Expression of p16 and p21 in the frontal association cortex of ALS/MND brains suggests neuronal cell cycle dysregulation and astrocyte senescence in early stages of the disease.

AIMS: Cellular senescence plays a role in organismal ageing and has been linked to persistent DNA damage in age-related diseases. Brain senescence has been described in astrocytes and microglia, but it is less well understood in neurones. Evidence suggests that neurones activate a senescence-like mechanism that could contribute to neurodegeneration. We aimed to determine whether a persistent DNA damage response (DDR) and senescence activation are features of motor neurone disease (amyotrophic lateral sclerosis, ALS/MND). METHODS: We examined expression of senescence (p16 and p21) and DNA damage markers (8-OHdG and γH2AX) in motor cortex (MCx), frontal association cortex (FACx) and occipital cortex (OCx) in post-mortem tissue donated by patients with ALS/MND and controls. RESULTS: Nuclear expression of p16 and p21 was detected in glial cells; double immunofluorescence for p16/p21 and glial fibrillary acidic protein (GFAP) suggested that some of these cells were GFAP+ astrocytes. p21 nuclear expression was also found in neurones. Higher levels of p16+ (glia, P = 0.028) and p21+ (glia, P = 0.003; neurones, P = 0.008) cells were found in the FACx of ALS/MND donors but not in the MCx or OCx. Expression of p16 and p21 did not correlate with 8-OHdG or γH2AX. CONCLUSIONS: Expression of p16 and p21 in glia, mainly in astrocytes, suggests senescence induction in these cells; however, neuronal p21 expression might reflect a more general mechanism of age-related cell cycle dysregulation. The significantly higher proportion of cells expressing either p16 or p21 in the FACx of ALS/MND donors could indicate senescence activation and cell cycle dysregulation in early stages of the disease.

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 Alzheimer's disease, to hyperphosphorylated tau and Aß, 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.

Calcium dysregulation in relation to Alzheimer-type pathology in the ageing brain.

AIMS: Calcium dyshomeostasis is implicated in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease. However, much of the previous research has focused on changes in neuronal calcium signalling. In a recent microarray study we identified dysregulation of several key signalling pathways including the Ca(2+) signalling pathway in astrocytes as Alzheimer-type pathology developed. In this study we sought to determine the expression of calpain-10 and calcium/calmodulin-dependent kinase alpha (CamKIIα) in relation to Alzheimer-type pathology in a population-based study. METHODS: Using post mortem temporal cortex samples derived from the Medical Research Council Cognitive Function and Ageing Study (MRC-CFAS) ageing brain cohort we examined calpain-10 and CamKIIα gene and protein expression using quantitative polymerase chain reaction and immunohistochemistry. RESULTS: We demonstrate that astrocytic expression of calpain-10 is up-regulated, and CamKIIα down-regulated with increasing Braak stage. Using immunohistochemistry we confirm protein expression of calpain-10 in astrocytes throughout the temporal cortex and demonstrate that calpain-10 immunoreactivity is correlated with both local and global measures of Alzheimer-type pathology. In addition, we identify a subpopulation of calpain-10 immunoreactive interlaminar astrocytes that extend processes deep into the cortex. CamKIIα is predominantly neuronal in localization and is associated with the presence of diffuse plaques in the ageing brain. DISCUSSION: Dysregulated expression of key calcium signalling molecules occurs with progression of Alzheimer-type pathology in the ageing brain, highlighting the need for further functional studies of astrocytic calcium signalling with respect to disease progression.

A size selective vascular barrier in the rat area postrema formed by perivascular macrophages and the extracellular matrix.

The fenestrated microvasculature of the area postrema shows a less restrictive blood-brain barrier than is found in other areas of the CNS. We have studied the expression and relationship of vascular endothelial tight junctional proteins, astrocytes, macrophages, and the extracellular matrix with the extravasation of fluorescently tagged dextrans and sodium fluorescein in the rat area postrema. Glial fibrillary acidic protein (GFAP) -positive astrocytes were present within the area postrema which was surrounded by a dense zone of highly GFAP-reactive astrocytes. Expression of the tight junction proteins claudin-5, -12 and occludin was absent, although diffuse cytoplasmic claudin-1 immunoreactivity was present. The extracellular matrix of the endothelium showed two non-fused thickened layers of laminin immunoreactivity. CD163 and CD169 immunoreactive perivascular macrophages were located within lacunae between these two laminin layers. Fluorescently tagged dextrans (10-70 kDa) passed from the vasculature but were retained between the inner and outer laminin walls and rapidly sequestered by the perivascular CD163 and CD169 macrophages. Three-kilodalton dextran diffused into the parenchyma, but was retained within the boundary of the area postrema at the interface with the highly reactive GFAP-astrocytes, while sodium fluorescein (0.3 kDa) passed from the area postrema into surrounding CNS areas. Our observations suggest that despite the absence of a tight blood-brain barrier, a size selective barrier restricting the movement of blood solutes into the parenchyma is present in the area postrema. We suggest that the rapid uptake by CD163 and CD169 macrophages together with the non-fused laminin immunoreactive layers of the extracellular matrix plays a size selective role in restricting movement of serum proteins and other blood borne macromolecules over 10 kDa in to the area postrema.

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

Alzheimer's disease (AD) is pathologically characterised by the age-dependent deposition of ß-amyloid (Aß) 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 Aß-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aß-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aß-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. Aß-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 Aß in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.

Attitudes and access to electronic exchange of information on occupational disease.

BACKGROUND: THOR is a network of work-related disease surveillance schemes dependent on volunteer case reporting by medical specialists. Data collection and dissemination has hitherto been paper-based. AIMS: To elicit the opinion of existing reporters in THOR on electronic exchange of information and to assess the practical capabilities of the same reporters to participate in electronic communication. METHODS: A mail-based questionnaire of randomly selected THOR reporters using closed format questions. RESULTS: The response rate to the questionnaire was 66% (253/383). Almost half (47%) of the responders wanted dissemination of information solely in an electronic form, 35% favoured paper-based reports, while 16% wanted both paper and electronic reports. Two-thirds (66%) would make use of electronic archives of reports and 59% would use this facility to resolve questions by accessing accumulated data. The majority (82%) read e-mail more than once a week and 34% browsed the web as frequently. However, 5% did not have e-mail and 6% never browsed the web. Most responders judged their internet connectivity to be rapid (68%) and convenient (83%), and 91% could receive e-mail attachments. CONCLUSIONS: Most responders have the skills and infrastructure required to engage in electronic information exchange, and are favourably disposed to electronic means of communication. However it is also relevant to note that one-third of responders have a preference for the existing paper-based system.

External urethral sphincter dilation for the management of high risk myelomeningocele: 15-year experience.

PURPOSE: We investigate the long-term outcome using external urethral sphincter dilation for high risk myelomeningocele. MATERIALS AND METHODS: Since 1984 external urethral sphincter dilation was performed in 25 patients with myelomeningocele who demonstrated passive leak point pressure greater than 40 cm. H2O and/or poor bladder compliance. Mean followup from the first dilation was 8.4 years. Overall 2.4 dilations were performed per patient (range 1 to 8). Cystometrography, imaging study and continence status were evaluated retrospectively. RESULTS: Overall external urethral sphincter dilation produced durable improvements in mean leak point pressure (60.9 versus 34.4 cm. H2O), capacity (119.8 versus 233.3 ml.), initial compliance (11.5 versus 28.4 ml./cm. H2O) and terminal compliance (1.1 versus 7.7 ml./cm. H2O). Categorical analysis revealed 3 groups in terms of outcome. Group 1 consisted of 11 patients (44%) who demonstrated durable improvements in urodynamic parameters as well as preservation of the upper tracts. These patients demonstrated a 2-step compliance pattern on pre-dilation cystometrography, in which elevated leak point pressure was associated with excellent initial compliance. Group 2 consisted of 5 patients (20%) who failed to maintain safe leak point pressure and whose upper tracts deteriorated, including 4 who eventually underwent augmentation cystoplasty. This group demonstrated a 1-step hypertonicity in which elevated leak point pressure was associated with a steep pressure increase during early filling. Group 3 consisted of 9 patients (36%) who responded minimally in terms of leak point pressure reduction but whose upper tracts remained well preserved. They demonstrated a high pressure instability pattern associated with excellent baseline compliance. CONCLUSIONS: External urethral sphincter dilation provides an effective long-term solution for select high risk myelomeningocele cases. Those who demonstrate elevated leak point pressure and poor bladder compliance at the time of external urethral sphincter dilation are less likely to respond, suggesting that the bladder may have already undergone irreversible changes due to high outlet resistance. Patients who demonstrate instability patterns are less likely to respond to external urethral sphincter dilation in terms of leak point pressure reduction but the upper tracts appear to be well preserved.