Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 3. PGC-1α increases the levels of mitochondrial transport proteins in the cortex of APP/PS1 mice. Immunohistochemistry was used to examine the expression patterns and levels of (Aa-a’) MFN2, (Ba-a’) KIF5A, and (Ca-a’) Dync1i1 in cortex samples from WT/2 × Tg-AD mice. The impact of treatment (AAV-Vector/AAV-PGC-1α) on the expression and levels of (Ab-b’) MFN2, (Bb- b’) KIF5A, and (Cb-b’) Dync1i1 in cortex samples from 2 × Tg-AD mice was also assessed using immunohistochemistry. Scale bars = 100 μm. The expression patterns and qualification of (Bc-c’) KIF5A in cortical samples from 2 × Tg-AD mice treated with AAV-Vector/AAV-PGC-1α were examined with immunoflu orescence. Scale bars = 200 μm. Green = HA-labeled PGC-1α; Red = KIF5A; Blue = DAPI. (Bd-d’) N2A cells were transfected with pEnCMV/PGC-1α plasmid and plasmid-encoding APPswe for 48 h. The expression patterns and quantifi cation of KIF5A in the cells were studied with western blot. For each group, n = 6. Values are expressed as the means ± S.E.M. Significance levels were set at ** p < 0.01 and *** p < 0.001 for noted differences between AAV-Vector- and AAV-PGC-1α-infused AD mice or pEnCMV- or PGC-1α-transfected APPswe cells. GAPDH was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Immunohistochemistry, Expressing, Plasmid Preparation, Labeling, Transfection, Western Blot, Control

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 5. RID3, but not RID2, of the PGC-1α region is indispensable for promoting retrograde transport of axonal mitochondria and enhancing mitochondrial auto phagic clearance. N2A cells were co-transfected with plasmid encoding APPswe and pEnCMV/PGC-1α/PGC-1αmL2/PGC-1αmL3 plasmids. The lysates were subjected to immunoblotting using the specified antibodies. Expression patterns and quantification of (A) the retrograde transport protein Dync1i1 and (B) mitophagy-relevant proteins, including (a) Parkin, (b) Pink, (c) LC3-I/II, (d) Beclin and (e) P62, were examined via western blot analysis. GAPDH was utilized as a loading control. Each group consisted of n = 6 samples. (C) Fluorescence confocal microscopy was employed to detect JC-1 signals in N2A cells. Data represent three independent measurements. Fluorescence was captured using excitation at 488 nm and adjusting the emission of confocal microscopy for J-monomers (visible as green) and J- aggregates (visible as red/orange). (D) The ratios of J-aggregates to J-monomers were quantified as an indicator of mitochondrial membrane potential (MMP). Each group included n = 6 samples. Scale bars = 10 μm. The data underwent One-way ANOVA analysis to assess the effects of individual factors in isolation from others. Different letters indicate significant differences in mean values between groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Fluorescence, Confocal Microscopy, Membrane, Isolation