Punc-7::gfp]): In this strain GFP is expressed in all cholinergic neurons revealing the cholinergic neuroanatomy. TJ356, zIs356 ([Pdaf-16::daf-16a/b-gfp; rol-6]): this strain expresses fluorescent DAF-16::GFP. All the strains were obtained from Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN). 3.4. In silico analysis of Human IDE: The human (identifier: P14735-1), Drosophila melanogaster (identifier: P22817) and Danio rerio (zebrafish; identifier: A5A8J7) IDE FASTA sequence was retrieved from UniProt (Universal Protein Resource) [26]. The human IDE isoform studied was Isoform 1 which was chosen because it represents the 'canonical' sequence. The C. elegans IDE orthologous genes were identified with multiple sequences searching tool BLASTp (NCBI) using human IDE as a template [27]. This resulted in seventeen C. elegans putative IDE candidates, listed in Table 1. All the C. elegans protein sequences were retrieved from wormbase [28]. Multiple sequence alignment was generated for the identified C. elegans IDE orthologous proteins using Clustal Omega alignment program [29]. The IDE highly conserved sequence motif: His-Xaa-Xaa-Glu-His was searched across all aligned sequences manually. For identification of conserved domain in protein NCBI protein database: “identify conserved domains” was used [30]. 3.5.RNAi induced gene silencing: For gene knockdown experiments, standard feeding protocol was followed as described previously [31]. Bacterial clones producing dsRNA against targeted genes were obtained from Ahringer RNAi library purchased from SA Biosciences (Cambridge, UK). Firstly, bacterial clone producing dsRNA was cultured in Luria Broth containing 50μg/ml ampicillin. A volume of 500 µl of the bacterial culture was seeded onto NGM-IPTG plates which

ACCEPTED MANUSCRIPT were prepared by adding 5mM isopropyl β-d-thiogalactoside (IPTG) and 25mg/L carbenicillin to cooled NGM media before pouring onto the plates. The seeded plates were incubated at 37°C overnight and were used thereafter.

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3.6. Aβ-Induced toxicity assay: For assaying Aβ-induced toxicity, transgenic C. elegans strain CL4176 was used. CL4176 is genetically engineered to express a temperature inducible human Aβ protein in the body wall muscle of the worm. The Aβ expression is muscle-directed by the use of myo-3 promoter. When the strain is propagated at 15oC there is no expression of Aβ, upshifting the temperature to 25oC induces the expression of human Aβ protein and worms get paralyzed. The extent of paralysis reflects the toxicity caused by the expression of Aβ. The method followed has been described previously [13]. Isolated embryos from CL4176 strain were placed onto the control and experimental plates and were incubated at 15°C for 48 h. After this the temperature for treatment plates was upshifted to 25°C and after 24 h the treatment plates were scored for paralyzed worms every 2-3 h. Worms were tapped on their head by the help of a platinum loop. Worms that did not show any movement were considered paralyzed. The reading presented is when worms of the control group exhibit a 50 % paralysis. Three replicates, each comprising of a minimum number of 30 worms, were scored for each experiment and the experiments were repeated thrice. The data for each experiment was derived from an N= 90 and Mean ± SE for representative experiment was plotted after analyzing statistical significance as described under the subsection “statistical analysis”.

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3.7. Image acquisition: Worms were washed from the treatment plates thrice using M9 to get rid of the adhering bacteria. Sodium azide (Sigma, cat No. 71289) 100 mM (final conc.) was added to the worm pellet in order to anesthetize the worms. Immobilized worms were mounted on agar padded glass slide. Finally, the images of the worms were grabbed using Carl Zeiss Axio Imager M2 microscope equipped with AxioCam digital camera at 20x or 40 x magnifications. ZEN2010 image acquiring/processing software was used for image acquisition. For each individual group a minimum of 10 images was analyzed. 3.8. Assay for cholinergic neuroanatomy and expression of synaptic vesicle acetylcholine transporter: VAChT is an Ach transporter which loads ACh into secretory vacuoles inside the neurons thus making ACh available for secretion. In C. elegans, VAChT is encoded by unc-17. For assaying cholinergic neuroanatomy and expression of VAChT, transgenic strain LX929 was employed. In LX929, GFP is expressed in all cholinergic neurons revealing the cholinergic neuroanatomy, and the fluorescence intensity represents the expression level of VAChT. The RNAi treatment plates along with control were prepared; isolated LX929 C. elegans embryos were placed onto it. After 48 h of incubation at 22 °C, fluorescence images were grabbed. Each assay plate contained a minimum of 50 worms and a set of three plates was studied for each condition. Imaging and quantification was carried out for a minimum of 10 worms from each group.

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3.9. Quantification of Acetylcholine level and Acetylcholinesterase activity: For Quantification of ACh level and AChE activity Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217; Invitrogen) was used. The RNAi treatment plates along with control were prepared, isolated wild-type C. elegans embryos were placed onto it in a way so as to have adequately populated worms. After 48 h of incubation at 22 °C, worms were washed thrice with sodium phosphate buffer at 1000 rpm for 2 min to clear off any adhering bacteria. Sonication was performed on ice at 25 % amplitude with pulse on time and off time of 15-s intervals for a span of 3 min. Homogenate was then centrifuged at 14,000 rpm for 30 min. The pellet was discarded and supernatant was assayed for ACh and AChE levels as provided in the manufacturer‟s manual. The final fluorescence reading was normalized with respect to protein content for each group. A set of three plates was used for each group and the experiment was carried out two times.

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3.10. Quantification of Reactive Oxygen Species: The alteration in the steady-state level of reactive oxygen species (ROS) was quantified by fluorescent 2‟,7‟–dichlorofluorescindiacetate (H2DCFDA) dye. The RNAi treatment plates along with control were prepared and isolated wild-type C. elegans embryos were placed onto it. After 48 h of incubation at 22°C, worms were washed thrice with M9 buffer and twice with Phosphate Buffer Saline. 100 worms per group (approx.) were transferred to Corning® 96 Well Black Flat Bottom Polystyrene NBS™ Microplate and the volume was made-up to 100 μl with PBS in each well. 50 μM (final concentration) H2DCFDA dye was added to each well. Fluorescence intensity was measured instantly, and after incubation of 1 h at room temperature using excitation wavelength of 485 nm and an emission wavelength of 520 nm. Final fluorescence intensity was determined for 100 worms in each group and final reading was calculated by subtracting 0 h reading from 1 h reading. This experiment required careful control on the number of worms studied within each group, hence the number of worms present in the final assay solution was maintained at approximately 100 worms by counting number of worms present in three equal-volume-drops of the solution. The mean number of worms was calculated and the volume that would contain 100 worms, was extrapolated accordingly. Triplicate wells were studied for each condition and the experiments were repeated thrice. 3.11. DAF-16 expression analysis: For analysis of DAF-16 expression, transgenic TJ356 strain was employed. TJ356 expresses DAF-16 tagged with GFP. The RNAi treatment plates along with control were prepared, isolated TJ356 C. elegans embryos were placed onto it. After 48 h of incubation at 22 °C, worms were washed thrice with M9 buffer. After 48 h of incubation at 22°C fluorescence images were grabbed. For the quantification of the DAF-16::GFP puncta, the number of GFP punctate structure (with boundaries distinguishable from surrounding fluorescence) were counted in the whole worm using DotCount software (Copyright (c) 20062015 Martin Reuter) with same size threshold for each image. For each individual group a minimum of 5 images was analyzed per treatment group. The data for each experiment was

ACCEPTED MANUSCRIPT derived from three individual experiments and Mean ± SE for representative experiment was plotted after analyzing statistical significance as described under the subsection “statistical analysis”.

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3.12. Statistical analysis: The data presented within the manuscript were derived from replicates as discussed for each individual endpoint. All the data are reported as mean ± standard error of the mean. The levels of significance between different groups were analyzed statistically by Student‟s t test using GraphPad prism.

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4. Discussion

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The interesting set of findings related to the ability of IDE in degrading Aβ, has stimulated an optimistic enthusiasm amongst neurobiologists who have been struggling towards obtaining a complete understanding of potential therapeutic interventions against Aβ aggregation and its associated effects. Aβ is a 42 amino acid peptide which is crucially involved in AD pathogenesis and is present as the main component of the amyloid plaques which is a characteristic diagnosis feature found in the brain of Alzheimer‟s patients [32]. Yester year studies have shown association between AD and IDE in human patients [33]. The single-nucleotide polymorphism studies carried out in human subjects, have demonstrated that the polymorphisms of the IDE gene are associated with late-onset AD [34]. There are more studies demonstrating the IDE, as a potential therapeutic genetic target for the treatment of AD[35-38]. Based on this interesting context we designed this study towards identification of the C. elegans IDE orthologous genes and towards testing them for their role against the AD related outcomes. These studies utilized bioinformatics, genetics and molecular biology approaches in identifying the putative IDE candidates in C. elegans and testing them against AD associated endpoints employing model system C. elegans. Our data revealed seventeen C. elegans putative IDE candidates. The highest homology of 99.2% was exhibited by C28F5.4 and lowest homology of 23.3% was exhibited by sma-9. The HXXEH motif, a characteristic feature of IDE that distinguishes IDE from the prototypic metalloprotease, was found to be present in seven genes viz ceIDE-1, C02G6.2, Y70C5C.1, F44E7.4, C02G6.1, C05D11.1 and mppb-1. In our analysis we found that HXXEH motif was conserved in Human, Drosophila, Zebrafish and C. elegans suggesting that IDE has retained its active site residues and catalytic property during the course of evolution. The histidine residues at 108 and 112 and glutamate at 189 position of human IDE, previously reported to participate in catalysis was also found to be conserved inC. elegans [39].The data obtained are further supported by the findings that these identified genes have been previously reported to act like nardilysin like protein in C. elegans [28]. The genes were studied further and assayed for Aβ-induced toxicity using CL4176 transgenic strain. CL4176 was first engineered by Link et al.; the strain expresses a human Aβ transgene in the body wall muscle and has been previously used to study the Aβ-induced toxicity of many agents [14, 40-43]. We observed that ceIDE-1 RNAi significantly decreased Aβ induced toxicity whereas others were insignificant in doing so. Deposition of Aβ in the AD patient brain and neuronal degeneration are characteristic

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hallmarks of the disease. It has been reported previously that Aβ induces neuronal degeneration, but the mechanism of neurotoxicity has not yet been fully elucidated [44]. The decreased paralysis phenotype may be attributed to the abolition of the cholinergic neuron structure or decreased Aβ expression. In the findings of Qui et al. it is hypothesized that presence of IDE could also result in oligomerization of monomeric Aβ into higher oligomeric forms [45]. As an interpretation to our studies, it will be naïve to comment whether paralysis phenotype is caused by Aβ expression or Aβ aggregation or neuronal degeneration. Knocking down IDE candidates ceIDE-1, C02G6.2, Y70C5C.1, C05D11.1 resulted in complete abolition of the cholinergic neuroanatomy and reduced GFP expression of UNC-17 while gene knockdown of F44E7.4, C02G6.1 and mppb-1 did not have any effect on these endpoints. As discussed earlier the abolition of the cholinergic neuroanatomy is an independent effect in AD and may or may not correspond to Aβ expression. Our observation also led to the same conclusion that Aβ expression and neurodegeneration can be independent effects and also highlights the potential role of ceIDE-1, C02G6.2, Y70C5C.1, C05D11.1 genes in neuronal survival. The pathogenesis of AD has been linked to deficiency of neurotransmitter ACh in the brain [46]. Subsequently, AChE inhibitors are used as drugs for the symptomatic treatment of AD [47, 48]. Considering the neuroprotective role displayed by C. elegans putative IDE candidates, we next endeavored to estimate the ACh levels and AChE activity. We observed that the knockdown of ceIDE-1, C02G6.2, C05D11.1 and F44E7.4 resulted in decreased ACh content whereas RNAi induced knockdown of none of the C. elegans IDE candidates altered AChE activity. This result again correlates with the findings of cholinergic neuroanatomy experiments. Later, we also analyzed the ROS content to study the oxidative stress after knocking down the C. elegans putative IDE candidates in wild type strain as increased oxidative stress may have an independent role in the pathogenesis of neuronal death than deposition of Aβ. Studies during recent years have proposed that the death of selective neurons in AD is due to the increased oxidative stress and may have a role in the pathogenesis of neuronal death. The evidence supporting the theory is based on the fact that high level of metals like Fe, Al, and Hg, increases lipid peroxidation. Free radicals are generated either by increased protein and DNA oxidation or by decrease in cytochrome-c oxidase, advanced glycation end products and SOD-1 in brain [23].The ROS level was found to be increased only in ceIDE-1 RNAi group, suggesting that the absence of ceIDE-1 resulted in increased oxidative stress inside the organism. The increased ROS content, which seems to be contrasting to its role in Aβ induced toxicity can be explained by the fact that the ROS was measured in wild type and not in CL4176 transgenic strain as we treated ROS as an independent parameter associated with AD. This observation explains that absence of ceIDE-1 may elevate ROS independently of Aβ. Lastly, we quantified the level of DAF-16, a critical regulator of the stress response. DAF-16 is a key transcription factor required for longevity of C .elegans. It codes for the Human forkhead box O (FOXO) homologue and acts in the insulin/IGF-1-mediated signaling pathway. DAF-16 regulates longevity, stress response, dauer formation, fat metabolism, and innate immunity [24]. Since, AD is an age-related disorder and DAF-16 governs insulin like signaling, we endeavored to study the effect of C. elegans IDE putative gene that is proposed to be a common link between

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AD and T2DM on DAF-16 expression and the result depicts that RNAi of C02G6.1 and C05D11.1 led to an increase in the DAF-16 GFP puncta. Amongst the identified C. elegans putative IDE candidates ceIDE-1, C02G6.2, Y70C5C.1, F44E7.4, C02G6.1, C05D11.1 and mppb-1 the best protective role against the AD parameters was displayed by ceIDE-1. Absence of ceIDE-1 modulated the AD associated conditions by leading to altered Aβ induced toxicity, abolition of the cholinergic neuroanatomy with reduced expression of UNC-17, decrease in ACh content, elevation in ROS levels and no effect on DAF-16 stress response protein. Our results are consistent with the findings hypothesized by performing blast homology search. The E-value predicted by blast homology search for ceIDE-1 against Human IDE sequence was 3e-179. The lowest E-value amongst the entire predicted C. elegans putative IDE candidates was of ceIDE-1. The lower the E-value, or the closer it is to zero, the more is the level of "significance", or, in case of present studies, it is of more relevance to the studied endpoints associated with Alzheimer‟s disease.

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5. Conclusions

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Our studies provide critical insight into the structural/functional orthology of IDEs across the human and nematode species. We identify functionally uncharacterized putative IDE in C. elegans and establish the role of IDE and insulin pathway in AD. Further studies in this direction could aid in identification of novel drug targets and in understanding of the common modulating factors between AD and T2DM. 6. Acknowledgement

This work was funded by CSIR-NWP EpiHeD (BSC0118). RH is supported by Senior Research Fellowship from UGC (Ref. No: 19-06/2011(i)EU-IV). Nematode strains used in the study were provided by Caenorhabditis Genetics Center (CGC), University of Minnesota, MN, USA, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). CSIR-CDRI communication number is XXXX. 7. References 1. 2.

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