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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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Non-lesional epilepsy and control groups. (A) Intense labelling of morphologically different cells types, including macrophages and multipolar cells (inset), around an organising intracranial electrode track mark. (B and C) Paired samples from regions of different ictal activity, based on intracranial monitoring: (B) is from case 34, sample 1 (ictal onset zone) where less pS6 labelling was seen with both markers (illustrated here with pS6 240/244) compared to (C) sample 2 which represented less active/seizure spreading zone where more pS6 labelling of small cells was observed as well as neuronal cells (inset). (D) Temporal lobe cortex with no specific pathology, adjacent to HS, where prominent ‘tramline’ labelling of cortical neurones was observed with pS6 240/244. (E) Another pathologically normal temporal lobe in epilepsy with a prominent perivascular labelling pattern of neuronal cells with pS6 235/236 (arrowhead) and (F) labelling of small glial like cells around vessels in the white matter and inset in the subpial layer (inset). Bar in A, B, C, E, F equivalent to approximately 50 microns and in d to 100 microns.
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Fig4: Non-lesional epilepsy and control groups. (A) Intense labelling of morphologically different cells types, including macrophages and multipolar cells (inset), around an organising intracranial electrode track mark. (B and C) Paired samples from regions of different ictal activity, based on intracranial monitoring: (B) is from case 34, sample 1 (ictal onset zone) where less pS6 labelling was seen with both markers (illustrated here with pS6 240/244) compared to (C) sample 2 which represented less active/seizure spreading zone where more pS6 labelling of small cells was observed as well as neuronal cells (inset). (D) Temporal lobe cortex with no specific pathology, adjacent to HS, where prominent ‘tramline’ labelling of cortical neurones was observed with pS6 240/244. (E) Another pathologically normal temporal lobe in epilepsy with a prominent perivascular labelling pattern of neuronal cells with pS6 235/236 (arrowhead) and (F) labelling of small glial like cells around vessels in the white matter and inset in the subpial layer (inset). Bar in A, B, C, E, F equivalent to approximately 50 microns and in d to 100 microns.

Mentions: The stage of inflammatory activity varied between the five cases (Table 1). In two cases with extensive tissue resections, areas with active inflammation alternated with stretches of cortex showing chronic scarring and quiescent inflammation as well as regions of more normal appearing cortex; the presence of hypertrophic, dysmorphic neurofilament-positive neurones was evident in the abnormal cortex. The presence of focal, active encephalitis was confirmed with the microglial marker, HLADR (for activated microglia and macrophages), which showed increased numbers in the region of more active inflammation (Figure 3A). CD163 labelling, which shows macrophages of haemopoietic origin, also showed scattered, immunopositive, rod-like cells in these regions (Figure 3A, inset) compared to less damaged cortex, where CD163 positive cells were limited to the perivascular spaces. In the regions of active encephalitis, there was evidence of increased labelling with both pS6 antibodies (Figure 3B,C). pS6 (ser 240/244) particularly highlighted DN, whose neuronal nature was confirmed by double labelling for SMI32 (Figure 3D). pS6 (ser235/236) showed prominent labelling of small multipolar (Figure 3E) and bipolar cells (Figure 3F) in the damaged cortex; these were of similar morphology to those observed in the dentate gyrus in HS. Double labelling studies suggested the majority of pS6-positive small cells were not GFAP-expressing astrocytes (Figure 3G); but rather showed more frequent co-localisation with Iba1 (Figure 4H), nestin (in both the cortex and white matter) (Figure 4L) and to a lesser extent, with DCX (Figure 4L inset). pS6 (ser240/244) labelling of cortical neurones, including pyramidal cells in all cortical layers, was also noted in the adjacent better-preserved cortex in larger resections.Figure 3


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)

Non-lesional epilepsy and control groups. (A) Intense labelling of morphologically different cells types, including macrophages and multipolar cells (inset), around an organising intracranial electrode track mark. (B and C) Paired samples from regions of different ictal activity, based on intracranial monitoring: (B) is from case 34, sample 1 (ictal onset zone) where less pS6 labelling was seen with both markers (illustrated here with pS6 240/244) compared to (C) sample 2 which represented less active/seizure spreading zone where more pS6 labelling of small cells was observed as well as neuronal cells (inset). (D) Temporal lobe cortex with no specific pathology, adjacent to HS, where prominent ‘tramline’ labelling of cortical neurones was observed with pS6 240/244. (E) Another pathologically normal temporal lobe in epilepsy with a prominent perivascular labelling pattern of neuronal cells with pS6 235/236 (arrowhead) and (F) labelling of small glial like cells around vessels in the white matter and inset in the subpial layer (inset). Bar in A, B, C, E, F equivalent to approximately 50 microns and in d to 100 microns.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230418&req=5

Fig4: Non-lesional epilepsy and control groups. (A) Intense labelling of morphologically different cells types, including macrophages and multipolar cells (inset), around an organising intracranial electrode track mark. (B and C) Paired samples from regions of different ictal activity, based on intracranial monitoring: (B) is from case 34, sample 1 (ictal onset zone) where less pS6 labelling was seen with both markers (illustrated here with pS6 240/244) compared to (C) sample 2 which represented less active/seizure spreading zone where more pS6 labelling of small cells was observed as well as neuronal cells (inset). (D) Temporal lobe cortex with no specific pathology, adjacent to HS, where prominent ‘tramline’ labelling of cortical neurones was observed with pS6 240/244. (E) Another pathologically normal temporal lobe in epilepsy with a prominent perivascular labelling pattern of neuronal cells with pS6 235/236 (arrowhead) and (F) labelling of small glial like cells around vessels in the white matter and inset in the subpial layer (inset). Bar in A, B, C, E, F equivalent to approximately 50 microns and in d to 100 microns.
Mentions: The stage of inflammatory activity varied between the five cases (Table 1). In two cases with extensive tissue resections, areas with active inflammation alternated with stretches of cortex showing chronic scarring and quiescent inflammation as well as regions of more normal appearing cortex; the presence of hypertrophic, dysmorphic neurofilament-positive neurones was evident in the abnormal cortex. The presence of focal, active encephalitis was confirmed with the microglial marker, HLADR (for activated microglia and macrophages), which showed increased numbers in the region of more active inflammation (Figure 3A). CD163 labelling, which shows macrophages of haemopoietic origin, also showed scattered, immunopositive, rod-like cells in these regions (Figure 3A, inset) compared to less damaged cortex, where CD163 positive cells were limited to the perivascular spaces. In the regions of active encephalitis, there was evidence of increased labelling with both pS6 antibodies (Figure 3B,C). pS6 (ser 240/244) particularly highlighted DN, whose neuronal nature was confirmed by double labelling for SMI32 (Figure 3D). pS6 (ser235/236) showed prominent labelling of small multipolar (Figure 3E) and bipolar cells (Figure 3F) in the damaged cortex; these were of similar morphology to those observed in the dentate gyrus in HS. Double labelling studies suggested the majority of pS6-positive small cells were not GFAP-expressing astrocytes (Figure 3G); but rather showed more frequent co-localisation with Iba1 (Figure 4H), nestin (in both the cortex and white matter) (Figure 4L) and to a lesser extent, with DCX (Figure 4L inset). pS6 (ser240/244) labelling of cortical neurones, including pyramidal cells in all cortical layers, was also noted in the adjacent better-preserved cortex in larger resections.Figure 3

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