Cora Sau-Wan Lai

Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinsons disease research.

Opposite effects of fear conditioning and extinction on dendritic spine remodelling

It is generally believed that fear extinction is a form of new learning that inhibits rather than erases previously acquired fear memories. Although this view has gained much support from behavioural and electrophysiological studies, the hypothesis that extinction causes the partial erasure of fear memories remains viable. Using transcranial two-photon microscopy, we investigated how neural circuits are modified by fear learning and extinction by examining the formation and elimination of postsynaptic dendritic spines of layer-V pyramidal neurons in the mouse frontal association cortex. Here we show that fear conditioning by pairing an auditory cue with a footshock increases the rate of spine elimination. By contrast, fear extinction by repeated presentation of the same auditory cue without a footshock increases the rate of spine formation. The degrees of spine remodelling induced by fear conditioning and extinction strongly correlate with the expression and extinction of conditioned fear responses, respectively. Notably, spine elimination and formation induced by fear conditioning and extinction occur on the same dendritic branches in a cue- and location-specific manner: cue-specific extinction causes formation of dendritic spines within a distance of two micrometres from spines that were eliminated after fear conditioning. Furthermore, reconditioning preferentially induces elimination of dendritic spines that were formed after extinction. Thus, within vastly complex neuronal networks, fear conditioning, extinction and reconditioning lead to opposing changes at the level of individual synapses. These findings also suggest that fear memory traces are partially erased after extinction.

Sleep promotes branch-specific formation of dendritic spines after learning

To sleep, perchance to remember Many researchers believe sleep helps us consolidate our memories, but no one knows quite how. Yang et al. investigated the precise role of sleep in changing mouse brain structures (see the Perspective by Euston and Steenland). When mice learned motor tasks, small protuberances—or “spines”—formed on some of the dendritic branches of specific brain neurons. These spines represent the physical correlate of a memory. But the neurons grew and retained these spines better when the mice slept after learning the task. Neurons that fired during learning fired again during subsequent slow-wave sleep, allowing the mice to conserve the newly formed spines—and memories. Science, this issue p. 1173; see also p. 1087 In mice, synaptic connectivity changes after motor learning are influenced by sleep. [Also see Perspective by Euston and Steenland] How sleep helps learning and memory remains unknown. We report in mouse motor cortex that sleep after motor learning promotes the formation of postsynaptic dendritic spines on a subset of branches of individual layer V pyramidal neurons. New spines are formed on different sets of dendritic branches in response to different learning tasks and are protected from being eliminated when multiple tasks are learned. Neurons activated during learning of a motor task are reactivated during subsequent non–rapid eye movement sleep, and disrupting this neuronal reactivation prevents branch-specific spine formation. These findings indicate that sleep has a key role in promoting learning-dependent synapse formation and maintenance on selected dendritic branches, which contribute to memory storage.

The pattern of cortical dysfunction in a mouse model of a schizophrenia-related microdeletion.

We used a mouse model of the schizophrenia-predisposing 22q11.2 microdeletion to evaluate how this genetic lesion affects cortical neural circuits at the synaptic, cellular, and molecular levels. Guided by cognitive deficits, we demonstrated that mutant mice display robust deficits in high-frequency synaptic transmission and short-term plasticity (synaptic depression and potentiation), as well as alterations in long-term plasticity and dendritic spine stability. Apart from previously reported reduction in dendritic complexity of layer 5 pyramidal neurons, altered synaptic plasticity occurs in the context of relatively circumscribed and often subtle cytoarchitectural changes in neuronal density and inhibitory neuron numbers. We confirmed the pronounced DiGeorge critical region 8 (Dgcr8)-dependent deficits in primary micro-RNA processing and identified additional changes in gene expression and RNA splicing that may underlie the effects of this mutation. Reduction in Dgcr8 levels appears to be a major driver of altered short-term synaptic plasticity in prefrontal cortex and working memory but not of long-term plasticity and cytoarchitecture. Our findings inform the cortical synaptic and neuronal mechanisms of working memory impairment in the context of psychiatric disorders. They also provide insight into the link between micro-RNA dysregulation and genetic liability to schizophrenia and cognitive dysfunction.

Antagonizing β-amyloid peptide neurotoxicity of the anti-aging fungus Ganoderma lucidum

Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi) is a medicinal fungus used clinically in many Asian countries to promote health and longevity. Synaptic degeneration is another key mode of neurodegeneration in Alzheimers disease (AD). Recent studies have shown the loss of synaptic density proteins in each individual neuron during the progression of AD. It was recently reported that beta-amyloid (Abeta) could cause synaptic dysfunction and contribute to AD pathology. In this study, we reported that aqueous extract of G. lucidum significantly attenuated Abeta-induced synaptotoxicity by preserving the synaptic density protein, synaptophysin. In addition, G. lucidum aqueous extract antagonized Abeta-triggered DEVD cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of c-Jun N-terminal kinase, c-Jun, and p38 MAP kinase was attenuated by G. lucidum in Abeta-stressed neurons. Taken together, the results prove a hypothesis that anti-aging G. lucidum can prevent harmful effects of the exterminating toxin Abeta in AD.

A pro-drug of the green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) prevents differentiated SH-SY5Y cells from toxicity induced by 6-hydroxydopamine

Regular consumption of green tea benefits people in prevention from cardiovascular disorders, obesity as well as neurodegenerative diseases. (-)-Epigallocatechin-3-gallate (EGCG) is regarded as the most biologically active catechin in green tea. However, the stability and bioavailability of EGCG are restricted. The purpose of the present study was to investigate whether a pro-drug, a fully acetylated EGCG (pEGCG), could be more effective in neuroprotection in Parkinsonism mimic cellular model. Retinoic acid (RA)-differentiated neuroblastoma SH-SY5Y cells were pre-treated with different concentrations of EGCG and pEGCG for 30 min and followed by incubation of 25 microM 6-hydroxydopamine (6-OHDA) for 24h. We found that a broad dosage range of pEGCG (from 0.1 to 10 microM) could significantly reduce lactate dehydrogenase release. Likewise, 10 microM of pEGCG was effective in reducing caspase-3 activity, while EGCG at all concentrations tested in the model failed to attenuate caspase-3 activity induced by 6-OHDA. Furthermore, Western-blot analysis showed that Akt could be one of the specific signaling pathways stimulated by pEGCG in neuroprotection. It was demonstrated that 25 microM of 6-OHDA significantly suppressed the phosphorylation level of Akt. Only pEGCG at 10 microM markedly increased its phosphorylation level compared to 6-OHDA alone. Taken together, as pEGCG has higher stability and bioavailability for further investigation, it could be a potential neuroprotective agent and our current findings may offer certain clues for optimizing its application in future.

Low molecular weight Aβ induces collapse of endoplasmic reticulum

The endoplasmic reticulum (ER) is a dynamic multifunction organelle that is responsible for Ca(2+) homeostasis, protein folding, post-translational modification, protein degradation, and transportation of nascent proteins. Disruption of ER architecture might affect the normal physiology of the cell. In yeast, expansion of the ER is observed under unfolded protein response (UPR) and subsequently induces autophagy initiated from the ER. Here, we found that soluble low molecular weight of Abeta disrupted the anchoring between ER and microtubules (MT) and induced collapse of ER. In addition, it decreased the stability of MT. Subsequently, low molecular weight Abeta triggered autophagy and enhanced lysosomal degradation, as shown by electron microscopy and live-cell imaging. Dysfunction of ER can be further proved in postmortem AD brain and transgenic mice bearing APP Swedish mutation by immunohistochemical analysis of calreticulin. Treatment with Taxol, a MT-stabilizing agent, could partially inhibit collapse of the ER and induction of autophagy. The results show that Abeta-induced disruption of MT can affect the architecture of the ER. Collapse/aggregation of the ER may play an important role in Abeta peptide-triggered neurodegenerative processes.

Significance of Molecular Signaling for Protein Translation Control in Neurodegenerative Diseases

It has long been known that protein synthesis is inhibited in neurological disorders. Protein synthesis includes protein transcription and translation. While many studies about protein transcription have been done in the last decade, we are just starting to understand more about the impact of protein translation. Protein translation control can be accomplished at the initiation or elongation steps. In this review, we will focus on translation control at initiation. Neurons have long neurites in which proteins have to be transported from the cell body to the end of the neurite. Since supply of proteins cannot meet the need of neuronal activity at the spine, protein locally translated at the spine will be a good solution to replace the turnover of proteins. Therefore, local protein translation is an important mechanism to maintain normal neuronal functions. In this notion, we have to separate the concept of global and local protein translation control. Both global and local protein translation control modulate normal neuronal functions from development to cognitive functions. Increasing lines of evidence show that they also play significant roles in neurodegenerative diseases, e.g. neuronal apoptosis, synaptic degeneration and autophagy. We summarize all the evidence in this review and focus on the control at initiation. The new live-cell imaging technology together with photoconvertible fluorescent probes allows us to investigate newly translated proteins in situ. Protein translation control is another line to modulate neuronal function in neuron-neuron communication as well as in response to stress in neurodegenerative diseases.

Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weight β-amyloid peptide

Alzheimers disease (AD) is an aging‐related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP‐ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up‐regulated the lysoso‐mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3‐methylade‐nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ‐induced apoptosis.

Treadmill exercise suppressed stress-induced dendritic spine elimination in mouse barrel cortex and improved working memory via BDNF/TrkB pathway

Stress-related memory deficit is correlated with dendritic spine loss. Physical exercise improves memory function and promotes spinogenesis. However, no studies have been performed to directly observe exercise-related effects on spine dynamics, in association with memory function. This study utilized transcranial two-photon in vivo microscopy to investigate dendritic spine formation and elimination in barrel cortex of mice under physical constrain or naive conditions, followed by memory performance in a whisker-dependent novel texture discrimination task. We found that stressed mice had elevated spine elimination rate in mouse barrel cortex plus deficits in memory retrieval, both of which can be rescued by chronic exercise on treadmill. Exercise also elevated brain-derived neurotrophic factor (BDNF) expression in barrel cortex. The above-mentioned rescuing effects for both spinognesis and memory function were abolished after inhibiting BDNF/tyrosine kinase B (TrkB) pathway. In summary, this study demonstrated the improvement of stress-associated memory function by exercise via facilitating spine retention in a BDNF/TrkB-dependent manner.Stress-related memory deficit is correlated with dendritic spine loss. Physical exercise improves memory function and promotes spinogenesis. However, no studies have been performed to directly observe exercise-related effects on spine dynamics, in association with memory function. This study utilized transcranial two-photon in vivo microscopy to investigate dendritic spine formation and elimination in barrel cortex of mice under physical constrain or naive conditions, followed by memory performance in a whisker-dependent novel texture discrimination task. We found that stressed mice had elevated spine elimination rate in mouse barrel cortex plus deficits in memory retrieval, both of which can be rescued by chronic exercise on treadmill. Exercise also elevated brain-derived neurotrophic factor (BDNF) expression in barrel cortex. The above-mentioned rescuing effects for both spinognesis and memory function were abolished after inhibiting BDNF/tyrosine kinase B (TrkB) pathway. In summary, this study demonstrated the improvement of stress-associated memory function by exercise via facilitating spine retention in a BDNF/TrkB-dependent manner.