Research Article: Diurnal Rhythms in Neurexins Transcripts and Inhibitory/Excitatory Synapse Scaffold Proteins in the Biological Clock

Date Published: May 25, 2012

Publisher: Public Library of Science

Author(s): Mika Shapiro-Reznik, Anje Jilg, Hadas Lerner, David J. Earnest, Nava Zisapel, Michael Hendricks.


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.

Partial Text

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]. In mammals, the neurexins are encoded by three genes NRXN1, NRXN2 and NRXN3, each has two promoters that generate longer(α) and shorter(β) forms [1], [2], [3]. The neurexin genes transcripts are edited by extensive alternative splicing at five canonical sites referred to as SS#1 to SS#5 in α neurexins, two of which (SS#4, SS#5) are shared with the β neurexins [3], [4]. Neurexins’ immunoreactivity has been localized mainly to pre-synaptic nerve terminals [5] while their known ligands are found predominantly at post-synaptic sites in target cells (i.e. neuroligins, leucine-rich repeat transmembrane neuronal proteins, GABAA receptors, Cbln1/Glutamate receptor delta2 and dystroglycan) or in the extracellular fluid (e.g. neurexophilins). Binding of neurexins to their ligands form trans-synaptic complexes which regulate glutamatergic and GABA-ergic transmission and subsequently the excitatory/inhibitory balance in brain networks [6]–[16].

This study demonstrates diurnal variations in expression and SS#3/SS#4 splicing of mNRXN1α and mNRXN2α (but not NRXN3α) neurexin-2α and postsynaptic scaffolding proteins (PSD-95 and gephyrin) levels in the mouse SCN in vivo. Neurexin-2α protein peak appeared ∼6 hours after the mRNA peak. Rhythmic expression and SS#3/SS#4 exons splicing of rNRXN1α and rNRXN2α (NRXN3α is not expressed) was also observed in rat SCN2.2 cells in vitro. Being found in SCN samples from mouse and SCN-derived cells from rat, these rhythms appear to be a general rather than species- specific trait of SCN cells.