Dopamine transporters are markedly reduced in Lesch-Nyhan disease in vivo.

Dopamine (DA) deficiency has been implicated in Lesch-Nyhan disease (LND), a genetic disorder that is characterized by hyperuricemia, choreoathetosis, dystonia, and compulsive self-injury. To establish that DA deficiency is present in LND, the ligand WIN-35,428, which binds to DA transporters, was used to estimate the density of DA-containing neurons in the caudate and putamen of six patients with classic LND. Comparisons were made with 10 control subjects and 3 patients with Rett syndrome. Three methods were used to quantify the binding of the DA transporter so that its density could be estimated by a single dynamic positron emission tomography study. These approaches included the caudate- or putamen-to-cerebellum ratio of ligand at 80-90 min postinjection, kinetic analysis of the binding potential [Bmax/(Kd x Vd)] using the assumption of equal partition coefficients in the striatum and the cerebellum, and graphical analysis of the binding potential. Depending on the method of analysis, a 50-63% reduction of the binding to DA transporters in the caudate, and a 64-75% reduction in the putamen of the LND patients was observed compared to the normal control group. When LND patients were compared to Rett syndrome patients, similar reductions were found in the caudate (53-61%) and putamen (67-72%) in LND patients. Transporter binding in Rett syndrome patients was not significantly different from the normal controls. Finally, volumetric magnetic resonance imaging studies detected a 30% reduction in the caudate volume of LND patients. To ensure that a reduction in the caudate volume would not confound the results, a rigorous partial volume correction of the caudate time activity curve was performed. This correction resulted in an even greater decrease in the caudate-cerebellar ratio in LND patients when contrasted to controls. To our knowledge, these findings provide the first in vivo documentation of a dopaminergic reduction in LND and illustrate the role of positron emission tomography imaging in investigating neurodevelopmental disorders.

cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein.

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha.

Cytokines are now recognized to play important roles in the physiology of the central nervous system (CNS) during health and disease. Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of several human CNS disorders including multiple sclerosis, AIDS dementia, and cerebral malaria. We have generated transgenic mice that constitutively express a murine TNF-alpha transgene, under the control of its own promoter, specifically in their CNS and that spontaneously develop a chronic inflammatory demyelinating disease with 100% penetrance from around 3-8 weeks of age. High-level expression of the transgene was seen in neurons distributed throughout the brain. Disease is manifested by ataxia, seizures, and paresis and leads to early death. Histopathological analysis revealed infiltration of the meninges and CNS parenchyma by CD4+ and CD8+ T lymphocytes, widespread reactive astrocytosis and microgliosis, and focal demyelination. The direct action of TNF-alpha in the pathogenesis of this disease was confirmed by peripheral administration of a neutralizing anti-murine TNF-alpha antibody. This treatment completely prevented the development of neurological symptoms, T-cell infiltration into the CNS parenchyma, astrocytosis, and demyelination, and greatly reduced the severity of reactive microgliosis. These results demonstrate that overexpression of TNF-alpha in the CNS can cause abnormalities in nervous system structure and function. The disease induced in TNF-alpha transgenic mice shows clinical and histopathological features characteristic of inflammatory demyelinating CNS disorders in humans, and these mice represent a relevant in vivo model for their further study.

Glial cell line-derived neurotrophic factor promotes the survival and morphologic differentiation of Purkinje cells.

Glial cell line-derived neurotrophic factor (GDNF) promotes survival of midbrain dopaminergic neurons and motoneurons. Expression of GDNF mRNA in cerebellum raises the possibility that cells within this structure might also respond to GDNF. To examine potential trophic activities of GDNF, dissociated cultures of gestational day 18 rat cerebellum were grown for < or = 21 days in the presence of factor. GDNF increased Purkinje cell number without affecting the overall number of neurons or glial cells. A maximal response (50% above control) was elicited with GDNF at 1 pg/ml. Effects of GDNF on Purkinje cell differentiation were examined by scoring the morphologic maturation of cells in treated and control cultures. GDNF increased the proportion of Purkinje cells that displayed relatively mature morphologies, characterized by dendritic thickening and the development of spines and filopodial extensions. Morphologic maturation of the overall neuronal population was unaffected. In sum, our data indicate that GDNF is a potent survival and differentiation factor for Purkinje cells, the efferent neurons of cerebellar cortex. Together with its other actions, these findings raise the possibility that GDNF might be a critical trophic factor at multiple loci in neuronal circuits that control motor function.

Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography.

Relative cerebral glucose metabolism was examined with positron-emission tomography (PET) as a measure of neuronal activation during performance of the classically conditioned eyeblink response in 12 young adult subjects. Each subject received three sessions: (i) a control session with PET scan in which unpaired presentations of the tone conditioned stimulus and corneal airpuff unconditioned stimulus were administered, (ii) a paired training session to allow associative learning to occur, and (iii) a paired test session with PET scan. Brain regions exhibiting learning-related activation were identified as those areas that showed significant differences in glucose metabolism between the unpaired control condition and well-trained state in the 9 subjects who met the learning criterion. Areas showing significant activation included bilateral sites in the inferior cerebellar cortex/deep nuclei, anterior cerebellar vermis, contralateral cerebellar cortex and pontine tegmentum, ipsilateral inferior thalamus/red nucleus, ipsilateral hippocampal formation, ipsilateral lateral temporal cortex, and bilateral ventral striatum. Among all subjects, including those who did not meet the learning criterion, metabolic changes in ipsilateral cerebellar nuclei, bilateral cerebellar cortex, anterior vermis, contralateral pontine tegmentum, ipsilateral hippocampal formation, and bilateral striatum correlated with degree of learning. The localization to cerebellum and its associated brainstem circuitry is consistent with neurobiological studies in the rabbit model of eyeblink classical conditioning and neuropsychological studies in brain-damaged humans. In addition, these data support a role for the hippocampus in conditioning and suggest that the ventral striatum may also be involved.

Identification of human brain regions underlying responses to resistive inspiratory loading with functional magnetic resonance imaging.

Compensatory ventilatory responses to increased inspiratory loading are essential for adequate breathing regulation in a number of pulmonary diseases; however, the human brain sites mediating such responses are unknown. Midsagittal and axial images were acquired in 11 healthy volunteers during unloaded and loaded (30 cmH2O; 1 cmH2O = 98 Pa) inspiratory breathing, by using functional magnetic resonance imaging (fMRI) strategies (1.5-tesla MR; repetition time, 72 msec; echo time, 45 msec; flip angle, 30 degrees; field of view, 26 cm; slice thickness, 5 mm; number of excitations, 1; matrix, 128 x 256). Digital image subtractions and region of interest analyses revealed significantly increased fMRI signal intensity in discrete areas of the ventral and dorsal pons, interpeduncular nucleus, basal forebrain, putamen, and cerebellar regions. Upon load withdrawal, certain regions displayed a rapid fMRI signal off-transient, while in others, a slower fMRI signal decay emerged. Sustained loading elicited slow decreases in fMRI signal across activated regions, while second application of an identical load resulted in smaller signal increases compared to initial signal responses (P < 0.001). A moderate inspiratory load is associated with consistent regional activation of discrete brain locations; certain of these regions have been implicated in mediation of loaded breathing in animal models. We speculate that temporal changes in fMRI signal may indicate respiratory after-discharge and/or habituation phenomena.

Deregulated expression of PAX5 in medulloblastoma.

Medulloblatoma is a pediatric brain tumor originating in the human cerebellum. A collection of 23 medulloblastomas was analyzed for expression of the developmental control genes of the PAX and EN gene families by RNase protection and in situ hybridization. Of all nine PAX genes investigated, only PAX5 and PAX6 were consistently expressed in most medulloblastomas (70 and 78% of all cases, respectively), as were the genes EN1 (57%) and EN2 (78%). EN1, EN2, and PAX6 genes were also expressed in normal cerebellar tissue, and their expression in medulloblastoma is consistent with the hypothesis that this tumor originates in the external granular layer of the developing cerebellum. PAX5 transcripts were, however, not detected in the neonatal cerebellum, indicating that this gene is deregulated in medulloblastoma. In the desmoplastic variant of medulloblastoma, PAX5 expression was restricted to the reticulin-producing proliferating tumor areas containing undifferentiated cells; PAX5 was not expressed in the reticulin-free nonproliferating islands undergoing neuronal differentiation. These data suggest that deregulated expression of PAX5 correlates positively with cell proliferation and inversely with neuronal differentiation in desmoplastic medulloblastoma.

Short-term and long-term verbal memory: a positron emission tomography study.

Short-term and long-term retention of experimentally presented words were compared in a sample of 33 healthy normal volunteers by the [15O]H2O method with positron emission tomography (PET). The design included three conditions. For the long-term condition, subjects thoroughly studied 18 words 1 week before the PET study. For the short-term condition, subjects were shown another set of 18 words 60 sec before imaging, with instructions to remember them. For the baseline condition, subtracted from the two memory conditions, subjects read a third set of words that they had not previously seen in the experiment. Similar regions were activated in both short-term and long-term conditions: large right frontal areas, biparietal areas, and the left cerebellum. In addition, the short-term condition also activated a relatively large region in the left prefrontal region. These complex distributed circuits appear to represent the neural substrates for aspects of memory such as encoding, retrieval, and storage. They indicate that circuitry involved in episodic memory has much larger cortical and cerebellar components than has been emphasized in earlier lesion studies.

Citrate modulates the regulation by Zn2+ of N-methyl-D-aspartate receptor-mediated channel current and neurotransmitter release.

The effect of the two metal-ion chelators EDTA and citrate on the action of N-methyl-D-aspartate (NMDA) receptors was investigated by use of cultured mouse cerebellar granule neurons and Xenopus oocytes, respectively, to monitor either NMDA-evoked transmitter release or membrane currents. Transmitter release from the glutamatergic neurons was determined by superfusion of the cells after preloading with the glutamate analogue D-[3H]aspartate. The oocytes were injected with mRNA isolated from mouse cerebellum and, after incubation to allow translation to occur, currents mediated by NMDA were recorded electrophysiologically by voltage clamp at a holding potential of -80 mV. It was found that citrate as well as EDTA could attenuate the inhibitory action of Zn2+ on NMDA receptor-mediated transmitter release from the neurons and membrane currents in the oocytes. These effects were specifically related to the NMDA receptor, since the NMDA receptor antagonist MK-801 abolished the action and no effects of Zn2+ and its chelators were observed when kainate was used to selectively activate non-NMDA receptors. Since it was additionally demonstrated that citrate (and EDTA) preferentially chelated Zn2+ rather than Ca2+, the present findings strongly suggest that endogenous citrate released specifically from astrocytes into the extracellular space in the brain may function as a modulator of NMDA receptor activity. This is yet another example of astrocytic influence on neuronal activity.