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Evidence for mTOR pathway activation in a spectrum of epilepsy-associated pathologies.

Liu J, Reeves C, Michalak Z, Coppola A, Diehl B, Sisodiya SM, Thom M - Acta Neuropathol Commun (2014)

Bottom Line: Immunohistochemistry for phospho-S6 (pS6) ser240/244 and ser235/236 and double-labelling for Iba1, neurofilament, GFAP, GFAPdelta, doublecortin, and nestin were performed.There was no difference in pS6 labelling in paired samples according to ictal activity.There was no definite evidence from our studies to suggest that pS6 expression is directly related to disease activity.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neuropathology, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK. m.thom@ucl.ac.uk.

ABSTRACT

Introduction: Activation of the mTOR pathway has been linked to the cytopathology and epileptogenicity of malformations, specifically Focal Cortical Dysplasia (FCD) and Tuberous Sclerosis (TSC). Experimental and clinical trials have shown than mTOR inhibitors have anti-epileptogenic effects in TS. Dysmorphic neurones and balloon cells are hallmarks of FCDIIb and TSC, but similar cells are also occasionally observed in other acquired epileptogenic pathologies, including hippocampal sclerosis (HS) and Rasmussen's encephalitis (RE). Our aim was to explore mTOR pathway activation in a range of epilepsy-associated pathologies and in lesion-negative cases.

Results: 50 epilepsy surgical pathologies were selected including HS ILAE type 1 with (5) and without dysmorphic neurones (4), FCDIIa (1), FCDIIb (5), FCDIIIa (5), FCDIIIb (3), FCDIIId (3), RE (5) and cortex adjacent to cavernoma (1). We also included pathology-negative epilepsy cases; temporal cortex (7), frontal cortex (2), paired frontal cortical samples with different ictal activity according to intracranial EEG recordings (4), cortex with acute injuries from electrode tracks (5) and additionally non-epilepsy surgical controls (3). Immunohistochemistry for phospho-S6 (pS6) ser240/244 and ser235/236 and double-labelling for Iba1, neurofilament, GFAP, GFAPdelta, doublecortin, and nestin were performed. Predominant neuronal labelling was observed with pS6 ser240/244 and glial labelling with pS6 ser235/236 in all pathology types but with evidence for co-expression in a proportion of cells in all pathologies. Intense labelling of dysmorphic neurones and balloon cells was observed in FCDIIb, but dysmorphic neurones were also labelled in RE and HS. There was no difference in pS6 labelling in paired samples according to ictal activity. Double-labelling immunofluorescent studies further demonstrated the co-localisation of pS6 with nestin, doublecortin, GFAPdelta in populations of small, immature neuroglial cells in a range of epilepsy pathologies.

Conclusions: Although mTOR activation has been more studied in the FCDIIb and TSC, our observations suggest this pathway is activated in a variety of epilepsy-associated pathologies, and in varied cell types including dysmorphic neurones, microglia and immature cell types. There was no definite evidence from our studies to suggest that pS6 expression is directly related to disease activity.

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pS6 in HS ILAE type 1 with (A-J) and without (K-L) dysmorphic changes. HS ILAE type 1 with dysmorphic changes (A-J) (A) The dentate gyrus in a case with ILAE HS type 1 and additional glassy balloon-like astroglial cells on H&E which show membranous positivity for CD34 (inset). (B) pS6 235/236 labelling of hypertrophic CA4 neurones and (C) in the granule cell layer was noted in HS cases with dysplasia features; inset shows co-localisation of labelling in a proportion of cells with the two pS6 antibodies. (D) pS6 235/236 also labelled small immature cells with bipolar or multipolar processes including in the basal layer of the dentate gyrus (D) as well as through the dentate gyrus (E). Co-localisation between doublecortin (DCX) and pS6 was noted in some of these small cells in the dentate gyrus (F) as well as with nestin (G); in addition both pS6 markers co-labelled a proportion of small multipolar cells in HS. (I) With pS6 240/244 prominent labelling of horizontal cells in the stratum moleculare of the hippocampus, in addition to more distinct labelling of pyramidal cells through hippocampal subfields was noted. HS ILAE type 1: Intense labelling of CA4 neurones but the the granule cells were more variably negative (K) or positive (L) with pS6 240/244. White arrowheads in all images indicate double-labelled cells. Bar in A, C, D, E, F, G, H and J equivalent to approximately 35 microns and in B, I, K and L approximately 50 microns.
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Fig1: pS6 in HS ILAE type 1 with (A-J) and without (K-L) dysmorphic changes. HS ILAE type 1 with dysmorphic changes (A-J) (A) The dentate gyrus in a case with ILAE HS type 1 and additional glassy balloon-like astroglial cells on H&E which show membranous positivity for CD34 (inset). (B) pS6 235/236 labelling of hypertrophic CA4 neurones and (C) in the granule cell layer was noted in HS cases with dysplasia features; inset shows co-localisation of labelling in a proportion of cells with the two pS6 antibodies. (D) pS6 235/236 also labelled small immature cells with bipolar or multipolar processes including in the basal layer of the dentate gyrus (D) as well as through the dentate gyrus (E). Co-localisation between doublecortin (DCX) and pS6 was noted in some of these small cells in the dentate gyrus (F) as well as with nestin (G); in addition both pS6 markers co-labelled a proportion of small multipolar cells in HS. (I) With pS6 240/244 prominent labelling of horizontal cells in the stratum moleculare of the hippocampus, in addition to more distinct labelling of pyramidal cells through hippocampal subfields was noted. HS ILAE type 1: Intense labelling of CA4 neurones but the the granule cells were more variably negative (K) or positive (L) with pS6 240/244. White arrowheads in all images indicate double-labelled cells. Bar in A, C, D, E, F, G, H and J equivalent to approximately 35 microns and in B, I, K and L approximately 50 microns.

Mentions: Lesional cases included patients with mesial temporal lobe epilepsy (mTLE) and HS ILAE type 1 with (n = 5) or without (n = 4) additional dysplasia-like cytopathological changes, including prominent dysmorphic, neurofilament-positive neuronal cells in CA4 and CD34-positive BC-like astrocytes in the dentate gyrus as previously described [15, 14, 13, 12] (Figure 1A). FCD was represented by type IIa (n = 1), type IIb (n = 5), type IIIa (dysplasia associated with HS; n = 5), type IIIb (dysplasia associated with dysembryoplastic neuroepithelial tumours (DNT)/CD34-positive long-term epilepsy-associated tumours (LEAT); n = 3), type IIId (dysplasia associated with an early infarct; n = 3) and a cavernoma with florid adjacent reactive gliosis (n = 1). In FCDIIb cases TSC was excluded clinically. We included resections from patients with a clinical and radiological diagnosis compatible with RE (n = 5) who had undergone either diagnostic biopsy or therapeutic neurosurgical procedure. The stages of inflammatory activity and scarring varied both within and between cases, as detailed in Table 1, and in two cases with RE, cortical neurones appeared hypertrophic and dysmorphic.Figure 1


Evidence for mTOR pathway activation in a spectrum of epilepsy-associated pathologies.

Liu J, Reeves C, Michalak Z, Coppola A, Diehl B, Sisodiya SM, Thom M - Acta Neuropathol Commun (2014)

pS6 in HS ILAE type 1 with (A-J) and without (K-L) dysmorphic changes. HS ILAE type 1 with dysmorphic changes (A-J) (A) The dentate gyrus in a case with ILAE HS type 1 and additional glassy balloon-like astroglial cells on H&E which show membranous positivity for CD34 (inset). (B) pS6 235/236 labelling of hypertrophic CA4 neurones and (C) in the granule cell layer was noted in HS cases with dysplasia features; inset shows co-localisation of labelling in a proportion of cells with the two pS6 antibodies. (D) pS6 235/236 also labelled small immature cells with bipolar or multipolar processes including in the basal layer of the dentate gyrus (D) as well as through the dentate gyrus (E). Co-localisation between doublecortin (DCX) and pS6 was noted in some of these small cells in the dentate gyrus (F) as well as with nestin (G); in addition both pS6 markers co-labelled a proportion of small multipolar cells in HS. (I) With pS6 240/244 prominent labelling of horizontal cells in the stratum moleculare of the hippocampus, in addition to more distinct labelling of pyramidal cells through hippocampal subfields was noted. HS ILAE type 1: Intense labelling of CA4 neurones but the the granule cells were more variably negative (K) or positive (L) with pS6 240/244. White arrowheads in all images indicate double-labelled cells. Bar in A, C, D, E, F, G, H and J equivalent to approximately 35 microns and in B, I, K and L approximately 50 microns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: pS6 in HS ILAE type 1 with (A-J) and without (K-L) dysmorphic changes. HS ILAE type 1 with dysmorphic changes (A-J) (A) The dentate gyrus in a case with ILAE HS type 1 and additional glassy balloon-like astroglial cells on H&E which show membranous positivity for CD34 (inset). (B) pS6 235/236 labelling of hypertrophic CA4 neurones and (C) in the granule cell layer was noted in HS cases with dysplasia features; inset shows co-localisation of labelling in a proportion of cells with the two pS6 antibodies. (D) pS6 235/236 also labelled small immature cells with bipolar or multipolar processes including in the basal layer of the dentate gyrus (D) as well as through the dentate gyrus (E). Co-localisation between doublecortin (DCX) and pS6 was noted in some of these small cells in the dentate gyrus (F) as well as with nestin (G); in addition both pS6 markers co-labelled a proportion of small multipolar cells in HS. (I) With pS6 240/244 prominent labelling of horizontal cells in the stratum moleculare of the hippocampus, in addition to more distinct labelling of pyramidal cells through hippocampal subfields was noted. HS ILAE type 1: Intense labelling of CA4 neurones but the the granule cells were more variably negative (K) or positive (L) with pS6 240/244. White arrowheads in all images indicate double-labelled cells. Bar in A, C, D, E, F, G, H and J equivalent to approximately 35 microns and in B, I, K and L approximately 50 microns.
Mentions: Lesional cases included patients with mesial temporal lobe epilepsy (mTLE) and HS ILAE type 1 with (n = 5) or without (n = 4) additional dysplasia-like cytopathological changes, including prominent dysmorphic, neurofilament-positive neuronal cells in CA4 and CD34-positive BC-like astrocytes in the dentate gyrus as previously described [15, 14, 13, 12] (Figure 1A). FCD was represented by type IIa (n = 1), type IIb (n = 5), type IIIa (dysplasia associated with HS; n = 5), type IIIb (dysplasia associated with dysembryoplastic neuroepithelial tumours (DNT)/CD34-positive long-term epilepsy-associated tumours (LEAT); n = 3), type IIId (dysplasia associated with an early infarct; n = 3) and a cavernoma with florid adjacent reactive gliosis (n = 1). In FCDIIb cases TSC was excluded clinically. We included resections from patients with a clinical and radiological diagnosis compatible with RE (n = 5) who had undergone either diagnostic biopsy or therapeutic neurosurgical procedure. The stages of inflammatory activity and scarring varied both within and between cases, as detailed in Table 1, and in two cases with RE, cortical neurones appeared hypertrophic and dysmorphic.Figure 1

Bottom Line: Immunohistochemistry for phospho-S6 (pS6) ser240/244 and ser235/236 and double-labelling for Iba1, neurofilament, GFAP, GFAPdelta, doublecortin, and nestin were performed.There was no difference in pS6 labelling in paired samples according to ictal activity.There was no definite evidence from our studies to suggest that pS6 expression is directly related to disease activity.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neuropathology, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK. m.thom@ucl.ac.uk.

ABSTRACT

Introduction: Activation of the mTOR pathway has been linked to the cytopathology and epileptogenicity of malformations, specifically Focal Cortical Dysplasia (FCD) and Tuberous Sclerosis (TSC). Experimental and clinical trials have shown than mTOR inhibitors have anti-epileptogenic effects in TS. Dysmorphic neurones and balloon cells are hallmarks of FCDIIb and TSC, but similar cells are also occasionally observed in other acquired epileptogenic pathologies, including hippocampal sclerosis (HS) and Rasmussen's encephalitis (RE). Our aim was to explore mTOR pathway activation in a range of epilepsy-associated pathologies and in lesion-negative cases.

Results: 50 epilepsy surgical pathologies were selected including HS ILAE type 1 with (5) and without dysmorphic neurones (4), FCDIIa (1), FCDIIb (5), FCDIIIa (5), FCDIIIb (3), FCDIIId (3), RE (5) and cortex adjacent to cavernoma (1). We also included pathology-negative epilepsy cases; temporal cortex (7), frontal cortex (2), paired frontal cortical samples with different ictal activity according to intracranial EEG recordings (4), cortex with acute injuries from electrode tracks (5) and additionally non-epilepsy surgical controls (3). Immunohistochemistry for phospho-S6 (pS6) ser240/244 and ser235/236 and double-labelling for Iba1, neurofilament, GFAP, GFAPdelta, doublecortin, and nestin were performed. Predominant neuronal labelling was observed with pS6 ser240/244 and glial labelling with pS6 ser235/236 in all pathology types but with evidence for co-expression in a proportion of cells in all pathologies. Intense labelling of dysmorphic neurones and balloon cells was observed in FCDIIb, but dysmorphic neurones were also labelled in RE and HS. There was no difference in pS6 labelling in paired samples according to ictal activity. Double-labelling immunofluorescent studies further demonstrated the co-localisation of pS6 with nestin, doublecortin, GFAPdelta in populations of small, immature neuroglial cells in a range of epilepsy pathologies.

Conclusions: Although mTOR activation has been more studied in the FCDIIb and TSC, our observations suggest this pathway is activated in a variety of epilepsy-associated pathologies, and in varied cell types including dysmorphic neurones, microglia and immature cell types. There was no definite evidence from our studies to suggest that pS6 expression is directly related to disease activity.

Show MeSH
Related in: MedlinePlus