Amelia Marutle

Laminar distribution of nicotinic receptor subtypes in cortical regions in schizophrenia

The laminar cortical distribution of the [125I]alpha-bungarotoxin, [3H]cytisine and [3H]epibatidine nicotinic acetylcholine receptor ligands was investigated by quantitative autoradiography in autopsy tissue from the cingulate, orbitofrontal and temporal cortices of control and schizophrenia subjects matched for age and smoking history. Different laminar binding patterns were observed for the various nicotinic ligands both in schizophrenic and control brains. [125I]alpha-Bungarotoxin binding was distributed homogeneously across all cortical layers in all three brain regions, with highest binding densities in the cingulate cortex. [3H]Cytisine and [3H]epibatidine binding varied across the cortical ribbon, with high binding in layers I, III, V and VI, within the three cortical regions. A significantly reduced [125I] alpha-bungarotoxin binding (-54%) was observed in the cingulate cortex of schizophrenia subjects, in comparison with normal individuals who smoked tobacco. In the same brain region also a significantly higher [3H]cytisine binding (48-77%) was observed in nearly all layers, except for layer I of the schizophrenia subjects, when compared to normal individuals with a history of tobacco use. No significant changes in [3H]epibatidine binding was observed within the individual cortical layers between control subjects and patients with schizophrenia, but when calculated as a whole region (i.e. measurements performed across the whole cortical ribbon), the temporal cortex showed a significant increase in [3H]epibatidine binding in schizophrenia subjects compared to control subjects. The results suggest opposite changes of the alpha4beta2 and alpha7 nicotinic receptor subtypes in the cingulate cortex of patients with schizophrenia which might reflect involvement of two different nicotinic receptor mechanisms in schizophrenia brain.

Regional distribution of subtypes of nicotinic receptors in human brain and effect of aging studied by (±)-[3H]epibatidine

Epibatidine, a potent nicotinic agonist, was used to study the regional distribution of nicotinic acetylcholine receptor binding sites in the human brain. Saturation studies performed in the human temporal cortex with (+/-)-[3H]epibatidine revealed binding to two binding sites with Kd and Bmax values of 0.018 and 4.2 nM, 12.7 and 15.4 fmol/mg protein, respectively. Competition studies with (+/-)-[3H]epibatidine/unlabelled nicotine or [3H]nicotine/unlabelled (+/-)-epibatidine showed binding to two binding sites in the human temporal cortex (Ki=0.16 and 12.6 nM; 0.007 and 0.3 nM, respectively). Similarly, when unlabelled nicotine was used to displace (+/-)-[3H]epibatidine, two binding sites were also revealed in the thalamus and the cerebellum of human brain (Ki=0.065 and 7.7 nM; 0.07 and 12.5 nM, respectively). The regional binding of (+/-)-[3H]epibatidine binding in human brain was somewhat different from that of [3H]nicotine. A proportionally higher binding was observed for (+/-)-[3H]epibatidine in the cerebellum and the thalamus compared to [3H]nicotine, probably reflecting different selectivity to nicotinic receptor subtypes. A marked significant age-related decrease in (+/-)-[3H]epibatidine binding was observed in the frontal and the temporal cortices (-79%, -84%, respectively) of human subjects between 56-85 years of age, which was similar to that of [3H]nicotine (-82%, -79%, respectively). The (+/-)-[3H]epibatidine binding in the cerebellum decreased significantly with age (-77%), while [3H]nicotine binding showed no significant age-related changes in this brain region. The findings indicate that a specifically modulate regional nicotinic receptors in human brain.

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 Alzheimers 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.

Different β-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 β-amyloid (Aβ) oligomer assemblies in autopsy brain with the levels of fibrillar Aβ and cholinergic synaptic function. Brain tissues obtained from the frontal cortex of 14 Alzheimers 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 Aβ oligomers in soluble (TBS), detergent soluble (TBST), and insoluble (GuHCl) fractions. The predominant oligomeric Aβ 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 Aβ oligomer assemblies and fibrillar Aβ 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 Aβ oligomers in the insoluble fraction in EOAD patients, and with decamers in the soluble fraction in LOAD patients. These novel findings suggest that distinct Aβ oligomers induce impairment of cholinergic neurotransmission in AD pathogenesis.

Selective Nicotinic Receptor Consequences in APPSWE Transgenic Mice

The nicotinic (nAChRs) and muscarinic (mAChRs) acetylcholine receptors and acetylcholinesterase (AChE) activity were studied in the brains of APP(SWE) transgenic mice (Tg+) and age-matched nontransgenic controls (Tg-) that were between 4 and 19 months of age. A significant increase in the binding of 125I-labeled alpha-bungarotoxin (alpha7 nAChRs) was observed in most brain regions analyzed in 4-month-old Tg+ mice, preceding learning and memory impairments and amyloid-beta (Abeta) pathology. The enhanced alpha7 receptor binding was still detectable at 17-19 months of age. Increase in [3H]cytisine binding (alpha4beta2 nAChRs) was measured at 17-19 months of age in Tg+ mice, at the same age when the animals showed heavy Abeta pathology. No significant changes in [3H]pirenzepine (M1 mAChRs) or [3H]AFDX 384 (M2 mAChRs) binding sites were found at any age studied. The upregulation of the nAChRs probably reflects compensatory mechanisms in response to Abeta burden in the brains of Tg+ mice.

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

Imaging fibrillar amyloid-β deposition in the human brain in vivo by positron emission tomography has improved our understanding of the time course of amyloid-β pathology in Alzheimers disease. The most widely used amyloid-β imaging tracer so far is (11)C-Pittsburgh compound B, a thioflavin derivative but other (11)C- and (18)F-labelled amyloid-β tracers have been studied in patients with Alzheimers disease and cognitively normal control subjects. However, it has not yet been established whether different amyloid tracers bind to identical sites on amyloid-β fibrils, offering the same ability to detect the regional amyloid-β burden in the brains. In this study, we characterized (3)H-Pittsburgh compound B binding in autopsied brain regions from 23 patients with Alzheimers 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 Alzheimers 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 Alzheimers 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 Alzheimers 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 Alzheimers 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 Alzheimers disease.

Neuronal Nicotinic Receptor Deficits in Alzheimer Patients with the Swedish Amyloid Precursor Protein 670/671 Mutation

Abstract : The influence of β‐amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (‐73 to ‐87%) as well as in sporadic Alzheimers disease (AD) cases (‐37 to ‐57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both α3 and α4 and to α4 nAChR subtypes, respectively. Saturation binding studies with [3epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (‐82%) for the high‐affinity site was observed in APP 670/671 subjects with no change in KD compared with controls (0.018 nM APP 670/671 ; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.

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 Alzheimers 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.

Age-Dependent Neuroplasticity Mechanisms in Alzheimer Tg2576 Mice Following Modulation of Brain Amyloid-β Levels

The objective of this study was to investigate the effects of modulating brain amyloid-β (Aβ) 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) APPSWE transgenic (Tg2576) mice were treated with the AD candidate drug (+)-phenserine for 16 consecutive days. We found significant reductions in insoluble Aβ1-42 levels in the cortices of both young and older transgenic mice, while significant reductions in soluble Aβ1-42 levels and insoluble Aβ1-40 levels were only found in animals aged 15–18 months. Autoradiography binding with the amyloid ligand Pittsburgh Compound B (3H-PIB) revealed a trend for reduced fibrillar Aβ deposition in the brains of older phenserine-treated Tg2576 mice. Phenserine treatment increased cortical synaptophysin levels in younger mice, while decreased interleukin-1β and increased monocyte chemoattractant protein-1 and tumor necrosis factor-alpha levels were detected in the cortices of older mice. The reduction in Aβ1-42 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 Aβ1-42 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 Aβ levels in Tg2576 mice when Aβ plaque pathology is prominent mainly alters the levels of proinflammatory cytokines and chemokines.

β-Amyloid 1-42 Oligomers Impair Function of Human Embryonic Stem Cell-Derived Forebrain Cholinergic Neurons

Cognitive impairment in Alzheimers 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 β-amyloid (Aβ) 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 Aβ1-40 and Aβ1-42 (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 Aβ1–40 increased the number of functional neurons, whereas oligomeric Aβ1–42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1–40 and Aβ1–42 induced gliogenesis. These findings indicate that Aβ1–42 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 Aβ.