RESUMO
Methotrexate (MTX) is an anti-metabolite that has been used for the treatment of patients of acute lymphocytic leukemia or non-Hodgikin lymphoma for decades. In some cases, MTX-treated patients suffer from neurological side effects, including seizures and cognitive dysfunctions. While most patients are at developmental stages, information of the mechanisms of the side effects of MTX treatment on the developing neurons has been limited. Neurons develop in five steps in the human brain: neurogenesis, polarity formation, dendrite and axon development, synapse formation, and neuronal death. Except for neurogenesis, these processes can be recapitulated in the primary culture system of cortical neurons. Using primary cultured cortical neurons, we studied the impact of MTX treatment on dendrite development, synapse formation, and neuronal death in the present report. MTX treatment impaired neuronal survival, dendrite development, and synapse formation. Interestingly, half maximal effective concentrations (EC50 s) of MTX for all three processes are at the similar range and lower than the MTX concentration in the cerebrospinal fluid in treated patients. Our results provide possible mechanisms of neurological side effects in treated patients.
Assuntos
Metotrexato , Neurônios , Humanos , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Neurônios/fisiologia , Neurogênese , Dendritos , SinapsesRESUMO
INTRODUCTION: Detection of drug effects on neuronal synapses is important for predicting their adverse effects. We have used drebrin as a marker to detect the synaptic changes in cultured neurons. High concentration of glutamate decreases the amount of drebrin in synapses. To increase the availability of this method for high throughput analysis, we applied the drebrin-based evaluation of synapses to high-content imaging analysis using microplates. METHODS: Three weeks old cultured neurons were fixed and processed for immunocytochemistry to visualize drebrin clusters, dendrites and neuronal cell bodies. After automated image acquisition, total number of drebrin clusters per fields, linear density of drebrin cluster along dendrites, dendrite length and neuron number were automatically measured by a custom-designed protocol. RESULTS: Automated image acquisition and analysis showed that dendrite length and drebrin cluster density along dendrites are measured consistently and reproducibly. In addition, application of 10-100⯵M glutamate for 10â¯min or 0.5-50⯵M latrunculin A for 5â¯min significantly decreased drebrin cluster density without affecting neuron number. These results were consistent with our previous results using manual image acquisition and analysis with regular fluorescence microscope and image analysis software. Furthermore, 0.3 or 1.0⯵M staurosporine for 24â¯h significantly decreased neuron number. DISCUSSION: The present study demonstrates that this high-throughput imaging analysis of drebrin cluster density along dendrites for detecting the effects of substances on synapses is sensitive enough to detect the effects of glutamate receptor activation and latrunculin A treatment, and indicates that this analysis will be useful for safety pharmacology study.