Impaired Cerebellar Development in Mice Overexpressing VGF

VGF nerve growth factor inducible (VGF) is a neuropeptide precursor induced by brain-derived neurotrophic factor and nerve growth factor. VGF is increased in the prefrontal cortex and cerebrospinal fluid in schizophrenia patients. In our previous study, VGF-overexpressing mice exhibited schizophrenia-like behaviors and smaller brain weights. Brain developmental abnormality is one cause of mental illness. Research on brain development is important for discovery of pathogenesis of mental disorders. In the present study, we investigated the role of VGF on cerebellar development. We performed a histological analysis with cerebellar sections of adult and postnatal day 3 mice by Nissl staining. To investigate cerebellar development, we performed immunostaining with antibodies of immature and mature granule cell markers. To understand the mechanism underlying these histological changes, we examined MAPK, Wnt, and sonic hedgehog signaling by Western blot. Finally, we performed rotarod and footprint tests using adult mice to investigate motor function. VGF-overexpressing adult mice exhibited smaller cerebellar sagittal section area. In postnatal day 3 mice, a cerebellar sagittal section area reduction of the whole cerebellum and external granule layer and a decrease in the number of mature granule cells were found in VGF-overexpressing mice. Additionally, the number of proliferative granule cell precursors was lower in VGF-overexpressing mice. Phosphorylation of Trk and Erk1 were increased in the cerebellum of postnatal day 3 VGF-overexpressing mice. Adult VGF-overexpressing mice exhibited motor disability. All together, these findings implicate VGF in the development of cerebellar granule cells via promoting MAPK signaling and motor function in the adult stage.

     

Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) regulates growth and patterning of the postnatal mouse cerebellum

COUP-TFII (also known as Nr2f2), a member of the nuclear orphan receptor superfamily, is expressed in several regions of the central nervous system (CNS), including the ventral thalamus, hypothalamus, midbrain, pons, and spinal cord. To address the function of COUP-TFII in the CNS, we generated conditional COUP-TFII knockout mice using a tissue-specific NSE-Cre recombinase. Ablation of COUP-TFII in the brain resulted in malformation of the lobule VI in the cerebellum and a decrease in differentiation of cerebellar neurons and cerebellar growth. The decrease in cerebellar growth in NSE^Cre/+/CII^F/F mice is due to reduced proliferation and increased apoptosis in granule cell precursors (GCPs). Additional studies demonstrated that insulin like growth factor 1 (IGF-1) expression was reduced in the cerebellum of NSE^Cre/+/CII^F/F mice, thereby leading to decreased Akt1 and GSK-3β activities, and the reduced expression of mTOR. Using ChIP assays, we demonstrated that COUP-TFII was recruited to the promoter region of IGF-1 in a Sp1-dependent manner. In addition, dendritic branching of Purkinje cells was decreased in the mutant mice. Thus, our results indicate that COUP-TFII regulates growth and maturation of the mouse postnatal cerebellum through modulation of IGF-1 expression.     

2-Deoxyglucose Enhances 1-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine-Induced ATP Loss in the Mouse Brain

Abstract: The effects of 2-deoxyglucose (2-DG), an inhibitor of the uptake and use of glucose, on ATP loss caused by the neurotoxicant 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) were determined in the mouse brain. 2-DG alone had no effect on brain ATP levels, but when administered 30 min before MPTP exposure, 2-DG significantly enhanced MPTP-induced ATP reduction. This was reflected as an increase in ATP loss in the striatum (from 15 to 27%) as well as a significant decrease in ATP in the cerebellar cortex, an area of the brain that was not affected after exposure to MPTP alone. In mice pretreated with 2-DG, striatal ATP levels remained significantly decreased for >8 h after MPTP administration. In contrast, ATP levels in the cerebellar cortex returned to normal values within 4 h from MPTP exposure. Mazindol, a catecholamine uptake blocker, completely protected against MPTP-induced loss of striatal ATP in the absence of 2-DG, but it only partially prevented striatal ATP decrease after administration of both 2-DG and MPTP; mazindol was also ineffective in protecting against ATP loss caused by 2-DG and MPTP in the cerebellar cortex. 2-DG/MPTP-induced ATP loss appeared to be associated with the presence of the 1 -methyl-4-phenylpyridinium (MPP⁺) metabolite because (1) the pattern of ATP recovery in the striatum and cerebellar cortex appeared to reflect the pattern of MPP⁺clearance from these areas of the brain (i.e., significant MPP⁺ levels persisted longer in the striatum than in the cerebellar cortex), and (2) ATP decrease was completely prevented by blocking the conversion of MPTP to MPP⁺with the monoamine oxidase B inhibitor deprenyl. Data indicate that impairment of glucose metabolism dramatically enhances the effects of MPTP/MPP⁺ on cerebral energy supplies, making these effects relatively nonselective for dopaminergic neurons of the nigrostriatal pathway.     

Morphine reduces cerebellar guanosine-3',5'-cyclic monophosphate content and elevates cerebrospinal fluid guanosine-3',5'-cyclic monophosphate content in rhesus monkey.

Morphine administration (20 mg/kg) to awake rhesus monkeys which had been chronically implanted with catheters for aspiration of cerebrospinal fluid (CSF) produced a significant elevation in the CSF level of guanosine-3′, 5′-cyclic monophosphate (cGMP). Additionally, biopsies of cerebral and cerebellar cortex were taken from anesthetized monkeys given 20 mg/kg of morphine sulfate. Only cerebellar cGMP levels changed significantly, showing a 35% decrease relative to anesthetized controls. Although the controlling factors of brain tissue and CSF cGMP levels are poorly understood, the possibility of a reciprocal relationship between cGMP levels in certain brain regions and in CSF under some conditions is discussed.     

Blood alcohol concentration and microencephaly: a dose-response study in the neonatal rat

The relationships among microencephaly, peak blood alcohol concentration (BAC), and dose of alcohol were examined in a rat model of third-trimester fetal alcohol effects. Ethyl alcohol was administered to neonatal rats from postnatal day 4 to day 10 during the brain growth spurt via an artificial rearing technique. Groups of rats received one of nine doses of alcohol (0.0, 2.5, 3.3, 4.0, 4.5, 5.3, 6.6, 7.5, or 8.5 g/kg body weight) administered in 8 hours each day. BACs were determined on postnatal days 6 and 7 at times corresponding to peak and trough BACs, respectively. On postnatal day 10, brains were removed, and total brain weights, cerebellar weights and brainstem weights were measured. Pups receiving 4.0 g/kg/day or less had mean peak BACs below 150 mg/dl and did not exhibit significant microencephaly when compared with controls. Higher dosages further increased the peak BAC and produced significant microencephaly. While a dose of 4.5 g/kg/day was sufficient to decrease significantly both total brain weight and cerebellar weight, a minimum dose of 6.6 g/kg/day was required for significant restriction of brainstem weight. The dose of 7.5 g/kg/day yielded a mean peak BAC of 420 mg/dl and reduced total brain weight, cerebellar weight, and brainstem weight by 33%, 52%, and 22%, respectively, relative to controls. Exposure to 8.5 g/kg/day was uniformly lethal. Peak BAC and total brain weight were highly correlated (r = -.916). As peak BAC increased, total brain weight decreased linearly. Comparisons with previous studies indicate that condensing the daily dose of alcohol effectively reduced the threshold doses for microencephaly and lethality.     

THE EFFECT OF PHYSOSTIGMINE AND NEOSTIGMINE ON THE CONCENTRATION OF GLYCOGEN IN VARIOUS BRAIN STRUCTURES OF THE RAT

Abstract—

• 1 Hypothalamus, mesencephalon, cerebral cortex, cerebellar cortex and medulla oblongata of the rat brain contain varying amounts of glycogen. The highest concentration was found in the medulla, and the lowest in the hypothalamus.
• 2 Low doses of physostigmine produced a significant decrease in the concentration of glycogen in mesencephalon, cerebral cortex, cerebellar cortex and medulla. Higher doses of physostigmine were necessary to produce glycogenolysis in the hypothalamus. In the first four structures glycogen stores were almost equally sensitive to the action of physostigmine. Neostigmine did not affect brain glycogen. The glycogenolytic effect of physostigmine was dose-dependent.
• 3 Both atropine and propranolol were found to block the glycogenolytic effect of physostigmine in brain.
• 4 It is concluded that probably both cholinergic and adrenergic processes participate in the glycogenolytic effect of physostigmine. It is suggested that physostigmine initiates the cholinergic processes which then trigger adrenergic processes.

     

Regional differences in the uptake of exogenous copper into rat brain after acute treatment with sodium diethyldithiocarbamate

Summary Since sodium diethyldithiocarbamate (SDEDTC) is known to increase the tissue uptake of copper, we have examined its effect on copper accumulation in the rat cerebellum, hypothalamus, parietal cortex and hippocampus by means of atomic absorption spectrophotometry. Acute SDEDTC (1000 mg/kg i.p.) administration alone did not alter the regional concentration of copper in the cerebellum, hypothalamus and parietal cortex, but significantly increased it in the hippocampus, 5 h after treatment. Copper acetate (5 mg/kg) given i.p. has a stimulatory effect on copper uptake only in the hypothalamus and hippocampus. When copper acetate was administered to rats which were pretreated with SDEDTC, an especially high significant increase in the hippocampal copper level could be observed (approximately 70%), while the enhancement in cerebellar copper concentration was much more lower (approximately 20%), but yet significant. These data suggest that SDEDTC enhances the uptake of exogenous copper in all brain regions examined since the lipophilic SDEDTC-copper complexes easily penetrate the cell membranes. Furthermore, our histochemical findings indicate that — under normal conditions — copper is stored predominantly in glial cells, while following an excessive uptake this metal is also accumulated in neurons (e.g. pyramidal cells of the hippocampus and cortex).     

Compromised mitochondrial complex II in models of Machado-Joseph disease

Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.     

Regional Specificity in Alterations of Rat Brain Copper and Catecholamines Following Perinatal Copper Deficiency

Abstract: Perinatal copper deficiency was studied in 1-month-old female and male Sprague-Dawley rat offspring to investigate regional changes in brain copper and catecholamine levels. Offspring of dams given the low copper treatment beginning at day 7 of gestation exhibited signs characteristic of deficiency such as impaired growth and 10-fold lower liver copper levels compared with copper-adequate controls. Regional analysis of brain copper by graphite furnace atomic absorption spectroscopy revealed uniform and severe reduction of copper to levels 20 ± 3% of controls in all regions, except the hypothalamus, where reductions to 56 and 28% of those in copper-adequate females and males, respectively, were measured. HPLC analysis revealed significant reductions in norepinephrine levels in cerebrum, midbrain, corpus striatum, cerebellum, and medulla-pons of copper-deficient offspring ranging between 39 and 67% of control values. There were no significant differences in norepinephrine concentration in the hypothalamus. There was a significant, one-third reduction of dopamine in the corpus striatum of copper-deficient male rats. Consistent with altered in vivo dopamine β-monooxygenase activity, there were five-, three-, and twofold elevations of dopamine in cerebellum, medulla-pons, and hypothalamus of copper-deficient rats. Spectrophotometric measurement of in vitro dopamine β-monooxygenase activity of brain and adrenal homogenates was higher in copper-deficient rats, confirming prior work. An explanation for the in vitro data is unclear. Changes in copper and catecholamine levels were influenced by diet and were regionally selective, especially in the hypothalamus.     

Effect of Chronic Fenvalerate Intoxication on Tissue Concentration of Copper in Goats and Further Exploration of Its Mechanism

This experiment was designed to assess the effect of chronic fenvalerate toxicity on tissue Cu concentration in goats and to explore the pathways responsible for it. A significant decrease in tissue Cu concentration of kidney, heart, and brain while an increase in the liver were recorded in fenvalerate intoxicated goats at 15 mg/kg b.w. orally daily for 270 days. Concentration of total Cu, protein-free Cu, and protein-bound Cu in the wet intestine of fenvalerate-treated goats revealed a significant decrease in Cu concentration of the intestine due to the decrease in trichloroacetic acid (TCA)-insoluble Cu, while TCA-soluble Cu remained almost unaffected. Rabbit duodenal loop technique was used to assess the relative absorption of nonisotopic copper in a living animal. This technique enabled to compare Cu absorption from the lumen of three closely associated loops, each receiving 100 µg of copper along with different doses (0, 15, and 30 µg) of fenvalerate. A significant dose-dependent decrease in Cu absorption from the lumen due to fenvalerate treatment was recorded. A decrease in total copper (TCA-insoluble fraction) suggested an interference in active transport of copper due to the inhibition of absorption of protein-bound copper. It was concluded that fenvalerate interfered in copper absorption mostly by inhibiting its active or mediated transport.     

DEVELOPMENTAL FEATURES OF CEREBELLAR HYPOPLASIA AND BRAIN BILIRUBIN LEVELS IN A MUTANT (GUNN) RAT WITH HEREDITARY HYPERBILIRUBINAEMIA 1

Abstract— The marked cerebellar hypoplasia found in the homozygous (jj) Gunn rat with hereditary unconjugated hyperbilirubinaemia may provide an explanation of bilirubin neurotoxicity in vivo. In the jj Gunn rat. Purkinje cells were nearly selectively affected in the cerebellar cortex, and the cerebellar weight showed no increase after 10 days of age. The development-dependency of the cerebellar lesion was supported by the observation that the cerebellar lobuli which developed earlier were less affected. Brain bilirubin in the developing jj Gunn rat was determined by a spectrophotometric method, and was found to be extremely low (1–3 μg). The level of brain bilirubin decreased after birth, and showed little correlation with the level of bilirubin free of albumin which correlated clearly with total serum bilirubin level even in the neonate. These findings suggest that there is an affinity of brain tissue for bilirubin associated with the blood-brain barrier to bilirubin. No significant difference was found between the levels of bilirubin in the cerebellum and those of other brain regions in jj Gunn rat. These results seem to imply that the development-dependency of cerebellar hypoplasia in the jj rat may be due to the characteristic nature of rat cerebellar development, i.e. the postnatal neurogenesis. and not to changes in brain bilirubin levels. In the jj Gunn rat. cerebellar cell proliferation appears to be in some way affected by bilirubin during cerebellar development.     

Neurodevelopment and brain growth in classic Menkes disease is influenced by age and symptomatology at initiation of copper treatment

Menkes disease is an X-linked recessive disorder of brain copper metabolism caused by mutations in an essential mammalian copper transport gene, ATP7A. Untreated affected individuals suffer failure to thrive and neurodevelopmental delays that usually commence at 6–8 weeks of age. Death by age three years is typical. While provision of working copies of ATP7A to the brain by viral vectors is a promising strategy under development, the only treatment currently available is subcutaneous copper injections. These can normalize circulating blood levels and may replete brain copper depending on the molecular context, e.g., the severity of ATP7A mutation and potential presence of mosaicism. In this paper, we summarize somatic growth and neurodevelopmental outcomes for 60 subjects enrolled in a recently concluded phase I/II clinical trial of copper histidine for Menkes disease (ClinicalTrials.gov Identifier: NCT00001262). Primary outcomes indicate highly statistically significant improvements in gross motor, fine motor/adaptive, personal-social, and language neurodevelopment in the cohort of subjects who received early treatment prior to onset of symptoms (n = 35). Correlating with these findings, quantitative parameters of somatic growth indicated statistically significant greater growth in head circumference for the initially asymptomatic group, whereas weight and height/length at age three years (or at time of death) did not differ significantly. Mortality at age 3 was higher (50%) in subjects older and symptomatic when treatment commenced compared to the asymptomatic group (28.6%). We conclude that early copper histidine for Menkes disease is safe and efficacious, with treatment outcomes influenced by the timing of intervention, and ATP7A mutation.     

Semaphorin-3F attracts the growth cone of cerebellar granule cells through cGMP signaling pathway

Growth cone extension is guided by extracellular factors during the brain development but the underlying cellular mechanisms remain largely unclear. Here, we examined the potential function of class-3 semaphorins in cultured cerebellar granule cells. We found neuropilin-2 (NP2), the high-affinity receptor for semaphorin-3F (Sema3F), is highly expressed in cerebellar granule cells. An extracellular gradient of Sema3F triggered an NP2-dependent attractive turning of the growth cone of cultured cerebellar granule cells. This Sema3F-triggered growth cone attraction was abolished by inhibition of the cGMP signaling pathway and reduced by elevating the intracellular cGMP level. Furthermore, Sema3F partially rescued the collapse induced by inhibition of basal cGMP in granule cells. Thus, Sema3F may act as a chemoattractant for the growth cone of cerebellar granule cells through cGMP signaling pathway.     

EFFECT OF COPPER STATUS ON BRAIN NEUROTRANSMITTER METABOLISM IN THE LAMB

Abstract— Ataxic and non-ataxic lambs reared under field conditions which gave rise to low copper status were treated with copper intravenously. Untreated ataxic animals served as controls. The neurotransmitter amines, dopamine, norepinephrine and serotonin, were determined in the anterior and posterior regions of the brain stem. Dopamine levels in the anterior region, including the corpus striatum, were significantly lower in the untreated animals than in those treated with copper. Norepinephrine levels were also lower but serotonin concentrations were not different. Plasma amine oxidase activity was markedly higher in the copper treated animals but monoamine oxidase activity in brain stem homogenates was not significantly affected. The monoamine oxidase activity in cortical and cerebellar homogenates was significantly lower in the treated animals than in the untreated animals.     

Effect of aging on neurotransmitter receptor binding in rat and human brain.

β-Adrenergic and GABA receptor binding were measured in brain areas of rats 3 to 24 months of age. While GABA receptor binding was not significantly different across age in any area, β-adrenergic receptor binding was significantly reduced in the cerebellum and brain stem, but not cerebral cortex, of 24-month-old animals. The loss in β-adrenergic receptor binding does not correlate in a temporal fashion with the reported decrease in norepinephrine-stimulated cyclic AMP accumulation in the cerebellum which occurs as early as 12 months of age. An age-related reduction in β-adrenergic binding was also noted in human cerebellar tissue obtained at autopsy, suggesting that the cerebellar dysfunction seen with aging may be related to a loss of cerebellar neurons which receive noradrenergic input.     

Effect of histamine on the development of astroglial cells in culture

The effect of histamine on different aspects of the growth of astrocytes was studied using primary cultures derived either from forebrain or from cerebellum of the rat. The influence on general growth and differentiation was monitored in terms of the activities of ornithine decarboxylase and glutamine synthetase enzymes, whereas [³H]thymidine incorporation into DNA was used as a specific index of cell proliferation. Treatment with 500 nM histamine of cells grown for 6 days in vitro, caused a time-dependent significant increase in ornithine decarboxylase activity of astrocytes from both sources. The maximum increase was observed at 4 h after histamine treatment, at that time the elevation in ornithine decarboxylase activity being about 80% and 300% over control values in the forebrain and the cerebellar astrocytes, respectively. Under similar experimental conditions, addition of histamine (500 nM) to medium resulted in a significant increase in [³H]thymidine incorporation into DNA in both types of cultures: in comparison with control, the elevation was about 45% at 48 h in forebrain astrocytes and at 24 h in cerebellar astrocytes. On the other hand, the specific activity of glutamine synthetase in cerebellar astrocytes was markedly enhanced (about 100%) by treatment with histamine (500 nM) for 4 days, but forebrain astrocytes were little affected. Addition of histamine to the culture medium produced no significant alteration in the activity of lactate dehydrogenase and protein content of either type of astroglial cells. The present findings, which support our earlier proposal that the biochemical properties of astrocytes differ between various brain regions, provide direct evidence for the involvement of histamine in the regulation of growth and development of astrocytes.     

Neurotoxic Effects of Prenatal Trichlorfon Administration in Pigs

Pregnant sows were administered trichlorfon (Neguvon®) during gestation. Examination of the offspring revealed clinical symptoms which included ataxia and tremor. The weights of the cerebrum and the celebellum were significantly reduced (67% and 41% of the control values). There was a clear correlation between the decrease in total brain weight and decrease in cerebellar weight (r = 0.94). Histological examination showed a well preserved lamination of the cerebral and cerebellar cortex. Periodical loss of Purkinje cells was the most notable finding in the cerebellum. The molecular layer in such regions was markedly thinned. Neurochemical investigations of the cerebellum showed a significant decrease in specific activity of the transmitter synthesizing enzymes choline acetyltransferase, glutamate decarboxylase and aromatic amino acid decarboxylase (61.6%, 30.3% and 47.1% of the control values, respectively). No effect in high affinity D-aspartate uptake was observed.     

Gender influences the effect of perinatal copper deficiency on cerebellar PKC gamma content

Change in cerebellar protein kinase C gamma (PKCgamma) content caused by perinatal copper (Cu) deficiency was determined in 22-day old rats. The offspring of dams with low Cu intake during gestation and lactation exhibited signs characteristic of Cu deficiency including anemia, greater than 90% reduction in liver Cu concentration, and undetectable serum ceruloplasmin. In addition, brain Cu concentrations were reduced 80%. No differences in the signs of Cu deficiency were observed between female and male offspring. However, cerebellar PKCgamma content was reduced 54% (P < 0.05, Tukey's test) in female offspring but only 18% (P > 0.05) in male offspring. Following 6 weeks of Cu supplementation, brain Cu concentrations remained depressed in female and male rats that experienced perinatal Cu deficiency, but cerebellar PKCgamma content was completely restored to control levels. Postnatal expression of PKCgamma in the cerebellum coincides with and regulates cerebellar maturation. The results of the present study indicate perinatal Cu deficiency may impair cerebellar maturation to a greater extent in females than in males. However, it is not clear whether suppression of PKCgamma by perinatal Cu deficiency produces permanent neuropathology in the cerebellum because the effects were reversed by Cu supplementation.     

Effect of Cerebellar Lesions on Monoamine Levels in Various Brain Areas of the Cat

Abstract: Modifications in the content of monoamines after different lesions of the cerebellar cortex were investigated in eight prosencephalic structures of cat's brain. Apart from other minor changes, lesions of the posterior vermis induced significant changes in the thalamus (decrease of DA and increase of 5-HT). Lesions of the cortex of a cerebellar hemisphere, on the other hand, produced an increase of 5-HT in the caudate nucleus and an increase of DA in the hippocampus in addition to a generalized increase of 5-HT in all the prosencephalic structures studied. These findings are discussed in relation to the anatomical connections of the lesioned areas and their expected role in the sleep-wakefulness cycle.     

Brain copper content and cuproenzyme activity do not vary with prion protein expression level

Prion diseases are neurodegenerative disorders that result from conformational transformation of a normal cell surface glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). Although the normal physiological function of PrP(C) has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of (64)Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrP(C) in copper metabolism.