Eric E. Abrahamson

Post-mortem correlates of in vivo PiB-PET amyloid imaging in a typical case of Alzheimer's disease

The positron emission tomography (PET) radiotracer Pittsburgh Compound-B (PiB) binds with high affinity to β-pleated sheet aggregates of the amyloid-β (Aβ) peptide in vitro. The in vivo retention of PiB in brains of people with Alzheimers disease shows a regional distribution that is very similar to distribution of Aβ deposits observed post-mortem. However, the basis for regional variations in PiB binding in vivo, and the extent to which it binds to different types of Aβ-containing plaques and tau-containing neurofibrillary tangles (NFT), has not been thoroughly investigated. The present study examined 28 clinically diagnosed and autopsy-confirmed Alzheimers disease subjects, including one Alzheimers disease subject who had undergone PiB-PET imaging 10 months prior to death, to evaluate region- and substrate-specific binding of the highly fluorescent PiB derivative 6-CN-PiB. These data were then correlated with region-matched Aβ plaque load and peptide levels, [3H]PiB binding in vitro, and in vivo PET retention levels. We found that in Alzheimers disease brain tissue sections, the preponderance of 6-CN-PiB binding is in plaques immunoreactive to either Aβ42 or Aβ40, and to vascular Aβ deposits. 6-CN-PiB labelling was most robust in compact/cored plaques in the prefrontal and temporal cortices. While diffuse plaques, including those in caudate nucleus and presubiculum, were less prominently labelled, amorphous Aβ plaques in the cerebellum were not detectable with 6-CN-PiB. Only a small subset of NFT were 6-CN-PiB positive; these resembled extracellular ‘ghost’ NFT. In Alzheimers disease brain tissue homogenates, there was a direct correlation between [3H]PiB binding and insoluble Aβ peptide levels. In the Alzheimers disease subject who underwent PiB-PET prior to death, in vivo PiB retention levels correlated directly with region-matched post-mortem measures of [3H]PiB binding, insoluble Aβ peptide levels, 6-CN-PiB- and Aβ plaque load, but not with measures of NFT. These results demonstrate, in a typical Alzheimers disease brain, that PiB binding is highly selective for insoluble (fibrillar) Aβ deposits, and not for neurofibrillary pathology. The strong direct correlation of in vivo PiB retention with region-matched quantitative analyses of Aβ plaques in the same subject supports the validity of PiB-PET imaging as a method for in vivo evaluation of Aβ plaque burden.

Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections.

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of the mammalian circadian timing system. The SCN is composed of two anatomically and functionally distinct subdivisions, designated core and shell, which can be distinguished on the basis of their chemoarchitecture and connections in the rat. In the present study, we examine the intrinsic organization and the afferent and efferent connections of the mouse SCN using immunocytochemistry and ocular injections of cholera toxin. Neurons of the SCN shell contain GABA, calbindin (CALB), arginine vasopressin (AVP), angiotensin II (AII) and met-enkephalin (mENK), and receive input from galanin (GAL) and vasoactive intestinal polypeptide (VIP) immunoreactive fibers. Neurons of the SCN core synthesize GABA, CALB, VIP, calretinin (CALR), gastrin releasing peptide (GRP), and neurotensin (NT), and receive input from the retina and from fibers that contain neuropeptide Y (NPY) and 5-hydroxytryptamine (5HT). Fibers projecting from SCN neurons that are immunoreactive for AVP and VIP exhibit a characteristic morphology, and project to the lateral septum, a series of medial hypothalamic areas extending from the preoptic to the posterior hypothalamic area and to the paraventricular thalamic nucleus. The organization of the mouse SCN, and its connections, are similar to that in other mammalian species.

Binding of the Positron Emission Tomography Tracer Pittsburgh Compound-B Reflects the Amount of Amyloid-β in Alzheimer's Disease Brain But Not in Transgenic Mouse Brain

During the development of in vivo amyloid imaging agents, an effort was made to use micro-positron emission tomography (PET) imaging in the presenilin-1 (PS1)/amyloid precursor protein (APP) transgenic mouse model of CNS amyloid deposition to screen new compounds and further study Pittsburgh Compound-B (PIB), a PET tracer that has been shown to be retained well in amyloid-containing areas of Alzheimers disease (AD) brain. Unexpectedly, we saw no significant retention of PIB in this model even at 12 months of age when amyloid deposition in the PS1/APP mouse typically exceeds that seen in AD. This study describes a series of ex vivo and postmortem in vitro studies designed to explain this low retention. Ex vivo brain pharmacokinetic studies confirmed the low in vivo PIB retention observed in micro-PET experiments. In vitro binding studies showed that PS1/APP brain tissue contained less than one high-affinity (Kd = 1-2 nm) PIB binding site per 1000 molecules of amyloid-β (Aβ), whereas AD brain contained >500 PIB binding sites per 1000 molecules of Aβ. Synthetic Aβ closely resembled PS1/APP brain in having less than one high-affinity PIB binding site per 1000 molecules of Aβ, although the characteristics of the few high-affinity PIB binding sites found on synthetic Aβ were very similar to those found in AD brain. We hypothesize that differences in the time course of deposition or tissue factors present during deposition lead to differences in secondary structure between Aβ deposited in AD brain and either synthetic Aβ or Aβ deposited in PS1/APP brain.

The posterior hypothalamic area: chemoarchitecture and afferent connections

This study provides an analysis of the chemoarchitecture of the posterior hypothalamic area (PHA) and a retrograde transport analysis of inputs to the PHA in the rat. The chemoarchitectural analysis reveals that the majority of PHA neurons contain glutamate. Hypocretin, melanin concentrating hormone, tyrosine hydroxylase, neuropeptide Y and gamma-aminobutyric acid are also found in subsets of PHA neurons, and fibers immunoreactive for these substances as well as for serotonin, dopamine-beta-hydroxylase and met-enkephalin are observed in the area and aid in the delineation of its borders. The retrograde tracing study demonstrates that the PHA receives input from multiple, diverse neuron populations. Descending projections to the PHA arise from the limbic forebrain (cingulate cortex and lateral septum) and both the medial and lateral hypothalamus. Subcortical visual nuclei, including the ventral lateral geniculate nucleus and intergeniculate leaflet, pretectal area, and superior colliculus, and the subthalamus (zona incerta, fields of Forel) also project to the PHA. Ascending projections to the PHA arise from brainstem cholinergic nuclei, the reticular formation, midbrain raphe nuclei, periaqueductal gray and parabrachial nucleus. Retrograde transport studies using the psuedorabies virus (PRV) demonstrate that the PHA receives input indirectly from the hippocampus, amygdala and suprachiasmatic nucleus through circuits including nuclei in the limbic forebrain and hypothalamus. These data suggest that the PHA is important in the neural control of behavioral state, modulating aspects of hippocampal, autonomic and cortical function as they relate to the elaboration of adaptive behavior.

Increased 5-Lipoxygenase Immunoreactivity in the Hippocampus of Patients With Alzheimer's Disease

The proinflammatory enzyme 5-lipoxygenase (5-LOX) is upregulated in Alzheimers disease (AD), but its localization and association with the hallmark lesions of the disease, β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs), is unknown. This study examined the distribution and cellular localization of 5-LOX in the medial temporal lobe from AD and control subjects. The spatial relationship between 5-LOX immunoreactive structures and AD lesions was also examined. We report that, in AD subjects, 5-LOX immunoreactivity is elevated relative to controls, and its localization is dependent on the antibody-targeted portion of the 5-LOX amino acid sequence. Carboxy terminus-directed antibodies detected 5-LOX in glial cells and neurons, but less frequently in neurons with dystrophic (NFT) morphology. In contrast, immunoreactivity observed using 5-LOX amino terminus-directed antibodies was virtually absent in neurons and abundant in NFTs, neuritic plaques, and glia. Double-labeling studies showed a close association of 5-LOX-immunoreactive processes and glial cells with Aβ immunoreactive plaques and vasculature and also detected 5-LOX in tau immunoreactive and amyloid containing NFTs. Different immunolabeling patterns with antibodies against carboxy vs amino terminus of 5-LOX may be caused by post-translational modifications of 5-LOX protein in Aβ plaques and NFTs. The relationship between elevated intracellular 5-LOX and hallmark AD pathological lesions provides further evidence that neuroinflammatory pathways contribute to the pathogenesis of AD.

Caspase inhibition therapy abolishes brain trauma-induced increases in Aβ peptide : Implications for clinical outcome

The detrimental effects of traumatic brain injury (TBI) on brain tissue integrity involve progressive axonal damage, necrotic cell loss, and both acute and delayed apoptotic neuronal death due to activation of caspases. Post-injury accumulation of amyloid precursor protein (APP) and its toxic metabolite amyloid-beta peptide (Abeta) has been implicated in apoptosis as well as in increasing the risk for developing Alzheimers disease (AD) after TBI. Activated caspases proteolyze APP and are associated with increased Abeta production after neuronal injury. Conversely, Abeta and related APP/Abeta fragments stimulate caspase activation, creating a potential vicious cycle of secondary injury after TBI. Blockade of caspase activation after brain injury suppresses apoptosis and improves neurological outcome, but it is not known whether such intervention also prevents increases in Abeta levels in vivo. The present study examined the effect of caspase inhibition on post-injury levels of soluble Abeta, APP, activated caspase-3, and caspase-cleaved APP in the hippocampus of nontransgenic mice expressing human Abeta, subjected to controlled cortical injury (CCI). CCI produced brain tissue damage with cell loss and elevated levels of activated caspase-3, Abeta(1-42) and Abeta(1-40), APP, and caspase-cleaved APP fragments in hippocampal neurons and axons. Post-CCI intervention with intracerebroventricular injection of 100 nM Boc-Asp(OMe)-CH(2)F (BAF, a pan-caspase inhibitor) significantly reduced caspase-3 activation and improved histological outcome, suppressed increases in Abeta and caspase-cleaved APP, but showed no significant effect on overall APP levels in the hippocampus after CCI. These data demonstrate that after TBI, caspase inhibition can suppress elevations in Abeta. The extent to which Abeta suppression contributes to improved outcome following inhibition of caspases after TBI is unclear, but such intervention may be a valuable therapeutic strategy for preventing the long-term evolution of Abeta-mediated pathology in TBI patients who are at risk for developing AD later in life.

Simvastatin therapy prevents brain trauma-induced increases in β-amyloid peptide levels†

Elevations in β‐amyloid peptide (Aβ) levels after traumatic brain injury (TBI) may confer risk for developing Alzheimers disease in head trauma patients. We investigated the effects of simvastatin, a 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitor, on hippocampal Aβ burden in a clinically relevant head injury/intervention model using mice expressing human Aβ. Simvastatin therapy blunted TBI‐induced increases in Aβ, reduced hippocampal tissue damage and microglial activation, and improved behavioral outcome. The ability of statins to reduce post‐injury Aβ load and ameliorate pathological sequelae of brain injury makes them potentially effective in reducing the risk of developing Alzheimers disease in TBI patients. Ann Neurol 2009;66:407–414

Precuneus amyloid burden is associated with reduced cholinergic activity in Alzheimer disease

Objective: This study examined the relationship between postmortem precuneus cholinergic enzyme activity, Pittsburgh compound B (PiB) binding, and soluble amyloid-β concentration in mild cognitive impairment (MCI) and Alzheimer disease (AD). Methods: Choline acetyltransferase (ChAT) activity, [3H]PiB binding, and soluble amyloid-β1–42 (Aβ42) concentration were quantified in precuneus tissue samples harvested postmortem from subjects with no cognitive impairment (NCI), MCI, and mild AD and correlated with their last antemortem Mini-Mental State Examination (MMSE) score and postmortem pathologic evaluation according to the National Institute on Aging–Reagan criteria, recommendations of the Consortium to Establish a Registry for Alzheimers Disease, and Braak stage. Results: Precuneus ChAT activity was lower in AD than in NCI and was comparable between MCI and NCI. Precuneus [3H]PiB binding and soluble Aβ42 levels were elevated in MCI and significantly higher in AD than in NCI. Across all case subjects, reduced ChAT activity was associated with increased [3H]PiB binding, increased soluble Aβ42, lower MMSE score, presence of the APOE*4 allele, and more advanced AD pathology. Conclusions: Despite accumulating amyloid burden, cholinergic enzyme activity is stable in the precuneus during prodromal AD. A decline in precuneus ChAT activity occurs only in clinical AD, when PiB binding and soluble Aβ42 levels are substantially elevated compared with those in MCI. Anti-amyloid interventions in MCI case subjects with a positive PiB PET scan may aid in reducing cholinergic deficits and cognitive decline later in the disease process.

X‐34 Labeling of Abnormal Protein Aggregates During the Progression of Alzheimer's Disease

Postmortem pathological diagnosis and basic research investigations of neurodegenerative disorders rely on histochemical staining procedures developed specifically to visualize abnormal protein conformation. In Alzheimers disease (AD), two major pathological hallmarks are required to confirm the clinical diagnosis. Both consist of abnormally aggregated proteins that share the structural and histological properties common to all amyloid deposits. Amyloid-beta peptide (Abeta) of extracellular senile plaques (SP) and hyperphosphorylated tau of intracellular neurofibrillary tangles (NFT) are assembled in the abnormal beta-pleated sheet (amyloid-like) structural conformation that can be visualized with histological staining procedures using Congo red or its derivatives. These histochemical dyes bind amyloid with high affinity and allow easy detection of amyloid structure in postmortem brain samples. This chapter focuses on the development and application of a histological protocol using the compound X-34, a highly fluorescent derivative of Congo red, for sensitive detection of pathological amyloid structures in histopathological investigations of postmortem brain tissue. This procedure provides a simple and effective method for detailed fluorescent visualization of the localization and distribution of the majority of currently known major histopathological structures in AD, including compact cored, neuritic, and diffuse-appearing SP, NFT, dystrophic neurites, neuropil threads, and cerebrovascular amyloidosis.

Lesions of suprachiasmatic nucleus efferents selectively affect rest-activity rhythm.

The suprachiasmatic nucleus (SCN) of the hypothalamus controls circadian rhythms in behavioral, neuroendocrine and physiological functions. In this study, we test the hypothesis that caudal SCN efferents to the subparaventricular zone (SPVZ) control the rhythm in rest-activity (R-A) through projections on posterior hypothalamic area arousal systems (PHA). Small electrolytic lesions of the ventral SPVZ cause a selective loss of the circadian R-A rhythm, sparing the core body temperature rhythm. In contrast, large excitotoxic lesions of the posterior hypothalamic area (PHA) that effectively ablate populations of hypocretin and melanin concentrating hormone neurons projecting to cortex and subcortical arousal areas decrease R-A rhythm amplitude but do not disrupt circadian regulation. Since dorsomedial hypothalamic nucleus lesions have effects similar to PHA lesions (Chou, T.C., Scammell, T.E., Gooley, J.J., Gaus, S.E., Saper, C.B., Lu, J., 2003. Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms. J. Neurosci. 23:10691-10702), these data support the view that the SPVZ is the principal relay nucleus for SCN signals to the multiple posterior hypothalamic arousal systems involved in generation of the R-A rhythm.