AAV5-hSyn-Cre

Clock neurons within the mammalian suprachiasmatic nucleus (SCN) encode circadian time using an interlocking transcription-translation feedback loop (TTFL) to drive rhythmic gene expression. However, the contribution of transcription factors beyond the circadian TTFL to SCN function remains unclear. Here, researchers report that the stem and progenitor transcription factor, sex-determining region Y-box 2 (SOX2), is expressed in adult SCN neurons and positively regulates transcription of the core clock gene Period2. Mice lacking SOX2 selectively in SCN neurons exhibit imprecise, poorly consolidated behavioral rhythms that fail to effectively synchronize to the ambient light cycle and are highly susceptible to sustained light-induced arrhythmias. RNA sequencing reveals that Sox2 deficiency alters the SCN transcriptome, reducing expression of core clock genes and neuropeptide receptor systems. By defining the transcriptional landscape within SCN neurons, SOX2 enables the generation of stable, trainable circadian rhythms that accurately reflect environmental time.

To determine whether the effects of Sox2 disruption on PER2 expression were intrinsic to the fully developed SCN, researchers used adeno-associated virus (AAV)-mediated delivery of cre recombinase to ablate the Sox2 gene in neurons in SCN tissue explants prepared from Sox2^fl/fl;Per2^Luc/+ pups at postnatal day (P) 12 (Figure 1K) and monitored PER2::LUCIFERASE rhythms in culture. As most developmental milestones of the mouse SCN have been reached by ∼P10, this approach could avoid any potential effects of Sox2 ablation on SCN development. The amplitude of PER2::LUC rhythms was significantly reduced in cultures transduced with AAV5-hSyn-Cre compared with SCN explants transduced with control AAV5-human synaptobrevin (hSyn)-TurboRFP (Figures 1L and 1M). Collectively, these data suggest that SOX2 is important for the generation of high-amplitude PER2 rhythms within the SCN.

Figure 1. (K) SOX2 (green) and RFP (magenta) expression in Sox2^fl/fl;Per2^Luc/+ SCN tissue explants transduced with AAV5-hSyn-TurboRFP or AAV5-hSyn-Cre. (L) Bioluminescence traces from Sox2^fl/fl;Per2^Luc/+ SCN tissue explants that had been transduced with AAV5-hSyn-TurboRFP or AAV5-hSyn-Cre on day 5 after initial plating. (M) Relative amplitude of bioluminescence rhythms of transduced Sox2^fl/fl;Per2^Luc/+ SCN tissue explants after medium change. (Cheng A H, et al., 2019)