Determined by Western blot with LC3 antibody was lower in ET cases than (-)-Calyculin A controls (0.8460.14 vs. 1.0060.14, p = 0.02)(Figure 1A, B). We used another LC3-II specific antibody (see Methods) and found the similar case-control differences (p = 0.01) and there was a high correlation between the LC3-II levels detected by two antibodies (r = 0.52, p = 0.01) Among ET cases, disease duration was inversely correlated with LC3-II protein level (r = 20.64, p = 0.046) (Figure 1C). The mean disease duration was 49.9622.2 years (range = 9?0 years). Based on the presence of a clear bimodal distribution in disease duration among ET cases in our sample (#40 years vs. .40 years), study subjects were stratified into 3 diagnosis-duration groups: controls, ET cases with shorter duration disease (n = 3, mean = 19.7612.2 years, range = 9?3 years), and ET cases with longer duration disease (n = 7, mean = 62.966.0 years, range = 55?0 years). The Licochalcone-A respective LC3-II protein levels were: 1.0060.14, 0.9560.14, and 0.7960.12, and in a linear regression model, LC3-II protein level declined by diagnosis-duration group (r2 = 0.37, p = 0.004) (Figure 1D). We also investigated calbindin level, a protein specifically expressed by 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]. We then investigated whether decrease in LC3-II was specific to the ET cerebellum. We determined LC3-II protein level in theoccipital cortex in 7 ET cases and 9 controls: ET cases had similar LC3-II level as controls (0.9860.13 vs. 1.0060.08) (Figure 1F, G). Cerebellar tissue was available for immunohistochemistry on 12 ET cases and 13 age-matched controls, who were similar with respect to age, 23977191 gender, brain weight and other variables of interest (Table 1). These included 6 of the 10 ET cases and 8 of 11 controls used in the Western blot analysis. We labeled the cerebellar sections with anti-LC3 and anti-calbindin antibodies to assess the LC3 content in PCs. PCs were found to have a robust autophagic activity, reflected by LC3 clustering; therefore, we used LC3 staining to assessed the autophagic activity in PCs [22]. We found that PCs in ET cases exhibited lower LC3 staining (Figure 2A ). We found that LC3 was present in punctate structures, which labels them as AVs: PCs in ET cases had strikingly fewer LC3 puncta than controls (Figure 
2I ). We quantified the fraction of PC bodies, excluding the nucleus, that was occupied by AVs (Figure 2M ). The percentage of cell bodies occupied by AVs was more than 4-fold lower in ET cases than controls (2.0363.45 vs. 8.8069.81, p = 0.03)(Figure 2P). The results from Western blot analyses (i.e., LC3-II protein levels), were highly correlated with these immunolabel results (r = 0.78, p = 0.001). Among the 12 ET cases with immunolabel results, the mean disease duration of the patients was 46.3622.1 years (range = 17?80 years). Disease duration was not correlated with the fraction of cell bodies occupied by AVs (r = 20.12, p = 0.70), yet when study subjects were stratified into 3 diagnosis-duration groups (controls; ET cases with shorter duration disease [n = 4, mean = 19.863.4 years]; and ET cases with longer duration disease [n = 8, mean = 59.6612.6 years]), the respective percentage of cell bodies occupied by AVs were: 8.8069.81, 3.2163.32, and 1.4463.56, and in a linear regression model, the f.Determined by Western blot with LC3 antibody was lower in ET cases than controls (0.8460.14 vs. 1.0060.14, p = 0.02)(Figure 1A, B). We used another LC3-II specific antibody (see Methods) and found the similar case-control differences (p = 0.01) and there was a high correlation between the LC3-II levels detected by two antibodies (r = 0.52, p = 0.01) Among ET cases, disease duration was inversely correlated with LC3-II protein level (r = 20.64, p = 0.046) (Figure 1C). The mean disease duration was 49.9622.2 years (range = 9?0 years). Based on the presence of a clear bimodal distribution in disease duration among ET cases in our sample (#40 years vs. .40 years), study subjects were stratified into 3 diagnosis-duration groups: controls, ET cases with shorter duration disease (n = 3, mean = 19.7612.2 years, range = 9?3 years), and ET cases with longer duration disease (n = 7, mean = 62.966.0 years, range = 55?0 years). The respective LC3-II protein levels were: 1.0060.14, 0.9560.14, and 0.7960.12, and in a linear regression model, LC3-II protein level declined by diagnosis-duration group (r2 = 0.37, p = 0.004) (Figure 1D). We also investigated calbindin level, a protein specifically expressed by 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]. We then investigated whether decrease in LC3-II was specific to the ET cerebellum. We determined LC3-II protein level in theoccipital cortex in 7 ET cases and 9 controls: ET cases had similar LC3-II level as controls (0.9860.13 vs. 1.0060.08) (Figure 1F, G). Cerebellar tissue was available for immunohistochemistry on 12 ET cases and 13 age-matched controls, who were similar with respect to age, 23977191 gender, brain weight and other variables of interest (Table 1). These included 6 of the 10 ET cases and 8 of 11 controls used in the Western blot analysis. We labeled the cerebellar sections with anti-LC3 and anti-calbindin antibodies to assess the LC3 content in PCs. PCs were found to have a robust autophagic activity, reflected by LC3 clustering; therefore, we used LC3 staining to assessed 
the autophagic activity in PCs [22]. We found that PCs in ET cases exhibited lower LC3 staining (Figure 2A ). We found that LC3 was present in punctate structures, which labels them as AVs: PCs in ET cases had strikingly fewer LC3 puncta than controls (Figure 2I ). We quantified the fraction of PC bodies, excluding the nucleus, that was occupied by AVs (Figure 2M ). The percentage of cell bodies occupied by AVs was more than 4-fold lower in ET cases than controls (2.0363.45 vs. 8.8069.81, p = 0.03)(Figure 2P). The results from Western blot analyses (i.e., LC3-II protein levels), were highly correlated with these immunolabel results (r = 0.78, p = 0.001). Among the 12 ET cases with immunolabel results, the mean disease duration of the patients was 46.3622.1 years (range = 17?80 years). Disease duration was not correlated with the fraction of cell bodies occupied by AVs (r = 20.12, p = 0.70), yet when study subjects were stratified into 3 diagnosis-duration groups (controls; ET cases with shorter duration disease [n = 4, mean = 19.863.4 years]; and ET cases with longer duration disease [n = 8, mean = 59.6612.6 years]), the respective percentage of cell bodies occupied by AVs were: 8.8069.81, 3.2163.32, and 1.4463.56, and in a linear regression model, the f.
