Motor deficits and hyperactivity in cerebral cortex-specific Dyt1 conditional knockout mice

DYT1 dystonia is a primary generalized early-onset torsion dystonia caused by mutations in DYT1 that codes for torsinA and has an autosomal dominant inheritance pattern with approximately 30% penetrance. Abnormal activity in the pallidal-thalamic-cortical circuit, especially in the globus pallidus internus, is the proposed cause of dystonic symptoms. However, recent neuroimaging studies suggest significant contribution of the cerebral cortex. To understand the contribution of the cerebral cortex to dystonia, we produced cerebral cortex-specific Dyt1 conditional knockout mice and analysed their behaviour. The conditional knockout mice exhibited motor deficits and hyperactivity that mimic the reported behavioural deficits in Dyt1 DeltaGAG knockin heterozygous and Dyt1 knockdown mice. Although the latter two mice exhibit lower levels of dopamine metabolites in the striatum, the conditional knockout mice did not show significant alterations in the striatal dopamine and its metabolites levels. The conditional knockout mice had well-developed whisker-related patterns in somatosensory cortex, suggesting formations of synapses and neural circuits were largely unaffected. The results suggest that the loss of torsinA function in the cerebral cortex alone is sufficient to induce behavioural deficits associated with Dyt1 DeltaGAG knockin mutation. Developing drugs targeting the cerebral cortex may produce novel medical treatments for DYT1 dystonia patients.     

Behavioral and Electrophysiological Characterization of Dyt1 Heterozygous Knockout Mice

DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinA^ΔE). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinA^ΔE does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia.     

Pre-Synaptic Release Deficits in a DYT1 Dystonia Mouse Model

DYT1 early-onset generalized torsion dystonia (DYT1 dystonia) is an inherited movement disorder caused by mutations in one allele of DYT1 (TOR1A), coding for torsinA. The most common mutation is a trinucleotide deletion (ΔGAG), which causes a deletion of a glutamic acid residue (ΔE) in the C-terminal region of torsinA. Although recent studies using cultured cells suggest that torsinA contributes to protein processing in the secretory pathway, endocytosis, and the stability of synaptic proteins, the nature of how this mutation affects synaptic transmission remains unclear. We previously reported that theta-burst-induced long-term potentiation (LTP) in the CA1 region of the hippocampal slice is not altered in Dyt1 ΔGAG heterozygous knock-in (KI) mice. Here, we examined short-term synaptic plasticity and synaptic transmission in the hippocampal slices. Field recordings in the hippocampal Schaffer collaterals (SC) pathway revealed significantly enhanced paired pulse ratios (PPRs) in Dyt1 ΔGAG heterozygous KI mice, suggesting an impaired synaptic vesicle release. Whole-cell recordings from the CA1 neurons showed that Dyt1 ΔGAG heterozygous KI mice exhibited normal miniature excitatory post-synaptic currents (mEPSC), suggesting that action-potential independent spontaneous pre-synaptic release was normal. On the other hand, there was a significant decrease in the frequency, but not amplitude or kinetics, of spontaneous excitatory post-synaptic currents (sEPSC) in Dyt1 ΔGAG heterozygous KI mice, suggesting that the action-potential dependent pre-synaptic release was impaired. Moreover, hippocampal torsinA was significantly reduced in Dyt1 ΔGAG heterozygous KI mice. Although the hippocampal slice model may not represent the neurons directly associated with dystonic symptoms, impaired release of neurotransmitters caused by partial dysfunction of torsinA in other brain regions may contribute to the pathophysiology of DYT1 dystonia.     

Dopamine Receptor and Gα(olf) Expression in DYT1 Dystonia Mouse Models during Postnatal Development

Background

DYT1 dystonia is a heritable, early-onset generalized movement disorder caused by a GAG deletion (ΔGAG) in the DYT1 gene. Neuroimaging studies and studies using mouse models suggest that DYT1 dystonia is associated with dopamine imbalance. However, whether dopamine imbalance is key to DYT1 or other forms of dystonia continues to be debated.

Methodology/Principal Findings

We used Dyt1 knock out (Dyt1 KO), Dyt1 ΔGAG knock-in (Dyt1 KI), and transgenic mice carrying one copy of the human DYT1 wild type allele (DYT1 hWT) or human ΔGAG mutant allele (DYT1 hMT). D1R, D2R, and Gα(olf) protein expression was analyzed by western blot in the frontal cortex, caudate-putamen and ventral midbrain in young adult (postnatal day 60; P60) male mice from all four lines; and in the frontal cortex and caudate putamen in juvenile (postnatal day 14; P14) male mice from the Dyt1 KI and KO lines. Dopamine receptor and Gα(olf) protein expression were significantly decreased in multiple brain regions of Dyt1 KI and Dyt1 KO mice and not significantly altered in the DYT1 hMT or DYT1 hWT mice at P60. The only significant change at P14 was a decrease in D1R expression in the caudate-putamen of the Dyt1 KO mice.

Conclusion/Significance

We found significant decreases in key proteins in the dopaminergic system in multiple brain regions of Dyt1 KO and Dyt1 KI mouse lines at P60. Deletion of one copy of the Dyt1 gene (KO mice) produced the most pronounced effects. These data offer evidence that impaired dopamine receptor signaling may be an early and significant contributor to DYT1 dystonia pathophysiology.     

DYT1 Knock-In Mice Are Not Sensitized against Mitochondrial Complex-II Inhibition

DYT1 is caused by a partly penetrant dominant mutation in TOR1A that leads to a glutamic acid deletion (ΔE) in torsinA. Identifying environmental factors that modulate disease pathogenesis and penetrance could help design therapeutic strategies for dystonia. Several cell-based studies suggest that expression of torsinA(ΔE) increases the susceptibility of neuronal cells to challenges to their oxidative/energy metabolism. Based on those reports, we hypothesized that mice expressing torsinA(ΔE) would be more susceptible than control littermates to the effects of oxidative stress and ATP deficits caused by disruption of the mitochondrial respiratory chain in neurons. To test this hypothesis, we administered 20 or 50 mg/kg/day of the irreversible complex-II inhibitor 3-nitropropionic acid (3-NP) intraperitoneally for 15 consecutive days to young heterozygote DYT1 knock-in (KI) mice and wild type littermates. Repeated phenotypic assessments were performed at baseline, during and after the injections. Animals were then sacrificed and their brains processed for protein analysis. The administration of 20 mg/kg 3-NP led to increased levels of torsinA in the striatum, the main target of 3-NP, but did not cause motor dysfunction in DYT1 KI or control mice. The administration of 50 mg/kg/day of 3-NP caused the death of ～40% of wild type animals. Interestingly, DYT1 KI animals showed significantly reduced mortality. Surviving animals exhibited abnormal motor behavior during and right after the injection period, but recovered by 4 weeks postinjection independent of genotype. In contrast to the findings reported in cultured cells, these studies suggest the DYT1 mutation does not sensitize central neurons against the toxic effects of oxidative stress and energy deficits.     

Dopamine release is impaired in a mouse model of DYT1 dystonia

Early onset torsion dystonia, the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein torsinA. This form of dystonia is referred to as DYT1. We have used a transgenic mouse model of DYT1 dystonia [human mutant-type (hMT)1 mice] to examine the effect of the mutant human torsinA protein on striatal dopaminergic function. Analysis of striatal tissue dopamine (DA) and metabolites using HPLC revealed no difference between hMT1 mice and their non-transgenic littermates. Pre-synaptic DA transporters were studied using in vitro autoradiography with [(3)H]mazindol, a ligand for the membrane DA transporter, and [(3)H]dihydrotetrabenazine, a ligand for the vesicular monoamine transporter. No difference in the density of striatal DA transporter or vesicular monoamine transporter binding sites was observed. Post-synaptic receptors were studied using [(3)H]SCH-23390, a ligand for D(1) class receptors, [(3)H]YM-09151-2 and a ligand for D(2) class receptors. There were again no differences in the density of striatal binding sites for these ligands. Using in vivo microdialysis in awake animals, we studied basal as well as amphetamine-stimulated striatal extracellular DA levels. Basal extracellular DA levels were similar, but the response to amphetamine was markedly attenuated in the hMT1 mice compared with their non-transgenic littermates (253 +/- 71% vs. 561 +/- 132%, p < 0.05, two-way anova). These observations suggest that the mutation in the torsinA protein responsible for DYT1 dystonia may interfere with transport or release of DA, but does not alter pre-synaptic transporters or post-synaptic DA receptors. The defect in DA release as observed may contribute to the abnormalities in motor learning as previously documented in this transgenic mouse model, and may contribute to the clinical symptoms of the human disorder.     

Overexpression of torsinA in PC12 cells protects against toxicity

Childhood-onset dystonia is an autosomal dominant movement disorder associated with a three base pair (GAG) deletion mutation in the DYT1 gene. This gene encodes a novel ATP-binding protein called torsinA, which in the central nervous system is expressed exclusively in neurons. Neither the function of torsinA nor its role in the pathophysiology of DYT1 dystonia is known. In order to better understand the cellular functions of torsinA, we established PC12 cell lines overexpressing wild-type or mutant torsinA and subjected them to various conditions deleterious to cell survival. Treatment of control PC12 cells with an inhibitor of proteasomal activity, an oxidizing agent, or trophic withdrawal, resulted in cell death, whereas PC12 cells that overexpressed torsinA were significantly protected against each of these treatments. Overexpression of mutant torsinA failed to protect cells against trophic withdrawal. These results suggest that torsinA may play a protective role in neurons against a variety of cellular insults.     

Loss of the dystonia-associated protein torsinA selectively disrupts the neuronal nuclear envelope

An enigmatic feature of many genetic diseases is that mutations in widely expressed genes cause tissue-specific illness. One example is DYT1 dystonia, a neurodevelopmental disease caused by an in-frame deletion (Deltagag) in the gene encoding torsinA. Here we show that neurons from both torsinA null (Tor1a(-/-)) and homozygous disease mutant "knockin" mice (Tor1a(Deltagag/Deltagag)) contain severely abnormal nuclear membranes, although non-neuronal cell types appear normal. These membrane abnormalities develop in postmigratory embryonic neurons and subsequently worsen with further neuronal maturation, a finding evocative of the developmental dependence of DYT1 dystonia. These observations demonstrate that neurons have a unique requirement for nuclear envelope localized torsinA function and suggest that loss of this activity is a key molecular event in the pathogenesis of DYT1 dystonia.     

Minimal Change in the Cytoplasmic Calcium Dynamics in Striatal GABAergic Neurons of a DYT1 Dystonia Knock-In Mouse Model

DYT1 dystonia is the most common hereditary form of primary torsion dystonia. This autosomal-dominant disorder is characterized by involuntary muscle contractions that cause sustained twisting and repetitive movements. It is caused by an in-frame deletion in the TOR1A gene, leading to the deletion of a glutamic acid residue in the torsinA protein. Heterozygous knock-in mice, which reproduce the genetic mutation in human patients, have abnormalities in synaptic transmission at the principal GABAergic neurons in the striatum, a brain structure that is involved in the execution and modulation of motor activity. However, whether this mutation affects the excitability of striatal GABAergic neurons has not been investigated in this animal model. Here, we examined the excitability of cultured striatal neurons obtained from heterozygous knock-in mice, using calcium imaging as indirect readout. Immunofluorescence revealed that more than 97% of these neurons are positive for a marker of GABAergic neurons, and that more than 92% are also positive for a marker of medium spiny neurons, indicating that these are mixed cultures of mostly medium spiny neurons and a few (~5%) GABAergic interneurons. When these neurons were depolarized by field stimulation, the calcium concentration in the dendrites increased rapidly and then decayed slowly. The amplitudes of calcium transients were larger in heterozygous neurons than in wild-type neurons, resulting in ~15% increase in cumulative calcium transients during a train of stimuli. However, there was no change in other parameters of calcium dynamics. Given that calcium dynamics reflect neuronal excitability, these results suggest that the mutation only slightly increases the excitability of striatal GABAergic neurons in DYT1 dystonia.     

Alteration of striatal dopaminergic neurotransmission in a mouse model of DYT11 myoclonus-dystonia

Background

DYT11 myoclonus-dystonia (M-D) syndrome is a neurological movement disorder characterized by myoclonic jerks and dystonic postures or movement that can be alleviated by alcohol. It is caused by mutations in SGCE encoding ε-sarcoglycan (ε-SG); the mouse homolog of this gene is Sgce. Paternally-inherited Sgce heterozygous knockout (Sgce KO) mice exhibit myoclonus, motor impairment and anxiety- and depression-like behaviors, modeling several clinical symptoms observed in DYT11 M-D patients. The behavioral deficits are accompanied by abnormally high levels of dopamine and its metabolites in the striatum of Sgce KO mice. Neuroimaging studies of DYT11 M-D patients show reduced dopamine D2 receptor (D2R) availability, although the possibility of increased endogenous dopamine, and consequently, competitive D2R occupancy cannot be ruled out.

Methodology/Principal Findings

The protein levels of striatal D2R, dopamine transporter (DAT), and dopamine D1 receptor (D1R) in Sgce KO mice were analyzed by Western blot. The striatal dopamine release after amphetamine injection in Sgce KO mice were analyzed by microdialysis in vivo. The striatal D2R was significantly decreased in Sgce KO mice without altering DAT and D1R. Sgce KO mice also exhibited a significant increase of dopamine release after amphetamine injection in comparison to wild-type (WT) littermates.

Conclusion/Significance

The results suggest ε-SG may have a role in the regulation of D2R expression. The loss of ε-SG results in decreased striatal D2R, and subsequently leads to increased discharge of dopamine which could contribute to the behavioral impairment observed in DYT11 dystonia patients and in Sgce KO mice. The results suggest that reduction of striatal D2R and enhanced striatal dopamine release may contribute to the pathophysiology of DYT11 M-D patients.     

Genetic background modulates the phenotype of a mouse model of DYT1 dystonia

DYT1 dystonia is a debilitating neurological disease characterized by involuntary twisting movements. The disease is caused by an in-frame deletion (GAG, "ΔE") mutation in the TOR1A gene that encodes the torsinA protein. Intriguingly, only 30% of mutation carriers exhibit motor symptoms despite the fact that functional brain imaging studies show abnormal brain metabolism in all carriers. Because genetic modifiers may be a determinant of this reduced penetrance, we examined the genetic contribution of three different inbred strains of mice on the DYT1 mutation in animals that are homozygous (Tor1a(ΔE/ΔE)) or heterozygous (Tor1a(ΔE/+); disease state) for the disease-causing ΔE mutation. We find that the DBA/2J, C57BL/6J, and CD1-ICR contribution of genes significantly alter lifespan in Tor1a(ΔE/ΔE) mice, which die during the first few days of life on the 129S6/SvEvTac (129) background. The C57BL/6J (B6) strain significantly decreases life expectancy of Tor1a(ΔE/ΔE) animals but, like 129S6/SvEvTac Tor1a(ΔE/+) mice, congenic C57BL/6J Tor1a(ΔE/+) mice do not exhibit any motor abnormalities. In contrast, the DBA/2J (D2) strain significantly increases life expectancy. This effect was not present in congenic DBA/2J Tor1a(ΔE/ΔE) mice, indicating that the extended lifespan of F2 129/D2 mice was due to a combination of homozygous and heterozygous allelic effects. Our observations suggest that genetic modifiers may alter the penetrance of the ΔE mutation, and that mapping these modifiers may provide fresh insight into the torsinA molecular pathway.     

Developmental profile of the aberrant dopamine D2 receptor response in striatal cholinergic interneurons in DYT1 dystonia

Background

DYT1 dystonia, a severe form of genetically determined human dystonia, exhibits reduced penetrance among carriers and begins usually during adolescence. The reasons for such age dependence and variability remain unclear.

Methods and Results

We characterized the alterations in D2 dopamine receptor (D2R) signalling in striatal cholinergic interneurons at different ages in mice overexpressing human mutant torsinA (hMT). An abnormal excitatory response to the D2R agonist quinpirole was recorded at postnatal day 14, consisting of a membrane depolarization coupled to an increase in spiking frequency, and persisted unchanged at 3 and 9 months in hMT mice, compared to mice expressing wild-type human torsinA and non-transgenic mice. This response was blocked by the D2R antagonist sulpiride and depended upon G-proteins, as it was prevented by intrapipette GDP-β-S. Patch-clamp recordings from dissociated interneurons revealed a significant increase in the Cav2.2-mediated current fraction at all ages examined. Consistently, chelation of intracellular calcium abolished the paradoxical response to quinpirole. Finally, no gross morphological changes were observed during development.

Conclusions

These results suggest that an imbalanced striatal dopaminergic/cholinergic signaling occurs early in DYT1 dystonia and persists along development, representing a susceptibility factor for symptom generation.     

4-Phenylbutyrate Attenuates the ER Stress Response and Cyclic AMP Accumulation in DYT1 Dystonia Cell Models

Dystonia is a neurological disorder in which sustained muscle contractions induce twisting and repetitive movements or abnormal posturing. DYT1 early-onset primary dystonia is the most common form of hereditary dystonia and is caused by deletion of a glutamic acid residue (302/303) near the carboxyl-terminus of encoded torsinA. TorsinA is localized primarily within the contiguous lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE), and is hypothesized to function as a molecular chaperone and an important regulator of the ER stress-signaling pathway, but how the mutation in torsinA causes disease remains unclear. Multiple lines of evidence suggest that the clinical symptoms of dystonia result from abnormalities in dopamine (DA) signaling, and possibly involving its down-stream effector adenylate cyclase that produces the second messenger cyclic adenosine-3′, 5′-monophosphate (cAMP). Here we find that mutation in torsinA induces ER stress, and inhibits the cyclic adenosine-3′, 5′-monophosphate (cAMP) response to the adenylate cyclase agonist forskolin. Both defective mechanins are corrected by the small molecule 4-phenylbutyrate (4-PBA) that alleviates ER stress. Our results link torsinA, the ER-stress-response, and cAMP-dependent signaling, and suggest 4-PBA could also be used in dystonia treatment. Other pharmacological agents known to modulate the cAMP cascade, and ER stress may also be therapeutic in dystonia patients and can be tested in the models described here, thus supplementing current efforts centered on the dopamine pathway.     

TorsinA protein degradation and autophagy in DYT1 dystonia

Early-onset generalized dystonia (DYT1) is a debilitating neurological disorder characterized by involuntary movements and sustained muscle spasms. DYT1 dystonia has been associated with two mutations in torsinA that result in the deletion of a single glutamate residue (torsinA DeltaE) and six amino-acid residues (torsinA Delta323-8). We recently revealed that torsinA, a peripheral membrane protein, which resides predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE), is a long-lived protein whose turnover is mediated by basal autophagy. Dystonia-associated torsinA DeltaE and torsinA Delta323-8 mutant proteins show enhanced retention in the NE and accelerated degradation by both the proteasome and autophagy. Our results raise the possibility that the monomeric form of torsinA mutant proteins is cleared by proteasome-mediated ER-associated degradation (ERAD), whereas the oligomeric and aggregated forms of torsinA mutant proteins are cleared by ER stress-induced autophagy. Our findings provide new insights into the pathogenic mechanism of torsinA DeltaE and torsinA Delta323-8 mutations in dystonia and emphasize the need for a mechanistic understanding of the role of autophagy in protein quality control in the ER and NE compartments.     

The AAA+ protein torsinA interacts with a conserved domain present in LAP1 and a novel ER protein

A glutamic acid deletion (DeltaE) in the AAA+ protein torsinA causes DYT1 dystonia. Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the DeltaE mutation enhances this interaction. Using a novel cell-based screen, we identify lamina-associated polypeptide 1 (LAP1) as a torsinA-interacting protein. LAP1 may be a torsinA substrate, as expression of the isolated lumenal domain of LAP1 inhibits the NE localization of "substrate trap" EQ-torsinA and EQ-torsinA coimmunoprecipitates with LAP1 to a greater extent than wild-type torsinA. Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA. Interestingly, LULL1 resides in the main ER. Consequently, torsinA interacts directly or indirectly with a novel class of transmembrane proteins that are localized in different subdomains of the ER system, either or both of which may play a role in the pathogenesis of DYT1 dystonia.     

TorsinA and dystonia: from nuclear envelope to synapse

A GAG deletion in the DYT1 gene is responsible for the autosomal dominant movement disorder, early onset primary torsion dystonia, which is characterised by involuntary sustained muscle contractions and abnormal posturing of the limbs. The mutation leads to deletion of a single glutamate residue in the C-terminus of the protein torsinA, a member of the AAA+ ATPase family of proteins with multiple functions. Since no evidence of neurodegeneration has been found in DYT1 patients, the dystonic phenotype is likely to be the result of neuronal functional defect(s), the nature of which is only partially understood. Biochemical, structural and cell biological studies have been performed in order to characterise torsinA. These studies, together with the generation of several animal models, have contributed to identify cellular compartments and pathways, including the cytoskeleton and the nuclear envelope, the secretory pathway and the synaptic vesicle machinery where torsinA function may be crucial. However, the role of torsinA and the correlation between the dysfunction caused by the mutation and the dystonic phenotype remain unclear. This review provides an overview of the findings of the last ten years of research on torsinA, a critical evaluation of the different models proposed and insights towards future avenues of research.     

Dtorsin, the Drosophila ortholog of the early-onset dystonia TOR1A (DYT1), plays a novel role in dopamine metabolism

Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.     

Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons

Septin 5, a parkin substrate, is a vesicle- and membrane-associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinson's disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron-specific toxicity in SN-DAergic neurons. In vitro and in vivo models were used to support this hypothesis. In our well-characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin-1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co-transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN-DAergic neurons. These mice acquired a progressive l-DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that l-DOPA-responsive motor deficits can occur even in the presence of elevated DA.     

Evaluation of TorsinA as a Target for Parkinson Disease Therapy in Mouse Models

Parkinson disease (PD) is a common and disabling disorder. No current therapy can slow or reverse disease progression. An important aspect of research in this field is target validation, a systematic approach to evaluating the likelihood that modification of a certain molecule, mechanism or biological pathway may be useful for the development of pharmacological or molecular treatments for the disease. TorsinA, a member of the AAA+ family of chaperone proteins, has been proposed as a potential target of neuroprotective therapy. TorsinA is found in Lewy bodies in human PD, and can suppress toxicity in cellular and invertebrate models of PD. Here, we evaluated the neuroprotective properties of torsinA in mouse models of PD based on intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as well as recombinant adeno associated virus (rAAV) induced overexpression of alpha-synuclein (α-syn). Using either transgenic mice with overexpression of human torsinA (hWT mice) or mice in which torsinA expression was induced using an rAAV vector, we found no evidence for protection against acute MPTP intoxication. Similarly, genetic deletion of the endogenous mouse gene for torsinA (Dyt1) using an rAAV delivered Cre recombinase did not enhance the vulnerability of dopaminergic neurons to MPTP. Overexpression of α-syn using rAAV in the mouse substantia nigra lead to a loss of TH positive neurons six months after administration, and no difference in the degree of loss was observed between transgenic animals expressing forms of torsinA and wild type controls. Collectively, we did not observe evidence for a protective effect of torsinA in the mouse models we examined. Each of these models has limitations, and there is no single model with established predictive value with respect to the human disease. Nevertheless, these data do seem to support the view that torsinA is unlikely to be successfully translated as a target of therapy for human PD.