Neural engineering with photons as synaptic transmitters.

Neuronal computation is achieved through connections of individual neurons into a larger network. To expand the repertoire of endogenous cellular communication, we developed a synthetic, photon-assisted synaptic transmission (PhAST) system. PhAST is based on luciferases and channelrhodopsins that enable the transmission of a neuronal state across space, using photons as neurotransmitters. PhAST overcomes synaptic barriers and rescues the behavioral deficit of a glutamate mutant with conditional, calcium-triggered photon emission between two neurons of the Caenorhabditis elegans nociceptive avoidance circuit. To demonstrate versatility and flexibility, we generated de novo synaptic transmission between two unconnected cells in a sexually dimorphic neuronal circuit, suppressed endogenous nocifensive response through activation of an anion channelrhodopsin and switched attractive to aversive behavior in an olfactory circuit. Finally, we applied PhAST to dissect the calcium dynamics of the temporal pattern generator in a motor circuit for ovipositioning. In summary, we established photon-based synaptic transmission that facilitates the modification of animal behavior.

FLInt: single shot safe harbor transgene integration via Fluorescent Landmark Interference.

The stable incorporation of transgenes and recombinant DNA material into the host genome is a bottleneck in many bioengineering applications. Due to the low efficiency, identifying the transgenic animals is often a needle in the haystack. Thus, optimal conditions require efficient screening procedures, but also known and safe landing sites that do not interfere with host expression, low input material and strong expression from the new locus. Here, we leverage an existing library of ≈300 different loci coding for fluorescent markers that are distributed over all 6 chromosomes in Caenorhabditis elegans as safe harbors for versatile transgene integration sites using CRISPR/Cas9. We demonstrated that a single crRNA was sufficient for cleavage of the target region and integration of the transgene of interest, which can be easily followed by loss of the fluorescent marker. The same loci can also be used for extrachromosomal landing sites and as co-CRISPR markers without affecting body morphology or animal behavior. Thus, our method overcomes the uncertainty of transgene location during random mutagenesis, facilitates easy screening through fluorescence interference and can be used as co-CRISPR markers without further influence in phenotypes.

Neuroprotective effects of a medium chain fatty acid, decanoic acid, isolated from H. leucospilota against Parkinsonism in C. elegans PD model.

Sea cucumbers are marine organism that have long been used for food and traditional medicine in Asian countries. Recently, we have shown that ethyl acetate fraction (HLEA) of the crude extract of the black sea cucumber, Holothuria leucospilota, could alleviate Parkinsonism in Caenorhabditis elegans PD models. In this study, we found that the effective neuroprotective activity is attributed to HLEA-P1 compound chemically isolated and identified in H. leucospilota ethyl acetate. We reported here that HLEA-P1 could attenuate DAergic neurodegeneration, improve DAergic-dependent behaviors, reduce oxidative stress in 6-OHDA-induced C. elegans. In addition, HLEA-P1 reduced α-synuclein aggregation, improved behavior deficit and recovered lipid deposition in transgenic C. elegans overexpressing α-synuclein. We also found that HLEA-P1 activates nuclear localization of DAF-16 transcription factor of insulin/IGF-1 signaling (IIS) pathway. Treatment with 25 µg/ml of HLEA-P1 upregulated transcriptional activity of DAF-16 target genes including anti-oxidant genes (such as sod-3) and small heat shock proteins (such as hsp16.1, hsp16.2, and hsp12.6) in 6-OHDA-induced worms. In α-synuclein-overexpressed C. elegans strain, treatment with 5 µg/ml of HLEA-P1 significantly activated mRNA expression of sod-3 and hsp16.2. Chemical analysis demonstrated that HLEA-P1 compound is decanoic acid/capric acid. Taken together, our findings revealed that decanoic acid isolated from H. leucospilota exerts anti-Parkinson effect in C. elegans PD models by partly modulating IIS/DAF-16 pathway.

An asymmetric mechanical code ciphers curvature-dependent proprioceptor activity.

A repetitive gait cycle is an archetypical component within the behavioral repertoire of many animals including humans. It originates from mechanical feedback within proprioceptors to adjust the motor program during locomotion and thus leads to a periodic orbit in a low-dimensional space. Here, we investigate the mechanics, molecules, and neurons responsible for proprioception in Caenorhabditis elegans to gain insight into how mechanosensation shapes the orbital trajectory to a well-defined limit cycle. We used genome editing, force spectroscopy, and multiscale modeling and found that alternating tension and compression with the spectrin network of a single proprioceptor encodes body posture and informs TRP-4/NOMPC and TWK-16/TREK2 homologs of mechanosensitive ion channels during locomotion. In contrast to a widely accepted model of proprioceptive "stretch" reception, we found that proprioceptors activated locally under compressive stresses in-vivo and in-vitro and propose that this property leads to compartmentalized activity within long axons delimited by curvature-dependent mechanical stresses.

Diterpene glycosides from Holothuria scabra exert the α-synuclein degradation and neuroprotection against α-synuclein-Mediated neurodegeneration in C. elegans model.

ETHNOPHARMACOLOGICAL RELEVANCE: Holothuria (Metriatyla) scabra Jaeger (H. scabra), sea cucumber, is the marine organism that has been used as traditional food and medicine to gain the health benefits since ancient time. Although our recent studies have shown that crude extracts from H. scabra exhibited neuroprotective effects against Parkinson's disease (PD), the underlying mechanisms and bioactive compounds are still unknown. AIM OF THE STUDY: In the present study, we examined the efficacy of purified compounds from H. scabra and their underlying mechanism on α-synuclein degradation and neuroprotection against α-synuclein-mediated neurodegeneration in a transgenic Caenorhabditis elegans PD model. MATERIAL AND METHODS: The H. scabra compounds (HSEA-P1 and P2) were purified and examined for their toxicity and optimal dose-range by food-clearance and lifespan assays. The α-synuclein degradation and neuroprotection against α-synuclein-mediated neurodegeneration were determined using transgenic C. elegans model, Punc-54::α-syn and Pdat-1:: α-syn; Pdat-1::GFP, respectively, and then further investigated by determining the behavioral assays including locomotion rate, basal slowing rate, ethanol avoidance, and area-restricted searching. The underlying mechanisms related to autophagy were clarified by quantitative PCR and RNAi experiments. RESULTS: Our results showed that HSEA-P1 and HSEA-P2 significantly diminished α-synuclein accumulation, improved motility deficits, and recovered the shortened lifespan. Moreover, HSEA-P1 and HSEA-P2 significantly protected dopaminergic neurons from α-synuclein toxicity and alleviated dopamine-associated behavioral deficits, i.e., basal slowing, ethanol avoidance, and area-restricted searching. HSEA-P1 and HSEA-P2 also up-regulated autophagy-related genes, including beclin-1/bec-1, lc-3/lgg-1, and atg-7/atg-7. RNA interference (RNAi) of these genes in transgenic α-synuclein worms confirmed that lc-3/lgg-1 and atg-7/atg-7 were required for α-synuclein degradation and DAergic neuroprotection activities of HSEA-P1 and HSEA-P2. NMR and mass spectrometry analysis revealed that the HSEA-P1 and HSEA-P2 contained diterpene glycosides. CONCLUSION: These findings indicate that diterpene glycosides extracted from H. scabra decreases α-synuclein accumulation and protects α-synuclein-mediated DAergic neuronal loss and its toxicities via lgg-1 and atg-7.

Anti-Parkinson activity of bioactive substances extracted from Holothuria leucospilota.

Parkinson's disease (PD) is a well-known neurodegenerative disorder characterized by dopaminergic (DA) neuron loss and α-synuclein aggregation. Recent study revealed that the extracts from sea cucumber, Holothuroidea spp., exhibited neuroprotective and lifespan extension effects in Caenorhabditis elegans model. Interestingly, the black sea cucumber, Holothuria leucospilota, possesses body wall and a specialized organ called cuvierian tubules containing high amount of bioactive compounds. In this study, the neuroprotective effects of the body wall (BW) and cuvierian tubules (CT) from this sea cucumber against PD were evaluated using C. elegans as a model. H. leucospilota were extracted using ethanol (ET), ethyl acetate (EA), butanol (BU) and aqueous (AQ) fractions. Extracts from these fractions were used to treat the 6-OHDA-induced BZ555 and α-synuclein expressing NL5901 strains of C. elegans. Treatment with ET, EA, BU and AQ fractions of H. leucospilota extracts could significantly prevent degeneration of DA neurons in 6-OHDA-induced worms, improve food-sensing behavior mediated by DA neurons, and up-regulate cat-2 and sod-3 gene expressions. These results indicate the neuroprotective activity of the extracts which may be attributed to the anti-oxidant activity of the bioactive compounds. Moreover, α-synuclein aggregation was significantly reduced together with the recovery of lipid deposition upon the treatment with H. leucospilota extracts. In addition, treatment with H. leucospilota extracts was able to increase the lifespan of 6-OHDA-induced N2. NMR analysis revealed the major chemical components in the effective EA fractions were terpenoids, steroids, saponins, and glycosides. In summary, H. leucospilota extracts exhibited anti-Parkinson effect in both toxin-induced and transgenic C. elegans models of PD. Further study will be performed to elucidate the most effective anti-PD molecules which will lead to the development of anti-PD drug.