Journal: Theranostics

Article Title: TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress

doi: 10.7150/thno.46566

Figure Lengend Snippet: SCI leads to autophagy flux blockade and ER stress-induced apoptosis. ( A ) Western blotting of autophagy flux markers, Beclin1, ATG5, VPS34, ATP6V1B2, LAMP2, C-CTSD, SQSTM1/p62, UB and LC3 in spinal cord tissue from Control and SCI mice at the indicated time points. ( B ) Representative Western blotting of LC3 in Control and SCI (Day1) spinal cord slides cultured in the presence or absence of CQ. ( C ) Densitometric analysis of Beclin1, ATG5, VPS34, ATP6V1B2, LAMP2, C-CTSD, SQSTM1/p62, UB and LC3II data from (A) normalized to loading control GAPDH. ( D ) Densitometric analysis of LC3II from (C) normalized to the loading control GAPDH. ( E ) Relative mRNA level of Sqstm1/p62 and Ctsd in the spinal cord from Control an SCI mice normalized to control β-actin at the indicated time points. ( F ) Western blot analysis of ER stress-induced apoptosis markers, GRP78, PDI, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP, CASP12, C-CASP12, CASP3 and C-CASP3 in Control and SCI groups. ( G ) Densitometric analysis of GRP78, PDI, p-PERK, p-eIF2α, ATF4, CHOP, C-CASP12 and C-CASP3 from (F) normalized to loading control GAPDH. n=6, ns stands for not significant, *P<0.05, **P<0.01.

Article Snippet: Primary antibodies against Beclin1 (Cat. No. 3738), ATG5 (Cat. No. 12994), ATP6V1B2 (Cat. No. 14617), Ubiquitin (Cat. No. 3936), ATF4 (Cat. No. 11815), CHOP (Cat. No. 2895), AMPKα (Cat. No. 5832), p-AMPKα (Cat. No. 2535) p-FOXO3a (Cat. No. 9466), p-EIF2α (Cat. No. 3398), p-4EBP1 (Cat. No. 9456), mTOR (Cat. No.2983), p-mTOR (Cat. No.5536) and CARM1 (Cat. No. 4438) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Cell Culture

Journal: Theranostics

Article Title: TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress

doi: 10.7150/thno.46566

Figure Lengend Snippet: ROS-induced lysosomal dysfuntion initiates autophagy flux blockade and ER stress-induced apoptosis after SCI. ( A ) ELISA of oxidation products 8-OHdG, AOPP, and MDA in spinal cord lesions from Control and SCI mice at the indicated time points. ( B ) ELISA of 8-OHdG, AOPP, and MDA in spinal cord lesions from mice grouped as indicated at Day1 after SCI. ( C ) Western blotting of Beclin1, VPS34, C-CTSD, SQSTM1/p62, UB and LC3 in spinal cord from non-SCI (Control) mice and SCI mice treated with MnTBAP or Vehicle1 at Day1 after SCI. ( D ) Western blot analysis of LC3 in SCI+Vehicle1, and SCI treated with MnTBAP spinal cord slides at Day1 cultured in the presence or absence of CQ. ( E ) Densitometric analysis of Beclin1, VPS34, ATP6V1B2, LAMP2, C-CTSD, SQSTM1/p62, UB and LC3II from (C) normalized to loading control GAPDH. ( F ) Densitometric analysis of LC3II from (D) normalized to the loading control GAPDH. ( G ) Western blot analysis of GRP78, PDI, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP, CASP12, C-CASP12, and C-CASP3 in spinal cord lesions from the grouped mice on Day3. ( H ) Densitometric analysis of GRP78, PDI, p-PERK, p-eIF2α, ATF4, CHOP, C-CASP12 and C-CASP3 from (G) normalized to loading control GAPDH. n=6, ns stands for not significant, *P<0.05, **P<0.01.

Article Snippet: Primary antibodies against Beclin1 (Cat. No. 3738), ATG5 (Cat. No. 12994), ATP6V1B2 (Cat. No. 14617), Ubiquitin (Cat. No. 3936), ATF4 (Cat. No. 11815), CHOP (Cat. No. 2895), AMPKα (Cat. No. 5832), p-AMPKα (Cat. No. 2535) p-FOXO3a (Cat. No. 9466), p-EIF2α (Cat. No. 3398), p-4EBP1 (Cat. No. 9456), mTOR (Cat. No.2983), p-mTOR (Cat. No.5536) and CARM1 (Cat. No. 4438) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture

Journal: Nature Communications

Article Title: Lysosomal exocytosis releases pathogenic α-synuclein species from neurons in synucleinopathy models

doi: 10.1038/s41467-022-32625-1

Figure Lengend Snippet: a Neuronal late-endosomes/lysosomes immunoisolated from the brains of 1- and 6-month-old Tg-αSyn A53T /Tg- HA LAMP1 Myc mice using anti-myc antibodies were analyzed by immunoblotting for levels of αSyn, αSyn pSer129 , αSyn Fila , and αSyn Amyl (Mono monomer, Aggr aggregates). The fraction of these αSyn species residing within neuronal lysosomes at 6 months of age (bottom graph) was back-calculated from the fraction of each αSyn species immunocaptured (αSyn species immunocaptured/total input), normalized to the fraction of neuronal late-endosomes/lysosomes captured—indicated by the fraction of HA LAMP1 Myc captured (HA blot immunocaptured/total input). Membrane-matched dot blots (β-Actin = loading control) are separated by dashed lines, and immunoblots against markers of lysosomes and of other organelles are also shown ( n = 8). b Electron micrographs showing increased αSyn aggregate-labeling within the lysosomal lumen in 6-month-old Tg-αSyn A53T brains (2 lysosomes shown with 12 and 5 gold particles) compared to WT brains (2 lysosomes shown with 1 possible and 0 gold particles). ( n = 50 lysosomes for WT and n = 55 lysosomes for Tg-αSyn A53T ). c Lysosomes isolated from the brains of 6-month-old Tg-αSyn A53T / HA LAMP1 Myc mice (as in a ) were subjected to on-bead limited proteolysis using the indicated concentrations of proteinase K (PK) in the absence or presence of 0.1% Triton X-100. Immunoblots for αSyn (αSyn, αSyn pSer129 , αSyn Fila , and αSyn Amyl ), mature cathepsin-L (Cath-L Mat ; lysosome lumen), and vATPase subunits ATP6V1B2 and ATP6V1E2 (lysosomal proteins exposed on the cytosolic side) ( n = 4 brains, isolation, and proteolysis). d Tg x2 -αSyn A53T primary cultures lentivirally expressing a synapsin-1 promoter-driven Apex-2 LAMP1 construct were subjected to proximity-labeling with biotin at 14 or 49 days in vitro (DIV). Labeled proteins were precipitated on streptavidin-coated magnetic beads and immunoblotted for the indicated pathogenic αSyn species, as well as for the marker proteins cathepsin-L (lysosomes), LAMP1 and LAMP2 (late-endosomes/lysosomes), TSG101 (exosomes), synaptogyrin-1 (synaptic vesicles), neuroserpin (non-lysosomal/constitutively secreted neuronal protein), and β-actin (cytosol). Membrane-matched dot blots (β-Actin = loading control) are separated by dashed lines. (representative of n = 3). e Tg x2 -αSyn A53T primary cultures were subjected to in situ proximity ligation assay (PLA) at either DIV14 (top) or at DIV49 (bottom), with primary antibodies against α-Syn Fila and cathepsin-D (Cath-D). Quantification of PLA puncta per nucleus is shown, performed identically between images from DIV14 and 49 (n = 4 cultures and PLA experiments; data from 3 images averaged for each n). Data represent means ± SEM. Each “n” corresponds to independently aged mouse littermates in a , lysosomes in b , independent mouse brains, immunoisolation and proteolysis experiments in c , independent primary neuron cultures in d , and independent pups, cultures, and PLA experiments in e . ****P < 0.0001 by 1-way ANOVA with Dunnett multiple-comparison correction and #### P < 0.0001 by non-parametric Kruskal–Wallis test with Dunn’s multiple-comparison adjustment; all tests comparing levels of each αSyn aggregate-type to αSyn monomer in a ; **** P < 0.0001 by unpaired 2-tailed Student’s t test and #### P < 0.0001 by non-parametric Mann–Whitney test in b ; 2-way ANOVA with Bonferroni multiple comparisons post-test (compared at each PK concentration) in c ; and ** P < 0.01 by unpaired 2-tailed Student’s t test and # P < 0.05 by non-parametric Mann–Whitney test in e .

Article Snippet: β-Actin: Sigma (A1978); A11 amyloid: Stressmarq (SPC-506D); ATP5G: Abcam (ab181243); ATP6V1B2: Cell Signaling (D2F9R); ATP6V1E2: St. John’s Labs (STJ191615); Calreticulin: Thermo Fisher (OTI15F5) and Novus (NB600-103); Cathepsin-D: R&D systems (AF1029); Cathepsin-L: Novus (JM10-78); CD81: Novus (SN206-01); EEA1: Thermo Fisher (MA5-14794); FLAG: Sigma Cl.

Techniques: Western Blot, Labeling, Isolation, Expressing, Construct, In Vitro, Magnetic Beads, Marker, In Situ, Proximity Ligation Assay, MANN-WHITNEY, Concentration Assay