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Dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in Alzheimer's disease.

Streit WJ, Braak H, Xue QS, Bechmann I - Acta Neuropathol. (2009)

Bottom Line: Although several works suggest that chronic neuroinflammation caused by activated microglia contributes to neurofibrillary degeneration, anti-inflammatory drugs do not prevent or reverse neuronal tau pathology.Deposits of amyloid-beta protein (Abeta) devoid of tau-positive structures were found to be colocalized with non-activated, ramified microglia, suggesting that Abeta does not trigger microglial activation.The results have far-reaching implications in terms of reevaluating current treatment approaches towards AD.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL 32610-0244, USA. streit@mbi.ufl.edu

ABSTRACT
The role of microglial cells in the pathogenesis of Alzheimer's disease (AD) neurodegeneration is unknown. Although several works suggest that chronic neuroinflammation caused by activated microglia contributes to neurofibrillary degeneration, anti-inflammatory drugs do not prevent or reverse neuronal tau pathology. This raises the question if indeed microglial activation occurs in the human brain at sites of neurofibrillary degeneration. In view of the recent work demonstrating presence of dystrophic (senescent) microglia in aged human brain, the purpose of this study was to investigate microglial cells in situ and at high resolution in the immediate vicinity of tau-positive structures in order to determine conclusively whether degenerating neuronal structures are associated with activated or with dystrophic microglia. We used a newly optimized immunohistochemical method for visualizing microglial cells in human archival brain together with Braak staging of neurofibrillary pathology to ascertain the morphology of microglia in the vicinity of tau-positive structures. We now report histopathological findings from 19 humans covering the spectrum from none to severe AD pathology, including patients with Down's syndrome, showing that degenerating neuronal structures positive for tau (neuropil threads, neurofibrillary tangles, neuritic plaques) are invariably colocalized with severely dystrophic (fragmented) rather than with activated microglial cells. Using Braak staging of Alzheimer neuropathology we demonstrate that microglial dystrophy precedes the spread of tau pathology. Deposits of amyloid-beta protein (Abeta) devoid of tau-positive structures were found to be colocalized with non-activated, ramified microglia, suggesting that Abeta does not trigger microglial activation. Our findings also indicate that when microglial activation does occur in the absence of an identifiable acute central nervous system insult, it is likely to be the result of systemic infectious disease. The findings reported here strongly argue against the hypothesis that neuroinflammatory changes contribute to AD dementia. Instead, they offer an alternative hypothesis of AD pathogenesis that takes into consideration: (1) the notion that microglia are neuron-supporting cells and neuroprotective; (2) the fact that development of non-familial, sporadic AD is inextricably linked to aging. They support the idea that progressive, aging-related microglial degeneration and loss of microglial neuroprotection rather than induction of microglial activation contributes to the onset of sporadic Alzheimer's disease. The results have far-reaching implications in terms of reevaluating current treatment approaches towards AD.

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Microglial degeneration can occur independent of age and is evident in cases of young subjects with minimal tau pathology. Double-label immunohistochemistry for microglia (iba1) and tau (AT8) is shown in the hippocampus of two young subjects with no (b) or minimal (d) tau pathology (case nos. 1, 5). a, c Focus series of four individual micrographs taken 10–15 μm apart, and reassembled into composite images in b and d. Note the difference in microglial morphology in the two young subjects, one of which shows minimal tau pathology evident as neuropil threads (arrows in d). Microglia in b show normal ramified appearance but are fragmented in d. Scale bars 20 μm (a, c); 10 μm (b, d)
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Fig3: Microglial degeneration can occur independent of age and is evident in cases of young subjects with minimal tau pathology. Double-label immunohistochemistry for microglia (iba1) and tau (AT8) is shown in the hippocampus of two young subjects with no (b) or minimal (d) tau pathology (case nos. 1, 5). a, c Focus series of four individual micrographs taken 10–15 μm apart, and reassembled into composite images in b and d. Note the difference in microglial morphology in the two young subjects, one of which shows minimal tau pathology evident as neuropil threads (arrows in d). Microglia in b show normal ramified appearance but are fragmented in d. Scale bars 20 μm (a, c); 10 μm (b, d)

Mentions: As a first step in our investigation it was essential to have a reliable histological method for demonstrating all microglial cells in archival human brain specimens stored for extended periods of time in formalin. To date no such method has been described, and published procedures demonstrate only partial visualization of microglial cells, due to loss or masking of antibody binding sites as a result of prolonged fixation [27]. In initial screening studies using 100-μm-thick sections of formalin-fixed human brains, we found that an antibody against the iba1 [19] provided excellent visualization of ramified microglia in normal, non-diseased control subjects (Fig. 1). Unlike antibodies against ferritin or HLA-DR antigens previously used for staining microglia in human brain, anti-iba 1 worked consistently well and labeled the entire microglial population, as judged by the great density of cells revealed throughout coronal sections of the temporal lobe (Fig. 1a, b). No special pretreatment of sections for antigen unmasking was required. Anti-iba1 staining also facilitated differentiating between resting and activated microglia using cell hypertrophy as the distinguishing feature (Fig. 1c, d). Negative control sections with the primary antibody omitted showed no staining at all. With this method, we set out to first study microglial morphology in cases of Alzheimer’s disease showing the classic pathology of senile plaques and neurofibrillary tangles (Braak stages V and VI, case nos. 10–13; Table 1). By combining anti-iba1 staining with anti-tau immunolabeling (AT8 antibody) we were able to generate clear representations of microglial cells and degenerating neuronal structures (Fig. 2). The extent of neurodegeneration was visible even to the naked eye on AT8-stained coronal sections encompassing the temporal lobe up to the middle temporal gyrus at the level of the uncus. Microscopically, anti-tau immunolabeling, as well as silver staining using the Gallyas method, prominently highlighted neurofibrillary tangles, neuritic plaques, and neuropil threads, which were readily distinguished from microglial cells in these double-stained preparations (Fig. 2a–d). Negative controls with mismatched secondary antibodies showed no staining at all. The visualization of microglia with anti-iba1 failed to show any evidence of microglial activation, such as hypertrophy of the cells’ processes or increased cell density due to proliferation. However, clusters of microglia which have been described consistently in the literature were abundant and always associated with neuritic plaques. On high magnification, the structure of microglial cells was strikingly abnormal in that the cytoplasm of nearly all cells was fragmented into many small pieces (Fig. 2). The use of 100-μm-thick sections allowed for 3-D analysis of microglial cell structure by taking photomicrographs in multiple focal planes (Fig. 2e) and then digitally reassembling the z-stack of four or more images into a composite final micrograph (Fig. 2f). This type of 3-D reconstruction clearly established that microglial fragmentation (cytorrhexis) was not due to selective focusing but instead represented the actual morphology of both clustered and surrounding cells. We therefore concluded that neurodegenerative changes of AD were associated not with microglial activation but with microglial fragmentation, suggesting ongoing degeneration. To further substantiate this idea we sought to corroborate an association of microglial fragmentation and tau pathology in other cases at extreme ends of the spectrum of neurofibrillary pathology, namely, in young individuals with minimal tau pathology and in subjects with Down’s syndrome (case nos. 5, 14, 15; Table 1) using the same double-staining and 3-D reconstruction procedures as for the AD cases. We were able to determine that an area of minimal tau pathology in the hippocampus of a young subject (case no. 5) was accompanied by selective microglial cytorrhexis, whereas a non-pathological young control subject (case no. 1) revealed intact ramified microglial cells in the same brain region (Fig. 3). On the opposite end of the spectrum were two cases of Down’s syndrome both of which demonstrated widespread neurofibrillary and amyloid pathology virtually identical to the most advanced forms of AD. As shown in Fig. 4, these subjects revealed complete destruction and total loss of microglial cell integrity to the point where hardly a single intact microglial cell could be identified anywhere in the section. Iba1-stained sections of both Down’s subjects were littered with microglial debris consisting of numerous cell fragments, spheroids, and severely atrophied and disfigured microglia. These findings clearly confirmed our notion that microglial degeneration and neurodegeneration progress in synchrony.Fig. 1


Dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in Alzheimer's disease.

Streit WJ, Braak H, Xue QS, Bechmann I - Acta Neuropathol. (2009)

Microglial degeneration can occur independent of age and is evident in cases of young subjects with minimal tau pathology. Double-label immunohistochemistry for microglia (iba1) and tau (AT8) is shown in the hippocampus of two young subjects with no (b) or minimal (d) tau pathology (case nos. 1, 5). a, c Focus series of four individual micrographs taken 10–15 μm apart, and reassembled into composite images in b and d. Note the difference in microglial morphology in the two young subjects, one of which shows minimal tau pathology evident as neuropil threads (arrows in d). Microglia in b show normal ramified appearance but are fragmented in d. Scale bars 20 μm (a, c); 10 μm (b, d)
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Fig3: Microglial degeneration can occur independent of age and is evident in cases of young subjects with minimal tau pathology. Double-label immunohistochemistry for microglia (iba1) and tau (AT8) is shown in the hippocampus of two young subjects with no (b) or minimal (d) tau pathology (case nos. 1, 5). a, c Focus series of four individual micrographs taken 10–15 μm apart, and reassembled into composite images in b and d. Note the difference in microglial morphology in the two young subjects, one of which shows minimal tau pathology evident as neuropil threads (arrows in d). Microglia in b show normal ramified appearance but are fragmented in d. Scale bars 20 μm (a, c); 10 μm (b, d)
Mentions: As a first step in our investigation it was essential to have a reliable histological method for demonstrating all microglial cells in archival human brain specimens stored for extended periods of time in formalin. To date no such method has been described, and published procedures demonstrate only partial visualization of microglial cells, due to loss or masking of antibody binding sites as a result of prolonged fixation [27]. In initial screening studies using 100-μm-thick sections of formalin-fixed human brains, we found that an antibody against the iba1 [19] provided excellent visualization of ramified microglia in normal, non-diseased control subjects (Fig. 1). Unlike antibodies against ferritin or HLA-DR antigens previously used for staining microglia in human brain, anti-iba 1 worked consistently well and labeled the entire microglial population, as judged by the great density of cells revealed throughout coronal sections of the temporal lobe (Fig. 1a, b). No special pretreatment of sections for antigen unmasking was required. Anti-iba1 staining also facilitated differentiating between resting and activated microglia using cell hypertrophy as the distinguishing feature (Fig. 1c, d). Negative control sections with the primary antibody omitted showed no staining at all. With this method, we set out to first study microglial morphology in cases of Alzheimer’s disease showing the classic pathology of senile plaques and neurofibrillary tangles (Braak stages V and VI, case nos. 10–13; Table 1). By combining anti-iba1 staining with anti-tau immunolabeling (AT8 antibody) we were able to generate clear representations of microglial cells and degenerating neuronal structures (Fig. 2). The extent of neurodegeneration was visible even to the naked eye on AT8-stained coronal sections encompassing the temporal lobe up to the middle temporal gyrus at the level of the uncus. Microscopically, anti-tau immunolabeling, as well as silver staining using the Gallyas method, prominently highlighted neurofibrillary tangles, neuritic plaques, and neuropil threads, which were readily distinguished from microglial cells in these double-stained preparations (Fig. 2a–d). Negative controls with mismatched secondary antibodies showed no staining at all. The visualization of microglia with anti-iba1 failed to show any evidence of microglial activation, such as hypertrophy of the cells’ processes or increased cell density due to proliferation. However, clusters of microglia which have been described consistently in the literature were abundant and always associated with neuritic plaques. On high magnification, the structure of microglial cells was strikingly abnormal in that the cytoplasm of nearly all cells was fragmented into many small pieces (Fig. 2). The use of 100-μm-thick sections allowed for 3-D analysis of microglial cell structure by taking photomicrographs in multiple focal planes (Fig. 2e) and then digitally reassembling the z-stack of four or more images into a composite final micrograph (Fig. 2f). This type of 3-D reconstruction clearly established that microglial fragmentation (cytorrhexis) was not due to selective focusing but instead represented the actual morphology of both clustered and surrounding cells. We therefore concluded that neurodegenerative changes of AD were associated not with microglial activation but with microglial fragmentation, suggesting ongoing degeneration. To further substantiate this idea we sought to corroborate an association of microglial fragmentation and tau pathology in other cases at extreme ends of the spectrum of neurofibrillary pathology, namely, in young individuals with minimal tau pathology and in subjects with Down’s syndrome (case nos. 5, 14, 15; Table 1) using the same double-staining and 3-D reconstruction procedures as for the AD cases. We were able to determine that an area of minimal tau pathology in the hippocampus of a young subject (case no. 5) was accompanied by selective microglial cytorrhexis, whereas a non-pathological young control subject (case no. 1) revealed intact ramified microglial cells in the same brain region (Fig. 3). On the opposite end of the spectrum were two cases of Down’s syndrome both of which demonstrated widespread neurofibrillary and amyloid pathology virtually identical to the most advanced forms of AD. As shown in Fig. 4, these subjects revealed complete destruction and total loss of microglial cell integrity to the point where hardly a single intact microglial cell could be identified anywhere in the section. Iba1-stained sections of both Down’s subjects were littered with microglial debris consisting of numerous cell fragments, spheroids, and severely atrophied and disfigured microglia. These findings clearly confirmed our notion that microglial degeneration and neurodegeneration progress in synchrony.Fig. 1

Bottom Line: Although several works suggest that chronic neuroinflammation caused by activated microglia contributes to neurofibrillary degeneration, anti-inflammatory drugs do not prevent or reverse neuronal tau pathology.Deposits of amyloid-beta protein (Abeta) devoid of tau-positive structures were found to be colocalized with non-activated, ramified microglia, suggesting that Abeta does not trigger microglial activation.The results have far-reaching implications in terms of reevaluating current treatment approaches towards AD.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL 32610-0244, USA. streit@mbi.ufl.edu

ABSTRACT
The role of microglial cells in the pathogenesis of Alzheimer's disease (AD) neurodegeneration is unknown. Although several works suggest that chronic neuroinflammation caused by activated microglia contributes to neurofibrillary degeneration, anti-inflammatory drugs do not prevent or reverse neuronal tau pathology. This raises the question if indeed microglial activation occurs in the human brain at sites of neurofibrillary degeneration. In view of the recent work demonstrating presence of dystrophic (senescent) microglia in aged human brain, the purpose of this study was to investigate microglial cells in situ and at high resolution in the immediate vicinity of tau-positive structures in order to determine conclusively whether degenerating neuronal structures are associated with activated or with dystrophic microglia. We used a newly optimized immunohistochemical method for visualizing microglial cells in human archival brain together with Braak staging of neurofibrillary pathology to ascertain the morphology of microglia in the vicinity of tau-positive structures. We now report histopathological findings from 19 humans covering the spectrum from none to severe AD pathology, including patients with Down's syndrome, showing that degenerating neuronal structures positive for tau (neuropil threads, neurofibrillary tangles, neuritic plaques) are invariably colocalized with severely dystrophic (fragmented) rather than with activated microglial cells. Using Braak staging of Alzheimer neuropathology we demonstrate that microglial dystrophy precedes the spread of tau pathology. Deposits of amyloid-beta protein (Abeta) devoid of tau-positive structures were found to be colocalized with non-activated, ramified microglia, suggesting that Abeta does not trigger microglial activation. Our findings also indicate that when microglial activation does occur in the absence of an identifiable acute central nervous system insult, it is likely to be the result of systemic infectious disease. The findings reported here strongly argue against the hypothesis that neuroinflammatory changes contribute to AD dementia. Instead, they offer an alternative hypothesis of AD pathogenesis that takes into consideration: (1) the notion that microglia are neuron-supporting cells and neuroprotective; (2) the fact that development of non-familial, sporadic AD is inextricably linked to aging. They support the idea that progressive, aging-related microglial degeneration and loss of microglial neuroprotection rather than induction of microglial activation contributes to the onset of sporadic Alzheimer's disease. The results have far-reaching implications in terms of reevaluating current treatment approaches towards AD.

Show MeSH
Related in: MedlinePlus