Two Human Metabolites Rescue A C. Elegans Model Of Alzheimer’s Disease Via A Cytosolic Unfolded Protein Response Ⅱ

Fig. 3 Carnosine and kynurenic acid prevent Aβ42 toxicity in a cistanche model of AD (GMC). In vivo screening strategy to identify endogenous metabolites that inhibit Aβ42 aggregation is illustrated in (a). We screened six identified candidate metabolites in a cistanche model of Alzheimer’s disease. Metabolites (depicted as M) were fed at the L4 stage of GMC worms and their effects were assessed at day 5 of adulthood through an (i) motility assay that determines the overall fitness of the worms, in terms of changes in motility, quantified as the body bends per minute (BPM) and (ii) quantification of NIAD-4-stained Aβ42 aggregates (screening data shown in Supplementary Fig. 1). Panels b–g show characterization of kynurenic acid and carnosine to rescue a cistanche model of AD. Panels (b and c) show the motility of worms, measured in body bends per minute (BPM) on Y-axis vs. days of adulthood on X-axis, treated with increasing concentrations of carnosine (blue) and kynurenic acid (red) compared to untreated worms (black). Increased thrashing frequency was observed across the lifespan of the worm up to 15 µM of carnosine and kynurenic acid (b and c). The motility at day 5 of adulthood, where phenotypic manifestations of Aβ42 are prominent in the GMC worm, is signifificantly improved by increasing carnosine up to 15 µM and for kynurenic acid at 10 µM (d and e). The radar chart shows the overall fitness of cistanche as a function of speed, bends per minute (BPM), and the live ratio as is seen on each of its axis (d and e). Aggregate staining was quantified after worms were incubated with the amyloidogenic-specific dye NIAD-4 (f–g) (scale bar, 80μm). White arrows in the panels (f–g) point to NIAD-4-stained Aβ42 aggregates, which appear orange-red in color. At all concentrations tested, carnosine treatment signifificantly inhibited Aβ42 aggregation compared to the GMC worms as shown in (f). N2 control worms, which do not express Aβ42, are shown for comparison (scale bar, 80 μm). Similar to carnosine, the aggregation of Aβ42 was inhibited by kynurenic acid treatment as shown in (g). For NIAD-4 screening of aggregates, approximately 15–23 animals were analyzed per condition for GMC (AD) worms and ∼10 animals per control (N2) worms. The beneficial effects of kynurenic acid and carnosine were observed in n = 3 biologically independent experiments. All error bars represent the standard error of the mean (SEM). Statistics were performed using one-way ANOVA, Dunnett’s multiple comparisons against the untreated Aβ42 group using GraphPad Prism, p-values are indicated on the plots.

Fig. 4 Carnosine and kynurenic acid do not have direct effects on Aβ42 aggregation. Kinetic profiles of the aggregation of a 2 μM Aβ42 sample in the absence (black) and the presence of increasing concentrations of carnosine or kynurenic acid (represented in different colors). The aggregation process of Aβ42 is not signifificantly accelerated or retarded by the presence of either of the metabolites (higher concentrations of the metabolites 20, 50, 100, and 500 μM are shown in Supplementary Fig. 3). Error bars are expressed as the standard deviations from three technical replicates.

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Carnosine and kynurenic acid do not directly inhibit Aβ42 aggregation. To test whether carnosine or kynurenic acid could directly inhibit Aβ42 aggregation, we carried out a ThT flfluorescence-binding chemical kinetics assay (see the “Methods” section). We did not observe, however, any direct effects of these two metabolites on the aggregation of Aβ42, at least over the relatively wide range of concentrations that we tested (Fig. 4 and Supplementary Fig. 3), which include physiologically relevant ones, indicating that another mechanism should be present that acts to clear the aggregates. Since the presence of this mechanism does not exclude the possibility that other endogenous metabolites may have a direct effect on Aβ42 aggregation, possibly by forming aggregates themselves or promoting phase separation of Aβ42 within the cytoplasm, we propose that it will be very interesting to test other endogenous metabolites for such a direct effect on protein aggregation.

Effect of Cistanche

Carnosine and kynurenic acid increase levels of HSF-1 and molecular chaperones. We then investigated the effects of carnosine and kynurenic acid on the expression of HSF-1, the master regulator of the HSR in cistanche. HSF-1 upregulates heat shock protein genes that act as molecular chaperones to maintain normal protein conformation under cellular stress, refold misfolded proteins, and target irreversibly damaged proteins for degradation42–44 (Fig. 5 and Supplementary Fig. 5). We used an antibody validated to identify cistanche HSF-1 by immunoprecipitation and mass spectrometry45, to probe for HSF-1 in lysates from worms treated with carnosine or kynurenic acid. We observed increased levels of HSF-1 following treatment with both the metabolites (Fig. 5a). Levels of HSF-1 increased in a dose-dependent manner in response to both carnosine and kynurenic acid treatment (Fig. 5a). In the cistanche lysate treated with the two metabolites, we observed the HSF-1 band at ∼100 kDa. HSF-1 primarily regulates the expression of genes encoding molecular chaperones, particularly the heat shock proteins HSP90, HSP70, and HSP40 that act as a cochaperone for HSP7046–48. We looked at the levels of core HSF-1-regulated chaperones and found that HSP90 and HSP70 levels were elevated in the GMC worms treated with 15 μM kynurenic acid, but not 15 μM carnosine as compared with untreated GMC and wild-type worms (Fig. 5b, c). Both DNJ-12 and DNJ-19 showed elevated levels in response to treatment with carnosine and kynurenic acid as compared to untreated GMC worms and treated wild-type N2 worms (Fig. 5d, e). In contrast, we did not observe signifificant changes in the levels of DNJ-13 upon metabolite treatment (Fig. 5f). DNJ-12 and DNJ-19 are class A J-protein (HSP40) cochaperones that have been characterized in vivo and in vitro for their protein disaggregate functions that promote organismal health48. Jproteins have also been previously characterized to have a disaggregase activity in a cistanche polyQ model, where complexed J-protein co-chaperones of class A (DNJ-12 and DNJ-19) and B (DNJ-13) enable disaggregase activity through associations with HSP-110 and HSP-70, both individually and in a synergistic cooperation48,49. Our data suggest that an increase in HSF-1 levels upon treatment with carnosine and 

kynurenic acid promotes increased levels of molecular chaperones and cochaperones, in particular, DNJ-12 and DNJ-19 to clear Aβ42 aggregates. We then asked whether the changes in protein levels were due to altered transcription by performing real-time quantitative PCR (RTqPCR). No signifificant fold changes in the mRNA levels of hsf- 1, daf-21, and hsp-70 were observed following treatment with either metabolite. However, consistent with our protein data, we observed that the mRNA levels of dnj-12 and dnj-19 were elevated by treatment with both metabolites. Intriguingly, the mRNA levels for dnj-13 were also elevated in carnosine, but not kynurenic acid, treated worms, despite the fact that no signifificant changes were observed at the protein level (Fig. 6a). We further asked if these metabolites triggered UPR related to the ER (UPRER) or mitochondria (UPRmt) by probing for mRNA levels of hsp-4 and hsp-6, the canonical markers of the UPRER and UPRmt, respectively50,51. There were no signifificant changes in these two markers compared with the controls (Fig. 6b), thus excluding these responses as underlying mechanisms for the clearance of aggregates in this study. To determine whether carnosine and kynurenic acid treatment suppress Aβ42 toxicity and aggregation through HSF-1 and

Fig. 5 Carnosine and kynurenic acid activate a cytosolic unfolded protein response through an HSF-1-dependent mechanism. Western blots show a differential increase in protein levels on treatment with the two metabolites (a–f). On treatment with carnosine and kynurenic acid, we observe a relative increase in the protein levels of molecular chaperones and their co-chaperones in worm lysates relative to the controls, untreated N2, and GMC. n = ∼3000 worms per condition; we measured 3–4 replicates per condition; only a representative western blot is shown for each condition. From the experiments described in Fig. 3, a signifificant increase in motility and a corresponding decrease in the NIAD-4-stained aggregates were observed at a dose of 15 μM of both metabolites. All error bars depict the standard error of mean (SEM). a–f show bands of HSF-1, downstream molecular chaperones, and J proteins namely HSP-90, HSP-70, DNJ-12, DNJ-19, and DNJ-13, respectively, for both N2 (wild type) and GMC (AD model) worms. For each Western blot experiment, we used Tubulin signal to normalize for total protein concentration in each lane. We then normalized each condition of the chaperone/co-chaperone band with its corresponding tubulin band from the same experiment run on a parallel gel, to plot the intensities in ImageJ. The gels for each condition were run, respectively, at the same time, using the same running buffer, in the same electrophoretic cell and the same western blot transfer sandwich onto membranes. These membranes were further developed using appropriate antibodies (see the “Methods“ section, Supplementary Fig. 5). Statistics are performed in GraphPad Prism using ordinary one-way ANOVA; we used Dunnett’s multiple comparisons test with untreated (H2O) for each N2 and GMC metabolite-treated groups; p-values are indicated in the figure sub-panels.

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