Ultrastructural analysis of nigrostriatal dopaminergic terminals in a knockin mouse model of DYT1 dystonia.

DYT1 dystonia is associated with decreased striatal dopamine release. In this study, we examined the possibility that ultrastructural changes of nigrostriatal dopamine terminals could contribute to this neurochemical imbalance using a serial block face/scanning electron microscope (SBF/SEM) and three-dimensional reconstruction to analyse striatal tyrosine hydroxylase-immunoreactive (TH-IR) terminals and their synapses in a DYT1(ΔE) knockin (DYT1-KI) mouse model of DYT1 dystonia. Furthermore, to study possible changes in vesicle packaging capacity of dopamine, we used transmission electron microscopy to assess the synaptic vesicle size in striatal dopamine terminals. Quantitative comparative analysis of 80 fully reconstructed TH-IR terminals in the WT and DYT1-KI mice indicate (1) no significant difference in the volume of TH-IR terminals; (2) no major change in the proportion of axo-spinous versus axo-dendritic synapses; (3) no significant change in the post-synaptic density (PSD) area of axo-dendritic synapses, while the PSDs of axo-spinous synapses were significantly smaller in DYT1-KI mice; (4) no significant change in the contact area between TH-IR terminals and dendritic shafts or spines, while the ratio of PSD area/contact area decreased significantly for both axo-dendritic and axo-spinous synapses in DYT1-KI mice; (5) no significant difference in the mitochondria volume; and (6) no significant difference in the synaptic vesicle area between the two groups. Altogether, these findings suggest that abnormal morphometric changes of nigrostriatal dopamine terminals and their post-synaptic targets are unlikely to be a major source of reduced striatal dopamine release in DYT1 dystonia.

Cell-specific Dyt1 ∆GAG knock-in to basal ganglia and cerebellum reveal differential effects on motor behavior and sensorimotor network function.

Dystonia is a neurological movement disorder characterized by repetitive, unintentional movements and disabling postures that result from sustained or intermittent muscle contractions. The basal ganglia and cerebellum have received substantial focus in studying DYT1 dystonia. It remains unclear how cell-specific ∆GAG mutation of torsinA within specific cells of the basal ganglia or cerebellum affects motor performance, somatosensory network connectivity, and microstructure. In order to achieve this goal, we generated two genetically modified mouse models: in model 1 we performed Dyt1 ∆GAG conditional knock-in (KI) in neurons that express dopamine-2 receptors (D2-KI), and in model 2 we performed Dyt1 ∆GAG conditional KI in Purkinje cells of the cerebellum (Pcp2-KI). In both of these models, we used functional magnetic resonance imaging (fMRI) to assess sensory-evoked brain activation and resting-state functional connectivity, and diffusion MRI to assess brain microstructure. We found that D2-KI mutant mice had motor deficits, abnormal sensory-evoked brain activation in the somatosensory cortex, as well as increased functional connectivity of the anterior medulla with cortex. In contrast, we found that Pcp2-KI mice had improved motor performance, reduced sensory-evoked brain activation in the striatum and midbrain, as well as reduced functional connectivity of the striatum with the anterior medulla. These findings suggest that (1) D2 cell-specific Dyt1 ∆GAG mediated torsinA dysfunction in the basal ganglia results in detrimental effects on the sensorimotor network and motor output, and (2) Purkinje cell-specific Dyt1 ∆GAG mediated torsinA dysfunction in the cerebellum results in compensatory changes in the sensorimotor network that protect against dystonia-like motor deficits.

Pathophysiology of Dyt1-Tor1a dystonia in mice is mediated by spinal neural circuit dysfunction.

Dystonia, a neurological disorder defined by abnormal postures and disorganized movements, is considered to be a neural circuit disorder with dysfunction arising within and between multiple brain regions. Given that spinal neural circuits constitute the final pathway for motor control, we sought to determine their contribution to this movement disorder. Focusing on the most common inherited form of dystonia in humans, DYT1-TOR1A, we generated a conditional knockout of the torsin family 1 member A (Tor1a) gene in the mouse spinal cord and dorsal root ganglia (DRG). We found that these mice recapitulated the phenotype of the human condition, developing early-onset generalized torsional dystonia. Motor signs emerged early in the mouse hindlimbs before spreading caudo-rostrally to affect the pelvis, trunk, and forelimbs throughout postnatal maturation. Physiologically, these mice bore the hallmark features of dystonia, including spontaneous contractions at rest and excessive and disorganized contractions, including cocontractions of antagonist muscle groups, during voluntary movements. Spontaneous activity, disorganized motor output, and impaired monosynaptic reflexes, all signs of human dystonia, were recorded from isolated mouse spinal cords from these conditional knockout mice. All components of the monosynaptic reflex arc were affected, including motor neurons. Given that confining the Tor1a conditional knockout to DRG did not lead to early-onset dystonia, we conclude that the pathophysiological substrate of this mouse model of dystonia lies in spinal neural circuits. Together, these data provide new insights into our current understanding of dystonia pathophysiology.

Pharmacological perturbation reveals deficits in D2 receptor responses in Thap1 null mice.

The primary dystonia DYT6 is caused by mutations in the transcription factor Thanatos-associated protein 1 (THAP1). To understand THAP1's functions, we generated mice lacking THAP1 in the nervous system. THAP1 loss causes locomotor deficits associated with transcriptional changes. Since many of the genes misregulated involve dopaminergic signaling, we pharmacologically challenged the two striatal canonical dopamine pathways: the direct, regulated by the D1 receptor, and the indirect, regulated by the D2 receptor. We discovered that depleting THAP1 specifically interferes with the D2 receptor responses, pointing to a selective misregulation of the indirect pathway in DYT6 with implications for pathogenesis and treatment.

Cell-specific effects of Dyt1 knock-out on sensory processing, network-level connectivity, and motor deficits.

DYT1 dystonia is a debilitating movement disorder characterized by repetitive, unintentional movements and postures. The disorder has been linked to mutation of the TOR1A/DYT1 gene encoding torsinA. Convergent evidence from studies in humans and animal models suggest that striatal medium spiny neurons and cholinergic neurons are important in DYT1 dystonia. What is not known is how torsinA dysfunction in these specific cell types contributes to the pathophysiology of DYT1 dystonia. In this study we sought to determine whether torsinA dysfunction in cholinergic neurons alone is sufficient to generate the sensorimotor dysfunction and brain changes associated with dystonia, or if torsinA dysfunction in a broader subset of cell types is needed. We generated two genetically modified mouse models, one with selective Dyt1 knock-out from dopamine-2 receptor expressing neurons (D2KO) and one where only cholinergic neurons are impacted (Ch2KO). We assessed motor deficits and performed in vivo 11.1 T functional MRI to assess sensory-evoked brain activation and connectivity, along with diffusion MRI to assess brain microstructure. We found that D2KO mice showed greater impairment than Ch2KO mice, including reduced sensory-evoked brain activity in key regions of the sensorimotor network, and altered functional connectivity of the striatum that correlated with motor deficits. These findings suggest that (1) the added impact of torsinA dysfunction in medium spiny and dopaminergic neurons of the basal ganglia generate more profound deficits than the dysfunction of cholinergic neurons alone, and (2) that sensory network impairments are linked to motor deficits in DYT1 dystonia.

Characterization of the direct pathway in Dyt1 ΔGAG heterozygous knock-in mice and dopamine receptor 1-expressing-cell-specific Dyt1 conditional knockout mice.

DYT1 dystonia is a movement disorder mainly caused by a trinucleotide deletion (ΔGAG) in DYT1 (TOR1A), coding for torsinA. DYT1 dystonia patients show trends of decreased striatal ligand-binding activities to dopamine receptors 1 (D1R) and 2 (D2R). Dyt1 ΔGAG knock-in (KI) mice, which have the corresponding ΔGAG deletion, similarly exhibit reduced striatal D1R and D2R-binding activities and their expression levels. While the consequences of D2R reduction have been well characterized, relatively little is known about the effect of D1R reduction. Here, locomotor responses to D1R and D2R antagonists were examined in Dyt1 KI mice. Dyt1 KI mice showed significantly less responsiveness to both D1R antagonist SCH 23390 and D2R antagonist raclopride. The electrophysiological recording indicated that Dyt1 KI mice showed a significantly increased paired-pulse ratio of the striatal D1R-expressing medium spiny neurons and altered miniature excitatory postsynaptic currents. To analyze the in vivo torsinA function in the D1R-expressing neurons further, Dyt1 conditional knockout (Dyt1 d1KO) mice in these neurons were generated. Dyt1 d1KO mice had decreased spontaneous locomotor activity and reduced numbers of slips in the beam-walking test. Dyt1 d1KO male mice showed abnormal gait. Dyt1 d1KO mice showed defective striatal D1R maturation. Moreover, the mutant striatal D1R-expressing medium spiny neurons had increased capacitance, decreased sEPSC frequency, and reduced intrinsic excitability. The results suggest that torsinA in the D1R-expressing cells plays an important role in the electrophysiological function and motor performance. Medical interventions to the direct pathway may affect the onset and symptoms of this disorder.

A2A Receptor Dysregulation in Dystonia DYT1 Knock-Out Mice.

We aimed to investigate A2A receptors in the basal ganglia of a DYT1 mouse model of dystonia. A2A was studied in control Tor1a+/+ and Tor1a+/- knock-out mice. A2A expression was assessed by anti-A2A antibody immunofluorescence and Western blotting. The co-localization of A2A was studied in striatal cholinergic interneurons identified by anti-choline-acetyltransferase (ChAT) antibody. A2A mRNA and cyclic adenosine monophosphate (cAMP) contents were also assessed. In Tor1a+/+, Western blotting detected an A2A 45 kDa band, which was stronger in the striatum and the globus pallidus than in the entopeduncular nucleus. Moreover, in Tor1a+/+, immunofluorescence showed A2A roundish aggregates, 0.3-0.4 µm in diameter, denser in the neuropil of the striatum and the globus pallidus than in the entopeduncular nucleus. In Tor1a+/-, A2A Western blotting expression and immunofluorescence aggregates appeared either increased in the striatum and the globus pallidus, or reduced in the entopeduncular nucleus. Moreover, in Tor1a+/-, A2A aggregates appeared increased in number on ChAT positive interneurons compared to Tor1a+/+. Finally, in Tor1a+/-, an increased content of cAMP signal was detected in the striatum, while significant levels of A2A mRNA were neo-expressed in the globus pallidus. In Tor1a+/-, opposite changes of A2A receptors' expression in the striatal-pallidal complex and the entopeduncular nucleus suggest that the pathophysiology of dystonia is critically dependent on a composite functional imbalance of the indirect over the direct pathway in basal ganglia.

TorsinA restoration in a mouse model identifies a critical therapeutic window for DYT1 dystonia.

In inherited neurodevelopmental diseases, pathogenic processes unique to critical periods during early brain development may preclude the effectiveness of gene modification therapies applied later in life. We explored this question in a mouse model of DYT1 dystonia, a neurodevelopmental disease caused by a loss-of-function mutation in the TOR1A gene encoding torsinA. To define the temporal requirements for torsinA in normal motor function and gene replacement therapy, we developed a mouse line enabling spatiotemporal control of the endogenous torsinA allele. Suppressing torsinA during embryogenesis caused dystonia-mimicking behavioral and neuropathological phenotypes. Suppressing torsinA during adulthood, however, elicited no discernible abnormalities, establishing an essential requirement for torsinA during a developmental critical period. The developing CNS exhibited a parallel "therapeutic critical period" for torsinA repletion. Although restoring torsinA in juvenile DYT1 mice rescued motor phenotypes, there was no benefit from adult torsinA repletion. These data establish a unique requirement for torsinA in the developing nervous system and demonstrate that the critical period genetic insult provokes permanent pathophysiology mechanistically delinked from torsinA function. These findings imply that to be effective, torsinA-based therapeutic strategies must be employed early in the course of DYT1 dystonia.

Disease Modeling with Human Neurons Reveals LMNB1 Dysregulation Underlying DYT1 Dystonia.

DYT1 dystonia is a hereditary neurologic movement disorder characterized by uncontrollable muscle contractions. It is caused by a heterozygous mutation in Torsin A (TOR1A), a gene encoding a membrane-embedded ATPase. While animal models provide insights into disease mechanisms, significant species-dependent differences exist since animals with the identical heterozygous mutation fail to show pathology. Here, we model DYT1 by using human patient-specific cholinergic motor neurons (MNs) that are generated through either direct conversion of patients' skin fibroblasts or differentiation of induced pluripotent stem cells (iPSCs). These human MNs with the heterozygous TOR1A mutation show reduced neurite length and branches, markedly thickened nuclear lamina, disrupted nuclear morphology, and impaired nucleocytoplasmic transport (NCT) of mRNAs and proteins, whereas they lack the perinuclear "blebs" that are often observed in animal models. Furthermore, we uncover that the nuclear lamina protein LMNB1 is upregulated in DYT1 cells and exhibits abnormal subcellular distribution in a cholinergic MNs-specific manner. Such dysregulation of LMNB1 can be recapitulated by either ectopic expression of the mutant TOR1A gene or shRNA-mediated downregulation of endogenous TOR1A in healthy control MNs. Interestingly, downregulation of LMNB1 can largely ameliorate all the cellular defects in DYT1 MNs. These results reveal the value of disease modeling with human patient-specific neurons and indicate that dysregulation of LMNB1, a crucial component of the nuclear lamina, may constitute a major molecular mechanism underlying DYT1 pathology.SIGNIFICANCE STATEMENT Inaccessibility to patient neurons greatly impedes our understanding of the pathologic mechanisms for dystonia. In this study, we employ reprogrammed human patient-specific motor neurons (MNs) to model DYT1, the most severe hereditary form of dystonia. Our results reveal disease-dependent deficits in nuclear morphology and nucleocytoplasmic transport (NCT). Most importantly, we further identify LMNB1 dysregulation as a major contributor to these deficits, uncovering a new pathologic mechanism for DYT1 dystonia.

Atypical presentations of DYT1 dystonia with acute craniocervical onset.

DYT1 gene mutations lead to early-onset dystonia that begins with focal limb onset and spreads to other body regions within 5 years, with typical sparing of the oromandibular muscles. In the present study, we describe two patients with an unusual presentation of the disease.

DYT-TUBB4A (DYT4 Dystonia): New Clinical and Genetic Observations.

OBJECTIVE: To report 4 novel TUBB4A mutations leading to laryngeal and cervical dystonia with frequent generalization. METHODS: We screened 4 families including a total of 11 definitely affected members with a clinical picture resembling the original description. RESULTS: Four novel variants in the TUBB4A gene have been identified: D295N, R46M, Q424H, and R121W. In silico modeling showed that all variants have characteristics similar to R2G. The variants segregate with the disease in 3 of the families with evidence of incomplete penetrance in 2 of them. All 4 variants would be classified as likely pathogenic. The clinical picture particularly included laryngeal dystonia (often the site of onset), associated with cervical and upper limb dystonia and frequent generalization. Laryngeal dystonia was extremely prevalent (>90%) both in the original cases and in this case series. The hobby horse gait was evident in only 1 patient in this case series. CONCLUSIONS: Our interpretation is that laryngeal involvement is a hallmark feature of DYT-TUBB4A. Nevertheless, TUBB4A mutations remain an exceedingly rare cause of laryngeal or other isolated dystonia.

Opposing patterns of abnormal D1 and D2 receptor dependent cortico-striatal plasticity explain increased risk taking in patients with DYT1 dystonia.

Patients with DYT1 dystonia caused by the mutated TOR1A gene exhibit risk neutral behaviour compared to controls who are risk averse in the same reinforcement learning task. It is unclear whether this behaviour can be linked to changes in cortico-striatal plasticity demonstrated in animal models which share the same TOR1A mutation. We hypothesised that we could reproduce the experimental risk taking behaviour using a model of the basal ganglia under conditions where cortico-striatal plasticity was abnormal. As dopamine exerts opposing effects on cortico-striatal plasticity via different receptors expressed on medium spiny neurons (MSN) of the direct (D1R dominant, dMSNs) and indirect (D2R dominant, iMSNs) pathways, we tested whether abnormalities in cortico-striatal plasticity in one or both of these pathways could explain the patient's behaviour. Our model could generate simulated behaviour indistinguishable from patients when cortico-striatal plasticity was abnormal in both dMSNs and iMSNs in opposite directions. The risk neutral behaviour of the patients was replicated when increased cortico-striatal long term potentiation in dMSN's was in combination with increased long term depression in iMSN's. This result is consistent with previous observations in rodent models of increased cortico-striatal plasticity at in dMSNs, but contrasts with the pattern reported in vitro of dopamine D2 receptor dependant increases in cortico-striatal LTP and loss of LTD at iMSNs. These results suggest that additional factors in patients who manifest motor symptoms may lead to divergent effects on D2 receptor dependant cortico-striatal plasticity that are not apparent in rodent models of this disease.

Improved survival and overt "dystonic" symptoms in a torsinA hypofunction mouse model.

DYT1 dystonia is an inherited movement disorder without obvious neurodegeneration. Multiple mutant mouse models exhibit motor deficits without overt "dystonic" symptoms and neurodegeneration. However, some mouse models do. Among the later models, the N-CKO mouse model, which has a heterozygous Tor1a/Dyt1 knockout (KO) in one allele and Nestin-cre-mediated conditional KO in the other, exhibits a severe lack of weight gain, neurodegeneration, overt "dystonic" symptoms, such as overt leg extension, weak walking, twisted hindpaw and stiff hindlimb, and complete infantile lethality. However, it is not clear if the overt dystonic symptoms were caused by the neurodegeneration in the dying N-CKO mice. Here, the effects of improved maternal care and nutrition during early life on the symptoms in N-CKO mice were analyzed by culling the litter and providing wet food to examine whether the overt dystonic symptoms and severe lack of weight gain are caused by malnutrition-related neurodegeneration. Although the N-CKO mice in this study replicated the severe lack of weight gain and overt "dystonic" symptoms during the lactation period regardless of culling at postnatal day zero or later, there was no significant difference in the brain astrocytes and apoptosis between the N-CKO and control mice. Moreover, more than half of the N-CKO mice with culling survived past the lactation period. The surviving adult N-CKO mice did not display overt "dystonic" symptoms, and in addition they still exhibited small body weight. The results suggest that the overt "dystonic" symptoms in the N-CKO mice were independent of prominent neurodegeneration, which negates the role of neurodegeneration in the pathogenesis of DYT1 dystonia.

Decreased number of striatal cholinergic interneurons and motor deficits in dopamine receptor 2-expressing-cell-specific Dyt1 conditional knockout mice.

DYT1 early-onset generalized torsion dystonia is a hereditary movement disorder characterized by abnormal postures and repeated movements. It is caused mainly by a heterozygous trinucleotide deletion in DYT1/TOR1A, coding for torsinA. The mutation may lead to a partial loss of torsinA function. Functional alterations of the basal ganglia circuits have been implicated in this disease. Striatal dopamine receptor 2 (D2R) levels are significantly decreased in DYT1 dystonia patients and in the animal models of DYT1 dystonia. D2R-expressing cells, such as the medium spiny neurons in the indirect pathway, striatal cholinergic interneurons, and dopaminergic neurons in the basal ganglia circuits, contribute to motor performance. However, the function of torsinA in these neurons and its contribution to the motor symptoms is not clear. Here, D2R-expressing-cell-specific Dyt1 conditional knockout (d2KO) mice were generated and in vivo effects of torsinA loss in the corresponding cells were examined. The Dyt1 d2KO mice showed significant reductions of striatal torsinA, acetylcholine metabolic enzymes, Tropomyosin receptor kinase A (TrkA), and cholinergic interneurons. The Dyt1 d2KO mice also showed significant reductions of striatal D2R dimers and tyrosine hydroxylase without significant alteration in striatal monoamine contents or the number of dopaminergic neurons in the substantia nigra. The Dyt1 d2KO male mice showed motor deficits in the accelerated rotarod and beam-walking tests without overt dystonic symptoms. Moreover, the Dyt1 d2KO male mice showed significant correlations between striatal monoamines and locomotion. The results suggest that torsinA in the D2R-expressing cells play a critical role in the development or survival of the striatal cholinergic interneurons, expression of striatal D2R mature form, and motor performance. Medical interventions to compensate for the loss of torsinA function in these neurons may affect the onset and symptoms of this disease.

Subtle changes in striatal muscarinic M1 and M4 receptor expression in the DYT1 knock-in mouse model of dystonia.

In early-onset generalized torsion dystonia, caused by a GAG deletion in TOR1A (DYT1), enhanced striatal cholinergic activity has been suggested to be critically involved. Previous studies have shown increased acetylcholine levels in the striatum of DYT1 knock-in (KI) mice. Ex vivo data indicated that muscarinic receptor antagonists normalize the activity of striatal cholinergic interneurons. Currently receptor subtype specific antagonists are developed for therapy, however, it is yet unknown whether the levels of targeted receptors are unaltered. In the present study, we firstly examined the expression of M1 and M4 receptors in DYT1 KI mice in comparison to wildtype mice. While no changes in mRNA were found in the motor cortex, the expression of M1 was higher in the striatum of DYT1 KI. However, M1 protein did not differ in striatum and cortex between the animal groups as shown by immunohistochemistry and western blot. M4 receptor protein, unaltered in the cortex, was slightly lower in lateral subparts of the striatum, but unchanged in somata of cholinergic interneurons and substance P immunoreactive projection neurons. Functional alterations of the cholinergic system and of aberrant striatal plasticity, demonstrated by previous studies, seem not to be related to overt changes in M1 and M4 expression. This critically informs the ongoing development of respective antagonists for therapy of dystonia.

Perturbed Ca2+-dependent signaling of DYT2 hippocalcin mutant as mechanism of autosomal recessive dystonia.

A recent report of autosomal-recessive primary isolated dystonia (DYT2 dystonia) identified mutations in HPCA, a gene encoding a neuronal calcium sensor protein, hippocalcin (HPCA), as the cause of this disease. However, how mutant HPCA leads to neuronal dysfunction remains unknown. Using a multidisciplinary approach, we demonstrated the failure of dystonic N75K HPCA mutant to decode short bursts of action potentials and theta rhythms in hippocampal neurons by its Ca2+-dependent translocation to the plasma membrane. This translocation suppresses neuronal activity via slow afterhyperpolarization (sAHP) and we found that the N75K mutant could not control sAHP during physiologically relevant neuronal activation. Simulations based on the obtained experimental results directly demonstrated an increased excitability in neurons expressing N75K mutant instead of wild type (WT) HPCA. In conclusion, our study identifies sAHP as a downstream cellular target perturbed by N75K mutation in DYT2 dystonia, demonstrates its impact on neuronal excitability, and suggests a potential therapeutic strategy to efficiently treat DYT2.

Loss of the dystonia gene Thap1 leads to transcriptional deficits that converge on common pathogenic pathways in dystonic syndromes.

Dystonia is a movement disorder characterized by involuntary and repetitive co-contractions of agonist and antagonist muscles. Dystonia 6 (DYT6) is an autosomal dominant dystonia caused by loss-of-function mutations in the zinc finger transcription factor THAP1. We have generated Thap1 knock-out mice with a view to understanding its transcriptional role. While germ-line deletion of Thap1 is embryonic lethal, mice lacking one Thap1 allele-which in principle should recapitulate the haploinsufficiency of the human syndrome-do not show a discernable phenotype. This is because mice show autoregulation of Thap1 mRNA levels with upregulation at the non-affected locus. We then deleted Thap1 in glial and neuronal precursors using a nestin-conditional approach. Although these mice do not exhibit dystonia, they show pronounced locomotor deficits reflecting derangements in the cerebellar and basal ganglia circuitry. These behavioral features are associated with alterations in the expression of genes involved in nervous system development, synaptic transmission, cytoskeleton, gliosis and dopamine signaling that link DYT6 to other primary and secondary dystonic syndromes.

Hypertrophy of nigral neurons in Torsin1A deletion (DYT1) carriers manifesting dystonia.

OBJECTIVE: To individuate morphometric changes and prevalent types of intraneuronal inclusions in nigral neurons of DYT1 dystonia autopsy-brains. METHODS: Using precise methods of quantification, such as unbiased stereology, we measured cellular and subcellular volumes of neuromelanin-containing (pigmented) neurons in the substantia nigra (SN) of DYT1 carriers with and without manifestation of generalized dystonia (manif-DYT1 and non-manif-DYT1, respectively), non-DYT1 carriers manifesting generalized dystonia (manif-non-DYT1) patients, and age-matched control subjects (controls). A total of four DYT1 carriers (two manif-DYT1 and two non-manif-DYT1), six manif-non-DYT1 carriers, and six controls autopsy-brains were available for these neuropathological-morphometric analyses. The search of brain lesions was performed for: tau neurofibrillary tangles and neurites, extracellular ß-amyloid deposits, Lewy bodies and neurites, TorsinA, Laminin A + C, Ubiquitin, p62, pTDP43 intraneuronal inclusions; and Negri, Bunina, Hirano, Marinesco, Nissl, and Buscaino bodies. RESULTS: An increased mean cell body, nuclear, and nucleolar volume of nigral neurons in manif-DYT1 vs. non-manif-DYT1 (p < 0.0001), manif-non-DYT1 (p < 0.0001), and controls (p < 0.00001) was found. Increased nuclear and nucleolar volumes in manif-non-DYT1 vs. controls were also found. None of the considered possible intraneuronal lesions were more frequent or prevalent in nigral neurons of manif-DYT1 vs. all the other groups. CONCLUSIONS: Unbiased stereology-based measurements of nigral neurons enlargement in manif-DYT1 in the absence of intraneuronal inclusions or neurodegenerative processes, is novel. These findings suggest distinct pathogenetic mechanisms between manif-DYT1 vs. non-manif-DYT1 and manif-non-DYT1 dystonia, especially in terms of possible nigral dopaminergic abnormalities. These data could open new pathophysiologic views on specific dopamino-associated pathomechanisms related to the clinical manifestation of generalized dystonia.

Efficient RNA interference-based knockdown of mutant torsinA reveals reversibility of PERK-eIF2α pathway dysregulation in DYT1 transgenic rats in vivo.

DYT1 dystonia is a neurological disease caused by a dominant mutation that results in the loss of a glutamic acid in the endoplasmic reticulum-resident protein torsinA. Currently, treatments are symptomatic and only provide partial relief. Multiple reports support the hypothesis that selectively reducing expression of mutant torsinA without affecting levels of the wild type protein should be beneficial. Published cell-based studies support this hypothesis. It is unclear, however, if phenotypes are reversible by targeting the molecular defect once established in vivo. Here, we generated adeno-associated virus encoding artificial microRNA targeting human mutant torsinA and delivered them to the striatum of symptomatic transgenic rats that express the full human TOR1A mutant gene. We achieved efficient suppression of human mutant torsinA expression in DYT1 transgenic rats, partly reversing its accumulation in the nuclear envelope. This intervention rescued PERK-eIF2α pathway dysregulation in striatal projection neurons but not behavioral abnormalities. Moreover, we found abnormal expression of components of dopaminergic neurotransmission in DYT1 rat striatum, which were not normalized by suppressing mutant torsinA expression. Our findings demonstrate the reversibility of translational dysregulation in DYT1 neurons and confirm the presence of abnormal dopaminergic neurotransmission in DYT1 dystonia.

The mutation responsible for torsion dystonia type 1 shows the ability to stimulate intracellular aggregation of mutant huntingtin.

OBJECTIVE: Introduction: Torsion dystonia type 1 is the most common form of early-onset primary dystonia. Previous reports have suggested that torsin 1A, a protein mutated in this disease, might function as a chaperone that prevents the toxic aggregation of misfolded polypeptides. The aim of the study: The aim of this study was to verify the chaperone function of torsin 1A by investigating its ability to prevent the aggregation of huntingtin model peptides. PATIENTS AND METHODS: Materials and methods: N-terminal mutant huntingtin fragments of different length were co-expressed in neuronal HT-22 and non-neuronal HeLa cells with either the wild-type or mutant (ΔE302/303) torsin 1A protein. The transfected cells were immunostained and analyzed for the presence of huntingtin aggregates using fluorescence microscopy. RESULTS: Results: The immunofluorescence analysis of huntingtin subcellular distribution within the transfected cells showed no significant difference between the huntingtin aggregation levels in cells co-expressing the wild-type torsin 1A and in control cells co-transfected with an empty vector. Instead, it was the increased level of huntingtin aggregation in the presence of the torsion dystonia-causing ΔE302/303 mutant that reached statistical significance in both neuronal and non-neuronal cells. CONCLUSION: Conclusions: Either torsin 1A does not function as a chaperone protein or huntingtin is not an efficient substrate for such a hypothetical chaperone activity. However, the ability of mutant torsin 1A to stimulate the accumulation of aggregation-prone polypeptides might constitute an important source of ΔE302/303 pathogenicity and thus a potential target for future therapy.