Targeted rescue of synaptic plasticity improves cognitive decline in sepsis-associated encephalopathy

Abstract

Sepsis-associated encephalopathy (SAE) is a frequent complication of severe systemic infection resulting in delirium, premature death, and long-term cognitive impairment. We closely mimicked SAE in a murine peritoneal contamination and infection (PCI) model. We found long-lasting synaptic pathology in the hippocampus including defective long-term synaptic plasticity, reduction of mature neuronal dendritic spines, and severely affected excitatory neurotransmission. Genes related to synaptic signaling, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and members of the transcription-regulatory EGR gene family, were downregulated. At the protein level, ARC expression and mitogen-activated protein kinase signaling in the brain were affected. For targeted rescue we used adeno-associated virus-mediated overexpression of ARC in the hippocampus in vivo. This recovered defective synaptic plasticity and improved memory dysfunction. Using the enriched environment paradigm as a non-invasive rescue intervention, we found improvement of defective long-term potentiation, memory, and anxiety. The beneficial effects of an enriched environment were accompanied by an increase in brain-derived neurotrophic factor (BDNF) and ARC expression in the hippocampus, suggesting that activation of the BDNF-TrkB pathway leads to restoration of the PCI-induced reduction of ARC. Collectively, our findings identify synaptic pathomechanisms underlying SAE and provide a conceptual approach to target SAE-induced synaptic dysfunction with potential therapeutic applications to patients with SAE.

Keywords: AAV-mediated overexpression; ARC; BDNF; enriched environment; hippocampus; memory dysfunction; sepsis-associated encephalopathy; synaptic plasticity.

Graphical abstract

Figure 1

Experimental sepsis causes neurocognitive dysfunction and anxiety, defective synaptic plasticity, and synaptic scaling in excitatory synapses (A) Experimental schematic. Acute phase of PCI is marked in red. Antibiotics (ATB) are delivered intraperitoneally until day 10, and behavioral testing (open field, OF; elevated plus maze, EPM; Barnes maze, BM) and ex vivo electrophysiological recordings (long-term potentiation, LTP; patch-clamp recordings, PC) are performed at the indicated time points. (B) (Left) Kaplan-Meier survival curve of all mice used in PCI experiments regardless of final experimental readouts. Mice intentionally taken out of the study, e.g., for experiments at early time points, were included up to the time when they were censored. (Right) Box plots and distribution of cumulative CSS on day 3 and day 5 after PCI (each circle represents an individual mouse after PCI). Note that subsets of PCI and SHAM mice were used in different experimental readouts in the study (detailed in the individual experiments). (C) In the BM, PCI mice (n = 30, red) show affected learning over the whole experimental period (two-tailed curve-permutation test) and a reduced memory recall in the probe trial (right; example tracking traces are shown, target quadrant marked in gray; SHAM mice n = 32, blue). (D) PCI mice spend less time in the EPM open arms (light gray). The locomotor activity as measured by total distance traveled within the EPM is unchanged (n = 12/7 SHAM/PCI). (E) PCI mice spent reduced time in the OF center (dark gray), whereas the distance traveled within the OF as a measure for locomotor activity was unchanged (n = 20/15 SHAM/PCI). (F) Long-term potentiation (LTP) in the hippocampal Schaffer collateral (SC)-CA1 pathway is severely impaired in PCI mice (n = 15/12 slices from 7/6 mice); two-tailed curve-permutation test, for time >0 min two-way repeated-measures ANOVA (F(1, 25) = 9,994), p = 0.0041). Two-tailed Student’s t test in the stable phase (min 25–30): p = 0.0123. Inset shows average traces of example recordings before and after theta burst stimulation. (G) Density of total synaptic spines and of mature mushroom spines in apical dendrites of CA1 neurons is reduced after PCI (n = 42/33 neurons). Scale bar, 3 μm. (H and I) Patch-clamp recordings of dentate gyrus granule cells indicates reduced frequency of miniature ESPCs (mESPCs) and spontaneous ESPCs (sEPSCs). (J) Peak amplitude of minimally evoked ESPCs (eEPSCs) is increased. PCI: n = 11/13/13, SHAM: n = 11/13/16 for mEPSCs, sEPSCs, and eEPSCs, respectively. Example traces in (H) and (I); average traces of minimal evoked eEPSCs in (H). Bar graphs show mean ± SEM; two-tailed Student’s t test, unless otherwise indicated.

Figure 2

Hierarchically clustered heatmap of DEGs in a murine sepsis model Expression of 144 DEGs (log₂FC > 1) in brain tissue on 3 days and 10 weeks after PCI with (color-coded) normalized Z-score visualization. Biological replicates from 3 days after PCI show a distinct and clustered expression profile (four lanes on the right).

Figure 3

PCI leads to distinct and time-dependent changes in gene expression in the brain Gene ontology (GO) terms enriched in 144 DEGs (log₂FC > 1) found by microarray analysis of brain tissue at 3 days and 10 weeks after PCI. DEGs were interrogated for enrichment in the gene sets of the GO definitions employing the piano package. Significantly enriched gene sets were clustered according to the score values of the five piano criteria distinct directional (up and down), mixed directional (up and down), and non-directional.

Figure 4

PCI induces downregulation of neuronal pathways relevant for synaptic plasticity (A) Interaction network of proteins corresponding to DEGs on day 3 after PCI. The node color depicts the respective log₂FC from microarray data. Proteins related to ARC synaptic signaling are enlarged for better visualization. (B) Expression of Arc and the six highlighted DEGs (in A) that are related to ARC signaling and synaptic function. Three days SHAM and PCI and 10 weeks PCI n = 4, 10 weeks SHAM n = 3. Box plots show median and 75^th to 25^th interquartile range, whiskers show respective maximum or minimum values within 1.5 times of the interquartile range; two-tailed Student’s t test. (C and D) Quantitative analysis of hippocampal ARC protein and pERK/ERK protein ratio (n = 8, each; normalized to SHAM ARC or SHAM pERK/ERK ratio, respectively) shows downregulation of protein signaling pathways in the network shown in (A). Bar graphs show mean ± SEM; two-tailed Student’s t test.

Figure 5

Hippocampal overexpression of Arc rescues inflammation-induced cognitive dysfunction and defective long-term potentiation (A) Experimental schedule indicating ARC overexpression in the hippocampus from day 10 after PCI. Stereotactic intrahippocampal microinjection of an adeno-associated virus (AAV) containing the bicistronic vector for ARC overexpression and Venus fluorescence under the synapsin promoter (bottom left). The injection coordinates are depicted on the bottom right. (B) Representative example of constitutive ARC overexpression in CA3 region of the hippocampus. Scale bars, 40 μm. (C) PCI mice receiving control AAV (PCI CTRL AAV; n = 17) show memory deficits in the Barnes maze compared to SHAM CTRL AAV (n = 18) as indicated by reduced time in the target zone in the probe trial. ARC overexpression in PCI mice (PCI ARC AAV; n = 18) results in rescue of learning and memory dysfunction, whereas memory after ARC overexpression in SHAM mice is unchanged (SHAM ARC AAV; n = 19). Example traces indicate tracks of individual mice during the probe trial; the target quadrant containing the open hole is marked in gray. Two-way ANOVA with Holm-Sidak post hoc test (F values: sepsis (1, 68) = 4.539, p = 0.368; ARC AAV (1, 68) = 0.887, p = 0.349; interaction (1, 68) = 8.791, p = 0.0042). (D) LTP as measured by slope of excitatory postsynaptic potentials (EPSPs) in single-cell whole-cell recordings in CA1 neurons after stimulation of Schaffer collaterals. Paired stimulation results in LTP in SHAM CTRL AAV (n = 11 slices from 4 mice) and PCI ARC AAV (n = 8 slices from 3 mice) mice compared to PCI mice with control virus injection (PCI CTRL AAV; n = 9 slices from 4 mice) and SHAM mice after ARC overexpression (SHAM ARC AAV; n = 11 from 5 mice), which show reduced LTP. Upper panel: average traces (blue: SHAM CTRL AAV; light blue: SHAM ARC AAV; red: PCI CTRL AAV; light red: PCI ARC AAV). Middle panel: LTP time course. Two-tailed curve-permutation tests with Benjamini-Hochberg correction applied on p values, for time >0 min. Lower panel: comparison of LTP at the indicated time points after theta burst stimulation. Two-way repeated-measures ANOVA with Tukey’s post hoc test (F values: 1^st 10 min: sepsis (1, 35) = 0.7281, p = 0.399; ARC AAV (1, 35) = 0.1733, p = 0.06797; interaction (1, 35) = 10.03, p = 0.0032; 3^rd 10 min: sepsis (1, 35) = 1.224, p = 0.2761; ARC AAV (1, 35) = 0.1566, p = 0.6947; interaction (1, 35) = 21.93, p < 0.0001). All graphs show mean ± SEM.

Figure 6

Enriched environment leads to improvement of synaptic pathology and cognitive dysfunction induced by PCI (A) Mice were subjected to enriched environment (EE) or standard housing (NE) from day 10 after PCI. Behavioral tests and LTP recordings were performed as indicated. (B) EE improves cognitive dysfunction in the BM after PCI (PCI EE, red framed bars) to control levels (SHAM EE, blue framed bars; SHAM NE, filled blue bars) as indicated by increased time in the BM target zone during the probe trial compared to PCI mice in standard care (PCI NE, filled red bars). Example traces indicate tracks of individual mice during the probe trial; target quadrant is marked in gray. Two-way ANOVA with Holm-Sidak post hoc test (F values: sepsis (1, 57) = 9.510, p = 0.0031; cage type (1, 57) = 3.811, p = 0.0558; interaction (1, 57) = 3.968, p = 0.0512.) (C) EE leads to reduced anxiety-related behavior in the EPM after PCI as shown by increased time in the open arms and reduced time in the closed arms. Example traces indicate tracks of individual mice in the EPM (n = 14/18/13/16; SHAM NE/SHAM EE/PCI NE/PCI EE in B and C). Two-way repeated-measures ANOVA with Tukey’s post hoc test (F values: open arm: sepsis (1, 53) = 5.835, p = 0.0192; EE (1, 53) = 2.940, p = 0.0922; interaction (1, 53) = 2.311, p = 0.1344. Closed arm: sepsis (1, 53) = 5.143, p = 0.0274; EE (1, 53) = 9.816, p = 0.0028; interaction (1, 53) = 0.6826, p = 0.4124). (D) LTP as evaluated by field potential recording in CA1 is improved in PCI mice after EE. Upper panel: average traces before (baseline traces) and after theta burst stimulation (SHAM EE, light blue; PCI EE, light red; amplitudes of the baseline traces of each group were scaled to the amplitude of the SHAM NE trace; dark blue). Middle panel: comparison of LTP at the indicated time points after theta burst stimulation (n slices/N mice: SHAM NE: 8/3; SHAM EE: 7/3; PCI NE: 10/4; PCI EE: 6/3). Two-way repeated-measures ANOVA with Tukey’s post hoc test (F values: time point (2, 54) = 3.033, p = 0.0564; group (3, 27) = 5.473, p = 0.0045). Bottom panel: LTP time course. Two-tailed curve-shuffling tests with Benjamini-Hochberg correction applied on p values for time >0 min. (E) Quantification of synaptic mushroom spines in apical dendrites of CA1 neurons. Example images are provided (scale bar, 5 μm). PCI leads to a reduction of mature mushroom spine density (see PCI NE), which is rescued to basal levels (as in SHAM NE) after enriched environment (see PCI EE); n = 39/18/33/33 for SHAM NE/SHAM EE/PCI NE/PCI EE. Two-way ANOVA with Tukey’s post hoc test (F values: sepsis (1, 119) = 17.94, p < 0.0001; EE (1, 119) = 0.0016; interaction (1, 119) = 0.413, p = 0.5214). All graphs show mean ± SEM.

Figure 7

ARC and BDNF levels are increased after enriched environment (A) Immunostaining for ARC in brain slices of post-septic mice with and without EE. Scale bar, 50 μm. (B) Increased density of ARC-positive granule cells in mice after EE. n = 7/8, two-tailed Student’s t test. (C) Arc expression increases in CA1 and CA3 pyramidal cell layer as measured from fluorescent intensity. n = 7/8, Mann-Whitney U test. (D) BDNF levels are increased in mouse brain tissue in SHAM and PCI mice after EE; n = 11/10/14/15 for SHAM NE/SHAM EE/PCI NE/PCI EE, pairwise permutation test with Benjamini-Hochberg correction. Bar graphs show mean ± SEM.