112 MARINE INGREDIENTS
muscle and binds to its clustered receptors (AChRs), starting the post-synaptic pathway and inducing muscle contraction.2 The novel ferment extract offers an
innovative and holistic approach to wrinkle expression by working along the three processes of neuromuscular communication: neuritogenesis and neuronal migration, synaptic vesicle recycling, and muscle contraction.
Neuritogenesis and neuronal migration Neuritogenesis must take place before the creation of neuromuscular connections. Initially, neurite projections sprout and extend throughout the surrounding environment. Following initial neurite outgrowth, a single axon is identified, and the remaining neurites mature into dendrites. The tip of the axon senses guidance cues that guide it toward cells with which it can establish multiple synaptic connections.3 The ferment extract was tested for its
ability to modulate neurite outgrowth in SH- SY5Y cells. Microscopy was used to measure neurite length, and pictures were acquired by fluorescent immunostaining of β-III tubulin (TUJ-1, a marker of neuronal cytoskeleton). The ferment extract significantly shortened neurite length by 17% (p<0.001) (Figure 2). The next step was to determine if the
ferment extract could modulate neuronal migration towards muscle cells, hence reducing the establishment of neuromuscular junctions (NMJs). This time, human skeletal muscle myoblasts and SH-SY5Y cells were allowed to differentiate in separate chambers of a neuronal device in the presence or absence of the ferment extract. These chambers were connected together
by many microchannels. Microscopy was used to assess the migration of neurons from their chamber to the chamber of myocytes after staining TUJ-1, actin and the nucleus. Neuronal migration in the channels was decreased by 13% (p<0.001) when the ferment extract was used (Figure 3).
Synaptic vesicles recycling Due to the fact that presynaptic terminals are small, the number of synaptic vesicles is
BASAL BASAL 0.05 mg/mL FERMENT EXTRACT
Figure 2: Neurite outgrowth reduction of SH-SY5Y cells (β-III tubulin in cyan, nuclei in blue)
Neurons in green
Neurons migrating towards myocytes (nuclei in blue)
neurons in green & myocytes
Migrated Basal
0.05 mg/mL
ferment extract
Figure 3: Neuronal migration decrease towards muscle cells in a neuronal device (neurons/β-III tubulin in green, nuclei in blue)
limited and they are rapidly recycled after each fusion event to guarantee that the synaptic transmission is maintained through multiple rounds of vesicle fusion. Otherwise, even at low levels of neuronal activity, they would be rapidly depleted.4 Clathrin is a three-legged protein that
covers synaptic vesicles, mediating recycling and thereby ensuring an ultrafast recharge of ACh. The absence of clathrin slows the recycling of synaptic vesicles, which means it take longer for the vesicles to be reloaded with
0.1 mg/mL FERMENT EXTRACT
neurotransmitters for a fresh release into the synapsis.4 After being treated with the ferment extract,
differentiated SH-SY5Y were stained for clathrin detection and images were acquired using a fluorescence microscope. Clathrin levels were lowered dose-dependently by the ferment extract, achieving a significant decrease of 34% (p<0.05) at the highest tested concentration (Figure 4). To assess the efficacy of the ferment extract
in regulating the release of ACh, human muscle cells and human motor neurons derived from fibroblast cells that had been reprogrammed into pluripotent (hiPS) cells were co-cultured for ten days. Under these circumstances, it is possible to generate muscle fibers that contract under the influence of motor neuron activity, therefore recreating NMJs with the same abilities as those found in vivo. After five days, spontaneous contractions
Figure 4: Fluorescent images of Clathrin levels decrease on SH-SY5Y cells (clathrin in green, nuclei in blue)
PERSONAL CARE April 2024
were observed. After that, the ferment extract was added and allowed to incubate with the co-culture for six hours. The release of ACh was then induced by adding glutamate for 30 minutes and acetylcholine release was evaluated by an ELISA test. The ferment showed to significantly reduce ACh release dose-dependently and reaching a decrease in the release of 73% (p<0.001) at the highest tested concentration.
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