Radioiodinated styrylbenzene derivatives as potential SPECT imaging agents for amyloid plaque detection in Alzheimer's disease

Development of probes for β-amyloid (Aβ) plaques, a critical factor associated with Alzheimer’s disease (AD), provides important tools for studying their role in AD. Previously, we reported [¹²⁵I]IMSB and [¹²⁵I]ISB as excellent probes for Aβ plaque labeling. Despite their exquisite in vitro binding characteristics, low brain uptakes (likely due to two ionizable carboxylic acid groups) limited their potential as in vivo imaging agents. To improve brain penetration, we have successfully prepared a neutral radioiodinated probe [¹²⁵I]3. The improved probe displayed good binding affinity for Aβ aggregates (K_i=2.0 ± 0.2 using Aβ40 aggregates). In addition, the brominated counterpart displayed fluorescent-staining properties of Aβ plaques in postmortem AD brain sections similar to BSB, a fluoroscent probe reported previously. [¹²⁵I]3 gave excellent plaque labeling by film autoradiography of AD brain sections. Unlike [¹²⁵I]IMSB (which preferentially detects Aβ40 plaques), the improved radioioinated probe, [¹²⁵I]3, can readily detect plaques containing aggregates of both Aβ40 and Aβ42. The initial brain uptake of [¹²⁵I]3 in normal mice at 2 min p.i. was moderate (0.18% ID) and displayed a very slow washout from the brain (0.11 %.ID at 4 h p.i). Taken together, these data suggest that [¹²⁵I]3 is useful for in vitro plaque detection, it may not be suitable for in vivo monitoring of Aβ progression and deposition.     

The relationship between amyloid and tau

Based on genetic findings, the relationship between the APP/Aβ and tau/tangle pathologies are discussed. It is argued that APP/Aβ is upstream of tau/tangle in the Alzheimer pathogenesis, and that the relationship between the pathologies are promiscuous in two ways: first, APP/Aβ can equally be seen to be upstream of synuclein/Lewy bodies in cell death pathways, and second, tau pathology can be initiated by genetic lesions in other pathways.     

Characterization of Aβ11-40/42 peptide deposition in Alzheimer’s disease and young Down’s syndrome brains: implication of N-terminally truncated Aβ species in the pathogenesis of Alzheimer’s disease

Senile plaques (SPs), one of two defining lesions of Alzheimer’s disease (AD), are composed of a mixture of full-length Aβ1-40/42, and N- or C-terminally truncated Aβ peptides, including Aβ11-40/42. Sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases produces Aβ1-40/42, but β-site APP-cleaving enzyme 1 (BACE1), the major β-secretase, also generates Aβ11-40/42, and BACE1 overexpression in cultured cells results primarily in secretion of Aβ11-40/42. The ratio of Aβ11-40/42 to Aβ1-40/42 depends on the ratio of BACE1 to APP, and Aβ11-40/42 can be generated from both full-length APP and its carboxy-terminal fragment (C99). Here, we investigated the role of Aβ11-40/42 in the pathogenesis of AD and Down’s syndrome (DS) brains. We demonstrated significant amount of Aβ11-42 in DS brains by Western blots. While pyroAβ11-42-modified Aβ species existed predominantly in mature SP cores in AD brain sections, both unmodified free Aβ11-40 and pyro-modified Aβ11-40 are detected in vascular amyloid deposits by immunohistochemistry. Using novel ELISAs for quantifying free Aβ11-40/42 and pyroAβ11-40/42, we showed that insoluble Aβ11-42 predominated in extracts of AD and DS brains. This is the first systematic study of Aβ11-40/42 in neurodegenerative Aβ amyloidosis implicating Aβ11-40/42 in SP formation of AD and DS brains. The detection of Aβ11-42 in young DS brain suggests an early role for this N-terminally truncated Aβ peptide in the pathogenesis of SPs in AD and DS.     

Microglial activation in early stages of amyloid β protein deposition

The present study was undertaken to investigate the relationship of microglial activation to amyloid β protein (Aβ) deposition, particularly at the early stage. Using single and double immunostaining methods with a panel of microglia markers and antibodies against Aβ and amyloid β protein precursor (APP), we examined the cerebrum and cerebella of both Alzheimer’s disease (AD) and non-demented subjects obtained at autopsy. In non-demented, middle-aged subjects that had small amounts of cerebral Aβ deposits, approximately 70% of the diffuse plaques contained ramified microglia. However, no evidence of microglial activation was found in diffuse plaques in any of the non-demented subjects. Dual immunostaining of sections of cerebral cortex using antibodies against Aβ and major histocompatibility complex class II antigen showed that in AD subjects, approximately 20% of total diffuse plaques contained a few, activated microglia. Most of these plaques were defined as a transitional form between diffuse and primitive plaques. Both primitive and classic plaques in the cerebral cortex of AD subjects consistently contained clusters of activated microglia. Subpial Aβ deposits without neuritic changes lacked microglial activation. In the cerebellum, all of the diffuse plaques lacked microglial activation, and activated microglia in the compact plaques were not as hypertrophic as those in cerebral primitive/classic plaques. Our findings indicate that microglial reactions are absent in the early stages of Aβ deposition, and it occurs during the transition from diffuse to primitive plaques, when amounts of Aβ deposits and the degree of neuritic changes increase. Received: 17 January 1997 / Revised, accepted: 7 April 1997     

Coexistence of Alzheimer-type neuropathology in Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) and Alzheimer’s disease (AD) share clinical, neuropathological, and pathogenetic features. To investigate eventual mutual influences, we screened prominently affected neocortex from 110 neuropathologically proven CJD patients for Alzheimer-type pathology with anti-β/A4, Bielschowsky and anti-tau (immuno)stains. The neuropathological classification of Alzheimer-type pathology was made according to the CERAD criteria. Results were controlled by comparison with Alzheimer-type changes in sections from the same cortical areas in 110 sex- and age-matched non-demented control patients. For comparison, the control patients were also classified according to the CERAD neuropathology criteria as if they had been demented. Alzheimer-type tissue changes as in definite and probable CERAD AD occur in 10.9% of the CJD patients and 19.1% of control patients (P = 0.11). The median age of CJD and control patients with CERAD AD is 72 and 68 years, respectively, which differs significantly from the median ages of 64 and 63 years, respectively, in the non-AD/CJD and non-AD control patients. Since CERAD criteria include “presence of other neuropathological lesions likely to cause dementia”, an AD diagnosis in CJD patients (all of whom are demented) is solely based on densities of neuritic plaques. Similar Alzheimer-type changes in even higher frequency, however, are also present in elderly non-demented controls. Thus, the coexistence of Alzheimer-type pathology in CJD most likely represents an age-related change. Deposits of prion protein (PrP) frequently accumulate at the periphery of β/A4 plaques. The presence of β/A4 amyloid in the brain may influence PrP morphogenesis. Received: 18 November 1997 / Revised, accepted: 11 February 1998     

Changes in localization of synaptophysin following fluid percussion injury in the rat brain

Traumatic brain injuries damage neurons and cause progressing dysfunctions of the brain. Synaptophysin (SYP), a major integral transmembrane protein of synaptic vesicles, provides a molecular marker for the synapse and serves as a functional marker of the brain. This study examined magnitude-dependent changes of SYP in the rat brain 2 days following low, moderate or high fluid percussion injuries and investigated time-dependent changes of SYP in the rat brain with moderate fluid percussion injury 2, 15 and 30 days after trauma using immunohistochemistry and Western blotting. SYP immunoreactivity increased in the lateral cortex and in the subcortical white matter, with increasing magnitude of injury and time after trauma. Increased SYP immunoreactivity was accompanied with degeneration of neuronal cell bodies, their processes and terminals as well as glial cell proliferations. Amounts of SYP measured by Western blotting remained unchanged in brains with moderate fluid percussion within 30 days after trauma. These findings indicate that trauma accumulates SYP at injured sites of neurons without changing SYP contents and that increased SYP immunoreactivity in the cerebral cortex following traumatic injury reflects an inhibition of synaptic vesicle transportation and dysfunction of synapses, thus providing a histological substrate for brain dysfunctions.     

Koch’s postulates and infectious proteins

Koch’s postulates were formulated in the late nineteenth century as guidelines for establishing that microbes cause specific diseases. Because the rules were developed for living agents—particularly bacteria—their applicability to inanimate pathogens such as viruses and infectious proteins has been problematic. The unorthodox mechanism by which prion diseases are transmitted, involving specific physicochemical characteristics of the protein as well as susceptibility traits of the host, has made these disorders refractory to analysis within the context of the original Koch’s postulates. In addition, evidence is accumulating that other proteopathies, such as AA amyloidosis, apolipoprotein AII amyloidosis, and cerebral Aβ amyloidosis, can be induced in vulnerable recipients by cognate proteinaceous agents. In light of the salient differences in the mode of disease-transmission by microbes and proteins, we propose modifications of Koch’s postulates that will specifically accommodate presumed infectious proteins.     

Mirror asymmetry of Category and Letter fluency in traumatic brain injury and Alzheimer's patients

In this study we contrasted the Category fluency and Letter fluency performance of 198 normal subjects, 57 Alzheimer’s patients and 57 patients affected by traumatic brain injury (TBI). The aim was to check whether, besides the prevalence of Category fluency deficit often reported among Alzheimer’s patients, the TBI group presented the opposite dissociation. According to some recent claims, in fact, the deficit of TBI would be equally severe for both fluency types. The inquiry followed different approaches for data analysis, including the evaluation of a unique index (Fluency Type Index or FTI), independent of the overall fluency and aimed at expressing at individual subject level the relationship between Category and Letter fluency. The results confirmed that Alzheimer’s patients are more defective on Category than Letter fluency, and also clearly indicated that an opposite pattern applies to TBI patients. TBI seems to cause a relatively more severe impairment of Letter than Category fluency, probably due to its impact on the frontal lobe structures. We discuss whether, on the basis of the statistical distribution of our data, it is worth considering as homogeneous populations broadly defined groups as Alzheimer’s or TBI patients.     

Quantitative analysis of tau protein-immunoreactive accumulations and β amyloid protein deposits in the cerebral cortex of the mouse lemur, Microcebus murinus

Recent studies have revealed the presence of tau protein-immunoreactive accumulations and β amyloid protein (Aβ) deposits in the cerebral cortex of the aged mouse lemur, Microcebus murinus. To examine the age-related evolution of these changes and compare their regional distribution to that reported for humans and nonhuman primates with Alzheimer’s disease lesions, we performed a quantitative analysis of a large series of mouse lemurs aged from 1 to 13 years. The prevalence and density of tau protein-immunoreactive accumulations in the neocortex of this prosimian increased steadily with age. Neocortical areas were frequently affected even in young mouse lemurs, whereas the subiculum and entorhinal cortex were only involved occasionally in animals older than 8 years. As in anthropoid primates, diffuse Aβ deposits were often observed in the cerebral cortex and amygdala of old mouse lemurs. Although all animals with diffuse Aβ deposits had tau protein-immunoreactive accumulations in the neocortex, no correlation was found between the densities of these lesions in each area and among the areas studied. The age-dependent progression of tau protein-immunoreactive accumulations indicates that this prosimian may represent a valuable model for the study of the biochemical mechanisms of brain aging, while the relative sparing of hippocampus in mouse lemurs contrasts sharply with previous reports on neurofibrillary tangle formation in humans, and suggests that this animal may also be useful to investigate the biological characteristics of neuroprotection in this area. Furthermore, the present data indicate that Aβ deposition in mouse lemurs is not age dependent, but occurs in a few vulnerable old animals. Received: 11 November 1996 / Revised, accepted: 20 January 1997     

Circulating beta amyloid protein is elevated in patients with acute ischemic stroke

Summary. Recent clinical and experimental studies suggest that ischemic strokes may play an important role in the pathogenesis of Alzheimer’s disease (AD). Beta amyloid (Aβ), a major component of senile plaque in AD, is known to be derived from ischemic brain or activated platelets. We prospectively enrolled 62 patients with acute ischemic stroke and 27 age-matched controls. The serum Aβ and P-selectin levels were determined using the Sandwich-ELISA. We divided ischemic strokes into subgroups according to the clinical syndrome, pathogenesis, and infarct size, and compared the Aβ level between each subgroup. The Aβ1–40 level was markedly elevated in ischemic stroke patients, as compared to controls (140.2 ± 54.0 vs 88.44 ± 34.96 pg/ml, p<0.001). Cardioembolic and larger artery atherosclerotic infarcts had higher Aβ1–40 level than small vessel disease (p = 0.001). Both infarct size and the initial NIHSS score had significantly positive correlations with the serum level of Aβ1–40 (r = 0.539, p<0.001 and r = 0.425, p = 0.001, respectively). However, the P-selectin level was not significantly correlated with serum Aβ1–40. Our data suggest that elevated circulating Aβ1–40 in ischemic stroke patients may be derived from brain as a consequence of ischemic insults.     

Diffuse plaques associated with astroglial amyloid β protein, possibly showing a disappearing stage of senile plaques

To clarify whether senile plaques disappear, we examined amyloid β protein (Aβ) deposits in non-demented subjects, and found novel diffuse plaques associated with astroglial Aβ. Formalin-fixed paraffin-embedded sections from cortical areas were immunolabeled with a panel of Aβ antibodies, and astroglial and microglial markers. Cerebral Aβ deposition was primarily found as diffuse plaques (DP) in these subjects. A subset of DP was associated with clusters of intensely Aβ-positive small granules. The clusters, which were located just adjacent to astroglial nucleus, had the characteristics of lipofuscin granules and, therefore, were quite different from “small stellate deposits”. Substantial amounts of Aβ-positive granules were found inside astrocytes by dual labeling of Aβ and glial fibrillary acid protein, and the majority of astroglial Aβ immunoreactivity was located on lipofuscin granules. Aβ-positive granules lacked immunoreactivity with antisera for the N-terminal region of Aβ. These peculiar DP showed a much weaker staining than ordinary DP. The DP associated with astroglial Aβ were found in about one third of the subjects, although the density varied widely among individuals. From these findings, we propose that DP, which are associated with the N-terminal truncated Aβ in astrocytes, represent the disappearing stage of senile plaques. Received: 1 July 1997 / Revised: 27 August 1997 / Accepted: 4 November 1997     

Synthesis and evaluation of 2-(3'-iodo-4'-aminophenyl)-6-hydroxybenzothiazole for in vivo quantitation of amyloid deposits in Alzheimer's disease

A potent and brain permeable amyloid ligand has been identified as a lead compound capable of I-123/125-labelling for single photon emission computed tomography (SPECT) imaging. In this study, we report the synthesis and I-125-radiolabelling of Compound 6 and its in vitro and in vivo properties. Compound 6 [2-(3′-iodo-4′-aminophenyl)-6-hydroxybenzothiazole] bound to synthetic Aβ_(1–40) fibrils in a saturable manner, exhibiting an affinity (Ki) of 11±1.1 nM in a competitive binding assay using a tritiated thioflavin T analog ([3H]BTA-1) as radioligand. [¹²⁵I]6 binding to synthetic Aβ_(1–40) fibrils fit a single-site model. [¹²⁵I]6 exhibited several-fold higher binding to homogenates of frontal cortex from post-mortem Alzheimer’s disease brain relative to age-matched control brain homogenates. No difference in binding was observed in cerebellum. The ratio of radioactivity concentration between frontal cortex and cerebellum was 6-fold higher in AD brain homogenates than the age-matched control. [¹²⁵I]6 also readily penetrated the blood-brain barrier in normal control mice with an average radioactivity concentration of 6.43 ± 0.62%ID/g detected in the whole brain at 2 min post i.v. injection. At 30 min, the radioactivity concentration decreased to 0.40 ± 0.05%ID/g, indicating good clearance in the absence of amyloid deposits in the brain. Dedicated to Dr. Hong-Sun Uh, President of E-Sung Chemicals Co. Ltd., on the occasion of his 65th birthday, with admiration of a great teacher and friend.     

Soluble amyloid precursor protein alpha reduces neuronal injury and improves functional outcome following diffuse traumatic brain injury in rats

Amyloid precursor protein (APP) has previously been shown to increase following traumatic brain injury (TBI). Whereas a number of investigators assume that increased APP may lead to the production of neurotoxic Abeta and be deleterious to outcome, the soluble alpha form of APP (sAPPalpha) is a product of the non-amyloidogenic cleavage of amyloid precursor protein that has previously been shown in vitro to have many neuroprotective and neurotrophic functions. However, no study to date has addressed whether sAPPalpha may be neuroprotective in vivo. The present study examined the effects of in vivo, posttraumatic sAPPalpha administration on functional motor outcome, cellular apoptosis, and axonal injury following severe impact-acceleration TBI in rats. Intracerebroventricular administration of sAPPalpha at 30 min posttrauma significantly improved motor outcome compared to vehicle-treated controls as assessed using the rotarod task. Immunohistochemical analysis using antibodies directed toward caspase-3 showed that posttraumatic treatment with sAPPalpha significantly reduced the number of apoptotic neuronal perikarya within the hippocampal CA3 region and within the cortex 3 days after injury compared to vehicle-treated animals. Similarly, sAPPalpha-treated animals demonstrated a reduction in axonal injury within the corpus callosum at all time points, with the reduction being significant at both 3 and 7 days postinjury. Our results demonstrate that in vivo administration of sAPPalpha improves functional outcome and reduces neuronal cell loss and axonal injury following severe diffuse TBI in rats. Promotion of APP processing toward sAPPalpha may thus be a novel therapeutic strategy in the treatment of TBI.     

Differential pattern of β-amyloid, amyloid precursor protein and apolipoprotein E expression in cortical senile plaques

Regional differences in senile plaques immunostained by antibodies against β-amyloid A4 (β-A4), amyloid precursor protein 695 (APP) and apolipoprotein E (apo E) were studied in the hippocampus and the entorhinal, temporal and occipital cortices both quantitatively and semiquantitatively with respect to the laminar cortical distribution of the plaques. These patterns were related to the staging of Alzheimer’s disease in regard to the distribution of neurofibrillary tangles [Braak and Braak (1991) Acta Neuropathol 82: 239–259]. In the hippocampus and especially in sector CA 1, no significant differences in the number of plaques visualized by the different antibodies were found. In contrast, there was a striking difference in neocortical regions. Here, significantly higher numbers of plaques positive for β-A4 than that for APP and apo E were present in all stages, except in the stages I and VI, and for apo E in stage II. The highest densities of β-A4-positive plaques were found in the isocortical layers III and V and in the entorhinal pre-α, pre-γ, pri-α and pri-β layers. The preferentially affected area, showing plaques positive for all three antibodies, was the entorhinal-hippocampal circuit with early affection of CA 1, which represents the direct and indirect target of the entorhinal neurons of the upper layers. Therefore, we suggest that plaques with dystrophic neurites, positive for APP, seem to be generated secondarily in afferent areas such as the hippocampus, which is the main afferent target of the entorhinal region. Diffuse plaques, negative for APP and apo E, are virtually absent in the CA 1 and seem to originate independently of afferent neuronal dysfunction, as indicated by neurofibrillary tangles. Received: 21 January 1996 / Revised: 18 July 1996, 13 November 1996 / Accepted: 27 November 1996     

p38 Mitogen-Activated Protein Kinase Regulates Myelination

Most neurodegenerative disorders are characterised by deposits of aggregated proteins that are readily visualised by light microscopy. Although the presence of such a bulky structure inside the cell or in the extracellular space is likely not to be healthy, over recent years the idea has emerged that these end-stage aggregates are a relatively safe way to deposit harmful aberrant proteins. Protein quality control is a multi-level security system to safeguard cells from aberrant proteins and is therefore a protective response. However, protein quality control may turn destructive in case of impairment of protein quality control for example by aging or because of overflow of the quality control systems due to prolonged exposure. In many cases the medicine is worse than the cause and the “protective” response of the cell to aggregates kills the cell, rather than the aggregate itself. Here we review the role of protein quality control in neurodegeneration and aim to distinguish protective and destructive responses to aggregates in order to find targets for therapeutic intervention.     

Intracerebral hemorrhage in two patients with Down’s syndrome and cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) is an important cause of spontaneous intracerebral hemorrhages in the elderly and is often seen in the brains of patients with Alzheimer’s disease, Down’s syndrome (DS), and hereditary cerebral hemorrhage with amyloidosis of the Dutch type. We report two patients with DS and extensive CAA who died of intracerebral hemorrhage; only two other such case reports exist in the literature. We believe the incidence of such cases is higher than is reported and that the likelihood of hemorrhage in the setting of CAA is independent of the patient’s underlying disease. Received: 27 March 1997 / Revised, accepted: 8 August 1997     

Immunohistochemical demonstration of tissue transglutaminase in amyloid plaques

The brain of Alzheimer’s disease patients contains deposits of the 39–42-amino acid (∼ 4 kDa) amyloid β-peptide, which is derived from the β-amyloid precursor protein. These pathological deposits have been shown to consist in part of insoluble 8- and 16-kDa aggregates of the amyloid β-peptide. This report confirms that the amyloid β-peptide is a substrate for tissue transglutaminase (TGase) and demonstrates that human brain preparations from Alzheimer’s disease patients and control patients form cross-linked dimers from added iodinated amyloid β-peptide. Immunohistochemical staining for TGase revealed its presence in tissue sections and isolated amyloid plaque cores obtained from brains of patients diagnosed as having Alzheimer’s disease. These results provide evidence that the previously described insoluble amyloid deposits in Alzheimer’s disease may involve TGase-mediated cross-linked amyloid β-peptide polymers, and suggest a potential role for TGase in the pathogenesis of this disease. Received: 23 September 1996 / Revised: 16 February 1998 / Accepted: 16 March 1998     

Should the guidelines for monitoring serum cholesterol levels in the elderly be re-evaluated?

Elevated circulating cholesterol can have profound effects on the health of an individual. Such excess cholesterol can promote coronary artery disease, production and accumulation of β-amyloid in the brain, and possibly Alzheimer’s disease (AD). In a clinical trial evaluating the benefit of a cholesterol-lowering drug in the treatment of AD, mean cholesterol levels at baseline among individuals participating in the trial were found to be relatively high. Based on this observation we suggest that cholesterol levels should be actively monitored in the elderly, as many individuals with AD are over 65 years of age and therefore excluded by currently accepted guidelines.     

A novel imaging probe for in vivo detection of neuritic and diffuse amyloid plaques in the brain

Extensive deposition of neuritic and diffuse amyloid plaques in the brain is a critical event for the pathogenesis of Alzheimer’s disease (AD) and considered to start before the appearance of clinical symptoms. In vivo detection of these brain β-amyloid (Aβ) deposits using positron emission tomography (PET), therefore, would be a useful marker for presymptomatic detection of AD. To develop a new agent for PET probe of imaging neuritic and diffuse amyloid deposits, novel fluorescent compounds, including styryl-fluorobenzoxazole derivatives, were examined. These compounds showed a high binding affinity for both synthetic Aβ1-40 and Aβ1-42 aggregates. Some of these compounds also displayed distinct staining of neuritic and diffuse amyloid plaques in AD brain sections. A biodistribution study of styryl-fluorobenzoxazole derivatives in normal mice exhibited excellent brain uptakes (4.5–5.5% injected dose/g at 2 min postinjection). Furthermore, iv administration of BF-145, a styryl-fluorobenzoxazole derivative, demonstrated specific in vivo labeling of compact and diffuse amyloid deposits in an APP23 transgenic mouse brain, in contrast to no accumulation in a wild-type mouse brain. These findings suggest that BF-145 is a potential candidate as a probe for imaging early brain pathology in AD patients.