Neuropathology

The histopathology of AD consists of four prominent hallmarks: senile amyloid (Aβ) plaques, neurofibrillary tangles (NFTs), brain atrophy and neuroinflammation (Figure 1).

Senile Aβ-plaques

Senile plaques were identified as extracellular aggregates of Aβ-peptides (Masters et al., 1985). Aβ is cleaved from the highly conserved integral membrane protein Amyloid Precursor Protein (APP), which is encoded by the APP gene located on chromosome 21 in humans. APP contains 18 exons with a total length of 290 kb (kilobases) (Yoshikai et al., 1990). Various splicing variants of APP can be found in different tissues and cell types in mammals. In human neurons the splicing variant APP695 is the most abundant one (reviewed by Matsui et al., 2007). Noteworthy, APP is extensively post-translationally modified. This includes amongst others glycosylation, sialylation and phosphorylation but also enzymatic processing (Kummer and Heneka, 2014). Enzymatic processing of APP can occur e.g. in an amyloidogenic or non-amyloidogenic manner, which will be described in detail in section 1.1.2.

Aβ-plaques can be categorized in dense-core also known as neuritic plaques and in diffuse plaques (Wisniewski et al., 1973). Dense-core Aβ-plaques consist of fibrillary amyloid clustering in a central core surrounded by loose Aβ-peptides, dystrophic neurites and gliosis. The dense core can be visualized by β-sheet binding dyes like Congo Red, Methoxy-XO4 or ThioflavinS (ThioS). Diffuse plaques are more amorphous lacking dystrophic neurites and a central core. Thus, they cannot be labeled with β-sheet binding dyes and need to be visualized by antibody staining (Selkoe, 2001).

Spreading of Aβ-plaques occurs in a distinct pattern, which can be categorized by the Thal Aβ phase (TAP) or the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) system (Mirra et al., 1991; Thal et al., 2002). The TAP system describes anatomical distribution of Aβ-plaques beginning in the neocortex (TAP 1), proceeding in the hippocampus, amygdala, allocortex and diencephalon (TAP 2+3) and further appears in the brain stem and cerebellum (TAP 4+5) according to immunohistochemical analysis (Thal et al., 2002) (Figure 2A). CERAD uses a semi-quantitative approach to assess neuritic plaques, ranging from none (0), sparse (1), moderate (2) to severe (3) deposition (Mirra et al., 1991).

Neurofibrillary tangles

Intraneuronal protein accumulations known as NFTs are found in post mortem tissue of AD patients. NFTs are built of paired helical filaments (PHFs), which consist of hyperphosphorylated Tau protein (pTau), a microtubule-associated protein (MAPT) (Goedert et al., 1988; Kidd, 1963;

Kopke et al., 1993). Under physiological conditions Tau is a modulator of the microtubule assembly and stabilization as well as of axonal transport (Goedert et al., 2006; Weingarten et al., 1975). Upon

Figure 1: Histopathological hallmarks of AD.

A) Atrophic hemibrain of a 70 year old AD patient (right) in comparison to a hemibrain of an age-matched healthy control (left). The cortex (C) shows extreme shriveling in the AD brain compared to the control brain. The hippocampus (H) also displays massive shrinkage, while the lateral ventricle (V) is prominently increased in the AD sample. B) Silver staining on post mortem brain tissue of the AD patient shows neuritic plaques (P) and neurofibrillary tangles (N).

C) Immunohistochemistry for MHC II (major histocompatibility complex II) in brown labels reactive microglia in AD post mortem tissue. D) Alongside astrogliosis is detcted by staining for GFAP (glial fibrillary acidic protein) in brown.

C+D) Blue counterstaining with haematoxylin labels nuclei. Images were modified after Gouw et al., (2008) and Wippold et al., (2008).

hyperphosphorylation Tau becomes dysfunctional, which is associated with synaptic dysfunction, altered intracellular trafficking and defective proteasomal degradation (Wang and Mandelkow, 2016).

Neither the pathological mechanisms causing Tau hyperphosphorylation, nor its interaction with Aβ-peptides are fully understood yet (Hochgrafe et al., 2013; Sydow et al., 2011). Aβ-pathology seems to be upstream of Tau pathology as studies using Tau knockout (KO) mice with overexpression of human APP found neuroprotection even though Aβ-burden was not altered (Roberson et al., 2007).

Furthermore, Aβ oligomerization can trigger pTau accumulation in neurons and thereby promote NTF formation (Ma et al., 2009; Oddo et al., 2003; Zempel et al., 2013).

Albeit NFTs are a hallmark of AD and mutations in the human MAPT gene are associated with within the frontal parts of the neocortex. NFT-burden in further parts of the neocortex is staged with Braak stages V and VI.

Brain atrophy

Brain atrophy is a very prominent feature of AD brains, correlating with NFT-burden and reflecting neuronal loss. Neuronal loss results in atrophy of hippocampus, temporal lobes and eventually Figure 2: Thal stages of amyloid (Aβ) and Braak stages of NFT pathology.

A) Thal stages of Aβ-plaque pathology are shown in blue. Phase 1 describes Aβ-deposits in the basal temporal cortex and in the orbitofrontal neocortex.

Phases 2 and 3 classify Aβ throughout the neocortex, in the hippocampus, the amygdala, the basal ganglia and the diencephalon. Phases 4 and 5 are used to describe Aβ-deposits in the mesencephalon, cerebral cortex and the lower brainstem. B) NFT pathology is shown in green. Stage I and II describes intraneuronal accumulations of hyperphophorylated Tau in the loculs coeruleus, the entorhinal and transentorhinal cortex.

Stage III and IV are used when NFTs are detected in the hippocampus and in the frontal neocortex. Stages V and VI define NFTs in neurons throughout the neocortex. Figure adapted from Goedert et al. (2015).

parietal cortex. Furthermore, it causes enlargement of ventricles (Figure 1A). Progressive reduction of brain volume due to neuronal loss can already be detected at early stages of the disease by MRI (Leung et al., 2013). Neuronal loss is preceded by synaptic dysfunction. All of these aspects lead to memory impairment.

Neuroinflammation

Another important finding in post mortem brains of AD patients is neuroinflammation. The term neuroinflammation describes activation of immune cells in the CNS as a consequence of brain injury, trauma or infection. Neuroinflammation is accompanied by reactive gliosis, which describes activation and proliferation of glia. Most commonly involved cell types in neuroinflammation are microglia and astrocytes. These cells are capable of clearing Aβ-deposits through phago-lysosomal degradation (Frackowiak et al., 1992; Wisniewski et al., 1991; Wyss-Coray et al., 2003). Briefly, phagocytosis is a specific form of endocytosis by which the cell membrane engulfs solid particles or whole microorganisms from the extracellular space. The engulfed debris is gradually transported within maturating endosomes, which eventually fuse with the lysosome for enzymatic degradation of the content. The detailed molecular mechanism underlying the endo-lysosomal pathway will be described in detail in section 1.2.4.

In microglia, e.g. this phago-lysosomal activation coincides with morphologic changes displayed by cell swelling, altered gene expression and the secretion of signaling molecules like cytokines to interact with the environment (Kettenmann et al., 2011). Moreover, there is emerging evidence that microglia can also directly interact with astrocytes and vice versa, suggesting a close link between gliosis and neuroinflammation (Liddelow et al., 2017). Throughout disease progression the blood brain barrier eventually breaks down which allows peripheral immune cells to enter the brain. This includes e.g. peripheral monocytes, neutrophils and T cells (Zenaro et al., 2017). How these cells contribute to neuroinflammation in AD is not well understood yet. Detailed characteristics of microgliosis in AD will be discussed in detail in section 1.2.3.