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High Resolution Dissection of Reactive Glial Nets in Alzheimer's Disease.

Bouvier DS, Jones EV, Quesseveur G, Davoli MA, A Ferreira T, Quirion R, Mechawar N, Murai KK - Sci Rep (2016)

Bottom Line: Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease.Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs).The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada.

ABSTRACT
Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

No MeSH data available.


Related in: MedlinePlus

Mouse RGNs in the CRND8 AD model share features with human RGNs and associate with neuronal pathology and Tau granules.(a) Time-course for the assembly of RGNs around Aβ deposits. At 2 months, low numbers of activated microglia and reactive astrocytes surround small Aβ deposits. At 4 months, well-constructed RGNs are found. By 12 months, organization of the RGN is degraded and amoeboid microglia and reactive astrocytes can be located distal to RGNs surrounding Aβ deposits. (b) 3D analysis of astrocyte and microglia position around Aβ deposits at mid-stages of the disease in the CRND8 model (3–5 months). (c) Graph showing the positive correlation between astrocyte and microglial cell number and Aβ deposit volume at mid-stages of the disease (r = 0.264 for total Iba1+ cells, r = 0.488 for Iba1+ cells within GFAP shell, and r = 0.561 for GFAP+ cells; pooled data, 3 to 5 month old mice). (d) Comparisons between plaques at mid- (3–5 months) and late-stages (8–9 months) of interval of inter-distance of GFAP+ cells from plaques (F(1, 57) = 14.635; p = 0.0003), numbers of Iba1+ cells within the astrocyte shell of the RGN (F(1, 57) = 7.725; p = 0.0074), and volume of the plaques (F(1, 57) = 5.425; p = 0.0234]. *p < 0.05, **p < 0.01 and ***p < 0.001). (e–g) Abnormal neuronal processes (e; SMI 312+; magenta) and granules of hyperphosphorylated Tau (f,g); detected by two different phospho-Tau antibodies PS422 (magenta) and AT8 (green), associated with RGNs. (h) Expression of IL-6 (upper panel, magenta) and IL-1β (lower panel, magenta) by GFAP+ astrocytes (green, arrows) in CRND8 mice at 9 months. (i) Western blot analysis of overall increases in IL-6 and IL-1β expression (mature form at 17 kDa, arrowhead) in the cortex of transgenic mice at 1, 4 and 9 months (with n = 3 for control and Tg+ at 1 month, n = 4 for control and n = 3 for Tg+ at 4 months, and n=4 for control and n = 4 for Tg+ at 9 months). (j) IL-1β clusters (green) are closely juxtaposed to hyperphosphorylated Tau granules (AT8; magenta) in CRND8 mice. Scale bars: 20 μm (a,e–h), 5 μm (j).
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f5: Mouse RGNs in the CRND8 AD model share features with human RGNs and associate with neuronal pathology and Tau granules.(a) Time-course for the assembly of RGNs around Aβ deposits. At 2 months, low numbers of activated microglia and reactive astrocytes surround small Aβ deposits. At 4 months, well-constructed RGNs are found. By 12 months, organization of the RGN is degraded and amoeboid microglia and reactive astrocytes can be located distal to RGNs surrounding Aβ deposits. (b) 3D analysis of astrocyte and microglia position around Aβ deposits at mid-stages of the disease in the CRND8 model (3–5 months). (c) Graph showing the positive correlation between astrocyte and microglial cell number and Aβ deposit volume at mid-stages of the disease (r = 0.264 for total Iba1+ cells, r = 0.488 for Iba1+ cells within GFAP shell, and r = 0.561 for GFAP+ cells; pooled data, 3 to 5 month old mice). (d) Comparisons between plaques at mid- (3–5 months) and late-stages (8–9 months) of interval of inter-distance of GFAP+ cells from plaques (F(1, 57) = 14.635; p = 0.0003), numbers of Iba1+ cells within the astrocyte shell of the RGN (F(1, 57) = 7.725; p = 0.0074), and volume of the plaques (F(1, 57) = 5.425; p = 0.0234]. *p < 0.05, **p < 0.01 and ***p < 0.001). (e–g) Abnormal neuronal processes (e; SMI 312+; magenta) and granules of hyperphosphorylated Tau (f,g); detected by two different phospho-Tau antibodies PS422 (magenta) and AT8 (green), associated with RGNs. (h) Expression of IL-6 (upper panel, magenta) and IL-1β (lower panel, magenta) by GFAP+ astrocytes (green, arrows) in CRND8 mice at 9 months. (i) Western blot analysis of overall increases in IL-6 and IL-1β expression (mature form at 17 kDa, arrowhead) in the cortex of transgenic mice at 1, 4 and 9 months (with n = 3 for control and Tg+ at 1 month, n = 4 for control and n = 3 for Tg+ at 4 months, and n=4 for control and n = 4 for Tg+ at 9 months). (j) IL-1β clusters (green) are closely juxtaposed to hyperphosphorylated Tau granules (AT8; magenta) in CRND8 mice. Scale bars: 20 μm (a,e–h), 5 μm (j).

Mentions: Mouse models of familial AD (FAD) have played an important role in studying pathways involved in AD. However, a better understanding of how the cellular and molecular alterations seen in mouse models reflect the pathology of AD is needed33. Guided by our analysis of human RGNs, we were interested in determining if RGN structures were conserved between AD and mouse model tissue and, if so, the spatio-temporal properties underlying their assembly. One particularly useful FAD mouse model is the CRND8Tg model that expresses the transgene human APP695 cDNA with double mutations at KM670/671/NL (Swedish mutation), along with the V717F (Indiana mutation) under the Syrian hamster prion promoter34. CRND8Tg is an early-onset FAD model showing Aβ deposits at 2 months, and Aβ plaques and neuritic pathology by 5 months, thus allowing the full time-course of AD-like disease to be monitored. Interestingly, we detected abundant RGNs at mid-stages (i.e. 4 months; Fig. 5a–c, Suppl. Fig. 6a) in CRND8Tg mice. Time-course analysis in cortical areas showed that starting at 2–3 months (“early-stage”), sparse Iba1+ microglia were found around small Aβ deposits. In some, but not all cases, single GFAP+ astrocytes were in close proximity (Fig. 5a). By 3–4 months (“mid-stage”), the first complete RGNs were present, with microglia encompassing larger Aβ plaques in a rosette conformation and with astrocytes forming an elaborate outer shell-like structure. Microglia extended elaborate processes that circumscribed plaques, similar to microglia around dense-core plaques in AD (Fig. 5a, Suppl. Fig. 6a). Reactive astrocytes surrounding the plaque showed a hypertrophic and highly polarized morphology with their processes creating a complex outer shell of the RGN centered about 40 μm from the Aβ dense-core (Fig. 5d). Microglia and astrocytes displayed a stereotypical arrangement at this time that was influenced by plaque volume (Fig. 5c). By 8 months+ (“late-stage”), an increased recruitment of microglia and astrocytes to RGNs was observed (Fig. 5a,d, Suppl. Fig. 6a,c,d), concomitant with increased plaque volume (n = 30). At this time, the properties of RGNs transformed to incorporate a larger number of activated microglia (Fig. 5d, Suppl. Fig. 6c,d) and encompassed a wider collection of GFAP+ astrocytes within an average of 60 μm inter-distance from the plaque center (Interval Max GFAP+) (Fig. 5d). The appearance of less compact RGNs at late stages in mice resembled the complexity of fibrillar-like plaque RGNs in AD. By 12–24 months, numerous activated microglia and reactive astrocytes adjacent to the RGN structure were frequent (Fig. 5a, Suppl. Fig. 6a) indicating more wide-spread glial reactivity beyond the boundaries of the RGN. Thus, despite differences in the level of overproduction of Aβ plaques in CRND8tg mice, and the temporal progression of AD-like disease, the principle features of RGNs are conserved between AD and an AD mouse model.


High Resolution Dissection of Reactive Glial Nets in Alzheimer's Disease.

Bouvier DS, Jones EV, Quesseveur G, Davoli MA, A Ferreira T, Quirion R, Mechawar N, Murai KK - Sci Rep (2016)

Mouse RGNs in the CRND8 AD model share features with human RGNs and associate with neuronal pathology and Tau granules.(a) Time-course for the assembly of RGNs around Aβ deposits. At 2 months, low numbers of activated microglia and reactive astrocytes surround small Aβ deposits. At 4 months, well-constructed RGNs are found. By 12 months, organization of the RGN is degraded and amoeboid microglia and reactive astrocytes can be located distal to RGNs surrounding Aβ deposits. (b) 3D analysis of astrocyte and microglia position around Aβ deposits at mid-stages of the disease in the CRND8 model (3–5 months). (c) Graph showing the positive correlation between astrocyte and microglial cell number and Aβ deposit volume at mid-stages of the disease (r = 0.264 for total Iba1+ cells, r = 0.488 for Iba1+ cells within GFAP shell, and r = 0.561 for GFAP+ cells; pooled data, 3 to 5 month old mice). (d) Comparisons between plaques at mid- (3–5 months) and late-stages (8–9 months) of interval of inter-distance of GFAP+ cells from plaques (F(1, 57) = 14.635; p = 0.0003), numbers of Iba1+ cells within the astrocyte shell of the RGN (F(1, 57) = 7.725; p = 0.0074), and volume of the plaques (F(1, 57) = 5.425; p = 0.0234]. *p < 0.05, **p < 0.01 and ***p < 0.001). (e–g) Abnormal neuronal processes (e; SMI 312+; magenta) and granules of hyperphosphorylated Tau (f,g); detected by two different phospho-Tau antibodies PS422 (magenta) and AT8 (green), associated with RGNs. (h) Expression of IL-6 (upper panel, magenta) and IL-1β (lower panel, magenta) by GFAP+ astrocytes (green, arrows) in CRND8 mice at 9 months. (i) Western blot analysis of overall increases in IL-6 and IL-1β expression (mature form at 17 kDa, arrowhead) in the cortex of transgenic mice at 1, 4 and 9 months (with n = 3 for control and Tg+ at 1 month, n = 4 for control and n = 3 for Tg+ at 4 months, and n=4 for control and n = 4 for Tg+ at 9 months). (j) IL-1β clusters (green) are closely juxtaposed to hyperphosphorylated Tau granules (AT8; magenta) in CRND8 mice. Scale bars: 20 μm (a,e–h), 5 μm (j).
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f5: Mouse RGNs in the CRND8 AD model share features with human RGNs and associate with neuronal pathology and Tau granules.(a) Time-course for the assembly of RGNs around Aβ deposits. At 2 months, low numbers of activated microglia and reactive astrocytes surround small Aβ deposits. At 4 months, well-constructed RGNs are found. By 12 months, organization of the RGN is degraded and amoeboid microglia and reactive astrocytes can be located distal to RGNs surrounding Aβ deposits. (b) 3D analysis of astrocyte and microglia position around Aβ deposits at mid-stages of the disease in the CRND8 model (3–5 months). (c) Graph showing the positive correlation between astrocyte and microglial cell number and Aβ deposit volume at mid-stages of the disease (r = 0.264 for total Iba1+ cells, r = 0.488 for Iba1+ cells within GFAP shell, and r = 0.561 for GFAP+ cells; pooled data, 3 to 5 month old mice). (d) Comparisons between plaques at mid- (3–5 months) and late-stages (8–9 months) of interval of inter-distance of GFAP+ cells from plaques (F(1, 57) = 14.635; p = 0.0003), numbers of Iba1+ cells within the astrocyte shell of the RGN (F(1, 57) = 7.725; p = 0.0074), and volume of the plaques (F(1, 57) = 5.425; p = 0.0234]. *p < 0.05, **p < 0.01 and ***p < 0.001). (e–g) Abnormal neuronal processes (e; SMI 312+; magenta) and granules of hyperphosphorylated Tau (f,g); detected by two different phospho-Tau antibodies PS422 (magenta) and AT8 (green), associated with RGNs. (h) Expression of IL-6 (upper panel, magenta) and IL-1β (lower panel, magenta) by GFAP+ astrocytes (green, arrows) in CRND8 mice at 9 months. (i) Western blot analysis of overall increases in IL-6 and IL-1β expression (mature form at 17 kDa, arrowhead) in the cortex of transgenic mice at 1, 4 and 9 months (with n = 3 for control and Tg+ at 1 month, n = 4 for control and n = 3 for Tg+ at 4 months, and n=4 for control and n = 4 for Tg+ at 9 months). (j) IL-1β clusters (green) are closely juxtaposed to hyperphosphorylated Tau granules (AT8; magenta) in CRND8 mice. Scale bars: 20 μm (a,e–h), 5 μm (j).
Mentions: Mouse models of familial AD (FAD) have played an important role in studying pathways involved in AD. However, a better understanding of how the cellular and molecular alterations seen in mouse models reflect the pathology of AD is needed33. Guided by our analysis of human RGNs, we were interested in determining if RGN structures were conserved between AD and mouse model tissue and, if so, the spatio-temporal properties underlying their assembly. One particularly useful FAD mouse model is the CRND8Tg model that expresses the transgene human APP695 cDNA with double mutations at KM670/671/NL (Swedish mutation), along with the V717F (Indiana mutation) under the Syrian hamster prion promoter34. CRND8Tg is an early-onset FAD model showing Aβ deposits at 2 months, and Aβ plaques and neuritic pathology by 5 months, thus allowing the full time-course of AD-like disease to be monitored. Interestingly, we detected abundant RGNs at mid-stages (i.e. 4 months; Fig. 5a–c, Suppl. Fig. 6a) in CRND8Tg mice. Time-course analysis in cortical areas showed that starting at 2–3 months (“early-stage”), sparse Iba1+ microglia were found around small Aβ deposits. In some, but not all cases, single GFAP+ astrocytes were in close proximity (Fig. 5a). By 3–4 months (“mid-stage”), the first complete RGNs were present, with microglia encompassing larger Aβ plaques in a rosette conformation and with astrocytes forming an elaborate outer shell-like structure. Microglia extended elaborate processes that circumscribed plaques, similar to microglia around dense-core plaques in AD (Fig. 5a, Suppl. Fig. 6a). Reactive astrocytes surrounding the plaque showed a hypertrophic and highly polarized morphology with their processes creating a complex outer shell of the RGN centered about 40 μm from the Aβ dense-core (Fig. 5d). Microglia and astrocytes displayed a stereotypical arrangement at this time that was influenced by plaque volume (Fig. 5c). By 8 months+ (“late-stage”), an increased recruitment of microglia and astrocytes to RGNs was observed (Fig. 5a,d, Suppl. Fig. 6a,c,d), concomitant with increased plaque volume (n = 30). At this time, the properties of RGNs transformed to incorporate a larger number of activated microglia (Fig. 5d, Suppl. Fig. 6c,d) and encompassed a wider collection of GFAP+ astrocytes within an average of 60 μm inter-distance from the plaque center (Interval Max GFAP+) (Fig. 5d). The appearance of less compact RGNs at late stages in mice resembled the complexity of fibrillar-like plaque RGNs in AD. By 12–24 months, numerous activated microglia and reactive astrocytes adjacent to the RGN structure were frequent (Fig. 5a, Suppl. Fig. 6a) indicating more wide-spread glial reactivity beyond the boundaries of the RGN. Thus, despite differences in the level of overproduction of Aβ plaques in CRND8tg mice, and the temporal progression of AD-like disease, the principle features of RGNs are conserved between AD and an AD mouse model.

Bottom Line: Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease.Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs).The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada.

ABSTRACT
Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

No MeSH data available.


Related in: MedlinePlus