Expression of the Fe65 adapter protein in adult and developing mouse brain.

Fe65 is a multimodular adaptor protein expressed mainly in the nervous system. Fe65 binds to the Alzheimer's disease amyloid precursor protein (APP) and the interaction is mediated via a phosphotyrosine binding domain in Fe65 and the carboxy-terminal cytoplasmic domain of APP. Fe65 modulates trafficking and processing of APP, including production of the beta-amyloid peptide that is believed to be central to the pathogenesis of Alzheimer's disease. Fe65 also facilitates translocation of a carboxy-terminal fragment of APP to the nucleus and is required for APP-mediated transcription events. In addition, Fe65 functions in regulation of the actin cytoskeleton and cell movement. Here we report the distribution profile of Fe65 immunoreactivity in adult mouse brain. Fe65 expression was found to be widespread in neurones in adult brain. The areas of highest expression included regions of the hippocampus in which the earliest abnormalities of Alzheimer's disease are detectable. Fe65 was also highly expressed in the cerebellum, thalamus and selected brain stem nuclei. Fe65 was evident in a sub-set of astrocytes within the stratum oriens and radiatum in the hippocampus. Expression of Fe65 was found to be developmentally regulated with levels reducing after embryonic day 15 and increasing again progressively from post-partum day 10 up to adulthood, a developmental pattern that partially parallels that of APP. These data indicate a widespread distribution of Fe65 in neurones throughout mouse brain and also suggest that Fe65 may have functions independent of APP and any potential role in the pathogenesis of Alzheimer's disease.

The expression of the glutamate re-uptake transporter excitatory amino acid transporter 1 (EAAT1) in the normal human CNS and in motor neurone disease: an immunohistochemical study.

A monoclonal antibody to excitatory amino acid transporter 1 (EAAT1) has been generated which robustly stains paraffin-embedded, formaldehyde-fixed as well as snap-frozen human post-mortem brain tissue. We have used this antibody to map the distribution of EAAT1 throughout normal human CNS tissue. In addition this antibody has been used to perform a semi-quantitative immunohistochemical analysis of the expression of EAAT1 in motor cortex and cervical cord tissue taken from motor neurone disease cases (n=17) and neurologically normal controls (n=12). By comparing the relative optical density measurements of identical regions of motor cortex and cervical spinal cord an increase in the expression levels of EAAT1 was observed in motor neurone disease tissue compared to the control tissue and in both motor cortex and cervical spinal cord (9-17% and 13-33% increases respectively). EAAT1 was observed to be the most abundant transporter in more "caudal" brain regions such as the diencephalon and brainstem and its expression in other regions was frequently more uniform than that of EAAT2. In the motor cortex, EAAT1 immunoreactivity was present in all grey matter laminae, with some staining of individual astrocytes in the white matter. In spinal cord, EAAT1 immunoreactivity was strongest in the substantia gelatinosa. In the ventral horn, motor neurones were surrounded with a dense rim of perisomatic EAAT1 immunoreactivity, and the neuropil showed diffuse staining. Additional studies using double-labelling immunocytochemistry demonstrated that astrocytic co-localisation of EAAT1 and EAAT2 may occasionally be seen, but was not widespread in the human CNS and that in general astrocytes were positive for either EAAT1 or EAAT2. These results demonstrate that the EAAT1 has a widespread abundance throughout all regions of the human CNS examined and that there exist discrete populations of astrocytes that are positive solely for either EAAT1 or EAAT2. Furthermore, there is evidence to suggest that altered EAAT1 expression in motor neurone disease follows a different pattern to the reported changes of EAAT2 expression in this condition, indicating that the role of glutamate transporters in the pathogenesis of motor neurone disease appears more complex than previously appreciated.

Mutation screening of manganese superoxide dismutase in amyotrophic lateral sclerosis.

Seventy-seven cases of ALS were screened for mutations in the manganese superoxide dismutase gene (SOD2). DNA was extracted from CNS tissue and screened using single stranded conformation polymorphism and heteroduplex analysis. No mutations were identified in the entire coding region of the SOD2 gene. The known polymorphism in the mitochondrial targeting sequence was identified. No association was found between this polymorphism and ALS. A further polymorphism was detected in the intronic sequence upstream of exon 4, though no association with ALS was demonstrated. We therefore conclude that mutations in SOD2 do not appear to cause ALS.

Screening of AP endonuclease as a candidate gene for amyotrophic lateral sclerosis (ALS).

DNA extracted from CNS tissue of 84 patients was screened by single-stranded conformation polymorphism (SSCP) and heteroduplex analysis for mutations in the apurinic/apyrimidinic endonuclease (APE) gene. One mutation was identified and characterized as a 4bp deletion in the 3'UTR. A rare polymorphism was identified in exon 3 and a common polymorphism in the coding region of exon 5. These results suggest that APE mutations do not account for a large number of ALS cases.

The expression of the glial glutamate transporter protein EAAT2 in motor neuron disease: an immunohistochemical study.

Emerging evidence suggests that a disturbance of the glutamate neurotransmitter system may be a contributory factor to motor neuron injury in motor neuron disease. Previous autoradiographic and immunoblotting studies have suggested that there may be reduced expression of glutamate transporter proteins in pathologically affected areas of the CNS in motor neuron disease. This study further explores the possible alteration in expression of the excitatory amino acid transporter protein EAAT2 in MND, by examining the protein expression in situ, in frozen sections, using immunohistochemistry. The aim of the study was to compare the distribution and density of EAAT2 in the motor cortex and spinal cord of MND cases (n = 16) compared with neurologically normal controls (n = 12), matched for relevant parameters. A novel, previously characterized, monoclonal antibody to EAAT2 was employed. EAAT2 immunoreactivity in motor neuron disease and control cases was compared using relative optical density measurements generated by computerized image analysis. In the motor cortex, EAAT2 immunoreactivity was laminated comprising a superficial intense band (corresponding to layers 1 and 2); a paler middle band (layer 3 and part of 5) and a more intense deep layer (layers 5 and 6). In the spinal cord, the ventral horn showed strong immunoreactivity with dense perisomatic staining around motor neuron cell bodies, the substantia gelatinosa showed moderate diffuse staining and the intermediate spinal laminae showed weak staining. This general pattern of immunoreactivity was preserved in the motor neuron disease cases. However, in the motor neuron disease cases compared with controls, the optical density values for EAAT2 immunoreactivity were significantly reduced in all grey matter regions of the lumbar spinal cord (P < 0.001) and were increased in the middle laminae of the motor cortex (P < 0.05). This study indicates that glutamate transporter pathology in motor neuron disease may be a more complex phenomenon than previously recognized.

Nucleoside diphosphate kinase and Cl(-)-sensitive protein phosphorylation in apical membranes from ovine airway epithelium.

We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl-], and anion species determine the steady-state phosphorylation of apical membrane proteins within human airway epithelium in vitro. We found that a Cl(-)-regulated 37-kD protein (p37) principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from sheep tracheal epithelium also contain a Cl(-)-regulated 37-kD phosphoprotein (p37s) and characterize one of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins with gamma[32P]GTP in the presence of MgCl2 showed that two proteins circa 19 and 21 kD (p19s and p21s) were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either gamma[32P]GTP or gamma[32P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical membrane proteins in the presence of gamma[32P]ATP and guanosine diphosphate (GDP) (but not GDPbetaS) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg2+, Mn2+, and Co2+ (but not Zn2+ or Ca2+). Our data show that ovine trachea is a good model for further characterization of the chloride-dependent cascade in airway epithelium.

Expression of the glial glutamate transporter EAAT2 in the human CNS: an immunohistochemical study.

Glutamate transporters play an essential role in terminating the excitatory glutamatergic signal at post-synaptic receptors and in protecting neurones from excitotoxic effects, as well as replenishing the neurotransmitter supply at glutamatergic synapses. The distribution and density of glutamate transporters may be important determinants of vulnerability to glutamate-mediated injury. There is emerging evidence that glutamate transporter dysfunction may be present in motor neurone disease (MND). In this study, a monoclonal antibody, suitable for immunohistochemistry (IHC) in human post-mortem tissue, was produced to the human astrocytic glutamate transporter EAAT2 (excitatory amino acid transporter 2). Western blotting of homogenates of human cortical tissue with the EAAT2 antibody produced a discrete band at 66 kDa. Detailed IHC analysis of the expression of the EAAT2 protein in the human CNS was undertaken. EAAT2 was exclusively localised to astrocytes, with preferential expression in the caudate nucleus, nucleus basalis of Meynert, spinal ventral horn, cerebral cortex and hippocampus, but with lower levels of expression throughout many other CNS regions. Motor neurone groups vulnerable to neurodegeneration in MND appeared distinctive in being surrounded by extensive, coarse, strongly immunoreactive perisomatic glial profiles. Motor neurone groups which tend to be spared in MND, such as those present in the oculomotor nucleus, showed a lower expression of EAAT2, with fewer perisomatic profiles. The EAAT2 antibody will provide a useful tool for increasing our understanding of the role of EAAT2 in excitatory neurotransmission in health and disease states.