Abstracts

Rationale: Arx is a multi-facetted transcription factor known to play a crucial role in interneuron development. Mutations in ARX lead to a spectrum of neurodevelopmental conditions including X-linked West syndrome. Postnatally Arx remains expressed in GABAergic interneurons such as parvalbumin (Parv) interneurons. We found that postnatal conditional ablation of Arx in parvalbumin interneurons resulted in a loss of parvalbumin basket cell markers such as Kv3.1 and the Cat-315 component of the perineuronal net. Further, intrinsic and extrinsic electrophysiological properties such as AP threshold, AP amplitude, and half-width duration are reduced in Arx-/--Parv-Tomato interneurons compared to controls. In addition, Arx-/--Parv-Tomato cells require higher current intensity (Rheobase) to reach depolarization threshold. However, the Arx-dependent transcriptional targets that are driving these changes remain to be elucidated. Methods: Arx was conditionally knocked out in parvalbumin interneurons by crossing a floxed Arx mouse (Arxfl/fl) with a Parv-Tomato Cre-recombinase (Parv-TomatoCre) mouse. After adult brain dissociation of hippocampal and cortical slices of adult mouse brains (P35-40) Tomato labelled parv interneurons were FACS sorted from the dissociated tissue. RNA from sorted cells was isolated using TrizolLS and the purified RNA sequenced on an Illumina sequencing platform (HiSeq 4000, Illumina). The gene expression profile of wildtype (n=3) and Arx knock-out (n=3) adult parvalbumin interneuron populations was subsequently analyzed using edgeR. Results: Comparison of wild type and Arx conditional knock out parvalbumin interneurons revealed 110 genes to be differentially expressed (p < 0.05; FDR < 0.1) of which 20 were down-regulated and 90 were up-regulated. Fold changes varied between -5 and 4. Pathway analysis on the 110 genes  demonstrated that several biological processes including nervous system development, synaptic transmission, and regulation of ion transmembrane transport were significantly enriched (p < 0.01). In addition, molecular pathways of the differentially expressed genes identified included calcium ion binding, ion channel activity, and voltage-gated channel activity (p < 0.0001). Further, transcripts related to the synaptic membrane, ionotropic glutamate receptor complex, and extracellular matrix (p < 0.01) were significantly dysregulated. Conclusions: Arx remains expressed in postnatal interneurons and appears to actively control transcription. Dysregulation of Arx, by conditional loss, results in alterations of basic neuronal functions such as synaptic transmission and ion channel activity which relates to the network and cellular phenotypes we have previously reported. These data will allow us to validate downstream targets of Arx that could be implemented as an approach to restoring normal function in patients with ARX mutations. To our knowledge, this is the first report of a systematic gene expression analysis performed on a complete hippocampal and cortical population of adult parvalbumin interneurons. Funding: RO1 NS082761-01 (Eric Marsh) and Stiftung zur Förderung der medizinischen Forschung der CAU zu Kiel (Markus von Deimling)