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To investigate whether longitudinal declines in cognition are associated with higher fibrillar amyloid-beta (A) deposition in vivo in individuals without dementia.

Aims: One promising approach for treatment of Alzheimers disease (AD) is use of anti-amyloid therapies, based on the hypothesis that increases in amyloid-beta (A) deposits in brain are a major cause of AD. Several groups have focused on A immunotherapy with some success. Small molecules derivatives of Congo red have been shown to inhibit A aggregation and protect against A neurotoxicity in vitro. The agents described here are all small molecule A- binding agents (SMABAs) derivatives of Congo red. Main Methods: Here, we have explored the anti-amyloid properties of these SMABAs in mice doubly transgenic for human prensenilin-1 (PS1) and APP gene mutations that cause early-onset AD. Mice were treated with either methoxy- X04, X:EE:B34 and X:034-3-OMe1. After treatment, brains were examined for A-deposition, using histochemistry, and soluble and insoluble A levels were determined using ELISA. Key Findings: A range of anti-amyloid activity was observed with these three compounds. PS1/APP mice treated with methoxy-X04 and X:EE:B34 showed decrease in total A load, a decrease in A fibril load, and a decrease in average plaque size. Treatment with methoxy-X04 also resulted in a decrease in insoluble A levels. The structurally similar compound, X:034:3-OMe1, showed no significant effect on any of these measures. The effectiveness of the SMABAs may be related to a combination of binding affinity for A and entry into brain, but other factors appear to apply as well. Significance: These data suggest that SMABAs may significantly decrease amyloid burden in brain during the pathogenesis of AD and could be useful therapeutics alone, or in combination with immunotherapy.

Aims: One promising approach for treatment of Alzheimer's disease (AD) is use of anti-amyloid therapies, based on the hypothesis that increases in amyloid-beta (Aβ) deposits in brain are a major cause of AD. Several groups have focused on Aβ immunotherapy with some success. Small molecules derivatives of Congo red have been shown to inhibit Aβ aggregation and protect against Aβ neurotoxicity in vitro. The agents described here are all small molecule Aβ- binding agents (SMAβBA's) derivatives of Congo red. Main Methods: Here, we have explored the anti-amyloid properties of these SMAβBA's in mice doubly transgenic for human prensenilin-1 (PS1) and APP gene mutations that cause early-onset AD. Mice were treated with either methoxy- X04, X:EE:B34 and X:034-3-OMe1. After treatment, brains were examined for Aβ-deposition, using histochemistry, and soluble and insoluble Aβ levels were determined using ELISA. Key Findings: A range of anti-amyloid activity was observed with these three compounds. PS1/APP mice treated with methoxy-X04 and X:EE:B34 showed decrease in total Aβ load, a decrease in Aβ fibril load, and a decrease in average plaque size. Treatment with methoxy-X04 also resulted in a decrease in insoluble Aβ levels. The structurally similar compound, X:034:3-OMe1, showed no significant effect on any of these measures. The effectiveness of the SMAβBA's may be related to a combination of binding affinity for Aβ and entry into brain, but other factors appear to apply as well. Significance: These data suggest that SMAβBA's may significantly decrease amyloid burden in brain during the pathogenesis of AD and could be useful therapeutics alone, or in combination with immunotherapy.

Extrapyramidal motor symptoms precede dementia in Parkinson disease (PDD) by many years, whereas dementia occurs early in dementia with Lewy bodies (DLB). Despite this clinical distinction, the neuropsychological and neuropathologic features of these conditions overlap. In addition to widespread distribution of Lewy bodies, both diseases have variable burdens of neuritic plaques and neurofibrillary tangles characteristic of Alzheimer disease (AD).

To compare brain β-amyloid (Aβ) burden measured with 11CPittsburgh Compound B (PIB) PET in normal aging, Alzheimer disease (AD), and other dementias.

The PET tracer 11C-labeled Pittsburgh Compound-B (11C-PIB) specifically binds fibrillar amyloid-beta (A) plaques and can be detected in Alzheimer disease (AD). We hypothesized that PET imaging with 11C-PIB would discriminate AD from frontotemporal lobar degeneration (FTLD), a non-A dementia.

Beta-amyloid (Aβ) plaques are the hallmark of Alzheimer disease (AD). A PET imaging tracer that binds to Aβ plaques in vivo, N-methyl-11C2-(4′-methylaminophenyl)-6-hydroxybenzothiazole (or 11CPIB for “Pittsburgh Compound-B”), has significantly higher binding in subjects diagnosed with dementia of the Alzheimer type (DAT) compared to nondemented controls. The authors used this imaging technique to investigate whether abnormal binding occurs in clinically normal individuals, prior to the development of cognitive changes.

The development of radioligands to image β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) in vivo in the aging human brain is an important and active area of radiopharmaceutical design. When used in combination with positron emission tomography (PET) or single photon emission computed tomography (SPECT), amyloid-imaging tracers could facilitate the evaluation of the efficacy of anti-amyloid therapies currently under intense development by many major pharmaceutical companies throughout the world. Amyloid-imaging agents could also serve as surrogate markers in early diagnosis and neuropathogenesis studies of Alzheimer's disease and other aging-related neurodegenerative disorders. In this review article, the design and biological evaluation of amyloid-imaging agents are discussed. The structures of these agents vary from large proteins and peptides such as radiolabeled Aβ peptides and monoclonal antibodies to small molecules derived from Congo red, Chrysamine-G, thioflavin-T, and Acridine Orange. In vitro studies indicate that amyloid plaques contain multiple binding sites that can accommodate structurally diverse compounds, providing flexibility for radiopharmaceutical design of amyloid imaging agents. Compared to large biomolecules, small molecule radiotracers are often readily accessible through chemical synthesis and can display superior brain permeability. Several small molecule amyloid-imaging radioligands display high binding affinities to Aβ and sufficient brain penetration for imaging studies. Recent studies demonstrate the feasibility of imaging amyloid plaques in vivo in human subjects with PET. Imaging NFTs, separately or in concert with Ab plaques, is not as far advanced as imaging Aβ plaques and remains to be fully characterized and demonstrated.

The development of radioligands to image -amyloid (A) plaques and neurofibrillary tangles (NFTs) in vivo in the aging human brain is an important and active area of radiopharmaceutical design. When used in combination with positron emission tomography (PET) or single photon emission computed tomography (SPECT), amyloid-imaging tracers could facilitate the evaluation of the efficacy of anti-amyloid therapies currently under intense development by many major pharmaceutical companies throughout the world. Amyloid-imaging agents could also serve as surrogate markers in early diagnosis and neuropathogenesis studies of Alzheimers disease and other aging-related neurodegenerative disorders. In this review article, the design and biological evaluation of amyloid-imaging agents are discussed. The structures of these agents vary from large proteins and peptides such as radiolabeled A peptides and monoclonal antibodies to small molecules derived from Congo red, Chrysamine-G, thioflavin-T, and Acridine Orange. In vitro studies indicate that amyloid plaques contain multiple binding sites that can accommodate structurally diverse compounds, providing flexibility for radiopharmaceutical design of amyloid imaging agents. Compared to large biomolecules, small molecule radiotracers are often readily accessible through chemical synthesis and can display superior brain permeability. Several small molecule amyloid-imaging radioligands display high binding affinities to A and sufficient brain penetration for imaging studies. Recent studies demonstrate the feasibility of imaging amyloid plaques in vivo in human subjects with PET. Imaging NFTs, separately or in concert with Ab plaques, is not as far advanced as imaging A plaques and remains to be fully characterized and demonstrated.

The synthesis and evaluation of a series of neutral analogues of thioflavin-T (termed BTA's) with high affinities for aggregated amyloid and a wide range of lipophilicities are reported. Radiolabeling with high specific activity [¹¹C]methyl iodide provided derivatives for in vivo evaluation. Brain entry in control mice and baboons was high for nearly all of the analogues at early times after injection, but the clearance rate of radioactivity from brain tissue varied by more than 1 order of magnitude. Upon the basis of its rapid clearance from normal mouse and baboon brain tissues, [N-methyl-¹¹C]2-(4‘-methylaminophenyl)-6-hydroxybenzothiazole (or [¹¹C]6-OH-BTA-1) was selected as the lead compound for further evaluation. The radiolabeled metabolites of [¹¹C]6-OH-BTA-1 were polar and did not enter brain. The binding affinities of [N-methyl-³H]6-OH-BTA-1 for homogenates of postmortem AD frontal cortex and synthetic Aβ(1−40) fibrils were similar (Kd = 1.4 nM and 4.7 nM, respectively), but the ligand-to-Aβ peptide binding stoichiometry was ~400-fold higher for AD brain than Aβ(1−40) fibrils. Staining of AD frontal cortex tissue sections with 6-OH-BTA-1 indicated the selective binding of the compound to amyloid plaques and cerebrovascular amyloid. The encouraging in vitro and in vivo properties of [¹¹C]6-OH-BTA-1 support the choice of this derivative for further evaluation in human subject studies of brain Aβ deposition.

Article abstract—

Article abstract—

To evaluate the inter-rater reliability and validity of clinical diagnostic criteria for neurodegenerative dementias.

Binding with low-affinity ligands, such as histological dyes, can be difficult to quantitate owing to the dissociation of bound ligand with washing or the retention of nonspecifically bound ligand because of incomplete washing. The present report describes two simple, rapid methods of discriminating bound from free ligand without the need for washing steps. One method is based on the spectral changes induced in a dye ligand, Congo red, on binding to the "receptor" insulin fibrils. This method discriminates spectrophotometrically between bound and free ligand without requiring any physical separation of the two forms. No radioactive ligands are necessary, and, by using disposable cuvettes, the entire binding assay can be done in a single container without the need for transfers. The second method employs a non-traditional filtration approach that avoids the need for a washing step by measuring the decrease in concentration of the dye ligand in the filtrate rather than by applying the usual approach of measuring the absolute amount of ligand bound to the precipitated "receptor." Both methods show saturation of binding sites and give similar values for the KD and Bmax.

The binding of Congo red to several purified amyloid-like peptides having a beta-pleated sheet conformation was quantitatively examined. Congo red binds preferentially to the beta-pleated sheet conformation of both insulin fibrils and poly-L-lysine. Congo red does not bind nearly so well to poly-L-serine or polyglycine, despite the fact that these peptides also have a beta-pleated sheet conformation. Binding to insulin fibrils was saturable with an apparent Bmax of 2 moles of Congo red per mole of insulin fibrils and an apparent KD of 1.75 x 10(-7) M. Binding to beta-poly-L-lysine was similar but had a much higher apparent Bmax of 43. Binding of Congo red to beta-poly-L-lysine was pH dependent and appeared to be determined by the number of protonated lysine residues in the 250 amino acid peptide. We present a new hypothesis in which Congo red binds to amyloid-like proteins via bonds between the two negatively charged sulfonic acid groups of Congo red and two positively charged amino acid residues of two separate protein molecules which are properly oriented by virtue of the beta-pleated sheet conformation of the peptide backbone.

Three-dimensional models of the picrotoxinin and alkyl-substituted gamma-butyrolactone (GBL) receptor sites were constructed with the aid of a molecular graphics computer system (MMS-X). These two independently derived models proved to be very compatible, which suggested that both types of compounds share a common site of action. Since picrotoxinin is known to act at gamma-aminobutyric acid-regulated chloride channels, a hypothesis was made and tested that the convulsant GBL and picrotoxinin analogues physically impede the passage of chloride ions through the channel. It was also shown that it was theoretically possible for the anticonvulsant GBLs to act at this same site without blocking chloride flux. Finally, the model was applied to several convulsant and anticonvulsant compounds of different chemical classes and was found to be of somewhat general applicability.

Derivatives of gamma-butyrolactone (GBL) substituted on the alpha- and/or gamma-positions were synthesized and tested for their effects on behavior in mice, on the electroencephalographs and blood pressure of paralyzed-ventilated guinea pigs, and on electrical activity of incubated hippocampal slices. Several compounds, including alpha-ethyl-alpha-methyl GBL (alpha-EMGBL), alpha, alpha-dimethyl GBL, alpha, gamma-diethyl-alpha, gamma-dimethyl GBL, and gamma-ethyl-gamma-methyl GBL, prevented seizures induced by pentylenetetrazol, beta-ethyl-beta-methyl-gamma-butyrolactone (beta-EMGBL), picrotoxin, or all three compounds in mice and guinea pigs but had no effect on seizures induced by maximal electroshock or bicuculline. Neither gamma-hydroxybutyrate (GHB) nor alpha-isopropylidine GBL had any anticonvulsant activity. The anticonvulsant alpha-substituted compounds had a potent hypotensive effect and antagonized the hypertensive effect of beta-EMGBL, alpha-EMGBL was tested in incubated hippocampal slices and was found to depress basal activity and antagonize excitation induced by beta-EMGBL. These results demonstrate that alpha-alkyl-substituted GBL and, to a lesser extent, gamma-substituted derivatives are anticonvulsant agents and that their effects are strikingly different from those of GHB or beta-alkyl-substituted GBLs, which are epileptogenic. Possibly beta- and alpha-substituted GBLs act at the same site as agonists and antagonists, respectively.

We used proton nuclear magnetic resonance spectroscopy in this preliminary study of perchloric acid extracts of 12 Alzheimer's disease (AD) and five control brain samples to measure the relative levels of taurine, aspartate, glutamine, glutamate, γ-aminobutyric acid (GABA), and the putative neuronal marker, N-acetyl-L-aspartate (NAA). We found no significant changes in taurine, aspartate, or glutamine. NAA was lower in AD compared with control, and this decrease correlated with the number of senile plaques and neurofibrillary tangles in adjacent tissue sections. GABA levels also were lower in AD brain. Glutamate levels were greater in AD than control and showed a close, inverse correlation with NAA levels. These findings suggest that the decrease in NAA reflects neuronal loss and that remaining neurons could be exposed to a relative excess of glutamate and a relative lack of GABA. If present in the neurotransmitter pool, this imbalance could result in neurotoxic cell damage. This hypothesis is further supported by in vitro and in vivo phosphorus 31 nuclear magnetic resonance findings.

We evaluated the reliability of clinical diagnoses using the recently standardized criteria for the diagnosis of vascular dementia (VaD) developed by the National Institute of Neurological Disorders and Stroke (NINDS) and the Association Internationale pour la Recherche et l'Enseignement en Neurosciences (AIREN). Two neurologists and two psychiatrists independently reviewed clinical data abstracted from those of 42 demented subjects participating in a longitudinal study of dementia at the University of Pittsburgh. For each patient we abstracted the clinical data on a standardized form. Each physician diagnosed each case according to the NINDS-AIREN criteria, using both clinical information and MRIs. We calculated the interrater agreement for all two-way combinations of clinicians with kappa statistics, which ranged from 0.46 (moderate agreement) to 0.72 (substantial agreement). The moderate reliability observed in this study may be attributable to patient-, clinician-, or criteria-centered sources of variance.

In vitroand in vivo31P magnetic resonance (MR) spectroscopy studies of Alzheimer's disease (AD) brain have revealed alterations in membrane phospholipid metabolism and high-energy phosphate metabolism. Mildly demented AD patients compared with control subjects have increased levels of phosphomonoesters, decreased levels of phosphocreatine and probably adenosine diphosphate and an increased oxidative metabolic rate. As the dementia worsens, levels of phosphomonoesters decrease and levels of phosphocreatine and adenosine diphosphate increase. The changes in oxidative metabolic rate suggest that the AD brain is under energetic stress. The phosphomonoester findings support our in vitrofindings and implicate basic defects in membrane metabolism in AD brain. MR spectroscopy provides new diagnostic insights and a noninvasive method to follow the progression of the disease and the metabolic response to therapeutic interventions.

ATP-dependent uptake of glutamate into synaptic vesicles has been well documented. Stimulation of glutamate uptake into synaptic vesicles by other high-energy phosphates has not been described. In this paper, we examine the stimulation of phosphocreatine (PCr)-induced glutamate uptake and determine whether this stimulation is secondary to conversion of PCr to ATP. We found the following. 1) PCr stimulates glutamate uptake into synaptic vesicles in the absence of added ATP. 2) At a glutamate concentration of 50 microM, no concentration of added ATP could produce the degree of stimulation seen in the presence of PCr. 3) 0.5 mM iodoacetamide completely inhibits synaptic vesicle creatine kinase activity but does not inhibit PCr-stimulated glutamate uptake. 4) PCr-dependent glutamate uptake, unlike ATP-dependent uptake, is not magnesium- or chloride-dependent. 5) 0.5 mM N-ethylmaleimide, a selective H+-ATPase inhibitor, completely inhibits ATP-dependent glutamate uptake but only slightly inhibits PCr-dependent glutamate uptake. 6) PCr-dependent glutamate uptake is sensitive to valinomycin, a K+/H+ translocator, whereas the ATP-dependent uptake is not. Therefore, it appears that in addition to the well-known ATP-dependent glutamate uptake system, there is a previously unreported PCr-dependent glutamate uptake system in synaptic vesicles. The total glutamate uptake by synaptic vesicles is likely the sum of both ATP- and PCr-dependent glutamate uptake.

Amyloid deposits in the form of senile plaques and fibrillary tangles have been targeted as a biomarker for the early diagnosis of Alzheimer's disease (AD) and evaluation of the efficacy of therapeutic treatments (1–3). For in vivo quantitation of amyloid deposits in AD, efforts have been made to develop radiopharmaceuticals for PET and/or SPECT imaging that readily penetrate the blood brain barrier and selectively localize in the area of beta‐amyloid deposits in AD brain. To date, these agents such as radiolabeled monoclonal antibodies (4), plaque‐related proteins (5, 6), analogues of Congo red (7–11), and membrane dyes (12) have found limited use due to either marginal brain uptake or high non‐specific binding.