The current findings show that the neurodegenerative changes that characterize AD are not accompanied by microglial activation but instead by microglial dystrophy, which likely reflects progressive degeneration of these cells [44
]. The significance of these findings is that they provide an explanation as to why anti-inflammatory drugs are ineffective at preventing or diminishing neurodegeneration and dementia. They serve to redirect thinking about AD pathogenesis away from inflammation-induced damage and towards an unexplored area of neuroscience, namely, processes or events that can damage microglial cells. One such process particularly relevant in the context of AD is chronological aging, and it has become clear in the recent years that microglia are subjected to aging-related changes, including telomere shortening [9
]. Truncated telomeres in peripheral blood leukocytes also have been identified as a possible marker of increased dementia risk [14
]. Since microglia are essential for providing neuroprotection [45
], aging-related weakening of microglial neuroprotective function is likely to have detrimental consequences for neurons. We propose here that one such negative consequence is development of neurofibrillary pathology by showing evidence that microglial degeneration likely precedes the onset of tau pathology.
How does one distinguish between resting, activated, and dystrophic microglia? The most reliable way of determination is by a qualitative assessment of cell morphology, which requires robust histochemical demonstration of the cells in situ.
The findings shown here are the results of applying an optimal method for staining microglia in human archival material using the iba1 antibody generated by Ito et al. [19
]. This method allows the demonstration of all morphological subtypes of microglia, including resting, activated and dystrophic cells which can be distinguished readily based on morphological grounds alone (Fig. ). Thus, unlike prior studies we have not relied on purportedly specific immunological markers for activated microglia, such as major histocompatibility complex (MHC) antigens, which have long been used to demonstrate ostensible microglial activation (and thus neuroinflammation) in AD and other neurodegenerative diseases [1
]. Although an upregulation of MHC antigens occurs on microglia activated after acute injuries, as shown in numerous animal studies, in human brain these antigens (HLA-DR) are widely expressed by non-activated, ramified (resting) microglia, as well as by dystrophic ones [10
], and thus anti-MHC staining is a questionable method for the identification of activated microglia [6
]. Qualitative morphological assessment also requires viewing microglia at the highest magnification possible for light microscopy and preferably in sections that measure at least 20 μm in thickness, which facilitates viewing in multiples focal planes. Often human brain tissue is examined at low magnification in thin sections of paraffin-embedded tissue, and while this type of analysis may be sufficient for making a diagnosis it does not always afford the level of detail necessary to distinguish between activated and dystrophic, or between resting and dystrophic microglia. The recent analysis by Sasaki et al. [40
] claiming a close association between activated microglia and tau pathology underscores this point.
The principal feature of microglial dystrophy described in this report is cytoplasmic fragmentation, or cytorrhexis, a process that has not yet been studied in great depth. Cytorrhexis appears to involve the pinching off or budding of cytoplasmic fragments and this phenomenon bears some resemblance to the cytoplasmic changes that occur during apoptosis [20
]. However, other prominent features of apoptosis, notably nuclear fragmentation (karyorrhexis), are not apparent during microglial cytorrhexis as the cells show intact nuclei and nucleoli associated with fragmented processes (e.g. Figs. , ). Our previous work describing microglial cytorrhexis in SOD1 transgenic rats also failed to produce evidence in favor of microglial apoptosis [8
], and thus we are reluctant to categorize microglial cytorrhexis as either apoptosis or as necrosis at this time. Additional studies are needed to further characterize this seemingly distinct mode of microglial degeneration involving primarily cytoplasmic deterioration. Microglial cytorrhexis occurs in the SOD1G93A
rat, a model of amyotrophic lateral sclerosis [8
]. Notably, in these animals which die at about 5–7 months of age due to motoneuron loss, the terminal stages of their neurodegenerative condition are marked by prominent microglial cytorrhexis in the spinal cord gray matter where motoneurons are degenerating [8
]. Although the mode of motoneuron cell death in SOD1G93A
rats remains enigmatic and does not appear to involve neurofibrillary pathology, this model provides an interesting parallel to the current findings in that here too microglial degeneration can be associated with neurodegeneration, thereby supporting our hypothesis that neurodegeneration may be secondary to microglial damage.
Although numerous reports have claimed a role for amyloid peptides in activating microglia and possibly stimulating phagocytosis by microglia (for reviews see [3
]), the current findings fail to corroborate the occurrence of either microglial activation or phagocytosis in brains marked by massive Aβ loads. The fact that we did not observe activated microglia in a spectrum of cases ranging from none to severe AD pathology suggests that neither soluble nor insoluble amyloid-beta proteins elicit microglial activation. Since levels of soluble Aβ have been correlated with cognitive impairments [25
], our observations do not support the suspected causality between soluble Aβ levels and impaired cognition from a neuroinflammation point of view. However, it is possible that soluble Aβ contributes to microglial degeneration, and this has been shown to occur in vitro under certain conditions [21
]. As for neuritic plaques, our findings clearly show that these are accompanied by dystrophic microglia (Fig. c), which needs an explanation for the many prior observations reporting activated microglia associated with these lesions [30
]. One possibility may be that in prior studies dystrophic microglia were misidentified as activated microglia. This could have been due to that antibodies used in earlier work produced incomplete visualization of microglial cells and also because microglial dystrophy had not yet been recognized. Another possibility may be related to the dynamic structure of Aβ plaques, that is, as these deposits evolve and undergo biochemical changes there may be a point where microglia do become activated and then progress to become dystrophic concurrently with the onset of tau pathology. Yet a third possibility is that the presence of systemic infectious disease may have influenced prior assessments of microglial activation and neuroinflammation in AD brain. Elderly, demented patients often have systemic comorbidities, such as pneumonia and other infections, and it has been shown that infectious disease outside the CNS can profoundly influence microglial activation [23
]. Our findings showing pervasive microglial activation in one subject who died with sepsis (case no. 6) seem to confirm this thought, and they underscore the need for more discerning studies of neuroinflammation that take into consideration the absence or presence of peripheral infections.
In summary, the current findings offer an alternative explanation for the involvement of microglia in AD pathogenesis, namely, that a loss of microglial cells contributes to the onset of neurodegeneration. This possibility is appealing because it takes into account old age as a primary factor in the pathogenesis of sporadic AD. According to the microglial dysfunction hypothesis, both microglia and neurons are subject to an aging-related decline in functions and these are exacerbated by genetic and epigenetic factors, including oxidative damage, which may be particularly detrimental to microglia [24
]. It remains unclear at this time how exactly damage in microglia is linked to tau pathology in neurons, but this may become a fertile area of investigation in future research. Notwithstanding this as of yet unexplored issue, it is worth noting that in the recent years studies have begun to emerge which provide evidence for microglial abnormalities and degeneration in other neurodegenerative diseases, including amyotrophic lateral sclerosis, Creutzfeldt–Jakob disease, and Huntington’s disease [8
], as well as in schizophrenia [48
]. Thus, breakdown of the brain’s immune system may be an important factor in the development of neurodegeneration.