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Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by the expansion of a glutamine repeat in the N-terminus of the huntingtin protein. To gain insight into the pathogenesis of HD, we generated transgenic mice that express a cDNA encoding an N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.

Objective: To provide the first comprehensive magnetic resonance imaging (MRI) assessment of brain in a series of patients with Lesch-Nyhan syndrome (LNS), with emphasis on basal ganglia measurements., Design: Routine readings of MRI studies, repeated reading in random order blinded to subject diagnosis, and 3-dimensional volumetric measures of basal ganglia regions., Setting: The Johns Hopkins Hospital, Baltimore, Md., Patients: Seven patients with LNS who have hypoxanthine guanine phosphoribosyltransferase levels less than 1.6% and characteristic clinical features of the disorder, which include hyperuricemia, cognitive impairment, and dystonic movement disorder, were compared with 7 age-matched control subjects. Five of the 7 patients demonstrated self-injurious behavior. MRI studies were performed using general anesthesia because of the severity of the movement disorder., Main Outcome Measures: Measurement of brain regions from MRI-obtained images., Results: Routine readings described mild cerebral atrophy in 2 of 7 patients, but no caudate or putamen abnormalities were reported. However, on the directed blinded rereading, small caudates were suspected in 5 of 7 cases, and abnormalities in cerebral size and cranium were identified. Volumetric studies of the patients with LNS confirmed a 34% decrease in caudate volume (P<.001), a 17% decrease in total cerebral volume (P<.03), and a 12% decrease in putamen volume (P=.19)., Conclusions: To our knowledge, this is the first demonstration of consistent neuroanatomic abnormalities in LNS. The findings of reduced basal ganglia volume are consistent with the dystonic movement disorder.

We have examined several aspects of neurotransmitter function in the brains of mice carrying a deletion mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). During the first 6 weeks of postnatal development, dopamine levels in whole-brain extracts from the mutant mice (HPRT-) failed to increase at rates comparable to normal animals, resulting in 40% lower dopamine levels throughout adulthood. Regional analysis in adult animals showed the caudoputamen to be the most severely affected region, with dopamine deficits of 48-64%. Dopamine levels in other regions were normal or less severely affected. The decrease in dopamine was accompanied by a decrease in tyrosine hydroxylase (TH) activity, the rate-limiting step in dopamine synthesis. Kinetic analysis of TH extracted from the caudoputamen of normal and HPRT- mice demonstrated a 45% decrease in Vmax with an increased affinity for the tetrahydropterin cofactor in the mutants. Labeling of midbrain dopamine neurons using TH immunohistochemistry revealed no obvious deficits in the number of midbrain dopamine neurons, but quantitative autoradiographic studies revealed significant reductions in the binding of 3H-N-[1-(2-benzo(beta)thiophenyl)cyclohexyl]piperidine (3H-BTCP) to dopamine uptake sites in the forebrain of the mutants. In contrast to these abnormalities of the dopamine systems in the mutant mice, other neurotransmitter systems appeared relatively unaffected. Norepinephrine, 5-HT, tryptophan hydroxylase, and glutamic acid decarboxylase were present at normal levels in the brains of the mutants. ChAT activity was slightly lower than normal in the caudoputamen of the mutant animals, but was normal in all other brain regions examined. These results indicate that HPRT deficiency is associated with a relatively specific deficit in basal ganglia dopamine systems that emerges during the first 2 months of postnatal development.

The gene encoding the synaptosomal-associated protein--25 kDa (SNAP-25) was mapped by analysis of somatic cell hybrids and an intersubspecies backcross to mouse Chromosome 2. To identify potential mutants for SNAP-25, mice bearing mutations mapping to this region of Chromosome 2 were screened for Snap gene abnormalities. Mice heterozygous for the semidominant mutation coloboma (Cm/+) were identified that carried a deletion of Snap gene sequence. Analysis of genomic DNA revealed that the Snap gene dosage in Cm/+ mice was 50% lower than control littermates. Additionally, SNAP-25 mRNA and protein expression were 50% lower in coloboma mice than control littermates. The coloboma mouse phenotype is characterized by small eyes and head bobbing; in addition, we observed that these mice were extremely hyperactive with spontaneous locomotor activity exceeding three times control mouse activity. The localization of the genetic abnormality in coloboma mice using the Snap gene marker will provide a powerful tool for studying the biologic basis of locomotor hyperactivity.

Congenital deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) in humans results in a severe neurogenetic disorder known as the Lesch-Nyhan syndrome. Since little information concerning the precise localization of HPRT in the brain is currently available, we have used in situ hybridization to examine the expression of HPRT mRNA in the mouse brain. The results showed that HPRT mRNA is expressed in many regions of the normal mouse brain, with high levels in most, but not all neurons. In contrast, glial cells did not express detectable levels of HPRT mRNA. No HPRT mRNA was detected in the brains of mutant mice carrying a deletion in the HPRT gene.

A combination of gene transfer and intracerebral grafting may provide a powerful technique for examining the role of discrete substances in the development or functioning of the brain. In the present study, primary fibroblasts obtained from a skin biopsy from inbred Fischer rats were used as donor cells for genetic modification and grafting. When grafted to the striatum of Fischer rats with a prior 6-hydroxydopamine lesion, primary fibroblasts containing a transgene for either tyrosine hydroxylase (TH) or beta-galactosidase survived for 10 weeks and continued to express the transgene. TH synthesized by the implanted fibroblasts appeared to convert tyrosine to L-dopa actively, as observed in vitro, and to affect the host brain, as assessed through a behavioral measurement. These results suggest that primary fibroblasts genetically altered to express TH have the capacity to deliver L-dopa locally to the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input.

In humans, deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) is associated with a disorder known as Lesch-Nyhan syndrome which includes severe neurobehavioral abnormalities. Several animal models which have been developed to examine the neurobiologic substrates of this disorder have suggested a role for abnormal function in purine/dopamine neurotransmission, but the relationship between HPRT-deficiency and these abnormalities remains unknown. Recently, HPRT-deficient mice have been produced which appear to have similar, though more subtle changes in brain dopamine function. These mice will be useful in elucidating the relationship between HPRT-deficiency and the neurological deficits observed in patients with this disorder.

Several methods for chronic delivery of compounds to the central nervous system (CNS) now exist. Peripheral drug administration is generally safest, but not always effective. If direct CNS delivery of a substance is required, then CNS implantation of drug-delivery systems or grafting of various cell types to the brain can be performed, although none of these interventions are yet of consistent, proven benefit in Alzheimer's disease and other neurodegenerative disorders. Grafting of genetically modified cells to the brain may be an alternative delivery system of some substances to the CNS.

GnRH stimulates LH release from pituitary gonadotropes. Prolonged exposure of these cells to GnRH results in decreased sensitivity to further stimulation by the releasing hormone both in vivo and in vitro. Chelation of extracellular Ca++ with EGTA blocks GnRH-stimulated LH release but does not prevent subsequent desensitization. Desensitization occurs when cells are preincubated in EGTA containing 10(-7) M GnRH for a variety of times (20 min to 12 h) or when cells are preincubated for 3 h in EGTA with 10(-10), 10(-9), or 10(-8) M GnRH. A GnRH antagonist does not cause desensitization to GnRH and blocks desensitization in response to GnRH in the Ca++-free medium. Preincubation in EGTA containing 10(-7) M GnRH for 3 h did not alter sensitivity of cells to sn 1,2 dioctanoylglycerol (a protein kinase C activator), Ca++ ionophore A23187, or veratridine (an activator of endogenous ion channels). These results suggest that desensitization results from occupancy of the GnRH receptor by an agonist and may be uncoupled from LH release.