**8.1 Alzheimer's disease (AD): microglia**

AD, the most common age-related neurodegenerative disease, is characterized by cognitive decline in people over 65years old. Pathologically, AD is presented with amyloid-β protein (Aβ) deposition (plaques), abnormal phosphorylation aggregation of the microtubule-associated protein tau (tangles), neuroinflammation, oxidative stress, and synaptic dysfunction. Neuroinflammation is underscored by microglial reaction and increased cytokine production. Microglia are also major sources of free radicals such as superoxide and nitric oxide in the brain. Microglia are considered the innate immune cells of the CNS and act as brain macrophages. They are mainly found in the subventricular and subgranular zone, where under physiological conditions self-renew over an organism's entire lifespan. Microglia are not uniformly distributed throughout the brain. A large number is present in the hippocampal dentate gyrus,

#### *Central Nicotinic and Muscarinic Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.112447*

substantia nigra, and parts of the basal ganglia. Interestingly, olfactory telencephalon in mice has the largest microglial population.

Microglia differ in size and ramification patterns within and between different histological layers of the cerebellar cortex. Substantia nigra contains the largest proportion of microglia (about 12%) compared to 5% in the cortex and corpus callosum. This regional heterogeneity is attributed to the residential environment, especially interactions with neurons or neural progenitor cells, as well as intrinsic mechanisms. Microglia are critical for regulation of the neuronal network as they support the development, maintenance, homeostasis, and repair of the brain by wiping out cell debris and phagocytizing viruses and bacteria. There are several stages in microglia morphology and function. For example, during the resting state, microglia are sensitive to environmental stimuli such as stress that can activate aberrant microglia functioning and lead to neurodegenerative and psychiatric disorders. Thus, it is critical to recognize microglial heterogeneity in identifying microglia-selectively therapies and uncover the underlying mechanisms that activate the reparative and regenerative functions of microglia [37, 38].

Pro-inflammatory microglia (M1-activated state) secrete proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase (iNOS), which typically lead to dysfunction following chronic activation. In contrast, neuroprotective microglia (M2 state) phagocytose cell debris and misfolded proteins, promote tissue repair and reconstruction of the extracellular matrix and support neuron survival mediated by neurotrophic factors [39].

AD is also associated with changes in neurovascular unit NVU, a structural and functional complex that maintains microenvironmental homeostasis and metabolic balance in CNS. Microglia are one of the most important components of the NVU. In AD, microglia may also cause blood–brain barrier (BBB) breakdown due to loss of pericytes. Pericytes are cells present at intervals along the walls of capillaries and are important for blood vessel formation, maintenance of BBB, regulation of immune cell entry into CNS and control of brain blood flow. Thus, BBB breakdown can lead to infiltration of peripheral white blood cells into CNS, abnormal contraction of cerebral vessels and neurovascular uncoupling (**Figure 2**) [40].

Family history is the second strongest risk factor following advanced age. Twin and family studies indicate that genetic factors are estimated to play a role in at least 80% of AD [41]. Moreover, autosomal dominant and late onset sporadic AD share a common pathophysiology [42]. Numerous sporadic AD risk genes including apolipoprotein E (ApoE) and complement receptor 1 (CR1), are highly expressed in microglia and affect microglial phagocytosis of amyloid-beta peptides. Actually, during the early stages of AD, microglia may provide protection against amyloid accumulation but in advanced AD stage they promote neuropathology. Indeed, approximately two-thirds of AD patients risk single nucleotide polymorphisms that are exclusively or dominantly expressed in microglia. Furthermore, AD is associated with increased microglial proliferation. It is noteworthy that microglia exert a "double-edged sword" effect involving neuroprotective or neurotoxic functions dependent on contextual factors as well as disease stage. Thus, at the early stage of AD, microglia are involved in clearance of Aβ and tau proteins from the brain, whereas in the later stages of AD, sustained microglial activation leads to chronic pro-inflammatory state, associated with increase production of pro-inflammatory cytokines, reactive oxygen species (ROS), and dysfunctional lysosomal deposits, all of which adversely affect neuronal survival by promoting protein aggregation and hence causing neuronal damage. Altogether, the findings suggest that further characterization of microglia and its detailed role in neurodegeneration, can lead to novel therapeutic targets for AD [38, 39, 43].

#### **8.2 AD: astrocytes**

It is believed that astrocytes are involved in many vital cognitive functions, including learning and memory. Moreover, astrocytes through production of antioxidant and anti-inflammatory proteins are involved in CNS protection. They also clean the extracellular environment and facilitate neuronal communication and help in maintenance of homeostasis. However, full exploitation of glial system as potential development of novel drugs and techniques to reverse oxidative stress and/or excess of inflammation that occurs in many CNS diseases, remains to be investigated (**Figure 2**) [44].

In contrast to microglia, astrocytes are brain cells that mainly control metabolic and redox homeostasis. Due to their swift response to brain pathology in the initial stages of the disease, their activation and differentiation are implicated in the pathogenesis of multiple neurodegenerative diseases, including AD [45]. Astrocytes play an important role in synaptic function, K+ buffering, BBB maintenance and neuronal metabolism. For example, BBB disruption occurs in the early stages of AD, which is associated with cognitive decline and might accelerate the disease progression. Reactive astrocytes denote astrocytes undergoing morphological, molecular and functional remodeling in response to pathological stimuli. During AD progression, reactive astrogliosis occur. For this reason, it has been suggested that manipulation of astrocytes and/or astrocytic biomarkers could be developed in diagnosis and/or treatment of AD (**Figure 2**) [46, 47].

Blood biomarkers have been investigated for the diagnosis, prognosis, and monitoring of AD. Although Aβ and tau are primarily blood biomarkers, recent studies have identified other reliable candidates such as glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal protein that can be detected in blood samples. Indeed, it has been suggested that GFAP levels can be used to detect early-stage AD. This is based on observations where GFAP level in the blood was higher in the Aβ-positive group than in the negative groups, and in individuals with AD or mild cognitive impairment (MCI) compared to the healthy controls [47]. Thus, astrocyte activation, accompanied by high levels of GFAP is often observed in AD patients. This elevated GFAP occurs around Aβ plaque, indicative of elevated phagocytosis. Structural alterations in AD astrocytes including swollen endfeet and soma shrinkage contribute to disruption in vascular integrity at capillary and arterioles levels. Astrocyte endfeet enwrap the entire vascular tree within CNS where they perform important functions in regulating BBB, cerebral blood flow, nutrient uptake, and waste clearance [48]. Like microglia in AD, astrocytes also are skewed into proinflammatory and oxidative profiles with increased secretions of vasoactive mediators inducing endothelial junction disruption and immune cell infiltration [49]. Regarding biomarkers, astrocytic α7nAChR levels or activity, was recently proposed as a marker since this receptor subtype is implicated in instigation and potentiation of early Aβ pathology. The same receptors could provide a target for therapeutic intervention in AD [46].

Recently, it has been proposed that the term "type III diabetes (T3DM)" be used in conjunction with AD as both conditions share similar molecular and cellular features. For example, T3DM is associated with insulin resistance and cognitive decline (memory deficits) in elderly individuals. Since astrocytes are involved in brain metabolism (e.g., glucose metabolism, lipid metabolism), neurovascular coupling, synapses, and synaptic plasticity, targeting them might be promising in alleviating neurodegeneration in these patients [50].

## **8.3 ACh-AChRs: microglia**

As mentioned above, neuroinflammation linked to glial function has been demonstrated to participate in the pathogenesis of AD (**Figure 2**). Moreover, anti-inflammatory and neuroprotective properties of ACh in several neurodegenerative disorders was also alluded to. More recently, specific influence of ACh on neuroinflammation and neurodegeneration in AD was investigated. It was reported that microglia played a key role in lipopolysaccharide (LPS)-induced hippocampal neuronal toxicity and that ACh, via activation of α7nAChR provided anti-inflammatory and neuroprotective effects. Furthermore, in neuron–microglia co-cultures, LPS increased the expression of pro-inflammatory factors, including iNOS, interleukin-1α, and tumor necrosis factor-α, and decreased expression of neurotrophic factors such as insulin-like growth factor-1, and neuronal apoptosis. However, ACh, via the action of α7nAChR on microglia, inhibited LPS-induced inflammatory response and provided neuroprotection, which was further enhanced by promoting microglial neurotrophic factor production [51]. Targeting microglia in age-related cognitive decline and AD, and bearing in mind the heterogeneity of microglia in these conditions and how pharmacological agents could target specific microglial states, has been recently reviewed [52]. Infiltration of immune cells into the brain might play a role in detrimental effects of activated microglia as this can lead to T-cell infiltration, which can induce tauopathy, another marker of AD neuropathology. Interestingly, drugs or antibodies that can result in death of microglia, have shown protection against brain atrophy in mice [53].

Microglia may contain both nAChRs and mAChRs. It is believed that a subpopulation of microglia that express functional mAChRs play a role in stroke and AD. These microglia tend to expand in these conditions, which are sensitive to blockers of protein synthesis and correlate with an upregulation of the M3 receptor subtype. Thus, carbachol, a mAChR agonist acts as a chemoattractant for microglia and reduces their phagocytic activity [54]. In addition to M3 receptor upregulation, there is an increased expression of major histocompatibility complex (MHC)-I and MHC-II. MHC molecules plays an important role in alerting the immune system to virally infected cells [55].

As mentioned above, nAChRs presence in microglia and consisting primarily of α7nAChR provide anti-inflammatory and neuroprotective effects. Hence, manipulation of microglial nAChRs and mAChRs may offer a new therapeutic strategy in neurodegenerative diseases in general, and AD, in particular.

### **8.4 ACh-AChRs: astrocytes**

ACh and AChRs are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. Astrocytes express mAChRs whose activation stimulates a robust intracellular signaling that regulate neurite outgrowth in hippocampal neurons, a system intimately involved in cognitive function. In fact, stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development [36]. Moreover, it was recently demonstrated that M1 muscarinic receptors in astrocytes mediate cholinergic regulation of adult hippocampal neurogenesis [56].

In CNS, as mentioned earlier, ACh is mainly present in interneurons. However, at least two important cholinergic pathways have also been identified. One is the cholinergic projection from the nucleus basalis of Meynert (in the basal forebrain) to the forebrain neocortex and associated limbic structures, degeneration of which is one of the pathologies associated with AD. The other is a projection from the medial septal and diagonal band region to limbic structures, commonly referred to as the septo-hippocampal pathway that is also involved in memory formation [57]. In both cases, both nAChRs and mAChRs mediate the effects of ACh, where nicotinic agonists including nicotine, have been shown to improve working memory, whereas muscarinic agonists may be more relevant to improvement of reference memory [58, 59].

AD patients have a substantial reduction in nAChRs in the cortex and hippocampus. Recently, using local cholinergic lesions it was possible to manipulate the cholinergic system more finely to determine the role of AChRs as well as nicotinicmuscarinic receptor interactions that can either synergize or antagonize the behavioral outcomes. Therefore, potential utility of combining selective nAChR subtypes as well selective mAChRs on memory and cognition warrants further investigation [60, 61]. It is noteworthy that along such selective agonists, manipulation of vesicular ACh transporter should also be considered [62].

That astrocytes express nAChRs was mentioned above. These receptors, predominantly α7nAChR and regulating calcium signaling, are likely mediators of nicotine's effects on morphological and functional changes of the astrocytes [63]. Interestingly, nicotine does not induce reactive astrocytosis even at high concentrations (10 μM) as determined by cytokine release and GFAP expression in-vitro. In vivo also, nicotine induces a change in the volume of astrocytes in the prefrontal cortex, CA1 of the hippocampus, and the substantia nigra. These and other findings indicate potential use of nicotine in neurodegenerative diseases including AD [10, 64, 65]. However, mode of nicotine administration appears to be an important factor in its therapeutic application. It is argued that pulsatile (e.g., via inhalation or nasal spray), rather than continuous administration of nicotine (e.g., via patch) would likely be effective for providing neuroprotection in any neurodegenerative disease [66].

Muscarinic M1 and M4 ACh receptors are also highly pursued drug targets for neurological diseases including AD. However, due to high sequence homology in M1-M5 mAChRs, selective targeting of any subtype through endogenous ligand binding site has been difficult to achieve. Recent discovery of highly subtype selective mAChR positive allosteric modulators has provided a new frontier in novel drug development. However, due to side effects, where M1 mAChR over-activation can have detrimental consequences, a drug candidate may need to exhibit a biased signaling profile. In this regard, recent studies in mice suggest that allosteric modulators for the M1 mAChR that bias signaling toward specific pathways may be therapeutically important [67].
