Abstract
Macroautophagy is a cellular mechanism for the clearance of protein aggregates and damaged organelles. Impaired macroautophagy has been observed in neurodegenerative disorders. We investigated the macroautophagy pathway in essential tremor (ET) cases compared to age-matched controls. We analyzed microtubule-associated protein light chain 3-II (LC3-II), S6K, phosphorylated S6K, beclin-1, and mitochondrial membrane proteins levels by Western blot in the post-mortem cerebellum of 10 ET cases and 11 controls. We also performed immunohistochemistry in 12 ET cases and 13 controls to quantify LC3 clustering in Purkinje cells (PCs). LC3-II protein levels were significantly lower in ET cases vs. controls on Western blot (0.84±0.14 vs. 1.00±0.14, p = 0.02), and LC3-II clustering in PCs by immunohistochemistry was significantly lower in ET cases vs. controls (2.03±3.45 vs. 8.80±9.81, p = 0.03). In ET cases, disease duration was inversely correlated with LC3-II protein level (r = −0.64, p = 0.046). We found that mitochondrial membrane proteins were accumulated in ET (TIM23: 1.36±0.11 in ET cases vs. 1.00±0.08 in controls, p = 0.02; TOMM20: 1.63±0.87 in ET cases vs. 1.00±0.14 in controls, p = 0.03). Beclin-1, which is involved in macroautophagy, was strikingly deficient in ET (0.42±0.13 vs. 1.00±0.35, p<0.001). Decreased macroautophagy was observed in the ET cerebellum, and this could be due to a decrease in beclin-1 levels, which subsequently lead to mitochondrial accumulation as a result of autophagic failure. This provides a possible means by which perturbed macroautophagy could contribute to PC pathology in ET.
Highlights
Essential tremor (ET) is among the most prevalent movement disorders [1]
Cerebellar tissue was available for Western blot analysis on ET cases and age-matched controls who were similar with respect to age, gender, brain weight and other variables of interest (Table 1)
We investigated calbindin level, a protein expressed by Purkinje cells (PCs) in the cerebellum, and found that ET cases had a lower level of calbindin than controls (0.5860.18 vs. 1.0160.26, p,0.01) (Figure 1E), consistent with our previous findings that ET cases had a lower number of PCs [3]
Summary
Essential tremor (ET) is among the most prevalent movement disorders [1]. In postmortem studies, degenerative changes in the cerebellum, including an increase in the number of Purkinje cell (PC) axonal torpedoes and PC loss have been reported [2,3]. Autophagy is of particular interest since many neurodegenerative diseases are characterized by autophagic alterations that are linked to proteinacious accumulations as well as neuronal death [10]. Mutations or overexpression in neurodegenerative disease genes, including presenilin [13], huntingtin (Htt) [14], a-synulcien [15,16], parkin, and PINK1 [17], have been reported to inhibit macroautophagy. These studies highlight the importance of autophagy in neuronal homeostasis and survival. We investigated whether changes in autophagy occur in the cerebellum of ET cases compared to that of age-matched controls
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