Chronic inflammation after double PolyI:C challenge
Double-challenged PolyI:C animals were injected in prenatal life at gestational day 17 and in the offspring at 2.5 months (PP). Animals were analyzed cross-sectionally at 3, 6, 9, and 16 months, taking saline-injected controls as reference (NN) (Fig. 1A). From 3 to 6 months of age levels of four circulating inflammatory cytokines (MCP-1, IL-6, TNF-α, and IL-10) are progressively higher in PP as compared to NN (Table 1). A two-way ANOVA highlights that the observed differences in the TNF α levels between treatment are due to the effect of age (F3,37=95, p<0.001) based on the interactions between age and treatment (F3,37=3, p<0.05), with higher levels in the 6 months PP mice than the other ages (F1,14=9.4, p<0.005). We also observed a significant effect on IL-6 expression after PolyI:C treatment with aging (F3,37=17, p<0.001) based on the interaction between treatment and age (F3,37=17.59, p<0.00005) with a peak of plasma IL-6 levels in 9 months PP mice (F1,8=71, p<0.00005). Later at 16 months, NN and PP mice show no difference in humoral immune factors (Table 1). Considering the upregulation of a large population of circulating chemokines in the blood of PP mice from 3 months of age, we examined the neutrophils, monocytes, and total polymorphonuclear cells (PMNs) from the freshly isolated whole blood and brain samples to study any infiltration of these leukocytes into the brain. Samples were analyzed using the flow cytometry for the expression of neutrophils and monocytes using antibodies stained for Ly6G, Siglec, F4/80, CD11b. Although no changes are observed at 3 months of age between groups, at 6 months PP mice exhibit elevated levels of neutrophils and PMNs in the systemic circulation but no significant differences are seen in the brain of PP mice as compared to NN animals (Suppl. Fig. 1A and B). On the other hand, the monocyte population differs neither in the blood nor in the brain (Suppl. Fig. 1C).
While there is no trace of infiltrating immune cells in the brain at 3 or 6 months, analysis by RT-PCR of IL-6 transcripts shows a significant different trend between PP and NN mice over time with aging (F3,30=4.4, p<0.05) based on the interaction of treatment with age (F3,30=10.9, p<0.005) After a peak at 6 months (F1,8=1.5; p=0.05), IL-6 decreases significantly in the brain of PP mice compared to saline controls (F1,8=5.25, p<0.005). Further, IL-1𝛽 expression in PP brains changes overtime with aging (F3,30=4.24, p<0.05), starting from lower levels in PP brains at 3 months (F1,8=6.44, p<0.01) followed by a peak at 9 months (F1,8=3.4, p<0.05). The effect of treatment on the expression of Interferon gamma (IFN𝛾), a cytokine critical against viral infections, showed a significant effect between the groups (F1,30=4.7, p<0.05) and a slight difference over time (F3,30=2.49, p<0.07) with a rise in expression of IFN𝛾 in PP brains at 6 months as compared to NN (F1,10=6.78, p<0.05).
We further analyzed how the inflammatory transcripts correlate with cell-type-specific transcripts for neurons (GRIN1, Glutamate Ionotropic Receptor NMDA Type Subunit 1), microglia (Iba1; Ionized calcium-binding adaptor molecule 1) and astroglia (GFAP; Glial Acidic Fibrillary Protein) in PP and NN animals. We observe that GRIN1 expression is stable except for a trending increase at 9 months (Fig. 1C), whereas Iba1 shows dynamic changes with a progressive increase from 6 to 9 months and drop at 16 months (F1,8=5.9, p<0.05) (Fig. 1D) similarly to the proinflammatory cytokines (IL-6, r16mo=0.77 and IL-1𝛽, r16mo=0.68). At 6 months (F1,8=1.7, p=0.19) and 9 months (F1,8=7.5, p<0.05), GFAP expression increases but decays to normal levels at 16 months (Fig. 1E). The temporal evolution of GFAP overlaps with that of IL-1𝛽, (rt =0.62, p<0.05; Fig. 1F). Our results show that the combination of prenatal and an early postnatal PolyI:C immune activation causes a prolonged and sustained systemic inflammatory response with the recruitment of circulating leukocytes of the innate immunity, and increased neuroinflammation and gliosis at mid-age.
Progressive tauopathy in PP mice
Inflammatory mechanisms are central to AD pathology involving both the interaction between inflammatory stimuli, proinflammatory cytokines/mediators, and AD pathological hallmarks such as aggregates of Aβ and hyperphosphorylated tau protein (p-tau) (Kinney et al. 2018), underlying neural network dysfunction and brain atrophy. We determined the impact of systemic immune challenge on the pre-tangle pathology by analyzing tau hyperphosphorylation at positions 231 and 205 using western blot (Fig. 2A) and immunohistochemistry (Fig. 2F and Suppl. Fig. 2A) on hippocampal tissue from 3 to 16 months of age. The content of hippocampal p-tau (pTau T231) relative to total tau (ptau/tau) is significantly different between PP and NN (F1,30=11.5, p<0.005) with aging (F3,30=6.7, p<0.005) based on the significant interaction between treatment and age (F3,30=5.6, p<0.005). Specifically, ptau/tau increases dramatically in PP mice at 6 months, (F1,7=21.7, p<0.00005), remains elevated at 9 months (F1,8=10.5, p<0.005) and then is indistinguishable from NN mice at 16 months (Fig. 2B and 2C). The progressive tauopathy is confirmed by immunolabeling of pTau fibers (pTau T205) in the hippocampal CA1 region and CA1 stratum lacunosum (Suppl. Fig. 2A), with a significant increase in pTau pixels in the CA1 region at 16 months (Suppl. Fig. 2B). Tau hyperphosphorylation and the formation of 𝛽-sheet fibrils, labeled by Thioflavin-S, can be observed in the CA3 field at 9 months in PP mice, in contrast to NN (Fig. 2F). To assess whether the progressive tauopathy in PP is associated with synaptic abnormalities and neuroinflammatory responses, we quantified the levels of synaptophysin and GFAP overtime (Fig. 2D). At 16 months in PP mice, we observe a non-significant drop in synaptophysin expression accompanied by significant increase in GFAP (F1,30=10.04, p<0.005) (Fig. 2E). The increase in GFAP immunoreactivity is confirmed by immunohistochemistry of the CA3 region showing the presence of amyloid-𝛽1-42 and p-tau small aggregates (Fig. 2G; insert). Further analysis of amyloid-𝛽 fibrils in the 16 months old mice indicates that A𝛽1-42 is also present in NN old mice with internalization in GFAP positive glial cells, which is more pronounced in PP mice (Fig. 3A, white stars). Besides, in 16 months old PP mice, A𝛽1-42 positive aggregates are visible in vessels ensheathed by astroglial endfeet reflecting CAA (Fig. 3A, white arrows). Increased colocalization between insoluble A𝛽1-42 and p-tau in 16 months old PP as compared to NN both in the hippocampus (Fig. 3A) and the entorhinal cortex (Fig. 3B). In the latter region, A𝛽1-42/p-tau positive aggregates display a stellate morphology resembling core-plaques (Fig. 3C, insert). The number of plaques rises 4 folds in PP as compared to NN (Fig. 3D), with a 70% larger amyloid-𝛽 burden in the area examined (Fig. 3E). Quantification of soluble A𝛽1-42 from the entorhinal cortex shows a peak in A𝛽1-42 release at 6 months in PP mice which later subdues (Fig. 3F), possibly as a result of insoluble aggregate formation in the brain.
PolyI:C-induced memory impairment
Based on the proteinopathy in the hippocampal and entorhinal cortex region following double PolyI:C injection, we assessed whether spatial working memory is affected. Previous work has shown that spontaneous alternation, a form of working memory, is entirely dependent on hippocampal synaptic function (Pioli et al. 2014; McHugh et al. 2008). Same set of PP and NN animals were subjected to the Y-maze spontaneous alternation task at 3, 6, 9, and 16 months. The number of arm entries and sequence were recorded, and the percent alternation was calculated (Fig. 3G). Although the activity of PP treated animals increases in terms of alterations at 3 months, the percentage of alternation did not change at this age as compared to NN (t(17= 0.76, P = 0.46; n = 10 for Control and n=9 for PolyI:C). On the other hand, deficits in the spontaneous alternation are observed in the PP group both at 6 months (t(17) = 3.21, p = 0.005, n = 10 for NN and n=9 for PP), 9 months (t(15) = 2.57, p = 0.013, n = 9 for NN and n=8 for PP) and 16 months (t(13) = 2.57, p = 0.02, n = 8 for NN and n=7 for PP). The number of arm entries in the Y maze task is not significantly different across all the age groups (Suppl. Table 4) indicating normal locomotory behavior representing a non-confounding factor for the percentage of an alternation. However, from 9 months of age both PP and NN display about half of the number of entries as compared to younger animals. Since early and late gestational PolyI:C treatment has been linked to stress and anxiety-like phenotypes relevant to schizophrenia (Hui et al. 2018; Silveira et al. 2017) we tested these mice for Light/dark, Elevated O-maze at 3 and 6 months and Open field tasks at all ages. We found no difference in the anxiety-like behaviour (Suppl. Table 5) disambiguating the spatial memory impairments observed. Together the data suggest that prenatal and early postnatal viral-like immune activation through PolyI:C treatment causes proteinopathy of the limbic regions with sustained working memory impairment.
Activation and phenotypic change in microglia of PP animals
Earlier studies have shown that systemic infections and the subsequent peripheral immune activation have a strong effect on brain function, glial response representing a risk for dementia (Cunningham and Hennessy 2015; Cunningham 2013). Microglia cells are the primary phagocytic innate immune cells of the brain that get stimulated upon immune activation. They normally exist in a resting state displaying a ramified morphology, while in intermediate states display a bipolar or rod-like phenotype (Davis et al. 2017) and in an activated state an amoeboid, irregular shape (Ling and Wong 1993). Quantification of Iba-1 positive cells per mm3 in the immunostained hippocampus (Fig 4A), indicates no difference between PP and NN at the different stages (Suppl. Table 6). Next, we assessed whether the PP brains show altered microglial cell morphologies reflecting an activated inflammatory status. We quantified morphological parameters like soma size, perimeter, circularity, skeleton analysis of Iba-1 positive microglia (Fig. 4), and finally we conducted the fractal analysis in NN & PP mice across staging. From 6 months of age, Iba-1 positive microglia show an increased ramified morphology as detected by immunofluorescence microscopy (Fig. 4A). Quantitative soma analysis shows a slight increase in the soma size, typical of activated microglia in PP mice at 3 months (22%, t(359)=4.7, p<0.005), which lasts up to 9 months (58%, t(109)=4.32, p<0.005) (Fig. 4B). The cell soma perimeter measurement shows dynamic changes at 3 months (11% increase, t(354)=2.7, p<0.01), and a significant increase at 9 months (57%, t(98)=4.5, p<0.005), while no changes are detected at 6 and 16 months (Fig. 4B). Circularity, which indicates the roundness index, shows a deviation from circularity in the intermediate ages at 6 months (10% decrease, t(300=3.15, p<0.005), 9 months (21% decrease, t(164)=3.9, p<0.005) and 16 months (10% increase, t(81)=2, p<0.05), indicating signs of progressive microglial activation with aging (Fig. 4B). These dynamic changes were further analyzed using the distribution curve analysis which indicates an overall shift in the number of cells with an increase in microglial cells soma size and perimeter (Suppl. Fig. 3A-3B) with irregularly shaped cell body during the adult and mid-aged intermediate stages (6 and 9 months) while at later stages (16 months), microglia become more circular or rounded suggesting morphological fluctuations across staging and signs of microglial activation (Suppl. Fig. 3C). To further characterize the phenotypic changes in microglia, reflecting their activation state, we performed the skeletal analysis of Iba-1 positive microglia, at 3, 6,9, and 16 months in PP and NN mice. Skeletonized Iba-1 renderings reveal a significant increase in the number of endpoints/ cell, maximum branch length/cell, and branch length/cell (Fig. 4C) in PP mice with age groups 3 months to 9 months indicating hyper ramified microglia. At 3 months (endpoints/cell t(94)=3.12, p<0.005; maximum branch length/cell t(59)=2.45, p<0.05; branch length/cell t(130)=0.4, p=0.6), 6 months (endpoints/cell t(101)=1.84, p<0.05; maximum branch length/cell t(116)=1.4, p=0.1; branch length/cell t(98)=2.5, p<0.05) and 9 months (endpoints/cell t(118)=3.52, p<0.005; maximum branch length/cell t(115)=3.4, p<0.005; branch length/cell t(118)=2.1, p<0.05). However, at 16 months, microglia in PP brains show a significant drop in the number of endpoints/cells (t(31)=2.7, p<0.05) and an overall decrease in the branch (t(41)=0.2, p=0.8) and maximum branch length (t(34)=1.48, p=0.1) (Fig. 4C). Therefore, prenatal and early postnatal systemic PolyI:C immune activation induces hyper ramified microglia in the offspring which later undergoes a transition from hyperactivated to more bushy or amoeboid with the reduction in microglial endpoints.
Having detected a microglia morphological transition in aged PP mice, confocal imaged microglia cells were further investigated using fractal analysis (FracLac plugin of ImageJ) which renders the shape of microglia cells and quantifies the parameters of cell area, cell perimeter, Span ratio, circularity, and fractal dimension, the latter reflecting pattern complexity. The fractal analysis revealed significant changes in hippocampal microglia parameters in the aged 16 months PP mice versus NN (Fig. 4D). Normal resting microglia are complex with a higher fractal dimension. In the PP mice total microglia cell surface area (t(80)=43.65, p<0.005) and the perimeter (t(75)=4.2, p<0.005) is reduced indicating a more compact shape (Fig. 4E-4F). Fractal dimension as a measure for complexity, circularity and as a measure for the roundness is also reduced (t(62)=4.9, p<0.005) after immune activation (Fig. 4H-4I). On the other hand, the span ratio is increased (t(79)=1.9, p=0.06) reflecting microglia elongation (ratio of cell length and width) (Fig. 4G). Span ratio and circularity are inversely proportional indicating an activated microglia state. Altogether, systemic inflammation through pre- and post-natal PolyI:C induces profound changes in microglia morphology indicating a shift from resting to an activated state in aged PP animals.
Dynamic remodeling of the hippocampal transcriptome
To understand the mechanisms underlying the spatial memory deficits and the pathophysiological processes associated with the proteinopathy, and neuroinflammation we performed a cross-sectional hippocampal bulk mRNA sequencing on 3, 6, 9, and 16 months PP and NN animals. The analysis reveals many differentially expressed genes (DEGs) in the hippocampus (log2 fold change cut-off:05, adjusted p<0.05) between treatments. The gene expression profiles as visualized in the volcano plot (Fig. 5A-5D), show that in aged animals differential expression is highest with a peak at 9 months, coinciding with the neuroinflammatory switch in microglia cells. A GO analysis contextualized to the synapse (SYNGO) (Koopmans et al. 2019) indicates a dynamic shift in DEGs from the presynaptic compartment at 3 months (Supp. Fig 4A) to the postsynaptic terminal at 9 and 16 months (Supp. Figure 4C and 4D), with a non-synaptic stage at 6 months (Supp. Fig 4B). At 3 months, out of 35 DEGs between PP and NN, 21 are downregulated, and 14 are upregulated (Fig. 5A). Gene ontology enrichment analysis (GEA) of the biological processes using a 5% false discovery rate indicates general repression in genes associated with synaptic transmission, calcium signaling, extracellular matrix organization, and secretion (Suppl. Table 7). Pathway analysis based on a composite KEGG, Reactome, WikiPathways dataset shows high interconnectivity between cellular cascades associated with extracellular matrix organization, chemical synaptic transmission, and calcium signaling (Suppl. Fig 5A). In the 6 months, out of 32 DEGs between PP and NN (Fig. 5B), 22 are downregulated, and 10 upregulated. A GEA indicates ongoing processes of morphogenesis and gliogenesis (Suppl. Table 7), with highly overlapping pathways (Suppl. Fig. 5B). At 9 months (Fig. 5C) out of 196 DEGs, 90 are downregulated, and 106 are upregulated. The GEA shows enrichment in several processes associated with vascular remodeling, neurogenesis, morphogenesis, cytokine response, and cell death (Suppl. Table 7). Pathway analysis indicates partially overlapping cellular cascades shared among focal adhesion, BMP signaling, MAPK signaling, and neuronal injury (Suppl. Fig. 5D) confirming the concurrent processes of morphogenesis and cell death at this stage. In the 16 months PP mice (Fig. 5D) from the 99 DEGs, 50 genes are downregulated and 49 genes are upregulated. GEA of the DEGs indicates enrichment in the response to metal ions, the reactive oxygen response, morphogenesis, the regulation of cellular proliferation, and the response to hormones (Suppl. Table 7). Pathways analysis shows less interconnected cascades implicated in potassium ion transmembrane transport, monocytes proliferation, lamellipodium organization, and glucose metabolism (Suppl. Fig. 5D).
To better understand the dynamic changes in the sterile infection model undergoing with age, we compared the significantly differentiated genes between PP and NN at the cross-sectional time points. As shown in the Venn diagram (Fig. 5E), the number of common genes that are uniquely and commonly affected in the hippocampus of 3, 6, 9, 16 months PP mice indicates a large number of overlapping gene sets between 9 and 16 months, while fewer genes are shared with earlier stages. In particular, 6 shared genes can be subdivided into 2 categories reflecting a progressive cell-communication dysfunction (I) and a proinflammatory drive (II) in aging PP mice. (I) Genes downstream of MAPK-signalling (c-Jun) (E. K. Kim and Choi 2010), NFKB-signaling (Egr2; Early growth response protein 2) (Williams et al. 1995), responsible for neuronal excitability (Kcnj2; Potassium Inwardly Rectifying Channel Subfamily J Member 2) (Binda et al. 2018), with reported association with synaptic dysfunction, are downregulated in PP mice starting from 6 months (Fig. 5F). (II) On the other hand, genes related to neuroinflammation such as the lipid-droplet dependent gene (Plin4; Perilipin 4) (Han et al. 2018), pro-inflammatory genes (H2-Aa; Immunohistocompatibility-complex) (Van Hove et al. 2019) and acute-phase proteins regulating to the inflammatory response (Lcn2; Lipocalin-2) (Dekens et al. 2020) are upregulated in aging PP mice, starting from 9 months (Fig. 5G). Overall, the gene remodeling across the aging continuum replicates processes typical of AD with neuronal network breakdown, altered immune response, chronic neuroinflammation and vascular dysfunction.
Substantial changes in brain metabolism and inflammation
We further validated the RNA-seq analysis via RT-PCR, on some of the significant DEG with a reported association to AD as well as genes belonging to the neuronal (I) (Fig 5H) and metabolic (II) (Fig 5I) gene groups. At 3 months, Cacna1g (Calcium Voltage-Gated Channel Subunit Alpha1 G), which was previously reported to decay with aging and regulate amyloid-𝛽 production (Rice et al. 2014), is unchanged (0.9) in contrast to the observed reduction via RNA-seq (Log2FC=-0.55), suggesting that aggregate changes rather than single-gene changes may contribute to the modeled conductivity dysfunction (Suppl. Tab. 7). As expected at this stage, Plin4 and Egr2 are unchanged, matching the RNAseq data (Table 2). At 6 months, we analyzed one of the genes with the strongest downregulation at the RNA-seq, with reported association with cognitive impairment in chronic cerebral hypoperfusion (Xie et al. 2018). The Glpr2 (Glucagon-Like Peptide 2 Receptor) decrease (80%), is confirmed (Table 2; p<0.05). Also, Ide (Insulin-degrading enzyme), which has been implicated in the clearance of insulin and amyloid-𝛽 (Qiu and Folstein 2006), show a comparable increase, between RNA-seq and RT-PCR analysis, but did not reach significance in the RT-PCR result (Table 2; p=0.16). Interestingly, Kcnj2 shows a significant 2 fold increase at 6 months (Table 2; p<0.05), suggesting a modulatory K+ currents effect. Plin4 remain unchanged between PP and NN at 6 months in both analyses (Table 2). At 9 months, the reduction in c-fos, c-Jun, Notch1, Kcnj2, Egr2, and the increase in Lcn2 can be confirmed at the RT-PCR, while Plin4 show a non-significant increase using RT-PCR (Table 2; p=0.24). At 16 months, Lcn2, Plin4 trends are reproduced according to the RT-PCR, while Kcnj2 shows a comparable but not significant decrease (Table 2; p=0.36). The data indicates that around ¾ of the RNA-seq outputs could be replicated via RT-PCR validation, confirming the robustness of the bulk RNAseq discovery method and emphasizing that aggregate dataset can explain ongoing cellular and molecular processes in such models. Validated DEG profiles of Kcnj2, Egr2, and Plin4 and Lcn2 are matched with the expression of genes specific for neurons (Grin1), microglia (Iba1) and astroglia (GFAP) at 9 and 16 months to investigate associations of the selected genes in specific cell types (Fig. 5J). Grin1, Iba1, and GFAP are positively associated with their peaking trend at these time points (Fig. 1C-E) supporting the neuro-glia interplay. On the other hand, Egr2 which decreases at 9 months and increases at 16 months is inversely correlated to Grin1, Iba1, and GFAP suggesting a potential ubiquitous regulation of this transcription factor on the cell-type-specific changes. Finally, Plin4 and Lcn2 are positively associated with their increasing trend from 9 to 16 months, confirming the RNAseq data and supporting the subsequent investigation of these neuroinflammatory markers in aged PP mice. Based on downregulation of Glpr2 in adulthood, indicating vascular hyperperfusion (Xie et al. 2018) and the subsequent upregulation of the vascular and inflammatory markers, Lcn-2, at 9 months, we investigated vascular integrity by validating the gene expression of Kruppel-like factor 4, Klf-4, at 9 and 16 months and observed a transient downregulation at 9 months (Table 2). At the same time point, angiogenesis markers such as Cytochrome P450 Family 1 Subfamily B Member 1, Cyp1b1, and Angiopoietin-like 4, Angptl4, with reported function in vascular homeostasis and alteration in AD (Chakraborty et al. 2018; Ghosh et al. 2016) showed a peak at 9 months, supporting pathological vascular processes in aged adult PP mice.
Nevertheless, an increase in Lcn2 has been reported in both AD brains (Dekens et al. 2018; Naudé et al. 2012) and CSF from vascular dementia patients (Cerebrospinal Fluid) (Llorens et al. 2020a) raising the possibility that this molecule can capture a mixed vascular-AD pathology. In line with our PolyI:C model of sterile infection, the increase in Lcn-2 is likely attributed to the production and release by activated microglia, reactive astrocytes, neurons, and endothelial cells in response to inflammatory and infectious insults (Jha et al. 2015). Our immunofluorescence analysis using an antibody specific for Lcn2 shows an increase in Lcn2 protein expression in the hippocampal CA3 field of the PolyI:C mice at 9 months and 16 months aged mice (Fig. 6A). At 16 months, small A𝛽1-42 aggregates are visible in the PP hippocampus in close association with Lcn2 positive cells (Fig, 6A, insert). Quantitative analysis of the Lcn2 signal shows a significant increase in the % of the Lcn2 stained area in PP mice in both age groups (9 months, t(25)=4.2, p<0.005; 16 months, t(62)=2, p<0.05) (Fig. 6B). To study lipid metabolism and intracellular lipid droplets accumulation, as a sign of neuroinflammation with aging, we have utilized the dye Nile Red which accumulates in lipids and emits red fluorescence (Greenspan, Mayer, and Fowler 1985). We observed more Nile Red-positive lipid droplets (LDs) in the hippocampal CA3 field with aging, which is even more evident in PP mice as compared to saline controls (Fig. 6C). Triple labeling with Nile Red, Neurofilament L-200, and Iba1, shows that both NF200 positive neurons and Iba1 positive microglial cells have increased lipid droplets (Fig 6D). Quantitative analysis of the LDs, represented as fold change between the PP and NN at the different time points indicates that the density of the LDs is significantly greater in PP mice starting from 3 months, resulting in increased stained area, peaking at 6 months (t(16)=2.6, p<0.05) (Fig. 6E). On the other hand, LDs’ size shows a small but significant expansion (30%, t(16)=3.14, p<0.05) in PP mice at 3 months but afterward remains unchanged between conditions (Fig. 6E). The histo-anatomical analysis confirms the presence of neuroinflammatory markers that contribute to an AD-like neuropathological progression.
Translation to Alzheimer’s disease
To assess whether the newly designed PolyI:C model is reproducing genetic changes in human AD, we performed targeted fingerprinting focusing on a cross-sectional cohort of post-mortem entorhinal cortices containing the hippocampus from age-matched subjects with mild-moderate AD, severe AD and age-matched healthy controls (CTL) (Table 3 and Suppl. Table 1). We first examined cell-type-specific genes, GFAP, Iba1, and MAP2. Consistently, with our model we observe a progressive increase in GFAP expression from Moderate to Severe AD (F2,21=9.29 p=0.012; Fig. 7A and Suppl. Table 8), while Iba1 and MAP2 remain unchanged (Fig. 7A and Suppl. Table 8). Next, we examined some of the relevant DEG with reported association with AD and divided them into functional categories. Glpr2 and Ide belonging to the glucose metabolism with differential expression in a 6-month-old PP adult, did not show any significant difference between the clinical groups and controls (Fig. 7A and Suppl. Table 8). We next examined Kcnj2 and Egr2 which show downregulation in the PP model at 9 months (Table 2 and Fig. 5H) and observed high variability with no changes across stages (Suppl. Table 8 and Fig 7C). Among the cellular signaling genes, with specific repression in the PP model, we detect an opposite increasing trend in c-Fos, c-Jun, and Notch1 to the PP model with a near to significant 3.1 upregulation of c-Fos in severe AD as compared to controls (F2,21=3.2 p=0.061; Suppl. Table 8 and Fig 7D). In the lipid metabolism group, Plin4 and Lcn2 show no change opposite to the PP model (Suppl. Table 8 and Fig 7E), suggesting that those molecules are more implicated in vascular inflammation. To validate this assumption, we analyzed the vascular genes, Klf4, Angptl4, Cyp1b1, which showed transient alterations in the PP mode, and observed no change across stages (Suppl. Table 8 and Fig 7F). To understand the dependencies between the examined genes, we performed a correlation analysis using the aggregate population. In general, all interactions are moderately significant (r>0.5) with positive associations between c-fos and GFAP, matching the increasing trend of the two transcripts, which suggest a cell-type-specific change, while c-fos is negatively associated with MAP2 (Fig. 7G). Kcnj2 is inversely associated with Ide reflecting their opposite trend, while Ide is positively correlated with Cyp1b1 (Fig. 7G), indicating potential dependencies. Other interactions among the studied genes are seen but too subtle to be of functional relevance. Overall, despite the PolyI:C model replicates some aspects of the AD proteinopathy and microglia changes, the gene expression between the mouse and humans differs substantially, raising the possibility of a mixed-vascular-AD model with diverse genetic fingerprints.
Translation into vascular dementia
To verify the hypothesis of a mixed vascular-AD phenotype, we performed gene targets’ validation on the second cohort of hippocampi from vascular dementia patients and age-matched healthy controls (Table 4 and Suppl. Table 2). Cell type-specific genes, GFAP and Iba1, indicate an increasing but not significant trend of microglia and astroglia, while MAP2 levels remain unchanged. Accordingly to our model, Glpr2 decreases by 90% in vascular dementia as compared to controls (F1,9=5.28 p=0.039; Fig. 7J and Suppl. Table 9) suggesting ongoing vascular hypoxia (Xie et al. 2018)). On the other hand, the inflammatory and vascular markers, Lcn2 (F1,9=4.96 p=0.05) and Cyp1b1 (F1,9=7.10 p=0.03) increase in vascular dementia as in the PP model (Fig. 7L and 7K and Suppl. Table 9). Opposite to the genetic expression in the mouse, Klf4, Notch1 and c-fos levels rise in vascular dementia (Fig. 7M and 7Y and Suppl. Table 9). Correlation analysis of the differentially expressed genes in the aggregate cohort indicates a positive association (r>0.6) among Lcn2, Notch1 and Klf4, implicating those factors in the vascular pathology. Whereas, along the mechanistic trajectory of vascular remodeling a negative association is observed between Glpr2 and the two angiogenesis genes, Klf4 and Cyp1b1. These results indicate that a handful of the gene targets that are significantly affected upon systemic inflammation in the brain of PP mice are reproduced in vascular neurodegenerative dementia.