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1.
Sci Rep ; 7(1): 14498, 2017 11 03.
Article in English | MEDLINE | ID: mdl-29101377

ABSTRACT

Loss of synapses or alteration of synaptic activity is associated with cognitive impairment observed in a number of psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. Therefore successful development of in vitro methods that can investigate synaptic function in a high-throughput format could be highly impactful for neuroscience drug discovery. We present here the development, characterisation and validation of a novel high-throughput in vitro model for assessing neuronal function and synaptic transmission in primary rodent neurons. The novelty of our approach resides in the combination of the electrical field stimulation (EFS) with data acquisition in spatially separated areas of an interconnected neuronal network. We integrated our methodology with state of the art drug discovery instrumentation (FLIPR Tetra) and used selective tool compounds to perform a systematic pharmacological validation of the model. We investigated pharmacological modulators targeting pre- and post-synaptic receptors (AMPA, NMDA, GABA-A, mGluR2/3 receptors and Nav, Cav voltage-gated ion channels) and demonstrated the ability of our model to discriminate and measure synaptic transmission in cultured neuronal networks. Application of the model described here as an unbiased phenotypic screening approach will help with our long term goals of discovering novel therapeutic strategies for treating neurological disorders.


Subject(s)
Drug Discovery/instrumentation , Neurons/physiology , Synapses/physiology , Synaptic Transmission/physiology , Voltage-Sensitive Dye Imaging , Animals , Calcium/metabolism , Cations, Divalent/metabolism , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/physiology , Electric Stimulation , Neurons/cytology , Neurons/drug effects , Neurotransmitter Agents/pharmacology , Primary Cell Culture , Rats, Sprague-Dawley , Synapses/drug effects , Synaptic Transmission/drug effects , Voltage-Sensitive Dye Imaging/instrumentation , Voltage-Sensitive Dye Imaging/methods
2.
J Alzheimers Dis ; 54(4): 1521-1538, 2016 10 18.
Article in English | MEDLINE | ID: mdl-27589517

ABSTRACT

Intracellular neurofibrillary tangles (NFTs) are the hallmark of Alzheimer's disease and other tauopathies in which tau, a microtubule-associated protein, loses its ability to stabilize microtubules. Several post-translational modifications including phosphorylation and truncation increase tau's propensity to aggregate thus forming NFTs; however, the mechanisms underlying tau conformational change and aggregation still remain to be defined. Caspase activation and subsequent proteolytic cleavage of tau is thought to be a potential trigger of this disease-related pathological conformation. The aim of this work was to investigate the link between caspase activation and a disease-related conformational change of tau in a neuroblastoma cell-based model of spontaneous tau aggregation. We demonstrated that caspase induction initiates proteolytic cleavage of tau and generation of conformationally altered and aggregated tau recognized by the MC1 conformational antibody. Most importantly, these events were shown to be attenuated with caspase inhibitors. This implies that therapeutics aimed at inhibiting caspase-mediated tau cleavage may prove beneficial in slowing cleavage and aggregation, thus potentially halting tau pathology and disease progression.


Subject(s)
Caspase Inhibitors/pharmacology , Caspases/metabolism , Copper-Transporting ATPases/metabolism , Protein Aggregation, Pathological/metabolism , tau Proteins/metabolism , Animals , Cell Line, Tumor , Copper-Transporting ATPases/chemistry , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Humans , Mice , Protein Aggregation, Pathological/pathology , Protein Conformation/drug effects , Staurosporine/pharmacology , tau Proteins/chemistry
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