Cannabidiol and Trazodone Ameliorate Neuronal Proteotoxicity in C. elegans

doi:10.21203/rs.3.rs-2351138/v1

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Cannabidiol and Trazodone Ameliorate Neuronal Proteotoxicity in C. elegans

https://doi.org/10.21203/rs.3.rs-2351138/v1

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Alzheimer’s disease (AD), typically characterized by the accumulation of the misfolded proteins Amyloid beta (Aβ1-42) and hyperphosphorylation of Tau (P-Tau), is the most common neurodegenerative disease. Although there is currently no cure, research with rodent models has found targeting the signaling pathways involved in reactive oxygen species (ROS) or the unfolded protein response (UPR) can diminish the toxic effects of AD (e.g., behavioral deficits, glial inflammation, and proteotoxicity). For this study, we hypothesized that treatment combining Cannabidiol (CBD) and Trazodone (TRA), would better mitigate neuronal dysfunction and extend the lifespan of nematodes engineered to aggregate toxic misfolded proteins. CBD and TRA have been individually found to diminish the deleterious effects of proteotoxicity in both in vivo and in vitro studies. The organisms utilized in the study included two strains of C. elegans genetically modified to express two pathological proteins of AD. Our experiments revealed that the motility and lifespan of C. elegans with proteotoxicity of either Aβ1-42 or P-tau were significantly improved by treatment with CBD and TRA in combination. We also confirmed aspects of existing research on the efficacy of CBD and TRA individually. Our data suggest that these benefits are seen with full-life, middle-age, and late-stage rescue utilizing CBD and TRA. These findings suggest the need for future experimentation incorporating CBD and TRA in treatment regiments in higher organisms, including neurodegenerative rodent models and aging canines with cognitive decline.

Alzheimer’s disease

neurodegenerative disorders

cannabidiol

trazodone

hyperphosphorylation of tau (p-tau)

Amyloid beta (Aβ1-42)

proteotoxicity

C. elegans

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, with over 6 million individuals in the United States living with its debilitating consequences, which most notably include cognitive impairment, loss of mobility, and shortened lifespan (“2021 Alzheimer's disease facts and figures,” 2021). Moreover, while other leading causes of death among Americans 65 years of age and older declined over the two decades between 2000 and 2019, deaths from AD increased by 145% (“2021 Alzheimer's disease facts and figures,” 2021). The pathological hallmarks of AD are the aggregation of misfolded proteins Amyloid beta 1–42 (Ab_1-42)and phosphorylated Tau (P-Tau) in the entorhinal cortex and hippocampal regions of the brain (Bloom, 2014). Here, we utilized strains of Caenorhabditis elegans (C. elegans) to study the ability of compounds to inhibit these proteins and the toxic effects they cause within the nematode.

In this research, the specific targets were the cellular stress pathways responsible for the generation of reactive oxygen species (ROS) and the repair of misfolded proteins through cotreatment with a combination of chemically unique drugs that individually address a variety of cellular stress avenues, cannabidiol (CBD) and trazodone (TRA). We hypothesize that the application of the two drugs in combination is a promising treatment for AD due to the unique properties of TRA in targeting the unfolded protein response (UPR) and of CBD as a ROS scavenger (Halliday et al., 2017; Zhang et al., 2022).

Misfolded Protein Pathological Markers of AD

Understanding how the accumulation of misfolded proteins occurs and the way these misfolded proteins behave is critical to the development of treatments and the understanding of the mechanisms behind the progression of these neurodegenerative disorders in general and AD in particular. Proteins have specific conformations that allow them to perform homeostatic functions; however, proteins can misfold and become toxic, which most often involves deviating from standard ɑ-helix configuration to one of 𝛽-sheets. When this occurs in the Aꞵ_1-42 configuration, it is damaging to the body (Gross, 2000). Normally, when proteins misfold, cellular translational control pathways—such as the UPR and protein chaperones—attempt to correct the issue by either correctly refolding them or degrading them. However, due to the exponential nature of AD and the typical degradation of cells in an aging organism, misfolded proteins are able to accumulate, degrading neurological function (Clark, 2004; Hoffman et al., 2020; Reynaud, 2010; Zheng et al., 2013).

Drugs of Interest

When identifying the compounds to employ for this treatment, we recruited two existing, pharmaceutical compounds with the aim of drug re-purposing. We also aimed to identify alternatives to existing products with narrow therapeutic range and/or high cost, e.g., donepezil and aducanumab, respectively (Chaudhuri & Paul, 2006; Halliday et al., 2017; Zhang et al., 2022). CBD is a phytochemical derived from the family Cannabis sativa. Metabolism of CBD in humans is largely associated with the endocannabinoid receptors 2-AG and N-arachidonoyl-ethanolamine (anandamide/AEA), which are present in nearly all mammals and in C. elegans (Land et al., 2021). CBD has shown to increase longevity in AD (Frandsen et al., 2022) and Ab pathologies (Wang et al., 2021) C. elegans models. C. elegans treated with CBD have increased longevity and higher heat stress tolerance over a wide range of dosages (0.4 µM to 4.0 mM; Land et al., 2021). Additionally, previous research has shown when neurodegenerative C. elegans models are treated with CBD, it ameliorated the deleterious effect of Aβ_1-42by acting as an ROS scavenger, likely due to the binding of the endocannabinoid receptors and the downstream activation of the CB1 receptor due to the phenolic hydroxyl group found in CBD (Bisogno et al., 2001; Land et al., 2021; Ryberg et al., 2007; Zhang et al., 2022), however more experimentation would need to be performed to address this.

Trazodone has been commercially available for over 50 years and was initially prescribed for depression as it is a serotonin antagonist and reuptake inhibitor (SARI; Agnoli, 1986). The drug is rapidly taken up and undergoes Phase I Biotransformation through the cytochrome P450 3A4 (CYP3A4) and cytochrome P450 2D6 (CYP2D6) isoenzyme (Krystal, 2010). Following the metabolism of these isoenzymes, TRA transforms into the more active metabolite meta-chlorophenylpiperazine (m-CPP; Melzacka et al., 1979). The metabolism of TRA is similar in the nematode, as their CYP13A7 is an ortholog to CYP3A4 (Chakrapani et al., 2008; Rotzinger et al., 1998). One study found a 3-year delay in symptoms of AD in adults taking SARIs with mild cognitive impairment (Bartels et al., 2018). In addition, one of the main mechanisms of interest is the UPR pathway, TRA has previously been shown to inhibit the PERK pathway and neurotoxic pathways in neurodegenerative rodent models. In prion-diseased mice and P-tauaggregated mouse models neuroprotection and reduction of behavioral deficits through the inhibition of the PERK/eIF2ɑ-P branch of the UPR during periods of chronic aggregation of misfolded proteins (Halliday et al., 2017; Moreno et al., 2013; Mori, 2000; Radford et al., 2015; Wood, 2017). Furthermore, in a controlled TRA clinical trial, subjects with frontotemporal dementia exhibiting related behavioral symptoms (e.g., agitation, irritation) as measured by the Neuropsychiatry Inventory (NPI), achieved significant improvement (up to 50% reduction in NPI score) with the drug (Lebert et al. 2004). Researchers also have found that TRA is a promising compound for rescuing motility in neurodegenerative C. elegans with Tau-related deficits (Wood, 2017). Each of these factors played a role in our decision to utilize CBD and TRA to protect the neurons in these AD-models of C. elegans.

2.1 Laboratory Organisms

The neurodegenerative C. elegans utilized in this study were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota and consisted of BR6427, BR5706, GRU101, GRU102, and N2. The N2 (Wild Type) is the ancestral strain of the nematode and does not have any associated disease or decreased lifespan. These nematode strains had either accumulated Aβ_1-42 or accumulated P-Tau. The GRU102 (Pro-Aβ Aggr.) strain is engineered to express Aβ_1-42 by injecting human minigene Aβ_1-42 into the nematode to express Aβ_1-42 in the neurons (Fong et al., 2016). The GRU101 strain (Anti-Aβ Aggr.) represented the control as it is genetically identical to our wild type, only differing by the presence of a YFP pharyngeal protein marker (Fong et al., 2016). For this reason, we utilized the N2 in the isolated gentle touch test, to confirm our Anti-Aβ and Anti-Tau nematodes had no difference in motility, but did not use it in the subsequent assays. The second neurotoxic nematode we utilized, BR5706 (Pro-Tau Aggr.), is engineered through a process of double-crossing two separate C. elegans that have mutated ΔK280 deletion and MAPT V337M (Fatouros et al., 2012). Both ΔK280 and MAPT are crucial for the formation of Tau mutation in this protein, which results in the misfolding of the protein (Spina et al., 2017; Strang et al., 2019). The culmination of these two deleterious genes results in the manifestation of P-tau in the GABAeric motor neurons of our nematode model. The control strain used in our tau model is the BR6427 (Anti-Tau Aggr.) C. elegans mutant, which is designed by double-crossing mutants resulting in a double knockout of ΔK280 and MAPT V337M (Fatouros et al., 2012).

The P-tau model, like the A𝛽_1-42 model, contains a control organism (that does not express the Tau protein) and an experimental organism (that does express p-Tau). The control strain, BR6427 (Anti-Tau Aggr.) is modified through double-crossing strains. This crossing of strains results in the organism’s inability to form Tau proteins (Fatouros et al., 2012). The experimental strain BR5706 (Pro-Tau Aggr.) is also created by crossing two separate strains of C. elegans. In both of our model sets of misfolded proteins, each strain contained an experimental and a control model.

2.1.1 Maintenance

The nematode strains were grown on Nematode Growth Media (NGM) agar plates and stored in a 20 °C incubator. Nematodes were fed 200 µL of OP50, a nonpathogenic Escherichia coli (E. coli) strain, once a week. The maintenance for all of the various strains was performed in the same manner. Chunking, which involves using a sterilized metal spatula to cut a small square in the NGM agar plate that is then placed onto a new NGM plate with OP50, to maintain strains.

2.1.2 Synchronization

For the longevity studies, we required age-specific model organisms. For this, the nematodes were first washed from the NGM plates using M9 buffer and glass serological pipettes after which they were collected in either a 50 mL or 15 mL conical. The nematodes were then synchronized using 1 M NaOH and 5.25% hypochlorite bleaching treatments in 1.5 mL microcentrifuge tubes and spun for 1 min at 2000 rpm. Nematode pellets were then transferred from the tubes onto Petri dishes containing agar with FUDR to prevent reproduction of nematodes using 5-in glass aspirator pipettes. Nematodes were then fed with 200 µL of OP50 for 24 hr to ensure all eggs hatched simultaneously so as to allow the animals to age at approximately the same rate.

2.2 Compounds and Other Materials

The study utilized the following:

Cannabidiol: Cayman Chemical Company, Batch – 0592969-62
Trazodone Hydrochloride: SIGMA, Lot #MKC
5-Fluoro-2^’-deoxyuridine (FUDR): Research Products International, CAS No. 50-91-9 Lot 119676-154090
S-Complete Buffer : , 5 ml 1 M Potassium citrate - pH 6.0, 5 ml Trace Metals solution 1.5 ml 1M MgCl2 1.5 ml 1M CaCl2 Trace Metals Solution Disodium EDTA (5 mM) FeSO4 7H2O (2.5 mM) MnCl2 4H2O (1 mM) ZnSO4 7H2O (1 mM) CuSO4 5H2O (0.1 mM) Dissolve in 1L H₂O
M9 Buffer (3 g KH₂PO₄, 6 g Na2HPO4, 5 g NaCl, and 1mL 1 M MgSO₄ in 1 L of H₂O)

2.3 Assays

2.3.1 Non-Synchronous Gentle Touch Test

The C. elegans strains utilized in this first assay were: N2, BR6427, BR5706, GRU101, and GRU102. Non-synchronous adult nematodes were exposed to NGM agar combined with soluble solutions of each of the drugs of interest in isolation for 1 week at varying doses and then subjected to the gentle touch test assay. Trazodone stocks were prepared in 10 mM solutions containing TRA dissolved in sterile water. Cannabidiol stocks were prepared in 10 mM solutions containing CBD dissolved in DMSO. For the TRA assay, the nematodes were exposed to doses of 50 µM, 100 µM, and 200 µM. For the CBD assay, the nematode were exposed to doses of 1 µM, 5 µM, and 10µM. Within both of these gentle touch test experiments, nematodes were exposed to the vehicle treatment used for both compounds. This was done to confirm not only that the doses elicited positive effect without toxicity but also that each drug’s vehicle did not have any toxic effect on the nematodes. The gentle touch test assay, which uses an eyelash, involves targeting the AVB, PVC, AVA, and AVD interneurons to test mobility (Goodman, 2006). AVB and PVC interneurons effect backward movement; AVA and AVD interneurons effect forward movement. Response to the gentle touch test of fleeing from both stimuli was recorded as a 100% response; failure to flee from only one or both stimuli was recorded as 0%.

2.3.2 Trazodone and CBD Longevity Studies

The protocol used for the longevity studies was adopted from Solis and Petrascheck (2011) and altered by adding the isolated drugs—CBD and TRA—at separate time points in the lifespan of the nematodes to correspond with full-life and then the approximate different stages of life of a patient with AD, i.e., full-life (starting exposure on Day 1), mid-life (starting exposure on Day 8), and late life (starting exposure on Day 16). The C. elegans were maintained in 96-well plates in S-complete buffer and treated with various concentrations of both drugs of interest individually. There were three cohorts of nematodes (n = 22–54 per well) for each compound. Each of the three cohorts was placed in a separate well and counted every 3–5 days. All the nematodes were fed 5 µL of 100 mg/mL OP50 once a week to keep growth of worms consistent.

2.3.3 Synchronous Gentle and Harsh Touch Test

To determine the most effective dosage(s) for the drug-stacking assay, we conducted longevity studies on the two compounds in isolation on non-synchronous adult C. elegans that included a neuronal function assay, as described above in Section 2.3.2. From the results of the gentle touch test, we determined the most promising dosages for our BR5706 and GRU102, which were 100 µM of TRA and 5 µM or 10 µM of CBD. We also considered other factors, including established efficacy in extending lifespan of C. elegans and the lack of toxicity of CBD to the nematodes across a range of dosages (Land et al., 2021).

Based on these findings, we set the combined dosage for the study at 100 µM TRA and 10 µM CBD, which was introduced into the NGM solution and used to produce the nematode growth plates. To determine the timing of treatments, we referenced the western blot data in the guidelines of the Aβ_1-42 engineers, which state the nematodes are middle-aged on Day 8 and show peak concentration of misfolded protein aggregation on Day 12 (Fong et al., 2016). To obtain a complete picture of the effects of the treatment with the isolated drugs and allow the analysis of full-life, middle-age, and late-stage exposure, the processes were as follow. Synchronous adult nematodes were exposed to NGM agar combined with the drugs of interest for either 4 or 6 days. To analyze mid-life rescue, nematodes were transferred to NGM agar plate containing 10 µM CBD and 100 µM TRA on Day 8 and subjected to both the gentle and harsh touch test on Day 12. For late-stage analysis, nematodes were transferred to NGM agar plate containing 10 µM CBD and 100 µM TRA on Day 12 and subjected to both touch tests on Day 16. As with the isolated touch test assay described in Section 3.3.1, we again targeted the AVB, PVC, AVA, and AVD interneurons to test mobility. The gentle touch test assay uses an eyelash; the harsh touch test assay uses a titanium pick. Response to touch test of fleeing from both stimuli was recorded as a 100% response; failure to flee from only one or both stimuli was recorded as 0%.

The findings of each of the assays are presented in the following order: (a) Gentle Touch Test Assay, which was used to establish the appropriate timing of treatment; (b) Longevity Study, which was conducted to establish the most promising dosages of each drug and the difference in motility response; and (c) Synchronous Gentle and Harsh Touch Test Assay, which examined the effects of the combinational treatment comprising 10 µM CBD and 100 µM TRA. The findings described in this section are presented on Figures 1, 3, 4 and 5.

3.1 Identification of a non-toxic dose of compounds

The gentle touch test assay with the isolated compounds was conducted to determine the appropriate and safe dosages for the combined treatment of TRA and CBD. Each group of three cohorts (n = 22–54 per plate) were exposed to varying concentrations of either TRA or CBD. The gentle touch tests in this phase were conducted after 1 week of exposure. Response to the gentle touch test of fleeing from both stimuli was recorded as a 100% response; failure to flee from only one or both stimuli was recorded as 0%.

The overall finding of this initial experimentation was that the experimental nematode strains had overall worse motility (p < .0001) compared to the control nematodes. Regarding the dosages, we found that 100 µM and 200 µM TRA (see Figure 1, Panels A and B) and 5 µM and 10 µM (see Figure 1, Panels C and D) provided the most benefit to the neurodegenerative nematode strains. Statistical analysis was performed using 2-way ANOVA, to compare the Pro-Tau and Pro-Aβ to their control counterparts.

3.2 Longevity Assays with varying treatment timing

This assay was conducted to determine the most appropriate timing of treatment. The nematodes were treated 1.) full-life, 2.) mid-life or day 8 prior to known aggregation of P-tau or Ab_1-42 and 3.) late-life day 16 with aggregation of P-tau or Ab_1-42. The C. elegans strains included in this second assay were: BR6427, BR5706, GRU101, GRU102, and N2. Each group of three cohorts (n = 22–54) were fed 10 mg/mL E. coli OP50 once a week and exposed to varying concentrations of either TRA or CBD in isolation. Mantel-Cox test was used to determine survival statistics.

3.2.1 Longevity Assay: Trazodone

The major finding of this longevity assay on the effects of TRA were that varying dosages of TRA (25 µM, 50 µM, and 100 µM) significantly improved lifespan of the experimental strains at all stages. Conversely, certain of the treatment timings of the control strains had no significant effect. These included: (a) the Day 8 TRA treatment of Anti-Aꞵ Aggr. (see Fig. 3, Panel C); (b) the Day 16 TRA treatment of Anti-Aꞵ Aggr. (see Fig. 3, Panel E); (c) the full-life TRA treatment of Anti-Tau Aggr. (see Fig. 3, Panel G); and (d) the Day 16 TRA treatment of Anti-Tau Aggr. (see Fig. 3, Panel K).

The treatments that used a dose of 100 µM TRA had the greatest incidence rate of significant effect, as follows: (a) full-life treatment of Anti-Aꞵ Aggr. (p < .010; see Fig. 3, Panel A); (b) Day 8 treatment of Anti-Tau Aggr. (p < .05; see Fig. 3, Panel I); (c) Day 8 treatment of Pro-Tau Aggr. (p < .001; see Fig. 3, Panel J); (d) Day 16 treatment of Pro-Tau Aggr. (p < .0001; see Fig. 3, Panel L); (e) full-life treatment of Pro-Aꞵ Aggr. (p < .05; see Fig. 3, Panel B); and (f) Day 8 treatment of Pro-Aꞵ Aggr. (p < .0001; see Fig. 3, Panels D and F). Significant effect with doses of 25 µM and 50 µM TRA were each seen in two cases. For the 50 µM dose, this was seen with the full-life treatment of Anti-Aꞵ Aggr. (p < .010; see Fig. 3, Panel A) and the full-life treatment of Pro-Tau Aggr. (p < .01; see Fig. 3, Panel H). For the 25 µM dose, this significant effect was seen with full-life treatment of Pro-Tau Aggr. (p < .01; see Fig. 3, Panel H) and the Day 8 treatment of Pro-Tau Aggr. (p < .05; see Fig. 3, Panel J). The full results of this section are presented on Figure 3.

3.2.2 Longevity Assay: Cannabidiol

The major finding of this isolated treatment longevity assay involving CBD was that varying dosages of CBD (1 µM, 5 µM, and 10 µM) significantly improved the lifespan of the experimental nematodes at all stages. We also found that certain timings of CBD treatment had no effect; these included: (a) the full-life (see Fig. 4, Panel A), Day 8 (see Fig. 4, Panel C), and Day 16 treatments of Anti-Aꞵ Aggr. (see Fig. 4, Panel E); and the full-life (see Fig. 4, Panel G) and Day 8 treatments of Anti-Tau Aggr. (see Fig. 4, Panel I).

Certain treatments achieved significant effect with all three dosages. For example, the Day 8 treatment of Pro-Aꞵ Aggr. C. (see Fig. 4, Panel D), achieved effect of p < .001 with 1 µM, p < .01 with 5 µM, and p < .0001 with 10 µM. This also occurred with the full-life treatment of Pro-Tau Aggr. (see Fig. 4, Panel H), where significant effect was seen with 1 µM (p < .05), with 5 µM (p < .05), and with 10 µM CBD (p < .0001). The Day 16 treatment of Pro-Aꞵ Aggr. had significant effect with both 5 µM (p < .01) and 10 µM (p < .0001), as shown on Figure 4, Panel F. The Day 8 treatment of Pro-Tau Aggr. (see Fig. 4, Panel J) had significant effect with the two lower doses of 1µM (p < .001) and 5 µM (p < .05). The remaining treatments only had significant effect with one dosage: (a) Day 16 treatment of Anti-Tau Aggr. with 10 µM (p < .05; see Fig. 4, Panel K); (b) the Day 16 treatment of Pro-Tau Aggr. with 5 µM (see Fig. 4, Panel L); and (c) the full-life treatment of 10 µM of Pro-Aꞵ Aggr. (p < .01; see Fig. 4, Panel B). The full results for this section are presented on Figure 4.

3.3 Combination of Treatments: Gentle and Harsh Touch Test Results

The final stage of the study was conducted using the dosages and timing of treatment established by the previous assays. For this experimentation, a combined dose of 10 µM CBD and 100 µM TRA was utilized. As previously described in Section 3, the four strains (i.e., BR6427, BR5706, GRU101, GRU102) of synchronized nematodes (n = 30) were fed 5 µL of 10 mg/mL E. coli OP50 once a week. The touch tests were conducted on the AVB and PVC interneurons, using an eyelash for the gentle touch test and a titanium pick for the harsh touch test. To obtain accurate data, touches for both tests were repeated to better observe neuronal integrity (McClanahan et al., 2017). Response to touch test of fleeing from both stimuli was recorded as a 100% response; failure to flee from only one or both stimuli was recorded as 0%. Statistical analysis was performed using a paired t-test.

The main findings of this assay were that after the periods of exposure to the combination of TRA and CBD, the motility of the Pro-Aꞵ Aggr. nematodes was found to be significantly improved as measured by their responses to both the gentle and harsh touch tests. The motility of the Pro-Tau Aggr. nematodes was also found to be significantly improved, but only as measured by the harsh touch test. The two treatment instances where no effect was seen based on response to gentle touch test only, were the Tau model C. elegans exposed on Day 8 with gentle touch test conducted on Day 12 (see Fig. 5, Panel B) and the Tau model C. elegans exposed on Day 16 as measured by gentle touch test on Day 18 (see Fig. 5, Panel D).

The significant findings regarding the combined treatment of CBD/TRA as demonstrated by gentle touch test were: Day 8 treatment of Aꞵ model with test conducted on Day 12 (p < .05; see Fig. 5, Panel A); and Day 16 treatment of Aꞵ model with test conducted on Day 18 (p < .05; see Fig. 5, Panel C). The significant findings here as demonstrated by harsh touch test were: (a) Day 8 treatment of Aꞵ model as demonstrated by test conducted on Day 12 (p < .01; see Fig. 5, Panel E); (b) the Day 8 treatment of Tau model as demonstrated by test conducted on Day 12 (p < .01; see Fig. 5, Panel F); (c) the Day 16 treatment of Aꞵ model as demonstrated by test conducted on Day 18 (p < .01; see Fig. 5, Panel G); and (d) the Day 16 treatment of Tau model as demonstrated by test conducted on Day 18 (p < .05; see Fig. 5, Panel H). These results are presented on Figure 5.

Alzheimer’s disease is characterized by the accumulation and aggregation of both Aβ_1-42 and P-tau within the brain (Ritchie et al., 2017). In this study, we propose a therapeutic model that utilizes transgenic C. elegans as a model organism to investigate the role of two drugs—CBD and TRA— individually and in combination. We hypothesized that these compounds in tandem would improve lifespan for C. elegans with proteotoxicity of either Aβ_1-42or P-Tau.

Our data support that both mid- and late-stage treatment with 10 µM CBD and 100 µM TRA in combinatio significantly improved the neuronal integrity of the nematodes as assessed with a touch test assay. In isolation, doses of 5 µM and 10 µM of CBD and 25 µM, 50 µM, and 100 µM of TRA extended the lifespan of our experimental C. elegans. The isolated gentle touch test on non-synchronous nematodes and the longevity analysis on synchronous nematodes, established 10 µM CBD and 100 µM TRA as the most promising combination of doses for a drug-stacking treatment. Individually, full-life exposure of either CBD and TRA are not toxic and also extend lifespan. While patients with neurodegenerative disorders like AD do not receive such a diagnosis until misfolded proteins manifest—typically in late middle-age to the senior years—these two examined drugs are far more tolerable and safer as compared to existing drugs for AD currently on the market that target acetylcholine or monoclonal antibody treatments (Briggs et al., 2016; Mimica & Presecki, 2009; Pasqualetti et al., 2015).

Although C. elegans differ from humans in many ways, it remains a beneficial model to study due to a shortened lifespan, homology to certain proteins, and fully mapped out connectome. From our work with the nematodes, we add to the existing research with our findings on how CBD and TRA in isolation positively affect the neuronal integrity and longevity of the animals at varying stages of life and times of exposure (Figure 2). In addition, we provide new insight into how these two drugs act when provided in combination. Moreover, unlike previous publications studying Alzheimer’s-modeled C. elegans that looked at only one or the other of the two proteins of interest, our study included both P-tau and Aβ_1-42. These findings ideally can be further implemented translationally in other models of AD to enable researchers to simultaneously target multiple pathways associated with the disease.

4.1 Limitations

As other researchers have noted, due to their less complex anatomy there are certain limitations to using nematodes in a survival analysis as compared to using higher organisms. However, utilizing this assay was advantageous for deducing appropriate concentrations of each drug for use in the drug-stacking treatment (Tissenbaum, 2015; Wang et al (2019). In addition, our Pro-Tau is not technically an AD-model, but it contains one of the proteins of interest. Pro-Tau Aggr. strain and its specific mutations— ΔK280 deletion and MAPT V337M—is also associated with frontal temporal dementia, which is sometimes misdiagnosed as AD (Spina et al., 2017). Another important limitation is that the timing of the treatment for both experimental strains is based on the aging of the Aβ_1-42 strain rather than on an aging timeline specific to the Tau model, which might mean while our experimental model was optimized for the Pro-Aβ Aggr. strain it was not necessarily optimized for the Pro-Tau Aggr. strain (Fong et al., 2016).

4.2 Future Recommendations

Since the mechanism of how CBD works in neurodegenerative disease is not well-established, one area for future research is the study of these strains of neurodegenerative nematodes to deduce the potential pathway of CBD by acquiring mutant C. elegans that are hypomorphic for Nrf-2 to deduce if CBD acts independently as an ROS scavenger or activates Nrf-2. Utilizing a higher order animal model is also necessary, and canines are considered a promising model, as they contain certain pathways that nematodes lack (e.g., NF-kB) and exhibit a similar neurodegenerative process as humans. Previous research in dogs has demonstrated tolerability and analyzed pharmacokinetic and pharmacodynamic parameters of CBD and TRA (Bartner et al., 2018; Jay et al., 2013). Additionally, future studies using higher order animals should include a full-life exposure set to further confirm the lack of toxicity of both compounds and also allow for the examination of the effects on more complex behaviors than those of C. elegans (Maccarrone, 2017). Given the findings on full-life treatment using the drug-stacking compound of CBD and TRA, another area that warrants investigation is that of the implications of full-life treatment for those at moderate- to high-risk for the development of neurodegenerative conditions.

4.3 Conclusions

Our data support that both mid- and late-stage treatment of 10 µM CBD and 100 µM TRA significantly improved the neuronal integrity of the nematodes as assessed with touch test assay. In addition, doses of 5 µM and 10 µM of CBD in isolation and doses of 25 µM, 50 µM, and 100 µM of TRA in isolation extended the lifespan of these C. elegans. Based on the survival analysis and neuronal integrity assay, both isolated treatments and combinational treatments of these known UPR-targeting and ROS-scavenging compounds may prove a unique avenue for the treatment of AD and ADRD characterized by protein misfolding.

Permission to reuse and Copyright

Figures, tables, and images will be published under a Creative Commons CC-BY licence and permission must be obtained for use of copyrighted material from other sources (including re-published/adapted/modified/partial figures and images from the internet). It is the responsibility of the authors to acquire the licenses, to follow any citation instructions requested by third-party rights holders, and cover any supplementary charges.

Resource Identification Initiative

- Cannabidiol Cayman Chemical Company: Batch – 0592969-62

- Trazodone Hydrochloride SIGMA: Lot # MKC

- 5-Fluoro-2^’-deoxyuridine Research Products International: CAS No. 50-91-9 Lot 119676-154090

- BR6427 (CGC, Species)

- BR5706 (CGC, Species)

- GRU101 (CGC, Species)

- GRU102 (CGC, Species)

- N2 (CGC, Species)

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethical Approval

The authors followed ethical procedures and the laboratory had Biosafety level 2 certification.

Author Contributions

V.S.G, S.M. and J.A.M conceive and designed the project. V.S.G, A.M.A, and A.V.A performed the experiments. V.S.G and J.A.M contributed to data analysis. V.S.G wrote the manuscript and all authors reviewed and edited the manuscript.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Acknowledgments

We would like to acknowledge Dr. Fred Hoerndli and his lab for assistance with the synchronization protocol for the C. elegans. We would also like to acknowledge both Arielle Hay and Amanda Latham for assistance with experimental troubleshooting.

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Cannabidiol and Trazodone Ameliorate Neuronal Proteotoxicity in C. elegans

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