Kaneyasu Nishimura

Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells

The balance of bone morphogenic protein (BMP), transforming growth factor‐β (TGFβ)/activin/nodal, and Wnt signals regulates the early lineage segregation of human embryonic stem cells (ESCs). Here we demonstrate that a combination of small‐molecule inhibitors of BMP (Dorsomorphin) and TGFβ/activin/nodal (SB431542) signals promotes highly efficient neural induction from both human ESCs and induced pluripotent stem cells (iPSCs). The combination of small molecules had effects on both cell survival and purity of neural differentiation, under conditions of stromal (PA6) cell coculture and feeder‐free floating aggregation culture, for all seven pluripotent stem cell lines that we studied, including three ESC and four iPSC lines. Small molecule compounds are stable and cost effective, so our findings provide a promising strategy for controlled production of neurons in regenerative medicine.

Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells

Summary Understanding human embryonic ventral midbrain is of major interest for Parkinson’s disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.

ANALYSIS OF MOTOR FUNCTION MODULATED BY CHOLINERGIC NEURONS IN PLANARIAN DUGESIA JAPONICA

Recent studies of the freshwater planarian Dugesia japonica have revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. Here, we identified the gene for planarian choline acetyltransferase (Djchat), which is essential for acetylcholine (ACh) biosynthesis. Immunofluorescence studies using anti-Dugesia japonica ChAT (DjChAT) antibody revealed that cholinergic neurons are widely distributed in the planarian nervous system, including the brain, ventral nerve cords, optic nerves, and pharyngeal nerve plexus. In order to investigate the function of cholinergic neurons in planarians, we used both pharmacological and RNA interference (RNAi) approaches. Administration of physostigmine (an acetylcholinesterase inhibitor) clearly elevated the amount of ACh, and then induced sudden muscle contraction behavior in a concentration-dependent manner. In addition, we found that pretreatment with tubocurarine (a muscle nicotinic ACh receptor antagonist) or atropine (a non-selective muscarinic ACh receptor antagonist), but not pretreatment with mecamylamine (a neural nicotinic ACh receptor antagonist), significantly extended the latency time for physostigmine-induced contraction behavior, suggesting that muscle nicotinic ACh receptors and muscarinic ACh receptors contribute to physostigmine-induced contraction behavior. We also confirmed that ACh biosynthesis ability and DjChAT-immunoreactivity were eliminated in Djchat(RNAi) planarians. Moreover, the decrease of the level of ACh induced by Djchat(RNAi) caused extension of the latency time for contraction behavior. Our findings support the possibility that the cholinergic functions of planarians are similar to those of vertebrates, suggesting that planarians are simple but useful model organisms for getting insight into the cholinergic nervous system in higher animals.

Identification and distribution of tryptophan hydroxylase (TPH)-positive neurons in the planarian Dugesia japonica.

We identified a full-length tryptophan hydroxylase (TPH) gene of planarian Dugesia japonica from a head EST database, and named it DjTPH. Based on whole-mount in situ hybridization and immunofluorescence analyses, DjTPH mRNA and protein were mainly expressed in the nervous system, especially ventral nerve cords and eye pigment cells. Furthermore, DjTPH immunoreactivity was clearly detected at commissure axonal connections in the ventral nerve cords. 5-HT was significantly decreased in DjTPH-knockdown planarians compared with control animals. These results suggest that DjTPH is required for 5-HT biosynthesis, and DjTPH antibody is a useful marker for serotonergic neurons in planarians.

Identification of glutamic acid decarboxylase gene and distribution of GABAergic nervous system in the planarian Dugesia japonica

The planarian Dugesia japonica has a relatively well-organized CNS that includes the brain and the ventral nerve cords, and also has high regenerative capacity derived from pluripotent stem cells present in the mesenchymal space throughout the body. Glutamic acid decarboxylase (GAD) is the enzyme that converts glutamic acid into GABA, a major inhibitory neurotransmitter. In this study, we first identified a full-length GAD gene (DjGAD, D. japonica glutamic acid decarboxylase) in the planarian D. japonica. Whole-mount in situ hybridization revealed that a few cells expressed DjGAD mRNA, and these cells were located in both the head and pharynx regions. In order to examine the distribution pattern of DjGAD protein, we generated a mouse monoclonal anti-DjGAD antibody. The distribution pattern of DjGAD protein was very similar to that of DjGAD mRNA. A neural network of DjGAD-immunopositive cells was also clearly observed. In addition, we examined the immunofluorescence during the process of regeneration of the head from the tail piece. At day 3 of regeneration, we could detect newly formed DjGAD-immunopositive neurons in the anterior region. During day 5-7 of regeneration, reconstruction of the neural network of DjGAD-immunopositive cells occurred. DjGAD-immunoreactivity was lost in DjGAD-knockdown planarians obtained by RNA interference. The amount of GABA was significantly decreased in DjGAD-knockdown planarians, which lost negative phototaxis but not locomotion activity. These results suggest that DjGAD is clearly required for GABA biosynthesis and photosensitivity in planarians, and expression of DjGAD as detected by anti-DjGAD antibody is a useful marker for GABAergic neurons.

Characterization of tyramine β-hydroxylase in planarian Dugesia japonica : Cloning and expression

The planarian Dugesia japonica has a relatively well-organized central nervous system (CNS) consisting of a brain and ventral nerve cords (VNCs), and can completely regenerate it CNS utilizing pluripotent stem cells present in the mesenchymal space. This remarkable capacity has begun to be exploited for research on neural regeneration. Recently, several kinds of molecular markers for labeling of neural subtypes have been reported in planarians. These molecular markers are useful for visualizing the distinct neural populations in planarians. In this study, we isolated a cDNA encoding tyramine beta-hydroxylase (TBH), an octopamine (OA) biosynthetic enzyme, by degenerate PCR in the planarian D. japonica, and named it DjTBH (D. japonica tyramine beta-hydroxylase). In order to examine whether DjTBH contributes to OA biosynthesis, we measured the OA content in DjTBH-knockdown planarians created by RNA interference. In addition, to examine the specificity of DjTBH for OA biosynthesis, we measured not only OA content but also noradrenaline (NA) content, because NA is synthesized by a pathway similar to that for OA. According to high-performance liquid chromatography analysis, the amount of OA, but not NA, was significantly decreased in DjTBH-knockdown planarians. In addition, we produced anti-DjTBH antibody to visualize the octopaminergic neural network. As shown by immunofluorescence analysis using anti-DjTBH antibody, DjTBH-immunopositive neurons were mainly distributed in the head region, and elongated their dendrites and/or axons along the VNCs. In order to visualize octopaminergic and dopaminergic nervous systems (phenolamine/catecholamine nervous system) in the planarian CNS, double-immunofluorescence analysis was carried out using both anti-DjTBH antibody and anti-DjTH (a planarian tyrosine hydroxylase) antibody. DjTBH-immunopositive neurons and DjTH-immunopositive neurons mainly formed distinct neural networks in the head region. Here, we demonstrated that DjTBH clearly contributes to OA biosynthesis, and DjTBH antibody is a useful tool for detecting octopaminergic neurons in planarians.

Planarians maintain a constant ratio of different cell types during changes in body size by using the stem cell system

Planarians change in body size depending upon whether they are in feeding or starving conditions. To investigate how planarians regulate this flexible system, the numbers of total cells and specific cell types were counted and compared among worms 2 mm to 9 mm in body length. The total cell number increased linearly with increasing body length, but the ratio of cell numbers between the head and the trunk portion was constant (1:3). Interestingly, counting the numbers of specific neurons in the eye and brain after immunostaining using cell type-specific antibodies revealed that the ratio between different neuron types was constant regardless of the brain and body size. These results suggest that planarians can maintain proportionality while changing their body size by maintaining a constant ratio of different cell types. To understand this system and reveal how planarians restore the original ratio during eye and brain regeneration, the numbers of specialized cells were Investigated during regeneration. The results further substantiate the existence of some form of “counting mechanism” that has the ability to regulate both the absolute and relative numbers of different cell types in complex organs such as the brain during cell turnover, starvation, and regeneration.

Regeneration in an evolutionarily primitive brain - the planarian Dugesia japonica model

A unique aspect of planarians is that they can regenerate a brain from somatic pluripotent stem cells called neoblasts, which have the ability to produce themselves (self‐renew) and to give rise to all missing cell types during regeneration. Recent molecular studies have revealed that the planarian brain is composed of many distinct neuronal populations, which are evolutionarily and functionally conserved ones, and acts as an information‐processing center to elicit distinct behavioral traits depending on a variety of signals arising from the external environment. How can planarians regenerate such a brain? On the basis of our recent findings, here we review the cellular and molecular mechanisms that regulate the stem cell dynamics involved in the brain regeneration of the planarian Dugesia japonica. Our findings suggest the possible value of in vivo planarian studies for guiding regenerative medicine to treat neurodegenerative diseases via interlinking stem cell biology and regeneration biology.

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain.

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07518.x

Serofendic acid prevents 6-hydroxydopamine-induced nigral neurodegeneration and drug-induced rotational asymmetry in hemi-parkinsonian rats.

Serofendic acid was recently identified as a neuroprotective factor from fetal calf serum. This study was designed to evaluate the neuroprotective effects of an intranigral microinjection of serofendic acid based on behavioral, neurochemical and histochemical studies in hemi‐parkinsonian rats using 6‐hydroxydopamine (6‐OHDA). Rats were injected with 6‐OHDA in the presence or absence of serofendic acid, or were treated with serofendic acid on the same lateral side, at 12, 24 or 72 h after 6‐OHDA lesion. Intranigral injection of 6‐OHDA alone induced a massive loss of tyrosine hydroxylase (TH)‐immunopositive neurons in the substantia nigra pars compacta (SNpc). Either simultaneous or 12 h post‐administration of serofendic acid significantly prevented both dopaminergic neurodegeneration and drug‐induced rotational asymmetry. Immunoreactivities for oxidative stress markers, such as 3‐nitrotyrosine (3‐NT) and 4‐hydroxy‐2‐nonenal (4‐HNE), were markedly detected in the SNpc of rats injected with 6‐OHDA alone. These immunoreactivities were markedly suppressed by the co‐administration of serofendic acid, similar to the results in vehicle‐treated control rats. In addition, serofendic acid inhibited 6‐OHDA‐induced α‐synuclein expression and glial activation in the SNpc. These results suggest that serofendic acid protects against 6‐OHDA‐induced SNpc dopaminergic neurodegeneration in a rat model of Parkinsons disease.