In vitro Characterization of the Regional Binding Distribution of Amyloid PET Tracer Florbetaben and the Glia Tracers Deprenyl and PK11195 in Autopsy Alzheimer's Brain Tissue.

BACKGROUND: Emerging evidence indicates a central role of gliosis in Alzheimer's disease (AD) pathophysiology. However, the regional distribution and interaction of astrogliosis and microgliosis in association with amyloid-ß (Aß) still remain uncertain. OBJECTIVE: Here we studied the pathological profiles in autopsy AD brain by using specific imaging tracers. METHODS: Autopsy brain tissues of AD (n = 15, age 70.4±8.5 years) and control cases (n = 12, age 76.6±10.9) were examined with homogenate binding assays, autoradiography for Aß plaques (3H-florbetaben/3H-PIB), astrogliosis (3H-L-deprenyl), and microgliosis (3H-PK11195/3H-FEMPA), as well as immunoassays. RESULTS: In vitro saturation analysis revealed high-affinity binding sites of 3H-florbetaben, 3H-L-deprenyl, and 3H-PK11195/3H-FEMPA in the frontal cortex of AD cases. In vitro3H-florbetaben binding increased across cortical and subcortical regions of AD compared to control with the highest binding in the frontal and parietal cortices. The in vitro3H-L-deprenyl binding showed highest binding in the hippocampus (dentate gyrus) followed by cortical and subcortical regions of AD while the GFAP expression was upregulated only in the hippocampus compared to control. The in vitro3H-PK11195 binding was solely increased in the parietal cortex and the hippocampus of AD compared to control. The 3H-florbetaben binding positively correlated with the 3H-L-deprenyl binding in the hippocampus and parietal cortex of AD and controls. Similarly, a positive correlation was observed between 3H-florbetaben binding and GFAP expression in hippocampus of AD and control. CONCLUSION: The use of multi-imaging tracers revealed different regional pattern of changes in autopsy AD brain with respect to amyloid plaque pathology versus astrogliosis and microgliosis.

Neural Stem Cell Transplant-Induced Effect on Neurogenesis and Cognition in Alzheimer Tg2576 Mice Is Inhibited by Concomitant Treatment with Amyloid-Lowering or Cholinergic α7 Nicotinic Receptor Drugs.

Stimulating regeneration in the brain has the potential to rescue neuronal networks and counteract progressive pathological changes in Alzheimer's disease (AD). This study investigated whether drugs with different mechanisms of action could enhance neurogenesis and improve cognition in mice receiving human neural stem cell (hNSC) transplants. Six- to nine-month-old AD Tg2576 mice were treated for five weeks with the amyloid-modulatory and neurotrophic drug (+)-phenserine or with the partial α7 nicotinic receptor (nAChR) agonist JN403, combined with bilateral intrahippocampal hNSC transplantation. We observed improved spatial memory in hNSC-transplanted non-drug-treated Tg2576 mice but not in those receiving drugs, and this was accompanied by an increased number of Doublecortin- (DCX-) positive cells in the dentate gyrus, a surrogate marker for newly generated neurons. Treatment with (+)-phenserine did however improve graft survival in the hippocampus. An accumulation of α7 nAChR-expressing astrocytes was observed around the injection site, suggesting their involvement in repair and scarring processes. Interestingly, JN403 treatment decreased the number of α7 nAChR-expressing astrocytes, correlating with a reduction in the number of DCX-positive cells in the dentate gyrus. We conclude that transplanting hNSCs enhances endogenous neurogenesis and prevents further cognitive deterioration in Tg2576 mice, while simultaneous treatments with (+)-phenserine or JN403 result in countertherapeutic effects.

Visualization of regional tau deposits using (3)H-THK5117 in Alzheimer brain tissue.

INTRODUCTION:  The accumulation of neurofibrillary tangles, composed of aggregated hyperphosphorylated tau protein, starts spreading early in specific regions in the course of Alzheimer's disease (AD), correlating with the progression of memory dysfunction. The non-invasive imaging of tau could therefore facilitate the early diagnosis of AD, differentiate it from other dementing disorders and allow evaluation of tau immunization therapy outcomes. In this study we characterized the in vitro binding properties of THK5117, a tentative radiotracer for positron emission tomography (PET) imaging of tau brain deposits. RESULTS: Saturation and competition binding studies of (3)H-THK5117 in post-mortem AD brain tissue showed the presence of multiple binding sites. THK5117 binding was significantly higher in hippocampal (p < 0.001) and temporal (p < 0.01) tissue homogenates in AD compared to controls. Autoradiography studies with (3)H-THK5117 was performed on large frozen brain sections from three AD cases who had been followed clinically and earlier undergone in vivo (18)F-FDG PET investigations. The three AD cases showed distinct differences in regional THK5117 binding that were also observed in tau immunohistopathology as well as in clinical presentation. A negative correlation between in vivo (18)F-FDG PET and in vitro (3)H-THK5117 autoradiography was observed in two of the three AD cases. CONCLUSIONS: This study supports that new tau PET tracers will provide further understanding on the role of tau pathology in the diversity of the clinical presentation in AD.

Astrocytosis precedes amyloid plaque deposition in Alzheimer APPswe transgenic mouse brain: a correlative positron emission tomography and in vitro imaging study.

PURPOSE: Pathological studies suggest that neuroinflammation is exacerbated by increased beta-amyloid (Aß) levels in the brain early in Alzheimer's disease (AD). The time course and relationships between astrocytosis and Aß deposition were examined using multitracer in vivo positron emission tomography (PET) imaging in an AD transgenic mouse model, followed by postmortem autoradiography and immunohistochemistry analysis. METHODS: PET imaging with the amyloid plaque tracer (11)C-AZD2184 and the astroglial tracer (11)C-deuterium-L-deprenyl ((11)C-DED) was carried out in APPswe mice aged 6, 8-15 and 18-24 months (4-6 animals/group) and in wild-type (wt) mice aged 8-15 and 18-24 months (3-6 animals/group). Tracer uptake was quantified by region of interest analysis using PMOD software and a 3-D digital mouse brain atlas. Postmortem brain tissues from the same APPswe and wt mice in all age groups were analysed for Aß deposition and astrocytosis by in vitro autoradiography using (3)H-AZD2184, (3)H-Pittsburgh compound B (PIB) and (3)H-L-deprenyl and immunostaining performed with antibodies for Aß42 and glial fibrillary acidic protein (GFAP) in sagittal brain sections. RESULTS: (11)C-AZD2184 PET retention in the cerebral cortices of APPswe mice was significantly higher at 18-24 months than in age-matched wt mice. Cortical and hippocampal (11)C-DED PET binding was significantly higher at 6 months than at 8-15 months or 18-24 months in APPswe mice, and it was also higher than at 8-15 months in wt mice. In vitro autoradiography (3)H-AZD2184 and (3)H-PIB binding confirmed the in vivo findings with (11)C-AZD2184 and demonstrated age-dependent increases in Aß deposition in APPswe cortex and hippocampus. There were no significant differences between APPswe and wt mice in (3)H-L-deprenyl autoradiography binding across age groups. Immunohistochemical quantification demonstrated more Aß42 deposits in the cortex and hippocampus and more GFAP(+) reactive astrocytes in the hippocampus at 18-24 months than at 6 months in APPswe mice. CONCLUSION: The findings provide further in vivo evidence that astrocytosis occurs early in AD, preceding Aß plaque deposition.

Glial Asthenia and Functional Paralysis: A New Perspective on Neurodegeneration and Alzheimer's Disease.

Neuroglia are represented by several population of cells heterogeneous in structure and function that provide for the homeostasis of the brain and the spinal cord. Neuroglial cells are also central for neuroprotection and defence of the central nervous system against exo- and endogenous insults. At the early stages of neurodegenerative diseases including Alzheimer's disease neuroglial cells become asthenic and lose some of their homeostatic, neuroprotective, and defensive capabilities. Astroglial reactivity, for example, correlates with preservation of cognitive function in patients with mild cognitive impairment and prodromal Alzheimer's disease. Here, we overview the experimental data indicating glial paralysis in neurodegeneration and argue that loss of glial function is fundamental for defining the progression of neurodegenerative diseases.

Fibrillar ß-amyloid 1-42 alters cytokine secretion, cholinergic signalling and neuronal differentiation.

Adult neurogenesis is impaired by inflammatory processes, which are linked to altered cholinergic signalling and cognitive decline in Alzheimer's disease. In this study, we investigated how amyloid beta (Aß)-evoked inflammatory responses affect the generation of new neurons from human embryonic stem (hES) cells and the role of cholinergic signalling in regulating this process. The hES were cultured as neurospheres and exposed to fibrillar and oligomeric Aß(1-42) (Aßf, AßO) or to conditioned medium from human primary microglia activated with either Aß(1-42) or lipopolysaccharide. The neurospheres were differentiated for 29 days in vitro and the resulting neuronal or glial phenotypes were thereafter assessed. Secretion of cytokines and the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and choline acetyltransferase (ChAT) involved in cholinergic signalling was measured in medium throughout the differentiation. We report that differentiating neurospheres released various cytokines, and exposure to Aßf, but not AßO, increased the secretion of IL-6, IL-1ß and IL-2. Aßf also influenced the levels of AChE, BuChE and ChAT in favour of a low level of acetylcholine. These changes were linked to an altered secretion pattern of cytokines. A different pattern was observed in microglia activated by Aßf, demonstrating decreased secretion of TNF-α, IL-1ß and IL-2 relative to untreated cells. Subsequent exposure of differentiating neurospheres to Aßf or to microglia-conditioned medium decreased neuronal differentiation and increased glial differentiation. We suggest that a basal physiological secretion of cytokines is involved in shaping the differentiation of neurospheres and that Aßf decreases neurogenesis by promoting a microenvironment favouring hypo-cholinergic signalling and gliogenesis.

³H-deprenyl and ³H-PIB autoradiography show different laminar distributions of astroglia and fibrillar ß-amyloid in Alzheimer brain.

BACKGROUND: The pathological features in Alzheimer's disease (AD) brain include the accumulation and deposition of ß-amyloid (Aß), activation of astrocytes and microglia and disruption of cholinergic neurotransmission. Since the topographical characteristics of these different pathological processes in AD brain and how these relate to each other is not clear, this motivated further exploration using binding studies in postmortem brain with molecular imaging tracers. This information could aid the development of specific biomarkers to accurately chart disease progression. RESULTS: In vitro binding assays demonstrated increased [³H]-PIB (fibrillar Aß) and [³H]-PK11195 (activated microglia) binding in the frontal cortex (FC) and hippocampus (HIP), as well as increased binding of [³H]-L-deprenyl (activated astrocytes) in the HIP, but a decreased [³H]-nicotine (α4ß2 nicotinic acetylcholine receptor (nAChR)) binding in the FC of AD cases compared to age-matched controls. Quantitative autoradiography binding studies were also performed to investigate the regional laminar distributions of [³H]-L-deprenyl, [³H]-PIB as well as [¹²5I]-α-bungarotoxin (α7 nAChRs) and [³H]-nicotine in hemisphere brain of a typical AD case. A clear lamination pattern was observed with high [³H]-PIB binding in all layers and [³H]-deprenyl in superficial layers of the FC. In contrast, [³H]-PIB showed low binding to fibrillar Aß, but [³H]-deprenyl high binding to activated astrocytes throughout the HIP. The [³H]-PIB binding was also low and the [³H]-deprenyl binding high in all layers of the medial temporal gyrus and insular cortex in comparison to the frontal cortex. Low [³H]-nicotine binding was observed in all layers of the frontal cortex in comparison to layers in the medial temporal gyrus, insular cortex and hippocampus. Immunohistochemical detection in the AD case revealed abundant glial fibrillary acidic protein positive (GFAP+) reactive astrocytes and α7 nAChR expressing GFAP+ astrocytes both in the vicinity and surrounding Aß neuritic plaques in the FC and HIP. Although fewer Aß plaques were observed in the HIP, some hippocampal GFAP+ astrocytes contained Aß-positive (6 F/3D) granules within their somata. CONCLUSIONS: Astrocytosis shows a distinct regional pattern in AD brain compared to fibrillar Aß, suggesting that different types of astrocytes may be associated with the pathophysiological processes in AD.

Amyloid tracers detect multiple binding sites in Alzheimer's disease brain tissue.

Imaging fibrillar amyloid-ß deposition in the human brain in vivo by positron emission tomography has improved our understanding of the time course of amyloid-ß pathology in Alzheimer's disease. The most widely used amyloid-ß imaging tracer so far is (11)C-Pittsburgh compound B, a thioflavin derivative but other (11)C- and (18)F-labelled amyloid-ß tracers have been studied in patients with Alzheimer's disease and cognitively normal control subjects. However, it has not yet been established whether different amyloid tracers bind to identical sites on amyloid-ß fibrils, offering the same ability to detect the regional amyloid-ß burden in the brains. In this study, we characterized (3)H-Pittsburgh compound B binding in autopsied brain regions from 23 patients with Alzheimer's disease and 20 control subjects (aged 50 to 88 years). The binding properties of the amyloid tracers FDDNP, AV-45, AV-1 and BF-227 were also compared with those of (3)H-Pittsburgh compound B in the frontal cortices of patients with Alzheimer's disease. Saturation binding studies revealed the presence of high- and low-affinity (3)H-Pittsburgh compound B binding sites in the frontal cortex (K(d1): 3.5 ± 1.6 nM; K(d2): 133 ± 30 nM) and hippocampus (K(d1):5.6 ± 2.2 nM; K(d2): 181 ± 132 nM) of Alzheimer's disease brains. The relative proportion of high-affinity to low-affinity sites was 6:1 in the frontal cortex and 3:1 in the hippocampus. One control showed both high- and low-affinity (3)H-Pittsburgh compound B binding sites (K(d1): 1.6 nM; K(d2): 330 nM) in the cortex while the others only had a low-affinity site (K(d2): 191 ± 70 nM). (3)H-Pittsburgh compound B binding in Alzheimer's disease brains was higher in the frontal and parietal cortices than in the caudate nucleus and hippocampus, and negligible in the cerebellum. Competitive binding studies with (3)H-Pittsburgh compound B in the frontal cortices of Alzheimer's disease brains revealed high- and low-affinity binding sites for BTA-1 (Ki: 0.2 nM, 70 nM), florbetapir (1.8 nM, 53 nM) and florbetaben (1.0 nM, 65 nM). BF-227 displaced 83% of (3)H-Pittsburgh compound B binding, mainly at a low-affinity site (311 nM), whereas FDDNP only partly displaced (40%). We propose a multiple binding site model for the amyloid tracers (binding sites 1, 2 and 3), where AV-45 (florbetapir), AV-1 (florbetaben), and Pittsburgh compound B, all show nanomolar affinity for the high-affinity site (binding site 1), as visualized by positron emission tomography. BF-227 shows mainly binding to site 3 and FDDNP shows only some binding to site 2. Different amyloid tracers may provide new insight into the pathophysiological mechanisms in the progression of Alzheimer's disease.

Age-dependent neuroplasticity mechanisms in Alzheimer Tg2576 mice following modulation of brain amyloid-ß levels.

The objective of this study was to investigate the effects of modulating brain amyloid-ß (Aß) levels at different stages of amyloid pathology on synaptic function, inflammatory cell changes and hippocampal neurogenesis, i.e. processes perturbed in Alzheimer's disease (AD). Young (4- to 6-month-old) and older (15- to 18-month-old) APP(SWE) transgenic (Tg2576) mice were treated with the AD candidate drug (+)-phenserine for 16 consecutive days. We found significant reductions in insoluble Aß1-42 levels in the cortices of both young and older transgenic mice, while significant reductions in soluble Aß1-42 levels and insoluble Aß1-40 levels were only found in animals aged 15-18 months. Autoradiography binding with the amyloid ligand Pittsburgh Compound B ((3)H-PIB) revealed a trend for reduced fibrillar Aß deposition in the brains of older phenserine-treated Tg2576 mice. Phenserine treatment increased cortical synaptophysin levels in younger mice, while decreased interleukin-1ß and increased monocyte chemoattractant protein-1 and tumor necrosis factor-alpha levels were detected in the cortices of older mice. The reduction in Aß1-42 levels was associated with an increased number of bromodeoxyuridine-positive proliferating cells in the hippocampi of both young and older Tg2576 mice. To determine whether the increased cell proliferation was accompanied by increased neuronal production, the endogenous early neuronal marker doublecortin (DCX) was examined in the dentate gyrus (DG) using immunohistochemical detection. Although no changes in the total number of DCX(+)-expressing neurons were detected in the DG in Tg2576 mice at either age following (+)-phenserine treatment, dendritic arborization was increased in differentiating neurons in young Tg2576 mice. Collectively, these findings indicate that reducing Aß1-42 levels in Tg2576 mice at an early pathological stage affects synaptic function by modulating the maturation and plasticity of newborn neurons in the brain. In contrast, lowering Aß levels in Tg2576 mice when Aß plaque pathology is prominent mainly alters the levels of proinflammatory cytokines and chemokines.

Neurotrophic and neuroprotective actions of (-)- and (+)-phenserine, candidate drugs for Alzheimer's disease.

Neuronal dysfunction and demise together with a reduction in neurogenesis are cardinal features of Alzheimer's disease (AD) induced by a combination of oxidative stress, toxic amyloid-ß peptide (Aß) and a loss of trophic factor support. Amelioration of these was assessed with the Aß lowering AD experimental drugs (+)-phenserine and (-)-phenserine in neuronal cultures, and actions in mice were evaluated with (+)-phenserine. Both experimental drugs together with the metabolite N1-norphenserine induced neurotrophic actions in human SH-SY5Y cells that were mediated by the protein kinase C (PKC) and extracellular signal-regulated kinases (ERK) pathways, were evident in cells expressing amyloid precursor protein Swedish mutation (APP(SWE)), and retained in the presence of Aß and oxidative stress challenge. (+)-Phenserine, together with its (-) enantiomer as well as its N1- and N8-norphenserine and N1,N8-bisnorphenserine metabolites, likewise provided neuroprotective activity against oxidative stress and glutamate toxicity via the PKC and ERK pathways. These neurotrophic and neuroprotective actions were evident in primary cultures of subventricular zone (SVZ) neural progenitor cells, whose neurosphere size and survival were augmented by (+)-phenserine. Translation of these effects in vivo was assessed in wild type and AD APPswe transgenic (Tg2576) mice by doublecortin (DCX) immunohistochemical analysis of neurogenesis in the SVZ, which was significantly elevated by 16 day systemic (+)-phenserine treatment, in the presence of a (+)-phenserine-induced elevation in brain- derived neurotrophic factor (BDNF).

Modulation of α7 nicotinic acetylcholine receptor and fibrillar amyloid-ß interactions in Alzheimer's disease brain.

The nicotinic receptors (nAChRs), which play a critical role in cognitive function, are impaired early in the course of Alzheimer's disease (AD). We have previously demonstrated that amyloid-ß (Aß) assemblies bind to α7 nAChRs and form complexes in AD brain, suggesting that this cooperative process may contribute to disruption of synaptic function in AD. In the current study, we further characterized the interaction between different nAChR subtypes and fibrillar Aß by binding assays in postmortem brain from AD and control cases using a wide range of drugs with different molecular targets, including selective nAChR subtype agonists, and the amyloid ligand Pittsburgh compound B (PIB) that binds with high (nanomolar) affinity to fibrillar Aß. The α7 nAChR agonists varenicline and JN403, but not the α4ß2 nAChR agonist cytisine, increased the 3H-PIB binding in autopsy tissue homogenates from AD and control frontal cortex. This effect was blocked in the presence of the α7 nAChR antagonists methyllycaconitine, α-bungarotoxin, and mecamylamine, but not by the α4ß2 nAChR antagonist dihydro-ß-erythroidine. Increases in (3)H-PIB binding evoked by varenicline and JN403 were also prevented by pre-incubation with another amyloid ligand, BF-227. The acetylcholinesterase inhibitor and allosteric nAChR modulator galantamine as well as the N-methyl-d-aspartate receptor blocker memantine did not significantly alter (3)H-PIB binding levels in AD brain. The present findings further support a specific interaction between fibrillar Aß and α7 nAChRs in the brain, suggesting that treatment with α7 nAChR stimulatory drugs can modulate Aß/α7 nAChR pathogenic signaling mechanisms in AD brain.

Different ß-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity.

In this study, we examined the relationship between various ß-amyloid (Aß) oligomer assemblies in autopsy brain with the levels of fibrillar Aß and cholinergic synaptic function. Brain tissues obtained from the frontal cortex of 14 Alzheimer's disease (AD) patients grouped into early-onset AD (EOAD) and late-onset AD (LOAD) and 12 age-matched control subjects were used to extract and quantify Aß oligomers in soluble (TBS), detergent soluble (TBST), and insoluble (GuHCl) fractions. The predominant oligomeric Aß assemblies detected were dodecamers, decamers, and pentamers, and different patterns of expression were observed between EOAD and LOAD patients. There was no association between any of the detected Aß oligomer assemblies and fibrillar Aß levels measured by N-methyl[(3)H] 2-(40-methylaminophenyl)-6-hydroxy-benzothiazole ([(3)H]PIB) binding. Levels of pentamers in the soluble fraction significantly correlated with a reduction in choline acetyltransferase activity in AD patients. The number of nicotinic acetylcholine receptors negatively correlated with the total amount of Aß oligomers in the insoluble fraction in EOAD patients, and with decamers in the soluble fraction in LOAD patients. These novel findings suggest that distinct Aß oligomers induce impairment of cholinergic neurotransmission in AD pathogenesis.

Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer's patients with the Swedish APP 670/671 mutation.

There is a significant flux of the neurotoxic oxysterol 27-hydroxycholesterol (27OHC) from the circulation across the blood-brain barrier. Because there is a correlation between 27OHC and cholesterol in the circulation and lipoprotein-bound cholesterol does not pass the blood-brain barrier, we have suggested that 27OHC may mediate the effects of hypercholesterolemia on the brain. We previously demonstrated a modest accumulation of 27OHC in brains of patients with sporadic Alzheimer's disease (AD), consistent with a role of 27OHC as a primary pathogenetic factor. We show here that there is a 4-fold accumulation of 27OHC in different regions of the cortexes of patients carrying the Swedish amyloid precursor protein (APPswe) 670/671 mutation. The brain levels of sitosterol and campesterol were not significantly different in the AD patients compared with the controls, suggesting that the blood-brain barrier was intact in the AD patients. We conclude that accumulation of 27OHC is likely to be secondary to neurodegeneration, possibly a result of reduced activity of CYP7B1, the neuronal enzyme responsible for metabolism of 27OHC. We discuss the possibility of a vicious circle in the brains of the patients with familial AD whereby neurodegenerative changes cause an accumulation of 27OHC that further accelerates neurodegeneration.

Positron emission tomography imaging and clinical progression in relation to molecular pathology in the first Pittsburgh Compound B positron emission tomography patient with Alzheimer's disease.

The accumulation of ß-amyloid in the brain is an early event in Alzheimer's disease. This study presents the first patient with Alzheimer's disease who underwent positron emission tomography imaging with the amyloid tracer, Pittsburgh Compound B to visualize fibrillar ß-amyloid in the brain. Here we relate the clinical progression, amyloid and functional brain positron emission tomography imaging with molecular neuropathological alterations at autopsy to gain new insight into the relationship between ß-amyloid accumulation, inflammatory processes and the cholinergic neurotransmitter system in Alzheimer's disease brain. The patient underwent positron emission tomography studies with (18)F-fluorodeoxyglucose three times (at ages 53, 56 and 58 years) and twice with Pittsburgh Compound B (at ages 56 and 58 years), prior to death at 61 years of age. The patient showed a pronounced decline in cerebral glucose metabolism and cognition during disease progression, while Pittsburgh Compound B retention remained high and stable at follow-up. Neuropathological examination of the brain at autopsy confirmed the clinical diagnosis of pure Alzheimer's disease. A comprehensive neuropathological investigation was performed in nine brain regions to measure the regional distribution of ß-amyloid, neurofibrillary tangles and the levels of binding of (3)H-nicotine and (125)I-α-bungarotoxin to neuronal nicotinic acetylcholine receptor subtypes, (3)H-L-deprenyl to activated astrocytes and (3)H-PK11195 to microglia, as well as butyrylcholinesterase activity. Regional in vivo (11)C-Pittsburgh Compound B-positron emission tomography retention positively correlated with (3)H-Pittsburgh Compound B binding, total insoluble ß-amyloid, and ß-amyloid plaque distribution, but not with the number of neurofibrillary tangles measured at autopsy. There was a negative correlation between regional fibrillar ß-amyloid and levels of (3)H-nicotine binding. In addition, a positive correlation was found between regional (11)C-Pittsburgh Compound B positron emission tomography retention and (3)H-Pittsburgh Compound B binding with the number of glial fibrillary acidic protein immunoreactive cells, but not with (3)H-L-deprenyl and (3)H-PK-11195 binding. In summary, high (11)C-Pittsburgh Compound B positron emission tomography retention significantly correlates with both fibrillar ß-amyloid and losses of neuronal nicotinic acetylcholine receptor subtypes at autopsy, suggesting a closer involvement of ß-amyloid pathology with neuronal nicotinic acetylcholine receptor subtypes than with inflammatory processes.

Functional interactions of fibrillar and oligomeric amyloid-ß with alpha7 nicotinic receptors in Alzheimer's disease.

Amyloid-ß (Aß) peptides in the brain of patients with Alzheimer's disease (AD) assemble into various aggregation forms that differ in size, structure, and functional properties. Previous studies have shown that Aß binds to nicotinic acetylcholine receptors (nAChRs) and activates signaling cascades that result in the disruption of synaptic functions. These findings suggest a possible link between impaired cholinergic neurotransmitter function in AD and Aß pathogenesis. However, it is not yet known how the different Aß assemblies interact with specific nAChR subtypes. In the present study, we demonstrate that neurotoxicity in neuronal cells in culture induced by fibrillar Aß(1-40) is prevented through an α7 nAChR-dependent mechanism. The α7 nAChR agonists varenicline and JN403 increased binding of the amyloid ligand [³H]PIB to fibrillar Aß in AD frontal cortex autopsy tissue. This suggests that the presence of nAChR agonists may inhibit interaction of Aß with α7 nAChRs and prevent the formation of Aß/α7 nAChR complexes. This interaction was confirmed in binding assays with [¹²5I]Aß(1-40) and α7 nAChRs in autopsy brain tissue homogenates from the frontal cortex. The functional effects of Aß fibrils and oligomers on nAChRs were examined by measuring intracellular calcium ([Ca(2+)](i) levels. Oligomeric, but not fibrillar Aß(1-40), increased [Ca(2+)](i) in neuronal cells, and this effect was attenuated by varenicline. Our findings demonstrate that fibrillar Aß exerts neurotoxic effects mediated partly through a blockade of α7 nAChRs, whilst oligomeric Aß may act as a ligand activating α7 nAChRs, thereby stimulating downstream signaling pathways.

Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation.

The amyloid precursor protein (APP) has been mainly studied in its role in the production of amyloid ß peptides (Aß), because Aß deposition is a hallmark of Alzheimer's disease. Although several studies suggest APP has physiological functions, it is still controversial. We previously reported that APP increased glial differentiation of neural progenitor cells (NPCs). In the current study, NPCs transplanted into APP23 transgenic mice primarily differentiated into glial cells. In vitro treatment with secreted APP (sAPP) dose-dependently increased glial fibrillary acidic protein (GFAP) immuno-positive cells in NPCs and over expression of APP caused most NPCs to differentiate into GFAP immuno-positive cells. Treatment with sAPP also dose-dependently increased expression levels of GFAP in NT-2/D1 cells along with the generation of Notch intracellular domain (NICD) and expression of Hairy and enhancer of split 1 (Hes1). Treatment with γ-secretase inhibitor suppressed the generation of NICD and reduced Hes1 and GFAP expressions. Treatment with the N-terminal domain of APP (APP 1-205) was enough to induce up regulation of GFAP and Hes1 expressions, and application of 22 C11 antibodies recognizing N-terminal APP suppressed these changes by sAPP. These results indicate APP induces glial differentiation of NPCs through Notch signaling.

Β-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons.

Cognitive impairment in Alzheimer's disease (AD) patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs). Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the ß-amyloid (Aß) peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES) cells with nerve growth factor (NGF) as well as with fibrillar and oligomeric Aß1-40 and Aß1-42 (nM-µM concentrations) and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aß1-40 increased the number of functional neurons, whereas oligomeric Aß1-42 suppressed the number of functional neurons. Interestingly, oligomeric Aß exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aß1-40 and Aß1-42 induced gliogenesis. These findings indicate that Aß1-42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aß.

Neurotrophic factors promote cholinergic differentiation in human embryonic stem cell-derived neurons.

Cholinergic neurotransmission is essential for many important functions in the brain, including cognitive mechanisms. Here we demonstrate that human embryonic stem (hES) cells differentiate into a population of neuronal cells that express the cholinergic enzyme choline acetyltransferase and homeobox proteins specifying neuronal progenitors of ventral telencephalic lineage. These differentiated cells express transcripts for cholinergic alpha(3), alpha(4) and alpha(7) nicotinic acetylcholine (ACh) receptor subunits and for M1, M2 and M3 muscarinic acetylcholine receptor (mAChR) subtypes. Stimulation with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor and nerve growth factor increases the proportion of cholinergic neurons. These cholinergic receptors also mediate ACh-evoked increase in cytosolic calcium levels, and this response was unaffected by extracellular calcium removal and was abolished by the mAChR antagonist scopolamine. Our findings demonstrate expression of functional cholinergic receptors on hES cell-derived neurons, which may provide a source of expandable cells to facilitate screening of novel cholinergic drugs and useful for evaluating cell transplantation in animal models of cholinergic dysfunction.

Modulation of human neural stem cell differentiation in Alzheimer (APP23) transgenic mice by phenserine.

In a previous study, we found that human neural stem cells (HNSCs) exposed to high concentrations of secreted amyloid-precursor protein (sAPP) in vitro differentiated into mainly astrocytes, suggesting that pathological alterations in APP processing during neurodegenerative conditions such as Alzheimer's disease (AD) may prevent neuronal differentiation of HNSCs. Thus, successful neuroplacement therapy for AD may require regulating APP expression to favorable levels to enhance neuronal differentiation of HNSCs. Phenserine, a recently developed cholinesterase inhibitor (ChEI), has been reported to reduce APP levels in vitro and in vivo. In this study, we found reductions of APP and glial fibrillary acidic protein (GFAP) levels in the hippocampus of APP23 mice after 14 days treatment with (+)-phenserine (25 mg/kg) lacking ChEI activity. No significant change in APP gene expression was detected, suggesting that (+)-phenserine decreases APP levels and reactive astrocytes by posttranscription regulation. HNSCs transplanted into (+)-phenserine-treated APP23 mice followed by an additional 7 days of treatment with (+)-phenserine migrated and differentiated into neurons in the hippocampus and cortex after 6 weeks. Moreover, (+)-phenserine significantly increased neuronal differentiation of implanted HNSCs in hippocampal and cortical regions of APP23 mice and in the CA1 region of control mice. These results indicate that (+)-phenserine reduces APP protein in vivo and increases neuronal differentiation of HNSCs. Combination use of HNSC transplantation and treatment with drugs such as (+)-phenserine that modulate APP levels in the brain may be a useful tool for understanding mechanisms regulating stem cell migration and differentiation during neurodegenerative conditions in AD.

Retinoic acid and nerve growth factor induce differential regulation of nicotinic acetylcholine receptor subunit expression in SN56 cells.

Retinoic acid (RA) and nerve growth factor (NGF) have multiple functions in the regulation of neuronal development. In the present study, we characterized the expression of different nicotinic acetylcholine receptor (nAChR) subtypes in the cholinergic SN56 cell line and investigated the roles of RA and NGF in the expression of choline acetyltransferase (ChAT) and different nAChR subtypes. The nAChR agonist [(3)H]epibatidine was bound to two sites, with apparent affinities of 13 and 380 pM. RT-PCR analysis revealed expression of alpha3, alpha4, alpha5, alpha7, beta2, and beta4 nAChR subunits. RA treatment induced morphological changes, and the mRNA level of ChAT was maximally elevated after 4 days of exposure. The density of [(3)H]epibatidine binding sites and the mRNA and protein level of the alpha3 and beta2 nAChR subunits were also increased by RA-induced differentiation. RA down-regulated the mRNA and protein level of the alpha4 nAChR subunit, whereas no significant change was observed in the mRNA and protein level of the alpha7 nAChR subunit. NGF treatment increased the mRNA and protein level of the alpha3 and beta2 nAChR subunits. No morphological effects of NGF were observed, and the mRNA level of ChAT and mRNA and protein level of the alpha4 and alpha7 nAChR subunits were not significantly altered. Validation was performed with real-time RT-PCR. The present results show that RA and NGF have different effects on the expression of ChAT and the morphology and the expression pattern of different nAChR subunits in cholinergic SN56 cells.