Region- and Cell-specific Aneuploidy in Brain Aging and Neurodegeneration.

Variations in genomic DNA content, or aneuploidy, are a well-recognized feature of normal human brain development. Whether changes in the levels of aneuploidy are a factor in Alzheimer's disease (AD) is less clear, as the data reported to date vary substantially in the levels of aneuploidy detected (0.7-11.5%), possibly due to methodological limitations, but also influenced by individual, regional and cellular heterogeneity as well as variations in cell subtypes. These issues have not been adequately addressed to date. While it is known that the DNA damage response increases with age, the limited human studies investigating aneuploidy in normal aging also show variable results, potentially due to susceptibility to age-related neurodegenerative processes. Neuronal aneuploidy has recently been reported in multiple brain regions in Lewy body disease, but similar genomic changes are not a feature of all synucleinopathies and aneuploidy does not appear to be related to alpha-synuclein aggregation. Rather, aneuploidy was associated with Alzheimer's pathology in the hippocampus and anterior cingulate cortex and neuronal degeneration in the substantia nigra. The association between Alzheimer's pathology and aneuploidy in regions with limited neurodegeneration is supported by a growing body of in vitro and in vivo data on aneuploidy and beta-amyloid and tau abnormalities. Large-scale studies using high-resolution techniques alongside other sensitive and specific methodologies are now required to assess the true extent of cell- and region-specific aneuploidy in aging and neurodegeneration, and to determine any associations with pathologies.

Relationship between neuronal loss and 'inflammatory plaques' in early onset Alzheimer's disease.

We have previously described a novel 'inflammatory plaque' in the cortex of early onset Alzheimer's disease (EOAD) cases with presenilin 1 mutations (PS1). These plaques are associated with a significant inflammatory infiltrate consisting of reactive microglia and astrocytes. We speculated that these inflammatory plaques might be responsible for the more severe disease process seen in EOAD. In the present study using the superior frontal cortex, 63 EOAD cases with mutations in PS1, presenilin 2 (PS2) and amyloid precursor protein (APP) were categorized as either having inflammatory plaques (13 cases, two APP and 11 PS1) or not. To determine the impact on cell loss, seven EOAD cases with inflammatory plaques (EOIP) and seven EOAD cases without (EONIP) were selected and neuronal cell counts performed. These were compared with neuronal counts taken from the same cortical region of seven control and six sporadic AD cases. Cases with EOAD had significantly less neurones per field compared with sporadic AD and control cases (EOAD = 19.5 +/- 0.8 neurones/field, spAD = 23.7 +/- 1.2 neurones/field, controls = 30.37 +/- 1.2 neurones/field). However, no significant difference in the number of neurones per field was seen in EOAD cases with or without inflammatory plaque pathology (EOIP = 19.2 +/- 1.4, EONIP = 19.7 +/- 0.8). These data demonstrate that EOAD cases exhibit greater neuronal cell loss in the superior frontal cortex than sporadic AD and that this effect is independent of the presence or absence of inflammatory plaque pathology.

Antigen-epitope retrieval to facilitate proteomic analysis of formalin-fixed archival brain tissue.

Formalin is a routine fixative facilitating tissue preservation and histopathology. Proteomic techniques require freshly frozen specimens, which are often difficult to procure, and methods facilitating proteomic analysis of archival formalin-fixed brain tissue are lacking. We employed antigen-epitope-retrieval principles to facilitate proteomic analysis of brain tissue that had been fixed and stored in formalin for 3-7 years. Twenty-micrometer-thick cryopreserved OCT-embedded sections from inferior temporal cortex of human (7 years in formalin) or mouse brain specimens (3 years in formalin) were hematoxylin-/eosin-stained. Approximately 16-64-mm2 areas of the tissue sections were manually scraped off slides, or approximately 2 mm2 of human brain cortex was captured off membrane-coated slides using laser microdissection. Tissue was treated using various pH and temperature conditions prior to trypsin digestion and nano-LC-MS/MS. The largest number of proteins were retrieved by solubilization at pH 9 at 95 degrees C for 1 h; treatments at pH 4 or 6 at 25 or 65 degrees C were generally ineffective. Three-year formalin-fixed murine tissue did not yield more proteins compared to human tissue. Use of formalin-fixed tissue for proteomics is an invaluable tool for medical research. The combination of proteomics and microdissection enables selective enrichment and identification of novel, unique, or abundant proteins that may be important in pathogenesis.

Inflammatory S100A9 and S100A12 proteins in Alzheimer's disease.

Inflammation, insoluble protein deposition and neuronal cell loss are important features of the Alzheimer's disease (AD) brain. S100B is associated with the neuropathological hallmarks of AD where it is thought to play a role in neuritic pathology. S100A8, S100A9 and S100A12 comprise a new group of inflammation-associated proteins that are constitutively expressed by neutrophils and inducible in numerous inflammatory cells. We investigated expression of S100B, S100A8, S100A9 and S100A12 in brain samples from sporadic and familial (PS-1) AD cases and controls using immunohistochemistry and Western blot analysis. S100B, S100A9 and S100A12, but not S100A8, were consistently associated with the neuropathological hallmarks of AD. Western blot analysis confirmed significant increases in soluble S100A9 in PS-1 AD compared to controls. S100A9 complexes that were resistant to reduction were also evident in brain extracts. A reactive component of a size consistent with hexameric S100A12 was seen in all cases. This study indicates a potential role for pro-inflammatory S100A9 and S100A12 in pathogenesis caused by inflammation and protein complex formation in AD.

Novel 'inflammatory plaque' pathology in presenilin-1 Alzheimer's disease.

Inflammation, in the form of reactive astrocytes and microglia, is thought to play an important role in Alzheimer's disease (AD) pathogenesis where it correlates with brain atrophy and disease severity. The Abeta protein, which comprises senile plaques, is thought to be responsible for initiating this inflammatory response. Despite having a more aggressive disease process and greater Abeta deposition, few studies have investigated inflammation in early onset AD cases with mutations in the presenilin-1 (PS-1) gene. In fact, many researchers place importance on a variant plaque pathology in PS-1 cases, known as cotton wool plaques, which lack significant inflammatory infiltrate. We investigated the association between inflammation and plaque pathology in PS-1 AD. Classic cored, cotton wool and diffuse Abeta plaques were observed in all cases. PS-1 cases also exhibited a novel plaque pathology with a significantly greater inflammatory response in the form of reactive microglia and astrocytes. These 'inflammatory plaques' consisted of a dense cresyl violet-, silver-, and thioflavin S-positive, but Abeta-, tau-, apolipoprotein E (ApoE)-, non-Abeta component of Alzheimer's disease amyloid (NAC)- and PS-1-negative core. These findings indicate potent stimulator(s) of inflammation that are not typical of the Abeta that accumulates in the pathological hallmarks of sporadic AD. Identification of this substance may be important for the development of future therapeutic strategies.

Histocompatibility antigens, aspirin use and cognitive performance in non-demented elderly subjects.

HLA genotype and anti-inflammatory drug use have independently been associated with a lower risk of Alzheimer's disease (AD). We recently reported a negative association between aspirin use and AD. To investigate this further, we performed a cross-sectional study to investigate cognitive performance in 151 non-demented individuals in relation to HLA-DRB1 genotype and aspirin use. Aspirin and HLA-DRB1*01 were positive predictors of performance on logical memory (aspirin, p=0.04) and verbal fluency tests (HLA-DRB1*01, p=0.018), respectively. HLA-DRB1*05 had a negative impact on the Boston naming test (p=0.002). Our results suggest that aspirin use and inflammatory genotype may influence cognition in non-demented subjects.

Staging disease severity in pathologically confirmed cases of frontotemporal dementia.

OBJECTIVE: To devise a staging scheme for addressing the severity of atrophy in patients with pathologically proven frontotemporal dementia (FTD) and determine any relationship with clinical indices. METHODS: Twenty-four cases with clinical and pathologic features of FTD were selected using standard inclusion and exclusion criteria from 125 dementia cases collected in Sydney, Australia, over an 8.5-year period. Patterns of gross atrophy were determined in two coronal brain slices. Reproducibility of a four-stage severity scheme was tested. Nonparametric statistics were used to determine relationships between the stage of atrophy and clinical indices (age at death, duration from diagnosis, and clinical severity at death). RESULTS: The FTD cases studied could be reliably grouped (kappa = 0.97) into four progressively severe stages of global atrophy. Initial mild atrophy occurred in the orbital and superior medial frontal cortices and hippocampus (stage 1), progressed to involve the other anterior frontal regions, temporal cortices, and basal ganglia (stage 2), then involved all remaining tissue in these coronal slices (stage 3), until very marked atrophy was observed in all areas (stage 4). These stages correlated with disease duration and clinical dementia severity, lending validity to the progressive nature of the staging scheme. CONCLUSIONS: The authors have identified a reproducible staging system for the severity of gross atrophy in cases of FTD. This staging scheme provides the required framework to compare different research indices and determine correlates relating to time and disease progression in FTD-information necessary to determine core disease processes and etiologic factors.

Relationship between DNA fragmentation, morphological changes and neuronal loss in Alzheimer's disease and dementia with Lewy bodies.

This aim of the present study was to identify whether apoptotic features relate to the degree of cortical neuronal loss in cases with variable cortical degeneration. Neuronal apoptosis was assessed using histochemical and morphological criteria in cases with Alzheimer's disease (AD, n=7) or Lewy bodies (n= 11) compared with controls (n=11). AD cases had both significant plaque and tangle formation but no Lewy bodies, while cases with Lewy bodies had significant plaque formation but no tangles. Cortical sections were stained using (TdT)-mediated UTP nick end labelling (TUNEL), propidium iodide, and cell and pathology-specific labels. Cells identified as non-neuronal were excluded. Large cortical nuclei were classified as abnormal if they were TUNEL-positive with DNA condensation across the nucleus and no visible nucleolus, and further subdivided according to the presence or absence of visible neuronal cytoplasm. Nuclei were considered morphologically normal regardless of TUNEL staining if they possessed a clear nucleolus. Cortical fields containing the greatest density of TUNEL-positive nuclei were regionally sampled and the proportion of all classified nuclei calculated. Analysis of variance was used to identify any significant relationships. Only AD cases had significant numbers of abnormal nuclei (23+/-6%, P=0.002) and a corresponding decrease in normal neurons (28+/-7% loss, P= 0.004). Absolute neuronal density was also decreased in AD (AD density 64+/-11% of other cases, P=0.02). Abnormal nuclei were not associated with plaque or tangle pathology. Our results suggest that nuclear abnormalities appear restricted to AD cases with substantial tau deposition and are related to the degree of neuronal degeneration.

Neurofilament-immunoreactive neurons are not selectively vulnerable in Alzheimer's disease.

Abnormal neurofilament protein distribution and phosphorylation contributes to the cytoskeletal pathology of Alzheimer's disease. Anatomical studies suggest that cortical neurons immunoreactive for nonphosphorylated 200-kDa neurofilament are most vulnerable. We repeated these studies in formalin-fixed temporal lobe tissue from five Alzheimer's disease cases with tissue volume loss compared to five controls without tissue loss. Immunohistochemistry for nonphosphorylated and phosphorylated forms of the neurofilament protein was counterstained for Nissl substance and immuno-positive and -negative pyramidal neurons quantified using areal fraction counts. Compared with controls, cases with Alzheimer's disease had similar numbers of neurons expressing the nonphosphorylated neurofilament protein, suggesting these neurons are largely spared by the disease process. In Alzheimer's disease there was a significant increase in neurons containing phosphorylated neurofilament and tau proteins and a decrease in neurons devoid of neurofilament protein. Our results challenge the theory that neurons containing 200 kDa neurofilament are selectively vulnerable in Alzheimer's disease.

Expression of amyloid precursor protein in human astrocytes in vitro: isoform-specific increases following heat shock.

The beta-amyloid protein deposited in senile plaques and cerebral blood vessels in the Alzheimer's disease brain is derived from the larger transmembrane spanning amyloid precursor protein. The present study investigates the effects of heat shock on the expression and processing of amyloid precursor protein in a normal human fetal astrocytic cell line CC2565 using reverse transcription-polymerase chain reaction, in situ hybridization histochemistry and western blot analysis. Heat shock led to an increase in the messenger RNA encoding Kunitz protease inhibitor isoforms of amyloid precursor protein, which peaked at 4h post-heat shock. This increase was confined to the messenger RNA encoding amyloid precursor protein-751, with a decrease in amyloid precursor protein-770 and no change in amyloid precursor protein-695. This shift in splicing was accompanied by a significant decrease in secreted amyloid precursor protein and an increase in beta-secretase processing within the cell. These findings demonstrate that astrocytes in vitro demonstrate a striking response to heat shock. This is unlikely to be due to a direct action on the promoter region of the gene, since the response is specific for one splice variant; amyloid precursor protein-751 messenger RNA. This increase in expression is further accompanied by a decrease in secretion of amyloid precursor protein, implying a shift in processing towards an intracellular route, possibly via the actions of the beta-secretase enzyme, which is known to be potentially amyloidogenic. Such a mechanism may contribute to amyloidosis in the intact brain in response to cellular stress, such as head injury.

Cortical inflammation in Alzheimer disease but not dementia with Lewy bodies.

BACKGROUND: There have been no previous studies on the role of inflammation in the brain for the second most common dementing disorder, dementia with Lewy bodies. OBJECTIVE: To investigate the degree of cortical inflammation in dementia with Lewy bodies (DLB) compared with Alzheimer disease (AD) and control brains. DESIGN AND MAIN OUTCOME MEASURES: Post-mortem tissue collection from a brain donor program using standardized diagnostic criteria. Brains collected from January 1, 1993, through December 31, 1996, were screened and selected only for the presence or absence of tau neuritic plaques. Results of immunohistochemistry for HLA-DR were quantified using area fraction counts. Counts were performed by investigators who were unaware of the diagnosis. Results were compared across groups using analysis of variance and posthoc testing. SETTING: A medical research institute in Sydney, Australia. PATIENTS: Eight brains with DLB and without the tau neuritic plaques typical of AD, 10 brains with AD and no Lewy bodies, and 11 nondemented controls without significant neuropathological features were selected from a consecutive sample. RESULTS: Compared with AD, DLB demonstrated significantly less inflammation in the form of HLA-DR-reactive microglia in all cortical regions (P<.001, posthoc). The level of inflammation in DLB was comparable to that seen in controls (P=.54, post hoc). CONCLUSIONS: Inflammation appears related to the tau neuritic plaques of AD. Despite similar clinical presentations, therapeutic anti-inflammatory strategies are not likely to be effective for pure DLB. Arch Neurol. 2000.

Effect of anti-inflammatory medications on neuropathological findings in Alzheimer disease.

BACKGROUND: There has been no analysis of brain tissue from longitudinally observed, cognitively tested patients to validate whether anti-inflammatory medications protect against the pathological changes of Alzheimer disease. OBJECTIVE: To investigate the role of anti-inflammatory medications in alleviating the pathological features of Alzheimer disease. DESIGN AND MAIN OUTCOME MEASURES: A 5-year postmortem tissue collection was performed after a case-control study of Alzheimer disease (approximately 90 [30%] of patients died during follow-up, of whom consent for autopsy was obtained in 44 [50%]). Cases were selected on the basis of (1) adequate clinical histories of nonsteroidal anti-inflammatory drug usage, (2) no neuropathological findings other than Alzheimer disease, and (3) no generalized sepsis at death. Variables analyzed included neuropsychological test scores and amount of tissue inflammation and Alzheimer-type pathological changes. Two-way analysis of variance was used to determine whether drug usage significantly affected these variables. SETTING: The Centre for Education and Research on Ageing and the Prince of Wales Medical Research Institute, Sydney, Australia. PATIENTS: Twelve patients with Alzheimer disease (5 taking anti-inflammatory drugs) and 10 nondemented controls (3 taking anti-inflammatory drugs) were selected (50% of available sample). RESULTS: Of the patients with Alzheimer disease, anti-inflammatory drug users performed better on neuropsychological test scores than did nonusers. However, there were no significant differences in the amount of inflammatory glia, plaques, or tangles in either diagnostic group. CONCLUSION: Long-term anti-inflammatory medications in patients with Alzheimer disease enhanced cognitive performance but did not alleviate the progression of the pathological changes. Arch Neurol. 2000.