Journal: International Journal of Molecular Sciences

Article Title: The E3 Ubiquitin Ligase PRAJA1: A Key Regulator of Synaptic Dynamics and Memory Processes with Implications for Alzheimer’s Disease

doi: 10.3390/ijms26072909

Figure Lengend Snippet: Pja1 Knockdown in CA1 Reduces Spine Density and Alters Synaptic Protein Expression. ( A ) Schematic of bilateral lentiviral injection targeting the CA1 region. ( B ) Representative immunofluorescence images of the CA1 region showing GFP expression (green), DAPI nuclear staining (blue), and merged images of mice injected with either scrambled shRNA (sc shRNA, top row) or Pja1 shRNA (bottom row) are shown. Scale bar: 100 μm. ( C ) Quantification of PRAJA1 protein levels following lentiviral injection, showing a significant reduction in the Pja1 shRNA group. ( n = 8 sc shRNA, n = 7 Pja1 shRNA, unpaired t -test, *** p = 0.0005). ( D ) Representative images of dendritic spines (top) and quantification of spine density (bottom), demonstrating a significant decrease in Pja1 knockdown mice. Scale bar: 5 μm. ( n = 33 spines from 3 mice per group; unpaired t -test; **** p < 0.0001). ( E ) Representative Western blots and quantification of pre-synaptic protein levels, showing significant reductions in Munc18-1 and SNAP-25 in the Pja1 shRNA group. (Munc18-1: n = 7 per group, * p = 0.025; SNAP-25: sc shRNA: n = 7; Pja1 shRNA: n = 9, p > 0.05, ** p = 0.006; syntaxin 1A: sc shRNA: n = 8; Pja1 shRNA: n = 7, p > 0.05 ns, unpaired t -test). ( F ) Representative Western blots and quantification of post-synaptic protein levels, revealing a significant reduction in PSD-95 in the Pja1 shRNA group (Synapsin (total and phosphorylated): sc shRNA: n = 8; Pja1 shRNA: n = 7, p > 0.05 ns; PSD-95: sc shRNA: n = 10; Pja1 shRNA: n = 11, p > 0.05, ** p = 0.008 unpaired t -test.

Article Snippet: The following antibodies were used: PRAJA1 (1:1000, ProteinTech, Chicago, IL, USA, 17687-1-AP), actin (1:1000, Cell Signaling, Danvers, MA, USA, CST:4970s), Cav1.2 (1:500, Alomone, Jerusalem, Israel, ACC-003), Cav1.3 (1:500, Alomone, Jerusalem, Israel, ACC-005), SNAP25 (1:1000, CST:5309s), Munc18-1 (1:1000, CST:d406v), rabbit anti-PSD-95 (1:1000, CST:2507s), spinophilin (1:1000, CST:14136s), and synapsin (1:1000, CST:2312s). p-synapsin (1:1000, Cell Signaling, Danvers, MA, USA, CST:2311s), syntaxin 1A (1:1000, CST: 13002s).

Techniques: Knockdown, Expressing, Injection, Immunofluorescence, Staining, shRNA, Western Blot