Chemical characterization of pro-inflammatory amyloid-beta peptides in human atherosclerotic lesions and platelets.

Amyloid-ß (Aß) peptides are intimately involved in the inflammatory pathology of atherosclerotic vascular disease (AVD) and Alzheimer's disease (AD). Although substantial amounts of these peptides are produced in the periphery, their role and significance to vascular disease outside the brain requires further investigation. Amyloid-ß peptides present in the walls of human aorta atherosclerotic lesions as well as activated and non-activated human platelets were isolated using sequential size-exclusion columns and HPLC reverse-phase methods. The Aß peptide isolates were quantified by ELISA and structurally analyzed using MALDI-TOF mass spectrometry procedures. Our experiments revealed that both aorta and platelets contained Aß peptides, predominately Aß40. The source of the Aß pool in aortic atherosclerosis lesions is probably the activated platelets and/or vascular wall cells expressing APP/PN2. Significant levels of Aß42 are present in the plasma, suggesting that this reservoir makes a minor contribution to atherosclerotic plaques. Our data reveal that although aortic atherosclerosis and AD cerebrovascular amyloidosis exhibit clearly divergent end-stage manifestations, both vascular diseases share some key pathophysiological promoting elements and pathways. Whether they happen to be deposited in vessels of the central nervous system or atherosclerotic plaques in the periphery, Aß peptides may promote and perhaps synergize chronic inflammatory processes which culminate in the degeneration, malfunction and ultimate destruction of arterial walls.

Collapsin response mediator protein-2 phosphorylation promotes the reversible retraction of oligodendrocyte processes in response to non-lethal oxidative stress.

The extension of processes of oligodendrocyte (OLG) and their precursor cells are crucial for migration, axonal contact and myelination. Here we show that a non-lethal oxidative stress induced by 3-nitropropionic acid (3-NP) elicited a rapid shortening of processes (~24%) in primary OLGs and in oligodendroglial cell line (OLN-93) cells (~36%) as compared with vehicle-exposed cells. This was reversible and prevented by antioxidants. Proteomics of OLG lysates with and without 3-NP treatment yielded collapsin response mediator protein 2 (CRMP-2) as a candidate effector molecule. Inhibition of rho kinase was sufficient to prevent process retraction in both OLGs and OLN-93 cells. Oxidative stress increased phosphorylation of CRMP-2 at T555 that was completely prevented by Y27632. Moreover, transfection of OLN-93 cells with the mutant CRMP-2 T555A which cannot be phosphorylated by rho kinase, prevented process shortening induced by 3-NP as compared with wild-type CRMP-2. Our results suggest a role for endogenous reactive oxygen species in a pathway that regulates OLG process extension. The vulnerability of late myelinated neurons in the adult brain and the presence of white matter pathology in human dementias warrant the study of this oligodendroglial pathway in the early stages of neurodegenerative conditions characterized by oxidative stress.

Is Alzheimer's disease amyloidosis the result of a repair mechanism gone astray?

Here, we synthesize several lines of evidence supporting the hypothesis that at least one function of amyloid-ß is to serve as a part of the acute response to brain hemodynamic disturbances intended to seal vascular leakage. Given the resilient and adhesive physicochemical properties of amyloid, an abluminal hemostatic repair system might be highly advantageous, if deployed on a limited and short-term basis, in young individuals. However, in the aged, inevitable cardiovascular dysfunction combined with brain microvascular lesions may yield global chronic hypoperfusion that may lead to continuous amyloid deposition and consequential negative effects on neuronal viability. A large body of experimental evidence supports the hypothesis of an amyloid-ß rescue function gone astray. Preventing or inducing the removal of amyloid in Alzheimer's disease (AD) has been simultaneously successful and disappointing. Amyloid deposits clearly play major roles in AD, but they may not represent the preeminent factor in dementia pathogenesis. Successful application of AD preventative approaches may hinge on an accurate and comprehensive view of comorbidities, including cardiovascular disease, diabetes, and head trauma.

Reduced clinical and postmortem measures of cardiac pathology in subjects with advanced Alzheimer's Disease.

BACKGROUND: Epidemiological studies indicate a statistical linkage between atherosclerotic vascular disease (ATH) and Alzheimer's disease (AD). Autopsy studies of cardiac disease in AD have been few and inconclusive. In this report, clinical and gross anatomic measures of cardiac disease were compared in deceased human subjects with and without AD. METHODS: Clinically documented cardiovascular conditions from AD (n = 35) and elderly non-demented control subjects (n = 22) were obtained by review of medical records. Coronary artery stenosis and other gross anatomical measures, including heart weight, ventricular wall thickness, valvular circumferences, valvular calcifications and myocardial infarct number and volume were determined at autopsy. RESULTS: Compared to non-demented age-similar control subjects, those with AD had significantly fewer total diagnosed clinical conditions (2.91 vs 4.18), decreased coronary artery stenosis (70.8 vs 74.8%), heart weight (402 vs 489 g for males; 319 vs 412 g for females) and valvular circumferences. Carriage of the Apolipoprotein E-ε4 allele did not influence the degree of coronary stenosis. Group differences in heart weight remained significant after adjustment for age, gender, body mass index and apolipoprotein E genotype while differences in coronary artery stenosis were significantly associated with body mass index alone. CONCLUSIONS: The results are in agreement with an emerging understanding that, while midlife risk factors for ATH increase the risk for the later development of AD, once dementia begins, both risk factors and manifest disease diminish, possibly due to progressive weight loss with increasing dementia as well as disease involvement of the brain's vasomotor centers.

Transcranial doppler ultrasound blood flow velocity and pulsatility index as systemic indicators for Alzheimer's disease.

BACKGROUND: Multiple lines of evidence suggest that cardiovascular co-morbidities hasten the onset of Alzheimer's disease (AD) or accelerate its course. METHODS: To evaluate the utility of cerebral vascular physical function and/or condition parameters as potential systemic indicators of AD, transcranial Doppler (TCD) ultrasound was used to assess cerebral blood flow and vascular resistance of the 16 arterial segments comprising the circle of Willis and its major tributaries. RESULTS: Our study showed that decreased arterial mean flow velocity and increased pulsatility index are associated with a clinical diagnosis of presumptive AD. Cerebral blood flow impairment shown by these parameters reflects the global hemodynamic and structural consequences of a multifaceted disease process yielding diffuse congestive microvascular pathology, increased arterial rigidity, and decreased arterial compliance, combined with putative age-associated cardiovascular output declines. CONCLUSIONS: TCD evaluation offers direct physical confirmation of brain perfusion impairment and might ultimately provide a convenient and a noninvasive means to assess the efficacy of medical interventions on cerebral blood flow or reveal incipient AD. In the near term, TCD-based direct assessments of brain perfusion might offer the prospect of preventing or mitigating AD simply by revealing patients who would benefit from interventions to improve circulatory system function.

Intracranial atherosclerosis as a contributing factor to Alzheimer's disease dementia.

BACKGROUND: A substantial body of evidence collected from epidemiologic, correlative, and experimental studies strongly associates atherosclerotic vascular disease (AVD) with Alzheimer's disease (AD). Depending on the precise interrelationship between AVD and AD, systematic application of interventions used to maintain vascular health and function as a component of standard AD therapy offers the prospect of mitigating the presently inexorable course of dementia. To assess this hypothesis, it is vital to rigorously establish the measures of AVD that are most strongly associated with an AD diagnosis. METHODS: A precise neuropathological diagnosis was established for all subjects, using a battery of genetic, clinical, and histological methods. The severity of atherosclerosis in the circle of Willis was quantified by direct digitized measurement of arterial occlusion in postmortem specimens and was compared between AD and nondemented control groups by calculating a corresponding index of occlusion. RESULTS: Atherosclerotic occlusion of the circle of Willis arteries was more extensive in the AD group than in the nondemented control group. Statistically significant differences were also observed between control and AD groups with regard to Braak stage, total plaque score, total neurofibrillary tangle score, total white matter rarefaction score, brain weight, Mini-Mental State Examination scores, and apolipoprotein E allelic frequencies. CONCLUSIONS: Our results, combined with a consideration of the multifaceted effects of impaired cerebral circulation, suggest an immediate need for prospective clinical trials to assess the efficacy of AD prevention using antiatherosclerotic agents.

Proteomics-derived cerebrospinal fluid markers of autopsy-confirmed Alzheimer's disease.

The diagnostic performance of several candidate cerebrospinal fluid (CSF) protein biomarkers in neuropathologically confirmed Alzheimer's disease (AD), non-demented (ND) elderly controls and non-AD dementias (NADD) was assessed. Candidate markers were selected on the basis of initial two-dimensional gel electrophoresis studies or by literature review. Markers selected by the former method included apolipoprotein A-1 (ApoA1), haemopexin (HPX), transthyretin (TTR) and pigment epithelium-derived factor (PEDF), while markers identified from the literature included Abeta1-40, Abeta1-42, total tau, phosphorylated tau, alpha-1 acid glycoprotein (A1GP), haptoglobin, zinc alpha-2 glycoprotein (Z2GP) and apolipoprotein E (ApoE). Ventricular CSF concentrations of the markers were measured by enzyme-linked immunosorbent assay (ELISA). The concentrations of Abeta1-42, ApoA1, A1GP, ApoE, HPX and Z2GP differed significantly among AD, ND and NADD subjects. Logistic regression analysis for the diagnostic discrimination of AD from ND found that Abeta1-42, ApoA1 and HPX each had significant and independent associations with diagnosis. The CSF concentrations of these three markers distinguished AD from ND subjects with 84% sensitivity and 72% specificity, with 78% of subjects correctly classified. By comparison, using Abeta1-42 alone gave 79% sensitivity and 61% specificity, with 68% of subjects correctly classified. For the diagnostic discrimination of AD from NADD, only the concentration of Abeta1-42 was significantly related to diagnosis, with a sensitivity of 58%, specificity of 86% and 86% correctly classified. The results indicate that for the discrimination of AD from ND control subjects, measurement of a set of markers including Abeta1-42, ApoA1 and HPX improved diagnostic performance over that obtained by measurement of Abeta1-42 alone. For the discrimination of AD from NADD subjects, measurement of Abeta1-42 alone was superior.

Patients with Alzheimer disease have altered transmitral flow: echocardiographic analysis of the vortex formation time.

OBJECTIVE: There is considerable epidemiologic evidence that Alzheimer disease (AD) is linked to cardiovascular risk factors and associated with an increased risk of symptomatic left ventricular (LV) dysfunction. Formation of a vortex alongside a diastolic jet signifies an efficient blood transport mechanism. The vortex formation time (VFT) is an index of optimal conditions for vortex formation. We hypothesized that AD and its associated cardiovascular risk factors impair diastolic transmitral flow efficiency and, therefore, shift the VFT value out of its optimal range. METHODS: Echocardiographic studies were performed on 45 participants in total: 22 patients with AD diagnosed according to the American Psychiatric Association's criteria and 23 age-matched individuals as a control group with cognitive function within normal limits. RESULTS: The echocardiographic ratio of the early to atrial phases of the LV filling velocities was significantly lower in the AD group (mean +/- SD, 0.67 +/- 14) when compared with the control individuals (0.79 +/- 0.14; P = .003). The interventricular septum diastolic thickness, left ventricular posterior wall diastolic thickness, and right ventricular end-diastolic diameter were significantly higher in the AD group (P

Impaired hepatic amyloid-beta degradation in Alzheimer's disease.

Extensive research strongly suggests that amyloid beta (Aß) aggregates in the brain have a central role in Alzheimer's disease (AD) pathogenesis. Pathological Aß deposition is likely due to an altered balance between overproduction and elimination. Rodent studies have suggested that the liver has a major role in Aß degradation. It is possible alterations of liver function could affect brain Aß levels through changes in blood Aß concentration. In this study, we hypothesized hepatic Aß degradation to be impaired in AD subjects. To test our hypothesis, an Aß degradation assay was developed using synthetic fluorescein-labeled Aß40 and Aß42 spiked into human liver homogenates. Aß degradation rates were lower in AD-derived homogenates as compared with those from non-demented (ND) control subjects, even after accounting for such covariates as age, sex, and APOE genotype. The protein expression of potential Aß-degrading enzymes were also examined. Neprilysin levels were not different in AD liver samples, while cathepsin D and insulin-degrading enzyme were significantly altered in AD subjects. The results support the possibility that impaired hepatic Aß degradation could be a factor contributing to increased brain Aß accumulation and AD.

Antemortem-Postmortem Correlation of Florbetapir (18F) PET Amyloid Imaging with Quantitative Biochemical Measures of Aß42 but not Aß40.

Amyloid imaging demonstrates the in vivo presence of amyloid-ß (Aß) deposits in the aging human brain but it is still unknown which structural forms and modifications of Aß are detected. In Alzheimer's disease, most amyloid deposits are predominantly composed of Aß ending at amino acid residues Val40 or Ala42. It has been reported that Aß40 is largely restricted to neuritic plaques while Aß42 may be deposited in amyloid plaques of all types, and is often the sole component of diffuse plaques. The distinction is important as it is mainly the neuritic plaques that correlate with cognitive impairment while diffuse plaques may be the initial type of Aß deposited. Whether PET amyloid ligands such as florbetapir-18F (Amyvid) are partially or wholly selective for brain deposits of Aß40 or Aß42 is currently unknown. We compared antemortem florbetapir PET cortical/cerebellar signal intensity (SUVr) of 55 subjects with postmortem biochemical (ELISA) measurements employing specific antibodies against Aß40 and Aß42. Spearman's univariable correlations were significant for both Aß40 and Aß42, but were much stronger for Aß42. Multiple linear regression showed significance only for Aß42. These results suggest that florbetapir binds only weakly, if at all, to Aß40. This may be in part due to the higher likelihood for Aß42 to be present in a ß-pleated sheet tertiary structure, or to differences between Aß40 and Aß42 in ß-pleated sheet tertiary or quaternary structure.

APP/Aß structural diversity and Alzheimer's disease pathogenesis.

The amyloid cascade hypothesis of Alzheimer's disease (AD) proposes amyloid- ß (Aß) is a chief pathological element of dementia. AD therapies have targeted monomeric and oligomeric Aß 1-40 and 1-42 peptides. However, alternative APP proteolytic processing produces a complex roster of Aß species. In addition, Aß peptides are subject to extensive posttranslational modification (PTM). We propose that amplified production of some APP/Aß species, perhaps exacerbated by differential gene expression and reduced peptide degradation, creates a diverse spectrum of modified species which disrupt brain homeostasis and accelerate AD neurodegeneration. We surveyed the literature to catalog Aß PTM including species with isoAsp at positions 7 and 23 which may phenocopy the Tottori and Iowa Aß mutations that result in early onset AD. We speculate that accumulation of these alterations induce changes in secondary and tertiary structure of Aß that favor increased toxicity, and seeding and propagation in sporadic AD. Additionally, amyloid-ß peptides with a pyroglutamate modification at position 3 and oxidation of Met35 make up a substantial portion of sporadic AD amyloid deposits. The intrinsic physical properties of these species, including resistance to degradation, an enhanced aggregation rate, increased neurotoxicity, and association with behavioral deficits, suggest their emergence is linked to dementia. The generation of specific 3D-molecular conformations of Aß impart unique biophysical properties and a capacity to seed the prion-like global transmission of amyloid through the brain. The accumulation of rogue Aß ultimately contributes to the destruction of vascular walls, neurons and glial cells culminating in dementia. A systematic examination of Aß PTM and the analysis of the toxicity that they induced may help create essential biomarkers to more precisely stage AD pathology, design countermeasures and gauge the impacts of interventions.

Familial Presenilin Mutations and Sporadic Alzheimer's Disease Pathology: Is the Assumption of Biochemical Equivalence Justified?

Studies of presenilin (PSEN) gene mutations producing early onset Alzheimer's disease (AD) have helped elucidate the pathogenic mechanisms of dementia and guided clinical trials of potential therapeutic interventions. Although familial and sporadic forms of AD share features, it is unclear if the two are precisely equivalent. In addition, PSEN mutations do not all produce a single phenotype, but exhibit substantial variability in clinical manifestations, which are related to the position and chemical nature of their amino acid substitutions as well as ratios of critical molecules such as Aß40 and Aß42. These differences complicate the interpretation of critical clinical trial results and their desired extrapolation to sporadic AD treatment. In this perspective, we examine differences between familial AD and sporadic AD as well as attributes shared by these uniquely arising disturbances in brain biochemical homeostasis that culminate in dementia.

Chemical and neuropathological analyses of an Alzheimer's disease patient treated with solanezumab.

INTRODUCTION: Based on the amyloid cascade hypothesis of Alzheimer's disease (AD) pathogenesis, a series of clinical trials involving immunotherapies have been undertaken including infusion with the IgG1 monoclonal anti-Aß antibody solanezumab directed against the middle of the soluble Aß peptide. In this report, we give an account of the clinical history, psychometric testing, gross and microscopic neuropathology as well as immunochemical quantitation of soluble and insoluble Aß peptides and other proteins of interest related to AD pathophysiology in a patient treated with solanezumab. MATERIALS AND METHODS: The solanezumab-treated AD case (SOLA-AD) was compared to non-demented control (NDC, n = 5) and non-immunized AD (NI-AD, n = 5) subjects. Brain sections were stained with H&E, Thioflavine-S, Campbell-Switzer and Gallyas methods. ELISA and Western blots were used for quantification of proteins of interest. RESULTS: The SOLA-AD subject's neuropathology and biochemistry differed sharply from the NDC and NI-AD groups. The SOLA-AD case had copious numbers of amyloid laden blood vessels in all areas of the cerebral cortex, from leptomeningeal perforating arteries to arteriolar deposits which attained the cerebral amyloid angiopathy (CAA) maximum score of 12. In contrast, the maximum CAA for the NI-AD cases averaged a total of 3.6, while the NDC cases only reached 0.75. The SOLA-AD subject had 4.4-fold more soluble Aß40 and 5.6-fold more insoluble Aß40 in the frontal lobe compared to NI-AD cases. In the temporal lobe of the SOLA-AD case, the soluble Aß40 was 80-fold increased, and the insoluble Aß40 was 13-fold more abundant compared to the non-immunized AD cases. Both soluble and insoluble Aß42 levels were not dramatically different between the SOLA-AD and NI-AD cohort. DISCUSSION: Solanezumab immunotherapy provided no apparent relief in the clinical evolution of dementia in this particular AD patient, since there was a continuous cognitive deterioration and full expression of amyloid deposition and neuropathology.

The Presence of Select Tau Species in Human Peripheral Tissues and Their Relation to Alzheimer's Disease.

Tau becomes excessively phosphorylated in Alzheimer's disease (AD) and is widely studied within the brain. Further examination of the extent and types of tau present in peripheral tissues and their relation to AD is warranted given recent publications on pathologic spreading. Cases were selected based on the presence of pathological tau spinal cord deposits (n = 18). Tissue samples from sigmoid colon, scalp, abdominal skin, liver, and submandibular gland were analyzed by western blot and enzyme-linked immunosorbent assays (ELISAs) for certain tau species; frontal cortex gray matter was used for comparison. ELISAs revealed brain to have the highest total tau levels, followed by submandibular gland, sigmoid colon, liver, scalp, and abdominal skin. Western blots with antibodies recognizing tau phosphorylated at threonine 231(pT231), serine 396 and 404 (PHF-1), and an unmodified total human tau between residues 159 and 163 (HT7) revealed multiple banding patterns, some of which predominated in peripheral tissues. As submandibular gland had the highest levels of peripheral tau, a second set of submandibular gland samples were analyzed (n = 36; 19 AD, 17 non-demented controls). ELISAs revealed significantly lower levels of pS396 (p = 0.009) and pT231 (p = 0.005) in AD cases but not total tau (p = 0.18). Furthermore, pT231 levels in submandibular gland inversely correlated with Braak neurofibrillary tangle stage (p = 0.04), after adjusting for age at death, gender, and postmortem interval. These results provide evidence that certain tau species are present in peripheral tissues. Of potential importance, submandibular gland pT231 is progressively less abundant with increasing Braak neurofibrillary tangle stage.

Characterizing Apolipoprotein E ε4 Carriers and Noncarriers With the Clinical Diagnosis of Mild to Moderate Alzheimer Dementia and Minimal ß-Amyloid Peptide Plaques.

IMPORTANCE: ß-Amyloid peptide (Aß) plaques are a cardinal neuropathologic feature of Alzheimer disease (AD), yet more than one-third of apolipoprotein E ε4 (APOE4) noncarriers with the clinical diagnosis of mild to moderate Alzheimer dementia may not meet positron emission tomographic criteria for significant cerebral amyloidosis. OBJECTIVES: To clarify the percentage of APOE4 carriers and noncarriers with the primary clinical diagnosis of mild to moderate Alzheimer dementia near the end of life and minimal Aß plaques noted at autopsy and the extent to which these cases are associated with appreciable neurofibrillary degeneration or a primary neuropathologic diagnosis other than AD. DESIGN, SETTING, AND PARTICIPANTS: Data on participants included in this study were obtained from the National Alzheimer Coordinating Center's Uniform Data Set, which comprises longitudinal clinical assessments performed at the AD centers funded by the National Institute on Aging. Neuropathology data are available for the subset of participants who died. A total of 100 APOE4 noncarriers and 100 APOE4 carriers had the primary clinical diagnosis of mild to moderate Alzheimer dementia at their last visit, known APOE4 genotype, died within the ensuing 24 months, and underwent neuropathologic evaluation on autopsy. The study was conducted from September 1, 2005, to September 1, 2012; analysis was performed from October 9, 2012, to March 20, 2015. MAIN OUTCOMES AND MEASURES: Standardized histopathologic assessments of AD neuropathologic changes were the primary measures of interest in this study, specifically Consortium to Establish a Registry for Alzheimer's Disease neuritic plaque density score, diffuse plaque density score, and Braak stage for neurofibrillary degeneration. The distributions of scores for these measures were the primary outcomes. RESULTS: Of the 37 APOE4 noncarriers with minimal neuritic plaques, 16 individuals (43.2%) had Braak stages III to VI ratings, and 15 of the others (75.0%) met neuropathologic criteria for other dementia-related diseases. Of the 13 APOE4 carriers with minimal neuritic plaques, 6 individuals (46.2%) had Braak stages III to VI ratings and met neuropathologic criteria for other dementia-related diseases. Similarly, of the 7 APOE4 carriers with minimal neuritic plaques and Braak stages 0 to II, 4 participants (57.1%) were thought to have pathologic changes and alterations resulting from non-AD neuropathologic features. CONCLUSIONS AND RELEVANCE: In this study, more than one-third of APOE4 noncarriers with the primary clinical diagnosis of mild to moderate Alzheimer dementia had minimal Aß plaque accumulation in the cerebral cortex and, thus, may show limited or no benefit from otherwise effective anti-Aß treatment. Almost half of the participants with a primary clinical diagnosis of mild to moderate Alzheimer dementia and minimal Aß plaque accumulation had an extensive topographic distribution of neurofibrillary degeneration. Additional studies are needed to better understand and provide treatment for patients with this unexpectedly common cliniconeuropathologic condition.

Adipose and leptomeningeal arteriole endothelial dysfunction induced by ß-amyloid peptide: a practical human model to study Alzheimer's disease vasculopathy.

BACKGROUND: Evidence point to vascular dysfunction and hypoperfusion as early abnormalities in Alzheimer's disease (AD); probing their mechanistic bases can lead to new therapeutic approaches. We tested the hypotheses that ß-amyloid peptide induces endothelial dysfunction and oxidative stress in human microvasculature and that response will be similar between peripheral adipose and brain leptomeningeal arterioles. NEW METHOD: Abdominal subcutaneous arterioles from living human subjects (n=17) and cadaver leptomeningeal arterioles (n=6) from rapid autopsy were exposed to Aß1-42 (Aß) for 1-h and dilation response to acetylcholine/papaverine were measured and compared to baseline response. Adipose arteriole reactive oxygen species (ROS) production and nitrotyrosine content were measured. COMPARISON WITH EXISTING METHODS: Methods described allow direct investigation of human microvessel functional response that cannot be replicated by human noninvasive imaging or post-mortem histology. RESULTS: Adipose arterioles exposed to 2 µM Aß showed impaired dilation to acetylcholine that was reversed by antioxidant polyethylene glycol superoxide dismutase (PEG-SOD) (Aß-60.9 ± 6%, control-93.2 ± 1.8%, Aß+PEGSOD-84.7 ± 3.9%, both p<0.05 vs. Aß). Aß caused reduced dilation to papaverine. Aß increased adipose arteriole ROS production and increased arteriole nitrotyrosine content. Leptomeningeal arterioles showed similar impaired response to acetylcholine when exposed to Aß (43.0 ± 6.2% versus 81.1 ± 5.7% control, p<0.05). CONCLUSION: Aß exposure induced adipose arteriole endothelial and non-endothelial dysfunction and oxidative stress that were reversed by antioxidant treatment. Aß-induced endothelial dysfunction was similar between peripheral adipose and leptomeningeal arterioles. Ex vivo living adipose and cadaver leptomeningeal arterioles are viable, novel and practical human tissue models to study Alzheimer's vascular pathophysiology.

Neuropathological and biochemical assessments of an Alzheimer's disease patient treated with the γ-secretase inhibitor semagacestat.

Amyloid deposition has been implicated as the key determinant of Alzheimer's disease (AD) pathogenesis. Interventions to antagonize amyloid accumulation and mitigate dementia are now under active investigation. We conducted a combined clinical, biochemical and neuropathological assessment of a participant in a clinical trial of the γ-secretase inhibitor, semagacestat. This patient received a daily oral dose of 140 mg of semagacestat for approximately 76 weeks. Levels of brain amyloid-ß (Aß) peptides were quantified using enzyme-linked immunosorbent assays (ELISA). Western blot/scanning densitometry was performed to reveal BACE1, presenilin1, amyloid precursor protein (APP) and its proteolysis-produced C-terminal peptides APP-CT99 and APP-CT83 as well as several γ-secretase substrates. To serve as a frame of reference, the ELISA and Western analyses were performed in parallel on samples from neuropathologically confirmed non-demented control (NDC) and AD subjects who did not receive semagacestat. Neuropathology findings confirmed a diagnosis of AD with frequent amyloid deposits and neurofibrillary tangles in most areas of the cortex and subcortical nuclei as well as cerebellar amyloid plaques. Mean levels of Tris-soluble Aß40 and glass-distilled formic acid (GDFA)/guanidine hydrochloride (GHCl)-extractable Aß40 in the frontal lobe and GDFA/GHCl-soluble Aß40 in the temporal lobe were increased 4.2, 9.5 and 7.7-fold, respectively, in the semagacestat-treated subject compared to those observed in the non-treated AD group. In addition, GDFA/GHCl-extracted Aß42 was increased 2-fold in the temporal lobe relative to non-treated AD cases. No major changes in APP, ß- and γ-secretase and CT99/CT83 were observed between the semagacestat-treated subject compared to either NDC or AD cases. Furthermore, the levels of γ-secretase substrates in the semagacestat-treated subject and the reference groups were also similar. Interestingly, there were significant alterations in the levels of several γ-secretase substrates between the NDC and non-treated AD subjects. This is the first reported case study of an individual enrolled in the semagacestat clinical trial. The subject of this study remained alive for ~7 months after treatment termination, therefore it is difficult to conclude whether the outcomes observed represent a consequence of semagacestat therapy. Additional evaluations of trial participants, including several who expired during the course of treatment, may provide vital clarification regarding the impacts and aftermath of γ-secretase inhibition.

Biochemical assessment of precuneus and posterior cingulate gyrus in the context of brain aging and Alzheimer's disease.

Defining the biochemical alterations that occur in the brain during "normal" aging is an important part of understanding the pathophysiology of neurodegenerative diseases and of distinguishing pathological conditions from aging-associated changes. Three groups were selected based on age and on having no evidence of neurological or significant neurodegenerative disease: 1) young adult individuals, average age 26 years (n = 9); 2) middle-aged subjects, average age 59 years (n = 5); 3) oldest-old individuals, average age 93 years (n = 6). Using ELISA and Western blotting methods, we quantified and compared the levels of several key molecules associated with neurodegenerative disease in the precuneus and posterior cingulate gyrus, two brain regions known to exhibit early imaging alterations during the course of Alzheimer's disease. Our experiments revealed that the bioindicators of emerging brain pathology remained steady or decreased with advancing age. One exception was S100B, which significantly increased with age. Along the process of aging, neurofibrillary tangle deposition increased, even in the absence of amyloid deposition, suggesting the presence of amyloid plaques is not obligatory for their development and that limited tangle density is a part of normal aging. Our study complements a previous assessment of neuropathology in oldest-old subjects, and within the limitations of the small number of individuals involved in the present investigation, it adds valuable information to the molecular and structural heterogeneity observed along the course of aging and dementia. This work underscores the need to examine through direct observation how the processes of amyloid deposition unfold or change prior to the earliest phases of dementia emergence.

Alzheimer disease periventricular white matter lesions exhibit specific proteomic profile alterations.

The white matter (WM) represents approximately half the cerebrum volume and is profoundly affected in Alzheimer's disease (AD). However, both the WM responses to AD as well as potential influences of this compartment to dementia pathogenesis remain comparatively neglected. Neuroimaging studies have revealed WM alterations are commonly associated with AD and renewed interest in examining the pathologic basis and importance of these changes. In AD subjects, immunohistochemistry and electron microscopy revealed changes in astrocyte morphology and myelin loss as well as up to 30% axonal loss in areas of WM rarefaction when measured against non-demented control (NDC) tissue. Comparative proteomic analyses were performed on pooled samples of periventricular WM (PVWM) obtained from AD (n=4) and NDC (n=5) subjects with both groups having a mean age of death of 86 years. All subjects had an apolipoprotein E ε3/3 genotype with the exception of one NDC subject who was ε2/3. Urea-detergent homogenates were analyzed using two different separation techniques: 2-dimensional isoelectric focusing/reverse-phase chromatography and 2-dimensional difference gel electrophoresis (2D-DIGE). Proteins with different expression levels between the 2 diagnostic groups were identified using MALDI-Tof/Tof mass spectrometry. In addition, Western blots were used to quantify proteins of interest in individual AD and NDC cases. Our proteomic studies revealed that when WM protein pools were loaded at equal amounts of total protein for comparative analyses, there were quantitative differences between the 2 groups. Molecules related to cytoskeleton maintenance, calcium metabolism and cellular survival such as glial fibrillary acidic protein, vimentin, tropomyosin, collapsin response mediator protein-2, calmodulin, S100-P, annexin A1, α-internexin, α- and ß-synuclein, α-B-crystalline, fascin-1, ubiquitin carboxyl-terminal esterase and thymosine were altered between AD and NDC pools. Our experiments suggest that WM activities become globally impaired during the course of AD with significant morphological, biochemical and functional consequential implications for gray matter function and cognitive deficits. These observations may endorse the hypothesis that WM dysfunction is not only a consequence of AD pathology, but that it may precipitate and/or potentiate AD dementia.

Molecular Differences and Similarities Between Alzheimer's Disease and the 5XFAD Transgenic Mouse Model of Amyloidosis.

Transgenic (Tg) mouse models of Alzheimer's disease (AD) have been extensively used to study the pathophysiology of this dementia and to test the efficacy of drugs to treat AD. The 5XFAD Tg mouse, which contains two presenilin-1 and three amyloid precursor protein (APP) mutations, was designed to rapidly recapitulate a portion of the pathologic alterations present in human AD. APP and its proteolytic peptides, as well as apolipoprotein E and endogenous mouse tau, were investigated in the 5XFAD mice at 3 months, 6 months, and 9 months. AD and nondemented subjects were used as a frame of reference. APP, amyloid-beta (Aß) peptides, APP C-terminal fragments (CT99, CT83, AICD), ß-site APP-cleaving enzyme, and APLP1 substantially increased with age in the brains of 5XFAD mice. Endogenous mouse tau did not show age-related differences. The rapid synthesis of Aß and its impact on neuronal loss and neuroinflammation make the 5XFAD mice a desirable paradigm to model AD.