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.

Neural expression of the transcription factor THAP1 during development in rat.

Loss of function mutations in THAP1 has been associated with primary generalized and focal dystonia in children and adults. THAP1 encodes a transcription factor (THAP1) that harbors an atypical zinc finger domain and plays a critical role in G(1)-S cell cycle control. Current thinking suggests that dystonia may be a neurodevelopmental circuit disorder. Hence, THAP1 may participate in the development of the nervous system. Herein, we report the neurodevelopmental expression patterns of Thap1 transcript and THAP1 protein from the early postnatal period through adulthood in the rat brain, spinal cord and dorsal root ganglia (DRG). We detected Thap1 transcript and THAP1-immunoreactivity (IR) in the cerebral cortex, cerebellum, striatum, substantia nigra, thalamus, spinal cord and DRG. Thap1 transcript expression was higher in the brain than in spinal cord and DRG at P1 and P7 and declined to similar levels at P14 and later time points in all regions except the cerebellum, where it remained high through adulthood. In the brain, THAP1 expression was highest in early development, particularly in the cerebellum at P7. In addition to Purkinje cells in the cerebellum, THAP1-IR was also localized to pyramidal neurons in the cerebral cortex, relay neurons in the thalamus, medium spiny and cholinergic neurons in the striatum, dopaminergic neurons in the substantia nigra, and pyramidal and interneurons in the hippocampus. In the cerebellar cortex, THAP1-IR was prominently distributed in the perikarya and proximal dendrites of Purkinje cells at early time-points. In contrast, it was more diffusely distributed throughout the dendritic arbor of adult Purkinje cells producing a moderate diffuse staining pattern in the molecular layer. At all time points, nuclear IR was weaker than cytoplasmic IR. The prominent cytoplasmic and developmentally regulated expression of THAP1 suggests that THAP1 may function as part of a cell surface-nucleus signaling cascade involved in terminal neural differentiation.

Incomplete nonsense-mediated decay facilitates detection of a multi-exonic deletion mutation in SGCE.

Mutations in SGCE represent the major cause of the myoclonus-dystonia syndrome (DYT11), an autosomal dominant disorder of reduced penetrance. Virtually all affected individuals have myoclonus, which is concentrated in the upper extremities, neck and trunk. Over half of patients have dystonia, usually affecting the neck or arms. SGCE is maternally imprinted. Of the more than 70 SGCE mutations reported in the literature, 18 are large deletions disrupting at least one exon. Therefore, testing for exonic deletions should be considered in individuals with a classic phenotype in whom Sanger sequencing is unrevealing. However, standard methodologies for detection of exonic deletion mutations are expensive, labor intensive and can produce false negatives. Herein, we report the use of cDNA derived from leukocyte RNA to identify a deletion mutation (exons 4 and 5) of SGCE in a family with DYT11. Residual RNA from incomplete nonsense-mediated decay permitted reverse transcription to cDNA. Breakpoints of the 8939 bp heterozygous deletion were then defined with long-range polymerase chain reaction and Sanger sequencing. Use of cDNA generated by reverse transcription of leukocyte RNA can reduce the costs associated with diagnostic genetic testing and can facilitate detection of deletion mutations.

The DYT1 carrier state increases energy demand in the olivocerebellar network.

DYT1 dystonia is caused by a GAG deletion in TOR1A, the gene which encodes torsinA. Gene expression studies in rodents and functional imaging studies in humans suggest that DYT1 dystonia may be a network disorder of neurodevelopmental origin. To generate high resolution metabolic maps of DYT1 dystonia and pinpoint dysregulated network elements, we performed 2-deoxyglucose autoradiography and cytochrome oxidase (CO) histochemistry in transgenic mice expressing human mutant (hMT1) torsinA and wild-type littermates. In comparison with controls, hMT1 mice showed increased glucose utilization (GU) in the inferior olive (IO) medial nucleus (IOM), IO dorsal accessory nucleus and substantia nigra compacta, and decreased GU in the medial globus pallidus (MGP) and lateral globus pallidus. The hMT1 mice showed increased CO activity in the IOM and Purkinje cell layer of cerebellar cortex, and decreased CO activity in the caudal caudate-putamen, substantia nigra reticulata and MGP. These findings suggest that (1) the DYT1 carrier state increases energy demand in the olivocerebellar network and (2) the IO may be a pivotal node for abnormal basal ganglia-cerebellar interactions in dystonia.

Huntington disease in a nonagenarian mistakenly diagnosed as normal pressure hydrocephalus.

In the absence of family history or overt chorea, the protean manifestations (cognitive, motor and behavioral) of Huntington disease (HD) may suggest alternative disease processes, particularly in elderly patients. Herein, we report on a nonagenarian with HD who did not manifest overt chorea until 91 years of age and was mistakenly diagnosed with normal pressure hydrocephalus at 89 years of age. The gait abnormalities seen in early HD should be readily distinguished from those of normal pressure hydrocephalus.

Novel THAP1 sequence variants in primary dystonia.

BACKGROUND: THAP1 encodes a transcription factor (THAP1) that harbors an atypical zinc finger domain and regulates cell proliferation. An exon 2 insertion/deletion frameshift mutation in THAP1 is responsible for DYT6 dystonia in Amish-Mennonites. Subsequent screening efforts in familial, mainly early-onset, primary dystonia identified additional THAP1 sequence variants in non-Amish subjects. OBJECTIVE: To examine a large cohort of subjects with mainly adult-onset primary dystonia for sequence variants in THAP1. METHODS: With high-resolution melting, all 3 THAP1 exons were screened for sequence variants in 1,114 subjects with mainly adult-onset primary dystonia, 96 with unclassified dystonia, and 600 controls (400 neurologically normal and 200 with Parkinson disease). In addition, all 3 THAP1 exons were sequenced in 200 subjects with dystonia and 200 neurologically normal controls. RESULTS: Nine unique melting curves were found in 19 subjects from 16 families with primary dystonia and 1 control. Age at dystonia onset ranged from 8 to 69 years (mean 48 years). Sequencing identified 6 novel missense mutations in conserved regions of THAP1 (G9C [cervical, masticatory, arm], D17G [cervical], F132S [laryngeal], I149T [cervical and generalized], A166T [laryngeal], and Q187K [cervical]). One subject with blepharospasm and another with laryngeal dystonia harbored a c.-42C>T variant. A c.57C>T silent variant was found in 1 subject with segmental craniocervical dystonia. An intron 1 variant (c.71+9C>A) was present in 7 subjects with dystonia (7/1,210) but only 1 control (1/600). CONCLUSIONS: A heterogeneous collection of THAP1 sequence variants is associated with varied anatomical distributions and onset ages of both familial and sporadic primary dystonia.

Glial elements contribute to stress-induced torsinA expression in the CNS and peripheral nervous system.

DYT1 dystonia is caused by a single GAG deletion in exon 5 of TOR1A, the gene encoding torsinA, a putative chaperone protein. In this study, central and peripheral nervous system perturbations (transient forebrain ischemia and sciatic nerve transection, respectively) were used to examine the systems biology of torsinA in rats. After forebrain ischemia, quantitative real-time reverse transcriptase-polymerase chain reaction identified increased torsinA transcript levels in hippocampus, cerebral cortex, thalamus, striatum, and cerebellum at 24 h and 7 days. Expression declined toward sham values by 14 days in striatum, thalamus and cortex, and by 21 days in cerebellum and hippocampus. TorsinA transcripts were localized to dentate granule cells and pyramidal neurons in control hippocampus and were moderately elevated in these cell populations at 24 h after ischemia, after which CA1 expression was reduced, consistent with the loss of this vulnerable neuronal population. Increased in situ hybridization signal in CA1 stratum radiatum, stratum lacunosum-moleculare, and stratum oriens at 7 days after ischemia was correlated with the detection of torsinA immunoreactivity in interneurons and reactive astrocytes at 7 and 14 days. Sciatic nerve transection increased torsinA transcript levels between 24 h and 7 days in both ipsilateral and contralateral dorsal root ganglia (DRG). However, increased torsinA immunoreactivity was localized to both ganglion cells and satellite cells in ipsilateral DRG but was restricted to satellite cells contralaterally. These results suggest that torsinA participates in the response of neural tissue to central and peripheral insults and its sustained up-regulation indicates that torsinA may contribute to remodeling of neuronal circuitry. The striking induction of torsinA in astrocytes and satellite cells points to the potential involvement of glial elements in the pathobiology of DYT1 dystonia.

Caytaxin deficiency disrupts signaling pathways in cerebellar cortex.

The genetically dystonic (dt) rat, an autosomal recessive model of generalized dystonia, harbors an insertional mutation in Atcay. As a result, dt rats are deficient in Atcay transcript and the neuronally-restricted protein caytaxin. Previous electrophysiological and biochemical studies have defined olivocerebellar pathways, particularly the climbing fiber projection to Purkinje cells, as sites of significant functional abnormality in dt rats. In normal rats, Atcay transcript is abundantly expressed in the granular and Purkinje cell layers of cerebellar cortex. To better understand the consequences of caytaxin deficiency in cerebellar cortex, differential gene expression was examined in dt rats and their normal littermates. Data from oligonucleotide microarrays and quantitative real-time reverse transcriptase-PCR (QRT-PCR) identified phosphatidylinositol signaling pathways, calcium homeostasis, and extracellular matrix interactions as domains of cellular dysfunction in dt rats. In dt rats, genes encoding the corticotropin-releasing hormone receptor 1 (CRH-R1, Crhr1) and plasma membrane calcium-dependent ATPase 4 (PMCA4, Atp2b4) showed the greatest up-regulation with QRT-PCR. Immunocytochemical experiments demonstrated that CRH-R1, CRH, and PMCA4 were up-regulated in cerebellar cortex of mutant rats. Along with previous electrophysiological and pharmacological studies, our data indicate that caytaxin plays a critical role in the molecular response of Purkinje cells to climbing fiber input. Caytaxin may also contribute to maturational events in cerebellar cortex.

Parasympathetic innervation of the meibomian glands in rats.

PURPOSE: To determine the location of parasympathetic neurons that innervate the meibomian glands in rats. METHODS: The B subunit of cholera toxin (CTB), fast blue, and a retrograde transneuronal tracer, the Bartha strain of pseudorabies virus (PRV-Ba), were injected into the upper eyelids of adult Sprague-Dawley rats after sectioning the ipsilateral branches of the facial nerve and resecting the superior cervical ganglia. Brains and orbital tissues were processed for the immunohistochemical detection of PRV-Ba and CTB. In selected cases, series of brain sections were double labeled for PRV-Ba and tyrosine hydroxylase to determine the relationship between the A5 noradrenergic cell group and superior salivatory nucleus, or for PRV-Ba and choline acetyltransferase to establish the neurochemical phenotype of parasympathetic preganglionic neurons. RESULTS: Labeled ganglionic cells were diffusely distributed within the ipsilateral pterygopalatine ganglion (PPG) and along the more proximal portions of the greater petrosal nerve (GPN). Labeled preganglionic neurons were cholinergic and were located immediately dorsolateral to the rostral-most portion of the facial nucleus and caudal superior olive, where they intermingled with A5 noradrenergic cells. CONCLUSIONS: The meibomian glands and other structures within the lid margin are subject to parasympathetic regulation by ganglion cells diffusely distributed within the PPG and along more proximal portions of the GPN. Cholinergic parasympathetic preganglionic neurons that project to meibomian gland-innervating ganglion cells are located immediately lateral, dorsal, and rostral to the facial motor nucleus in the region commonly referred to as the superior salivatory nucleus.

Regulation of the tyrosine hydroxylase and dopamine beta-hydroxylase genes by the transcription factor AP-2.

The retinoic acid-inducible and developmentally regulated transcription factor AP-2 plays an important role during development. In adult mammals, AP-2 is expressed in both neural and non-neural tissues. However, the function of AP-2 in different neuronal phenotypes is poorly understood. In this study, transcriptional regulation of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) genes by AP-2 was investigated. AP-2 binding sites were identified in the upstream regions of both genes. Electrophoretic mobility shift assays (EMSA) and DNase I footprinting analyses indicate that the AP-2 interaction with these motifs is more prominent in catecholaminergic SK-N-BE(2)C and CATH.a than in non-catecholaminergic HeLa and HepG2 cell lines. Exogenous expression of AP-2 robustly transactivated TH and DBH promoter activities in non-catecholaminergic cell lines. While AP-2 regulates the DBH promoter activity via a single site, transactivation of the TH promoter by AP-2 appears to require multiple sites. In support of this, mutation of multiple AP-2 binding sites but not that of single site diminished the basal promoter activity of the TH gene in cell lines that express TH and abolished transactivation by exogenous AP-2 expression in cell lines that do not express TH. In contrast, mutation of a single AP-2 binding site of the DBH gene completely abolished transactivation by AP-2. Double-label immunohistochemistry showed that AP-2 is coexpressed with TH in noradrenergic and adrenergic neurons in both the central and peripheral nervous systems of adult rodents. Numerous non-catecholaminergic cell groups within the spinal cord, medulla, cerebellum, and pons also express AP-2. The concentration of AP-2 in dorsomedial locations along the neuraxis suggests a regionally specific role for this transcription factor in the regulation of neuronal function. Based on these findings we propose that AP-2 may coregulate TH and DBH gene expression and thus participate in expression/maintenance of neurotransmitter phenotypes in (nor)adrenergic neurons and neuroendocrine cells.

Serotonergic modulation of eye blinks in cat and monkey.

Serotonergic modulation of spontaneous and reflexive blinking was studied in four cats and one monkey. In cats, facial nucleus injections of the type-2 serotonin receptor (5-HT2) antagonist ketanserin tended to increase the latency of the first (R1) and second (R2) components of the blink reflex to supraorbital nerve stimulation. Injections of serotonin tended to increase and of ketanserin, to decrease the duration and amplitude of R2. Serotonin also produced unilateral blepharospasm and hemifacial spasm. In the monkey, the 5-HT2 agonist 2,5-dimethoxy-4-iodoamphetamine increased spontaneous blink frequency while ketanserin decreased both peak blink velocity and spontaneous blink frequency. These findings in cat and monkey indicate that serotonergic innervation of the facial nucleus has a behaviorally important role in modulation of spontaneous and reflexive blinks and suggest that dysfunction of serotonergic systems could be important to the pathophysiology of some cases of blepharospasm.

Single-unit activity of cerebellar nuclear cells in the awake genetically dystonic rat.

The purpose of this study was to characterize neuronal activity in the deep cerebellar nuclei of the unanesthetized genetically dystonic rat during the neonatal period when the clinical signs of the dystonic syndrome first appear. Previous lesion studies have established cerebellar output as critical to the expression of the dystonic rat's motor syndrome, a disorder that closely resembles generalized dystonia in humans. In the dystonic rat, both cerebellectomy and selective lesions of the deep cerebellar nuclei decrease the frequency of abnormal motor signs and improve performance on tests of motor function. Single-unit activity was recorded from the medial, interpositus and lateral cerebellar nuclei in awake normal (N=49) and dystonic (N=54) rats at postnatal days 12-26. One hundred and eighty-three cells were isolated, 91 from normal and 92 from dystonic rats. Interspike interval histograms, autocorrelations and ratemeter histograms were generated for each cell's spike train. Interspike interval histograms were modeled with single and double gamma distributions. Cells from dystonic rats as young as 12 days of age showed bursting firing patterns, positively skewed or bimodal interspike interval histograms, and sinusoidal autocorrelations. Bursting activity increased linearly with postnatal age in dystonic rats. Cells from normal rats demonstrated non-sinusoidal autocorrelations and unimodal interspike interval histograms. Spike frequency increased linearly with postnatal age in both normal and dystonic rats. There were no statistically significant group differences in spike frequency between normal and dystonic rats. These findings show that functional neuropathology can be detected at the level of single neurons in the deep cerebellar nuclei at the earliest behavioral stages of the dystonic rat's movement disorder. The degree of abnormality in spike train parameters correlates with the severity of the movement disorder. Independent of neuronal firing rates, abnormal neuronal firing patterns can serve as a guide to the localization of pathological cell populations within the central nervous system. These results provide additional evidence that abnormal cerebellar output plays a critical role in the pathophysiology of the dystonic rat's motor syndrome.

Blink reflex to supraorbital nerve stimulation in the cat.

Neurophysiological studies of the blink reflex to supraorbital nerve stimulation were conducted in eight alert, adult male cats. The cat, like other mammals, shows both short-latency (R1) and long-latency (R2) orbicularis oculi electromyographic (OOemg) components. Measures of OOemg latency, duration, integrated area, and maximum amplitude (MA) were obtained at a stimulus magnitude of 1.5xR2 threshold. The mean (+/-SE) minimal latencies for R1 and R2 were 8.26+/-0.85 and 22.97+/-1.53 ms, respectively. On average, R1 MA was larger than R2 MA. R1 and R2 area measures were similar. Three stimulus paradigms were tested. In a paired-stimulus paradigm, the interstimulus interval (ISI) was randomly varied from 100 to 1200 ms. Ratios were constructed for the OOemg area and MA by dividing the test response by the conditioning response. In this paradigm, although a significant linear relationship was observed only between ISI and R2 MA, conditioning effects were noted on both R1 and R2 area and MA test responses at several ISIs. In a habituation paradigm, both R2 and R1 showed habituation at stimulus frequencies from 0.5 to 2 Hz. In a stimulus-response paradigm, stimulus magnitude was randomly varied between threshold and 2xthreshold. In this paradigm, OOemg area and MA of both R1 and R2 were linearly related to stimulus magnitude. Neither the systemically administered centrally acting alpha2-adrenergic antagonist yohimbine nor agonist clonidine had significant effects on blink reflex parameters, habituation, or the paired-stimulus paradigm. Overall, these results suggest that there are important similarities in the control and modulation of the R1 and R2 components of the blink reflex to supraorbital nerve stimulation in cats.

Selective elimination of cerebellar output in the genetically dystonic rat.

The genetically dystonic (dt) rat, an autosomal recessive mutant, exhibits a progressive motor syndrome that resembles the generalized idiopathic dystonia seen in humans. Even with supportive measures, dt rats die before reaching maturity. A total cerebellectomy that includes the dorsal portions of the lateral vestibular nuclei (dLV) eliminates the dystonic motor syndrome of the dt rats, greatly improves motor function, and prevents early death. The selective elimination of cerebellar nuclei was used to determine the cerebellar components critical to the mutant's motor syndrome. Bilateral electrolytic and/or excitatory amino acid lesions of the medial cerebellar nucleus, nucleus interpositus, lateral cerebellar nucleus and dLV were created in separate groups of 15-day-old dt rats. Rats were observed for the presence of abnormal motor signs (falls, twists, clasps, pivots) and tested on several measures of motor performance (activity, climbing, righting, homing, hanging) before surgery and again on Postnatal Day 20. All nuclear lesions produced significant improvements in motor function and decreases in the frequency of abnormal motor signs. Electrolytic lesions of the dLV were associated with the greatest improvements.

SPECT abnormalities in generalized dystonia.

A patient with severe, generalized dystonia and 6 age range-matched controls were studied with the regional cerebral blood flow tracer technetium-99m hexamethylpropyleneamine oxime by single-photon emission computed tomography to test the hypothesis that cerebellar function is abnormal in dystonia. Analysis was performed by drawing regions of interest around the caudate head nuclei, hemithalami, deep cerebellar nuclei, and cerebellar hemicortices. The counts in each region of interest were normalized to whole brain cerebral blood flow in an identical manner for each subject. The dystonic patient had a difference in regional cerebral blood flow between the right and left deep cerebellar nuclei, increased regional cerebral blood flow in subcortical motor structures, and an abnormal relationship between right cerebellar cortical and right deep cerebellar nuclear regional cerebral blood flow. The findings in this patient provide evidence that the cerebellum may play a role in the pathophysiology of motor signs in some patients with dystonia.

Inferior olive serotonin and norepinephrine levels during development in the genetically dystonic rat.

The dystonic (dt) rat is an autosomal recessive mutant with a motor syndrome that shares several features with idiopathic torsion dystonia in humans. In the dt rats, marked biochemical and physiological abnormalities have been localized to the olivo-cerebellar system. At the pharmacological level, the dt rats exhibit enhanced sensitivity to the behavioral effects of serotonergic (5HT) agonists, including quipazine, a drug that activates the neurons of the inferior olive (IO). High performance liquid chromatography with electrochemical detection was used to assay 5-HT, 5-hydroxyindoleacetic acid (5HIAA), and norepinephrine (NE) in micropunches of the IO in normal and dt rats at 14, 18 and 22 days of age. Samples of the rostral frontal lobes were used as internal controls. Significant age-dependent effects were seen on 5-HT and 5-HIAA levels in the IO, but not the frontal cortex, in both groups. Although both groups reached similar 5-HT levels by postnatal day 22, a significant interaction effect between age and phenotype indicated a difference in the pattern of development. Administration of quipazine (10 mg/kg, IP) to 18-day-old normal and dt rats 1 h prior to sacrifice caused significant reductions in NE, 5-HIAA and the ratio of 5-HIAA to 5-HT; however, no phenotypic differences were detected. The findings do not suggest that the differential behavioral responses to 5-HT agonists seen in normal and dt rats are the result of global abnormalities in 5-HT systems, nor do they suggest the presence of presynaptic defects in the IO.(ABSTRACT TRUNCATED AT 250 WORDS)