William J. Lipinski

Augmented Senile Plaque Load in Aged Female β-Amyloid Precursor Protein-Transgenic Mice

Transgenic mice (Tg2576) overexpressing human beta-amyloid precursor protein with the Swedish mutation (APP695SWE) develop Alzheimers disease-like amyloid beta protein (Abeta) deposits by 8 to 10 months of age. These mice show elevated levels of Abeta40 and Abeta42, as well as an age-related increase in diffuse and compact senile plaques in the brain. Senile plaque load was quantitated in the hippocampus and neocortex of 8- to 19-month-old male and female Tg2576 mice. In all mice, plaque burden increased markedly after the age of 12 months. At 15 and 19 months of age, senile plaque load was significantly greater in females than in males; in 91 mice studied at 15 months of age, the area occupied by plaques in female Tg2576 mice was nearly three times that of males. By enzyme-linked immunosorbent assay, female mice also had more Abeta40 and Abeta42 in the brain than did males, although this difference was less pronounced than the difference in histological plaque load. These data show that senescent female Tg2576 mice deposit more amyloid in the brain than do male mice, and may provide an animal model in which the influence of sex differences on cerebral amyloid pathology can be evaluated.

The role of microglial cells and astrocytes in fibrillar plaque evolution in transgenic APPSW mice

Ultrastructural reconstruction of 27 fibrillar plaques in different stages of formation and maturation was undertaken to characterize the development of fibrillar plaques in the brains of human APP(SW) transgenic mice (Tg2576). The study suggests that microglial cells are not engaged in Abeta removal and plaque degradation, but in contrast, are a driving force in plaque formation and development. Fibrillar Abeta deposition at the amyloid pole of microglial cells appears to initiate three types of neuropil response: degeneration of neurons, protective activation of astrocytes, and attraction and activation of microglial cells sustaining plaque growth. Enlargement of neuronal processes and synapses with accumulation of degenerated mitochondria, dense bodies, and Hirano-type bodies is the marker of toxic injury of neurons by fibrillar Abeta. Separation of amyloid cores from neurons and degradation of amyloid cores by cytoplasmic processes of hypertrophic astrocytes suggest the protective and defensive character of astrocytic response to fibrillar Abeta. The growth of cored plaque from a small plaque with one microglial cell with an amyloid star and a few dystrophic neurites to a large plaque formed by several dozen microglial cells seen in old mice is the effect of attraction and activation of microglial cells residing outside of the plaque perimeter. This mechanism of growth of plaques appears to be characteristic of cored plaques in transgenic mice. Other features in mouse microglial cells that are absent in human brain are clusters of vacuoles, probably of lysosomal origin. They evolve into circular cisternae and finally into large vacuoles filled with osmiophilic, amorphous material and bundles of fibrils that are poorly labeled with antibody to Abeta. Microglial cells appear to release large amounts of fibrillar Abeta and accumulate traces of fibrillar Abeta in a lysosomal pathway.

Axonopathy, tau abnormalities, and dyskinesia, but no neurofibrillary tangles in p25-transgenic mice.

Neurofibrillary tangles, one of the pathologic hallmarks of Alzheimers disease (AD), are composed of abnormally polymerized tau protein. The hyperphosphorylation of tau alters its normal cellular function and is thought to promote the formation of neurofibrillary tangles. Growing evidence suggests that cyclin‐dependent kinase 5 (cdk5) plays a role in tau phosphorylation, but the function of the enzyme in tangle formation remains uncertain. In AD, cdk5 is constitutively activated by p25, a highly stable, 25kD protein thought to be increased in the AD brain. To test the hypothesis that p25/cdk5 interactions promote neurofibrillary pathology, we created transgenic mouse lines that overexpress the human p25 protein specifically in neurons. Mice with high transgenic p25 expression have augmented cdk5 activity and develop severe hindlimb semiparalysis and mild forelimb dyskinesia beginning at approximately 3 months of age. Immunohistochemical and ultrastructural analyses showed widespread axonal degeneration with focal accumulation of tau in various regions of the brain and, to a lesser extent, the spinal cord. However, there was no evidence of neurofibrillary tangles in neuronal somata or axons, nor were paired helical filaments evident ultrastructurally. These studies confirm that p25 overexpression can lead to tau abnormalities and axonal degeneration in vivo but do not support the hypothesis that p25‐related induction of cdk5 is a primary event in the genesis of neurofibrillary tangles. J. Comp. Neurol. 446:257–266, 2002.

Exogenous induction of cerebral β-amyloidosis in βAPP-transgenic mice

A key commonality of most age-related neurodegenerative diseases is the accumulation of aggregation-prone proteins in the brain. Except for the prionoses, the initiation and propagation of these proteopathies in vivo remains poorly understood. In a previous study, we found that the deposition of the amyloidogenic peptide Abeta can be induced by injection of dilute extracts of Alzheimeric neocortex into the brains of Tg2576 transgenic mice overexpressing the human beta-amyloid precursor protein. The present study was undertaken to assess the pathology after long-term (12 months) incubation, and to clarify the distinctive anatomical distribution of seeded Abeta-immunoreactivity. All mice were injected at 3 months of age; 5 months later, as expected, Abeta deposits were concentrated mostly in the injected hemisphere. After 12 months, abundant, transgene-derived Abeta deposits were present bilaterally in the forebrain, but plaque load was still clearly greater in the extract-injected hemisphere. There was also evidence of tau hyperphosphorylation in axons of the corpus callosum that had been injured by the injection, most prominently in transgenic mice, but also, to a lesser degree, in non-transgenic mice. Five months following injection of AD-extract, an isolated cluster of Abeta-immunoreactive microglia was sometimes evident in the ipsilateral entorhinal cortex; the strong innervation of the hippocampus by entorhinal cortical neurons suggests the possible spread of seeded pathology from the injection site via neuronal transport mechanisms. Finally, using India Ink to map the local dispersion of injectate, we found that Abeta induction is especially potent in places where the injectate is sequestered. The AD-seeding model can illuminate the emergence and spread of cerebral beta-amyloidosis and tau hyperphosphorylation, and thus could enhance our understanding of AD and its pathogenic commonalties with other cerebral proteopathies.

The synthesis and structure-activity relationship of substituted N-phenyl anthranilic acid analogs as amyloid aggregation inhibitors

It is believed that beta-amyloid aggregation is an important event in the development of Alzheimers disease. In the course of our studies to identify beta-amyloid aggregation inhibitors, a series of N-phenyl anthranilic acid analogs were synthesized and studied for beta-amyloid inhibition activity. The synthesis, structure-activity relationship, and in vivo activity of these analogs are discussed.

Aging, gender and APOE isotype modulate metabolism of Alzheimer's Aβ peptides and F2‐isoprostanes in the absence of detectable amyloid deposits

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimers disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by‐products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid β (Aβ) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEɛ3 or human APOEɛ4 alleles]. Aging‐, gender‐, and APOE‐genotype‐dependent changes were observed for endogenous mouse brain Aβ40 and Aβ42 peptides. Levels of the oxidized lipid F2‐isoprostane (F2‐isoPs) in the brains of the same animals as those used for the Aβ analyses revealed aging‐ and gender‐dependent changes in APOE KO and in human APOEɛ4 transgenic KO mice. Human APOEɛ3 transgenic KO mice did not exhibit aging‐ or gender‐dependent increases in F2‐isoPs. In general, the changes in the levels of brain F2‐isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Aβ levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging‐dependent, APOE‐genotype‐sensitive rise in murine brain Aβ levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEɛ3, but not human APOEɛ4, can apparently prevent the aging‐dependent rise in murine brain Aβ levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Aβ/F2‐isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2‐isoPs can stimulate Aβ generation and aggregation.

Modeling Alzheimer's disease and other proteopathies in vivo: is seeding the key?

Summary. Protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including Alzheimers disease (AD), Parkinsons disease, triplet repeat disorders, tauopathies, and prion diseases. In many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. Possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide range of diseases. However, testing compounds preclinically will require disease-relevant animal models. Numerous transgenic mouse models of β-amyloidosis, tauopathy, and other aspects of AD have now been produced, but none of the existing models fully recapitulates the pathology of AD. In an attempt to more faithfully replicate the human disease, we infused dilute AD-brain extracts into Tg2576 mice at 3-months of age (i.e. 5–6 months prior to the usual onset of β-amyloid deposition). We found that intracerebral infusion of AD brain extracts results in: 1) Premature deposition of β-amyloid in eight month-old, β-amyloid precursor protein (βAPP)-transgenic mice (Kane et al., 2000); 2) augmented amyloid load in the injected hemisphere of 15 month-old transgenic mice; 3) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 4) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. The seeding of β-amyloid in vivo by AD brain extracts suggests pathogenic similarities between β-amyloidoses such as AD and other cerebral proteopathies such as the prionoses, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases.

In vitro and in vivo evaluation of the subtype-selective muscarinic agonist PD 151832

PD 151832 is a potent partial muscarinic agonist that displays a high level of functional selectivity for the muscarinic m1 receptor subtype, as evidenced by its selective stimulation of PI turnover and cellular metabolic activity in transfected Hm1-CHO cells at concentrations that produce minimal stimulation of other cloned human muscarinic receptors. PD 151832 enhanced the amplification of Hm1-transfected NIH-3T3 cells at concentrations lower than those required to produce similar effects in Hm2 or Hm3-transfected cells. The functional m1 selectivity of PD 151832 is consistent with its improvement of mouse water maze performance at doses far lower than those required to produce peripheral parasympathetic side effects.

Chapter 54: Subtype selective muscarinic agonists: potential therapeutic agents for Alzheimer's disease

Publisher Summary Senile dementia of Alzheimer type (AD) is a progressive neurodegenerative disease of unknown etiology. AD and related dementias are characterized by significant neuronal pathology in discrete cortical and subcortical brain regions. The basal forebrain cholinergic system, among other neurotransmitter systems, is severely affected in Alzheimers disease. Significant loss of forebrain cholinergic neurons accompanied by decreased neocortical choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, the major anabolic and catabolic enzymes for acetylcholine, is consistently seen in the brains of demented subjects. Moreover, reduction in ChAT and AChE activity are correlated with the degree of dementia and severity of neuropathological hallmarks of AD. A close association, therefore, exists between cholinergic biochemical abnormalities and this disease. Replacement of lost cholinergic function should provide palliative relief from the cognitive symptoms accompanying AD. Several approaches to cholinergic replacement have been tried in AD. Clinical studies with cholinesterase inhibitors and acetylcholine releasing agents have shown significant, albeit weak activity. The activity of AChE inhibitors and ACh releasing agents is dependent on the state of forebrain cholinergic neurons. In contrast, postsynaptic muscarinic receptors on cholinoceptive neurons in the neocortex and hippocampus are relatively spared in AD. Agents acting directly at these sites and mimicking the actions of acetylcholine should restore lost cholinergic function and retain efficacy throughout AD. Unfortunately, reducing theory to practice in this area has been difficult and the clinical efficacy of muscarinic agonists in AD is equivocal. The limited clinical utility of muscarinic agonists can be attributed to their propensity to induce unwanted cholinergic-mediated parasympathetic effects— that is, (nausea, hypersalivation, sweating), poor oral bio-availability, short durations of action and perhaps subtype selectivity for receptors mediating unwanted effects. This limited clinical utility should not be surprising since these are old agents that were not specifically designed for the use in treating the cognitive decline associated with aging and dementia. Newer muscarinic agonists with improved efficacy to side effect ratios and optimized durations of action are needed.

PD 142676 (CI 1002), a novel anticholinesterase and muscarinic antagonist

Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associated with Alzheimers disease (AD). However, unwanted peripheral side effects often limit the usefulness of the available anticholinesterases. Recently, we identified a dihydroquinazoline compound, PD 142676 (CI 1002) that is a potent anticholinesterase and a functional muscarinic antagonist at higher concentrations. Peripherally, PD 14276, unlike other anticholinesterases, inhibits gastrointestinal motility in rats, an effect consistent with its muscarinic antagonist properties. Centrally, the compound acts as a cholinomimetic. In rats, PD 142676, decreases core body temperature. It also increases neocortical arousal, as measured by quantitative electroencephalography, and cortical acetylcholine levels, measured by in vivo microdialysis. The compound improves the performance of C57/B10j mice in a water maze task and of aged rhesus monkeys in a delayed match-to-sample task involving short-term memory. The combined effect of AChE inhibition and muscarinic antagonism distinguishes PD 142676 from other anticholinesterases and may be useful in treating the cognitive dysfunction of AD and produce fewer peripheral side effects.