Yt Cheung

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.

Filtering of ineffective siRNAs and improved siRNA design tool

MOTIVATION Short interfering RNAs (siRNAs) can be used to suppress gene expression and possess many potential applications in therapy, but how to design an effective siRNA is still not clear. Based on the MPI (Max-Planck-Institute) basic principles, a number of siRNA design tools have been developed recently. The set of candidates reported by these tools is usually large and often contains ineffective siRNAs. In view of this, we initiate the study of filtering ineffective siRNAs. RESULTS The contribution of this paper is 2-fold. First, we propose a fair scheme to compare existing design tools based on real data in the literature. Second, we attempt to improve the MPI principles and existing tools by an algorithm that can filter ineffective siRNAs. The algorithm is based on some new observations on the secondary structure, which we have verified by AI techniques (decision trees and support vector machines). We have tested our algorithm together with the MPI principles and the existing tools. The results show that our filtering algorithm is effective. AVAILABILITY The siRNA design software tool can be found in the website http://www.cs.hku.hk/~sirna/ CONTACT smyiu@cs.hku.hk

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.

A reciprocal relationship between reactive oxygen species and mitochondrial dynamics in neurodegeneration

Mitochondrial fragmentation due to fission/fusion imbalance has often been linked to mitochondrial dysfunction and apoptosis in neurodegeneration. Conventionally, it is believed that once mitochondrial morphology shifts away from its physiological tubular form, mitochondria become defective and downstream apoptotic signaling pathways are triggered. However, our study shows that beta-amyloid (Aβ) induces morphological changes in mitochondria where they become granular-shaped and are distinct from fragmented mitochondria in terms of both morphology and functions. Accumulation of mitochondrial reactive oxygen species triggers granular mitochondria formation, while mitoTEMPO (a mitochondria-targeted superoxide scavenger) restores tubular mitochondrial morphology within Aβ-treated neurons. Interestingly, modulations of mitochondria fission and fusion by genetic and pharmacological tools attenuated not only the induction of granular mitochondria, but also mitochondrial superoxide levels in Aβ−treated neurons. Our study shows a reciprocal relationship between mitochondrial dynamics and reactive oxygen species and provides a new potential therapeutic target at early stages of neurodegenerative disease pathogenesis.

Disruptions of endoplasmic reticulum and mitochondria prime mTOR suppression in low molecular weight Aβ-induced autophagy

Background: Recent studies have demonstrated that soluble Ab dimers isolated from AD brains inhibit long-term potentiation (LTP), reduce dendritic spine density in vitro and upon direct injection into the brain of normal rats disrupt memory (Shankar et al., 2008). In the current study we evaluated the acquisition performance of PSAPP mice in a Morris water maze, and searched for Ab oligomers in brains of PSAPP transgenic mice to determine whether the soluble Ab in the transgenic mice has similar biochemical properties to human Ab extracted from AD brains. Methods: Wild type (wt) and heterozygous PSAPP mice were trained in the Morris water maze (4 trials/day for 9 days) and the latency to reach the platform was recorded for each trial. Soluble Ab oligomers were measured from the brains of the PSAPP transgenic mice using a sensitive immunoprecipitation/Western blotting protocol after homogenizing the whole cerebra in Tris-buffered saline. Results: Aged mice (>12 months) exhibit an increase in plaque load and have deficits in the Morris water maze. We observe high levels of Ab dimers in young transgenic mice (2-3 months of age) which decline before increasing again from 7-12 months. No overt cognitive deficits were seen in the 3 and 9 month old mice suggesting that dimers need to reach a critical threshold before memory deficits, in this specific cognitive paradigm, are observed. Both Ab1-40 and Ab1-42 are present in soluble extracts from PSAPP brains. However, in human AD brains Ab1-42 is the predominant species in the soluble extract. Conclusions: These studies demonstrate that the biochemical and, potentially, the pathophysiological properties of Ab in young PSAPP mice are different from that of Ab extracted from elderly human AD patients.