A novel conditional knock-in approach defines molecular and circuit effects of the DYT1 dystonia mutation.

DYT1 dystonia, the most common inherited form of primary dystonia, is a neurodevelopmental disease caused by a dominant mutation in TOR1A. This mutation ('ΔE') removes a single glutamic acid from the encoded protein, torsinA. The effects of this mutation, at the molecular and circuit levels, and the reasons for its neurodevelopmental onset, remain incompletely understood. To uniquely address key questions of disease pathogenesis, we generated a conditional Tor1a knock-in allele that is converted from wild-type to DYT1 mutant ('induced' ΔE: Tor1a(i-ΔE)), following Cre recombination. We used this model to perform a gene dosage study exploring the effects of the ΔE mutation at the molecular, neuropathological and organismal levels. These analyses demonstrated that ΔE-torsinA is a hypomorphic allele and showed no evidence for any gain-of-function toxic properties. The unique capabilities of this model also enabled us to test a circuit-level hypothesis of DYT1 dystonia, which predicts that expression of the DYT1 genotype (Tor1a(ΔE/+)) selectively within hindbrain structures will produce an overtly dystonic animal. In contrast to this prediction, we find no effect of this anatomic-specific expression of the DYT1 genotype, a finding that has important implications for the interpretation of the human and mouse diffusion tensor-imaging studies upon which it is based. These studies advance understanding of the molecular effects of the ΔE mutation, challenge current concepts of the circuit dysfunction that characterize the disease and establish a powerful tool that will be valuable for future studies of disease pathophysiology.

Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.

NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones.

Inherited dystonias: clinical features and molecular pathways.

Recent decades have witnessed dramatic increases in understanding of the genetics of dystonia - a movement disorder characterized by involuntary twisting and abnormal posture. Hampered by a lack of overt neuropathology, researchers are investigating isolated monogenic causes to pinpoint common molecular mechanisms in this heterogeneous disease. Evidence from imaging, cellular, and murine work implicates deficiencies in dopamine neurotransmission, transcriptional dysregulation, and selective vulnerability of distinct neuronal populations to disease mutations. Studies of genetic forms of dystonia are also illuminating the developmental dependence of disease symptoms that is typical of many forms of the disease. As understanding of monogenic forms of dystonia grows, a clearer picture will develop of the abnormal motor circuitry behind this relatively common phenomenology. This chapter focuses on the current data covering the etiology and epidemiology, clinical presentation, and pathogenesis of four monogenic forms of isolated dystonia: DYT-TOR1A, DYT-THAP1, DYT-GCH1, and DYT-GNAL.