Claire J. Garwood

Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture.

Alzheimers disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase C gamma 1, Grb2, and Src family kinases

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 p85α subunit of phosphatidylinositol 3-kinase, phospholipase Cγ1, 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.

Microarray analysis of the astrocyte transcriptome in the aging brain: relationship to Alzheimer's pathology and APOE genotype

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 Alzheimers 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.

Minocycline reduces the development of abnormal tau species in models of Alzheimer’s disease

Alzheimers disease (AD) is characterized by the presence of neurofibrillary tangles of hyperphosphorylated, aggregated tau protein and extracellular deposits of β‐amyloid peptide. Increased β‐amyloid levels are thought to precede tangle formation, but tau pathology is more closely related to neuronal death. Minocycline, a tetracycline derivative, has potent antiinflammatory, antiapoptotic, and neuroprotective effects in several models of neurodegenerative disease, including models of AD with amyloid pathology. We have used both in vitro and in vivo models of AD to determine whether minocycline may have therapeutic efficacy against tau pathology. In primary cortical neurons, minocycline prevents β‐amyloid‐induced neuronal death, reduces caspase‐3 activation, and lowers generation of caspase‐3‐cleaved tau fragments. Treatment of tangle‐forming transgenic mice (htau line) with minocycline results in reduced levels of tau phosphorylation and insoluble tau aggregates. The in vivo effects of minocycline are also associated with reduced caspase‐3 activation and lowered tau cleavage by caspase‐3. In tau mice, we find that conformational changes in tau are susceptible to minocycline treatment, but are not directly associated with the amount of tau fragments produced, highlighting a dissociation between the development of these pathological tau species. These results suggest a possible novel therapeutic role for minocycline in the treatment of AD and related tauopathies.— Noble, W., Garwood, C., Stephenson, J., Kinsey, A. M., Hanger, D. P., Anderton, B. H. Minocycline reduces the development of abnormal tau species in models of Alzheimers disease. FASEB J. 23, 739–750 (2009)

Alterations in the blood brain barrier in ageing cerebral cortex in relationship to Alzheimer-type pathology: A study in the MRC-CFAS population neuropathology cohort

Impairment of the blood brain barrier (BBB) in human brain ageing and its relationship to Alzheimer-type pathology remains poorly defined. We have investigated the BBB in temporal cortex of brain donations from a population-representative sample of 92 participants from the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS), a longitudinal study with a programme of brain donation. BBB alteration was investigated by immunohistochemistry to albumin and fibrinogen and to the tight junction proteins claudin-5, zonula occludens-1 (ZO-1) and occludin. BBB leakage showed wide population-variation and increased with progression of Alzheimer-type pathology, though with considerable overlap between different levels of Alzheimer-type pathology. This was accompanied by increased mean vascular density, but not by down-regulation of tight junction proteins. ZO-1 and occludin were also expressed in glia. Mechanisms leading to BBB leakage in brain ageing remain to be defined, but the population-variation in BBB changes and its early increase in relationship to Alzheimer-type pathology progression suggest that BBB dysfunction contributes to brain ageing.

Kinase activities increase during the development of tauopathy in htau mice.

Hyperphosphorylated tau aggregates are the core constituent of neurofibrillary tangles. Recent research has shown a division between the presence of tangles, neurodegeneration and subsequent memory impairment, raising the possibility that an earlier pre‐aggregated form of tau may be toxic. To gain further insight into the relationship between abnormal forms of tau, we have analyzed pathological changes in tau during tauopathy development in tangle‐forming transgenic mice. In addition, we have quantified changes in the endogenous levels of a panel of protein kinases. We show progressive increases in aggregated tau and disease‐specific conformational change, with hyperphosphorylation occurring in an age‐dependent manner at specific sites. There were significant correlations between specific phosphorylation changes and amounts of aggregated tau and and abnormal tau conformations. Of the protein kinases tested, we found increases in phosphorylated (activated) p38 and the cyclin‐dependent kinase‐5 neuronal activators, p35 and p25, with aging, in the htau line, but not in non‐tangle‐forming control mice. Changes in tau kinases correlated with the amount of tau present in abnormal conformations and with insoluble tau in htau mice. These data suggest that cdk5 and p38 may be associated with pathological changes in wild‐type human tau during the progressive development of tauopathy.

Minocycline as a potential therapeutic agent in neurodegenerative disorders characterised by protein misfolding

Many neurodegenerative disorders share common features including the accumulation of aggregated misfolded proteins, neuroinflammation and apoptotic neuronal death. 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 characterised by the presence of misfolded proteins.

Anti-Inflammatory Impact of Minocycline in a Mouse Model of Tauopathy

Alzheimers disease (AD) is characterized by the extracellular deposition of β-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 Huntingtons disease, Parkinsons disease and motor neuron disease. Recently, we demonstrated that minocycline reduces the development of abnormal tau species in the htau mouse model of Alzheimers 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 Alzheimers disease and related disorders.

Astrocytes and neuroinflammation in Alzheimer's disease

Increased production of amyloid β-peptide (Aβ) and altered processing of tau in Alzheimers 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.

Review: Astrocytes in Alzheimer's disease and other age-associated dementias; a supporting player with a central role.

Astrocytes have essential roles in the central nervous system and are also implicated in the pathogenesis of neurodegenerative disease. Forming non‐overlapping domains, astrocytes are highly complex cells. Immunohistochemistry to a variety of proteins can be used to study astrocytes in tissue, labelling different cellular components and sub‐populations, including glial fibrillary acidic protein, ALDH1L1, CD44, NDRG2 and amino acid transporters, but none of these labels the entire astrocyte population. Increasing heterogeneity is recognized in the astrocyte population, a complexity that is relevant both to their normal function and pathogenic roles. They are involved in neuronal support, as active components of the tripartite synapse and in cell interactions within the neurovascular unit (NVU), where they are essential for blood–brain barrier maintenance and neurovascular coupling. Astrocytes change with age, and their responses may modulate the cellular effects of neurodegenerative pathologies, which alone do not explain all of the variance in statistical models of neurodegenerative dementias. Astrocytes respond to both the neurofibrillary tangles and plaques of Alzheimers disease, to hyperphosphorylated tau and Aβ, eliciting an effect which may be neuroprotective or deleterious. Not only astrocyte hypertrophy, in the form of gliosis, occurs, but also astrocyte injury and atrophy. Loss of normal astrocyte functions may contribute to reduced support for neurones and dysfunction of the NVU. Understanding how astrocytes contribute to dementia requires an understanding of the underlying heterogeneity of astrocyte populations, and the complexity of their responses to pathology. Enhancing the supportive and neuroprotective components of the astrocyte response has potential translational applications in therapeutic approaches to dementia.