CBD induces neuronal autophagy
Autophagy eliminates defective cellular molecules via lysosome-mediated degradation during aging. Compromised autophagy is a hallmark of aging . We measured autophagy during C. elegans aging with or without CBD treatment. The binding of the autophagy receptors is facilitated by ATG8/LC3 family proteins. LGG-1 is the C. elegans ortholog of mammalian LC3 and yeast ATG8. We first examined the induction of autophagy after CBD treatment using transgenic worms expressing GFP-tagged LGG-1, an autophagy reporter, visualizes autophagic structures as fluorescent punctae . The number of APs was measured by counting GFP::LGG-1 positive punctae in nerve-ring neurons, intestine, body-wall muscle, and the pharynx . During normal aging without CBD treatment, the nerve-ring neuron was the only tissue among all measured tissues, significantly reducing APs from day 1 to day 5 (− 44.28%, p < 0.001). One-day CBD treatment significantly increased APs in nerve-ring neurons (34.14%, p < 0.001) but not any other tissues compared with the controls (Fig. 1AD). Furthermore, a 5-day CBD treatment significantly increased APs in nerve-ring neurons, body-wall muscle, and pharynx (78.25%, 44.92%, 59.48%, all p < 0.01), but not intestine, compared with controls (Fig. 1AD).
To further validate the neuronal effect, we treated SH-SY5Y neurons with CBD. The result showed that CBD modulated the autophagy in SH-SY5Y neurons in a time-dependent manner. It is that CBD for 48 h increased LC3-II/LC3-I (53.19%, p < 0.05, Fig. 1E and F) but decreased SQSTM1 (− 34.22%, p < 0.01, Fig. 1E and F), indicating that CBD induces autophagy in SH-SY5Y neurons. Similarly, the mouse primary hippocampal neurons showed activated autophagy after CBD treatment (Fig. 1G). Hippocampal neurons treated with CBD showed increased LC3 intensity compared with the control untreated group (24.1%, p < 0.05, Fig. 1H). In addition, CBD treatment led to increased co-localization of LC3 and LAMP1, lysosomal membrane, in primary hippocampal neurons (86.9%, p < 0.001, Fig. 1I). These results based on in vivo and in vitro studies indicate that CBD induces neuronal autophagy.
CBD promotes autophagic flux in neurons
Autophagic flux is an indication of autophagy completion, including autophagosome and autophagolysosome formation. Autophagic flux was detected by dual-florescent mCherr::GFP::LGG-1 reporter monitors for APs and autolysosome (AL) formation . GFP and mCherry have different sensitivities to the acidic pH of the lysosome, which enables ALs (mCherry-only puncta) to be distinguished from APs (GFP and mCherry positive puncta, which appear yellow) (Fig. 2A). We used transgenic worms expressing mCherry::GFP::LGG-1 from a pan-neuronal rgef-1 promoter to specifically visualize autophagic flux in neurons (Fig. 2B). Interestingly, the nerve-ring neurons showed an age-associated decrease in the number of APs (− 69.76%, p < 0.001) and ALs (− 77.31%, p < 0.001) when assessed with the mCherry::GFP::LGG-1 reporter (Fig. 2C and D). However, more APs and ALs were observed in the nerve-ring neurons after CBD treatment compared with control worms on day 7 (AP: 91.1%, p < 0.05; AL: 106.34%, p < 0.001; Fig. 2C and D).
To further validate autophagic flux in neurons, we monitored SH-SY5Y cells transfected with mCherry-GFP-LC3 plasmid using the IncuCyte live imaging system, similar to the experiment performed above in C. elegans. To demonstrate the capacity of this system to monitor autophagic flux, we performed two control treatments. Rapamycin treatment was used as a positive control; more red puncta are observed in the presence of rapamycin because of the increased fusion of autophagosomes with lysosomes. Meanwhile, inhibition of autophagic flux using chloroquine (CQ) treatment, which blocks the binding of autophagosomes to lysosomes, showed more yellow puncta (Fig. 2E). Similar to the effect of rapamycin treatment, CBD treatment led to the observation of more red puncta than yellow puncta, suggesting that CBD promotes autophagic flux (200.94%, p < 0.05, Fig. 2F). These findings indicate that autophagic flux decreased during aging in the neurons of C. elegans. However, CBD promotes autophagic flux in neurons.
CBD-mediated longevity requires autophagy integrity
The rate of autophagic flux decreases with advancing age and a lifespan extension in C. elegans [33, 34]. The autophagy genes are the basic requirement for autophagic flux. In worms, bec-1 is the ortholog of the yeast and mammalian autophagy proteins Atg6/Vps30 and Beclin 1, which interacts with the class III PI3 kinase vps-34, an essential protein required for autophagy, membrane trafficking, and endocytosis . Autophagy can degrade cargos with the help of selective autophagy receptors such as p62/SQSTM1 . The lifespan was significantly increased in C. elegans treated with 1 μM (p < 0.001, Fig. 3A, Table S2) and also 5 and 10 μM CBD (both p < 0.01, Fig. S1A and B). To further understand the effects of CBD on the crosstalk between autophagy and longevity, we knocked down autophagy genes sqst-1, vps-34, and bec-1 by RNAi and assessed lifespan in these experimental groups. We found that bec-1 or vps-34 RNAi treatment significantly shortened lifespan compared with the control (empty vector, EV) RNAi group (all p < 0.01). Furthermore, CBD treatment failed to extend lifespan in bec-1 and vps-34 RNAi groups (Fig. 3B and C). When we performed sqst-1 RNAi, this did not affect lifespan compared with the EV RNAi group; however, CBD failed to extend lifespan in the sqst-1 RNAi group (Fig. 3D). These results suggest that bec-1 and vps-34, which induce autophagic vesicle nucleation, were required for a normal lifespan while sqst-1 was not. All essential autophagy genes (bec-1, vps-34, and sqst-1) were required for the lifespan increase mediated by CBD treatment.
CBD improves healthspan and neuronal morphology associated with aging in C. elegans
Aging can lead to poor health, movement retardation, and neuronal degeneration . The life quality of aging is equally important as living longer. Whether or not CBD improves healthspan is not known. The healthspan indicator used in this study includes pharyngeal pumping rate, hermaphrodite reproductive capacity, and locomotion in C. elegans as per previous studies [38, 39]. The declines of these parameters cause a decline in survival probability . CBD treatment significantly increased the pumping rate on days 3 and 5 compared to the controls (day 3: 34.95%, p < 0.001; day 5: 53.10%, p < 0.001; Fig. 4A). Substantially, more eggs were laid on day 5 of C. elegans treated with CBD compared with controls (266.67%, p < 0.01, Fig. S1C), although there was no difference between the two groups on day 1 (Fig. S1C). Total brood size (the number of progeny) was increased after treatment with CBD (11.89%, p < 0.05, Fig. 4B). Body movements as the body bend in 20 s were declined with increasing age in C. elegans measured on days 1, 3, 5, 7, 9, and 11 of adulthood (day 3: 0.3%; day 5: 7.6%; day 7: 27.20%; day 9: 36.77%; day 11: 45.89%, compared with day 1; Fig. S1D). Overall, CBD treatment increased body bends over 11 days, with day 7 showing a significant increase (24.72%, p < 0.05, Fig. S1D).
A feature of neuronal aging is morphological changes, which are progressive changes associated with brain function alteration [41,42,43]. The age-associated neuronal morphological changes are frequently studied in C. elegans including branching, beading, and blebbing in the anterior and posterior touch receptor neurons (ALM, PLM), which are also referred to as “defective neurons.” Using a transgenic line expressing a TRN-specific mec-4p::gfp transgene, we measured the morphological changes in ALM and PLM neurons in young and aged C. elegans (Fig. 4C,D, and F). In ALM neurons, C. elegans significantly increased the number of irregularly shaped soma on day 8 compared with day 1 (179.04%, p < 0.001, Fig. 4E). CBD treatment significantly reduced these changes on both days 1 and 8 compared with controls (day 1: 22.36%, p < 0.05; day 8: 12.97%, p < 0.001; Fig. 4E). In PLM neurons, C. elegans significantly increased the branching, beading, and blebbing on day 8 compared to day 1 (Fig. 4G). CBD treatment for 8 days significantly reduced the proportion of defective PLM processes (65.09%, p < 0.001, Fig. 4G). The results suggest that CBD slows down neuronal aging.
CBD requires autophagy to protect age-associated neuronal morphological changes
Since autophagy genes (bec-1, vps-34, and sqst-1) were required for CBD-mediated longevity, we investigated whether autophagy processes were involved in regulating age-associated morphological changes. In young (day 1 adult) worms in which key autophagy genes (bec-1, vps-34, and sqst-1) were knocked down with RNAi, no significant changes in ALM defects were observed compared with the EV group, except for the bec-1 RNAi cohort, which showed a significant increase in ALM defects (78.57%, p < 0.001, Fig. 5B). In old (day 8 adult) worms, knockdown of bec-1(12.93%, p < 0.01) and sqst-1 (12.93%, p < 0.01) by RNAi led to a significant increase in ALM defects compared with controls (Fig. 5A and C). On day 8, 40.9% of the control RNAi (EV) group showed a defective PLM process, compared with worms on day 1 (Fig. 5D,E and F). Similar to effects on ALM, the bec-1 RNAi treatment showed higher defective PLM neurons on day 1 (11.11%, p < 0.001) and day 8 (49.38%, p < 0.001) (Fig. 5D). These data indicate that bec-1 RNAi leads to more defective in ALM and PLM neurons, while sqst-1 increases the percentage of defective ALM soma, but not PLM on day 8.
We then tested whether autophagy genes might modulate ALM and PLM during aging after CBD treatment. RNAi EV + no CBD treatment control shows age-related ALM/PLM defects. RNAi EV + CBD treatment shows a lower proportion of ALM/PLM defects on day 8, showing that CBD treatment protects from neuronal aging phenotypes (ALM: 12.97%, p < 0.001; PLM: 65.07%, p < 0.001; Fig. 5C and F). Furthermore, CBD treatment in experimental groups in which bec-1 and sqst-1 were knocked down with RNAi showed similar levels of ALM defects compared with RNAi EV + CBD group on day 1 and day 8. However, CBD treatment showed a lower proportion of both ALM and PLM defects in vps-34 RNAi group in day 1 (ALM: 54.75%, p < 0.001; PLM: 42.85%, p < 0.01) and day 8 (ALM: 10.24%, p < 0.05; PLM: 44.10%, p < 0.001; Fig. 5B,C,D, and F) suggesting that vps-34 is not required for CBD-mediated effects on neuronal aging. These data suggest that bec-1 and sqst-1 were required for the action of CBD on neuronal aging.
CBD requires sir-2.1 to improve lifespan, autophagy, and neuronal aging
SIRT1, a class III histone deacetylase, links to the extension of lifespan and age-related cellular mechanisms . The ortholog of SIRT1 in C. elegans is sir-2.1 . To determine whether CBD regulates lifespan extension in C. elegans via the sir-2.1/aak-2 pathway, we performed a lifespan assay in worms in which sir-2.1 had been knocked down via RNAi, or in aak-2 mutant animals. We found that the lifespan of the sir-2.1 RNAi group was significantly shorter than the control RNAi (EV) group (p < 0.001, Fig. 6A). We also found that the aak-2 mutant was significantly short-lived compared with wild-type controls (p < 0.001, Fig. 6B). CBD treatment did not affect the shortened lifespan of sir-2.1 (RNAi) or aak-2 mutant groups (Fig. 6A and B). The results suggest that sir-2.1 and aak-2 are required for CBD-induced longevity.
Transgenic overexpression or pharmacological activation of SIRT1 stimulates autophagic flux in both worms and human cells . We examined autophagic flux in nerve-ring neurons expressing mCherry::GFP::LGG-1 of the worms with or without sir-2.1 RNAi (Fig. 6C). CBD increased the number of APs and ALs, but the effect disappeared in the sir-2.1 RNAi worms compared to controls (Fig. 6D and E).
We examined whether sir-2.1 is required for CBD-dependent effects on age-associated neuronal morphological changes. We examined ALM and PLM neurons of worms with or without sir-2.1 RNAi treatment from the embryo/egg stage and then treated with or without CBD at day 1 and day 8 (Fig. 6F). There were age-dependent defects in both ALM and PLM neurons in wild-type worms. Compared with the controls, sir-2.1 RNAi significantly increased ALM soma defects at day 8, but not PLM (17.33%, p < 0.001, Fig. 6G). We next examined whether CBD affects neuronal aging through sir-2.1. We found that sir-2.1 RNAi + CBD treatment shows a higher proportion of ALM/PLM defects at day 8 (ALM: 12.04%, p < 0.01; PLM: 270.6%, p < 0.001; Fig. 6G), compared with the EV RNAi + CBD group. In addition, CBD-mediated age-associated morphological changes in PLM neurons were significantly abrogated by RNAi of sir-2.1 on day 8 (Fig. 6G).
Overall, these results suggest that the protective role of CBD in C. elegans lifespan, autophagy, and neuronal aging involves sir-2.1. A deficiency of sir-2.1 results in shortened lifespan, impaired autophagic flux, and triggered early neuronal aging.
SIRT1 is required for CBD-enhanced neurite outgrowth and spine density
To further validate the neuronal effect, we examined if knocking down SIRT1 alters the neurite outgrowth induced by CBD in SH-SY5Y neurons. Compared with the control group, CBD treatment upregulated the expression of SIRT1 (57.1%, p < 0.01) and p-AMPK/AMPK (224.1%, p < 0.001, Fig. 7A and B). We measured the neurite length in real time and showed that the neurite outgrowth of CBD treatment was reduced when SIRT1 levels were downregulated by shRNA-SIRT1 (27.04%, p < 0.001, Fig. S2A and B). We also confirmed these findings in primary hippocampal neurons with or without CBD treatment. Hippocampal neurons at 7 days in vitro (DIV) pre-treated with EX-527 for a half-hour, a selective inhibitor of SIRT1, were then exposed to CBD for 48 h. Neurite length and axon length were visualized by staining these neurons with a MAP2 antibody and quantified by ImageJ (Fig. 7C). We observed increased total neurite length (37.19%, p < 0.001, Fig. 7D) as well as mean neurite length after CBD treatment (16.21%, p < 0.001, Fig. S2C). Furthermore, the axon length in the CBD group was also longer than in the control (no CBD/EX-527 treatment) group (25.18%, p < 0.001, Fig. 7D). Conversely, a SIRT1 inhibitor EX-527 attenuated CBD’s effect on neurite length while not on axon length. Furthermore, the control group and CBD group showed higher distribution in 3 neurites and 4 neurites per neuron. However, the higher distribution of neurite number per neuron was changed to 2 and 3 in the EX-527 group treated and untreated with CBD (Fig. S2D). We also examined spine density in CBD- and/or EX-527-treated neurons. Hippocampal neurons were treated with EX-527 at DIV11 and then exposed to CBD for 3 days. The neurons were fixed at DIV14 and the morphology of the spines was visualized by confocal microscopy (Fig. 7E). Treatment with CBD significantly increased dendritic spine density compared with the control group (62.15%, p < 0.01, Fig. 7F). Neurons treated with EX-527 blocked CBD’s effect to increase dendritic density (Fig. 7F). Together, these results indicate that CBD can enhance neurite length and increase spine density; however, the effects were abolished by blocking SIRT1.