Limbic system synaptic dysfunctions associated with prion disease onset

Abstract

Misfolding of normal prion protein (PrP^C) to pathological isoforms (prions) causes prion diseases (PrDs) with clinical manifestations including cognitive decline and mood-related behavioral changes. Cognition and mood are linked to the neurophysiology of the limbic system. Little is known about how the disease affects the synaptic activity in brain parts associated with this system. We hypothesize that the dysfunction of synaptic transmission in the limbic regions correlates with the onset of reduced cognition and behavioral deficits. Here, we studied how prion infection in mice disrupts the synaptic function in three limbic regions, the hippocampus, hypothalamus, and amygdala, at a pre-clinical stage (mid-incubation period) and early clinical onset. PrD caused calcium flux dysregulation associated with lesser spontaneous synchronous neuronal firing and slowing neural oscillation at the pre-clinical stage in the hippocampal CA1, ventral medial hypothalamus, and basolateral amygdala (BLA). At clinical onset, synaptic transmission and synaptic plasticity became significantly disrupted. This correlated with a substantial depletion of the soluble prion protein, loss of total synapses, abnormal neurotransmitter levels and synaptic release, decline in synaptic vesicle recycling, and cytoskeletal damage. Further, the amygdala exhibited distinct disease-related changes in synaptic morphology and physiology compared with the other regions, but generally to a lesser degree, demonstrating how different rates of damage in the limbic system influence the evolution of clinical disease. Overall, PrD causes synaptic damage in three essential limbic regions starting at a preclinical stage and resulting in synaptic plasticity dysfunction correlated with early disease signs. Therapeutic drugs that alleviate these early neuronal dysfunctions may significantly delay clinical onset.

Keywords: Cognition; Emotion; Limbic system; Neurodegeneration; Prion disease; Synaptic transmission.

Fig. 1

Prion propagation in the limbic regions during disease. a An illustration of reduced nesting and mood disorder-like symptoms in a mouse with prion disease (middle, right) compared with a normal healthy mouse (left). b A schematic diagram illustrating the timeline of the study, where weanling wild-type mice were inoculated with RML prions, and ex vivo fresh coronal slices of the limbic regions (hippocampus, hypothalamus, amygdala) were used for the assessments in this study at a pre-clinical stage or mid-incubation period (50% of the disease progression to terminal stage disease at ~ 80 days post-inoculation, dpi) and at clinical onset (~ 70% of disease progression or at ~ 108 dpi). c Prion seeding activity (logSD50) in the limbic regions at the pre-clinical and clinical onset (Unpaired Student’s t-test was used to compare prion seeding activity between two disease time points within a region). d Western blotting for PrP (6D11 antibody) after an ultracentrifugation separation of soluble from insoluble isoforms, relative to the total PrP, in the three limbic regions in samples from uninfected controls (UN) and the two disease time points. The bottom panels are Coomassie stains of total protein for the total PrP blots. Molecular weight markers are on the right. e The quantification of the blots in d. PrP at each time point was compared to the UN control by unpaired Student’s t-test. c, e Each dot represents a mouse, and data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Dysfunction of basal neuronal activity. a–f Spontaneous synchronous neuronal firing in hippocampal CA1 (a, b), ventral medial hypothalamus (VMH; c, d), and basolateral amygdala (BLA; e, f) at the pre-clinical stage (a, c, e) and at clinical onset (b, d, f) measured by MEA (left panels of a, c, e; black dots are electrodes with the regions of interest marked by the dotted box). Raster plots (middle panels) present the neuronal firing (black dash) and burst (red line) with the quantification of burst rate (scatterplot on the right), comparing uninfected controls (UN) and infected mice (INF). See supplementary file 2 for representative burst traces. g–i Relative oscillatory powers of delta and gamma oscillations in UN versus INF at the pre-clinical stage and clinical onset in the CA1 (g), VMH (h), and BLA (i). Upper panels are representative traces of local field potentials (LFP; light blue) and delta waves from UN (dark blue) and INF (red). See supplementary file 3 for delta and gamma peak frequency. j–l Calcium flux analysis in the three limbic regions (hippocampus, hypothalamus, amygdala) at the pre-clinical stage and clinical onset (n = 3 per group). m–o Input–output curves measuring the magnitude of field excitatory post-synaptic potential (fEPSP) responses to increasing strength of stimuli to assess the impact of prion disease on the strength of synaptic transmission in the three limbic regions (CA1, VMH, BLA) at the pre-clinical stage (left panels; n = 8 for UN; n = 4 for IN) and clinical onset (right panels; n = 8 for UN; n = 4 for IN). Representative traces of fEPSPs are presented in each plot (vertical bars represent 100 µV and horizontal bars show 10 ms). Data were analyzed by unpaired Student’s t test (a–i) and Two-way ANOVA (assuming sphericity) with repeated measures and Sidak corrections for multiple comparisons. Data (biological replicates) are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Synaptic changes in limbic regions during disease. a Subregions within the three limbic regions (schematic diagram), including the hippocampal CA1, ventral medial hypothalamus (VMH), and basolateral amygdala (BLA), were assessed by immunofluorescence (IF) labelling for synapses. The right panels display the subregions labeled with phalloidin (green), where the dotted boxes mark the specific regions assessed by higher magnification confocal microscopy. b Immunofluorescence assessment of total synapses by MAP2 and synaptophysin in the CA1, VMH, and BLA in uninfected controls (UN) relative to infected (INF) samples from the pre-clinical and clinical onset phases. c, d The quantification of MAP2 (c) and synaptophysin (d) in multiple fields of view of immunofluorescence images represented in b. Each dot represents a field of view. e Western blotting analysis of synaptophysin in the three brain regions at both time points with the lower panels showing the Coomassie stain for total protein as the loading control. f The quantification of synaptophysin normalized to the total protein. g Western blotting analysis of PSD95 in the three brain regions at both disease time points with the bottom panels showing Coomassie stain for loading control. h The quantification of PSD95 after normalizing to total protein. c, d, f, h Unpaired Student’s t-test was used to analyze the disease-related change in each marker relative to its respective UN control. Data (biological replicates) are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Cytoskeletal damage that may impede synaptic transmission in the limbic regions during prion disease. a A schematic diagram (Biorender) of a neuron showing the cytoskeletal markers analyzed, including filamentous actin (F-actin), microtubules (beta-tubulin), and neurofilament (NF-L). b–d Immunofluorescence analysis of the cytoskeletal markers in the hippocampal CA1, ventral medial hypothalamus (VMH), and basolateral amygdala (BLA) in uninfected controls (UN) versus infected mice at the pre-clinical stage and clinical onset. e–g The quantification of phalloidin for F-actin (e), NF-L (f), and beta tubulin (g) in multiple fields of view of immunofluorescence images represented in b–d. Each dot represents a field of view. h Western blotting analysis of NF-L in the hippocampus, hypothalamus, and amygdala (containing all subregions) at the pre-clinical time point and clinical onset. Coomassie stain for total protein (TP) was the loading control. Bottom panels show the quantifications of NF-L after normalizing to TP. Each dot represents a biological replicate. e–h Unpaired Student’s t-test was used to compare the level of each cytoskeletal marker between INF and UN control in each region. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Neurotransmitter level and synaptic release during the disease transition from pre-clinical to clinical onset. a Bubble plots showing fold change of neurotransmitters in scrapie-infected (INF) hippocampus, hypothalamus, and amygdala (containing all subregions) at the pre-clinical disease and clinical onset relative to the age-matched uninfected (UN) controls (n = 4). b, c Western blotting analysis of synaptojanin 1 in each limbic region at the preclinical stage (b) and clinical onset (c) with the mean synaptojanin 1 level relative to its age-matched UN control compared by unpaired Student’s t test. d–f Paired-pulse (PP) ratios measured by paired-pulse test (See Methods) to estimate the probability of neurotransmitter release in hippocampal CA1 (d), ventral hypothalamus (e), and basolateral amygdala (f) of normal UN controls and INF mice at the two disease time points. Top panels are representative traces of field excitatory postsynaptic potentials (fEPSPs) evoked by the first pulse (p1) and the second pulse (p2). Vertical bar represents 100 µV and horizontal bar shows 20 ms. The mean 1/PP ratio was compared between UN and disease time points by Kruskal Wallis testing with Dunn’s correction for multiple comparisons. g TEM images in the hippocampus, hypothalamus, and amygdala in normal UN controls and INF mice with the pre-synaptic terminals are highlighted, harboring synaptic vesicles of neurotransmitters at the release sites (white arrows), adjacent the post-synaptic terminals (black arrow). h Bar graphs (by Microsoft Excel) displaying the scoring of TEM images or fields of view (FOV) based on the integrity of post-synapse and size of the readily releasable pool (RRP) of synaptic vesicles. This analysis (see Methods for more details) estimated levels of normal and damaged synapses and numbers of FOV with these phenotypes in uninfected and infected samples. The total FOV per group are listed on the right. Data in b–f (biological replicates) are presented as mean ± SEM. *p < 0.05

Fig. 6

NMDA receptor-dependent long-term synaptic plasticity in limbic regions at the transition from pre-clinical to clinical disease. a–c Long-term synaptic plasticity induced by trains of high-frequency stimulation in the hippocampal CA1, ventral medial hypothalamus (VMH), and basolateral amygdala (BLA) at pre-clinical stage and clinical onset. Unpaired Student’s t-test was used to compare the mean field excitatory postsynaptic potential (fEPSP) of the last 10 min of the recording between uninfected controls (UN) and infected (INF) samples. Data are presented as mean ± SEM with n = 4 mice per group. d Representative immunofluorescence (IF) images of PSD95, MAP2, and synaptophysin in UN and INF CA1, VMH, and BLA after tetani induction of synaptic plasticity. e Estimates of various features of synapses by images (as represented in d) using segmentation approaches (n = 3/group). f–h The inverted paired pulse ratio (PPR) that estimates of the probability of neurotransmitter release before and after the synaptic plasticity induction (PPR1 and PPR2), indicating the release efficacy in the UN versus the INF samples at the pre-clinical stage and clinical onset. Each dot represents a mouse. Paired Student t-test was used to compare PPR1 and PPR2. Data (biological replicates) are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001