Elsevier

Behavioural Brain Research

Volume 274, 1 November 2014, Pages 128-136
Behavioural Brain Research

Research report
Deletion of KCC3 in parvalbumin neurons leads to locomotor deficit in a conditional mouse model of peripheral neuropathy associated with agenesis of the corpus callosum

https://doi.org/10.1016/j.bbr.2014.08.005Get rights and content

Abstract

Hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC or ACCPN) is an autosomal recessive disease caused by the disruption of the SLC12A6 gene, which encodes the K–Cl cotransporter-3 (KCC3). A ubiquitous deletion of KCC3 in mice leads to severe locomotor deficits similar to ACCPN patients. However, the underlying pathological mechanism leading to the disease remains unclear. Even though a recent study suggests that the neuropathic features of ACCPN are mostly due to neuronal loss of KCC3, the specific cell type responsible for the disease is still unknown. Here we established four tissue specific KCC3 knockout mouse lines to explore the cell population origin of ACCPN. Our results showed that the loss of KCC3 in parvalbumin-positive neurons led to significant locomotor deficit, suggesting a crucial role of these neurons in the development of the locomotor deficit. Interestingly, mice in which KCC3 deletion was driven by the neuron-specific enolase (NSE) did not develop any phenotype. Furthermore, we demonstrated that nociceptive neurons targeted with Nav1.8-driven CRE and Schwann cells targeted with a desert hedgehog-driven CRE were not involved in the development of ACCPN. Together, these results establish that the parvalbumin-positive neuronal population is an important player in the pathogenic development of ACCPN.

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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