Autophagy impairment in a mouse model of neuropathic pain

Laura Berliocchi, Rossella Russo, Maria Maiarù, Alessandra Levato, Giacinto Bagetta, Maria Tiziana Corasaniti, Laura Berliocchi, Rossella Russo, Maria Maiarù, Alessandra Levato, Giacinto Bagetta, Maria Tiziana Corasaniti

Abstract

Autophagy is an intracellular membrane trafficking pathway controlling the delivery of cytoplasmic material to the lysosomes for degradation. It plays an important role in cell homeostasis in both normal settings and abnormal, stressful conditions. It is now recognised that an imbalance in the autophagic process can impact basal cell functions and this has recently been implicated in several human diseases, including neurodegeneration and cancer.Here, we investigated the consequences of nerve injury on the autophagic process in a commonly used model of neuropathic pain. The expression and modulation of the main autophagic marker, the microtubule-associated protein 1 light chain 3 (LC3), was evaluated in the L4-L5 cord segment seven days after spinal nerve ligation (SNL). Levels of LC3-II, the autophagosome-associated LC3 form, were markedly higher in the spinal cord ipsilateral to the ligation side, appeared to correlate with the upregulation of the calcium channel subunit α2δ-1 and were not present in mice that underwent sham surgery. However, LC3-I and Beclin 1 expression were only slightly increased. On the contrary, SNL promoted the accumulation of the ubiquitin- and LC3-binding protein p62, which inversely correlates with autophagic activity, thus pointing to a block of autophagosome turnover.Our data showed for the first time that basal autophagy is disrupted in a model of neuropathic pain.

Figures

Figure 1
Figure 1
Spinal nerve ligation induced a severe mechanical allodynia. A severe and persistent mechanical hypersensitivity developed and maintained over time following SNL and was absent in animals that underwent sham surgery. Data were expressed as mean ± SEM of 50% of pain threshold and were normalized to the baseline of each animal (sham: n = 6; SNL: n = 10). For all time points, p < 0.001.
Figure 2
Figure 2
LC3 expression in the spinal cord following spinal nerve ligation. (A) LC3 expression was analyzed by Western blot in the hemi-cord contralateral (C) and ipsilateral (I) to the side of ligation, 7 days after surgery. SNL animals showed higher LC3-I expression than sham animals (A). A clear trend towards an increased LC3-I expression in the ipsilateral (I) compared to the contralateral (C) side, together with the appearance of LC3-II, was observed after ligation (A) and confirmed by densitometric analysis (B and C). The slight increase in LC3-I levels and the apparent formation of LC3-II well correlated with α2δ-1 upregulation (A and D). Signals from each band were normalized towards the corresponding GADPH signal. One representative Western blot is shown. Sham: n = 5, SNL: n = 6.
Figure 3
Figure 3
Beclin 1 expression in the spinal cord following spinal nerve ligation. (A) Beclin 1 expression was analyzed by Western blot in the hemi-cord contralateral (C) and ipsilateral (I) to the side of ligation, 7 days after surgery. SNL animals showed higher Beclin 1 expression than sham animals (A). A clear trend towards an increase in Beclin 1 expression in the ipsilateral (I) compared to the contralateral (C) side was observed after ligation (A) and confirmed by densitometric analysis (B). The slight increase in Beclin 1 expression well correlated with α2δ-1 upregulation (A and C). Signals from each band were normalized towards the corresponding GAPDH signal. One representative Western blot is shown. Sham: n = 5, SNL: n = 6.
Figure 4
Figure 4
Statistical analysis. Quantitative analysis of Western blots by densitometry confirmed a trend towards an increase in LC3-I (A) and Beclin 1 (C) expression in the ipsilateral (I) compared to the controlateral (C) side of SNL animals 7 days after surgery. In the same animals, this was accompanied by a marked increase in LC3-II levels (B) and associated to strong α2δ-1 upregulation (D). For LC3-I, LC3-II, Beclin 1 and α2δ-1: n = 5. Signals from each band were normalized towards the corresponding GAPDH signal. Values were expressed as mean ± SEM, * p < 0.05.
Figure 5
Figure 5
p62 accumulation in the spinal cord following spinal nerve ligation. (A) p62 expression was analyzed by Western blot in the hemi-cord contralateral (C) and ipsilateral (I) to the side of ligation, 7 days after surgery. The minor band detected by the antibody together with the main upper band has been previously suggested as a p62 splicing variant or a partially cleaved product [19]. SNL animals showed higher p62 expression than sham animals (A). A clear trend towards an increased p62 expression in the ipsilateral (I) compared to the contralateral (C) side was observed after ligation (A) and confirmed by densitometric analysis (B). The increase in p62 levels correlated with α2δ-1 upregulation (A and C) and was confirmed by quantitative analysis (n = 5, D). Signals from each band were normalized towards the corresponding GAPDH signal. Values were expressed as mean ± SEM, * p < 0.05.

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