Lary C. Walker

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.

Accelerated Glial Reactivity to Stroke in Aged Rats Correlates With Reduced Functional Recovery

Following cerebral ischemia, perilesional astrocytes and activated microglia form a glial scar that hinders the genesis of new axons and blood vessels in the infarcted region. Since glial reactivity is chronically augmented in the normal aging brain, the authors hypothesized that postischemic gliosis would be temporally abnormal in aged rats compared to young rats. Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3- and 20-month-old male Sprague Dawley rats. The functional outcome was assessed in neurobehavioral tests at 3, 7, 14, and 28 days after surgery. Brain tissue was immunostained for microglia, astrocytes, oligodendrocytes, and endothelial cells. Behaviorally, aged rats were more severely impaired by stroke and showed diminished functional recovery compared with young rats. Histologically, a gradual activation of both microglia and astrocytes that peaked by days 14 to 28 with the formation of a glial scar was observed in young rats, whereas aged rats showed an accelerated astrocytic and microglial reaction that peaked during the first week after stroke. Oligodendrocytes were strongly activated at early stages of infarct development in all rats, but this activation persisted in aged rats. Therefore, the development of the glial scar was abnormally accelerated in aged rats and coincided with the stagnation of recovery in these animals. These results suggest that a temporally anomalous gliotic reaction to cerebral ischemia in aged rats leads to the premature formation of scar tissue that impedes functional recovery after stroke.

Calcium channel alpha2-delta type 1 subunit is the major binding protein for pregabalin in neocortex, hippocampus, amygdala, and spinal cord: An ex vivo autoradiographic study in alpha2-delta type 1 genetically modified mice

Pregabalin is a synthetic amino acid compound effective in clinical trials for the treatment of post-herpetic neuralgia, diabetic peripheral neuropathy, generalized anxiety disorder and adjunctive therapy for partial seizures of epilepsy. However, the mechanisms by which pregabalin exerts its therapeutic effects are not yet completely understood. In vitro studies have shown that pregabalin binds with high affinity to the alpha(2)-delta (alpha(2)-delta) subunits (Type 1 and 2) of voltage-gated calcium channels. To assess whether alpha(2)-delta Type 1 is the major central nervous system (CNS) binding protein for pregabalin in vivo, a mutant mouse with an arginine-to-alanine mutation at amino acid 217 of the alpha(2)-delta Type 1 protein (R217A mutation) was generated. Previous site-directed mutagenesis studies revealed that the R217A mutation dramatically reduces alpha(2)-delta 1 binding to pregabalin in vitro. In this autoradiographic analysis of R217A mice, we show that the mutation to alpha(2)-delta Type 1 substantially reduces specific pregabalin binding in CNS regions that are known to preferentially express the alpha(2)-delta Type 1 protein, notably the neocortex, hippocampus, basolateral amygdala and spinal cord. In mutant mice, pregabalin binding was robust throughout regions where the alpha(2)-delta Type 2 subunit mRNA is abundant, such as cerebellum. These findings, in conjunction with prior in vitro binding data, provide evidence that the alpha(2)-delta Type 1 subunit of voltage-gated calcium channels is the major binding protein for pregabalin in CNS. Moreover, the distinct localization of alpha(2)-delta Type 1 and mutation-resistant binding (assumed to be alpha(2)-delta Type 2) in brain areas subserving different functions suggests that identification of subunit-specific ligands could further enhance pharmacologic specificity.

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.

The cerebral proteopathies: neurodegenerative disorders of protein conformation and assembly.

The abnormal assembly and deposition of specific proteins in the brain is the probable cause of most neurodegenerative disease afflicting the elderly. These “cerebral proteopathies” include Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), prion diseases, and a variety of other disorders. Evidence is accumulating that the anomalous aggregation of the proteins, and not a loss of protein function, is central to the pathogenesis of these diseases. Thus, therapeutic strategies that reduce the production, accumulation, or polymerization of pathogenic proteins might be applicable to a wide range of some of the most devastating diseases of old age.

Animal models of cerebral β-amyloid angiopathy

Abstract Cerebral amyloid angiopathy (CAA) is a significant risk factor for hemorrhagic stroke in the elderly, and occurs as a sporadic disorder, as a frequent component of Alzheimers disease, and in several rare, hereditary conditions. The most common type of amyloid found in the vasculature of the brain is β-amyloid (Aβ), the same peptide that occurs in senile plaques. A paucity of animal models has hindered the experimental analysis of CAA. Several transgenic mouse models of cerebral β-amyloidosis have now been reported, but only one appears to develop significant cerebrovascular amyloid. However, well-characterized models of naturally occurring CAA, particularly aged dogs and non-human primates, have contributed unique insights into the biology of vascular amyloid in recent years. Some non-human primate species have a predilection for developing CAA; the squirrel monkey ( Saimiri sciureus ), for example, is particularly likely to manifest β-amyloid deposition in the cerebral blood vessels with age, whereas the rhesus monkey ( Macaca mulatta ) develops more abundant parenchymal amyloid. These animals have been used to test in vivo β-amyloid labeling strategies with monoclonal antibodies and radiolabeled Aβ. Species-differences in the predominant site of Aβ deposition also can be exploited to evaluate factors that direct amyloid selectively to a particular tissue compartment of the brain. For example, the cysteine protease inhibitor, cystatin C, in squirrel monkeys has an amino acid substitution that is similar to the mutant substitution found in some humans with a hereditary form of cystatin C amyloid angiopathy, possibly explaining the predisposition of squirrel monkeys to CAA. The existing animal models have shown considerable utility in deciphering the pathobiology of CAA, and in testing strategies that could be used to diagnose and treat this disorder in humans.

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.

Upregulation of MAP1B and MAP2 in the rat brain after middle cerebral artery occlusion: effect of age.

Although stroke in humans usually afflicts the elderly, most experimental studies on the nature of cerebral ischemia have used young animals. This is especially important when studying restorative processes that are age dependent. To explore the potential of older animals to initiate regenerative processes after cerebral ischemia, the authors studied the expression of the juvenile-specific cytoskeletal protein, microtubule-associated protein (MAP) 1B, and the adult-specific protein, MAP2, in male Sprague-Dawley rats at 3 months and 20 months of age. The levels of MAP1B and MAP2 transcripts and the corresponding proteins declined with increasing age in the hippocampus. In the cortex, the levels of the transcripts did not change significantly with age, but the morphologic features of immunostained fibers were clearly affected by age; that is, cortical MAP1B fibers became thicker, and MAP2 fibers, more diffuse, in aged rats. Focal cerebral ischemia, produced by reversible occlusion of the right middle cerebral artery, resulted in a large decrease in the expression of both MAP1B and MAP2 in the infarct core at the messenger ribonucleic acid and protein levels. However, at 1 week after the stroke, there was vigorous expression of MAP1B and its messenger ribonucleic acid, as well as MAP2 protein, in the border zone adjacent to the infarct of 3-month-old and 20 month-old male Sprague-Dawley rats. The upregulation of these key cytologic elements generally was diminished in aged rats compared with young animals, although the morphologic features of fibers in the infarct border zone were similar in both age groups. These results suggest that the regenerative potential of the aged rat brain appears to be competent, although attenuated, at least with respect to MAP1B and MAP2 expression up to 20 months of age.

Intra-arterial infusion of [125I]A beta 1-40 labels amyloid deposits in the aged primate brain in vivo.

Alzheimers disease is characterized by extracellular amyloid deposits in the brain at both vascular sites (cerebrovascular amyloid, CVA) and within the neuropil (plaques). In the present study we demonstrated that brain amyloid of aged non-human primates is efficiently detected by [125I]A beta in vitro, and assessed the detection of that amyloid in vivo by intravascular infusion of [125I]A beta. Aged squirrel monkeys (Saimiri sciureus) were anesthetized and infused intra-arterially with [125I]A beta, and sacrificed 2 h later. Analysis of the anterior frontal and temporal cortices by autoradiography demonstrated that [125I]A beta was deposited on CVA and that essentially every amyloid deposit which could be detected with thioflavin S or anti-A beta antibodies was also labeled by [125I]A beta. These experiments suggest that intravascular infusion of radiolabeled A beta can be used to detect and image amyloid deposits in the human AD brain.

Primate-like amyloid-β sequence but no cerebral amyloidosis in aged tree shrews

A central pathological feature of Alzheimers disease is the profuse deposition of amyloid-beta protein (Abeta) in the brain parenchyma and vessel walls. Abeta also forms deposits in the brains of a variety of mammals, including all aged non-human primates studied to date. The sequence of Abeta in these animals is identical to that in humans. No Abeta deposits have been found in the brains of wild-type rats and mice, suggesting that the three amino acid differences between their Abeta and that of amyloid-bearing mammals impedes the fibrillogenicity of Abeta. Analysis of the primary sequence of the beta-amyloid precursor protein in tree shrews revealed a 98% similarity and 97% identity with the human protein. Furthermore, the predicted amino acid sequence of Abeta in tree shrews is identical to that in humans. However, immunohistochemical analysis failed to reveal beta-amyloid deposits in the neural parenchyma or vasculature of eight aged (7-8 years) tree shrews (Tupaia belangeri). The lack of correlation between the Abeta sequence and amyloid formation suggests that other factors contribute to cerebral amyloid deposition in aged animals.