M. Svedberg, C. Unger, A. Nordberg
INTRODUCTION
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by mem- ory impairments and increasingly severe dementia. Alois Alzheimer described the two major pathological hallmarks present in the AD brain: amyloid plaques, which are mainly com- posed of amyloid !(A!), and neurofibrillary tangles, consisting of hyperphosphorylated tau protein. Although genetic and biochemical studies have suggested a cardinal role for A!in AD, the underlying mechanism(s) of how A!induces degeneration in the central nervous system is still unclear [1]. The aggregated form of A!(i.e. insoluble), soluble as well as intra/extracellular deposition, seems to be an important factor. The exact identity of the active species is still unclear. Fibrils, smaller peptide oligomers, water-soluble non-filamentous forms of A!, dimeric and trimeric species have all been suggested to be the toxic form [2–6].
During the last 10–15 years there has been intensive ongoing research with the aim of finding the underlying pathophysiological mechanisms of AD and how these abnormal processes could be influenced in order to prevent or slow down the progression of the disease [7].
Figure 10.1 shows several pathological processes that might be involved in the ongoing neurodegenerative processes in AD, including inflammatory processes, oxidative stress mechanisms, growth factor activation, microglial activation and synaptic impairments. The final cognitive disturbances are strongly related to impairment of the cholinergic system.
NEURONAL NICOTINIC RECEPTORS
The cholinergic system in the brain is involved in higher cognitive functions and mediates its effect via the muscarinic and nicotinic acetylcholine receptors (nAChRs): the nAChRs seem to play an important role in AD. The nAChRs are ion channel receptors present in neuronal and non-neuronal cells. They belong to the same receptor family as glutamate,
"-aminobutyric acid (GABA) and serotonin 3 (5-HT3) receptors that transduce cations, Na# and Ca2#[8, 9]. The nAChRs are transmembrane allosteric proteins formed by five subunits arranged around a central core that is perpendicular to the membrane [10].
The neuronal nAChRs are composed of $and !subunits. To date, six different $subunits ($2–$7), and three !subunits (!2–!4) have been cloned and sequenced from human brain tissue. The neuronal nAChRs can be homo-oligomers composed of $7, or hetero-oligomers,
Marie Svedberg, PhD, Researcher, Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, Division of Molecular Neuropharmacology, Karolinska University, Hospital Huddinge, Stockholm, Sweden Christina Unger, PhD, Researcher, Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, Division of Molecular Neuropharmacology, Karolinska University, Hospital Huddinge, Stockholm, Sweden
Agneta Nordberg, MD, PhD, Professor, Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, Division of Molecular Neuropharmacology, Karolinska University, Hospital Huddinge, Stockholm, Sweden
©Atlas Medical Publishing Ltd 2007
composed of different $subunits or both $and !subunits; each combination appears to dictate particular pharmacological and physiological functions [9, 11].
The most abundant nAChR subtype is the $4-nAChR subtype, but also the $3- and
$7-nAChRs subtypes are common in the human brain. The $4!2-nAChR binds nicotine with high affinity, while the $7-nAChR subtype binds $ bungarotoxin [8]. The $4!2-nAChR channels recognize and bind agonists with high affinity and are not as rapidly desensitized as the $7-nAChRs, which have a high Ca2#permeability, short open time and recognize and bind with low affinity all nicotinic agonists [12]. It has been proposed that the influx of Ca2#
regulated by nAChR activation may elicit a number of downstream intracellular events, including activation of protein kinases, initiation of immediate early genes and new protein synthesis, ultimately leading to changes in synaptic plasticity and neuronal remodelling. A single neurone often expresses several nAChR subtypes. Both the $4!2- and $7-nAChR subtypes are present in neurones of other systems including the glutamatergic, dopaminer- gic, serotonergic, noradrenergic and GABAergic neurotransmitter systems [13–17].
The nAChR subtypes studied in human post-mortem brain indicate a different regional distribution for the $4!2- and $7-nAChR subtypes. The $4- and $7-receptor subtypes, meas- ured by [3H]nicotine, [3H]cytisine and [3H]epibatidine binding in human post-mortem brain tissue, showed abundant binding in cortical regions with different laminar distributions [18], whereas the $7-receptor subtypes, measured by [125I]$ bungarotoxin binding, show high density in the hippocampus [19]. Recently, it has been shown that the $7-nAChRs are not only distributed on neuronal cells but also in glial cells, thus the nAChRs may have a different role in these type of tissues [20–22].
APP N C Ageing
Genetics Lifestyle
A!
Inflammatory
processes Oxidative stress
Microglial activation Growth factors
Neurofibrillary tangles A! plaque
Synaptic and neurotransmittor
dysfunction Pathological processes in AD
Figure 10.1 Schematic picture of the suggested pathological processes in AD. Age, genetics and lifestyle are all important factors that might determine the risk of developing AD. The increased production of A!causes accumulation of A!into plaques and hyperphosphorylation of tau, which eventually leads to synaptic and neurotransmittor dysfunction as well as microglia activation, inflammatory processes, oxidative stress and disturbances in the release of growth factors.
There is a significant loss of nAChRs in the human brain during normal ageing. Receptor binding analysis in post-mortem brain tissue obtained from subjects with no neurological or psychiatric history have revealed an 80% reduction in nAChRs in the cortex between 56 and 85 years of age [19].
NICOTINIC RECEPTOR CHANGES IN AD
There is a widespread reduction in the $4-nAChR subtype but also of the $3in AD brains.
Also the expression of the $7-nAChR is changed, but to a smaller extent, although an increase in mRNA encoding the $7-nAChR subtype was observed in hippocampus in post-mortem brain tissue from AD patients [23–25]. It has been unknown how these changes are associ- ated with neurones and/or astrocytes. Ligand binding assays and Western blotting only pro- vide information concerning the total binding capacity or total level of the protein, respectively, but give no information about the possible expression of the receptor proteins on astrocytes and neurones. When the presence of $7-nAChR was measured in neurones and astrocytes, respectively, a significant decrease was observed in the neurones while there was a significant increase in astrocytes [22]. The decrease in receptors on neurone may thus be levelled out by the increases in $7 on astrocytes of AD brain [22] (Figure 10.2), when recep- tors are quantified by binding studies in homogenates. The amyloid precursor protein (APP) 670/671 (Swedish) mutation (APPswe) [26], produces more A!1–42and subsequently larger plaques compared to sporadic AD [27, 28]. Although receptor analysis in post-mortem tissue from AD patients with the Swedish mutation (APPswe) showed similar reductions in nAChR binding sites as in sporadic AD [29], the loss in nAChRs was more pronounced in APP 670/671 brain due to the younger age-matched controls compared to sporadic AD. An ele- vated total number of astrocytes were observed in the hippocampus and temporal cortex of both APPswe and sporadic AD patients, while the increase in the level of expression of
$7-nAChRs on astrocytes was more pronounced in the Swedish mutation than in the sporadic AD brain [22]. In addition, a reduction in nAChRs has also been found in non-neuronal periph- eral cells, lymphocytes, from AD patients at both mRNA and binding site level [24, 30].
It has been possible to visualize the nAChRs by using 11C-nicotine and positron emission tomography (PET). A lower 11C-nicotine binding has been observed in cortical brain regions and hippocampus of AD patients compared to age-matched controls [31]. When a kinetic model is used for measuring the 11C-nicotine binding a positive correlation is observed Figure 10.2 $7-nAChRs are present on astrocytes, surrounding the plaques. Double immunolabelling for A!- and $7-nAChRs shows $7-nAChR positive astrocytes surrounding neuritic plaques. Scale bar%20 &m.
Reproduced with permission from [22].
between cognition (measured as Mini-mental State Examination [MMSE]) and 11C-nicotine binding in the temporal cortex [31]. Recently, a new amyloid PET ligand, the Pittsburgh Compound-B (PIB), has shown promising results in visualizing amyloid in AD patients [32, 33]. In cortical regions, such as the temporal–parietal association cortex, where there is a lower 11C-nicotine uptake and also lower cerebral glucose metabolism (18F-FDG uptake) an increased retention is observed for 11C-PIB as a sign of high amyloid. PIB imaging will be a valuable tool for studies of anti-amyloid drug effects.
APP/A!MECHANISMS IN AD
A!is produced via proteolytic processing of the APP. There are two variants of A!(A!1–40 and A!1–42). Ninety per cent of the A!produced is A!1–40, but A!1–42is more prone to aggre- gate as fibrils [34]. A!1–42is the major A!species found in cerebral plaques [35]. A!has been identified in humans in a fibrilar form within the plaques [36], but also in a soluble form as stable dimers [37, 38]. It has been shown that soluble A!has a synaptotoxic effect at early age, prior to plaque formation, in the brain of different animal models of AD [4, 39, 40].
There is also evidence that soluble oligomers of A!, but not monomers or insoluble A!fib- rils, may be responsible for synaptic dysfunction in AD patients [3, 41, 42].
One of the major targets in treatment strategies today is to lower the A!load in the AD brain [43] and by affecting the production, aggregation or clearance of A!, a modifying effect on disease progression is expected. One strategy for the development of anti-amyloid drugs is to block the production of A!. This effect might be obtained by inhibiting !- or
"-secretase activities, or by stimulating $-secretase activity [44, 45]. Blocking the aggregation of A!or enhancing the clearance of A!are other possible therapeutic strategies. Immune- based therapies directed against the A!peptide have been shown to ameliorate A!path- ology and reverse cognitive behavioural deficits in transgenic mice models [46–50]. Based on the successful clearance of A!, an active immunization trial was performed in AD patients. However, the trial was terminated due to the development of meningoencephali- tis in a subset of AD patients [51]. Brain autopsies of a few patients who died since receiv- ing the vaccine showed lower-than-expected levels of plaques, while no reduction of A!in the vessels or tau pathology was found [52, 53]. Greater decrease in brain volume and greater ventricular enlargement, as well as a reduction of cerebrospinal fluid (CSF) tau, in the antibody responders compared to placebo patients have been reported, while the cogni- tive improvements were modest [54, 55]. As a result, passive immunization with anti-A!
monoclonal antibodies has been suggested as a potentially safer alternative [56].
NEUROPROTECTIVE MECHANISMS AND THE nAChRs
The protection of neurones and their synapses against damage and death with preservation of function in neurodegenerative diseases, such as AD, is of great therapeutic importance.
Since nAChRs play a role in cognition and their expression is adversely affected by choliner- gic degeneration, a link between nAChR expression/function and other pathological fea- tures of AD, such as A!deposition/toxicity, might be plausible. Experimental in vitrostudies have shown neuroprotective effects viathe nAChRs (Table 10.1). It has been demonstrated that oestrogen exerts neuroprotective properties via the $7-nAChRs and there is promotion of additional neuroprotection by cholinesterase inhibitors like tacrine and donepezil [57].
Nicotine has been shown to protect against cytotoxicity induced by excitotoxins and A!in both in vitro and in vivosystems [58]. Several studies suggest that both the $7- and the
$4-nAChRs are involved in this neuroprotection. Selective nAChR agonists are candidates for symptomatic and neuroprotective AD therapy [7] and numerous investigations, both in vivo and in vitro, indicate that nicotine can enhance neurone survival in response to a range of neu- rotoxic insults. nAChR stimulation has been shown to increase neurotrophic factors in the
brain, increase the expression of nerve growth factor (NGF) receptors [6], and to protect neural cells against glutamate-induced toxicity [59]. nAChR stimulation also offers protection against trophic factor-deprivation-induced toxicity [60, 61] and A!-induced toxicity [62]. It has been suggested that stimulation of the $4!2-nAChRs and the $7-nAChRs may be responsible for neuroprotective effects against A!cytotoxicity [62, 63].
Due to these observations in experimental studies, it has been suggested that the
$7-nAChRs might be a potential therapeutic target for treatment of neurodegenerative dis- orders. Epidemiological studies have indicated that tobacco smoking may be associated with a reduced risk of developing AD and a delayed onset of familial AD, although these findings are controversial (for review, see [64]). Post-mortem investigations have shown reduced levels of A!in smoking AD patients and controls [65–68]. Recent studies in the Tg2576 (APPswe) transgenic mice have revealed that both short- (10 days) and long-term treatment (5.5 months) with nicotine drastically reduce the levels of aggregated (guan- idinium-soluble) A!1–40and A!1–42by 40–80% in the brain of these mice [66, 69, 70] (Figure 10.3). Treatment with nicotine for 10 days also resulted in less GFAP immunoreactive astrocytes around the plaques, increased levels of synaptophysin and increased number of
Toxic Neuroprotective
Model system Drug insult effect Reference
PC12 cells Tacrine/ A! ↓cell death via nAChRs [57, 83]
donepezil/
oestrogen
SH-SY5Y cells Galantamine A!or okadaic ↓A!or okadaic acid [84]
acid toxicity via $7-nAChR and PI3K-Akt pathway
SH-SY5Y cells Donepezil A!or okadaic ↓A!or okadaic acid toxicity [84]
acid via $7-nAChR and PI3K-Akt pathway
SH-SY5Y cells Galantamine A!or Prevention of apoptotic [85]
thapsigargin cell death via
$7-nAChRs
Primary rat Galantamine A! #glutamate ↓ A!enhanced glutamate [86]
cortical toxicity via $7-nAChR
neurones to PI3K cascade
Primary rat Donepezil Glutamate ↑ cell viability via both [87]
cortical culture the $4- and $7-nAChRs
Human cortical Nicotine A! #glutamate ↓ A!enhanced glutamate [88]
neurones toxicity via $7-nAChR
to PI3K cascade
Primary rat Nicotine Glutamate ↓ glutamate excitotoxicity [89]
cortical neurones via nAChRs
Primary rat Nicotine A! ↓ of A!excitotoxicity [62]
cortical neurones via$7-nAChRs
Rat hippocampal A! ↓ of A! [90]
culture Nicotine excitotoxicity via nAChRs
Differentiated Nicotine NGF and serum ↑ cell viability via [91]
PC12 cells deprivation $7-nAChRs
Table 10.1 Neuroprotective effects via the nAChRs in cell lines
$7-nAChRs in the cortex of APPswe transgenic mice [70]. On the contrary, chronic nicotine treatment (5 months) in the triple transgenic mouse model, expressing PS1M146V, APPswe and tauP301L(3'Tg-AD mice) resulted in an increase in hyperphosphorylation and aggrega- tion of tau [71]. Nicotine has also been shown to increase tau in cell cultures [72]. Thus, the different effects observed on A!and tau following nicotine exposure call for further studies when considering nicotinic agonists as a possible therapy for AD.
It is important to determine whether the clearance of A!might be due to the !-sheet breaking activity of nicotine or whether nicotine can activate astrocytes, and thereby pro- mote increased clearance of A!. It was recently demonstrated that L-(-)-nicotine not only inhibits the aggregation of A!1–40and A!1–42, but can also disaggregate fibrils preformed from both of these peptides [73]. Furthermore, it has also been shown that both enantiomers of nicotine (D-(#)- and L-(-)-nicotine) can affect the early stages of A!aggregation, delaying oligomerization and fibril formation and thereby maintaining a population of less toxic A!
species [74]. This suggests that this effect might not be due to a specific binding interaction between nicotine and A!, as previously thought, but could be due instead to a weaker, rela- tively non-specific binding or it could be due to the anti-oxidant or metal-chelating properties of nicotine. Since nicotine does not change the activities of cortical $-, !- or "-secretase in APPswe transgenic mice or non-transgenic controls [75], and the levels of intracellular A!
were not reduced [70], it is tempting to suggest that the action of nicotine (or one of its metabolites) might primarily be via degradation of insoluble A!deposits, rather than affect- ing the accumulation of the peptide. Further studies of the interaction of intracellular and extracellular A!at synapses as well as activation of $7-nAChRs in microglial cells may lead to a better understanding of the interactive effect of nicotine on AD pathology and may pro- mote new therapeutic strategies for the disease.
INTERACTION BETWEEN A!AND "7-nAChRs
Several experimental studies suggest that there is an interaction between A! and the
$7-nAChRs in AD. A possible mechanism for how the $7-nAChRs can regulate the accumula- tion and formation of A!plaques in the brain and thereby play an important role in AD pathogenesis is as follows: A!1–42binds with high affinity to the $7-nAChRs and thereby a gradual intracellular accumulation of A!1–42by endocytosis occurs [76–79].
The binding of the A!1–42peptide to the receptor results in the formation of an A!1–42/
$7 - nAChR complex, which might be an important step in the accumulation of A!1–42in the
0 20 40 60 80 100
0 20 40 60 80 100
0 20 40 60 80 100
A!1–40 A!1–42 5.5 months’ treatment 10 days’ treatment 10 days’ treatment
** **
* *
*
*
Reduction compared to sucrose/saline treated mice (%)
Figure 10.3 The effect of nicotinic treatment on the A!levels in APPswe transgenic mice. (A) Reduction (%) in guanidinium soluble A!1–40and A!1–42levels following treatment with nicotine for 5.5 months (compared to sucrose treated animals) and 10 days (compared to saline treated animals) respectively.
Reproduced with permission from reference [69]. *P(0.05, **P(0.01. Student t-test (from refs [69] and [75]). Non-parametric Kruskal-Wallis test followed by Mann-Whitney test (from ref [70]).
neurones [77]. The consequence of accumulated A!1–42in the cell will eventually result in the cell undergoing lysis, following a selective loss of neurones and dispersal of their cytoplasmic contents, including the accumulated A!1–42, into the surrounding extra- cellular space to form amyloidogenic plaques that disrupt neural and synaptic function in the brain (for review, see [80]). The A!1–42/$7 - nAChR complex is expected to be most abundant on neurones particularly those with a high $7-nAChR expression (e.g. cortical pyramidal cells).
Glial cell
Presynaptic terminal
Synapse
Postsynaptic terminal A!
A!
A!
A!
$7-nAChR
$7-nAChR
Altered synaptic plasticity Ca2+
A!
A!
Increased endocytosis of
membrane NMDARs NMDA A!
AMPA AMPA
A!
$7-nAChR A!
Extracelluar Intracelluar
A! A! A!
$7-nAChR
Figure 10.4 Schematic picture of tentative interactions between A!- and $7-nAChRs. Several studies have shown a direct binding between A!and the $7-nAChR. The binding of A!peptide to the $7-nAChR results in the formation of an A!/$7 nAChR complex, which might be an important step in the accumulation of A!in the neurones [77]. It is also plausible that A!could activate intracellular signalling pathways, like the ERK2 MAPK cascade, which causes an influx of Ca2#into the cell leading to altered synaptic plasticity [76]. Furthermore, it has been proposed that A!might directly modulate the nAChRs by blocking the $7-nAChR-like currents in hippocampal interneurones [78]. A!may, by binding to $7-nAChRs, impair glutamatergic transmission, compromise synaptic function and reduce LTP and thereby promoting endocytosis of NMDA receptors [82]. The elevated expression of
$7-nAChR on astrocytes might participate in A!cascade and formation of neuritic plaques [22].
It is also plausible that A!has a direct interaction with the nAChRs and that the A!1–42
thereby could activate intracellular signalling pathways, like the ERK2 MAPK cascade, which causes an influx of Ca2#into the cell and leads to an increase in the $7-nAChRs in the hippocampus [76]. Furthermore, it has been proposed that A!1–42might directly modulate the nAChRs by blocking the $7-nAChR-like currents in hippocampal interneurones [78].
Figure 10.4 is a schematic picture of the possible interactions and consequences between A!
and the $7-nAChRs.
A direct involvement of glial cells, notably astrocytes and microglia, has been apparent in plaque formation in AD brains. The $7-nAChRs are expressed on glial cells [20–22] and the elevated expression of $7-nAChR on astrocytes might participate in the A!cascade and the formation of neuritic plaques. Thus, the A!1–42peptide is abundant in astrocytic intracellu- lar deposits and in amyloid plaques throughout AD brains. The detection of microglial cells, astrocyte processes and choline acetyltransferase- (ChAT-) positive fibres around !-amyloid plaques in transgenic APPswe mice suggest a close connection between cholinergic term- inals and microglial cells [81].
Several lines of evidence suggest that the A!toxicity might be related to elevated levels of glutamate and/or over-activity of the NMDA receptors. The observation that A!reduces long-term potentiation (LTP) and facilitates long-term depression suggests a role for A!in regulating trafficking of glutamate receptors. A signalling pathway, where A!1–42may impair glutamatergic transmission, compromize synaptic function and reduce LTP by binding to $7-nAChRs thereby promoting endocytosis of NMDA receptors in cortical neu- rones, was recently reported [82]. Neuronal cell cultures from APPswe transgenic mice showed a reduction in surface-expressed NMDA receptors, while no change was observed in total receptor numbers [82].
The nAChRs (especially the $7 subtype) seem, according to the literature, to be of import- ance in neuroprotection. Whether the beneficial effects of nicotine in experimental studies reflect a possible pharmacological approach in the treatment of AD and the effect on cogni- tion when A!is reduced, remains to be further studied. According to the hypothesis that A!binds to the $7-nAChR subtype, an $7 nicotinic antagonist might be of preference to reduce the A!levels in the AD brain. Furthermore, the observation that nicotine can dra- matically decrease parenchymal A!and, in contrast to A!vaccination, is also able to reduce A!deposits associated with blood vessels, might be of importance for new therapeutic strat- egies. Increased understanding of the neuroprotective mechanisms and beneficial effects of nicotine, its interaction with A!(directly or viathe nicotinic receptors) and the effect of A!
on neurotransmission in the brain will eventually lead to a beneficial treatment of AD.
ACKNOWLEDGEMENTS
Mrs. Marianne Grip is acknowledged for her professional help with the illustrations.
Financial support was provided by the Swedish Research Council (project no. 05817).
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