Roger G. Sutcliffe

A candidate gene for human neurodegenerative disorders: a rat PKCγ mutation causes a Parkinsonian syndrome

Rats harboring the agu mutation have altered behavior and brain pathology resembling human Parkinsonian syndromes; notably, they have a movement disorder and age-progressive dysfunction and death of neurons in the midbrain (substantia nigra pars compacta) that use dopamine as a neurotransmitter. We present evidence that this phenotype is due to a mutation in the rat protein kinase Cγ (in rat, Prkcg; in mouse, Prkcc; in human, PRKCG) gene, which generates a premature stop codon, drastically reducing the level of synthesis of the catalytic domain of the brain-specific protein kinase Cγ protein.

The AS/AGU rat: a spontaneous model of disruption and degeneration in the nigrostriatal dopaminergic system

The AS/AGU rat provides an alternative to experimentally produced laboratory models of basal ganglia disorders. This mutant is characterised by disturbances of movement including clumsy gait, whole body tremor, rigidity and difficulty in initiating movement. From an early age, there is a profound depletion of extracellular dopamine in the dorsal caudate‐putamen as measured via in vivo microdialysis; levels are only 10–20% of those found in the parent Albino Swiss (AS) strain. Subsequently a depletion of whole tissue dopamine levels occurs and, later still, loss of dopaminergic cells in the substantia nigra pars compacta. The dysfunction in movement and the nigrostriatal dopaminergic system are clearly linked, since movement can be ameliorated by L‐DOPA administration. Furthermore, there are depletions in glucose utilisation in several regions of the basal ganglia circuitry, including the substantia nigra pars compacta, the subthalamic nucleus and the ventrolateral thalamus. The AS/AGU rat represents a unique opportunity to investigate the intrinsic factors controlling the integrity of dopaminergic systems and the recent successful positional cloning of the agu gene will allow the molecular mechanisms underlying this interesting phenotype to be analysed.

Neostriatal dopamine depletion and locomotor abnormalities due to the Albino Swiss rat agu mutation

The sub-strain of Albino Swiss rat (AS/AGU) is a spontaneous mutation characterised by an ungainly, staggering gait, hindlimb rigidity, whole body tremor and (when symptoms are fully developed) difficulty in initiating movement; it exhibits a progressive decrease in dopaminergic cells within the substantia nigra. A breeding programme involving Albino Swiss (AS) and AS/AGU parent rats was used to produce the F1 offspring of AS x AS/AGU matings and, subsequently, F1 x AS/AGU back crosses. When adult, the movement of all animals was assessed blind by observers on three occasions, each animal being identifiable by a subcutaneous transponder implanted before weaning. All AS/AGU and half the F1 x AS/AGU back cross animals had abnormal gait, while all AS, F1 and the remaining F1 x AS/AGU backcross animals showed normal gait, implying that the mutation is recessive. Brains of males aged 12-15 months (n = 10 per group) were sectioned transversely on a cryostat (-20 degrees C) to produce a cut face just caudal to the anterior commissure (approximately Bregma -0.5 mm) and 1 mm diameter x 1 mm deep micropunches were taken from three areas of the caudate-putamen. Levels of dopamine were measured in all samples by high performance liquid chromatography with electrochemical detection (HPLC-ECD) followed by protein estimation. Levels of dopamine in the dorsal and middle caudate-putamen varied according to a simple inheritance pattern, being high in males from AS, F1 and F1 x AS/AGU back crosses without locomotor impairment, but lower in AS/AGU and F1 x AS/AGU back crosses with disordered gait. Dopamine levels in the ventral caudate-putamen did not show such a clear variation.

Age changes in dopamine levels in the corpus striatum of Albino Swiss (AS) and AS/AGU mutant rats

The AS/AGU rat is characterised by an ungainly, staggering gait, hind-limb rigidity, whole body tremor and (in older animals) difficulty in initiating movement. Brains of AS and AS/AGU males aged between 3 and 12 months (n = 10 per group) were sectioned transversely on a cryostat (-20 degrees C) to produce two successive cut faces (corresponding approximately to Bregma +1.2 and -0.5 mm) and 1 mm diameter x 1 mm deep micropunches were taken from four areas of the caudate-putamen. Levels of dopamine in all four areas (measured by HPLC-ECD followed by protein estimation) peaked at around 6 months and then declined in AS and AS/AGU rats. In the dorsal and lateral caudate-putamen, dopamine levels were significantly reduced in AS/AGU rats compared to AS controls from 6 months onwards. This provides further evidence that the AS/AGU mutant has impairment of its striatal dopaminergic systems.

Regional chromosomal assignment of human 3-beta-hydroxy-5-ene steroid dehydrogenase to 1p13.1 by non-isotopic in situ hybridisation

SummaryIn situ hybridisation using a biotinylated 1.2-kb human cDNA clone for human 3-beta-hydroxy-5-ene steroid dehydrogenase (HSD) supports the provisional regional localisation of the HSD gene to chromosome 1p13 and refines this localisation to 1p13.1.

Polymorphisms of the glucocorticoid receptor gene in laboratory and wild rats: steroid binding properties of trinucleotide CAG repeat length variants.

The polyglutamine tract, beginning at codon 75 in the N-terminal modulatory domain of rat glucocorticoid receptor (rGR), was analyzed in 61 inbred strains and 155 wild caught Rattus norvegicns. A discontinuous distribution of repeat lengths was found (7, 17–23 repeats). To investigate the possible significance of this distribution, full-length rGR cDNAs with 7, 18, 20, and 21 CAG repeats were expressed in CV-1 cells, and the resulting GR protein analyzed by Western blots and extensive Scatchard analyses. The quantity and steroid binding capacity of GR, together with the binding affinities for dexamethasone and corticosterone, were found to be indistinguishable for the four repeat alleles. From the sequencing of four inbred strains CAG repeat variants were found to be flanked by silent allelic substitutions at nucleotide positions 198, 531, and 711. The four variable sites extended over 471–519 bp of coding sequence, forming six Grl haplotypes. The results are discussed in the light of genetic studies on the Milan hypertensive and normotensive strains of rat. Codon sequence of rat GR required amendment at the following residues: D98, G226, D260, P600, and F602.

A novel homozygous nonsense mutation E135* in the type II 3β-hydroxysteroid dehydrogenase gene in a girl with salt-losing congenital adrenal hyperplasia

Mutations in the 3β‐hydroxysteroid dehydrogenase (3β‐HSD) type II gene have been reported in a small number of affected females. We report a 46,XX girl born to consanguineous parents from Chile. At birth, she had normal but hyperpigmented female external genitalia. At 60 days she presented salt loss. At 20 months, the diagnosis of classic salt‐losing 3β‐HSD deficiency was made based on an elevated serum 17‐hydroxypregnenolone concentration and a high 17 hydroxypregnenolone/17‐hydroxyprogesterone ratio. Genomic DNA was amplified by PCR and screened for mutations by denaturing gradient gel electrophoresis and directly sequenced. A novel homozygous E135* mutation was found in the 3β‐HSD type II gene of the patient while her parents were heterozygotes. This novel nonsense homozygous E135* mutation led to encode a predicted truncated 134 amino acid protein instead of the native 371 amino acid 3β‐HSD type II protein. This predicted product is consistent with the severe 3β‐HSD deficiency in this girl. Hum Mutat 12:139, 1998.

No linkage to the 3?-HSD gene cluster in a kindred affected with 3?-hydroxy-?5-C27steroid dehydrogenase deficiency and early onset hepatic failure

We studied the segregation of the genes for 3β-hydroxy-C19/21-steroid dehydrogenase types I and II (3β-HSD I and II) in a consanguineous family affected with 3β-hydroxy-Δ5-C27steroid dehydrogenase (3β-OH-C27-SD) deficiency. The results show that the C27 and C19/21 steroid dehydrogenase activities are encoded by distinct genes that are not in genetic linkage. Further kindreds would assist in screening for linkage of 3β-OH-C27-SD to other members of the 3β-hydroxysteroid dehydrogenase gene family.

Local cerebral glucose utilization in the AS/AGU rat: a mutant with movement disorders

The AS/AGU mutant rat is characterized by a wide staggering gait and a movement disorder of the hindlimbs. Local cerebral glucose utilization in the brain was investigated using the [14C]2‐deoxyglucose autoradiographic technique to map any functional alterations in the mutant AS/AGU (agu/agu) compared with Albino Swiss controls (+/+). Locomotor tests were also performed to confirm the phenotypic assignment of the animals. Statistically significant reductions in glucose utilization were apparent in 12 of the 44 regions examined in the AS/AGU animals. The regions showing the most significant differences (P < 0.01) from the control AS strain were the substantia nigra pars compacta (–23%) and medial geniculate body (–17%). Statistically significant decreases (P < 0.05 and P < 0.02) in glucose utilization ranging from −15 to −26% were also displayed in the superior colliculus superficial layer, auditory cortex, ventroposterior nucleus of the thalamus, molecular layer of the hippocampus, dentate gyrus, medial amygdaloid nucleus, median raphe nucleus, subthalamic nucleus, medial preoptic area of the hypothalamus and anterior hypothalamus. In no region studied was the mean value of glucose use in the AS/AGU rat greater than in the control animals. The results of this study complement previous behavioural and neurochemical characterization studies of this mutant, confirm that the disorder involves functional disturbances of the basal ganglia, and demonstrate the involvement of the limbic system and some sensory systems.

Active Learning in a Large First Year Biology Class: A Collaborative Resource-Based Study Project on "AIDS in Science and Society".

SUMMARY Didactic instruction encourages passive learning and deters students from developing inquisitive‐ness, reasoning, problem solving and teamwork. We therefore introduced a Study Project, AIDS in Science and Society, to our first year Biology students (n = 550 to 750). For 30 to 40 hours of resourced‐based learning, students worked in autonomous study groups, preparing for inter‐group debates and the submission of personal written work. No academic lecture support for AIDS was provided. Independent staff assessed the quality of written work and held interviews and questionnaire sessions with students and tutors. The quality of student written work was highly acceptable, and tutors supported the exercise. The participation rate by students was very high (93%) and 89 to 92% of students stated that the project was a good way to learn. Students without prior qualifications in biology completed the project as satisfactoraly as others. A resource bibliography is included.