**5. Does expression of antigen correlate with identifiable function**

immunoreactivity to brain microglia even though protein and mRNA expression of CD200R

**Figure 2.** Approaches to microglial phenotyping in Alzheimer's disease brains. (A) and (B) Immunohistochemistry for a new marker for microglia (toll-like receptor-3: TLR-3 in human brains. Double immunostaining for TLR-3 (purple) colocalizing with IBA-1 microglia in (A) non-demented control middle temporal gyrus and (B) Alzheimer's disease case. See text for further explanation. These findings were obtained using R&D Systems antibody (AF1868). (C) Western blot of human brain samples for TLR-3. This panel illustrates that protein bands other than full length peptides can be present in biological samples. (D) and (E) Absence of alternatively activated microglia expressing CD206 in ND (D) or AD (E) temporal cortex brain sections but positive expression in perivascular/vascular macrophages. (F) and (G) The proinflammatory marker CD14 does show increased expression by microglia in AD cases (purple) colocalizing with IBA-1 immunoreactivity brain Strong positive staining is present in perivascular/vascular macrophages (purple) is also

One marker that seems to have been overlooked in microglial profiling in tissue is CD14, the LPS co- receptor. This receptor is a classical M1-like activation marker with upregulation

are detectable in human brains [58].

48 Alzheimer's Disease - The 21st Century Challenge

a feature.

How does antigen expression relate to demonstrated microglial function? With the exception of HLA-DR and IBA-1, most studies of microglia in human brains have not been adequately replicated. Immunohistochemistry is not a quantitative technique due to the amplification technologies used along with the non-availability of standards for comparison, but the studies by Boche and colleagues using semi-quantitative measures deserve mention [10, 14, 66, 67]. Using a large series of brain samples and the expression of different markers, including CD64, MSR-A, CD68, HLA-DR and IBA-1, it was shown that microglia could be subtyped depending on their profile. These studies set the standards for microglial profiling in human brains. These studies employed two measures for analysis; the area of immunoreactivity and the numbers of positive cells. These studies attempted to assign phagocytic function or activation function to the microglia in relation to the type of pathology. One interesting observation was the lack of significant correlation between expression levels of these different antigens by microglia. These markers are related to different functions, with CD64, MSR-1, and C68 being related to phagocytosis, HLA-DR with antigen presentation and IBA-1 with microglial motility. Studies of TREM-2 and CD33 in AD brains demonstrated upregulated expression in AD brains, but both receptors induce inhibitory signaling when activated [55, 56]. There is evidence that upregulation of such activated microglial proteins encountered in disease tissue could be to have an inhibitory effect on inflammation, not amplifying inflammatory responses.

to the human disease, not to models of disease in a transgenic animal or cell culture. AD is a uniquely human disease of the elderly, with pathology having developed for years before dementia becomes observable. In transgenic models, disease pathology can develop over weeks. There have been many agents that can reverse plaque development and inflammation in AD mice models that have failed to be effective in humans. There are many challenges involved in human focused studies, but the benefits of having human disease targets validated in human tissue could involve significant saving in time and resources from pursuing the wrong approaches. Immunohistochemistry is not considered a state of art technique in the twenty-first century as the technologies have not much changed in 30 years, but ultimately it is required to show that gene discovery findings are valid. The need for large numbers of high quality human tissue samples has been one limitation, but this can be addressed by collaborative studies. Brain tissue that has been consistently prepared with appropriate clinical and pathological records allow studies involving progressive changes in pathology from negligible to severe rather than the less useful classification of control or AD. There is also a need for improved expectations on the performance of antibodies. The performance and reproducibility of antibodies in all biological experiments has been a growing concern [70], but ultimately it is the responsibility of the experimenter/pathologist to determine the suitability of antibodies used to make a unique observation. The field of neuroinflammation in AD has been reinvigorated with discoveries about TREM-2 having a direct link to AD risk. To be able to reliably identify TREM-2 positive microglia in brain is needed to fully understand its role in sporadic AD, and validate the large numbers of model studies that have proposed

Defining Microglial Phenotypes in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.75511 51

Microglia represent approximately 10% of the total cell population in human brain, but it is now appreciated how complex their responses are to pathological stimuli and for maintaining healthy neurons. Treating pathological inflammation in AD with broad spectrum agents (e.g., cyclooxygenase inhibitors) may do more harm than good. If the microglial responses to pathology are highly dependent on the microenvironment; for example one microglia may be producing excess TNF-α while an adjacent one is attempting to remove the pathological stimuli, then treatments need to be targeted appropriately. This will only be possible if the

The work on TLR-3 was supported by a grant to author from National Institutes of Health, National Institutes on Aging (1R21AG044068). The human brain sections were provided to author from the Banner Sun Health Research Institute Brain and Body Donation Program. The operation of the Banner Sun Health Research Institute Brain and Body Donation Program has been supported by the National Institute of Neurological Disorders and Stroke (U24

therapeutic strategies for AD focused on TREM-2.

microglia actively involved in AD can be adequately profiled.

**7. Conclusions**

**Acknowledgements**

#### **5.1. Where are the non-activated microglia in AD or aging brains?**

In recent years, gene discovery methodology (RNA sequencing, microarrays, single cell sorting) has been applied to studies of microglia. One particularly interesting marker identified is the purinergic ADP/ATP receptor P2RY12. This was shown to be highly expressed by microglia (human and rodent) compared to macrophages. In addition, it appears to be a marker of non-activated microglia as expression becomes rapidly downregulated upon inflammatory activation with LPS [68]. P2RY12 has been defined as a M2 marker as it is unregulated in vitro in human microglia by treatment with IL-4 [69]. A common concept over the years about neuroinflammation and AD is that widespread proinflammatory activation is a significant and extensive feature. The expression that the brain is on fire has been used more than once in review articles of AD inflammation. If inflammation is widespread in pathological affected tissues, one would expect that P2RY12 expression would be very low or absent in AD brains. Our preliminary findings show that this is not the situation; there were many P2RY12 microglia in regions with severe AD pathology. Although western blot and mRNA studies showed no difference in expression of this gene between AD and control samples, however by immunohistochemistry, there was a very specific distribution of P2RY12 positive and negative microglia in brains in relation to pathological structures.
