Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins

Background The lack of physiologically relevant and predictive cell-based assays is one of the major obstacles for testing and developing botulinum neurotoxins (BoNTs) therapeutics. Human-induced pluripotent stem cells (hiPSCs)-derivatives now offer the opportunity to improve the relevance of cellular models and thus the translational value of preclinical data. Methods We investigated the potential of hiPSC-derived motor neurons (hMNs) optical stimulation combined with calcium imaging in cocultured muscle cells activity to investigate BoNT-sensitivity of an in vitro model of human muscle-nerve system. Results Functional muscle-nerve coculture system was developed using hMNs and human immortalized skeletal muscle cells. Our results demonstrated that hMNs can innervate myotubes and induce contractions and calcium transient in muscle cells, generating an in vitro human motor endplate showing dose-dependent sensitivity to BoNTs intoxication. The implementation of optogenetics combined with live calcium imaging allows to monitor the impact of BoNTs intoxication on synaptic transmission in human motor endplate model. Conclusions Altogether, our findings demonstrate the promise of optogenetically hiPSC-derived controlled muscle-nerve system for pharmaceutical BoNTs testing and development.

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system

Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions—however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

The role, carrier and mission of neural crest cells in the skin

The neuro crest arising from the ectoderm is a transient structure and disappears as the neurocrest cells leave these places to invade the whole embryo. The epidermis develops from the ectoderm in the fourth embryonal weeks. The embryos consist of cranial-,vagal-, truncal and sacral segments and the neuro crest cells migrate from these places to form various structures, including the peripheral nerve system, the craniofacial bones and cartilages, etc. The neuro crest cells degrade the basal membrane of neural tube and thereafter migrate through the extracellular matrix in ventromedial and dorsolateral direction. Neural crest cells use various cell adhesion molecules and diferent proteaes. The invasive capacity of these cells is infuenced by aquaporin-1 , too. . The sensory nerves developig from the neuro- crest cells can be found in the epidermis and its appendicular organ, the dermal autonomic nerves in the dermis. The epidermal melanocytes develop partly from the neural crest cells, partly from the Schwann cells of the sensory nerves. The cutaneous nerves produce and secrete neuropeptides thus contributing to the development of the skin into a neuroimmuno-endocrin organ.

Electroacupuncture could balance the gut microbiota and improve the learning and memory abilities of Alzheimer’s disease animal model

Alzheimer’s disease (AD), as one of most common dementia, mainly affects older people from the worldwide. In this study, we intended to explore the possible mechanism of improving cognitive function and protecting the neuron effect by electroacupuncture. Method: We applied senescence-accelerated mouse prone 8 (SAMP8) mice as AD animal model, used Morris water maze, HE staining, 16S rDNA amplicon sequencing of gut microbiota and ELISA to demonstrate our hypothesis. Results: electroacupuncture improved the learning and memory abilities in SAMP8 mice (P<0.05) and could protect the frontal lobe cortex and hippocampus of SAMP8 mice; electroacupuncture significantly decreased the expression of IL-1β (P<0.01), IL-6 (P<0.01) and TNF-α (P<0.01 in hippocampus, P<0.05 in serum) in serum and hippocampus; electroacupuncture balanced the quantity and composition of gut microbiome, especially of the relative abundance in Delta-proteobacteria (P<0.05) and Epsilon-proteobacteria (P<0.05). Conclusion: electroacupuncture treatment could inhibit the peripheral and central nerve system inflammatory response by balancing the gut microbiota.

A Case Report of Hyperhidrosis with Sympathicotonia Detected by Iris Diagnosis

Objective: The aim of this case study was to describe a case of iris diagnosis of primary hyperhidrosis and the use of Korean medicine.Methods: A patient with symptoms of hyperhidrosis was diagnosed as having Taeeumin after assessment using the four basic Korean diagnostic methods. Iris diagnosis was used for further examination. The images obtained showed a remarkably defined collarette and increased nerve rings, which suggested an overactive sympathetic nerve system. Under the diagnosis of Taeeum, a Korean herbal medicine was prescribed with additional herbs to help alleviate the hyperactivity of the sympathetic nervous system.Results: The patient had been receiving treatment for hyperhidrosis for >30 years, with various medical attempts to relieve her symptoms, which were ineffective. She showed signs of improvement from day 4 into the treatment, and 80% of her symptoms were improved after completing a 6-week treatment course.Conclusion: The herbal medicine prescribed to the patient proved effective for reducing her chronic symptoms that had been unresponsive to previous medical treatments.

Dissecting the Roles of the Autonomic Nervous System and Physical Activity on Circadian Heart Rate Fluctuations in Mice

Heart rate (HR) and blood pressure as well as adverse cardiovascular events show clear circadian patterns, which are linked to interdependent daily variations in physical activity and cardiac autonomic nerve system (ANS) activity. We set out to assess the relative contributions of the ANS (alone) and physical activity to circadian HR fluctuations. To do so, we measured HR (beats per minute, bpm) in mice that were either immobilized using isoflurane anesthesia or free-moving. Nonlinear fits of HR data to sine functions revealed that anesthetized mice display brisk circadian HR fluctuations with amplitudes of 47.1±7.4bpm with the highest HRs in middle of the dark (active) period (ZT 18: 589±46bpm) and lowest HRs in the middle of the light (rest) period (ZT 6: 497±54bpm). The circadian HR fluctuations were reduced by ~70% following blockade of cardiac parasympathetic nervous activity (PNA) with atropine while declining by &lt;15% following cardiac sympathetic nerve activity (SNA) blockade with propranolol. Small HR fluctuation amplitudes (11.6±5.9bpm) remained after complete cardiac ANS blockade. Remarkably, circadian HR fluctuation amplitudes in freely moving, telemetrized mice were only ~32% larger than in anesthetized mice. However, after gaining access to running wheels for 1week, circadian HR fluctuations increase to 102.9±12.1bpm and this is linked directly to increased O2 consumption during running. We conclude that, independent of physical activity, the ANS is a major determinant of circadian HR variations with PNA playing a dominant role compared to SNA. The effects of physical activity to the daily HR variations are remarkably small unless mice get access to running wheels.

Function of Oncogene Mycn in Adult Neurogenesis and Oligodendrogenesis

Human MYCN is an oncogene amplified in neuroblastoma and many other tumors. Both human MYCN and mouse Mycn genes are important in embryonic brain development, but their functions in adult healthy nerve system are completely unknown. Here, with Mycn-eGFP mice and quantitative RT-PCR, we found that Mycn was expressed in specific brain regions of young adult mice, including subventricular zone (SVZ), subgranular zone (SGZ), olfactory bulb (OB), subcallosal zone (SCZ), and corpus callosum (CC). With immunohistochemistry (IHC), we found that many Mycn-expressing cells expressed neuroblast marker doublecortin (DCX) and proliferation marker Ki67. With Dcx-creER and Mki67-creER mouse lines, we fate mapped Dcx-expressing neuroblasts and Mki67-expressing proliferation cells, along with deleting Mycn from these cells in adult mice. We found that knocking out Mycn from adult neuroblasts or proliferating cells significantly reduced cells in proliferation in SVZ, SGZ, OB, SCZ, and CC. We also demonstrated that the Mycn-deficient neuroblasts in SGZ matured quicker than wild-type neuroblasts, and that Mycn-deficient proliferating cells were more likely to survive in SVZ, SGZ, OB, SCZ, and CC compared to wild type. Thus, our results demonstrate that, in addition to causing tumors in the nervous system, oncogene Mycn has a crucial function in neurogenesis and oligodendrogenesis in adult healthy brain.

Nerve-Sparing Radical Hysterectomy: Kobayashi's Method

After Prof. S. Okabayashi introduced Okabayashi Operation in 1921, several surgeons introduced numerous improvements in Japan. One of them is so-called the Tokyo Method which was improved and revised by Dr. Kyusaku Ogino (1950), Prof. Takashi Kobayashi, University of Tokyo (1961, 1970), and Prof. Shoichi Sakamoto, University of Tokyo (1981). The nerve-sparing radical hysterectomy without sacrificing radicality was introduced in 19611 and improved in 1970 by Prof. Kobayashi.2 The autonomic nerve pathway including hypogastric nerve (sympathetic nerve), pelvic splanchnic nerve (parasympathetic nerve), and pelvic nerve plexus as a junction of the two nerves and the branch of the plexus to the bladder (vesical nerve branch) are preserved except in advanced cases. He divided the process of nerve-sparing surgery into four steps for separating the autonomic nerve pathway from adjacent tissues along the pathway consisting of cardinal, sacrouterine, rectouterine/vaginal, and vesicouterine ligaments. The first step is separation of the cardinal ligament (deep uterine vessels) from the pelvic splanchnic nerve. The second step is separation of the medial side of severed cardinal ligament from the pelvic nerve plexus. The first and second steps are performed in the lateral side of the autonomic nerve system. The third step is separation of sacrouterine and rectouterine/vaginal ligaments from hypogastric nerve and pelvic nerve plexus. The third step is necessary for achieving high radicality, namely, for severing the sacrouterine and rectouterine/vaginal ligaments near the rectum without damage to the pelvic nerve plexus. The fourth step is separation of paravaginal tissues and posterior (deep) layer of the vesicouterine ligament from the vesical nerve branches of the plexus. The third and fourth steps are performed in the medial side of the autonomic nerve system.

4.1N-Mediated Interactions and Functions in Nerve System and Cancer

Scaffolding protein 4.1N is a neuron-enriched 4.1 homologue. 4.1N contains three conserved domains, including the N-terminal 4.1-ezrin-radixin-moesin (FERM) domain, internal spectrin–actin–binding (SAB) domain, and C-terminal domain (CTD). Interspersed between the three domains are nonconserved domains, including U1, U2, and U3. The role of 4.1N was first reported in the nerve system. Then, extensive studies reported the role of 4.1N in cancers and other diseases. 4.1N performs numerous vital functions in signaling transduction by interacting, locating, supporting, and coordinating different partners and is involved in the molecular pathogenesis of various diseases. In this review, recent studies on the interactions between 4.1N and its contactors (including the α7AChr, IP3R1, GluR1/4, GluK1/2/3, mGluR8, KCC2, D2/3Rs, CASK, NuMA, PIKE, IP6K2, CAM 1/3, βII spectrin, flotillin-1, pp1, and 14-3-3) and the 4.1N-related biological functions in the nerve system and cancers are specifically and comprehensively discussed. This review provides critical detailed mechanistic insights into the role of 4.1N in disease relationships.