**5. Clearance mechanisms**

possessing N-terminal pyroglutamic acid (pyroE), generated by glutamic acid [21]. The Nterminal truncated Aβ3-42/Aβ3 is generated by the zinc-metalloprotease neutral endopepti‐ dase or neprilysin (NeP)-40 cleaving Aβ between Arg-2 and Glu-3. On the other hand, BACE-1 is also capable of cleaving between Tyr-10 and Glu-11, leading to the release of Aβ11-42/ Aβ11-40 peptides [22]. Then, the GluN-terminal undergoes N-terminal pyroglutamate (pGlu)

The native conformation of Aβ is an unfolded protein. Aβ forms amyloid fibrils by folding from the native random-coil-rich state to α-helical-rich intermediate, and finally to a β-sheet-

Another protein closely related to AD pathology is the tau protein. This is encoded in mapt gene, located on chromosome 17q21. Several tau isoforms are generated by alternative splicing, creating high and low isoforms. The human central nervous system expresses six low molec‐

Tau is a neuronal cytosolic protein whose function is to promote microtubules polymerization and stabilization. In addition, Tau has importance in maintaining an appropriate morphology

Domains of Tau are defined on the basis of their microtubule interaction and their amino acid character. The C-terminal domain (assembly domain) binds microtubules while the Nterminal domain projects away from microtubule (projection domain). The overall amino acid tau composition is hydrophilic, consistent with its unfolded character; however, the Nterminal is predominantly acidic and the C-terminal roughly neutral, which is important for microtubule interaction. The middle region is a proline-rich domain which is targets of many proline-directed kinases and binding sites for proteins with SH3 domains [28]. Tau is highly regulated and is subject to multiple post-translational modifications. Phosphorylation is the most common tau post-translational modification described resulted from the equilibrium

Hyperphosphorylation of tau is not only associated with the disease, but is also employed by the neuron to downregulate its activity transiently and reversibly where required, for example during development, anesthesia, and hypothermia. It is the nonreversible nature of the abnormal hyperphosphorylation of tau in AD and other tauopathies that results in an involuntary slowing down of neuronal activity and a consequent chronic progressive neuro‐ degeneration [29]. Increased tau phosphorylation decreased its affinity for microtubules resulting in an abnormal increase in the levels of the free (unbound) Tau fraction; next small nonfibrillary tau deposits (normally referred to as "pretangles") are formed followed by structural rearrangement involving the formation of the characteristic pleated β-sheet struc‐

modification catalyzed by glutaminylcyclase (**Figure 2**) [23].

rich amyloid monomer that self-assembles into the fibrils [24].

ular weight isoform ranging from 352 to 441 amino acids [25].

of neurons and it appear to modulate axonal transport [26, 27].

between the amount and activity of protein kinases and phosphatases.

tures, which finally form the neurofibrillary tangles by self-assemble [26].

**4. Aβ structure**

174 Update on Dementia

#### **5.1. Alzheimer's disease clearance: is microglia involved?**

Since Alois Alzheimer described the disease in 1907 several therapeutic options have been developed. The therapeutic treatments available today treat the symptoms without targeting the cause of the disease [30] and as a consequence the disease follows it's natural course [31]. Cholinesterase inhibitors and memantine are FDA-approved therapies against the cognitive symptoms for AD [32]. These drugs favor short-term cognitive benefits, so even though patients are receiving the ideal treatment, they will return to their baseline cognitive decline [33].

Since the amyloid cascade was described research for a disease modifying therapy is being aimed toward the study of Aβ. β-Secretase inhibitors have been tested in an attempt to reduce the production of Aβ. It was demonstrated that β-secretase inhibitors reduced plasma and CSF levels of Aβ but concerns have emerged about potential side effects with chronic administra‐ tion [34].

One of the most recent strategies, known for its ability to reduce the accumulation of Aβ and promote a cognitive benefit in preclinical trials, is the immunotherapy. The immunothera‐ peutic approach can be classified as either active or passive. Passive immunotherapy refers to the administration of anti-Aβ antibodies, bypassing the patient need to mount an immuno‐ logical response toward Aβ. Active immunotherapy involves the administration of full length Aβ or peptide fragments conjugated to a carrier protein, with a T-cell epitope and with an adjuvant in order to stimulate the patient own immune response. The basis of both immuno‐ therapeutic approaches relies on the recognition of Aβ aggregates by specific anti-Aβ anti‐ bodies [35].

Extensive studies of active and passive immunization with Aβ showed promising benefits. Schenk et al. were the first to report the beneficial effects of Aβ immunotherapy in a preclinical study with active immunization in PDAPP mouse. The immunization with Aβ reduced levels of cerebral amyloid and produced high serum antibody titers. A year later Morgan et al. reported that Aβ immunization improved behavioral performance in learning and memory tasks [36]. Passive immunization studies showed that antibodies were able to enter the central nervous system, 0.01% of the peripherally administered antibodies, bind plaques, and induce clearance of preexisting amyloid lesions. Passive immunization of PDAPP mice led to reduce plaque burden, increase blood circulating Aβ, and improve cognitive performance [37]. Immunotherapy via active or passive immunization against Aβ peptides has shown to be very successful in reducing Aβ aggregates in AD animal models [38–41]. The immunotherapeutic approach was translated to clinical trials by ELAN/Wyeth in 2000. After a few immunized subjects, the trial was stopped due to the development of meningoencephalitis in 6% of immunized AD patients [42]. The postmortem analysis of participants, who died from causes not related to the immunization, showed patchy clearance of amyloid plaques in the brain [43]. These areas of clearance were accompanied by Aβ immunoreactive microglia cells, supporting the hypothesis that Aβ-specific antibodies may lead to the phagocytosis of Aβ by microglia cells [39].

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].

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

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 TNF-α and IL-1β [52].

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 kill activated microglia in APP mice. The elimination of activated microglia reduced Aβ clearance by anti-Aβ antibodies, although appreciable clearance was still present [53]. This suggests that microglia, dependent and independent mechanisms, are likely involved in the clearance of amyloid aggregates following immunotherapy [54]. Additional mechanisms for clearance of amyloid peptides are possible *in vivo*. If Fab2 fragments, which fail to activate microglia, are injected in transgenic mice, the clearance of Aβ deposits is blocked, but the clearance of diffuse aggregates is unaffected [46]. These data suggest that the clearance of diffuse aggregates may proceed by the catalytic dissolution mechanism proposed by Solomon [50], who postulated that direct interaction of antibodies with Aβ may lead to the disruption of aggregates [55]. This process does not depend on FcR activation. Even though there are several non-Fc-dependent mechanisms for the removal of Aβ aggregates, previous studies demonstrate, through the analysis of microglial cells and frozen tissue sections, that the Fcmediated mechanism is dominant in the removal of amyloid deposits [39].

Even though the involvement of microglia is important for the removal of amyloid deposits, the activation of microglia by anti Aβ antibodies is accompanied with microhemorrhages and edema. It has been proposed that microglia activation by antibodies induces damage to the vasculature and to the neurons [50]. The prevention of microhemorrhages is achieved when antibodies with reduced affinity for FcγR are used. This was shown by using Crenezumab, a humanized antibody with lower affinity for all FcγR; this antibody promotes a reduction in microglial activation, limiting the release of inflammatory cytokines to avoid side effects, such as vasogenic edema. In the phase I of the safety trial, no vasogenic edema or microhemorrhage were found (Clinicaltrials.gov). The current immunotherapeutic approaches for AD are directed to the design of an optimized antibody that could separate the phagocytic and inflammatory response, promoting an efficient clearance of Aβ aggregates the induction of the detrimental proinflammatory citokyne release [56].
