**6. Intracellular mechanisms**

the hypothesis that Aβ-specific antibodies may lead to the phagocytosis of Aβ by microglia

The involvement of microglia in clearance of Aβ aggregates after immunotherapy has been demonstrated through several studies. After a single injection of anti-Aβ antibody to APP mice, the antibodies were found associated not only with amyloid deposits but also with microglia surrounding the plaques [44]. Wilcock et al. reported that 24 and 72 hours after the injection of anti-Aβ antibodies to Tg2576 APP mice there was a reduction in fibrillar amyloid deposits and showed an increase in microglial activation, evaluated by CD45, a protein tyrosine phosphatase commonly used as a marker for microglia activation, and MHC-II staining. Intracraneal injections of anti-Aβ antibodies to APP mice demonstrated that the increase in CD45 expression of microglia is evident after the clearance of diffuse deposits and is parallel with the clearance of fibrillar deposits [36]. The temporal association of fibrillar amyloid loss with microglia activation suggests some causal role for microglial activation in the process [45]. In 2004 Wilcock reported that 1 month after the administration of anti-Aβ antibodies to APP transgenic mice an increase in CD45 expression on microglia surrounding amyloid deposits in both the hippocampus and frontal cortex. After 2 months of treatment there was an additional increase in CD45 not only in microglia surrounding amyloid plaques but also in microglia associated with soluble aggregate [46, 47]. This microglial activation also takes form

of an increased transcript level of proinflammatorycyctokines and iNOS [47].

mouse treated with the full-length antibody [49].

TNF-α and IL-1β [52].

The role of microglia in the clearance of amyloid deposits after the administration of anti-Aβ antibodies was analyzed *in vivo* through the generation of the CX3CR1-GFP protein. CX3XR1 is a gene specifically expressed in microglia in the CNS [48]. After administration of an anti-Aβ antibody that recognizes both aggregated and soluble Aβ, PDAPP mice contained more levels of CX3CR1-GFP positive cells and these cells had twice as many protruding processes from their cell bodies. These changes were detected surrounding amyloid plaques and amyloid deposits associated with blood vessels. These changes were also seen with the microglia marker Iba-1 and with CD45 staining [49]. When Fab fragments of the antibody were injected there was no effect on the number of microglia CX3CR1-GFP positive cells or on microglia morphology, suggesting that the Fc is required to elicit the microglial changes observed in the

One of the mechanisms for plaques clearance is by anti-Aβ immunotherapy through FcγRmediated phagocytosis of plaques by microglia [50]. After administration of anti-Aβ antibodies to APP mice there was an increase in FcRII and FcRIII on microglia. The microglia expressing the FcR were associated with amyloid plaques and with diffuse aggregates [46]. When examined in *an ex vivo* assay with sections of PDAPP or AD brain tissue, antibodies against Aβ-activated microglia cells clear amyloid plaques through FcR-mediated phagocytosis and subsequent peptide degradation [39]. Anti-Aβ antibodies with binding affinity for FcγR increased the Aβ oligomer induced p38MAPK activity in microglia [51]. The p38MAPK pathway is responsible for the upregulation of proinflamatory cytokines in microglia, such as

After the role of microglia in anti-Aβ antibodies amyloid clearance was proposed, and the effect of microglia inhibition was assessed. The anti-Mac-1-saporin immunotoxin was used to

cells [39].

176 Update on Dementia

#### **6.1. βA peptides degradation by autophagy**

Cellular homeostasis largely depends on the proteostasis network. Under normal conditions, this network senses and rectifies disturbances in the proteome to restore homeostasis in cells. The main players in proteostasis maintenance are chaperones and two proteolitic systems: the ubiquitin-proteasome and the autophagy system.

Although there are some differences in this proteolitic systems, substrates of the ubiquitinproteasomes pathway are predominantly short-live proteins and misfolded or damaged proteins. Meanwhile, the autophagy substrates are long-live proteins and multiple proteins organized into oligomeric complex or aggregates that cannot be degraded by others systems [53].

In this sense, macroautophagy (hereafter referred to as autophagy) has been characterized as a catabolic process that engulfs aberrant organelles, misfolded proteins, and protein aggregates into double membrane vesicles (named autophagosomes) and delivers it to lysosomas [54]. The correct function of this catabolic process is very important because it is the only known mechanism that eukaryotic cells possess to degrade protein aggregates and the only one by which entire organelles such as mitochondria and peroxisomes are recycled. Several studies proposed that autophagy helps to relieve the proteotoxic stress of misfolded proteins by degrading toxic oligomers in the cytosol [55].

Posmitotic cells like neurons are highly dependent on autophagy. Mainly because once neurons mature and become postmitotic, they lose their ability to dilute insults by cell division. Thus, neuronal survival heavily depends on housekeeping processes to maintain cellular quality control [56]. In this regard, the loss of autophagy particularly in neurons causes the accumulation of ubiquitin-positive inclusion bodies and triggers a process of neurodegener‐ ation [57]. Evidence points that dysfunction in the autophagy processes is part of Alzheimer's disease pathogenesis [58] and the clearance of autophagic vacuoles and lysosomal degradation of Aβ could prevent the intracellular accumulation.

#### **6.2. Autophagy**

Nowadays it is recognized that autophagy has a fundamental role in homeostasis through the degradation of components that would be toxics for the cell.

**Figure 3.** Autophagy elimination of Aβ plaques. Autophagy is the only known mechanisms that eukaryotic cells pos‐ sess to degrade Aβ aggregates.

Autophagy is a complex process that requires a series of coordinated steps. In the first place, the formation of a vesicle isolation called phagophore is described. After the phagophore formation, it elongates around the cytoplasmatic components selected for degradation. The recognition of the components that will be degraded and the closing of the vesicle are de‐ pendent on the lipidated form of LC3 protein (microtubule-associated protein light chain 3). The lipidated form of LC3 is associated with the outer and inner membranes of the autopha‐ gosome and has become a reliable method for monitoring autophagy and autophagy-related processes [59].

Finally, the late stage of autophagy or maturation depends on the successful fusion of autophagosome with lysosome. This fusion allows contact of autophagosome cargo with lysosomal hydrolases and consequently the degradation of the components that in some cases are recycled.

These steps are fundamentaly important for the autophagic flux (defined as the continuous series of events since the cargo is engulf until it is degraded). Any event that alters the autophagic flux also alters the degradation process and consequently leads to the accumulation of autophagosomes (**Figure 3**).

#### **6.3. Autophagy and Alzheimer's disease**

into double membrane vesicles (named autophagosomes) and delivers it to lysosomas [54]. The correct function of this catabolic process is very important because it is the only known mechanism that eukaryotic cells possess to degrade protein aggregates and the only one by which entire organelles such as mitochondria and peroxisomes are recycled. Several studies proposed that autophagy helps to relieve the proteotoxic stress of misfolded proteins by

Posmitotic cells like neurons are highly dependent on autophagy. Mainly because once neurons mature and become postmitotic, they lose their ability to dilute insults by cell division. Thus, neuronal survival heavily depends on housekeeping processes to maintain cellular quality control [56]. In this regard, the loss of autophagy particularly in neurons causes the accumulation of ubiquitin-positive inclusion bodies and triggers a process of neurodegener‐ ation [57]. Evidence points that dysfunction in the autophagy processes is part of Alzheimer's disease pathogenesis [58] and the clearance of autophagic vacuoles and lysosomal degradation

Nowadays it is recognized that autophagy has a fundamental role in homeostasis through the

**Figure 3.** Autophagy elimination of Aβ plaques. Autophagy is the only known mechanisms that eukaryotic cells pos‐

Autophagy is a complex process that requires a series of coordinated steps. In the first place, the formation of a vesicle isolation called phagophore is described. After the phagophore

degrading toxic oligomers in the cytosol [55].

of Aβ could prevent the intracellular accumulation.

degradation of components that would be toxics for the cell.

**6.2. Autophagy**

178 Update on Dementia

sess to degrade Aβ aggregates.

Autophagic vacuoles are uncommon in neurons of the healthy brain because this process is constitutive active in neurons and the efficient clearance of autophagosomes keeps their presence low [60]. Moreover, in AD there is an accumulation of autophagic vesicles preferen‐ tially in dystrophic neuritis [61]. This evidence suggests that some of the later steps in the autophagic process is altered; this idea is supported by the observation that the lysosomal hidrolases are increased and abnormally distributed in AD brain indicating the defective maturation of autophagosomes [62]. Additionally, it was observed that acidification of lysosomes causes an autophagosome accumulation without altering the induction [62].

#### **6.4. Why autophagy is altered in AD?**

It is not clear, but it has been demonstrated, that a large number of autophagic vacuoles are observed in dystrophic neuritis before extracellular Aβ deposition in neurons of AD patients and transgenic mouse models [63, 64]. This suggests that autophagy dysfunction leads to the accumulation of Aβ, avoiding proper degradation.

Inductors of autophagy as trehalosa (a natural disaccharide that block glucose transporters) could rescue the AD-like phenotype in APP/PS1 transgenic mice. In this sense, trehalosa treatment significantly improves the performance of memory and learning tasks. In accord‐ ance with behavioral test, Aβ deposits were significantly reduced in hippocampus [65].

In addition, the induction of autophagy by rapamicyn (an mTORC1 complex inhibitor) in PD/ APP transgenic mice improves the cognitive performance through the degradation of extrac‐ ellular Aβ depositions.

In this model, autophagy induction was higher in the hippocampus of transgenic mice compared with nontransgenic mice [66]. This suggests that autophagy dysfunctions could be reversed through the pharmacological stimulation and these inductions have beneficial effects by promoting Aβ degradation. Different studies have been performed finding that rapamycin reduces the accumulation of Aβ levels and fibrillar aggregates approximately 40–50% in 3XAD-Tg mice and APP transgenic mice [67].

Morphological evidence shows that APP and Aβ peptide are colocalized with LC3-positive autophagosomes and autophagy induction shows a greater colocalization of Aβ in autophagic vacuoles, suggesting a more active degradation [68]. However, the mechanism by which autophagy can degrade extracellular amyloid plaque content is unknown. But autophagic process of microglia (the resident macrophages in the brain) seems to play an important role.

The degradation of extracellular amyloid content through microglia involves at the first step phagocytosis; once Aβ peptide is in the cytosol it is exposed to be recognized by LC3-II via optineurin (an adaptor protein). LC3-II/OPTN recognition allows Aβ degradation via the autophagic–lysosomal system [69].

The exact pathology of AD is still unknown, but it is widely believed that the deposition of Aβ is one of the main causes leading to the degeneration and death of neurons. So, finding alternatives that avoid Aβ accumulation or enhance its degradation could be a strong thera‐ peutic target. In this sense, autophagy seems to be the first line of defense to face accumulation but it is not clear how autophagy dysfunctions are related to Aβ aggregation or if Aβ over‐ production directly induces autophagy defects.

#### **6.5. Differential autophagy activation by monomers and oligomers**

Some observations have demonstrated that a large number of autophagic vacuoles are observed in dystrophic neuritis before extracellular Aβ depositions in the neurons of AD patients and murine models of AD [70], but recently it has been demonstrated that Aβ monomers and oligomers differentially modulate autophagy in neurons. In a different way, monomers stimulate autophagy increasing autophagosome rates and the elevation of LC3-II protein levels, but at the same time monomers impaired lysosomal pathway affecting the autophagy flux. These events resulted in autophagosome accumulation. On the other hand, Aβ oligomers cause a less pronounced increase in LC3-II protein levels and does not affect the autophagy flux [58], suggesting that defects in autophagy could be the result of an increase in amyloid monomers.

Enhancing autophagic clearance of toxic protein aggregates through rapamycin or trehalosa ameliorates protein aggregation, neuron survival, and this is reflected in the improvement of cognitive skills. However, converging evidence suggests that improvement in autophagic flux through stimulation is a promising therapeutic intervention, and this field is still developing.

In other words, there are barely some studies showing the degradation by autophagy of the N-truncated beta amyloid peptide [71–74]. Recently, it has been determined that pE3-Aβ is strongly reduced in the TgCRND8 mice fed with a normal diet supplemented with the antioxidant Oleurupein (OLE) and that such a decrease likely reflects the parallel reduction of QC expression. In addition, their model of (Aβ) peptide deposition displayed strongly improved performance in behavioral and cognitive tests, reduced inflammatory response, and recovered dysfunctions of transgene-induced long-term potentiation (LTP) in the CA1 hippocampal area. These effects were induced, at least in part, by a strong activation of autophagy. All these results suggest new perspectives for AD not only at the prevention but also at the therapeutic level [75–77].
