Patent Application
20220008449
The following specification particularly describes the invention and the manner in which it is to be performed
The present disclosure is generally related to the field of molecular biology. Specifically, the present disclosure provides a method of increasing lifespan by restricting diet which improves proteostasis in a subject. More specifically, the present disclosure provides a method of inducing an early and transient upregulation of the Unfolded Protein response (UPRER) in a subject by either generating a nutrient restriction or administering pharmaceutical reagent. Also, the present disclosure provides a method of treating protein folding disorders.
A vast majority of the secreted as well as membrane proteins fold and mature in the endoplasmic reticulum (ER) before they are exported to their destinations. The protein folding capacity of the ER is carefully monitored and calibrated by three conserved signal transduction pathways, collectively called the Unfolded Protein Response (UPRER) regulators, that ensure protein homeostasis (proteostasis) and proper cellular function. This is achieved by activating the proteostasis network (PN) consisting of molecular chaperons, protein degradation machinery and stress response pathways that act to resolve consequences of protein misfolding in the ER. In metazoan ER, the three arms of UPRER, namely IRE1, PERK and ATF6 function in parallel to trigger the PN and counteract ER stress by a) increasing folding capacity through the expression of various chaperons, b) attenuating translation to reduce protein load in ER, c) regulated IRE1-dependent decay of mRNA (RIDD), or d) activating ER-associated proteasomal degradation (ERAD) to remove terminally misfolded proteins.
Aging is characterized by a catastrophic collapse of the proteostasis network due to the loss of protein quality control machineries across the cellular compartments. With age, the ER structure begins to deteriorate, and the ER health fails as it is unable to mount an optimal UPRER.
Expression levels of key ER mRNA and proteins as well as activities of ER resident proteins, including BiP, PDI, calnexin and GRP94 are known to decline with age. As a possible consequence, many diseases of protein misfolding including Alzheimer's, Parkinson's and Huntington's diseases have an age-onset.
The flagging cellular proteostasis can be improved simply by exposing cells or organisms to moderate stress before they encounter acute stress. This process is called hormesis and has been shown to be beneficial to life span and health.
Hormesis can be affected in a compartment-specific manner in a cell. For example, in C. elegans, Drosophila and human fibroblasts, hormetic heat shock upregulates cytosolic molecular chaperones that further protect against acute heat stress. In C. elegans, glucose restriction increases mitochondrial respiration and ROS production, which results in protection against oxidative stress and enhanced longevity, a process termed mitohormesis. Mitohormesis has also been implicated in other long-lived mutants in worms and fly. Similarly, ER hormesis has been observed in Drosophila carrying mutations associated with ER protein folding/degradation machinery that induce cytoprotective response towards subsequent ER insults. In mammalian cells, ER stress preconditioning protects against brain ischemia and attenuates heart ischemia/reperfusion injury. ER preconditioning is also neuroprotective in Drosophila and mice models of Parkinson's disease. The mutation in the C. elegans heterochromatin protein like-2 (HPL-2), induces hormetic induction of stress resistance dependent on the IRE-1 branch of UPRER. Further, expression of xbp-1s in neurons has been shown to increase longevity in a cell non-autonomous manner, suggesting a possible role of ER hormesis in life span regulation.
Dietary Restriction (DR) is a conserved intervention that can delay proteostasis, activate cytoprotective pathways and increase life span across species. A wealth of evidence suggests that dietary restriction may act as a mild stress that enhances lifespan through hormesis. The fact that glucose restriction that is akin to DR increases life span through mitohormesis attests to this. However, the involvement of ER hormesis in DR is not known.
The present invention shows that dietary restriction transiently upregulates UPRER during early larval development. Mimicking this response with a pharmacological glycosylation inhibitor, tunicamycin or a non-hydrolysable glucose analog, increases life span. Both transient UPRER and increased life span depend on the ER stress sensor, IRE-1.
The present application shows that the transient ER stress leads to better iUPRER at adulthood and better proteostasis. This hormetic dose of ER stress during development upregulates ERAD genes leading to efficient degradation of ER resident proteins and thus, contributes towards enhanced proteostasis.
The present disclosure is related to a method of inducing an early and transient upregulation of Unfolded Protein Response (UPRER) in a subject by either generating a nutrient restriction or administering pharmaceutical reagent, wherein said method mimics the pro-longevity effects of the dietary restrictions (DR). It is shown in the present invention that a transient pharmacological ER stress, if imposed early in development on Caenorhabditis elegans, enhances proteostasis, prevents iUPRER decline with age, and increases adult life span Importantly, dietary restriction (DR), that has a conserved positive effect on life span, employs this mechanism of ER hormesis for longevity assurance.
The disclosure may be more fully understood by reading the following detailed description of the embodiment, with reference made to the accompanying drawings as follows:
FIG. S (supplementary) 1: (A) Representative images of Phsp-4:gfp(zcIs4) worms, grown on control or drl-1 RNAi, at 32 hours post-bleaching. Densitometric quantification averaged over 2 biological repeats, shows the transient upregulation of basal UPRER. Error bars-SEM, *p<0.05 as determined by student's t test. (B) Representative images of Phsp-4:gfp(zcIs4) worms growing on control or drl-1 RNAi at indicated larval stages and in gravid adults. Densitometric quantification showing drl-1 knockdown worms having no increase in basal UPRER response as compared to control RNAi in these stages. Basal UPRER is lower in drl-1 RNAi worms at gravid adult stage. Average of 2 biological replicates. Error bars are SD. *p<0.05 by student's t test. (C) drl-1 knockdown does not affect cytosolic heat shock response (HSR; upper panel). Representative images of Phsp-16.2:gfp(dvIs70) worms, representing HSR, grown on control or drl-1 RNAi at 32 hours post-bleaching. (lower panel) drl-1 knockdown does not affect mitochondrial stress response (lower panel). Representative images of Phsp-6:gfp(zcIs9) worms, representing UPRmt, grown on control or drl-1 RNAi at 32 hours post-bleaching. Densitometric quantification averaged over 2 biological repeats do not show any difference between drl-1 knockdown and control RNAi, ns-not significant. (D) UPRER up-regulation in L2 larval stage on drl-1 knockdown is dependent on ire-1. Representative images of Phsp-4:gfp(zcIs4) and ire-1(zc14);Phsp-4:gfp worms growing on control or drl-1 RNAi at 32 hours post-bleaching. Densitometric quantification of GFP fluorescence averaged over 2 biological repeats is shown in the lower panel. *p<0.05 as determined by Two-way Annova.
At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.
Throughout the description, the phrases “comprise” and “contain” and variations of them mean “including but not limited to”, and are not intended to exclude other moieties, additives, components, integers or steps. Thus, the singular encompasses the plural unless the context otherwise requires. Wherever there is an indefinite article used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification including any accompanying claims, abstract and drawings or any parts thereof, or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation.
Unless contraindicated or noted otherwise, throughout this specification, the terms “a” and “an” mean one or more, and the term “or” means and/or.
Unfolded protein response (UPR) of the endoplasmic reticulum (UPRER) helps maintain proteostasis in the cell. The ability to mount an effective UPR' to external stress (iUPRER) decreases with age and is linked to the pathophysiology of multiple age-related disorders. The inventors in the present disclosure have shown that a transient pharmacological ER stress, imposed early in development on Caenorhabditis elegans, enhances proteostasis, prevents iUPRER decline with age, and increases adult life span. Importantly, dietary restriction (DR), that has a conserved positive effect on life span, employs this mechanism of ER hormesis for longevity assurance.
The present inventors found that only the IRE-1-XBP-1 branch of UPRER is required for the longevity effects, resulting in increased ER-associated degradation (ERAD) gene expression and degradation of ER resident proteins during DR. Further, both ER hormesis and DR protect against polyglutamine aggregation in an IRE-1-dependent manner. It has been shown that the DR-specific FOXA transcription factor PHA-4 transcriptionally regulates the genes required for ER homeostasis and is required for ER preconditioning-induced life span extension. Together, the present invention provides a mechanism by which DR offers its benefits and opens the possibility of using ER-targeted pharmacological interventions to mimic the prolongevity effects of DR.
The endoplasmic reticulum (ER) deteriorates with age and fails to mount an effective stress response against misfolded proteins (UPRER), leading to protein folding disorders. However, preconditioning the ER using a mild ER stress (ER hormesis) can protect against future insults.
It has been shows that dietary restriction is an intervention that protects against protein misfolding disorders and increases life span across species, uses ER hormesis as a mechanism of action. Mimicking the ER hormesis in Caenorhabditis elegans by transient treatment with pharmacological reagent leads to delayed age-onset failure of UPRER, better capacity to process misfolded proteins, and increased life span.
In one embodiment, the present invention provides a method of inducing an early and transient upregulation of Unfolded Protein Response (UPRER) in a subject by either generating a nutrient restriction or administering pharmaceutical reagent, wherein said method mimics the pro-longevity effects of the dietary restriction (DR).
In another embodiment, said UPRER is upregulated in early stage of the life cycle of the subject.
In yet another embodiment, said UPRER is endoplasmic reticulum specific.
In an embodiment, the nutrient restriction is transient glucose deprivation.
In another embodiment, said pharmaceutical reagent is tunicamycin or 2-deoxyglucose.
In one embodiment, the present invention provides a method, wherein the mimicking effect of the dietary restrictions (DR) includes the following mechanism:
In another embodiment, wherein the mimicking effect of the dietary restrictions (DR) increases the ability of the subject to mount an efficient UPRER during adulthood when challenged with an acute dose of tunicamycin or 2-deoxy glucose.
In yet another embodiment, the mimicking further includes a DR-specific transcription FOXA factor (PHA-4) for the transient UPRER during development as well as the upregulation of ERAD genes in adulthood in subjects undergoing DR.
In one embodiment, the present invention provides the method of treating protein mis-folding disorders and increasing the life span of a subject by applying the method as defined in the present specification.
In yet another embodiment, the present invention provide a pharmaceutical reagent or dietary restriction for inducing an early and transient upregulation of Unfolded Protein Response (UPRER) in a subject.
In an embodiment, the present invention provides use of pharmaceutical reagent or dietary restriction for inducing an early and transient upregulation of Unfolded Protein Response (UPRER) in a subject.
In a further embodiment, the present invention provides a kit claim comprising a pharmaceutical reagent and instruction manual for performing the method of inducing an early and transient upregulation of Unfolded Protein Response (UPRER) in a subject.
The following examples serve to illustrate certain embodiments and aspects of the present disclosure and are not to be considered as limiting the scope thereof.
Material and Methods:
Detailed Experimental Procedures
C. elegans Strain Maintenance
Unless otherwise mentioned, all the C. elegans strains were maintained and propagated at 20° C. on E. coli OP50 using standard procedures. The strains used in this study were: N2 Bristol (wild-type), eat-2(ad1116)II, Phsp-4:gfp(zcIs4), rrf-3(pk1426)II, ire-1(v33)II, atf-6(ok551)X, pek-1(ok275)X, ire-1(ok799)II, xbp-1(zc12)III, xbp-1(tm2457)III, xbp-1(tm2482)III, hsp-4(gk514)II, Phsp-16.2;gfp(dvIS70), Phsp-6;gfp(zcIs9), ire-1(zc14)II;Phsp-4:gfp, rrf-3(pk1426)II, Punc-54:Q40:yfp(rmIs133). The above-mentioned strains were obtained from Caenorhabditis Genetics Centre, University of Minnesota, USA. The other strains including eat-2(ad1116)II;Phsp-4:gfp(zcIs4), eat-2(ad1116)II; rrf-3(pk1426)II, pek-1(ok275)X;Phsp-4:gfp(zcIs4), ire-1(v33)II;Punc-54:Q40:yfp(rmIs133) and eat-2(ad1116)II;Punc-54:Q40:yfp(rmIs133) were generated in-house using standard mating techniques.
Preparation of RNAi Plates
RNAi plates were poured using autoclaved (NGM) nematode growth medium supplemented with 100 μg/ml ampicillin and 2 mM IPTG. Plates were dried at room temperature for 1-2 days. Bacterial culture harbouring RNAi construct was grown in Luria Bertani (LB) media supplemented with 100 μg/ml ampicillin and 12.5 μg/ml tetracycline, overnight at 37° C. in a shaker incubator. Saturated cultures were re-inoculated the next day in fresh LB media containing 100 μg/ml ampicillin by using 1/100th volume of the primary inoculum and grown in 37° C. shaker until OD600 reached 0.5-0.6. The bacterial cells were pelleted down by centrifuging the culture at 5000 r.p.m for 10 minutes at 4° C. and resuspended in 1/10th volume of M9 buffer containing 100 μg/ml ampicillin and 1 mM IPTG. Around 350 μl of this resuspension was seeded onto RNAi plates and left at room temperature for 2 days for drying, followed by storage at 4° C. till further use.
Pellet from Part A was resuspended in 1×M9 buffer containing 100 μg/ml ampicillin and 1 mM IPTG to ½ of its volume (25 ml, if the initial culture volume of Part A was 50 ml). This resuspended bacterial culture was used as feed during the Tm treatment.
Hypochlorite Treatment to Obtain Eggs and Synchronizing Worm Population
Gravid adult worms, initially grown on E. coli OP50 bacteria were collected using M9 buffer in a 15 ml falcon tube. Worms were washed thrice by first centrifuging at 1800 r.p.m for 60 seconds followed by resuspension of the worm pellet in 1×M9 buffer. After the third wash, worm pellet was resuspended in 3.5 ml of 1×M9 buffer and 0.5 ml 5N NaOH and 1 ml of Sodium hypochlorite solution were added. The mixture was vortexed for 7-10 minutes until the entire worm bodies dissolved, leaving behind the eggs. The eggs were washed 5-6 times, by first centrifuging at 2500 r.p.m, decanting the 1×M9, followed by resuspension in 1×M9 buffer to remove traces of bleach and alkali. After the final wash, eggs were resuspended in approximately 100-200 μl of M9 and added to different RNAi plates
RNAi Life Span
Gravid adult worms, initially grown on E. coli OP50 were bleached and eggs were hatched on different RNAi plates. On reaching adulthood, 50-60 young adult worms were transferred to the similar RNAi plates in triplicates and overlaid with Fluoro-deoxyuridine (FudR) to final concentration of 0.1 mg/ml of agar (2). At the 7th Day of adulthood, sick, sluggish and slow dwelling worms were removed from the life span population and the remaining were considered as the number of subjects ‘N’. Following this, number of dead worms were scored every alternate day and plotted as % survival against the number of days. Statistical analysis for survival was conducted using Mantel-Cox Log Rank test using Oasis software. Average life span was also determined using the same method and represented as Mean life span±Standard Error Mean (S.E.M).
Measurement of Basal UPRER During Larval Development
Transgenic worms expressing GFP under hsp-4 (mammalian ortholog GRP78/Bip) promoter [Phsp-4:gfp(zcIs4) and eat-2(ad1116);Phsp-4:gfp(zcIs4] were bleached and eggs were hatched on control RNAi. Fifty L3, L4, young adult or Day 1 gravid adult worms were immobilized on glass slides coated with 2% agarose using 20 mM sodium azide and visualized under Axio-imager M2 epifluorescent microscope (Carl Zeiss, Germany) equipped with a monochromatic camera lens (MRm) and GFP filter set. Fluorescence of ≥20 worms at different time points was quantified using NIH Image J software and represented as arbitrary units (AU). Basal UPRER in other strains like ire-1(zc14);Phsp-4:gfp(zcIs4) and on different RNAi like ire-1 or drl-1 was measured similarly.
Measurement of Induced UPRER Efficiency with Age
Transgenic Phsp-4:gfp(zcIs4) and eat-2(ad1116);Phsp-4:gfp(zcIs4) were bleached and eggs were hatched on control RNAi. Worms were allowed to grow till adulthood and then transferred onto plates overlaid with FuDR to a final concentration of 0.1 mg/ml. At each successive day (day 1 till day 4 of adulthood), approximately 100 worms were transferred to plates supplemented with 5 or 10 μg/ml tunicamycin (Tm) and incubated for 6 hours at 20° C. Since Tm is dissolved in 0.05% DMSO, it was used as vehicle control. After 6 hours, 50 worms from each treatment were mounted onto 2% agarose slides and visualized using Axio-Imager M2 epifluorescent microscope with a GFP filter set (Carl Zeiss, Germany) Fluorescence of ≥20 Tm-treated and untreated worms was quantified using NIH ImageJ software. Average fluorescence of treated worms was normalized to untreated worms and plotted as normalized GFP fold induction at different days of adulthood. Similar procedure was followed to compare iUPRER efficiency of Phsp-4:gfp(zcIs4) on control and drl-1 RNAi on different days of adulthood.
Tunicamycin Hormesis Treatment
Preparation of Feed:
Overnight grown bacterial culture expressing Control or test RNAi were re-inoculated in 200 ml fresh LB media and grown at 37° C. until OD600 reached 0.5-0.6. The culture was divided in two parts (A and B) in a ratio of 1:3 and cells are pelleted separately using a centrifuge (4810R, Eppendorf, Germany) at 5000 r.p.m for 10 minutes at 4° C. Pellet from part B (150 ml) of the culture was resuspended in 1×M9 buffer containing 100 μg/ml ampicillin and 1 mM IPTG to 1/10th of its initial culture volume (for 150 ml culture, 15 ml of resuspension buffer was used) and 350 μl was seeded onto each RNAi plate and left for drying at room temperature for 24 hours.
Preparation of Worms
Gravid adult worms initially grown on E. coli OP50 bacteria were bleached and eggs were re-suspended in 100-200 ul of 1×M9 buffer and standardized for the number of eggs present in a particular volume.
Preparation of the Cocktail
An intermediate stock solution of Tunicamycin (10 μg/ml) was prepared in MQ using a stock of 25 mg/ml (Sigma Aldrich, USA). In the final reaction mixture, this was diluted so as to treat the eggs with a variety of Tm concentrations ranging from 0.062 to 1 μg/ml. Entire composition of cocktail (total volume 1 ml) is mentioned as follows:
Eggs were left on rotation in this cocktail at a slow speed for 24 hrs. Following this, the hatched L1 larvae were washed 3-4 times with 1×M9 buffer to remove traces of tunicamycin and added onto seeded control RNAi or test RNAi plates containing no reagent. They were allowed to grow till adulthood when 100 worms belonging to each treatment regime were transferred onto FuDR containing RNAi plates and life span was scored as mentioned above. iUPRER efficiency was also measured on Day 2 of adulthood, as mentioned previously.
DAVID Analysis
The RNA-seq data for synchronised Day1 adult worms [WT and eat-2(ad1116)] grown on E. coli OP50 (submitted to NCBI with the BioProject ID PRJNA342407) was analyzed using DAVID functional annotation tool. The listed pathways have p<0.05 and FDR<10 (Bonferroni Corrected p-value—0.0011).
ChIP-Seq Data Analysis
The ChIP-seq data analysis was performed using parameters mentioned in Singh A, et al. (2016) [A chromatin modifier integrates insulin/IGF-1 signalling and dietary restriction to regulate longevity. Aging Cell 15(4):694-705]. PHA-4 ChIP-seq data (SRA-NCBI GSE50301) was downloaded from modENCODE in .sra format. Downloaded data was converted into fastq format using NCBI-recommended SRA toolkit (version 2.2.2a). Converted fastq of replicates were merged and used for further analysis. Reads were aligned to the C. elegans genome (WS230) using Bowtie (v0.12.7) with the following parameters: -q -m 1 --best --strata. Mapped reads were used for peak calling and calculation of read density. Enriched peaks were identified using the peak calling algorithm MACS (v1.4.2) using following parameters: --mfold=5,30 --bw=175 -w. Statistically significant peaks (P<1×10-5) were used for further analysis. To find target genes, PeakAnalyzer (v1.4) program was used and all genes having peaks within 2 kb of the promoter region were considered for further analysis. UCSC genome browser was used for visualizing aligned data as wig files.
DR Triggers a Transient Up-Regulation of UPRER During Early Larval Development
In order to elucidate the kinetics of UPRER during nutrient stress, the levels of UPRER in the two genetic paradigms of DR was evaluated, namely eat-2(ad1116) and drl-1 RNAi. For this, Phsp-4:gfp(zcIs4) transgenic strain was used, where the promoter of hsp-4 was transcriptionally fused to gfp and GFP fluorescence gives a quantitative readout of UPRER. The GFP fluorescence in Phsp-4:gfp(zcIs4) and eat-2(ad1116);Phsp-4:gfp(zcIs4) at 32-35 hours post-bleaching was compared and it was found that DR triggered a transient up-regulation of UPRER specifically during the L2 larval stage of eat-2(ad1116) (
In the present invention media supplemented with 2% glucose was used and Phsp-4:gfp(zcIs4) or eat-2(ad1116);Phsp-4:gfp(zcIs4) worms are grown on the same. It was observed that the transient UPRER upregulation is mitigated on glucose-containing media in eat-2(ad1116) (
Life Span Extension by DR is Dependent on IRE-1
Knocking down the eat-2 gene or implementing BDR has been shown to decrease proteotoxicity and enhance longevity in adult worms. Since in higher eukaryotes, ER is responsible for folding around 70% of the proteome, the importance of the UPRER machinery in proteo-protective, longevity benefits conferred by DR has been investigated in the present invention. Towards this end, life span analysis was performed in two genetic mimics of DR, in the presence or absence of ire-1, atf-6 or pek-1, the ER membrane proteins that function to sense misfolded protein stress. Interestingly, knocking down ire-1 led to a significant suppression in life span of eat-2(ad1116) (
Early and Transient Up-Regulation of UPRER is Sufficient for Life Span Extension
Since transient up-regulation of UPRER was observed, experiments were conducted to see its causal role in life span extension, as observed during DR. For this, the transient UPRER up-regulation was mimicked through an external supplementation of a small dose of Tunicamycin (Tm) for 24 hrs during hatching of the eggs in liquid culture. After the pharmaceutical reagent was washed off, the worms were grown on solid NGM media and post-adult life span was recorded. Interestingly, it was found that exposing WT larvae to 0.125 μg/ml of Tm during the first 24 hours of its post-embryonic life led to significant extension of life span (
DR and a Hormetic Dose of ER Stress Delays the Age-Related Decline in iUPRER Efficiency
The efficiency of the ER stress response decreases with age, resulting in a progressively dysfunctional ER. Not only the levels of various UPRER target genes decrease in old rats and mice, the efficiency of mounting UPRER in response to various insults also decrease with age. To test whether the iUPRER efficiency in maintained in the genetic models of DR on different days of adulthood, this experiment was conducted. Also, a decline in iUPRER efficiency on the second and third day of adulthood in WT worms, respectively [
Since it was observed that transient ER stress using Tm at early post-embryonic stage increased life span, it was tested whether this treatment could also lead to a better iUPRER efficiency with age. Towards this end, the iUPRER efficiency of transiently Tm-treated worms on day 2 of adulthood was checked and it was found that these worms displayed higher iUPRER efficiency (
DR Increases the Expression of Genes Responsible for Protein Homeostasis within ER
The data obtained suggests that DR may help to partly combat the proteostasis collapse that occurs within the first two days of adult life, through a hormetic upregulation of UPRER during development. In an effort to better understand this process during DR, the changes in the global transcriptome profile of wild-type and eat-2(ad1116) at day 1 of adulthood was assessed). It was determined that the Gene-Ontology terms and KEGG pathways for the genes that were up regulated >2 folds (p-value≤0.05) in eat-2(ad1116) mutant as compared to WT and it was found that genes associated with protein folding functions within the ER were significantly enriched in this set (
The quantitative real-time PCR (QRT-PCR) analysis was performed to validate the sequencing results. There was an increase in the mRNA levels in five of the UPR target genes; sel-1,sel-11, ufd-2, pdr-1 and dnj-1 in eat-2 mutant worms as compared to WT (
This increase in expression starts early in development in eat-2 mutant worms undergoing DR and is sustained till adulthood (
ERAD in DR Worms Efficiently Degrades ER Proteins
ERAD is an adaptive mechanism of the ER that regulates protein homeostasis within the organelle. It involves degradation of the misfolded proteins so as to prevent their accumulation inside the ER lumen. In the DR transcriptome mentioned above, genes pertaining to ERAD pathway formed almost half of the enriched ER gene set (19 out of 41 up-regulated transcripts) (
Life Span Extension by DR is Dependent on ERAD Genes
Upon finding evidence of an up-regulated ERAD during DR, experiments were conducted to see whether it is required for promoting increased longevity. In this regard a life span analysis of WT and eat-2(ad1116) worms was performed on bacteria expressing RNAi against two of the up-regulated ERAD genes, ufd-2 and sel-1. SEL-1 (orthologue of mammalian HRD3) is a vital component of HRD-3/HRD-1 E3 ubiquitin ligase complex while UFD-2 functions in complex with CDC48 to regulate the length of polyubiquitin chains attached to proteins. It was found that life span extension of eat-2(ad1116) was partially suppressed on knocking down ufd-2 or sel-1 (
FOXA Transcription Factor PHA-4 Controls Important Aspects of ER Homeostasis.
The C. elegans FOXA transcription factor PHA-4 is required for increased life span that is observed in multiple genetic as well as nongenetic paradigms of DR. Hence, the inventor tested whether up-regulation of the ER protein processing genes in an eat-2 mutant worm is dependent on PHA-4.
Using metadata analysis of published PHA-4 ChIP-seq data, it was found that PHA-4 binds to the promoter proximal sites of all of the 5 ER protein processing genes that are up-regulated in eat-2(ad1116) (
DR Alleviates Polyglutamine Aggregation in an Ire-1 and ERAD-Dependent Manner
In the context of protein homeostasis, a better health-span will translate into fewer deleterious proteins accumulating or aggregating within the cell. Proteins that carry a stretch of polyglutamine residue over a threshold length are known to aggregate with age. These form the basis for various polyglutamine expansion disorders like Huntington's and Spinocerebellar ataxias. The exposed hydrophobic patches formed by multiple glutamine residues, typically more than 35, increases the chances of protein aggregation. Inefficient degradation of these proteins with increasing age exacerbates aggregation and associated diseases. Supporting this, is the observation that impaired ERAD and ER stress are primary events that augment polyglutamine toxicity. C. elegans is used as a model organism to study polyglutamine aggregation owing to the availability of fluorescent reporters that help in visualizing aggregate formation in different tissues, in real time.
In order to evaluate the role of DR in augmenting the ERAD and its physiological consequences, a reporter was used that expresses a stretch of 40 glutamines (Q40) tagged with a yellow fluorescent protein in C. elegans muscle. This reporter strain was crossed with eat-2(ad1116) mutant worms to generate worms carrying both the reporter transgene and the eat-2 mutation (hereafter, referred to as eat-2;PolyQ). It was observed that incorporating eat-2 mutation led to 60-80% suppression in the number of aggregates at all age points, supporting the earlier observation of alleviation in the age-associated increase in PolyQ aggregation during DR (
Finally, to further evaluate the role of ERAD machinery in DR-induced suppression of polyglutamine aggregation, eat-2;PolyQ worms were grown on control, ufd-2 or sel-1 RNAi. it was found that eat-2(ad1116) worms grown on ERAD gene RNAi had significantly less suppression of PolyQ puncta compared to control RNAi-grown worms (
This suggests that DR leads to a healthier ER that reduces polyglutamine aggregation in adult worms in a manner dependent on ire-1 and ERAD.
A Hormetic Dose of ER Stress, During Early Life, Up-Regulates ERAD and Suppresses polyQ Aggregation
It was reported that an atypical up-regulation of UPRER in larvae contributes towards increased life span during DR. It was shown that pharmaceutically mimicking the transient UPRER up-regulation during early larval life augmented the iUPRER efficiency with age and increased life span. To determine whether such hormetic intervention can protect against the age-related proteostasis collapse, WT worms were exposed with a range of concentrations of Tm only during the start of larval development, as mentioned before. The untreated and Tm-treated PolyQ:yfp worms were compared for the number of aggregates on different days of adulthood and witnessed a suppression in the treated worms (
| Number | Date | Country | Kind |
|---|---|---|---|
| 201811047507 | Dec 2018 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2019/050917 | 12/13/2019 | WO | 00 |
