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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 FCD subtypes. FCD II: (A) Intense labelling was confirmed in dysmorphic neurones in FCDIIa and FCD IIb (B) as anticipated, highlighting the tigroid appearance of the cytoplasm (B). Dysmorphic neurones were also intensely positive with pS6 235/6 (C), although the cytoplasm of balloon cells appeared somewhat weaker. (D) Double labelling of pS6 with DCX confirmed the balloon cells as pS6 positive, with some balloon cells co-labelling with DCX (arrowhead, inset); small DCX bipolar cells were not always pS6 labelled (arrow) and wrapping of a DCX-positive cell processes around a pS6-positive balloon cell (inset) was also noted. FCD IIIa: (E) FCD type IIIa (adjacent to HS) with laminar neuronal loss demonstrated with NeuN labelling and clusters of neurones in layer II accompanied by mark superficial cortical gliosis (F); in these cases labelling of the residual neurones in layer II with pS6 ser 235/6 (G) and pS6 ser 240/244 (H) was noted. Double labelling studies confirmed co-localisation of pS6 240/244 with GFAPdelta isoform in the cortex (I) and white matter (J) and between nestin and pS6 235/6 in small glial cells particularly in perivascular regions (K), as well as focally between DCX-positive small cells and pS6 235/236 (L). FCD IIIb: pS6 highlighted intense labelling of scattered cortical neurones in adjacent dyslaminar cortex, as well as neurones trapped within the tumour (inset), but negative labelling of the small tumour cells (M). FCDIIId: Disrupted cortex adjacent to an old perinatal infarct in this case showed a prominent ‘tramline’ labelling pattern of pyramidal cells (N); inset shows prominent labelling of astroglial cells in the region of chronic cortical scarring in an FCD IIId case. Bar in A, B, C, D, I, J, K and L equivalent to approximately 35 microns, in e,f,g,h and m to 50 microns and in n to 100 microns.
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Fig2: pS6 in FCD subtypes. FCD II: (A) Intense labelling was confirmed in dysmorphic neurones in FCDIIa and FCD IIb (B) as anticipated, highlighting the tigroid appearance of the cytoplasm (B). Dysmorphic neurones were also intensely positive with pS6 235/6 (C), although the cytoplasm of balloon cells appeared somewhat weaker. (D) Double labelling of pS6 with DCX confirmed the balloon cells as pS6 positive, with some balloon cells co-labelling with DCX (arrowhead, inset); small DCX bipolar cells were not always pS6 labelled (arrow) and wrapping of a DCX-positive cell processes around a pS6-positive balloon cell (inset) was also noted. FCD IIIa: (E) FCD type IIIa (adjacent to HS) with laminar neuronal loss demonstrated with NeuN labelling and clusters of neurones in layer II accompanied by mark superficial cortical gliosis (F); in these cases labelling of the residual neurones in layer II with pS6 ser 235/6 (G) and pS6 ser 240/244 (H) was noted. Double labelling studies confirmed co-localisation of pS6 240/244 with GFAPdelta isoform in the cortex (I) and white matter (J) and between nestin and pS6 235/6 in small glial cells particularly in perivascular regions (K), as well as focally between DCX-positive small cells and pS6 235/236 (L). FCD IIIb: pS6 highlighted intense labelling of scattered cortical neurones in adjacent dyslaminar cortex, as well as neurones trapped within the tumour (inset), but negative labelling of the small tumour cells (M). FCDIIId: Disrupted cortex adjacent to an old perinatal infarct in this case showed a prominent ‘tramline’ labelling pattern of pyramidal cells (N); inset shows prominent labelling of astroglial cells in the region of chronic cortical scarring in an FCD IIId case. Bar in A, B, C, D, I, J, K and L equivalent to approximately 35 microns, in e,f,g,h and m to 50 microns and in n to 100 microns.

Mentions: In the single case with FCDIIa pathology, prominent labelling of DN was seen with both pS6 antibodies, highlighting the region of dysplasia (Figure 2A), with less intense labelling of reactive glia in the region of the dysplasia; labelling of neurones in the adjacent non-dysplastic cortex was, however, also noted. In five cases of FCDIIb, DN showed a striking tigroid cytoplasmic labelling pattern with both pS6 markers, sometimes condensing in the perinuclear zone (Figure 2B,C). BC showed immunopositivity with both markers but these cells were, in general, less intensely labelled than the DN (Figure 2C). In addition, labelling of small glial-like cells (multipolar and bipolar cells) was noted in the region of dysplasia, and in morphologically normal neurones in the adjacent cortex. Double labelling with anti-pS6 (ser 235/236) and DCX showed the co-labelling of some BC, although many small DCX-positive cells were not labelled (Figure 2D).Figure 2


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 FCD subtypes. FCD II: (A) Intense labelling was confirmed in dysmorphic neurones in FCDIIa and FCD IIb (B) as anticipated, highlighting the tigroid appearance of the cytoplasm (B). Dysmorphic neurones were also intensely positive with pS6 235/6 (C), although the cytoplasm of balloon cells appeared somewhat weaker. (D) Double labelling of pS6 with DCX confirmed the balloon cells as pS6 positive, with some balloon cells co-labelling with DCX (arrowhead, inset); small DCX bipolar cells were not always pS6 labelled (arrow) and wrapping of a DCX-positive cell processes around a pS6-positive balloon cell (inset) was also noted. FCD IIIa: (E) FCD type IIIa (adjacent to HS) with laminar neuronal loss demonstrated with NeuN labelling and clusters of neurones in layer II accompanied by mark superficial cortical gliosis (F); in these cases labelling of the residual neurones in layer II with pS6 ser 235/6 (G) and pS6 ser 240/244 (H) was noted. Double labelling studies confirmed co-localisation of pS6 240/244 with GFAPdelta isoform in the cortex (I) and white matter (J) and between nestin and pS6 235/6 in small glial cells particularly in perivascular regions (K), as well as focally between DCX-positive small cells and pS6 235/236 (L). FCD IIIb: pS6 highlighted intense labelling of scattered cortical neurones in adjacent dyslaminar cortex, as well as neurones trapped within the tumour (inset), but negative labelling of the small tumour cells (M). FCDIIId: Disrupted cortex adjacent to an old perinatal infarct in this case showed a prominent ‘tramline’ labelling pattern of pyramidal cells (N); inset shows prominent labelling of astroglial cells in the region of chronic cortical scarring in an FCD IIId case. Bar in A, B, C, D, I, J, K and L equivalent to approximately 35 microns, in e,f,g,h and m to 50 microns and in n to 100 microns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: pS6 in FCD subtypes. FCD II: (A) Intense labelling was confirmed in dysmorphic neurones in FCDIIa and FCD IIb (B) as anticipated, highlighting the tigroid appearance of the cytoplasm (B). Dysmorphic neurones were also intensely positive with pS6 235/6 (C), although the cytoplasm of balloon cells appeared somewhat weaker. (D) Double labelling of pS6 with DCX confirmed the balloon cells as pS6 positive, with some balloon cells co-labelling with DCX (arrowhead, inset); small DCX bipolar cells were not always pS6 labelled (arrow) and wrapping of a DCX-positive cell processes around a pS6-positive balloon cell (inset) was also noted. FCD IIIa: (E) FCD type IIIa (adjacent to HS) with laminar neuronal loss demonstrated with NeuN labelling and clusters of neurones in layer II accompanied by mark superficial cortical gliosis (F); in these cases labelling of the residual neurones in layer II with pS6 ser 235/6 (G) and pS6 ser 240/244 (H) was noted. Double labelling studies confirmed co-localisation of pS6 240/244 with GFAPdelta isoform in the cortex (I) and white matter (J) and between nestin and pS6 235/6 in small glial cells particularly in perivascular regions (K), as well as focally between DCX-positive small cells and pS6 235/236 (L). FCD IIIb: pS6 highlighted intense labelling of scattered cortical neurones in adjacent dyslaminar cortex, as well as neurones trapped within the tumour (inset), but negative labelling of the small tumour cells (M). FCDIIId: Disrupted cortex adjacent to an old perinatal infarct in this case showed a prominent ‘tramline’ labelling pattern of pyramidal cells (N); inset shows prominent labelling of astroglial cells in the region of chronic cortical scarring in an FCD IIId case. Bar in A, B, C, D, I, J, K and L equivalent to approximately 35 microns, in e,f,g,h and m to 50 microns and in n to 100 microns.
Mentions: In the single case with FCDIIa pathology, prominent labelling of DN was seen with both pS6 antibodies, highlighting the region of dysplasia (Figure 2A), with less intense labelling of reactive glia in the region of the dysplasia; labelling of neurones in the adjacent non-dysplastic cortex was, however, also noted. In five cases of FCDIIb, DN showed a striking tigroid cytoplasmic labelling pattern with both pS6 markers, sometimes condensing in the perinuclear zone (Figure 2B,C). BC showed immunopositivity with both markers but these cells were, in general, less intensely labelled than the DN (Figure 2C). In addition, labelling of small glial-like cells (multipolar and bipolar cells) was noted in the region of dysplasia, and in morphologically normal neurones in the adjacent cortex. Double labelling with anti-pS6 (ser 235/236) and DCX showed the co-labelling of some BC, although many small DCX-positive cells were not labelled (Figure 2D).Figure 2

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