Fig 2: Accumulation of aggregate-prone proteins and deficits in spatial learning and memory in SGPL1fl/fl/Nes mice. Representative western blot images and graphs showing mean ± SEM in brain extracts of control and SGPL1fl/fl/Nes mice of the indicated ages for: (A) APP-FL (full length) and APP-CTFs (C-terminal fragments) (n ≥ 3; 2-way ANOVA, Pgenotype, APP-FL = 0.0034, Ptime, APP-CTFs = 0.0453, Pgenotype, APP-CTFs = 0.0359). (B) SNCA (n ≥ 3; 2-way ANOVA, Pgenotype = 0.0050). Western blots of ACTB are shown in all panels as loading control. (C to E) Hidden version of the Morris water maze; TQ, target quadrant with hidden platform; OQ, other quadrants. (C) Time of quadrant occupancy (2-way ANOVA, P = 0.001); (D) number of target crossings after completion of training (2-way ANOVA, P = 0.001); (E) time spent in the target area expressed as distance from the target (2-way ANOVA, P = 0.043). (F) Fear conditioning test. Shown is the relative time of activity expressed as the activity suppression ratio. Baseline activity was determined 2 min before aversive stimulus whereas time of activity was determined 1 d after associative training in a context fear-conditioning paradigm (unpaired t test, P = 0.0053). a.u., arbitrary units.

Fig 3: Effects of Anesthesia/Surgery/ICU on expression of synuclein alpha, neurotrophic receptor tyrosine kinase 1 and syntaxin 1a genes. (A) Snca α, Ntrk1, and STX1a mRNA levels were significantly impaired in A/S/I mice relative to controls following ICU environment (–3.785, –1.498, and –2.268 fold decrease, respectively). Hippocampal total RNA was extracted from A/S/I animals (pooled N = 3) and controls (pooled N = 3). (B–E) Protein levels of STX1a (B: P < 0.012), Ntrk1 (C: P = 0.039), and Snca α (D: P = 0.017) were significantly decreased in A/S/I mice relative to controls. Snca phosphorylated at serine residue 129 was also significantly down-regulated in insult-exposed mice (E: P = 0.008). Two-tailed unpaired t-test (N = 7 A/S/I and N = 7 control mice). STX1a, syntaxin 1a; NtrK1, neurotrophic tropomyosin receptor kinase 1; Snca α, synuclein alpha; p-Snca α, synuclein alpha phosphorylated at serine residue 129. ∗P < 0.05 and ∗∗P < 0.01, respectively.

Fig 4: Effects of Anesthesia/Surgery/ICU on synaptophysin, microtubule-associated protein 2 and postsynaptic density protein 95 levels. (A–C) Protein levels of Syp (A: P = 0.002), MAP2 (B: P = 0.013), and PSD-95 (C: P = 0.003) were significantly decreased in A/S/I mice relative to controls. Two-tailed unpaired t-test (N = 5 A/S/I and N = 5 control mice). Syp, synaptophysin; MAP2, microtubule-associated protein 2; PSD-95, postsynaptic density protein 95. (D) Representative 20× magnification images of the distribution of PSD-95 immunostaining in sagittal CA1 and CA3 hippocampal sections from two control and two A/S/I mice. Arrows highlight differences in PSD-95 immunofluorescent staining. (E) Plot of STX1a, Ntrk1, Snca α, Syp, MAP2, and PSD-95 protein levels against individual mouse Z scores. Z scores from behavioral assessments obtained at the 24 h time point were used, since tissue collection for protein studies was performed immediately after this behavioral assessment. The vertical dotted line represents a protein level of 85% (expressed as change from control). ∗P < 0.05 and ∗∗P < 0.01, respectively.

Fig 5: Workflow for the acquisition and overall description of proteomes. (a) Overview of proteomic workflow. The midbrain and striatum from the brains of two mice were dissected and combined to generate one sample replicate. The ipsilateral injected and the contralateral non-injected sides were kept separate and processed throughout the workflow independently. Immobilized metal affinity chromatography (IMAC) was used to enrich for phosphorylated peptides. (b) Venn diagrams depicting the number of proteins and phosphopeptides quantified in 4 independent sample replicates in the injected side and non-injected side in wild type mice. The acquired proteomes in wild type and Snca−/− showed typical distribution in terms of molecular weight, cellular location and major biological processes when compared to a whole mouse brain proteome: (b) Distribution of the proteins quantified in wild type and Snca−/− mice based on protein molecular weight. (d) Gene ontology enrichment analysis using cellular components (e) Gene enrichment using biological process and KEGG pathways.