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.

Identification of a novel region of the GABA(B2) C-terminus that regulates surface expression and neuronal targeting of the GABA(B) receptor.

GABA(B) is a G protein-coupled receptor composed of two subunits, GABA(B1) and GABA(B2). GABA(B1) contains an endoplasmic reticulum-retention sequence and is trafficked to the cell surface only in association with GABA(B2). To determine whether the C-terminus of GABA(B2) regulates GABA(B) trafficking, we constructed forms of GABA(B2) with various C-terminal truncations and examined their surface expression. Truncation of GABA(B2) after residue 841 significantly reduced surface expression of both the subunit and the heterodimerized receptor. Turnover of the Delta841 construct, however, did not differ from that of full-length GABA(B2). To determine whether the C-terminus of GABA(B2) might target GABA(B) to neurites, cultured hippocampal neurons were transfected with the truncated GABA(B2) constructs. Truncation of GABA(B2) at residue 841 resulted in primarily somatic localization; furthermore, axonal trafficking of this construct was significantly more restricted than dendritic trafficking. Finally, to biochemically assess trafficking of the truncated GABA(B2) constructs, we digested transfected HEK293 cell lysates with endoglycosidase H. When GABA(B2) was truncated at residue 841, it became sensitive to digestion by this enzyme, indicating incomplete trafficking. Taken together, these data show that the region of the GABA(B2) C-terminus between residues 841 and 862 is important for regulating forward trafficking and neuronal targeting of the GABA(B) receptor.

Comparison between margin-growing algorithms in radiotherapy software environments.

Margin-growing algorithms are commonly used tools that are available within virtual simulation and treatment planning software. We report on the accuracy of the margin-growing algorithms available in six commercially available radiotherapy software environments. A phantom containing two differently sized spheres and two rods (one level and one inclined) was constructed and scanned by CT with 1.25 mm, 2.5 mm, 3.75 mm and 5 mm slice thicknesses. The objects were outlined on a GE Advantage Simulator, and the outlined volumes recorded. Images and structures were transferred to MasterPlan, Xio, Pinnacle, Eclipse and Prosoma, where imported volumes were recorded. The contours on each system were grown isotropically by 10 mm, 20 mm and 30 mm, and volumes for each grown contour were recorded. Transfer of structure sets created in GE Advantage Simulator to the other software environments showed that the reported volumes of the four structures differ on each system. Results showed no correlation between volume accuracy and slice thickness. In general, margin growth of up to 30 mm for the rods and spheres is shown to be consistent between systems to within 1.33 mm for all slice thicknesses. Slice thickness did not appear to influence the accuracy of margin growth. Although this work highlights apparent differences in the reported volumes grown from the same original structure sets, the significance of this aspect of the planning process needs to weighed against reported intra- and inter-clinician variability in contour definition. It is not unreasonable, however, to expect that software packages should at least be consistent in volume information provided to the user.

Uridine enhances neurite outgrowth in nerve growth factor-differentiated PC12 [corrected].

During rapid cell growth the availability of phospholipid precursors like cytidine triphosphate and diacylglycerol can become limiting in the formation of key membrane constituents like phosphatidylcholine. Uridine, a normal plasma constituent, can be converted to cytidine triphosphate in PC12 [corrected] cells and intact brain, and has been shown to produce a resulting increase in phosphatidylcholine synthesis. To determine whether treatments that elevate uridine availability also thereby augment membrane production, we exposed PC12 [corrected] cells which had been differentiated by nerve growth factor to various concentrations of uridine, and measured the numbers of neurites the cells produced. After 4 but not 2 days uridine significantly and dose-dependently increased the number of neurites per cell. This increase was accompanied by increases in neurite branching and in levels of the neurite proteins neurofilament M [corrected] and neurofilament 70. Uridine treatment also increased intracellular levels of cytidine triphosphate, which suggests that uridine may affect neurite outgrowth by enhancing phosphatidylcholine synthesis. Uridine may also stimulate neuritogenesis by a second mechanism, since the increase in neurite outgrowth was mimicked by exposing the cells to uridine triphosphate, and could be blocked by various drugs known to antagonize P2Y receptors (suramin; Reactive Blue 2; pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid). Treatment of the cells with uridine or uridine triphosphate stimulated their accumulation of inositol phosphates, and this effect was also blocked by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid. Moreover, degradation of nucleotides by apyrase blocked the stimulatory effect of uridine on neuritogenesis. Taken together these data indicate that uridine can regulate the output of neurites from differentiating PC12 [corrected] cells, and suggest that it does so in two ways, i.e. both by acting through cytidine triphosphate as a precursor for phosphatidylcholine biosynthesis and through uridine triphosphate as an agonist for P2Y receptors.