Research reportDeletion of KCC3 in parvalbumin neurons leads to locomotor deficit in a conditional mouse model of peripheral neuropathy associated with agenesis of the corpus callosum
Introduction
Peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN) is an autosomal recessive disease characterized by progressive sensorimotor neuropathy, mental retardation, dysmorphic features (high arched palate, hypertelorism, and syndactyly), and complete or partial agenesis of the corpus callosum [2], [16]. Genetic linkage in 14 families mapped the autosomal recessive disorder to chromosome 15q [7] and several mutations in SLC12A6, the human gene which encodes the K–Cl cotransporter-3 (KCC3), were later identified in HMSN/ACC patients [5], [13], [23], [28]. In a mouse model where the KCC3 gene was disrupted to produce a global knockout, an early onset and severe locomotor deficit similar to the crippling human peripheral neuropathy disorder was observed [13]. The mice also developed high blood pressure [1], [5], age-related deafness [5], and renal dysfunction [30]. At the ultra-structural level, KCC3-deficient mice exhibited axonal and peri-axonal swelling, suggesting both neuronal and Schwann cell defects [6].
In a recent study, Shekarabi et al. used a synapsin 1 promoter-CRE mouse to drive deletion of KCC3 in neurons, and demonstrated that loss of neuronal KCC3 reproduced the neuropathy phenotype observed in the KCC3 knockout mouse [25]. However, since synapsin 1 promoter is present in all neurons, it remains to be determined which specific neuronal cell type play underlies the development of the peripheral neuropathy, and whether KCC3 in Schwann cells are also involved in the ontogeny of the disease.
In the present study, we created several novel mouse models to target deletion of KCC3 in specific cell types by using specific transgenic CRE lines. CRE recombinase is an enzyme derived from the P1 bacteriophage which catalyzes the specific recombination between two 34 bp DNA recognition sites (loxP sites). The artificial loxP sites are introduced in the target gene, Slc12a6 in this case, and a mutant mouse is produced. The recombinase which is under a tissue-specific promoter is expressed from DNA (transgene) inserted in the genome of a separate mouse. The Sc12a6 gene with loxP sites flanking an exon of interest and the recombinase transgene are brought together by crossing the two lines of mice. Deletion of KCC3 in small sensory neurons, driven by the Nav1.8 Na+ channel promoter, resulted in mice that exhibited no motor coordination phenotype. In contrast, deletion of KCC3 in larger type-Ia proprioceptive afferent neurons driven by parvalbumin resulted in a significant loss of locomotion. In these mice, there was also a trend towards a hyperactivity phenotype. Surprisingly, deletion of KCC3 driven by enolase-2, which is supposedly expressed in most mature neurons, failed to recapitulate this phenotype. The KCC3 deletion in the Schwann cells driven by desert hedgehog also failed to induce a locomotor phenotype. Histology analysis showed that parvalbumin-positive neurons and enolase-2 positive neurons in dorsal root ganglion had different expression profiles, consistent with the distinct performances in locomotor tests. Finally, histological analysis revealed a significant pathology associated with cells that were immunoreactive to parvalbumin. Therefore, our study demonstrates that parvalbumin-positive neurons played a key role in producing the ACCPN-like locomotor phenotype.
Section snippets
Generation of tissue-specific KCC3 knockout mice
All procedures performed with mice were approved by the Vanderbilt University Institutional Animal Care and Use Committee. We disrupted the mouse Slc12a6 gene by inserting loxP sites around exon 7 (131 bp), followed by a neomycin resistance gene cassette flanked by FRT (Flippase Recombination Target) sites. The targeting vector was constructed using recombineering techniques to drop the short 5′ end and long 3′ end arms of recombination from Bacterial Artificial Chromosome (BAC) clone bMQ-302F12
Results
To determine the cellular origin of HSMN/ACC, we created several conditional KCC3 knockout mouse lines by using CRE mediated recombination under the control of tissue-specific promoters. First, we created a mouse in which exon 7 of the Slc12a6 gene was flanked by loxP sites. A construct targeting exon 7 was engineered (Fig. 1) and electroporated in mouse embryonic stem cells. After germline transmission and elimination of the neomycin-resistance gene cassette, a 3 kb fragment was PCR-amplified
Discussion
To address the cellular origin of the locomotor deficit associated with the disruption of KCC3 in mice and the development of the early onset peripheral neuropathy disorder observed in HSN/ACC patients [5], [13], we created several tissue-specific knockout lines. Only when KCC3 was targeted in parvalbumin-positive neurons did we detect a locomotor deficit similar to the one observed in the global knockout animals. Parvalbumin is a member of the large family of EF-hand calcium-binding proteins,
Acknowledgments
We thank Thomas M. Austin for the statistical analysis of the rotarod data. This work was supported by NIH grants NS036758 and GM074771 to ED. The Translational Pathology Shared Resource supported by NCI/NIH Cancer Center Support Grant 2P30 CA068485-14) and the Vanderbilt Mouse Metabolic Phenotyping Center Grant 5U24DK059637-13.
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