Androgen-controlled specific gene expression in neuroblastoma cells.

In Kennedy's syndrome, a mutation in the androgen receptor (AR) gene leads to sensory and lower motor neurone degeneration, therefore genes that are regulated by androgens in neurones may be important in the process of motor neurone cell death. The aim of this study was to identify androgen-inducible genes in the human neuroblastoma cell line SH-SY5Y. We have shown that SH-SY5Y cells expressed the AR by Northern blot analysis using a 32P-labelled probe derived from a human AR cDNA. Differential display of mRNAs has been used to identify and clone putative genes that are regulated by androgens in SH-SY5Y cells. Nine cDNA fragments, ranging in size from 180 bp to 480 bp, corresponding to mRNA species which appear to be differentially expressed in response to 5 alpha dihydrotestosterone (DHT), have been successfully cloned. These are now being sequenced and used as hybridisation probes for Northern blot analysis in order to confirm their induction by DHT. Characterisation of these genes may provide clues to the mechanisms of motor neurone degeneration.

Superoxide dismutase activity in lymphoblastoid cells from motor neurone disease/amyotrophic lateral sclerosis (MND/ALS) patients.

Point mutations in the gene encoding Cu,Zn superoxide dismutase (SOD1) are associated with autosomal dominant familial amyotrophic lateral sclerosis (FALS). We have measured Cu,Zn SOD activity in lymphoblastoid cells from affected and at risk FALS patients carrying mutations in the SOD1 gene, FALS patients without mutations in the SOD1 gene, individuals affected by the sporadic form of the disease (SALS), normal controls and individuals with other neurological abnormalities. The results show a significant decrease in Cu,Zn SOD activity in affected and at risk FALS individuals as compared to FALS patients without mutations, SALS individuals, normal and neurological controls. It is concluded that decreased SOD activity may contribute, together with other as yet unknown factors, to motor neurone demise.

Androgen receptor gene polymorphisms in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is more common in men than in women (male to female ratio of approximately 2:1), suggesting a role for a sex-linked factor in the disease. The recent identification of a mutation of the androgen receptor gene in Kennedy's disease or X-linked bulbospinal neuronopathy, a rare form of progressive lower motor neurone degeneration, also associated with clinical signs of androgen insensitivity, raises the possibility that androgen function may be disturbed in other motor neurone disorders, including ALS. The Kennedy's disease mutation consists of an increased size of a highly polymorphic CAG repeat sequence in the first exon of the androgen receptor gene, coding for a polyglutamine tract. We have analysed this CAG repeat sequence in a large number of patients with typical sporadic ALS and in normal controls, in order to test the hypothesis that this polymorphism of the androgen receptor gene may influence susceptibility for ALS. We report that the distribution of alleles relating to the size of the CAG repeat sequence of the androgen receptor gene is similar in ALS and controls, indicating that polymorphisms of the CAG repeat sequence of the androgen receptor gene play a limited role, if any, in susceptibility to ALS.

Studies on the compartmentation of DOG metabolism in the brain.

Using 31P-NMR studies we have observed that 1. 2-Deoxyglucose leads into the brain in vivo and in superfused cortical slices in vitro to a maximum concentration at between 45 and 60 min, when 80% of the material is in the phosphorylated form. 2. The phosphorylated DOG6P disappears from the n.m.r. spectra with a half-life of ca 130 min. 3. Two resonances of DOG6P are observed in the actively metabolising tissue, whereas only one is visible in deproteinised tissue extracts. This suggests that the DOG6P is in two separate compartments which differ in pH. 4. Compartmentation between mitochondria, nerve endings and cytoplasm was concluded to be unlikely from subcellular fractionation studies, but the possibility of compartmentation between neurones and glia could not be so clearly assessed.

Heat shock protein expression in corpora amylacea in the central nervous system: clues to their origin.

Small bodies expressing epitopes of the 72 kD heat shock protein (HSP) have been identified in the brain and spinal cord in normal and neurologically abnormal individuals. These bodies resemble the 'pre-corpora amylacea' (pre-CA), thought to be the primary structure in the development of the mature body. Corpora amylacea are laminated hyaline bodies composed of polyglucosans. They are found in larger numbers with increasing age in the brain and spinal cord. Mature, histologically 'classical', corpora amylacea express epitopes of HSP chiefly at the periphery of the corpus, whilst smaller immature 'pre-corpora' stain intensely throughout the entire structure. A heat shock or stress response in neurons and glial cells may be part of the cellular reaction to accumulation of abnormal products.

Ubiquitin-immunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis. Morphology, distribution, and specificity.

Antibodies to ubiquitin have been used to search for evidence of abnormal protein degradation in amyotrophic lateral sclerosis--motor neuron disease (ALS). Anterior horn cell ubiquitin-immunoreactive (IR) inclusions were present in all of 31 ALS cases but in none of 23 neurologically normal and in only 1 of 22 neurologically abnormal controls. These inclusions, which were present in familial and sporadic ALS cases, and in cases with dementia, took the form of dense rounded or irregular ubiquitin-IR cytoplasmic inclusions (dense bodies), or loosely arranged bundles ('skeins') of filamentous-appearing material. The presence of ubiquitin-IR inclusions corresponded to the pattern of selective neuronal vulnerability in ALS, although inclusions in pyramidal neurons of the motor cortex were infrequent and were noted in only a minority of cases. Ubiquitin-IR inclusions were more prevalent than Bunina bodies. The latter were present in 67% of ALS cases but were seldom labelled by antibodies to ubiquitin. Intraneuronal inclusions resembling Lewy bodies were present in 23% of ALS cases and were often identified by antibodies to ubiquitin. We conclude that the presence of ubiquitin-IR inclusions in lower motor neurons represents a characteristic pathological feature of ALS in its various clinical forms. Ubiquitin-IR inclusions in ALS differ from ubiquitinated inclusions in other neuronal degenerations in that they are not readily identified by antibodies to cytoskeletal proteins. They may represent accumulations of altered or abnormal neuronal proteins resistant to degradation via the ubiquitin proteolytic pathway.

Stress protein inclusions in cerebral vessels in dialysis encephalopathy.

Dialysis encephalopathy, a complication of long-term haemodialysis, is a syndrome characterized by progressive dementia, myoclonus, dysarthria and ataxia associated with high serum and brain levels of aluminium. Expression of heat-shock or stress proteins, including ubiquitin can be induced in cell culture experiments by aluminium. We report immunohistochemical studies of heat shock protein (HSP) expression in the frontal cortex of three patients with dialysis dementia. Immunolabelling with antibody to the 72 kD heat shock protein revealed punctate granules in most endothelial cells of cortical vessels in patients with dialysis encephalopathy. These granules, 1-5 microns in diameter, aggregated to form inclusions that resembled stress-granules, typically induced in plant or animal cell culture by repeated insult. These granules did not express epitopes of ubiquitin. They were rare in endothelial cells in the brains of subjects dying with other neurological disorders or of non-neurological causes. We suggest that these stress granules represent a toxic response of endothelial cells in the brain to aluminium.

Ubiquitin and heat shock protein expression in amyotrophic lateral sclerosis.

The expression of two heat shock proteins, HSP72 and p57, in addition to ubiquitin, has been studied immunocytochemically in nine amyotrophic lateral sclerosis (ALS) cases and 10 age-matched controls. HSP72 and p57 antibodies did not identify the characteristic ubiquitin-immunoreactive inclusions present in anterior horn cells in ALS spinal cord. Antibodies to HSP72, but not to p57 or ubiquitin, strongly labelled structures corresponding to polyglucosan bodies in spinal grey matter. Such immunoreactive profiles were more abundant in ALS cases, although they were also present in control material. They were sometimes identified by haematoxylin and eosin and periodic acid Schiff reaction, but were not labeled by phosphotungstic acid haematoxylin or by antibodies to glial fibrillary acidic protein. Although ubiquitin, HSP72 and p57 are stress-induced proteins, they are expressed differently and might therefore have different significance in neuronal degeneration.

Spinal cord trauma in man: studies of phosphorylated neurofilament and ubiquitin expression.

Changes in neuronal proteins are a major feature of the neurodegenerative disorders, but the biological significance of these changes is not understood. We describe alterations in neuronal proteins as a result of surgical tractotomy on the human spinal cord at 3, 5, 7, 14 and 72 days after operation. Antibodies to phosphorylated neurofilaments labelled neuronal cell bodies in the nucleus dorsalis and anterior horns. Axonal swellings showed positive staining for ubiquitin, but ubiquitinated neuronal inclusions were not evident. The presence of phosphorylated neurofilaments in the neuronal cell bodies and of ubiquitination of axonal swellings may be indicators of interrupted axonal transport. The changes found in intracellular proteins in neurons after trauma do not resemble those found in motor neuron disease, suggesting that the intraneuronal ubiquitinated inclusions may have a unique significance in the cytopathology of that disorder.

Microdissection: a novel method for the study of intracellular inclusion bodies.

The purification of many intracellular and extracellular inclusions is often difficult to achieve due to the low concentration of the abnormalities in the tissue under study, or due to the degradation of components during extraction. We describe the use of microdissection for the isolation of neurons and intraneuronal inclusion bodies. The resulting suspension may be used for biochemical, immunological or ultrastructural studies. The technique is applicable to the study of a wide range of cellular abnormalities.

Brain tissue concentrations of ATP, phosphocreatine, lactate, and tissue pH in relation to reduced cerebral blood flow following experimental acute middle cerebral artery occlusion.

Local CBF (LCBF) was compared with the corresponding local tissue concentration of ATP, phosphocreatine (PCr), and lactate in anaesthetized baboons subjected to focal ischaemia produced by middle cerebral artery occlusion (MCAO). LCBF hydrogen electrodes were implanted in cortical regions where MCAO had been previously shown to produce severe and penumbral ischaemia and in posterior regions where blood flow is not altered. Metabolites were assayed in small tissue samples collected either by cryoprobe biopsy in the regions where LCBFs were measured (series 1) or by sampling appropriate regions of the rapidly frozen brain (series 2). Subsequent topographical study of brain tissue pH with umbelliferone was performed in this latter series. The results from these two series are compared and discussed in terms of the more appropriate way to perform simultaneous electrode measurements and analysis of tissue samples for studying focal ischaemia in the primate brain. They confirm that the concentrations of ATP and PCr decrease, and that lactate level increases, with decreasing blood flow. These metabolites tended to change more rapidly below a blood flow threshold, rather than showing a steady decrease as the blood flow was reduced, although the variability of the data precluded us from establishing this with confidence. Topographical study of tissue pH often showed sharp boundaries between zones of very low pH and regions with normal pH.

In situ freezing of the brain for metabolic studies: evaluation of the "box" method for large experimental animals.

The box method of freezing the brain in situ was assessed in baboons. The cooling rate of the tissue was monitored in several regions located at various depths from the skull surface. These measurements allowed us to examine the time required for the tissue to reach 0 degree C, in relation to its depth measured from the top of the skull. To define brain regions with proven ischaemia, frozen tissue sections were surveyed for areas of decreased pH. In addition, concentrations of ATP, phosphocreatine, and lactate were determined in gray matter located at various depths from the top of the brain surface. Normal tissue pH and low lactate concentration, without any significant decrease in high-energy phosphate levels, were found in regions at a depth less than approximately 10 mm from the brain surface. Deep structures including the inferiomedial aspect of the temporal lobe, the lateral geniculate body, and the limbic system (hippocampus) consistently showed mild tissue acidosis, indicating that these regions were subjected to some degree of ischaemia before they were reached by the freezing front. In some cases, acidosis was also detectable in the thalamus, basal ganglia, and in the deeper part of some sulci. We conclude that, with baboons, in situ freezing using the box method is valid for metabolic studies of the cerebral cortex and structures located at a depth less than approximately 10 mm from the top of the brain surface.

Brain levels of NADH and NAD+ under hypoxic and hypoglycaemic conditions in vitro.

The effects of hypoxia and hypoglycaemia on the redox state in vitro have been studied. NADH and NAD+ were extracted simultaneously from superfused cerebral cortex slices and assayed by bioluminescence. The results show a nonsignificant increase in NADH and the redox ratio in "mild hypoxia," whereas "severe hypoxia" produced an increase of over 200% in NADH and in the NADH/NAD+ ratio. When the glucose in the incubation medium was reduced from its control value of 10 mM to 0.5 mM, significant decreases in NADH and the redox ratio to 60% of control value were observed. Further decreasing the glucose to 0.2 mM gave lower levels of NADH and the redox ratio (40% of control). The effects on the redox state of alternative substrates to glucose were also tested. Replacement of glucose by 10 mM pyruvate decreased the NADH by 77% and the NADH/NAD+ ratio by 79%. Replacement of glucose with 10 mM lactate gave decreases of 70% and 71%, respectively, whereas in the presence of 15 mM 2-deoxyglucose and 5 mM glucose, the NADH was decreased by 56% and the ratio by 50%. The results are discussed in relation to levels of creatine phosphate and ATP, as well as evoked action potentials, observed from parallel studies.