Glycine-induced activation of GPR158 increases the intrinsic excitability of medium spiny neurons in the nucleus accumbens.

It has been recently established that GPR158, a class C orphan G protein-coupled receptor, serves as a metabotropic glycine receptor. GPR158 is highly expressed in the nucleus accumbens (NAc), a major input structure of the basal ganglia that integrates information from cortical and subcortical structures to mediate goal-directed behaviors. However, whether glycine modulates neuronal activity in the NAc through GPR158 activation has not been investigated yet. Using whole-cell patch-clamp recordings, we found that glycine-dependent activation of GPR158 increased the firing rate of NAc medium spiny neurons (MSNs) while it failed to significantly affect the excitability of cholinergic interneurons (CIN). In MSNs GPR158 activation reduced the latency to fire, increased the action potential half-width, and reduced action potential afterhyperpolarization, effects that are all consistent with negative modulation of potassium M-currents, that in the central nervous system are mainly carried out by Kv7/KCNQ-channels. Indeed, we found that the GPR158-induced increase in MSN excitability was associated with decreased M-current amplitude, and selective pharmacological inhibition of the M-current mimicked and occluded the effects of GPR158 activation. In addition, when the protein kinase A (PKA) or extracellular signal-regulated kinase (ERK) signaling was pharmacologically blocked, modulation of MSN excitability by GPR158 activation was suppressed. Moreover, GPR158 activation increased the phosphorylation of ERK and Kv7.2 serine residues. Collectively, our findings suggest that GPR158/PKA/ERK signaling controls MSN excitability via Kv7.2 modulation. Glycine-dependent activation of GPR158 may significantly affect MSN firing in vivo, thus potentially mediating specific aspects of goal-induced behaviors.

Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases.

Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.