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prominent activation of microglia IR for both β1i and β5i was also observed in cells of the microglia/macrophage lineage (HLA-II; Fig. 2 and 4). Balloon (FCD IIb; Fig. 2E) and giant cells (TSC; Fig. 4 G) displayed β1i and β5i IR as well (Table 2). Co-localization was observed for both β1i and β5i with pS6 (Fig. 2 and Fig. 4). β1i and β5i expression in neu- rons was positively associated with pS6 expression within our MCD cohort (β1i cyto- plasm, r= 0.5905, p=0.030; β1i nucleus, r= 0.6244, p=0.0014; β5i cytoplasm, r= 0.4510, p=0.0065). A positive correlation was detected between β1i and β5i expression in neu- rons and glial cells and IL-1β IRS within the dysplastic region (neuronal β1i cytoplasm, r= 0.4287, p=0.0413; neuronal β1i nucleus, r= 0.5090, p=0.0131; glia β1i cytoplasm, r= 0.5298, p=0.0093; glia β1i nucleus, r= 0.6091, p=0.0003; neuronal β5i cytoplasm, r= 0.7322, p= < 0.001; glia β5i cytoplasm, r= 0.7005, p= < 0.001; glia β5i nucleus, r= 0.4210, p=0.0455).
Immunoproteasome subunit expression and clinical features
We found no statistically significant association between the IRS of β1, β1i, β5 or β5i and clinical features, such gender, age at surgery, location of the lesion or duration of epilepsy. However, a positive correlation was observed between nuclear glial and neu- ronal subunit expression and the pre-operative seizure frequency (β1 and β1i neuron: τ=0.639 and τ=0.633, p<0.001; β1 and β1i glia: τ=0.479, p=0.005 and τ=0.65, p<0.001; β1 and β1i neuron: τ=0.550, p=0.004 and τ=0.417, p=0.016; β1 and β1i glia: τ=0.570, p=0.001; τ=0.586, p=0.001).
Regulation of immunoproteasome subunit expression in human glial cells in culture
Since IL-1β is known to be strongly upregulated in FCD and TSC human brain specimens 27, 34, 35 and to play a key pathogenic role in human epilepsy (for review see 20, 36, we also investigated whether this inflammatory cytokine could play a role in the regulation of the expression and cellular localization of immunoproteasome subunits. qPCR analysis of astrocyte-enriched human fetal cell cultures demonstrated that exposure to IL-1β did not modify the expression of the constitutive subunits (Fig. 5 A, D), but did consistently increase the expression of both immunoproteasome subunits β1i and β5i (Fig. 5 B, E), increasing the β1/ β1i and β5/β5i ratios (Fig. 5 C, F). Treatment with lipopolysaccharide (LPS), also a potent inducer of the immune response, gave comparable results (Fig. 5). Exposure to IL-1β and LPS did not significantly affect the expression of IFN-γ in these cultures. Immunohistochemistry showed a translocation of the β1i and β5i subunits, shifting from cytoplasmic to perinuclear-nuclear expression following IL-1β treatment (Fig. 6 D, H).
Effects of rapamycin on proteasome subunit expression in FCD II derived astrocytes
Since both FCD II and TSC are associated with constitutive activation of the mTOR pathway 19, 20 we investigated whether the canonical and allosteric mTOR kinase rapamy- cin modulates the immunoproteasome in cell cultures derived from FCD II specimens. Western blot analysis confirmed that 100 nM rapamycin reduced the phosphorylation of S6 (pS6) in human astrocytes, an indicator of mTOR activation (supplementary Fig. 2). Pretreatment with 100 nM rapamycin reduced the mRNA expression of β1 and β1i sub-


























































































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