Cerebellar Granule Cells Research Articles

Cerebellar granule cell migration and folia development require Mllt11/Af1q/Tcf7c.

The organization of neurons into distinct layers, known as lamination, is a common feature of the nervous system. This process, which arises from the direct coupling of neurogenesis and neuronal migration, plays a crucial role in the development of the cerebellum, a structure exhibiting a distinct folding cytoarchitecture with cells arranged in discrete layers. Disruptions to neuronal migration can lead to various neurodevelopmental disorders, highlighting the significance of understanding the molecular regulation of lamination. We report a role Mllt11/Af1q/Tcf7c (myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 fused gene from chromosome 1q, also known as Mllt11 transcriptional cofactor 7; henceforth referred to Mllt11) in the migration of cerebellar granule cells (GCs). We now show that Mllt11 plays a role in both the tangential and radial migration of GCs. Loss of Mllt11 led to an accumulation of GC precursors in the rhombic lip region and a reduction in the number of GCs successfully populating developing folia. Consequently, this results in smaller folia and an overall reduction in cerebellar size. Furthermore, analysis of the anchoring centers reveals disruptions in the perinatal folia cytoarchitecture, including alterations in the Bergmann glia fiber orientation and reduced infolding of the Purkinje cell plate. Lastly, we demonstrate that Mllt11 interacts with non-muscle myosin IIB (NMIIB) and Mllt11 loss-reduced NMIIB expression. We propose that the dysregulation of NMIIB underlies altered GC migratory behavior. Taken together, the findings reported herein demonstrate a role for Mllt11 in regulating neuronal migration within the developing cerebellum, which is necessary for its proper neuroanatomical organization.

Joubert syndrome-derived induced pluripotent stem cells show altered neuronal differentiation in vitro.

Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the "molar tooth sign." Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.

Gap Junctions May Have A Computational Function In The Cerebellum: A Hypothesis

In the cerebellum, granule cells make parallel fibre contact on (and excite) Golgi cells and Golgi cells inhibit granule cells, forming an open feedback loop. Parallel fibres excite Golgi cells synaptically, each making a single contact. Golgi cells inhibit granule cells in a structure called a glomerulus almost exclusively by GABA spillover acting through extrasynaptic GABAA receptors. Golgi cells are connected dendritically by gap junctions. It has long been suspected that feedback contributes to homeostatic regulation of parallel fibre signals activity, causing the fraction of the population that are active to be maintained at a low level. We present a detailed neurophysiological and computationally-rendered model of functionally grouped Golgi cells which can infer the density of parallel fibre signals activity and convert it into proportional modulation of inhibition of granule cells. The conversion is unlearned and not actively computed; rather, output is simply the computational effect of cell morphology and network architecture. Unexpectedly, the conversion becomes more precise at low density, suggesting that self-regulation is attracted to sparse code, because it is stable. A computational function of gap junctions may not be confined to the cerebellum.

Simple spike patterns and synaptic mechanisms encoding sensory and motor signals in Purkinje cells and the cerebellar nuclei

Whisker stimulation in awake mice evokes transient suppression of simple spike probability in crus I/II Purkinje cells. Here, we investigated how simple spike suppression arises synaptically, what it encodes, and how it affects cerebellar output. Invitro, monosynaptic parallel fiber (PF)-excitatory postsynaptic currents (EPSCs) facilitated strongly, whereas disynaptic inhibitory postsynaptic currents (IPSCs) remained stable, maximizing relative inhibitory strength at the onset of PF activity. Short-term plasticity thus favors the inhibition of Purkinje spikes before PFs facilitate. Invivo, whisker stimulation evoked a 2-6ms synchronous spike suppression, just 6-8ms (∼4 synaptic delays) after sensory onset, whereas active whisker movements elicited broadly timed spike rate increases that did not modulate sensory-evoked suppression. Firing in the cerebellar nuclei (CbN) inversely correlated with disinhibition from sensory-evoked simple spike suppressions but was decoupled from slow, non-synchronous movement-associated elevations of Purkinje firing rates. Synchrony thus allows the CbN to high-pass filter Purkinje inputs, facilitating sensory-evoked cerebellar outputs that can drive movements.

Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey’s fibres and not by its mineral phase

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey’s fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Parvalbumin, calbindin, and calretinin immunostaining of 10 neural structures within the cerebellar cortex of 143 mammal species

In the current study we employed immunohistochemical techniques to investigate the localisation of three calcium-binding proteins (parvalbumin, calbindin, and calretinin) in 10 neuronal structures of the cerebellar cortex (stellate cells, basket cells, parallel fibres, climbing fibres, Purkinje cells, granule cells, Golgi type II cells, Lugaro neurons, unipolar brush neurons, and mossy fibres) in 143 species from across the mammalian radiation. Most often, parvalbumin was localised in the neural structures of the molecular and Purkinje cell layers but was absent in the granule cell layer. Calbindin was most often immunolocalised in the neural structures of the Purkinje cell layer and mossy fibres, whereas calretinin was most often immunolocalised in the climbing fibres of the molecular layer and all neural structures of the granule cell layer. Despite this general consistency, variations in the localisation of these three calcium-binding proteins were found in every lineage, and almost every species, the one exception being the western tree hyrax that showed the full suite of most often observed calcium-binding protein chemoarchitecture for the mammalian cerebellar cortex. These consistencies and variances in the calcium-binding protein chemoarchitecture of the cerebellar cortex of mammals may play significant roles in the species-specific learning and refining of motor, perceptual, and cognitive skills and capacities required to survive in the environments they inhabit.

A Microfluidic Perfusion Culture Setup to Investigate Cell Migration in 3D Constrictions

AbstractCell migration is a fundamental process underlying the morphological maturation of organs, but also in disease‐related conditions such as cancer. Cells are able to migrate through crowded space and a tight extracellular matrix (ECM). Passing through a constriction, a cell deforms strongly, including its nucleus. Such nuclear deformation can lead to changes in the 3D‐genomic architecture, and putatively, DNA methylation. However, the specific effects of deformation on cells are not well understood. It is highly desired to establish an ex vivo methodology to induce well‐defined cell deformation in complex geometrical constrictions. This study introduces a microfluidic system for the study of migrating cells in precisely controlled geometrical confinement. A procedure for coating, seeding of cerebellar granule cells, and perfusion culture is presented. By leveraging direct laser writing, channels with smooth, anisotropically curved surfaces on the cell‐scale can be fabricated. The system consists of constriction channels with a radius of 2 or 4 µm for the cells to pass through. This corresponds to a compression of the nucleus to 3.5% and 14.2% of its undeformed cross‐sectional area, respectively. The system can be used to investigate the influence of confinement geometry on the migration behavior and transcriptome of various cell types.

Lateral Ankle Ligaments: An Insight Into Their Functional Anatomy, Variations, and Surgical Importance.

Ankle sprains are prevalent injuries leading to functional impairment. The lateral ankle ligament complex (LLC), comprising the anterior talofibular ligament (ATFL), posterior talofibular ligament (PTFL), and calcaneofibular ligament (CFL), is weak and prone to injury. The morphometric data of these ligaments are essential for orthopedic practices, including techniques like direct repair or ATFL reconstruction with autograft/allograft, which are limited in the literature. The present study aims to document the anatomy and morphometry of the LLC. Fifteen adult Indian-origin embalmed cadavers were selected for the study. Ankles with antemortem or postmortem injuries or previous surgical interventions were excluded from the study. After precise dissection of the ankle's anterior and lateral aspects as per Cunningham's dissection manual, ligaments were exposed. Length and width were measured using a digital vernier caliper. Morphological attributes such as shape, orientation, and inter-fiber angles were documented. The most common shape in ATFL was a single band (53.33%). Inner ATFL fibers merged with the ankle joint capsule in 73.33%. ATFL mean length and width were 14 ± 2.4 mm and 7.6 ± 2.0 mm. The angle between the fibula's long axis and ATFL fibers was 107 ± 22°, and the angle between tibiotalar joint lines and parallel ATFL fibers was 30 ± 9.5°. A single band of CFL was predominant (73.33%). The mean length and width ofCFL were 18.4 ± 3.9 mm and 5.2 ± 1.3 mm; the angle between the anterior fibula border's long axes and parallel CFL line was 131°. PTFL length was 20.9 ± 3.3 mm and width was 6.2 ± 1.4 mm. The mean length and width of the anterior inferior talofibular ligament (AiTFL) were 11.7 ± 2.6 mm and 9.5 ± 1.6 mm, and of the posterior inferior talofibular ligament (PiTFL) were 12.8 ± 2.1 mm and 10.4 ± 2 mm. Comprehensive knowledge of these ligaments' anatomy and relationships is vital for clinical examination and ultrasonography. Understanding LLCdetails aids radiologists and orthopedic surgeons in graft selection, sizing, and precise anatomical structure placement during surgical reconstruction.

Local synaptic inhibition mediates cerebellar granule cell pattern separation and enables learned sensorimotor associations.

The cerebellar cortex has a key role in generating predictive sensorimotor associations. To do so, the granule cell layer is thought to establish unique sensorimotor representations for learning. However, how this is achieved and how granule cell population responses contribute to behavior have remained unclear. To address these questions, we have used in vivo calcium imaging and granule cell-specific pharmacological manipulation of synaptic inhibition in awake, behaving mice. These experiments indicate that inhibition sparsens and thresholds sensory responses, limiting overlap between sensory ensembles and preventing spiking in many granule cells that receive excitatory input. Moreover, inhibition can be recruited in a stimulus-specific manner to powerfully decorrelate multisensory ensembles. Consistent with these results, granule cell inhibition is required for accurate cerebellum-dependent sensorimotor behavior. These data thus reveal key mechanisms for granule cell layer pattern separation beyond those envisioned by classical models.

Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Targeted therapy improves cellular dysfunction, ataxia, and seizure susceptibility in a model of a progressive myoclonus epilepsy

The recurrent variant KCNC1-p.Arg320His causes progressive myoclonus epilepsy (EPM) type 7, defined by progressive myoclonus, epilepsy, and ataxia, and is without effective treatment. KCNC1 encodes the voltage-gated potassium channel subunit Kv3.1, specifically expressed in high-frequency-firing neurons. Variant subunits act via loss of function; hence, EPM7 pathogenesis may involve impaired excitability of Kv3.1-expressing neurons, while enhancing Kv3 activity could represent a viable therapeutic strategy. We generate a mouse model, Kcnc1-p.Arg320His/+, which recapitulates the core features of EPM7, including progressive ataxia and seizure susceptibility. Kv3.1-expressing cerebellar granule cells and neocortical parvalbumin-positive GABAergic interneurons exhibit abnormalities consistent with Kv3 channel dysfunction. A Kv3-specific positive modulator (AUT00206) selectively enhances the firing frequency of Kv3.1-expressing neurons and improves motor function and seizure susceptibility in Kcnc1-Arg320His/+ mice. This work identifies a cellular and circuit basis of dysfunction in EPM7 and demonstrates that Kv3 positive modulators such as AUT00206 have therapeutic potential for the treatment of EPM7.

RhoGDI phosphorylation by PKC promotes its interaction with death receptor p75NTR to gate axon growth and neuron survival

How receptors juggle their interactions with multiple downstream effectors remains poorly understood. Here we show that the outcome of death receptor p75NTR signaling is determined through competition of effectors for interaction with its intracellular domain, in turn dictated by the nature of the ligand. While NGF induces release of RhoGDI through recruitment of RIP2, thus decreasing RhoA activity in favor of NFkB signaling, MAG induces PKC-mediated phosphorylation of the RhoGDI N-terminus, promoting its interaction with the juxtamembrane domain of p75NTR, disengaging RIP2, and enhancing RhoA activity in detriment of NF-kB. This results in stunted neurite outgrowth and apoptosis in cerebellar granule neurons. If presented simultaneously, MAG prevails over NGF. The NMR solution structure of the complex between the RhoGDI N-terminus and p75NTR juxtamembrane domain reveals previously unknown structures of these proteins and clarifies the mechanism of p75NTR activation. These results show how ligand-directed competition between RIP2 and RhoGDI for p75NTR engagement determine axon growth and neuron survival. Similar principles are likely at work in other receptors engaging multiple effectors and signaling pathways.

Experimental study on a parallel optical fiber Sagnac loops-based sensor with the advantages of both high sensitivity and ultra-wide measurement range

The Vernier effect (VE) in optical interferometers has been used to improve the sensitivity in the measurements of strain, temperature, refractive index, etc. However, as the wavelength shifts beyond a free spectral range (FSR), it is difficult to determine the magnitude of the physical quantity and the measurement range is usually narrow. To overcome this problem, we constructed a fiber optic sensor combining two parallel Sagnac interferometers (PSIs) with a fiber Bragg grating (FBG). The sensitivity of temperature and strain measurements were enhanced about 5 times due to the VE effect in PSIs. FBG spectrum, which showed a monotonic and linear variation trend along with the variation of strain and temperature, was used to estimate the magnitude of the physical quantity beyond the FSR. Finally, an average strain sensitivity of −104.4 pm/µε was obtained in a wide measuring range of 0–5440 µε, while an average temperature sensitivity of 6.12 nm/°C was obtained in the temperature range of 2–58 °C. The measurement range has been extended 6 times and 4 times respectively compared to a single FSR. The integration of PSIs and FBG sensors offers not only high sensitivity, but also a wide measuring range. It can be used in many areas including medical equipment, automotive, and aerospace to make accurate strain and temperature measurements.

Comparison of the filtration performance of konjac glucomannan-based aerogel and cellulose acetate

Abstract Konjac glucomannan (KGM)-based aerogel was used as filtration material to compare filtration performance with conventional cellulose acetate (CA). KGM-based aerogel showed a three-dimensional network structure and higher specific surface area and porosity, providing a longer channel and larger adsorption interface for smoke stream and promoting the multidirectional interception for particulate matters (PMs), which was superior to the parallel fiber structure of CA. The smoke analyzer based on leaf burning was utilized to detect the filtration performance. KGM-based aerogel was significantly better than CA for total PMs, nicotine, and tar interception, with an increase of 11.6%, 22.4%, and 10.3%, respectively.

Why Can Modulation of α6-Containing GABAA Receptors Reduce the Symptoms of Multiple Neuropsychiatric Disorders?

α6-containing GABAA receptors (α6GABAARs) are strongly expressed in cerebellar granule cells, where they mediate a correctly timed and precise coordination of all muscle groups that execute behavior and protect the brain from information overflow. Recently, it was demonstrated that positive modulators with a high selectivity for α6GABAARs (α6-modulators) can reduce the symptoms of multiple neuropsychiatric disorders in respective animal models to an extent comparable with established clinical therapeutics. Here, these incredible findings are discussed and explained. So far, the beneficial actions of α6-modulators and their lack of side effects have only been demonstrated in animal models of the respective disorders. Preclinical studies have demonstrated their suitability for further drug development. Future human studies have to investigate their safety and possible side effects, and to clarify to which extent individual symptoms of the respective disorders can be reduced by α6-modulators in patients during acute and chronic dosing. Due to their broad therapeutic potential, α6-modulators might become a valuable new treatment option for multiple neuropsychiatric disorders.

Bite Force Mapping Based on Distributed Fiber Sensing Network Approach.

Bite force measurements are crucial in the realm of biomedical research, particularly in the areas of dentistry and orthodontic care. Various intraoral devices have been used to assess biting force, but each has limitations and drawbacks. Fiber optic sensors (FOSs) offer advantages such as electrical inertness, immunity to electromagnetic interference, and high sensitivity. Distributed fiber optic sensing allows an increase in the number of sensing points and can interrogate numerous reflections from scattering events within an optical fiber. We present four dental bites with heights of 6 mm, which enabled bilateral measurements. U-shaped sensors were prepared by embedding fibers into silicone by folding a single-mode fiber into four lines and multiplexing eight parallel nanoparticle-doped fibers. Dental bite models were created using two silicone materials (Sorta Clear 18 and Sorta Clear 40). The developed sensors were calibrated by applying weights up to 900 g, resulting in a linear response. Experiments were conducted to compare the efficacy of the dental bites. The collection of massive data was enabled by constructing a 2D map of the dental bites during multi-point sensing.

Copper Ions Reduced Toxicity of Sodium Azide and Lipopolysaccharide on Cultured Cerebellar Granule Neurons

Introduction. Copper ions (Cu2+) are structural elements of proteins such as cytochrome с oxidase (Complex IV), an enzyme that catalyzes the final step of electron transfer to oxygen during oxidative phosphorylation in the mitochondria. With Cu2+ homeostasis being of utmost importance, its disturbances in the central nervous system are involved in the mechanisms of many neurodegenerative and other brain disorders.
 This study aimed to assess the effects of non-toxic copper ion levels on death of cerebellar granule neurons associated with lipopolysaccharide (LPS; in vitro inflammation model) or azide sodium (NaN3; cytochrome с oxidase inhibitor).
 Materials and methods. LPS (10 μg/mL) or NaN3 (250 μM) was added on day 7 to 8 to the culture medium with rat cerebellar cells for 24 hours in vitro. Nitrite concentrations were measured in the culture medium by Griess assay; absorbance was recorded with a spectrophotometer at 540 nm, and morphologically intact cells were counted as survived neurons.
 Results. Added to the culture medium, LPS or NaN3 reduced neuron survival to 15 ± 2% or 20 ± 3% vs. control, respectively. Cu2+ (0.5 to 5.0 μM) increased neuron survival in a dose-dependent manner to 78 ± 4% with toxic levels of LPS and to 86 ± 6% with NaN3 with 5 μM Cu2+. The concentration of nitrites in the control culture medium was 2.0 ± 0.2 μM. Added to the cell cultures, LPS increased the concentration of nitrites to 8.5 ± 0.5 μM. Cu2+ 5 μM did not show any significant effects on nitrite accumulation in the culture medium.
 Conclusions. We showed that copper ions can exert protective effects on neurons against LPS-induced or NaN3-induced toxicity. This protection is likely to be associated rather with Cu2+ interaction with Complex IV of the electron transfer chain in the mitochondria than with inhibition of NO production. Effects of Cu2+ on apoptosis pathway proteins also cannot be ruled out.

Choline supplementation mitigates effects of bilirubin in cerebellar granule neurons in vitro.

Premature infants may suffer from high levels of bilirubin that could lead to neurotoxicity. Bilirubin has been shown to decrease L1-mediated ERK1/2 signaling, L1 phosphorylation, and L1 tyrosine 1176 dephosphorylation. Furthermore, bilirubin redistributes L1 into lipid rafts (LR) and decreases L1-mediated neurite outgrowth. We demonstrate that choline supplementation improves L1 function and signaling in the presence of bilirubin. Cerebellar granule neurons (CGN) were cultured with and without supplemental choline, and the effects on L1 signaling and function were measured in the presence of bilirubin. L1 activation of ERK1/2, L1 phosphorylation and dephosphorylation were measured. L1 distribution in LR was quantified and neurite outgrowth of CGN was determined. Forty µM choline significantly reduced the effect of bilirubin on L1 activation of ERK1/2 by 220% (p = 0.04), and increased L1 triggered changes in tyrosine phosphorylation /dephosphorylation of L1 by 34% (p = 0.026) and 35% (p = 0.02) respectively. Choline ameliorated the redistribution of L1 in lipid rafts by 38% (p = 0.02) and increased L1-mediated mean neurite length by 11% (p = 0.04). Choline pretreatment of CGN significantly reduced the disruption of L1 function by bilirubin. The supplementation of pregnant women and preterm infants with choline may increase infant resilience to the effects of bilirubin. This article establishes choline as an intervention for the neurotoxic effects of bilirubin on lipid rafts. This article provides clear evidence toward establishing one intervention for bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into the mechanism of the ameliorative effect of choline on bilirubin neurotoxicity.

Coumarin-azasugar-benzyl conjugates as non-neurotoxic dual inhibitors of butyrylcholinesterase and cancer cell growth.

We have applied the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to prepare a library of ten coumarin-azasugar-benzyl conjugates and two phthalimide-azasugar-benzyl conjugates with potential anti-Alzheimer and anti-cancer properties. The compounds were evaluated as cholinesterase inhibitors, demonstrating a general preference, of up to 676-fold, for the inhibition of butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE). Nine of the compounds behaved as stronger BuChE inhibitors than galantamine, one of the few drugs in clinical use against Alzheimer's disease. The most potent BuChE inhibitor (IC50 = 74 nM) was found to exhibit dual activities, as it also showed high activity (GI50 = 5.6 ± 1.1 μM) for inhibiting the growth of WiDr (colon cancer cells). In vitro studies on this dual-activity compound on Cerebellar Granule Neurons (CGNs) demonstrated that it displays no neurotoxicity.