Absence of Apolipoprotein E is associated with exacerbation of prion pathology and promotes microglial neurodegenerative phenotype

Prion diseases or prionoses are a group of rapidly progressing and invariably fatal neurodegenerative diseases. The pathogenesis of prionoses is associated with self-replication and connectomal spread of PrPSc, a disease specific conformer of the prion protein. Microglia undergo activation early in the course of prion pathogenesis and exert opposing roles in PrPSc mediated neurodegeneration. While clearance of PrPSc and apoptotic neurons have disease-limiting effect, microglia-driven neuroinflammation bears deleterious consequences to neuronal networks. Apolipoprotein (apo) E is a lipid transporting protein with pleiotropic functions, which include controlling of the phagocytic and inflammatory characteristics of activated microglia in neurodegenerative diseases. Despite the significance of microglia in prion pathogenesis, the role of apoE in prionoses has not been established. We showed here that infection of wild type mice with 22L mouse adapted scrapie strain is associated with significant increase in the total brain apoE protein and mRNA levels and also with a conspicuous cell-type shift in the apoE expression. There is reduced expression of apoE in activated astrocytes and marked upregulation of apoE expression by activated microglia. We also showed apoE ablation exaggerates PrPSc mediated neurodegeneration. Apoe−/− mice have shorter disease incubation period, increased load of spongiform lesion, pronounced neuronal loss, and exaggerated astro and microgliosis. Astrocytes of Apoe−/− mice display salient upregulation of transcriptomic markers defining A1 neurotoxic astrocytes while microglia show upregulation of transcriptomic markers characteristic for microglial neurodegenerative phenotype. There is impaired clearance of PrPSc and dying neurons by microglia in Apoe−/− mice along with increased level of proinflammatory cytokines. Our work indicates that apoE absence renders clearance of PrPSc and dying neurons by microglia inefficient, while the excess of neuronal debris promotes microglial neurodegenerative phenotype aggravating the vicious cycle of neuronal death and neuroinflammation. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01261-z.


Fig. S1
Infection of Apoe -/-mice with ME7 mouse adapted scrapie strain causes significant shortening of the prion disease incubation period. To assure the effect of Apoe -/-on the prion pathology is not specific to the 22L strain we intraperitonealy inoculated 8 -10 week-old WT and Apoe -/-mice of both sexes ( 50%:50% female to male ratio) with ME7 infectious and normal brain homogenate (NBH). Unlike the 22L strain, the ME7 strain does not replicate in non-neuronal cells and has slightly longer incubation period. Shown is Kaplan-Meier estimator of the incubation time in ME7 and NBH inoculated WT and Apoe -/-mice. The x-axis denotes days post inoculation (dpi) while the y-axis a percent of animals, which remain asymptomatic from the initial groups of 11 -13 ME7 and 22 -23 NBH inoculated WT and Apoe -/-mice. p < 0.0001 denotes the significance between 22L WT and 22L Apoe -/-groups (Log-rank test). Differences between 22L Apoe -/-and NBH Apoe -/-and between 22L WT and NBH WT, which are not shown on the graph also are significant at p < 0.0001. The difference between NBH WT and NBH Apoe -/-is not statistically significant.

ApoE Modulates Prion Pathology
Pankiewicz, JE et al. and e Body condition, which were assigned based on the following criteria 0 = normal, 1 = subtle, 1.5 = mild, 2 = moderate, 2.5= advanced, and 3 = severe. The tally of all subscores makes the Total Scrapie Score depicted in Fig. 2b. The mice were serially assessed starting from the 100 th day post inoculation by two independent examiners blinded to the animal genotype. All data represent mean  SEM from n = 11 -12 mice per group. a -e p < 0.0001 (2-way ANOVA).

Fig. S3
PrP deposition has predilection to the thalamus and to the layer V of the neocortex and is more prominent in 22L Apoe -/-mice. Shown are representative microphotographs of coronal brain section from NBH and 22L inoculated WT and Apoe -/-mice at 23 wpi. The sections were immunostained against PrP. Scale bar: 350 μm. Abbreviations: Hip -hippocampus, L V -layer V, S1 Ctx -primary somatosensory cortex, and Th -thalamus.  Apoe -/-astrocytes (e -/-), which were resolved using native-PAGE and SDS-PAGE, respectively. c and d Show is immunoblot analysis of the total PrP protein and that of proteinase K (PK) resistant PrP Sc in N2A/22L cells treated with astrocytic media containing natively lipidated apoE4 or control media from Apoe -/-astrocytes for 96 hrs., respectively. Also included is -actin as the loading control in c.

Fig. S5
Prion related expression of C3 by astrocytes is upregulated in the absence of apoE.
Shown are representative epifluorescent microphotographs of astrocytes in the layer V of the S1 cortex from mice of indicated experimental groups, which were double immunostained against C3 and GFAP. There is no C3 expression in astrocytes from control, NBH inoculated WT and Apoe -/-mice. At 15 wpi, C3 expression is detectable in astrocytes of 22L Apoe -/-mice but not in 22L WT mice, while at 23 wpi it is detectable in both genotypes but is significantly higher in 22L Apoe -/-mice. Scale bar: 40 μm.  Abbreviations: Hip -hippocampus, L V -layer V, S1 Ctx -primary somatosensory cortex, and Th -thalamus. Asterisks demarcates limits of the ventroposterior thalamic nucleus (VPN), which along with the S1 cortex was selected for the quantitative analysis of Iba + microglia load.

Fig. S7
Apoe -/-is associated with upregulation of P2RY12 and TMEM119 homeostatic microglia markers. Analysis of a P2ry12 and b Tmem119 mRNA level. The qRT-PCR results are presented as the ΔC T values (n = 3 -11 mice/group). c Shown are representative epifluorescent microphotographs of microglia in the layer V of the S1 cortex from mice of indicated experimental groups, which were immunostained against TMEM119 and d the quantitative analysis of TMEM119 load in the S1 cortex, respectively (n = 5 -7 mice/group). a, b, and d p < 0.0001 (ANOVA); *p < 0.05, **p < 0.01, and ****p < 0.0001 (Holm's-Sidak's post hoc test). Values in a, b, and d represent mean + SEM. Scale bar: 40 μm in c.