Abstract
The neurexin genes (NRXN1/2/3) encode two families (α and β) of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4). Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN) act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively) were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic scaffolding proteins in SCN across the 24-h cycle. NRXNs gene transcripts may have a role in coupling the circadian clock to diurnal rhythms in excitatory/inhibitory synaptic balance.
Highlights
Neurexins are neuron-specific cell-surface proteins [1,2] that act in the vertebrate nervous system as trans-synaptic receptors and have an important role in cognition [3]
We show here that these cells show circadian variations in rNRXN1/2 expression and SS#3/SS#4 exons splicing and in PSD-95 and gephyrin levels and intracellular localization
There were no significant rhythms in mNRXN3 a transcripts in the suprachiasmatic nuclei of the hypothalamus (SCN) (Fig. 1E)
Summary
Neurexins are neuron-specific cell-surface proteins [1,2] that act in the vertebrate nervous system as trans-synaptic receptors and have an important role in cognition [3]. Binding of neurexins to their ligands form transsynaptic complexes which regulate glutamatergic and GABA-ergic transmission and subsequently the excitatory/inhibitory balance in brain networks [6,7,8,9,10,11,12,13,14,15,16]. We have recently demonstrated in one day-old rat brain neurons, that activation (by depolarization) induced a reversible, calcium-dependent splicing repression of the rNRXN2a SS#3 exon [17]. Fear conditioning training in rats induced in vivo significant and transient repressions of hippocampal rNRXN1/2/3a, SS#4 splicing, attenuated levels of the excitatory postsynaptic scaffolding protein PSD-95 and the inhibitory postsynaptic scaffolding protein gephyrin in the hippocampus and fear memory formation [18]. Induced neuronal activity may affect NRXNs transcripts and subsequently synaptic fine tuning
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