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Since 2020, synchrotron radiation facilities in several Asia-Pacific countries have been collaborating in a major project called "SYNAPSE"(Synchrotrons for Neuroscience: an Asia-Pacific Scientific Enterprise). They use x-ray imaging to attack in a coordinated fashion one of the major issues in modern science: the structure of animal and human brains, including neurons and connections. The objective is to develop Google-like maps also including detailed structural and functional information for selected regions of interest. The sheer mass of data needed for the objective poses huge problems for the acquisition, processing, storage and use of images. In order to complete the task within a reasonable time, the key element of the SYNAPSE strategy is the parallel and coordinated work of several facilities on the same specimens. This article reviews different aspects of the enterprise, including the foundations of synchrotron radiation, coherence and of its role in advanced imaging, electron accelerators, x-ray optics and detectors. This will provide the foundation for an extensive presentation of the different components of SYNAPSE, with an overview of results already obtained within the consortium.

Mapping the large neural networks of animal and human brains is a fundamental but so far elusive task, because of the massive amount of data and the consequent prohibitively long image taking and processing times. We developed an effective strategy called “AXON” (Accelerated X-ray Observation of Neurons) to solve this problem. AXON can achieve comprehensive whole-brain mapping within a reasonable time by combining fast image taking and processing, plus two other critical performances: three-dimensional (3D) imaging with high and isotropic spatial resolution, and multi-scale resolution. We successfully tested this strategy with coordinated experiments at four synchrotron facilities in Japan, Taiwan, Singapore and Korea on two animal models, Drosophila and mouse. Its performances notably allowed full 3D mapping of the Drosophila brain in a few days. With reasonable improvements, AXON can deliver full mapping of large animal and human brains on a realistic time scale of a few years.

Cytosolic phospholipase A2 (cPLA2) is an enzyme that releases arachidonic acid (AA) for the synthesis of eicosanoids and lysophospholipids which play critical roles in the initiation and modulation of oxidative stress and neuroinflammation. In the central nervous system, cPLA2 activation is implicated in the pathogenesis of various neurodegenerative diseases that involves neuroinflammation, thus making it an important pharmacological target. In this paper, a new class of arachidonic acid (AA) analogues was synthesized and evaluated for their ability to inhibit cPLA2. Several compounds were found to inhibit cPLA2 more strongly than arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor that is commonly used in the study of cPLA2-related neurodegenerative diseases. Subsequent experiments concluded that one of the inhibitors was found to be cPLA2-selective, non-cytotoxic, cell and brain penetrant and capable of reducing reactive oxygen species (ROS) and nitric oxide (NO) production in stimulated microglial cells. Computational studies were employed to understand how the compound interacts with cPLA2.

Rapid advances in sequencing technology have led to an explosive increase in the number of genetic variants identified in patients with neurological disease and have also enabled the assembly of a robust database of variants in healthy individuals. A surprising number of variants in the GRIN genes that encode N-methyl-D-aspartate (NMDA) glutamatergic receptor subunits have been found in patients with various neuropsychiatric disorders, including autism spectrum disorders, epilepsy, intellectual disability, attention-deficit/hyperactivity disorder, and schizophrenia. This review compares and contrasts the available information describing the clinical and functional consequences of genetic variations in GRIN2A and GRIN2B. Comparison of clinical phenotypes shows that GRIN2A variants are commonly associated with an epileptic phenotype but that GRIN2B variants are commonly found in patients with neurodevelopmental disorders. These observations emphasize the distinct roles that the gene products serve in circuit function and suggest that functional analysis of GRIN2A and GRIN2B variation may provide insight into the molecular mechanisms, which will allow more accurate subclassification of clinical phenotypes. Furthermore, characterization of the pharmacological properties of variant receptors could provide the first opportunity for translational therapeutic strategies for these GRIN-related neurological and psychiatric disorders.

Arachidonic acid derivatives equipped with either one or two fluorescent groups attached to the tip of the alkyl chains were synthesized and shown to function as inhibitor and substrate probes of cPLA2. The inhibitor probe was demonstrated to perform dual functions of inhibition and imaging while the substrate probe could be used for activity assay.

The nucleus incertus (NI), located in the caudal brainstem, mainly consists of GABAergic neurons with widespread projections across the brain. It is the chief source of relaxin-3 in the mammalian brain and densely expresses corticotropin-releasing factor type 1 (CRF1) receptors. Several other neurotransmitters, peptides and receptors are reportedly expressed in the NI. In the present investigation, we show the expression of dopamine type-2 (D2) receptors in the NI by reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting (WB) and immunofluorescence (IF). RT-PCR did not show expression of D3 receptors. D2 receptor short isoform (D2S)-like, relaxin-3, CRF1/2 receptor and NeuN immunoreactivity were co-expressed in the cells of the NI. Behavioural effects of D2 receptor activation by intra-NI infusion of quinpirole (a D2/D3 agonist) were evaluated. Hypolocomotion was observed in home cage monitoring system (LABORAS) and novel environment-induced suppression of feeding behavioural paradigms. Thus the D2 receptors expressed in the NI are likely to play a role in locomotion. Based on its strong bidirectional connections to the median raphe and interpeduncular nuclei, the NI was predicted to play a role in modulating behavioural activity and the present results lend support to this hypothesis. This is the first evidence of expression of a catecholamine receptor, D2-like immunoreactivity, in the NI.

The iron siderophore binding protein lipocalin 2 (LCN2, also known as 24p3, NGAL and siderocalin) may be involved in iron homeostasis, but to date, little is known about expression of its putative receptor, brain-type organic cation transporter (BOCT, also known as BOCT1, 24p3R, NGALR and LCN2R), in the brain during neurodegeneration. The present study was carried out to elucidate the expression of LCN2 and BOCT in hippocampus after excitotoxicity induced by the glutamate analog, kainate (KA) and a possible role of LCN2 in neuronal injury. As reported previously, a rapid and sustained induction in expression of LCN2 was found in the hippocampus after intracerebroventicular injection of KA. BOCT was expressed in neurons of the saline-injected control hippocampus, and immunolabel for BOCT protein was preserved in pyramidal neurons of CA1 at 1 day post-KA injection, likely due to the delayed onset of neurodegeneration after KA injection. At 3 days and 2 weeks after KA injections, loss of immunolabel was observed due to degenerated neurons, although remaining neurons continued to express BOCT, and induction of BOCT was found in OX-42 positive microglia. This resulted in an overall decrease in BOCT mRNA and protein expression after KA treatment. Increased expression of the pro-apoptotic marker, Bim, was found in both neurons and microglia after KA injection, but TUNEL staining indicating apoptosis was found primarily in Bim-expressing neurons, but not microglia. Interaction between LCN2 and BOCT was found by DuoLink assay in cultured hippocampal neurons. Apo-LCN2 without iron caused no significant differences in neuronal Bim expression or cell survival, whereas holo-LCN2 consisting of LCN2:iron:enterochelin complex increased Bim mRNA expression and decreased neuronal survival. Together, results suggest that LCN2 and BOCT may have a role in neuronal injury.

Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original “straw-in-straw” method (250 μl sterile straw placed in 500 μl straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.

Cytosolic phospholipase A2 (cPLA2) is an enzyme that releases arachidonic acid (AA) for the synthesis of eicosanoids and lysophospholipids which play critical roles in the initiation and modulation of oxidative stress and neuroinflammation. In the central nervous system, cPLA2 activation is implicated in the pathogenesis of various neurodegenerative diseases that involves neuroinflammation, thus making it an important pharmacological target. In this paper, a new class of arachidonic acid (AA) analogues was synthesized and evaluated for their ability to inhibit cPLA2. Several compounds were found to inhibit cPLA2 more strongly than arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor that is commonly used in the study of cPLA2-related neurodegenerative diseases. Subsequent experiments concluded that one of the inhibitors was found to be cPLA2-selective, non-cytotoxic, cell and brain penetrant and capable of reducing reactive oxygen species (ROS) and nitric oxide (NO) production in stimulated microglial cells. Computational studies were employed to understand how the compound interacts with cPLA2.

Arachidonic acid derivatives equipped with either one or two fluorescent groups attached to the tip of the alkyl chains were synthesized and shown to function as inhibitor and substrate probes of cPLA

Amyloid precursor protein (APP) is commonly associated with Alzheimer disease, but its physiological function remains unknown. Nav1.6 is a key determinant of neuronal excitability in vivo. Because mouse models of gain of function and loss of function of APP and Nav1.6 share some similar phenotypes, we hypothesized that APP might be a candidate molecule for sodium channel modulation. Here we report that APP colocalized and interacted with Nav1.6 in mouse cortical neurons. Knocking down APP decreased Nav1.6 sodium channel currents and cell surface expression. APP-induced increases in Nav1.6 cell surface expression were Go protein-dependent, enhanced by a constitutively active Go protein mutant, and blocked by a dominant negative Go protein mutant. APP also regulated JNK activity in a Go protein-dependent manner. JNK inhibition attenuated increases in cell surface expression of Nav1.6 sodium channels induced by overexpression of APP. JNK, in turn, phosphorylated APP. Nav1.6 sodium channel surface expression was increased by T668E and decreased by T668A, mutations of APP695 mimicking and preventing Thr-668 phosphorylation, respectively. Phosphorylation of APP695 at Thr-668 enhanced its interaction with Nav1.6. Therefore, we show that APP enhances Nav1.6 sodium channel cell surface expression through a Go-coupled JNK pathway.

Local Chinese family enterprises (CFEs) are an integral part of Singapore's economy. The objective of this study is to understand the growth and development of CFEs. The study looks at case studies of eight CFEs that are listed on the Stock Exchange of Singapore. In-depth interviews and documentary analyses are used as research instruments. The research uncovers several interesting findings. For example, CFEs experience four stages of development in a process that leads to public listing: Start-Up, Survival, Stability, and Success. The study uses McKinney's seven S factors of Structure, Strategy, System, Staff, Skill, Style (leadership), and Shared Values to examine the characteristics of each stage. It is found that each stage is dominated by one or two distinct S factors that propel the growth of the firm. There is a significant difference in terms of the roles the S factors play, the forms of the S factor, as well as the dominant S factor of each stage. The findings of the study are significant to CFE owners, entrepreneurs, and educators of cross-cultural management.

We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a straw-in-straw technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.

Relaxin-3 or insulin-like peptide 7 (INSL7) is the most recently discovered relaxin/insulin-like family peptide. Mature relaxin-3 consists of an A chain and a B chain held by disulphide bonds. According to structure activity relationship studies, the relaxin-3 B chain is more important in binding and activating the receptor. RXFP3 (also known as Relaxin-3 receptor 1, GPCR 135, somatostatin- and angiotensin- like peptide receptor or SALPR) was identified as the cognate receptor for relaxin-3 by expression profiles and binding studies. Recent studies imply roles of this system in mediating stress and anxiety, feeding, metabolism and cognition. Stapling of peptides is a technique used to develop peptide drugs for otherwise undruggable targets. The main advantages of stapling include, increased activity due to reduced proteolysis, increased affinity to receptors and increased cell permeability. Stable agonists and antagonists of RXFP3 are crucial for understanding the physiological significance of this system. So far, agonists and antagonists of RXFP3 are peptides. In this study, for the first time, we have introduced stapling of the relaxin-3 B chain at 14th and 18th positions (14s18) and 18th and 22nd position (18s22). These stapled peptides showed greater helicity than the unstapled relaxin-3 B chain in circular dichroism analysis. Both stapled peptides bound RXFP3 and activated RXFP3 as observed in an inhibition of forskolin-induced cAMP assay and a ERK1/2 activation assay, although with different potencies. Therefore, we conclude that stapling of the relaxin3 B chain does not compromise its ability to activate RXFP3 and is a promising method for developing stable peptide agonists and antagonists of RXFP3 to aid relaxin-3/RXFP3 research.

The new Brain Imaging Beamline (BIB) of the Taiwan Photon Source (TPS) has been commissioned and opened to users. The BIB and in particular its endstation are designed to take advantage of bright unmonochromatized synchrotron X-rays and target fast 3D imaging, ∼1 ms exposure time plus very high ∼0.3 µm spatial resolution. A critical step in achieving the planned performances was the solution to the X-ray induced damaging problems of the detection system. High-energy photons were identified as their principal cause and were solved by combining tailored filters/attenuators and a high-energy cut-off mirror. This enabled the tomography acquisition throughput to reach >1 mm3 min−1, a critical performance for large-animal brain mapping and a vital mission of the beamline.