Prion infection increases brain apoE level and causes cell-type shift in the apoE expression
We first compared the total brain apoE level and its expression by astrocytes and microglia in wild-type (WT) B6 mice, which were intraperitoneally inoculated with 22L mouse adapted scrapie strain or NBH. 22L inoculated B6 mice were killed at 15 and 23 week post inoculation (wpi), when animals remain neurologically asymptomatic or show overt clinical symptoms of prion disease, respectively. NBH controls were killed at 23 wpi and they were clinically asymptomatic. At 15 wpi the apoE protein level in the brain cortex of 22L inoculated mice is comparable to that of NBH inoculated controls; however, at 23 wpi 22L inoculated mice show 1.3-fold increase in the level of apoE protein (p < 0.001) and 1.6-fold increase in the level of apoE mRNA (p < 0.05) relative to NBH controls, respectively (Fig. 1a–c). Cell-type specific determination of apoE expression was performed in the brain cortex by apoE CTCF analysis in apoE/GFAP and apoE/Iba1 double immunostained astrocytes and microglia, respectively. It shows 0.74-fold decrease in the apoE signal in astrocytes (p < 0.0001) (Fig. 1d, e) and 24-fold increase in the apoE signal in microglia (p < 0.0001) (Fig. 1f, g) in 22L mice relative to NBH controls.
Absence of apoE shortens prion disease incubation time and increases pathology burden
The length of the prion disease incubation period was determined by longitudinal locomotor behavioral testing using the parallel bar crossing test [5, 64, 65]. Starting from 10 wpi all mice were tested weekly by an observer blinded to their genotype and the type of inoculum they received. Mice, which display gross motor incompetence for three weeks in a row are considered symptomatic and the first day they score positively is considered in retrospect the disease onset. Kaplan-Meyer estimator was used to analyze differences in the prion incubation time across the animal groups. 22L inoculated Apoe−/− mice have 20 days shorter median incubation time than 22L inoculated WT mice (p < 0.0001) (Fig. 2a). No neurological symptoms were identified in either NBH inoculated WT or Apoe−/− mice by 23 wpi. To assure the effect of apoE absence on the prion incubation time is not strain specific we also inoculated mice with the ME7 strain and likewise noticed statistically significant difference in the median disease incubation period between ME7 WT mice and ME7 Apoe−/− mice, which was 26 days shorter in the latter group (p < 0.0001) (Additional file 1: Fig. S1). Separate cohorts of 22L inoculated WT and Apoe−/− mice also were subjected to longitudinal assessment of the Total Scrapie Score, which gives a quantitative measure of disease progression and severity. The individual components of the Scrapie Score were assessed from 12 wpi onward by two independent examiners who were blinded to the animal genotype (Additional file 1: Fig. S2a-e). As shown in Fig. 2b in 22L Apoe−/− mice the symptoms occur earlier and their course is more aggressive (p < 0.0001). At 23 wpi when mice are killed the Total Scrapie Score in 22L Apoe−/− mice is by 4.1 points higher than that in 22L WT mice.
Detailed neuropathological analysis was carried out in symptomatic animals killed at 23 wpi. In 22L inoculated Apoe−/− mice the load of spongiform lesions in the M1 primary motor cortex is 1.5-fold higher (p < 0.0001) (Fig. 2c, d) and the integrated density of immunostained PrP deposits in the brain cortex is 1.7-fold higher (p < 0.0001) (Fig. 2e–g) relative to those in 22L inoculated WT mice. PrP deposition shows a particular predilection to the layer V of the neocortex (Fig. 2e, Additional file 1: Fig. S3), the thalamus (Additional file 1: Fig. S3), the entorhinal cortex, the globus pallidus, and the amygdala (not shown). In affected structures, PrP forms conspicuous clusters (Additional file 1: Fig. S3), which tend to merge covering the entire cross-sectional area of the structure. There are no anatomical differences in the PrP distribution between 22L WT and 22L Apoe−/− mice. The main dissimilarity is that in the former group the intensity of anti-PrP immunostaining is more pronounced and the PrP clusters appear more conspicuous (Additional file 1: Fig. S3).
In parallel, we determined changes in the total PrP protein level and that of PK-resistant PrPSc in the brain cortex homogenate at 23 wpi by quantitative WB densitometry. Total PrP level is 1.1-fold increased (p < 0.05) (Fig. 3a, b) and PK-resistant PrPSc level is 1.4-fold increased (p < 0.05) (Fig. 3c, d) in 22L inoculated Apoe−/− mice relative to 22L inoculated WT animals, respectively. To determine whether apoE may directly affect PrPSc replication we treated 22L infected N2A cells with natively lipidated apoE complexes derived from astrocytes expressing human apoE4 or with control media from Apoe−/− astrocytes (Additional file 1: Fig. S4a, b). This supplementary experiment showed no apparent effect of apoE presence or absence on the total cellular PrP level or the level of PrPSc in PrPSc replicating N2A/22L cells (Additional file 1: Fig. S4c, d).
Lack of apoE promotes astrogliosis and the induction of A1 neurotoxic phenotype
Astrogliosis and upregulation of GFAP level are hallmarks of prion pathology. We quantified the load of reactive astrocytes (defined as the % of cross-sectional area with GFAP+ immunostaining) in the ventral posterior thalamic nucleus (VPN) and in the S1 primary somatosensory cortex, as the thalamus and the brain cortex are affected in sequential order in the intraperitoneal prion inoculation model. In 22L Apoe−/− mice, astrogliosis occurs earlier and it is more robust compared to 22L WT mice. At 15 wpi both 22L WT and 22L Apoe−/− mice show significant increase in the GFAP+ load in the VPN compared to their genotype-matched NBH controls (Fig. 4a, b) but only 22L Apoe−/− mice show significantly elevated GFAP+ load in the S1 cortex (Fig. 4c, d). At 23 wpi both 22L WT and 22L Apoe−/− mice have significantly increased GFAP+ load in the VPN and in the S1 cortex over their NBH controls. At either time point the GFAP+ load in 22L Apoe−/− mice is higher than that in 22L WT mice. At 15 wpi it is 3.2-fold higher in the VPN (p < 0.0001) and 8.4-fold higher in the S1 cortex (p < 0.0001) relative to 22L WT mice, while at 23 wpi it is 1.2-fold (p < 0.0001) and 1.7-fold (p < 0.0001) higher in the respective structures. Consistently, there are changes in the level of GFAP protein in the brain cortex revealed by quantitative WB. At 15 wpi only 22L Apoe−/− mice show significant increase in the GFAP protein level, while at 23 wpi it is significantly increased both in 22L WT and 22L Apoe−/− mice relative to their NBH controls, (Fig. 4e, f). At 15 wpi and 23 wpi the GFAP level in 22L Apoe−/− mice is twofold (p < 0.001) and 1.7-fold (p < 0.0001) higher relative to that in 22L WT mice, respectively. Commensurate changes in the Gfap mRNA level across the experimental groups additionally were confirmed by qRT-PCR (Fig. 4g). There are no significant differences in the GFAP load in the VPN and in the S1 cortex and in the level of GFAP protein or in the Gfap mRNA level between NBH inoculated Apoe−/− and WT mice (Fig. 4a–g).
To gain insight into the profile of astrocyte activation we analyzed expression of the complement component 3 (C3) protein, which is a histological marker of A1 (neurotoxic) astrocyte phenotype [38, 49]. A robust expression of C3 protein in cortical astrocytes already is seen in 22L Apoe−/− mice at 15 wpi but not in 22L WT mice (Additional file 1: Fig. S5). At 23 wpi cortical astrocytes both in 22L Apoe−/− and 22L WT mice highly express C3 protein, yet the C3/GFAP ratio is 1.5-fold higher in 22L Apoe−/− mice relative to 22L WT mice (p < 0.0001) (Fig. 4, h, i). Also at 23 wpi C3 expression calculated from qRT-PCR data is 7.4-fold higher in 22L Apoe−/− mice than that in 22L WT mice (p < 0.0001) (Fig. 4j). C3+ astrocytes are undetectable in NBH inoculated WT and Apoe−/− mice (Additional file 1: Fig. S5).
Transcriptomic profile of astrocytes was characterized using the nanoStringTM nCounter® technology in animals killed at 23 wpi. Hierarchical cluster analysis of astrocytic transcript shows strong clustering of 22L Apoe−/− animals and their separation from other experimental groups both for PAN-reactive and A1-reactive cassettes, while 22L WT mice cluster together and separate from other groups only for the PAN-reactive but not for the A1-reactive cassette (Fig. 5a, b). Direct comparison of the nCounter® values shows significant upregulation of the following PAN-reactive markers in 22L Apoe−/− mice relative to the averaged NBH controls: Aldh1l1 (2.7-fold, p < 0.001), Gfap (29.0-fold, p < 0.0001), Serpina3n (16.4-fold, p < 0.01), and Vim (6.8-fold, p < 0.05) (Fig. 5d). In 22L WT mice a significant increase concerns only Aldh1l1 (1.6-fold, p < 0.05), and Gfap (5.8-fold, p < 0.05). Numerous A1 markers also are significantly elevated in 22L Apoe−/− mice relative to the averaged NBH controls: Fbln5 (2.8-fold, p < 0.01), Ggta1 (3.7-fold, p < 0.001), H2-D1 (8.0-fold, p < 0.001), H2-T23 (3.7-fold, p < 0.0001), Psmb8 (6.6-fold, p < 0.0001), Serping1 (3.4-fold, p < 0.001) and Srgn (2.9-fold, p < 0.05) (Fig. 5d). Likewise, modest upregulation of these markers is observed in 22L WT mice but reaches statistical significance only for H2-T23 (1.3-fold, p < 0.01), and Psmb8 (2.1-fold, p < 0.05). In direct comparison of nCounter® values between 22L Apoe−/− and 22L WT mice, there is a statistical post-hoc significance for all PAN- and A1-reactive markers with the former group showing numerical advantage (Fig. 5d). A2-reactive cassette markers demonstrate no specific clustering at group level (Fig. 5c) or statistically significant increase in either 22L WT or 22L Apoe−/− group relative to the averaged NBH controls (Fig. 5d).
Absence of apoE exaggerates microglia activation and their MGnD transition
Parallel to astrogliosis we analyzed activation of microglia at 15 wpi and 23 wpi. We quantified the load of Iba1+ and CD68+ cells in the VPN and in the S1 cortex as general and phagocytic microglia activation markers, respectively [57]. Similarly, to the extent of GFAP+ astrocytosis, at 15 wpi both 22L WT and 22L Apoe−/− mice show significantly increased Iba1+ load in the VPN compared to their genotype-matched NBH controls (Fig. 6a, b) but only 22L Apoe−/− mice show significantly elevated Iba1+ load in the S1 cortex (Fig. 6c, d). At 23 wpi both 22L WT and 22L Apoe−/− mice show significantly higher Iba1+ load in the VPN and the S1 cortex over their NBH controls (Fig. 6a–d; Additional file 1: Fig. S6). At either time point, the Iba1+ load in 22L Apoe−/− mice is higher relative to that in 22L WT mice. At 15 wpi it is 1.6-fold higher in the VPN (p < 0.0001) and 1.5-fold higher in the S1 cortex (p < 0.0001), while at 23 wpi 1.5-fold higher in the VPN (p < 0.0001) and 2.1-fold higher in the S1 cortex (p < 0.0001). In addition to the Iba1+ load, we enumerated density of Iba1+ microglial cells in the layer V of the S1 cortex, which shows the greatest microglia proliferation, consistently with the patterns of PrP deposition (Additional file 1: Fig. S6). In 22L Apoe−/− mice the numerical density of microglia in the layer V is 1.5-fold higher at 15 wpi (p < 0.0001) and 1.7-fold higher at 23 wpi (p < 0.0001) relative to those in 22L WT mice, respectively (Fig. 6e, f).
CD68+ load shows significant increase in the VPN and in the S1 cortex already at 15 wpi both in 22L WT and 22L Apoe−/− mice compared to their genotype-matched NBH controls (Fig. 6g–j). However, unlike Iba1+ load, there is no significant difference in the numerical values of the CD68+ load between 22L WT and 22L Apoe−/− mice at 15 wpi. At 23 wpi the CD68+ load in both groups increases further, but in 22L Apoe−/− mice represents 0.8-fold (p < 0.0001) and 0.7-fold (p < 0.0001) of those in 22L WT mice in the VPN and in the S1 cortex, respectively. These observations suggest considerably lower phagocytic activity of microglia in 22L Apoe−/− mice relative to their overall activation compared to 22L WT mice. There is no significant difference in the Iba1+ load, numerical density of Iba1+ microglia, and CD68+ load in the VPN and in the S1 cortex between NBH inoculated WT and Apoe−/− mice (Fig. 6a–j; Additional file 1: Fig S6).
Microglia specific transcripts split into MGnD—specific, and M0 (Homeostatic)—specific cassettes was characterized in animals killed at 23 wpi using the nanoStringTM nCounter® technology. Hierarchical cluster analysis of MGnD specific cassette shows clustering at individual group level (NBH WT, NBH Apoe−/−, 22L WT, and 22L Apoe−/−) and distinct separation of the 22L Apoe−/− animals from other groups (Fig. 7a). Direct comparison of the nCounter® values reveals robust increase of MGnD markers in 22L Apoe−/− mice relative to the averaged NBH controls: Aif1 (3.1-fold, p < 0.0001), Axl (2.7-fold, p < 0.001), Bhlhe41 (1.5-fold, p < 0.001), Cd9 (3.6-fold, p < 0.01), Clec7a (5.5-fold, p < 0.001), Csf1 (2.5-fold, p < 0.001), Cst7 (43.6-fold, p < 0.0001), Ctsb (1.9-fold, p < 0.01), Ctsd (4.8-fold, p < 0.01), Ctsl (2.8-fold, p < 0.05), Ctss (7.5-fold, p < 0.01), Gpnmb (3.2-fold, p < 0.01), Lag3 (15.8-fold, p < 0.01), Lyz2 (6.5-fold, p < 0.05), Trem2 (7.5-fold, p < 0.05), and Tyrobp (6.2-fold, p < 0.01) (Fig. 7c). In 22L WT mice the increase in some of MGnD markers also is observed but it is more modest compared to the degree of their upregulation in 22L Apoe−/− mice. Statistically significant differences relative to the pooled NBH controls are noted for Apoe (1.6-fold, p < 0.05), Aif1 (1.5-fold, p < 0.05), Csf1 (1.6-fold, p < 0.05), and Cst7 (8.0-fold, p < 0.05) (Fig. 7c). In direct comparison of the nCounter® values between 22L Apoe−/− and 22L WT mice, there is a statistical post-hoc significance for all MGnD markers with the former group showing numerical advantage, except for Apoe, which is not expressed by 22L Apoe−/− mice (Fig. 7c). Hierarchical cluster analysis of M0 specific cassette shows clustering within NBH WT and 22L Apoe−/− groups with salient separation of the 22L Apoe−/− animals from other experimental groups (Fig. 7b). Direct comparison of the nCounter® values shows significantly increased expression of several gene markers typical for M0 homeostatic microglia in 22L Apoe−/− mice relative to the averaged NBH controls: Csf1r (3.7-fold, p < 0.001), Gpr34 (2.8-fold, p < 0.001), Hexb (4.2-fold, p < 0.001), Jun (1.4-fold, p < 0.01), Olfml3 (3.9-fold, p < 0.0001), P2ry12 (1.9-fold, p < 0.001), and Tmem119 (3.7-fold, p < 0.001) (Fig. 7c). In contrast, in 22L WT mice only Olfml3 expression is significantly increased (1.7-fold, p < 0.01) (Fig. 7c), however other M0 markers show no significant repression. Values in the parentheses both for 22L Apoe−/− and 22L WT mice denote fold change and statistical significance relative to the averaged NBH controls. In direct comparison of the nCounter® values between 22L Apoe−/− and 22L WT mice, there is a statistical post-hoc significance for all M0 markers with the former group showing numerical advantage (Fig. 7c). Significant upregulation of the P2ry12 and Tmem119 transcript in 22L Apoe−/− mice at 23 wpi relative to NBH Apoe−/− and 22L WT mice was independently confirmed by qRT-PCR (Additional file 1: Fig. S7a, b). Likewise the load of TMEM119+ microglia in the S1 cortex in 22L Apoe−/− mice is 1.4-fold higher (p < 0.0001) than that in 22L WT mice at 23 wpi (Additional file 1: Fig. S7c, d). 22L infected mice of both genotypes feature significant increase in the TMEM119+ load compared to their genotype matched NBH controls. NBH Apoe−/− mice also shows modestly higher TMEM119+ load compared to NBH WT mice.
In prion infected mice lacking apoE activated microglia show impaired phagocytic activity
We analyzed several aspects of microglial phagocytosis including quantification of the CD68 and TREM2 expression in the context of their Iba1 expression in the S1 cortex. 22L Apoe−/− mice show significant reduction in the CD68:Iba1 ratio relative to 22L WT mice—0.6-fold change (p < 0.05) at 15 wpi and 0.3-fold change (p < 0.0001) at 23 wpi (Fig. 8a, b; Additional file 1: Fig. S8). The TREM2:Iba1 ratio also is reduced in 22L Apoe−/− mice—0.4-fold change relative to 22L WT animals at 23 wpi (p < 0.01) (Fig. 8c, d; Additional file 1: Fig. S9). We also enumerated TREM2+ microglia in the layer V of the S1 cortex at 23 wpi. In 22L Apoe−/− mice 20.0 ± 1.1% of microglia are TREM2+ while in 22L WT mice 67.2 ± 1.9% show immunodetectable TREM2 expression (3.4-fold difference, p < 0.0001) (Fig. 8e; Additional file 1: Fig. S9). Though there is reduction in the expression of CD68 and TREM2 protein by microglia, both CD68 and Trem2 transcripts are significantly upregulated in 22L Apoe−/− mice relative to 22L WT animals (Figs. 7c, 8f, and 9f). Expression of CD68 in NBH inoculated mice is very low (CD68:Iba1 ratio ~ 0.03) and shows no significant differences between WT and Apoe−/− animals (Fig. 8b, Additional file 1: Fig. S8), while expression of TREM2 is not detectable by immunohistochemistry in NBH inoculated mice of either genotype (Additional file 1: Fig. S9). In addition, we enumerated neurons, which are opsonized by phagocytically activated microglia, defined as being CD68+, and used this count as a surrogate marker of microglia neuronophagy. There is 2.2-times more neurons opsonized by microglia in 22L WT mice than in 22L Apoe−/− mice in the layer V of the S1 cortex at 23 wpi (Fig. 9a, b) (p < 0.0001). Qualitative differences in the opsonization between the genotypes were further explored using LSCM imaging. It shows that in 22L WT mice opsonizing microglia tightly envelop neurons and feature high level of CD68 expression, while in 22L Apoe−/− mice microglia/neuronal associations are loose while CD68 expression remains low (Fig. 9c, d). No close neuronal/microglia contacts are observed in NBH inoculated WT and Apoe−/− controls (Fig. 9a, b).
Hierarchical cluster analysis of the nanoStringTM nCounter® gene expression data for the genes involved in the efferocytosis related signaling [28] and those involved in the endo/lysosomal pathway shows clear clustering within 22L WT and 22L Apoe−/− groups and salient separation of 22L Apoe−/− mice from all other animals (Fig. 9e). Direct analysis of the nCounter® values shows a significant upregulation in the group of “find me/eat me” signaling genes in 22L Apoe−/− mice, which change relative to the averaged NBH controls is denoted along with the p value in the parentheses: Axl (2.7-fold, p < 0.001), Cx3cr1 (3.2-fold, p < 0.001), Mertk (2.6-fold, p < 0.001), P2ry6 (2.1-fold, p < 0.001), P2ry12 (1.9-fold, p < 0.001), and Stab1 (2.1-fold, p < 0.0001) (Fig. 9e, f). Inversely, expression of the Cd47 gene relaying “do not eat me” signal is significantly repressed in 22L Apoe−/− mice—0.85-fold change, relative to the NBH control (p < 0.05). In 22L WT mice expression of all “find me/eat me” signaling genes is modestly elevated but apart from Stab1 (1.8-fold change relative to the NBH; p < 0.0001) it is not statistically significant (Fig. 9e, f). 22L WT mice also show reduction in Cd47 expression (0.9-fold change relative to the NBH) which does not reach statistical significance. Differences in the expression of all “find me/eat me” genes between 22L Apoe−/− and 22L WT groups are statistically significant with the former group showing greater degree of change (Fig. 9f). Expression of Cd47 does not significantly differ between 22L inoculated WT and Apoe−/− mice.
We also used the nanoStringTM nCounter® analysis to determine differences in the expression of genes involved in the endo/lysosomal pathway. In 22L Apoe−/− mice there is a significant upregulation, denoted as a change relative to the averaged NBH controls, of lysosomal hydrolases Ctsb (1.9-fold, p < 0.01), Ctsd (4.8-fold, p < 0.01), and Ctsf (1.3-fold, p < 0.05) and lysosomal transport and metabolism markers CD68 (2.9-fold, p < 0.01), Lamp1 (1.6-fold, p < 0.01), and Lamp2 (1.7-fold, p < 0.0001). In contrast expression of the genes encoding proteins responsible for lysosomal acidification (Atp6v0d1, Atp6v1a, Atp6v1b2, and Atp6v1h) is modestly reduced relative to the averaged NBH controls (0.76 – 0.92-fold change range) reaching statistical significance for Atp6v1a (p < 0.05) and Atp6v1h (p < 0.01) (Fig. 9e, f). In 22L WT mice lysosomal hydrolases and lysosomal transport and metabolism markers are only modestly upregulated relative to the NBH controls. The statistical significance is reached only for CD68 (2.0-fold, p < 0.05) and Lamp2 (1.2-fold change, p < 0.05). In direct comparison of the lysosomal hydrolases and the lysosomal transport and metabolism markers between 22L Apoe−/− and 22L WT mice there is a statistical significance for all the genes with the former genotype showing greater degree of increase relative to the NBH controls (Fig. 9f). In contrast to 22L Apoe−/− mice, the lysosomal acidification markers in 22L WT mice are either comparable to or higher than those in the averaged NBH controls (Atp6v1b2; 1.13-fold change, p < 0.05). In direct comparison between the 22L Apoe−/− and WT groups, there is a statistical significance for all lysosomal acidification markers but Atp6v1a due to the opposite direction of change from the NBH control (Fig. 9f). This altered gene expression pattern indicates upregulation of neuronal/microglia signaling underlying neuronophagy or efferocytosis during prion pathogenesis, along with consistent increase in lysosomal hydrolases and lysosomal transport and metabolism markers with all changes enhanced in the absence of apoE. However, 22L Apoe−/− mice also show oppositional reduction in the expression of lysosomal acidification markers, which suggests that absence of apoE renders lysosomal degradation inefficient.
Absence of apoE aggravates inflammatory response associated with prion pathology
Increased brain level of inflammatory markers is an inherent feature of prion pathology. We used Mouse Inflammatory Cytokines Multi-Analyte ELISArray to compare the levels of inflammatory cytokines in the brain cortex homogenate between 22L WT and 22L Apoe−/− mice killed at 23 wpi. Figure 10a depicts cytokine level in the brain cortex of 22L Apoe−/− mice relative to those in 22L WT mice. There is significant increase in key pro-inflammatory cytokines IL-1α, IL-1β, IL-17A, IFN-γ, and TNF-α, which ranges from 1.1-fold to 1.4-fold (Fig. 10a). In addition, we detailed the level of IL-1β, which is a prime pro-inflammatory cytokine, in the brain cortex across all experimental groups (Fig. 10b). In mice killed at 15 wpi IL-1β level in 22L WT and 22L Apoe−/− mice is comparable to those in genotype-matched NBH controls, while in mice killed at 23 wpi the IL-1β level is significantly increased both in 22L WT and 22L Apoe−/− mice relative to the NBH controls (p < 0.0001). In 22L Apoe−/− mice the IL-1β level is 1.4-fold higher relative to 22L WT mice (p < 0.0001).
Hierarchical cluster analysis of the nanoStringTM nCounter® gene expression data for the inflammatory response genes shows clear clustering of 22L Apoe−/− animals and their salient separation from all other groups (Fig. 10c). Direct comparison of the nCounter® values between 22L Apoe−/− mice and the averaged NBH controls shows a robust increase in the expression of genes encoding components of the C1 and the C4 complement complexes C1qa (8.2-fold, p < 0.01), C1qb (9.0-fold, p < 0.001), C1qc (8.3-fold, p < 0.01), C4a/b (15.9-fold, p < 0.0001), Complement Component 3a Receptor 1 (C3ar1) (7.3-fold, p < 0.05), Ccl12 (10.1-fold, p < 0.0001), Colony Stimulating Factor 3 Receptor (Csf3r) (3.2-fold, p < 0.01), Il18 (1.4-fold, p < 0.05), and Toll-like receptors Tlr2 (5.2-fold, p < 0.05), Tlr3 (2.4-fold, p < 0.001), and Tlr4 (2.5-fold, p < 0.05) (Fig. 10d). In 22L WT mice upregulation of these genes is more modest and significant only for C4a/b (3.5-fold, p < 0.05), and Tlr3 (1.6-fold, p < 0.05). The fold change and statistical significance in the parentheses denotes the difference relative to the averaged NBH controls. In direct comparison between 22L Apoe−/− and 22L WT groups significant difference is noted for the expression of all the genes but Il18 with the former group always showing greater degree of increase relative to the NBH controls (Fig. 10d).
Prion induced neuronal loss and degeneration are aggravated by the lack of apoE
We enumerated pyramidal neurons in the layer V of the S1 cortex at 23 wpi and determined neuronal loss in the course of prion disease in 22L Apoe−/− mice is nearly twice higher than that in 22L WT mice. In 22L Apoe−/− mice there is 22% reduction in neuronal density (p < 0.0001), while in 22L WT mice the reduction is 13% (p < 0.0001) relative to their genotype-matched NBH control groups respectively (Fig. 11a, b). Of note, NBH inoculated Apoe−/− mice have 5% reduced density of the layer V neurons compared to NBH inoculated WT mice (p < 0.01) (Fig. 11a, b). We also used staining with FJC, an anionic florescent dye, which selectively labels degenerating neurons [27, 67]. At 23 wpi 22L Apoe−/− mice shows 2.6-times more FJC+ neurons in the layer V of the S1 cortex than 22L WT mice (p < 0.0001) which represents a higher rate of neuronal demise and degeneration in the former group (Fig. 11c, d). FJC staining of neurons in NBH Apoe−/− and WT control groups was negligible.