RESEARCH ARTICLE

Presence of Severe Neuroinflammation Does Not Intensify Neurofibrillary Degeneration in Human Brain Wolfgang J. Streit,1 Qing-Shan Xue,1 Heiko Braak,2 and Kelly del Tredici2 This study investigated the allegedly causal relationship between microglial activation and neurofibrillary degeneration (NFD) typical of Alzheimer’s disease (AD) by determining if presence of extreme microglial activation coincides with intensified NFD. We performed comparative histopathological analyses of NFD and microglial reactivity in 18 primary subjects ranging from 4 to 51 years of age. Ten of these subjects (median age 34) died from infectious disease (HIV, sepsis) and CNS trauma, while eight subjects (median age 32.5) died from non-infectious conditions (controls). Second, we also examined two 52-year-old subjects with Down syndrome where one had comorbid sepsis and the other one did not. We found that all 10 subjects with infectious=traumatic diagnoses showed severe neuroinflammation, while the 8 control subjects completely lacked neuroinflammatory changes. However, all 18 primary subjects were found to show the same early-stage, pretangle neuropathology of Braak stage 1a and 1b, that is, they exhibited primarily subcortical NFD in the locus coeruleus and sporadic lesions in the transentorhinal cortex. Similarly, the two subjects with Down syndrome showed the same high levels of NFD (Braak stage VI) irrespective of the comorbid sepsis-related neuroinflammation present in one of these individuals. Collectively, our findings show that despite rampant microglial activation in all subjects with neuroinflammatory conditions the extent of NFD is at the same level as seen in non-inflamed controls. These findings demonstrate that microglial activation does not initiate or exacerbate NFD, and we conclude that CNS inflammation is unlikely to be causally involved in the development of NFD characteristic of AD dementia. GLIA 2014;62:96–105

Key words: microglial activation, sepsis, infection, Alzheimer, Down syndrome

Introduction

A

large body of literature has accumulated implicating a chronic neuroinflammatory response in the causation of Alzheimer’s disease (AD) neurodegeneration and dementia (Akiyama et al., 2000; Eikelenboom et al., 2006; Griffin et al., 1989; Hardy and Selkoe, 2002; McGeer and McGeer, 2001; Rogers et al., 2002). Microglia are the principal cells involved in any neuroinflammatory reaction and they typically become activated during reactive responses following CNS injury (Graeber and Streit, 2010; Saijo et al., 2013). Presence of activated microglia has been described as a prominent feature in AD and Down syndrome (DS) brain (Dickson et al., 1993; Griffin et al., 1989; Lott and Head, 2005; Mrak, 2012; Stoltzner et al., 2000), yet the hypothesis that activated microglia are involved in the causation of neurofi-

brillary degeneration has not been proven (Eikelenboom et al., 2006). Much of the work conducted thus far has revolved around the relationship between microglia and amyloid-beta (Ab) protein deposits as the amyloid cascade hypothesis continues to be a major driving force in the AD field (Floden et al., 2005; Rogers et al., 2002). Specifically, it is thought that Ab is inflammatogenic and triggers production of free radicals, cytokines, and other neurotoxic substances in activated microglia which then cause neuronal death, evident in the AD brain as neurofibrillary degeneration (NFD; tau pathology). The proposed causal relationship between microglial activation and development of tau pathology is largely conjectural and not supported by direct evidence such as, for example, in situ demonstration of neurotoxin-producing microglial

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22589 Published online November 4, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received May 24, 2013, Accepted for publication Oct 2, 2013. Address correspondence to Wolfgang J. Streit, Department of Neuroscience, P.O. Box 100244, University of Florida, Gainesville, Florida 32610-0244, USA. Email: [email protected] or [email protected] From the 1Department of Neuroscience, University of Florida College of Medicine and McKnight Brain Institute, Gainesville, Florida; 2Clinical Neuroanatomy, Department of Neurology, University of Ulm, Ulm, Germany.

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cells in close proximity to neurofibrillary tangles in human brain. In addition, there are a number of rather compelling caveats from both clinical and histopathological perspectives. Clinical trials with nonsteroidal anti-inflammatory drugs have failed to stop progression of tau pathology or slow the development of dementia (Arvanitakis et al., 2008; Heneka et al., 2011; Martin et al., 2008). Tau pathology occurs in non-AD tauopathies in the absence of Ab deposits, and it can be detected in subcortical sites in some rather young individuals without concurrent presence of Ab protein (Braak and Del Tredici, 2011b). In AD, the distribution of intraneuronal neurofibrillary lesions differs substantially from the distribution of Ab plaques (Braak and Braak, 1991a), and development of tau pathology usually precedes the formation of aggregated Ab (Braak and Del Tredici, 2004; Duyckaerts and Hauw, 1997). Reliable and consistent identification of activated, let alone neurotoxic microglia, in human brain is challenging and poses significant difficulties related to the fact that activated morphology or neurotoxic functions are often not demonstrated unequivocally in vivo (Griffin et al., 1989; Sasaki et al., 2008; Sheng et al., 1997). Ever since microglial activation was first reported in AD (McGeer et al., 1987), immunoreactivity for HLA-DR (MHC) antigens has been used to identify activated, and presumably neurotoxic microglia, yet MHC antigens are found also on non-activated resting microglial cells (Craggs and Webster, 1985; Hayes et al., 1987) and using solely MHC expression to identify activated microglia is unreliable (Croisier et al., 2005; Graeber, 2010). Lastly, the very existence of neuroinflammatory reactions in the AD brain is questionable since recent work has shown not only absence of activated microglia in both aged as well as AD and DS brain, but instead a prevalence of degenerating (dystrophic) microglia particularly in cortical areas prone to first developing tau pathology, such as the entorhinal cortex (Streit et al., 2009, 2004; Xue and Streit, 2011). In one of our prior studies (Streit et al., 2009), anecdotal evidence (n 5 1) suggested the possibility that when indeed activated microglia are present in the human brain this could be due to peripheral infections and therefore be unrelated to CNS endogenous neurodegeneration. In an effort to substantiate this idea, we now present a systematic study using a larger number of cases with rather severe neuroinflammatory disease further examining the allegedly causal relationship between neuroinflammation and tau pathology. Importantly, our prior work (Streit et al., 2009) had also shown that despite presence of substantial Ab deposits many subjects do not develop neuroinflammation or NFD. Thus, in this study all subjects, except those with DS, were characterized by a complete absence of amyloid-beta (Ab) protein deposits (Table 1) allowing us to examine directly the purported causal link between neuroinflammation and NFD. January 2014

Materials and Methods Selection of Cases Autopsy material was supplied by the Braak Collection (Goethe University Frankfurt), as described previously (Braak and Braak, 1991a; Braak and Del Tredici, 2004, 2011a, b; Braak et al., 2011; Streit et al., 2009). No patient data other than age, gender, and diagnosis were disclosed. Ten cases with a medical record suggestive of neuroinflammation (sepsis, HIV infection, toxoplasmosis, head injury; cases 1–10 in Table 1) were selected from a larger cohort of subjects that had been previously characterized in terms of presence and extent of AD-type neuropathology (Braak and Del Tredici, 2011a,b; Braak et al., 2011). Eight additional cases that were of similar age and where the medical record suggested absence of an infectious and=or inflammatory etiology were selected as controls from the same cohort (cases 11–18 in Table 1). Furthermore, two cases of Down syndrome were included because they were of the same age, gender, and Braak stage (stage VI), where the only significant known difference between them was presence of sepsis in one individual. All brains had been fixed and stored by immersion in 4% paraformaldehyde solution for variable lengths of time beginning at the time of removal from the skull.

Tissue Processing and Immunohistochemistry Tissue blocks of brainstem and temporal lobe were embedded in polyethylene glycol (PEG 1000, Merck), and sections were cut on a macrotome at 100 lm in the frontal plane. Sections were processed for immunohistochemical staining using primary antibodies Iba1 (rabbit polyclonal, 1:1,000, Wako) for detecting microglia, AT8 (mouse monoclonal, 1:2,000, Pierce Endogen) for detecting human PHF-tau, and anti-CD68 (mouse monoclonal, 1:200, Dako). Primary antibody binding sites were visualized using biotinylated secondary anti-mouse or anti-rabbit antibodies made in goat (Vector, 1:200) followed by strepavidin-peroxidase. Peroxidase substrates were diaminobenzidine (DAB-H2O2) and Vector SG substrate (Cat. No. SK-4700), to yield brown and blue-gray reaction products, respectively. For double immunohistochemical staining, the DAB substrate reaction was always performed first and sections were rinsed thoroughly in phosphate buffered saline, pH 7.2–7.4, before proceeding with the second immunostaining reaction. Negative controls consisted of omitting either the primary antibodies or incubating primary antibodies with mismatched secondary antibodies (e.g., primary mouse with biotinylated goat-anti-rabbit). Preparations were examined and photographed using an Olympus Vanox microscope.

Results The essential finding of this study is that the severity of tau pathology does not differ across our primary cohort of 18 subjects with and without neuroinflammatory pathologies (Table 1). All 18 subjects revealed a level of tau pathology that was staged 1b maximally, which is below Braak stage I and signifies presence of AT8-positive neurites and=or AT8positive nerve cells in the coeruleus=subcoeruleus complex and other non-thalamic nuclei projecting to the cerebral cortex, as well as AT8-positive neurites and isolated AT8-positive 97

TABLE 1: Patient Data and Neuropathology of Cases Studied

Case

Gender

Age

AT8

Iba1

CD68

Ab deposits

Diagnosis

1

M

32

0

111

111

0

HIV, CMV, toxoplasmosis

2

F

43

1b

11

111

0

Sepsis, toxoplasmosis

3

M

37

1a

111

111

0

HIV

4

M

47

1a

111

11

0

AIDS=Kaposi

5

M

26

1a

11

11

0

HIV

6

M

37

1a

11

111

0

HIV

7

M

8

0

11

11

0

Sepsis

8

M

6

1a

1

11

0

Sepsis

9

F

26

1a

11

111

0

TBI

10

M

36

1a

1

11

0

HIV

11

M

4

0

0

1

0

Primary CNS tumor

12

M

26

1b

0

1

0

Acute trauma (thorax, mandible)

13

M

26

1a

0

1

0

Peptic ulcer, coma since age 12

14

M

34

1b

0

1

0

Aortic valve disease, Cardiac arrest

15

F

31

1b

0

1

0

Fatal status asthmaticus

16

M

36

1a

0

1

0

Metastatic soft tissue sarcoma

17

M

44

1b

0

1

0

Malignant neoplasm (oral cavity, tongue)

18

M

51

1b

0

11

0

Malignant neoplasm (bronchial)

19

M

52

VI

111

111

4

Down syndrome, sepsis

20

M

52

VI

0

11

4

Down syndrome

Scoring of microglial responses (Iba1): 0, ramified and=or dystrophic cells only; 1, activated (hypertrophic) cells and clustering; 11, mononuclear cell infiltration and=or rounded brain macrophages; 111, necrotic septic foci and=or perivascular microgliosis. Scoring of CD68 staining intensity: 1, minimal staining; in isolated perivascular and=or microglial cells; 11, intermediate staining; in moderate numbers of microglial, perivascular, and=or infiltrating mononuclear cells; 111, intense staining; in numerous microglial, perivascular, and=or infiltrating mononuclear cells. Staging of Ab deposits: 0, absent; 4, maximal.

pyramidal cells in the transentorhinal cortex (Braak and Del Tredici, 2011a,b; Braak et al., 2011). Figure 1 shows examples of stage 1b cortical tau pathology observed in two control subjects exhibiting no neuroinflammatory changes. It is worth noting that in the current cohort stage 1b was more frequent in the 8 controls (cases 11–18) than in the 10 subjects with neuroinflammation (cases 1–10) where cortical pretangles were often undetectable (Table 1). Figure 1 shows that stage 1b tau pathology was accompanied by a lack of microglial activation and that existing microglia were either of the resting (ramified) or dystrophic (fragmented) type. Among the subjects with neuroinflammation several had been diagnosed with sepsis, and this was reflected by widespread presence of Iba1-positive, hypertrophic microglia in all CNS regions examined (Fig. 2). Many of these activated microglia 98

were also positive for the intracytoplasmic lysosomal macrophage antigen, CD68, contrasting with control cases which showed only weak CD68 staining in isolated microglial and perivascular cells (Fig. 3A). In some microscopic fields of Iba1-stained sections from neuroinflammation cases, hypertrophic microglia were intermingled with dystrophic microglia which were identified based on the fact that their cytoplasmic processes were fragmented and discontinuous. Ostensibly such dystrophic cells were distributed at random with no particular predilection for any brain region; they were seen in both cortical and brainstem regions. Occasionally, small clusters of microglia were observed and these also appeared to occur at random in both white and gray matter and were not correlated with presence of Ab deposits. The extent of tau pathology was generally more conspicuous in the Volume 62, No. 1

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FIGURE 1: Neurodegeneration occurs in the absence of microglial activation in non-inflamed control cases. Shown is the maximal level of tau pathology (stage 1b) observed in any of the 18 primary cases studied using double-label immunohistochemistry for hyperphosphorylated tau (AT8) and microglia (Iba1). (A) Case 18; AT8-positive pyramidal neuron with AT8-positive processes (arrow; black reaction product) is shown in the entorhinal cortex; microglial cells (brown reaction product) are ramified and nonactivated. (B) Case 12; pretangles (arrows) and ramified, nonactivated microglia are evident in the entorhinal cortex. Scale bar: 100 lm.

coeruleus=subcoeruleus complex than in cortical regions, and there it was manifest mostly as AT8-positive neurites with occasional pretangle neurons (Fig. 2A, B). Extreme inflammation was evident in one case of traumatic brain injury (TBI; case 9) as well as in several HIV cases. Here, immunoreactivity for CD68 was upregulated dramatically and many cells were aggregated, likely due to formation of clusters of phagocytic microglia (Fig. 3). Similarly, Iba1 immunoreactivity was intense and revealed an abundance of activated microglia as well as many small rounded cells that represented infiltrating blood-borne leukocytes (monocytes and lymphocytes). Subjects with HIV infection presented with fulminant neuroinflammation, often including multiple intense foci and January 2014

perivascular microgliosis (Fig. 4). These severe neuroimmune responses were likely caused by opportunistic infections some of which, for example, Toxoplasma gondii, had been documented in the patients’ records. In one case (case 6), there was widespread presence of fragmented, hypertrophic microglia in the hippocampus, which was unusual because cytoplasmic fragmentation thus far had only been observed in nonhypertrophic cells. The significance of this phenomenon remains to be determined. Overall, AT8 staining in HIV subjects was not very prominent and mostly limited to neurites in the locus coeruleus area; cortical pretangles were quite rare. Comparison of neuropathological features in the two DS subjects revealed widespread tau pathology consisting of neuropil threads, neurofibrillary tangles, and neuritic plaques in both individuals. The level of neurofibrillary degeneration was essentially the same in both subjects and they were assigned Braak stage VI. However, there were remarkable differences in terms of microglial phenotypes and neuroinflammatory changes (Fig. 5). Although in case 20 (non-inflamed) all microglia were either of the ramified or dystrophic, fragmented type and hypertrophic cells were absent, the latter were present in case 19 (inflamed) indicating ongoing microglial activation, consistent with all other sepsis cases. Similar to other sepsis cases, case 19 also showed multiple foci of necrosis which were conspicuously revealed by intense Iba1 and CD68 immunoreactivity. Although levels of CD68 staining were not dramatically different between the two DS subjects (Table 1), case 19 did show more CD68-positive cells than case 20. The relatively small difference in CD68 staining may be explained by the observation that microglial senescence, which was prevalent in case 20, is accompanied by an increase in CD68 antigen expression (Perry et al., 1993; Wong et al., 2005).

Discussion Our study has revealed that there is no correlation between presence of neuroinflammatory histopathology and the severity of neurofibrillary degeneration (tau pathology) in human brain. These findings are well aligned with the results of pathological and clinical studies using anti-inflammatory drugs, which also show that progression of tau pathology and dementia are unaffected by anti-inflammatory treatments (Arvanitakis et al., 2008; Halliday et al., 2000; Martin et al., 2008). Furthermore, our results are supported by studies in AD mouse models which demonstrate that despite large amyloid burdens due to APP overexpression and ensuing neuroinflammation neurofibrillary degeneration is absent (Irizarry et al., 1997; Schwab et al., 2004; Xu et al., 2002). The results of our study are important because neurofibrillary degeneration provides the best correlate to clinical symptoms of dementia (Arriagada et al., 1992; Braak and Braak, 1991b; 99

FIGURE 2: Neuroinflammation is apparent during sepsis (case 2); AT8=Iba1 double-labeling (A–D); anti-CD68 (E, F). (A and B) AT8positive neurites are present in the locus coeruleus (top left, arrows in A), and to a lesser extent in the surrounding brainstem (arrows in B). Both fields show presence of hypertrophic, activated microglia. In the entorhinal cortex activated microglia are prevalent either as single cells (C), or in occasional clusters (D). In C, a few dystrophic cells marked by cytoplasmic fragmentation (arrowheads) are intermingled with activated microglia. Many activated microglia are positive also for the CD68 antigen in both brainstem (E) and hippocampus (F). Scale bars: 200 lm (A, F); 100 lm (B–E).

Grober et al., 1999; Riley et al., 2002; Thal et al., 1998), and it therefore seems obvious that continued pursuit of antiinflammatory strategies for treatment or prevention of AD dementia is unlikely to be effective. The intensity of neuroinflammation described in this study far exceeds the level of neuroinflammation described in AD where at most a mild glial reaction occurs, evident primarily as microglial clustering around Ab deposits (GrundkeIqbal et al., 1990; Sasaki et al., 1997). Infiltration of bloodborne leukocytes, which is a hallmark feature of most inflammatory reactions, is virtually non-existent in AD. In the cases presented here, the level of neuroinflammation shown was quite severe and possibly near its maximal theoretical limit 100

since in some of our sepsis cases it produced discernible tissue damage that was evident in what appeared to be necrotic foci. Such focal tissue damage has been reproduced experimentally in animal studies where potently inflammatogenic microbial cell wall components, such as zymosan or LPS, were injected directly into the CNS (Popovich et al., 2002). This type of extreme immune stimulation with microbial products, which is commonly used to simulate microglial activation in vitro (Colton and Gilbert, 1987), generates superactivated microglia capable of producing a variety of bactericidal substances that are potentially neurotoxic and can apparently result in axonal and neuronal necrosis. However, as shown here in our sepsis cases, even such extreme microglial activation does not Volume 62, No. 1

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FIGURE 3: Massive neuroinflammation occurs after traumatic brain injury. (A and B) control (case 17) shows minimal expression of CD68 in both brainstem (A) and hippocampus (B). (C and D) The CD68 antigen is dramatically and widely upregulated in the hippocampus after head injury (case 9). Many CD68-positive cells are clustered. (E and F) There is widespread microglial activation as well as mononuclear cells infiltration in the hippocampus after head injury (Iba1 staining). At high magnification, infiltrating leukocytes appear as small rounded cells (F). Scale bars: 100 lm (A, B, D, F); 500 lm (C, E). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

initiate or exacerbate neurofibrillary degeneration, making it very unlikely that the mild gliosis associated with AD and aging would do so. If indeed neuroinflammation was causally involved in triggering neurofibrillary degeneration, one would have expected to see at least some exacerbation of tau pathology in our “inflamed” cases compared with controls, which was not the case. In fact, the level of tau pathology was slightly higher in control, non-inflamed cases (Table 1). Thus, in contrast to previously published papers which based on cell culture studies have claimed a role for neurotoxinproducing activated microglia in AD neurodegeneration [reviewed in (Streit, 2010)], our current findings in human brain show that microglial activation is highly unlikely to play a role in the slowly progressing neurofibrillary degeneraJanuary 2014

tion typical of AD. It is now known that initial stages of tau pathology occur quite early in the lives of some individuals and that this pathology likely progresses to more advanced neurodegeneration over a lifetime (Braak and Del Tredici, 2011b; Braak et al., 2011). If neuroinflammation was the cause or driving force behind neurofibrillary degeneration, it would mean that incessant damaging brain inflammation would have to persist over decades to promote formation of more advanced lesions. From a clinical perspective this is an extremely remote possibility. Humans cannot endure decades of damaging brain inflammation, which even when it occurs only briefly can be fatal as evidenced by the outcome of Ab vaccination trials (Munch and Robinson, 2002). In contrast, a recent study reports that some changes related to 101

FIGURE 4: HIV infection causes fulminant neuroinflammation as seen with AT8=Iba1 double immunolabeling. (A–C) Case 4; widespread microglial activation is present throughout the parahippocampal gyrus (A, B) with a hyperintense focus (arrow in A). (B) High magnification shows extremely dense accumulation of activated microglia and possibly ongoing tissue necrosis. (C) Additional foci of hyperintense microglial activation are apparent in the subiculum. No AT8-positive structures are visible. Scale bars: 500 lm (A); 200 lm (C); 100 lm (B).

102

neuroinflammation induced by acute head trauma, i.e., increased numbers of HLA-DR and CD68-positive microglia=macrophages, can persist in the corpus callosum for years after a single traumatic event (Johnson et al., 2013). However, it is difficult to reconcile such prolonged lingering of macrophages in the corpus callosum with the onset of NFD in the temporal lobe, or with the effects of aging which include naturally increased expression of these antigens on senescent microglial cells (Perry et al., 1993; Wong et al., 2005). At this point, a brief comment regarding nomenclature seems appropriate. It is unfortunate that use of the term “neuroinflammation” has evolved inappropriately to describe diseases which are not truly inflammatory in nature and which most pathologists would classify as degenerative. Yet the idea that neuroinflammation is a factor in neurodegenerative disease pathogenesis has been gaining popularity and the notion is being propagated even further (and rather indiscriminately) to an ever expanding list of CNS pathologies. We are at a point where just about any time anybody sees, or thinks they see activated microglial cells (e.g., MHC-positive ones) “neuroinflammation” is promptly implicated. The problem with this is that the term neuroinflammation carries a negative connotation and implies a chronically detrimental process, which is of course misleading. Microglial activation is beneficial to the brain because it constitutes a cellular response to restore homeostasis after injury has occurred. Thus, if one uncouples “neuroinflammation” from its implicitly negative connotation and uses it objectively to describe a glial response to CNS injury there is no problem with using the term. In AD, the timing of onset of allegedly detrimental microglial activation is unknown and its chronic presence merely assumed. Its detrimental character is based on in vitro studies using extreme immune stimulation and its causative role in the development of tau pathology is speculative and unsubstantiated by observations in human brain or in AD animal models. Histopathologically, the features that have been used to support a role for harmful inflammation in AD neurodegeneration are, (a) presence of HLA-DR-positive microglia (McGeer et al., 1987), (b) presence of microglial clusters around Ab deposits (Sasaki et al., 1997), and (c) presence of interleukin-1 (IL-1) immunoreactivity (Griffin et al., 1989; Sheng et al., 1997). There are problems with interpreting all of these features as evidence for detrimental neuroinflammation. HLA-DR-positivity is present in normal brain and not a specific marker for neurotoxic or even activated microglia. Widespread expression of HLA-DR antigens on microglia in normal white matter (Hayes et al., 1987) does not mean that normal white matter is chronically inflamed and is being damaged. Microglial clustering around Ab Volume 62, No. 1

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FIGURE 5: Comparison of microglial staining in the temporal cortex of DS subjects without (A–C; case 20) and with (D–F; case 19) comorbid sepsis. AT8=Iba1 double immunolabeling (A, B, D, E) and CD68 staining (C, F). (A and D) Both DS subjects show presence of neurofibrillary tangles (arrows). In case 20 (A) tangles are surrounded by dystrophic, fragmented microglia while in case 19 (D) tangles are surrounded by hypertrophic, activated microglia. (B) Numerous neuritic plaques (arrowheads) are surrounded by ramified, occasionally fragmented (arrow) microglia. (E) A necrotic focus showing intense Iba1 immunoreactivity is present amidst abundant tau-positive neurons and neurites throughout the field. Several rounded brain macrophages are apparent (arrowheads). (C and F) CD68 staining of an occasional neuritic plaques (arrow in C) in case 20 is contrasted with widespread CD68 immunoreactivity in activated microglia in case 19. Scale bar: 100 lm.

deposits could mean that cells are attracted there to remove amyloid via phagocytosis; it could also mean that cells become aggregated there due to a loss of contact inhibition that normally keeps them apart and evenly spaced. It does not mean that the cells are neurotoxic and cause neurofibrillary degeneration. Similarly, presence of IL-1 immunoreactivity also does not constitute evidence for neurotoxicity; quite to the contrary, IL-1 is typically upregulated during regenerative responses in the CNS (Fagan and Gage, 1990). Thus, in contrast to longstanding speculation regarding a potentially pivotal role of activated, neurotoxic microglia in the causation January 2014

of AD neurodegeneration, the current findings offer conclusive evidence in human subjects to justify eliminating inflammation from the pathogenetic series of events that may lead to AD neurodegeneration. As an alternative to the neuroinflammation theory, we propose that NFD occurs because of deteriorating microglial cell functions and progressive microglial degeneration (microglial dystrophy), a topic discussed previously as the microglial dysfunction hypothesis (Conde and Streit, 2006; Streit, 2002, 2004, 2006). By studying tissues from both AD and DS subjects, we have found a rather consistent association between 103

presence of tau pathology and presence of dystrophic rather than activated microglia (Streit et al., 2009; Xue and Streit, 2011), findings that strongly support the microglial dysfunction theory. Moreover, prior and current observations from our laboratory (Streit et al., 2009), as well as from others (Lemstra et al., 2007; Mattiace et al., 1990), have shown that microglial activation occurs in the CNS of subjects with sepsis. Peripheral infections are common in the elderly, and it has been found that the most common cause of death in patients with dementia disorders is bronchopneumonia (Brunnstrom and Englund, 2009). Thus, one can readily envision how in the past presence of peripheral infectious disease might have confounded histopathological analyses of neuroinflammation in the context of neurodegenerative diseases. Failure to differentiate between AD subjects with and without concurrent infections in previous neuropathological studies focused on examining microglial activation has likely contributed to the idea that inflammation is involved in AD pathogenesis. Our current data on DS subjects corroborate this point by showing that advanced, Braak stage VI neurofibrillary pathology occurs both in the presence and absence of neuroinflammation. Very recently, additional genetic evidence has emerged to support microglial dysfunction in the context of AD pathogenesis, namely, that variants of the triggering receptor expressed on myeloid cells 2 (TREM 2) constitute a risk factor for AD (Neumann and Daly, 2013).

Acknowledgment

Braak H, Del Tredici K. 2011a. Alzheimer’s pathogenesis: Is there neuron-toneuron propagation? Acta Neuropathol 121:589–595. Braak H, Del Tredici K. 2011b. The pathological process underlying Alzheimer’s disease in individuals under thirty. Acta Neuropathol 121:171–181. Braak H, Thal DR, Ghebremedhin E, Del Tredici K. 2011. Stages of the pathologic process in Alzheimer disease: Age categories from 1 to 100 years. J Neuropathol Exp Neurol 70:960–969. Brunnstrom HR, Englund EM. 2009. Cause of death in patients with dementia disorders. Eur J Neurol 16:488–492. Colton CA, Gilbert DL. 1987. Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett 223:284–288. Conde JR, Streit WJ. 2006. Microglia in the aging brain. J Neuropathol Exp Neurol 65:199–203. Craggs RI, Webster HD. 1985. IA antigens in the normal rat nervous system and in lesions of experimental allergic encephalomyelitis. Acta Neuropathol 68:263–272. Croisier E, Moran LB, Dexter DT, Pearce RK, Graeber MB. 2005. Microglial inflammation in the parkinsonian substantia nigra: Relationship to alphasynuclein deposition. J Neuroinflammation 2:14. Dickson DW, Lee SC, Mattiace LA, Yen SH, Brosnan C. 1993. Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer’s disease. Glia 7:75–83. Duyckaerts C, Hauw JJ. 1997. Prevalence, incidence and duration of Braak’s stages in the general population: Can we know? Neurobiol Aging 18:362– 369; discussion 389–392. Eikelenboom P, Veerhuis R, Scheper W, Rozemuller AJ, van Gool WA, Hoozemans JJ. 2006. The significance of neuroinflammation in understanding Alzheimer’s disease. J Neural Transm 113:1685–1695. Fagan AM, Gage FH. 1990. Cholinergic sprouting in the hippocampus: A proposed role for IL-1. Exp Neurol 110:105–120. Floden AM, Li S, Combs CK. 2005. Beta-amyloid-stimulated microglia induce neuron death via synergistic stimulation of tumor necrosis factor alpha and NMDA receptors. J Neurosci 25:2566–2575. Graeber MB. 2010. Changing face of microglia. Science 330:783–788.

The authors wish to thank the Cooper family of Indialantic, FL for their support of dementia research. The authors also thank Simone Feldengut for expert technical assistance.

References Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom P, Emmerling M, Fiebich BL, Finch CE, Frautschy S, Griffin WS, Hampel H, Hull M, Landreth G, Lue L, Mrak R, Mackenzie IR, McGeer PL, O’Banion MK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, Rydel R, Shen Y, Streit W, Strohmeyer R, Tooyoma I, Van Muiswinkel FL, Veerhuis R, Walker D, Webster S, Wegrzyniak B, Wenk G, Wyss-Coray T. 2000. Inflammation and Alzheimer’s disease. Neurobiol Aging 21:383–421. Arriagada PV, Growdon JH, Hedley-Whyte ET, Hyman BT. 1992. Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer’s disease. Neurology 42:631–639. Arvanitakis Z, Grodstein F, Bienias JL, Schneider JA, Wilson RS, Kelly JF, Evans DA, Bennett DA. 2008. Relation of NSAIDs to incident AD, change in cognitive function, and AD pathology. Neurology 70:2219–2225. Braak H, Braak E. 1991a. Demonstration of amyloid deposits and neurofibrillary changes in whole brain sections. Brain Pathol 1:213–216.

Graeber MB, Streit WJ. 2010. Microglia: Biology and pathology. Acta Neuropathol 119:89–105. Griffin WS, Stanley LC, Ling C, White L, MacLeod V, Perrot LJ, White CL III, Araoz C. 1989. Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc Natl Acad Sci USA 86:7611– 7615. Grober E, Dickson D, Sliwinski MJ, Buschke H, Katz M, Crystal H, Lipton RB. 1999. Memory and mental status correlates of modified Braak staging. Neurobiol Aging 20:573–579. Grundke-Iqbal I, Fleming J, Tung YC, Lassmann H, Iqbal K, Joshi JG. 1990. Ferritin is a component of the neuritic (senile) plaque in Alzheimer dementia. Acta Neuropathol 81:105–110. Halliday GM, Shepherd CE, McCann H, Reid WG, Grayson DA, Broe GA, Kril JJ. 2000. Effect of anti-inflammatory medications on neuropathological findings in Alzheimer disease. Arch Neurol 57:831–836. Hardy J, Selkoe DJ. 2002. The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics. Science 297:353–356. Hayes GM, Woodroofe MN, Cuzner ML. 1987. Microglia are the major cell type expressing MHC class II in human white matter. J Neurol Sci 80:25–37.

Braak H, Braak E. 1991b. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82:239–259.

Heneka MT, Kummer MP, Weggen S, Bulic B, Multhaup G, Munter L, Hull M, Pflanzner T, Pietrzik CU. 2011. Molecular mechanisms and therapeutic application of NSAIDs and derived compounds in Alzheimer’s disease. Curr Alzheimer Res 8:115–131.

Braak H, Del Tredici K. 2004. Alzheimer’s disease: Intraneuronal alterations precede insoluble amyloid-beta formation. Neurobiol Aging 25:713–718; discussion 743–746.

Irizarry MC, McNamara M, Fedorchak K, Hsiao K, Hyman BT. 1997. APPSw transgenic mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in CA1. J Neuropathol Exp Neurol 56:965–973.

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Volume 62, No. 1

Streit et al.: Neuroinflammation and Tau Pathology Johnson VE, Stewart JE, Begbie FD, Trojanowski JQ, Smith DH, Stewart W. 2013. Inflammation and white matter degeneration persist for years after a single traumatic brain injury. Brain 136:28–42.

Sasaki A, Yamaguchi H, Ogawa A, Sugihara S, Nakazato Y. 1997. Microglial activation in early stages of amyloid beta protein deposition. Acta Neuropathol 94:316–322.

Lemstra AW, Groen in’t Woud JC, Hoozemans JJ, van Haastert ES, Rozemuller AJ, Eikelenboom P, van Gool WA. 2007. Microglia activation in sepsis: A case-control study. J Neuroinflammation 4:4.

Schwab C, Hosokawa M, McGeer PL. 2004. Transgenic mice overexpressing amyloid beta protein are an incomplete model of Alzheimer disease. Exp Neurol 188:52–64.

Lott IT, Head E. 2005. Alzheimer disease and Down syndrome: Factors in pathogenesis. Neurobiol Aging 26:383–389. Martin BK, Szekely C, Brandt J, Piantadosi S, Breitner JC, Craft S, Evans D, Green R, Mullan M. 2008. Cognitive function over time in the Alzheimer’s disease anti-inflammatory prevention trial (ADAPT): Results of a randomized, controlled trial of naproxen and celecoxib. Arch Neurol 65:896–905. Mattiace LA, Davies P, Dickson DW. 1990. Detection of HLA-DR on microglia in the human brain is a function of both clinical and technical factors. Am J Pathol 136:1101–1114. McGeer PL, Itagaki S, Tago H, McGeer EG. 1987. Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR. Neurosci Lett 79:195–200. McGeer PL, McGeer EG. 2001. Inflammation, autotoxicity and Alzheimer disease. Neurobiol Aging 22:799–809. Mrak RE. 2012. Microglia in Alzheimer brain: A neuropathological perspective. Int J Alzheimers Dis 2012:165021. Munch G, Robinson SR. 2002. Potential neurotoxic inflammatory responses to Abeta vaccination in humans. J Neural Transm 109:1081–1087. Neumann H, Daly MJ. 2013. Variant TREM2 as risk factor for Alzheimer’s disease. N Engl J Med 368:182–184. Perry VH, Matyszak MK, Fearn S. 1993. Altered antigen expression of microglia in the aged rodent CNS. Glia 7:60–67. Popovich PG, Guan Z, McGaughy V, Fisher L, Hickey WF, Basso DM. 2002. The neuropathological and behavioral consequences of intraspinal microglial=macrophage activation. J Neuropathol Exp Neurol 61:623–633. Riley KP, Snowdon DA, Markesbery WR. 2002. Alzheimer’s neurofibrillary pathology and the spectrum of cognitive function: Findings from the Nun Study. Ann Neurol 51:567–577. Rogers J, Strohmeyer R, Kovelowski CJ, Li R. 2002. Microglia and inflammatory mechanisms in the clearance of amyloid beta peptide. Glia 40:260–269. Saijo K, Crotti A, Glass CK. 2013. Regulation of microglia activation and deactivation by nuclear receptors. Glia 61:104–111. Sasaki A, Kawarabayashi T, Murakami T, Matsubara E, Ikeda M, Hagiwara H, Westaway D, George-Hyslop PS, Shoji M, Nakazato Y. 2008. Microglial activation in brain lesions with tau deposits: Comparison of human tauopathies and tau transgenic mice TgTau(P301L). Brain Res 1214:159–168.

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Sheng JG, Mrak RE, Griffin WS. 1997. Glial-neuronal interactions in Alzheimer disease: Progressive association of IL-1alpha1 microglia and S100beta1 astrocytes with neurofibrillary tangle stages. J Neuropathol Exp Neurol 56: 285–290. Stoltzner SE, Grenfell TJ, Mori C, Wisniewski KE, Wisniewski TM, Selkoe DJ, Lemere CA. 2000. Temporal accrual of complement proteins in amyloid plaques in Down’s syndrome with Alzheimer’s disease. Am J Pathol 156:489– 499. Streit WJ. 2002. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia 40:133–139. Streit WJ. 2004. Microglia and Alzheimer’s disease pathogenesis. J Neurosci Res 77:1–8. Streit WJ. 2006. Microglial senescence: Does the brain’s immune system have an expiration date? Trends Neurosci 29:506–510. Streit WJ. 2010. Microglial activation and neuroinflammation in Alzheimer’s disease: A critical examination of recent history. Front Aging Neurosci 2:22. Streit WJ, Braak H, Xue QS, Bechmann I. 2009. Dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in Alzheimer’s disease. Acta Neuropathol 118: 475–485. Streit WJ, Sammons NW, Kuhns AJ, Sparks DL. 2004. Dystrophic microglia in the aging human brain. Glia 45:208–212. Thal DR, Arendt T, Waldmann G, Holzer M, Zedlick D, Rub U, Schober R. 1998. Progression of neurofibrillary changes and PHF-tau in end-stage Alzheimer’s disease is different from plaque and cortical microglial pathology. Neurobiol Aging 19:517–525. Wong AM, Patel NV, Patel NK, Wei M, Morgan TE, de Beer MC, de Villiers WJ, Finch CE. 2005. Macrosialin increases during normal brain aging are attenuated by caloric restriction. Neurosci Lett 390:76–80. Xu G, Gonzales V, Borchelt DR. 2002. Abeta deposition does not cause the aggregation of endogenous tau in transgenic mice. Alzheimer Dis Assoc Disord 16:196–201. Xue QS, Streit WJ. 2011. Microglial pathology in Down syndrome. Acta Neuropathol 122:455–466.

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