Functional characterization of Caspr2 in a mouse model of sciatic nerve injury

Abstract

BACKGROUND: The intrinsic molecular mechanisms and regulatory networks of peripheral nervous system injury remain to be systematically analyzed. Caspr2, as a cell adhesion molecule specifically expressed on the surface of neuronal axons, plays an important role in the myelination of nerve fibers, but the dynamic regulatory mechanism of Caspr2 in peripheral nerve regeneration has not been clarified. OBJECTIVE: To focus on the functional characterization of Caspr2 in a sciatic nerve injury model, reveal the molecular mechanism by which Caspr2 regulates axonal regeneration of dorsal root ganglion neurons, and provide a new perspective for developing precise repair strategies for peripheral nerve injury. METHODS: An ICR mouse model of sciatic nerve crush injury was established. The transcriptional and protein expression characteristics of Caspr2 in dorsal root ganglion tissue were analyzed by qRT-PCR and western blot, and the expression of Caspr2 in dorsal root ganglion neurons was analyzed by immunofluorescence staining. An intervention model was constructed using primary dorsal root ganglion neuron culture system: Caspr2 siRNA negative control group, Caspr2 siRNA group; Caspr2 empty vector control group, Caspr2 overexpression group. Axonal regeneration dynamics were quantitatively evaluated by Tuj1 immunofluorescence staining and AxioVision image analysis system. RESULTS AND CONCLUSION: (1) Injury response characteristics: On day 3 after sciatic nerve injury, the mRNA and protein expression levels of Caspr2 in dorsal root ganglion tissue decreased, and further decreased on day 7 after injury. In addition, the expression of Caspr2 in dorsal root ganglion neurons also significantly decreased on day 7 after injury. (2) Loss-of-function effect: Caspr2 siRNA treatment significantly promoted neuronal axon growth. (3) Overexpression effect: Caspr2 overexpression significantly inhibited neuronal axon extension. These results indicate that Caspr2, as a key inhibitory factor in peripheral nerve regeneration, participates in the nerve repair process by bidirectionally regulating axonal regeneration ability.

1. Introduction

Peripheral nerve injury can affect motor, sensory, and autonomic functions, and chronic cases are more likely to progress to irreversible disabilities such as denervated muscle atrophy [1-4]. Although the peripheral nervous system possesses some repair capacity, complete functional recovery is extremely rare. Regulation of axonal regeneration ability is a key scientific issue in nerve repair. Adult mammalian peripheral nervous system neurons retain limited regenerative potential, but the molecular regulatory network has not been fully elucidated [5-6]. Current research focuses on two consensus mechanisms: inhibitory microenvironment barriers and the decline of intrinsic regenerative capacity in mature neurons [7-8]. Although microenvironmental regulation strategies have achieved staged progress, clinical translation results remain unsatisfactory [9-10]. In contrast, activating the intrinsic regenerative program of neurons through epigenetic regulation or transcriptional reprogramming shows better prospects, suggesting that enhancing the autonomous regenerative ability of neurons may be the key to breaking through the bottleneck of injury repair.

Studies have found that Caspr2 (Contactin-associated protein-like 2), a member of the neurexin superfamily, plays an important role in neuronal development, synaptic plasticity, and neural circuit function [11]. Caspr2 affects the development of the nervous system by regulating the axon diameter, myelin thickness, and intrinsic excitability of cortical neurons in the cerebral commissural fibers. In gene knockout mice, morphological abnormalities in these structures persist from embryonic to adult stages [12]. Caspr2 plays an important role from axonal guidance during development to synaptic plasticity and behavioral regulation in adulthood [13]. After Caspr2 gene knockout, the number of neurite branches decreases [14], suggesting that Caspr2 may play a role in axonal regeneration after peripheral nerve injury. This study aims to investigate the expression changes of Caspr2 in neurons after peripheral nerve injury and the effects of silencing or overexpressing Caspr2 on injured axonal regeneration, laying an important theoretical foundation for developing targeted Caspr2 strategies to promote peripheral nerve regeneration.