Dystonia as a network disorder: what is the role of the cerebellum?

The dystonias are a group of disorders defined by sustained or intermittent muscle contractions that result in involuntary posturing or repetitive movements. There are many different clinical manifestations and causes. Although they traditionally have been ascribed to dysfunction of the basal ganglia, recent evidence has suggested dysfunction may originate from other regions, particularly the cerebellum. This recent evidence has led to an emerging view that dystonia is a network disorder that involves multiple brain regions. The new network model for the pathogenesis of dystonia has raised many questions, particularly regarding the role of the cerebellum. For example, if dystonia may arise from cerebellar dysfunction, then why are there no cerebellar signs in dystonia? Why are focal cerebellar lesions or degenerative cerebellar disorders more commonly associated with ataxia rather than dystonia? Why is dystonia more commonly associated with basal ganglia lesions rather than cerebellar lesions? Can answers obtained from animals be extrapolated to humans? Is there any evidence that the cerebellum is not involved? Finally, what is the practical value of this new model of pathogenesis for the neuroscientist and clinician? This article explores potential answers to these questions.

Neuropathology of cervical dystonia.

The aim of this study was to search for neuropathological changes in postmortem brain tissue of individuals with cervical dystonia (CD). Multiple regions of formalin-preserved brains were collected from patients with CD and controls and examined with an extensive battery of histopathological stains in a two-stage study design. In stage one, 4 CD brains underwent a broad screening neuropathological examination. In stage two, these 4 CD brains were combined with 2 additional CD brains, and the subjective findings were quantified and compared to 16 age-matched controls. The initial subjective neuropathological assessment revealed only two regions with relatively consistent changes. The substantia nigra had frequent ubiquitin-positive intranuclear inclusions known as Marinesco bodies. Additionally, the cerebellum showed patchy loss of Purkinje cells, areas of focal gliosis and torpedo bodies. Other brain regions showed minor or inconsistent changes. In the second stage of the analysis, quantitative studies failed to reveal significant differences in the numbers of Marinesco bodies in CD versus controls, but confirmed a significantly lower Purkinje cell density in CD. Molecular investigations revealed 4 of the CD cases and 2 controls to harbor sequence variants in non-coding regions of THAP1, and these cases had lower Purkinje cell densities regardless of whether they had CD. The findings suggest that subtle neuropathological changes such as lower Purkinje cell density may be found in primary CD when relevant brain regions are investigated with appropriate methods.

Primary episodic ataxias: diagnosis, pathogenesis and treatment.

Primary episodic ataxias are autosomal dominant channelopathies that manifest as attacks of imbalance and incoordination. Mutations in two genes, KCNA1 and CACNA1A, cause the best characterized and account for the majority of identified cases of episodic ataxia. We summarize current knowledge of clinical and genetic diagnosis, genotype-phenotype correlations, pathophysiology and treatment of episodic ataxia syndromes. We focus on unresolved issues including phenotypic and genetic heterogeneity, lessons from animal models and technological advancement, rationale and feasibility of various treatment strategies, and shared mechanisms underlying episodic ataxia and other far more prevalent paroxysmal conditions such as epilepsy and migraine.

Expression of catecholaminergic mRNAs in the hyperactive mouse mutant coloboma.

The SNAP-25 deficient mouse mutant coloboma (Cm/+) is an animal model for investigating the biochemical basis of locomotor hyperactivity. The spontaneous hyperactivity exhibited by coloboma is three times greater than control mice and is a direct result of the SNAP-25 deletion. SNAP-25 is a presynaptic protein that regulates exocytotic neurotransmitter release; coloboma mice express only 50% of normal protein concentrations. Previous research has determined that there is an increase in the concentration of norepinephrine but a decrease in dopamine utilization in the striatum and nucleus accumbens of coloboma mice. In situ hybridization analysis revealed that there were corresponding increases in tyrosine hydroxylase (TH) mRNA expression in noradrenergic cell bodies of the locus coeruleus of Cm/+ mice. In contrast, TH mRNA expression in substantia nigra appeared normal in the mutant mouse. alpha(2)-Adrenergic receptors are important modulators of central noradrenergic function and dopamine release. In situ hybridization data revealed that alpha(2A)-adrenergic receptor mRNA expression is upregulated in Cm/+ mice. These results suggest an underlying abnormality in noradrenergic regulation in this hyperactive mouse mutant.

Iron deficiency decreases dopamine D1 and D2 receptors in rat brain.

Iron deficiency (ID) in early life is known to alter neurological development and functioning, but data regarding specific effects on dopamine biology are lacking. The objective of this study was to determine the extent of functional alterations in dopamine receptors in two dopaminergic tracts in young, growing, iron-deficient rats. Forty male and 40 female weanling Sprague-Dawley rats were fed either an iron-deficient (ID) diet or control (CN) diet for 6 weeks. ID decreased densities of D(1) and D(2) receptors in the caudate-putamen and decreased D(2) receptor densities in the nucleus accumbens. There were no apparent effects of ID on the affinities for the ligands in either receptor in several brain regions. In situ hybridization studies for both dopamine receptors revealed no significant effect of ID on mRNA expression for either receptor. Iron-deficient rats had a significantly higher ED(50) for raclopride-induced hypolocomotion in male and female rats compared to control rats of each sex. The loss of iron in the striatum due to dietary ID was significantly correlated with the decrease in D(2) receptor density; however, this relationship was not apparent in other brain regions. These experiments thus demonstrate abnormal dopamine receptor density and functioning in several brain regions that are related to brain regional iron loss. Importantly, the impact of ID on dopamine was more pronounced in males than females, demonstrating sex-related different sensitivities to nutrient deprivation.

Abnormal presynaptic catecholamine regulation in a hyperactive SNAP-25-deficient mouse mutant.

The consequences of a reduction in the presynaptic protein, SNAP-25, were investigated to determine the neurochemical basis of the marked hyperlocomotor activity in coloboma (Cm/+) mice. SNAP-25 is part of the minimal presynaptic machinery necessary for exocytotic neurotransmitter release. Reserpine treatment was used to deplete vesicular stores of catecholamines. Coloboma mice were more sensitive to the effects of reserpine than control mice. However, presynaptic regulation of dopamine (DA) release, as assessed by low-dose apomorphine challenge, was intact. There were region-specific reductions in in vivo tyrosine hydroxylation and the DA metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum and nucleus accumbens of Cm/+ mice. While hyperactivity is often associated with changes in DA concentration, norepinephrine (NE) concentration was significantly increased in the striatum and nucleus accumbens of the hyperactive mutant. The increase in NE may regulate the hyperactivity in these mice, as suggested by current hypotheses of the mechanisms underlying attention-deficit hyperactivity disorder (ADHD) and Tourette's syndrome.

Amperometric analysis of exocytosis at chromaffin cells from genetically distinct mice.

Amperometry is a very powerful technique for investigating the role(s) specific proteins play in exocytosis at the single-cell level. In this study, amperometry has been used to investigate possible changes in exocytosis at chromaffin cells isolated from coloboma and tottering mutant mice. Coloboma mice possess a deletion mutation that encompasses the gene for the presynaptic protein SNAP-25 and tottering mice carry a mutation of the alpha(1A) subunit gene, which encodes the pore-forming region of P/Q-type calcium channels. Although amperometric data measured from tottering and coloboma cells are not significantly different from that measured at wild-type control cells, significant differences are found when groups of wild-type chromaffin cells are analyzed at room temperature and at 37 degrees C. Due to the large variability inherent to amperometric data, it is possible that changes in release resulting from some genetic differences cannot be detected. To fully exploit the technical advantages of using mouse chromaffin cells, experimental guidelines are described which should maximize changes in release resulting from genetic differences and increase the likelihood of detecting a change in amperometric data.

Calcium channel agonists and dystonia in the mouse.

Systemic administration of the L-type calcium channel agonists +/-Bay K 8644 or FPL 64176 causes a characteristic pattern of motor dysfunction in normal C57BL/6J mice that resembles generalized dystonia. There is no associated change in the electroencephalogram, confirming that the motor disorder does not reflect epileptic seizures. However, the electromyogram reveals an increase in baseline motor unit activity with prolonged phasic discharges consistent with dystonia. The duration and severity of dystonia is dependent on the dose administered and the age of the animal at testing. The effects are transient, with the return of normal motor behavior 1-4 hours after treatment. Similar effects can be provoked by intracerebral administration of small amounts of the drugs, indicating a centrally mediated response. Dystonia can be attenuated by co-administration of dihydropyridine L-type calcium channel antagonists (nifedipine, nimodipine, and nitrendipine) but not by non-dihydropyridine antagonists (diltiazem, verapamil, and flunarizine). These results implicate abnormal function of L-type calcium channels in the expression of dystonia in this model.

Quantitative and statistical analysis of the shape of amperometric spikes recorded from two populations of cells.

Previously used methods of comparing amperometric spike characteristics from two separate groups of cells have entailed pooling all the values for a spike characteristic from each group of cells and then statistically comparing the two samples. Although this approach has indicated that there are significant differences between the spike characteristics from coloboma and control mouse chromaffin cells, the results are not consistent between experiments. We have reexamined the assumptions of the statistical tests used as well as the variability inherent in amperometric data measured from two groups of cells. Our findings indicate that when comparing amperometric spike characteristics between groups of cells, it is more appropriate to compare samples of mean spike values. This method consistently indicates that there is no difference between coloboma and control amperometric spikes. These results have been validated by using samples of mean spike characteristics to detect changes in the shape of amperometric spikes from both mouse chromaffin cells at 37 degrees C and PC12 cells previously exposed to 50 microM L-3,4-dihydroxyphenylalanine and by the use of an additional analysis method, the nested ANOVA. Together, these results indicate that pooled samples of amperometric spike characteristics can give results that may confound the interpretation of amperometric data.

Characterization and distribution of ferritin binding sites in the adult mouse brain.

Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using 125I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant (K(D)) of 4.65 x 10(-9) M and a binding site density (Bmax of 17.9 fmol bound/microg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and white matter.

Influence of age and strain on striatal dopamine loss in a genetic mouse model of Lesch-Nyhan disease.

Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Affected individuals exhibit a characteristic pattern of neurological and behavioral features attributable in part to dysfunction of basal ganglia dopamine systems. In the current studies, striatal dopamine loss was investigated in five different HPRT-deficient strains of mice carrying one of two different HPRT gene mutations. Caudoputamen dopamine concentrations were significantly reduced in all five of the strains, with deficits ranging from 50.7 to 61.1%. Mesolimbic dopamine was significantly reduced in only three of the five strains, with a range of 31.6-38.6%. The reduction of caudoputamen dopamine was age dependent, emerging between 4 and 12 weeks of age. Tyrosine hydroxylase and aromatic amino acid decarboxylase, two enzymes responsible for the synthesis of dopamine, were reduced by 22.4-37.3 and 22.2-43.1%, respectively. These results demonstrate that HPRT deficiency is strongly associated with a loss of basal ganglia dopamine. The magnitude of dopamine loss measurable is dependent on the genetic background of the mouse strain used, the basal ganglia subregion examined, and the age of the animals at assessment.

L-type calcium channels contribute to the tottering mouse dystonic episodes.

Tottering mice inherit a recessive mutation of the calcium channel alpha1A subunit that causes ataxia, polyspike discharges, and intermittent dystonic episodes. The calcium channel alpha1A subunit gene encodes the pore-forming protein of P/Q-type voltage-dependent calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons with moderate expression in hippocampus and inferior colliculus. Because calcium misregulation likely underlies the tottering mouse phenotype, calcium channel blockers were tested for their ability to block the motor episodes. Pharmacologic agents that specifically block L-type voltage-dependent calcium channels, but not P/Q-type calcium channels, prevented the inducible dystonia of tottering mutant mice. Specifically, the dihydropyridines nimodipine, nifedipine, and nitrendipine, the benzothiazepine diltiazem, and the phenylalkylamine verapamil all prevented restraint-induced tottering mouse motor episodes. Conversely, the L-type calcium channel agonist Bay K8644 induced stereotypic tottering mouse dystonic at concentrations significantly below those required to induce seizures in control mice. In situ hybridization demonstrated that L-type calcium channel alpha1C subunit mRNA expression was up-regulated in the Purkinje cells of tottering mice. Radioligand binding with [3H]nitrendipine also revealed a significant increase in the density of L-type calcium channels in tottering mouse cerebellum. These data suggest that although a P/Q-type calcium channel mutation is the primary defect in tottering mice, L-type calcium channels may contribute to the generation of the intermittent dystonia observed in these mice. The susceptibility of L-type calcium channels to voltage-dependent facilitation may promote this abnormal motor phenotype.

Tottering mouse motor dysfunction is abolished on the Purkinje cell degeneration (pcd) mutant background.

Tottering (tg) mice inherit a recessive mutation of the calcium channel alpha 1A subunit gene, which encodes the pore-forming protein of P/Q-type voltage-sensitive calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons. The phenotypic consequences of the tottering mutation include ataxia, polyspike discharges, and an intermittent motor dysfunction best described as paroxysmal dystonia. These dystonic episodes induce c-fos mRNA expression in the cerebellar circuitry, including cerebellar granule and Purkinje neurons, deep cerebellar nuclei, and the postsynaptic targets of the deep nuclei. Cellular abnormalities associated with the mutation include hyperarborization of brainstem nucleus locus ceruleus axons and abnormal expression of L-type calcium channels in cerebellar Purkinje cells. Here, the role of these two distinct neural pathways in the expression of tottering mouse intermittent dystonia was assessed. Lesion of locus ceruleus axons with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine (DSP-4) did not affect the frequency of tottering mouse dystonic episodes. In contrast, removal of cerebellar Purkinje cells with the Purkinje cell degeneration (pcd) mutation by generation of tg/tg; pcd/pcd double mutant mice completely eliminated tottering mouse dystonia. Further, the c-fos expression pattern of tg/tg; pcd/pcd double mutants following restraint was indistinguishable from that of wild-type mice, suggesting that the pcd lesion eliminated an essential link in this abnormal neural network. These data suggest that the cerebellar cortex, where the mutant gene is abundantly expressed, contributes to the expression of tottering mouse dystonic episodes.

Cerebellar circuitry is activated during convulsive episodes in the tottering (tg/tg) mutant mouse.

Tottering (tg) is an autosomal recessive mutation of the calcium channel alpha1A subunit in the mouse that results in epileptic spike and wave discharges, mild ataxia and paroxysmal episodes of involuntary spasms of the limbs, trunk and face. These convulsions have been especially difficult to characterize because of their unpredictable occurrence and lack of electroencephalographic correlates. However, it is, in fact, possible to induce these convulsions, making this facet of the tottering phenotype amenable to controlled experimentation for the first time. Here, the neuroanatomical basis of the convulsions in tottering mice has been identified using in situ hybridization for c-fos messenger RNA to chart abnormal neuronal activity. Convulsion-induced c-fos messenger RNA expression was most prominent in the cerebellum of convulsing tottering mice. Additionally, cerebral cortex and principal cerebellar relay nuclei were also activated during a convulsion. The c-fos activation in the cerebellum temporally preceded expression in cerebral cortex, suggesting that cerebral cortex is not driving the expression of convulsions. These results suggest that the cerebellum, a region not classically associated with paroxysmal events, is important in the generation and/or maintenance of the intermittent convulsions in tottering mutant mice.

Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis.

Spontaneous neurologic mutations in the mouse provide powerful tools for the study of mammalian central nervous system development. The study of mouse neurologic mutants has led to a better understanding of the complex mechanisms involved in the development of the nervous system. Because few of these mutations have been identified, molecular probes distinguishing heterozygotes from homozygotes are generally unavailable. Further, most neurologic mouse mutants breed poorly as homozygotes, making it necessary to breed heterozygotes and select homozygous mutant progeny based on phenotype. The requirement for heterozygous breeding and the lack of molecular markers specific for the mutation have hampered developmental studies because the underlying neurologic perturbations occur before the mutant mice can be identified by phenotype. The recent identification and chromosomal assignment of simple sequence repeats (SSRs), repetitive sequences of DNA found at a high density throughout the mouse genome, provide the tools for mapping mutations in the mouse and for subsequent genotyping of potential mutants prior to phenotype onset. The SSRs are useful because these markers are polymorphic (for review see Weber, J.L., Human DNA polymorphisms based on length variations in simple-sequence tandem repeats. In: K.E. Davies and S.M. Tilghman (Eds.), Genetic and Physical Mapping. Genome Analysis, Vol. I, Cold Spring Harbor Laboratory Press, Plainview, NY, 1990, pp. 159-181 [16]), that is, the size of the individual SSRs differs among strains of mice. Following polymerase chain reaction (PCR) amplification of an SSR and separation of PCR products by polyacrylamide gel electrophoresis, one can easily visualize differences in the size of the PCR product between mouse strains. Many mutations in the mouse arose spontaneously on inbred strains and were subsequently backcrossed onto a different strain. After many generations of congenic backcrosses, the only DNA retained from the original mutant strain is composed of the mutant gene and closely linked regions. Thus, it is possible to cross the mutant strain to a different mouse strain and map the mutation by correlating mutant phenotype to SSRs the same size as the original mutant strain. We have mapped the tottering (tg), Purkinje cell degeneration (pcd), and nervous (nr) mutations using SSRs in backcrossed mouse strains. The SSRs distinguishing mutant from normal strains can then be used to genotype potential mutant pups before the onset of the mutant phenotype. The protocol described below can be adapted to almost any mutation congenically inbred for genotyping. Here we describe a method for selecting primers appropriate for genotyping potential mouse mutants and a rapid protocol for genotype screening. Even with SSRs distinguishing mutant from normal mice, genotyping several mice simultaneously can be a daunting task. This is primarily because the protocols available for preparing DNA for PCR amplification are time-consuming, requiring several purification steps including phenol extractions. Although kits are commercially available for DNA preparation without organic extractions, these kits tend to be expensive. The protocol described is a rapid, inexpensive method of determining the genotype of mice using PCR analysis of dried blood spots. The protocol only requires PCR primers distinguishing among alleles and is therefore ideal for the rapid identification of potential mutants for those mouse mutations which have been mapped using microsatellite markers. The DNA preparation protocol may also be used in rapid screening of potential transgenic mice.

Cloning and characterization of the Bg/II restriction-modification system reveals a possible evolutionary footprint.

Bg/II, a type II restriction-modification (R-M) system from Bacillus globigii, recognizes the sequence 5'-AGATCT-3'. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R-M system. Oligonucleotide probes specific for the 5' end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R-M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R-M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R-M system.

Cloning and sequence comparison of AvaI and BsoBI restriction-modification systems.

AvaI and BsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5'CYCGRG3' and cleave between the first C and second Y to generate a four-base 5' extension. The AvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned into Escherichia coli by the methylase selection method. The BsoBI restriction endonuclease gene (bsoBIR) and part of the BsoBI methylase gene (bsoBIM) were cloned by the "endo-blue" method (SOS induction assay), and the remainder of bsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated that AvaI and BsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. The avaIM gene precedes avaIR in the AvaI RM system, while the bsoBI R gene is located upstream of bsoBI M in the BsoBI RM system. Both AvaI and BsoBI methylases contain motifs conserved among the N4 cytosine methylases.