Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 19 de 19
Filter
Add more filters










Publication year range
1.
J Neurochem ; 160(4): 434-453, 2022 02.
Article in English | MEDLINE | ID: mdl-34767256

ABSTRACT

Although controversial, the amyloid cascade hypothesis remains central to the Alzheimer's disease (AD) field and posits amyloid-beta (Aß) as the central factor initiating disease onset. In recent years, there has been an increase in emphasis on studying the role of low molecular weight aggregates, such as oligomers, which are suggested to be more neurotoxic than fibrillary Aß. Other Aß isoforms, such as truncated Aß, have also been implicated in disease. However, developing a clear understanding of AD pathogenesis has been hampered by the complexity of Aß biochemistry in vitro and in vivo. This review explores factors contributing to the lack of consistency in experimental approaches taken to model Aß aggregation and toxicity and provides an overview of the different techniques available to analyse Aß, such as electron and atomic force microscopy, nuclear magnetic resonance spectroscopy, dye-based assays, size exclusion chromatography, mass spectrometry and SDS-PAGE. The review also explores how different types of Aß can influence Aß aggregation and toxicity, leading to variation in experimental outcomes, further highlighting the need for standardisation in Aß preparations and methods used in current research.


Subject(s)
Alzheimer Disease/genetics , Amyloid beta-Peptides/genetics , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Animals , Humans
2.
Eur J Neurosci ; 54(9): 6987-7005, 2021 11.
Article in English | MEDLINE | ID: mdl-34536321

ABSTRACT

Oxidative DNA damage induces changes in the neuronal cell cycle and activates a DNA damage response (DDR) to promote repair, but these processes may be altered under a chronic oxidative environment, leading to the accumulation of unrepaired DNA damage and continued activation of a DDR. Failure to repair DNA damage can lead to apoptosis or senescence, which is characterized by a permanent cell cycle arrest. Increased oxidative stress and accumulation of oxidative DNA damage are features of brain ageing and neurodegeneration, but the effects of persistent DNA damage in neurons are not well characterized. We developed a model of persistent oxidative DNA damage in immortalized post-mitotic neurons in vitro by exposing them to a sublethal concentration of hydrogen peroxide following a 'double stress' protocol and performed a detailed characterization of the neuronal transcriptome using microarray analysis. Persistent DNA damage significantly altered the expression of genes involved in cell cycle regulation, DDR and repair mechanisms, and mitochondrial function, suggesting an active DDR response to replication stress and alterations in mitochondrial electron transport chain. Quantitative polymerase chain reaction (qPCR) and functional validation experiments confirmed hyperactivation of mitochondrial Complex I in response to persistent DNA damage. These changes in response to persistent oxidative DNA damage may lead to further oxidative stress, contributing to neuronal dysfunction and ultimately neurodegeneration.


Subject(s)
DNA Damage , Transcriptome , Cell Cycle , Mitochondria/metabolism , Neurons/metabolism , Oxidative Stress
3.
Int J Mol Sci ; 21(21)2020 Oct 28.
Article in English | MEDLINE | ID: mdl-33126586

ABSTRACT

Hypoxia is a feature of neurodegenerative diseases, and can both directly and indirectly impact on neuronal function through modulation of glial function. Astrocytes play a key role in regulating homeostasis within the central nervous system, and mediate hypoxia-induced changes in response to reduced oxygen availability. The current study performed a detailed characterization of hypoxia-induced changes in the transcriptomic profile of astrocytes in vitro. Human astrocytes were cultured under normoxic (5% CO2, 95% air) or hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 h, and the gene expression profile assessed by microarray analysis. In response to hypoxia 4904 genes were significantly differentially expressed (1306 upregulated and 3598 downregulated, FC ≥ 2 and p ≤ 0.05). Analysis of the significant differentially expressed transcripts identified an increase in immune response pathways, and dysregulation of signalling pathways, including HIF-1 (p = 0.002), and metabolism, including glycolysis (p = 0.006). To assess whether the hypoxia-induced metabolic gene changes observed affected metabolism at a functional level, both the glycolytic and mitochondrial flux were measured using an XF bioanalyser. In support of the transcriptomic data, under physiological conditions hypoxia significantly reduced mitochondrial respiratory flux (p = 0.0001) but increased basal glycolytic flux (p = 0.0313). However, when metabolically stressed, hypoxia reduced mitochondrial spare respiratory capacity (p = 0.0485) and both glycolytic capacity (p = 0.0001) and glycolytic reserve (p < 0.0001). In summary, the current findings detail hypoxia-induced changes in the astrocyte transcriptome in vitro, identifying potential targets for modifying the astrocyte response to reduced oxygen availability in pathological conditions associated with ischaemia/hypoxia, including manipulation of mitochondrial function, metabolism, and the immune response.


Subject(s)
Astrocytes/pathology , Hypoxia/physiopathology , Immunity/genetics , Mitochondria/pathology , Transcriptome , Astrocytes/immunology , Astrocytes/metabolism , Cells, Cultured , Glycolysis , Homeostasis , Humans , In Vitro Techniques , Mitochondria/immunology , Mitochondria/metabolism
4.
J Neuropathol Exp Neurol ; 79(9): 950-958, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32766675

ABSTRACT

Diabetes mellitus is a risk factor for dementia, and nonenzymatic glycosylation of macromolecules results in formation of advanced glycation end-products (AGEs). We determined the variation in AGE formation in brains from the Cognitive Function and Ageing Study population-representative neuropathology cohort. AGEs were measured on temporal neocortex by enzyme-linked immunosorbent assay (ELISA) and cell-type specific expression on neurons, astrocytes and endothelium was detected by immunohistochemistry and assessed semiquantitatively. Fifteen percent of the cohort had self-reported diabetes, which was not significantly associated with dementia status at death or neuropathology measures. AGEs were expressed on neurons, astrocytes and endothelium and overall expression showed a positively skewed distribution in the population. AGE measures were not significantly associated with dementia. AGE measured by ELISA increased with Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neurofibrillary tangle score (p = 0.03) and Thal Aß phase (p = 0.04), while AGE expression on neurons (and astrocytes), detected immunohistochemically, increased with increasing Braak tangle stage (p < 0.001), CERAD tangle score (p = 0.002), and neuritic plaques (p = 0.01). Measures of AGE did not show significant associations with cerebral amyloid angiopathy, microinfarcts or neuroinflammation. In conclusion, AGE expression increases with Alzheimer's neuropathology, particular later stages but is not independently associated with dementia. AGE formation is likely to be important for impaired brain cell function in aging and Alzheimer's.


Subject(s)
Aging/metabolism , Aging/pathology , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Glycation End Products, Advanced/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Cohort Studies , Dementia/metabolism , Dementia/pathology , Female , Humans , Male , Neurofibrillary Tangles/metabolism , Neurofibrillary Tangles/pathology , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology
5.
Int J Mol Sci ; 21(1)2020 Jan 04.
Article in English | MEDLINE | ID: mdl-31947996

ABSTRACT

Astrocytes play a major role in the pathogenesis of a range of neurodegenerative diseases, including Alzheimer's disease (AD), undergoing dramatic morphological and molecular changes that can cause potentially both beneficial and detrimental effects. They comprise a heterogeneous population, requiring a panel of specific phenotype markers to identify astrocyte subtypes, changes in function and their relation to pathology. This study aimed to characterise expression of the astrocyte marker N-myc downstream regulated gene 2 (NDRG2) in the ageing brain, investigate the relationship between NDRG2 and a panel of astrocyte markers, and relate NDRG2 expression to pathology. NDRG2 specifically immunolabelled the cell body and radiating processes of astrocytes in the temporal cortex of the Cognitive Function and Ageing Study (CFAS) neuropathology cohort. Expression of NDRG2 did not correlate with other astrocyte markers, including glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 2 (EAAT2) and glutamine synthetase (GS). NDRG2 showed a relationship to AT8+ neurofibrillary tangles (p = 0.001) and CD68+ microglia (p = 0.047), but not ß-amyloid plaques or astrocyte nuclear γH2AX immunoreactivity, a marker of DNA damage response. These findings provide new insight into the astrocyte response to pathology in the ageing brain, and suggest NDRG2 may be a potential target to modulate this response.


Subject(s)
Aging , Alzheimer Disease/pathology , Brain/metabolism , Microglia/metabolism , Neurofibrillary Tangles/metabolism , Tumor Suppressor Proteins/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Astrocytes/cytology , Astrocytes/metabolism , Brain/pathology , DNA Damage , Excitatory Amino Acid Transporter 2/metabolism , Gene Expression Regulation , Glial Fibrillary Acidic Protein/metabolism , Glutamate-Ammonia Ligase/metabolism , Humans , Microglia/pathology , Tumor Suppressor Proteins/genetics , tau Proteins/metabolism
6.
Glia ; 66(11): 2316-2323, 2018 11.
Article in English | MEDLINE | ID: mdl-30098078

ABSTRACT

Oxidative stress and oxidative DNA damage are early features of mild cognitive impairment and Alzheimer's disease (AD), occurring before the formation of classical AD neuropathology, and resulting from an imbalance between pro- and anti-oxidants. Astrocytes play a major neuroprotective role, producing high levels of anti-oxidants including metallothionein-I and -II (MT-I/II). In the present study we characterized the immunoreactive profile of MT-I/II in the temporal cortex of the Cognitive Function and Ageing Study (CFAS) aging population-representative neuropathology cohort, and examined H2 O2 -modulation of MT transcription by human astrocytes. MT-I/II is primarily expressed by astrocytes in the aging brain, but is also associated with pyramidal neurons in a small proportion of cases. Astrocyte expression of MT-I/II does not correlate with Alzheimer-type pathology (Aß plaques and neurofibrillary tangles) but does relate to astrocyte oxidative DNA damage (rs = .312, p = .006) and the astrocyte response to oxidative DNA damage in vivo (rs = .238, p = .04), and MT gene expression is significantly induced in human astrocytes response to oxidative stress in vitro (p = .01). In contrast, neuronal MT-I/II does not relate to oxidative DNA damage or the neuronal DNA damage response, but is significantly higher in cases with high levels of local tangle pathology (p = .007). As MT-I/II is neuroprotective against oxidative stress, modulation of MT-I/II expression is a potential therapeutic target to treat the onset and progression of cognitive impairment.


Subject(s)
Aging/pathology , Alzheimer Disease/pathology , Astrocytes/metabolism , Brain/metabolism , DNA Damage/physiology , Metallothionein/metabolism , Aged , Aged, 80 and over , Astrocytes/drug effects , Astrocytes/pathology , Brain/pathology , Cells, Cultured , Female , Gene Expression Regulation/drug effects , Humans , Hydrogen Peroxide/pharmacology , Male , Metallothionein/genetics , Neurons/metabolism , Tauopathies/metabolism , Tauopathies/pathology , Time Factors
7.
Neuroscience ; 390: 46-59, 2018 10 15.
Article in English | MEDLINE | ID: mdl-30056117

ABSTRACT

The insulin/insulin-like growth factor 1 (IGF1) signaling pathways are implicated in longevity and in progression of Alzheimer's disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signaling transcripts are reduced in astrocytes in human brain with progression of Alzheimer's neuropathology and developed a model of IGF1 signaling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signaling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.


Subject(s)
Astrocytes/metabolism , Electron Transport Complex I/metabolism , Neurons/metabolism , Receptors, Somatomedin/metabolism , Antibodies, Monoclonal/administration & dosage , Coculture Techniques/methods , Energy Metabolism , Humans , Neuronal Outgrowth , Oxidative Stress , Primary Cell Culture , Receptor, IGF Type 1 , Receptors, Somatomedin/immunology , Transcriptome
8.
Eur J Neurosci ; 47(12): 1444-1456, 2018 06.
Article in English | MEDLINE | ID: mdl-29738614

ABSTRACT

Occludin is a component of tight junctions, which are essential structural components of the blood-brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull-down proteins from 1321N1 cell lysates and protein-binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states.


Subject(s)
Astrocytes/metabolism , Astrocytoma/metabolism , Brain/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Endothelial Cells/metabolism , Occludin/metabolism , RNA/metabolism , Cell Line, Tumor , Cells, Cultured , Cytological Techniques , Fetus , Humans , Mass Spectrometry , Proteomics
9.
Neuropathol Appl Neurobiol ; 42(2): 167-79, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26095650

ABSTRACT

AIMS: Oxidative damage and an associated DNA damage response (DDR) are evident in mild cognitive impairment and early Alzheimer's disease, suggesting that neuronal dysfunction resulting from oxidative DNA damage may account for some of the cognitive impairment not fully explained by Alzheimer-type pathology. METHODS: Frontal cortex (Braak stage 0-II) was obtained from the Medical Research Council's Cognitive Function and Ageing Study cohort. Neurones were isolated from eight cases (four high and four low DDR) by laser capture microdissection and changes in the transcriptome identified by microarray analysis. RESULTS: Two thousand three hundred seventy-eight genes were significantly differentially expressed (1690 up-regulated, 688 down-regulated, P < 0.001) in cases with a high neuronal DDR. Functional grouping identified dysregulation of cholesterol biosynthesis, insulin and Wnt signalling, and up-regulation of glycogen synthase kinase 3ß. Candidate genes were validated by quantitative real-time polymerase chain reaction. Cerebrospinal fluid levels of 24(S)-hydroxycholesterol associated with neuronal DDR across all Braak stages (rs = 0.30, P = 0.03). CONCLUSIONS: A persistent neuronal DDR may result in increased cholesterol biosynthesis, impaired insulin and Wnt signalling, and increased GSK3ß, thereby contributing to neuronal dysfunction independent of Alzheimer-type pathology in the ageing brain.


Subject(s)
Aging/metabolism , Alzheimer Disease/metabolism , Brain/metabolism , Cholesterol/metabolism , DNA Damage/physiology , Neurons/metabolism , Aged, 80 and over , Aging/pathology , Alzheimer Disease/pathology , Blotting, Western , Brain/pathology , Female , Humans , Immunohistochemistry , Laser Capture Microdissection , Male , Neurons/pathology , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Signal Transduction/physiology , Transcriptome
10.
Neurosci Lett ; 609: 11-7, 2015 Nov 16.
Article in English | MEDLINE | ID: mdl-26455863

ABSTRACT

The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council's Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment.


Subject(s)
Astrocytes/metabolism , Brain/metabolism , Cell Nucleus/metabolism , DNA Damage , Dementia/metabolism , Forkhead Transcription Factors/metabolism , Glutamate-Ammonia Ligase/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Dementia/pathology , Female , Forkhead Box Protein O3 , Gliosis/metabolism , Gliosis/pathology , Humans , Male , Temporal Lobe/metabolism , Temporal Lobe/pathology
11.
Mol Brain ; 8: 51, 2015 Aug 22.
Article in English | MEDLINE | ID: mdl-26297026

ABSTRACT

BACKGROUND: The insulin/IGF1 signalling (IIS) pathways are involved in longevity regulation and are dysregulated in neurons in Alzheimer's disease (AD). We previously showed downregulation in IIS gene expression in astrocytes with AD-neuropathology progression, but IIS in astrocytes remains poorly understood. We therefore examined the IIS pathway in human astrocytes and developed models to reduce IIS at the level of the insulin or the IGF1 receptor (IGF1R). RESULTS: We determined IIS was present and functional in human astrocytes by immunoblotting and showed astrocytes express the insulin receptor (IR)-B isoform of Ir. Immunocytochemistry and cell fractionation followed by western blotting revealed the phosphorylation status of insulin receptor substrate (IRS1) affects its subcellular localisation. To validate IRS1 expression patterns observed in culture, expression of key pathway components was assessed on post-mortem AD and control tissue using immunohistochemistry. Insulin signalling was impaired in cultured astrocytes by treatment with insulin + fructose and resulted in decreased IR and Akt phosphorylation (pAkt S473). A monoclonal antibody against IGF1R (MAB391) induced degradation of IGF1R receptor with an associated decrease in downstream pAkt S473. Neither treatment affected cell growth or viability as measured by MTT and Cyquant® assays or GFAP immunoreactivity. DISCUSSION: IIS is functional in astrocytes. IR-B is expressed in astrocytes which differs from the pattern in neurons, and may be important in differential susceptibility of astrocytes and neurons to insulin resistance. The variable presence of IRS1 in the nucleus, dependent on phosphorylation pattern, suggests the function of signalling molecules is not confined to cytoplasmic cascades. Down-regulation of IR and IGF1R, achieved by insulin + fructose and monoclonal antibody treatments, results in decreased downstream signalling, though the lack of effect on viability suggests that astrocytes can compensate for changes in single pathways. Changes in signalling in astrocytes, as well as in neurons, may be important in ageing and neurodegeneration.


Subject(s)
Astrocytes/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin/metabolism , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolism , Signal Transduction , Aged , Astrocytes/drug effects , Brain/drug effects , Brain/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Female , Fructose/pharmacology , Humans , Immunohistochemistry , Insulin/pharmacology , Male , Middle Aged , Phosphorylation/drug effects , Protein Isoforms/metabolism , Protein Transport/drug effects , Signal Transduction/drug effects , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism
12.
PLoS One ; 10(2): e0118463, 2015.
Article in English | MEDLINE | ID: mdl-25707004

ABSTRACT

AIMS: ß-amyloid (Aß) plaques are a key feature of Alzheimer's disease pathology but correlate poorly with dementia. They are associated with astrocytes which may modulate the effect of Aß-deposition on the neuropil. This study characterised the astrocyte response to Aß plaque subtypes, and investigated their association with cognitive impairment. METHODS: Aß plaque subtypes were identified in the cingulate gyrus using dual labelling immunohistochemistry to Aß and GFAP+ astrocytes, and quantitated in two cortical areas: the area of densest plaque burden and the deep cortex near the white matter border (layer VI). Three subtypes were defined for both diffuse and compact plaques (also known as classical or core-plaques): Aß plaque with (1) no associated astrocytes, (2) focal astrogliosis or (3) circumferential astrogliosis. RESULTS: In the area of densest burden, diffuse plaques with no astrogliosis (ß = -0.05, p = 0.001) and with focal astrogliosis (ß = -0.27, p = 0.009) significantly associated with lower MMSE scores when controlling for sex and age at death. In the deep cortex (layer VI), both diffuse and compact plaques without astrogliosis associated with lower MMSE scores (ß = -0.15, p = 0.017 and ß = -0.81, p = 0.03, respectively). Diffuse plaques with no astrogliosis in layer VI related to dementia status (OR = 1.05, p = 0.025). In the area of densest burden, diffuse plaques with no astrogliosis or with focal astrogliosis associated with increasing Braak stage (ß = 0.01, p<0.001 and ß = 0.07, p<0.001, respectively), and ApoEε4 genotype (OR = 1.02, p = 0.001 and OR = 1.10, p = 0.016, respectively). In layer VI all plaque subtypes associated with Braak stage, and compact amyloid plaques with little and no associated astrogliosis associated with ApoEε4 genotype (OR = 1.50, p = 0.014 and OR = 0.10, p = 0.003, respectively). CONCLUSIONS: Reactive astrocytes in close proximity to either diffuse or compact plaques may have a neuroprotective role in the ageing brain, and possession of at least one copy of the ApoEε4 allele impacts the astroglial response to Aß plaques.


Subject(s)
Aging/pathology , Astrocytes/pathology , Cognition Disorders/pathology , Plaque, Amyloid/pathology , Aged , Aged, 80 and over , Amyloid beta-Peptides/metabolism , Cognition Disorders/metabolism , Female , Glial Fibrillary Acidic Protein/metabolism , Humans , Male , Plaque, Amyloid/metabolism
13.
Biochem Soc Trans ; 42(5): 1321-5, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25233410

ABSTRACT

Increased production of amyloid ß-peptide (Aß) and altered processing of tau in Alzheimer's disease (AD) are associated with synaptic dysfunction, neuronal death and cognitive and behavioural deficits. Neuroinflammation is also a prominent feature of AD brain and considerable evidence indicates that inflammatory events play a significant role in modulating the progression of AD. The role of microglia in AD inflammation has long been acknowledged. Substantial evidence now demonstrates that astrocyte-mediated inflammatory responses also influence pathology development, synapse health and neurodegeneration in AD. Several anti-inflammatory therapies targeting astrocytes show significant benefit in models of disease, particularly with respect to tau-associated neurodegeneration. However, the effectiveness of these approaches is complex, since modulating inflammatory pathways often has opposing effects on the development of tau and amyloid pathology, and is dependent on the precise phenotype and activities of astrocytes in different cellular environments. An increased understanding of interactions between astrocytes and neurons under different conditions is required for the development of safe and effective astrocyte-based therapies for AD and related neurodegenerative diseases.


Subject(s)
Alzheimer Disease/pathology , Astrocytes/pathology , Neurons/pathology , Alzheimer Disease/immunology , Alzheimer Disease/metabolism , Animals , Astrocytes/immunology , Astrocytes/metabolism , Cell Communication , Humans , Neurons/immunology , Neurons/metabolism , Signal Transduction
14.
Neuropathol Appl Neurobiol ; 40(7): 802-14, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24861546

ABSTRACT

AIMS: Abnormalities of the brain microvasculature in Alzheimer's disease have led to the vascular hypothesis of the disease, which predicts that vascular changes precede neuronal dysfunction and degeneration. To determine the spectrum of endothelial injury in the elderly and its relation to Alzheimer-type neuropathology we investigated DNA damage in a population-based sample derived from the Medical Research Council Cognitive Function and Ageing Study. METHODS: We examined endothelial damage in frontal and temporal cortex (n = 97) using immunohistochemistry for γH2AX and DNA-protein kinase (DNA-PKcs). To determine the effects of endothelial DNA damage at the earliest stages of Alzheimer's pathology we further focused our analysis on cases classified as Braak 0-II and examined endothelial senescence using histochemistry for ß-galactosidase and the expression of genes related to DNA damage and senescence using quantitative polymerase chain reaction (qPCR). RESULTS: We demonstrated large variation in endothelial DNA damage which was not associated with Alzheimer's neuropathology. Endothelial DNA-PKcs correlated with neuronal and glial DNA-PKcs counts. Focusing our further analysis on Braak 0-II cases, qPCR analysis demonstrated a trend to increased TP53 (P = 0.064) in cases with high compared with low endothelial DNA damage which was supported by immunohistochemical analysis of p53. Endothelial ß-galactosidase expression was associated with increased neuronal (P = 0.033) and glial (P = 0.038), but not endothelial DNA-PKcs expression. CONCLUSIONS: Damage to brain endothelial cells occurs early in relation to, or independently of, Alzheimer pathology, and parallels that in neurones and glia. Endothelial DNA damage and senescence are a brain ageing process that may contribute to dysfunction of the neurovascular unit in some elderly individuals.


Subject(s)
Alzheimer Disease/genetics , Cellular Senescence/genetics , Cerebral Cortex/blood supply , Cerebral Cortex/metabolism , DNA Damage , Endothelial Cells/metabolism , Aged , Aged, 80 and over , Disease Progression , Frontal Lobe/blood supply , Frontal Lobe/metabolism , Humans , Microvessels/metabolism , Temporal Lobe/metabolism
15.
Aging Cell ; 13(1): 49-59, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23919677

ABSTRACT

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of ß-amyloid (Aß) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase-3, activity is a prominent feature of AD brain. In addition, we observe increased calpain-mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aß1-42. We also show that exposure of primary cortical neurons to oligomeric Aß1-42 results in calpain-dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aß toxicity. Our findings suggest that Aß mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.


Subject(s)
Alzheimer Disease/enzymology , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Calpain/metabolism , Sodium-Calcium Exchanger/metabolism , Aged , Aged, 80 and over , Amyloid beta-Peptides/toxicity , Animals , Brain/drug effects , Brain/enzymology , Brain/pathology , Calcium-Binding Proteins/metabolism , Caspase 3/metabolism , Cells, Cultured , Female , Gene Knockdown Techniques , Humans , Male , Middle Aged , Oligonucleotides, Antisense/pharmacology , Postmortem Changes , Protein Subunits/metabolism , Rats , Spectrin/metabolism , Substrate Specificity/drug effects , Tauopathies/enzymology , Tauopathies/pathology
16.
Neurobiol Aging ; 32(10): 1795-807, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21705112

ABSTRACT

Astrocytes contribute to a variety of functions in the brain, including homeostasis, synapse formation, plasticity, and metabolism. Astrocyte dysfunction may disrupt their normal role, including neuronal support, thereby contributing to neurodegenerative pathologies, including Alzheimer's disease (AD). To understand the role of astrocytes in the pathogenesis of age-related disorders, we isolated astrocytes by laser capture microdissection, using glial fibrillary acidic protein (GFAP) as a marker, and characterized the astrocyte transcriptome at different Braak neurofibrillary tangle stages in postmortem temporal cortex samples derived from the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS) cohort, using microarray analysis. The largest number of significant, differentially expressed genes were identified when the expression profile of astrocytes from isocortical stages of neurofibrillary tangle pathology (Braak stages V-VI) were compared with entorhinal stages (Braak stages I-II). Dysregulation of genes associated with the actin cytoskeleton, proliferation, apoptosis, and ubiquitin-mediated proteolysis occurred at low Braak stages, while altered regulation of intracellular signaling pathways, including insulin, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinase (MAPK) pathways were primarily associated with high levels of Alzheimer-type pathology, and occurred at lower Braak stages in individuals with the APOEε4 allele. Our findings implicate astrocyte dysfunction in the pathogenesis of neurodegenerative pathology in the aging brain, and provide a basis for future candidate studies based on specific pathways.


Subject(s)
Aging/pathology , Apolipoproteins E/genetics , Astrocytes/metabolism , Gene Expression Regulation/genetics , Temporal Lobe/pathology , Transcriptome/physiology , Actins/metabolism , Aged , Aged, 80 and over , Astrocytes/pathology , Female , Genotype , Glial Fibrillary Acidic Protein/metabolism , Humans , Male , Microarray Analysis/methods , Microdissection/methods , Postmortem Changes , Reproducibility of Results , Signal Transduction/genetics
17.
Front Psychiatry ; 1: 136, 2010.
Article in English | MEDLINE | ID: mdl-21423446

ABSTRACT

Alzheimer's disease (AD) is characterized by the extracellular deposition of ß-amyloid in senile plaques, the intraneuronal accumulation of hyperphosphorylated tau aggregates as neurofibrillary tangles, and progressive neuronal loss leading to the onset of dementia. Increasing evidence suggests that neuroinflammatory processes contribute to the progression of AD. Minocycline is a semi-synthetic tetracycline derivative commonly used in the treatment of acne. Many studies have revealed that minocycline also has potent anti-inflammatory actions that are neuroprotective in rodent models of Huntington's disease, Parkinson's disease and motor neuron disease. Recently, we demonstrated that minocycline reduces the development of abnormal tau species in the htau mouse model of Alzheimer's disease. We have now extended these findings by examining the impact of minocycline on inflammatory processes in htau mice. Immunohistochemical analysis revealed that minocycline treatment resulted in fewer activated astrocytes in several cortical regions of htau mice, but did not affect astrocytosis in the hippocampus. We found htau mice have significantly elevated amounts of several cortical pro-inflammatory cytokines. In addition, we find that minocycline treatment significantly reduced the amounts of several inflammatory factors, including monocyte chemoattractant proteins 1 and 5, interleukins -6 and -10, eotaxin, and I-309. Furthermore, the reduced amounts of these cytokines significantly correlated with the amount of tau phosphorylated at Ser396/404 in the cortex of htau mice. These results may reveal new cytokine targets of minocycline that could be associated with its inhibition of tau pathology development in vivo. It is possible that further investigation of the role of these cytokines in neurodegenerative processes may identify novel therapeutic targets for Alzheimer's disease and related disorders.

18.
Prion ; 3(2): 78-83, 2009.
Article in English | MEDLINE | ID: mdl-19458490

ABSTRACT

Many neurodegenerative disorders share common features including the accumulation of aggregated misfolded proteins, neuroinflammation and the induction of apoptosis. While the contributions of each of these individual elements to neuronal death remain unclear, a commonly used antibiotic, minocycline, has been shown to reduce the progression and severity of disease in several models of neurodegeneration by variously downregulating these molecular pathways. Here we discuss the evidence for the potential of minocycline as a broad-specificity therapeutic agent for those neurodegenerative diseases that are characterized by the presence of misfolded proteins.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Minocycline/therapeutic use , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/metabolism , Animals , Anti-Bacterial Agents/chemistry , Humans , Minocycline/chemistry , Molecular Structure , Protein Folding
19.
J Biol Chem ; 283(26): 18177-86, 2008 Jun 27.
Article in English | MEDLINE | ID: mdl-18467332

ABSTRACT

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.


Subject(s)
GRB2 Adaptor Protein/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma/metabolism , src-Family Kinases/metabolism , tau Proteins/chemistry , Alzheimer Disease/metabolism , Amino Acid Sequence , Humans , Molecular Conformation , Molecular Sequence Data , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Structure, Tertiary , src Homology Domains
SELECTION OF CITATIONS
SEARCH DETAIL
...