# Chloroquine Causes Aging-like Changes in Diaphragm Neuromuscular Junction Morphology in Mice

## TL;DR

Chloroquine causes changes in mouse diaphragm neuromuscular junctions similar to those seen in aging, suggesting a link between autophagy and NMJ health.

## Contribution

This study demonstrates that chloroquine induces aging-like morphological changes in neuromuscular junctions in mice.

## Key findings

- Chloroquine treatment reduced pre-synaptic volume at diaphragm neuromuscular junctions by 12%.
- Chloroquine increased the proportion of partially denervated neuromuscular junctions by 2.7-fold.
- The observed changes resembled those in 18-month-old mice, indicating aging-like effects.

## Abstract

Autophagy impairments have been implicated in various aging conditions. Previous studies in cervical motor neurons show an age-dependent increase in the key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. Chloroquine is commonly used to inhibit autophagy by preventing autophagosome–lysosome fusion and may thus emulate the effects of aging on the neuromuscular system. Indeed, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, consistent with aging effects. We hypothesized that chloroquine alters diaphragm muscle neuromuscular junction (NMJ) morphology and increases denervation. Adult male and female C57BL/6 × 129J mice between 5 and 8 months of age were used to examine diaphragm muscle NMJ morphology and denervation following daily intraperitoneal injections of chloroquine (10 mg/kg/d) or vehicle for 7 days. The motor end-plates and pre-synaptic terminals were fluorescently labeled with α-bungarotoxin and anti-synaptophysin, respectively. Confocal microscopy was used to assess pre- and post-synaptic morphology and denervation. At diaphragm NMJs, chloroquine treatment decreased pre-synaptic volume by 12% compared to the vehicle (p < 0.05), with no change in post-synaptic volume. Chloroquine treatment increased the proportion of partially denervated NMJs by 2.7-fold compared to vehicle treatment (p < 0.05). The morphological changes observed were similar to those previously reported in the diaphragm muscles of 18-month-old mice. These findings highlight the importance of autophagy in the maintenance of the structural properties at adult NMJs in vivo.