Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.

Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease1 and is also linked to its idiopathic form2. LRRK2 has been proposed to function in membrane trafficking3 and colocalizes with microtubules4. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson's disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure5. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold.

Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted with spatial and temporal precision. To investigate the mechanisms that coordinate motor ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical-polarity motors, motor number had minimal affect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.

Structural basis for microtubule binding and release by dynein.

Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nanometers separating dynein's track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein's microtubule-binding domain bound to microtubules by cryo-electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein's affinity for microtubules. Our results provide a molecular model for how dynein's binding to microtubules is communicated to the rest of the motor.

Biocompatible semiconductor optoelectronics.

We investigate optoelectronic properties of integrated structures comprising semiconductor light-emitting materials for optical probes of microscopic biological systems. Compound semiconductors are nearly ideal light emitters for probing cells and other microorganisms because of their spectral match to the transparency wavelengths of biomolecules. Unfortunately, the chemical composition of these materials is incompatible with the biochemistry of cells and related biofluids. To overcome these limitations, we investigate functionalized semiconductor surfaces and structures to simultaneously enhance light emission and the flow of biological fluids in semiconductor microcavities. We have identified several important materials problems associated with the semiconductor/biosystem interface. One is the biofluid degradation of electroluminescence by ionic diffusion into compound semiconductors. Ions that diffuse into the active region of a semiconductor light emitter can create point defects that degrade the quantum efficiency of the radiative recombination process. In this paper we discuss ways of mitigating these problems using materials design and surface chemistry, and suggest future applications for these materials.

The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors.

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.

Focal microinjection of carbachol into the periaqueductal gray induces seizures in the forebrain of the rat.

Previous studies have reported that the repetition of running-bouncing and tonic-clonic seizures mediated by brainstem structures eventually elicits seizure activity in the forebrain. The purpose of the present study was to determine if the periaqueductal gray (PAG) region is a component of the neural network through which brainstem seizures elicit forebrain seizures. Bilateral microinjection of 40 nmol carbachol into the PAG region of rats induced arrested, staring behavior accompanied by epileptiform electrocorticogram (ECoG) afterdischarge recorded from the parietal cortex. In two animals limbic seizure activity similar to kindled amygdala seizures was also induced. The carbachol effect was dose-related as the 40 nmol dose induced a significantly greater duration of ECoG afterdischarge than a 20 nmol dose. The carbachol effect was mediated by muscarinic receptors as bilateral 50 nmol atropine microinjection 1 min prior to 40 nmol carbachol microinjection inhibited all seizure activity. Immunohistochemical detection of the proto-oncogene c-fos was used to verify that seizure activity was induced in forebrain regions. Rats with seizures induced by PAG carbachol microinjections exhibited dense c-fos-like immunoreactivity in the dentate gyrus but not the CA(1) or CA(3) regions, amygdala, piriform cortex, perirhinal cortex or hypothalamus. In addition, PAG microinjection of 10 nmol N-methyl-D-aspartic acid (NMDA) induced wild-running convulsions while 400 pmol bicuculline induced clonic spasms, myoclonic activity or limbic seizures. These results indicate that stimulation of the PAG, a brainstem structure, is sufficient to induce forebrain seizures. Since the forebrain seizures were induced by a single carbachol administration, it is proposed that the PAG serves as a pathway for caudal-rostral seizure generalization.

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae.

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.

Seizures induced by theophylline and isoniazid in mice.

Isoniazid-induced seizures respond poorly to anticonvulsants but well to pyridoxine (Vitamin B6); theophylline produces difficult-to-treat seizures with substantial morbidity and mortality. Theophylline therapy depresses plasma pyridoxal-5'-phosphate (PLP), the active metabolite of pyridoxine, suggesting that theophylline-induced seizures might be amenable to treatment with pyridoxine. Our study established the dose-response relationship for convulsions due to isoniazid and theophylline in mice and determined if pyridoxine antagonized such seizures. Female CD-1 outbred mice weighing 25 to 30 g were used. Clonic seizures had clonic activity lasting 5 sec; tonic seizures had loss of the righting reflex with tonic hindlimb extension. Groups of 10 mice received single doses of 50, 100, 150, 200, 250 or 300 mg aminophylline/kg i.p. or 100, 150, 200, 250, 300 or 350 mg isoniazid/kg i.p. and were observed for seizures or death. Pyridoxine or saline with aminophylline or isoniazid were administered simultaneously. The LD50 for aminophylline was 266 mg/kg; for isoniazid it was 160 mg/kg. Doses of 150 mg aminophylline/kg or 100 mg isoniazid/kg did not induce seizures. Pyridoxine with aminophylline or isoniazid did not alter the frequency or time of onset of seizures or death. This was unexpected because pyridoxine antagonizes theophylline-induced seizures in mice and reverses isoniazid-induced seizures in humans. We found no evidence that PLP depletion in mice is a mechanism for seizures induced by isoniazid or aminophylline in a fashion similar to isoniazid in humans.

The tail of a yeast class V myosin, myo2p, functions as a localization domain.

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN). Overexpression of the tail domain of Myo2p results in a dominant-negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2-66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth. Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 x g pellet. The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.

Muscarinic receptors mediate carbachol-induced inhibition of maximal electroshock seizures in the nucleus reticularis pontis oralis.

PURPOSE: Previous reports from this laboratory indicated a role for N-methyl-D-aspartic acid (NMDA) and gamma-aminobutyric acid (GABA) receptors among the neuronal mechanisms of the nucleus reticularis pontis oralis (RPO) that regulate the tonic hindlimb extension (THE) component of maximal electroshock seizures (MESs) in rats. This study was intended to determine the role of cholinergic mechanisms in the RPO regulation of THE. METHODS: Rats were surgically prepared with microinjection guide cannulas for the focal administration of drug solutions directly into the RPO. MES was induced with corneal electrodes. RESULTS: RPO microinjection of carbachol significantly inhibited the incidence of THE. RPO microinjection of atropine by itself had no effect on the seizure response but significantly antagonized the anticonvulsant effect induced by RPO microinjection of carbachol. The selective nicotinic agonist dimethylpiperizinium (DMPP) by itself had no effect on THE. RPO microinjection of 10 ng pertussis toxin by itself had no effect on THE but significantly antagonized the anticonvulsant effect induced by RPO microinjection of carbachol. CONCLUSIONS: RPO microinjection of carbachol inhibited the THE component of MESs in rats. The carbachol effect appeared to be mediated by muscarinic receptors as the anticonvulsant activity was antagonized by atropine, and the selective nicotinic agonist DMPP induced no anticonvulsant activity. Because pertussis toxin acts to inhibit muscarinic receptor-linked G proteins, the pertussis toxin antagonism of carbachol also supports a muscarinic mechanism of action.

Asymmetric synthesis of the balanol heterocycle via a palladium-mediated epimerization and olefin metathesis.

[formula: see text] The enantioselective formal synthesis of balanol, a potent protein kinase C inhibitor, was accomplished from D-serine utilizing a Pd-catalyzed equilibration of diastereomeric 5-vinyloxazolines to set the stereochemistry of the vicinal amino and hydroxyl groups. A ruthenium-catalyzed ring-closing metathesis was employed to form the seven-membered nitrogen heterocyle.

Neuronal degeneration in rat forebrain resulting from D-amphetamine-induced convulsions is dependent on seizure severity and age.

Neuronal damage and degeneration in the rat forebrain was characterized by B4 isolectin and Fluoro-Jade labeling techniques after 4 doses of 15 mg/kg amphetamine i.p. in 70- and 180-day-old Sprague-Dawley rats. In amphetamine-dosed rats some seizure activity occurred in all rats exhibiting pronounced hyperthermia but the degree of seizure activity varied greatly between individual rats. Over 90% of the rats in both age groups that showed behavioral signs of limbic seizures had somatic degeneration in the taenia tecta within 3 days of amphetamine exposure. Degenerating small star-shaped cells were seen in the septum and hippocampus in 70-day-old rats having extensive seizure activity. Although somatic degeneration only sporadically occurred in the piriform cortex of the younger rats, extensive B4 isolectin binding to activated microglia was observed in this area. In older rats prominent somatic degeneration was seen in the piriform cortex and orbital and insular areas of the frontal cortex of rats having seizures. Damage to the basal ganglia and related areas, including the thalamus, parietal cortex and dorsal medial striatum, occurred in rats with pronounced hyperthermia but only correlated with seizures in older rats. In the more severe cases of thalamic damage the highest density of neurodegeneration was localized perivascularly. Thus, amphetamine can produce notable damage to the limbic system when seizures occur and to the basal ganglia and related areas when hyperthermia occurs but the neurotoxicity profiles in these areas are age-dependent and not produced solely by hyperthermia. Further studies to determine whether neuronal damage is the result of or the cause of amphetamine-induced seizures are necessary.

Evaluating a more cost-efficient alternative to providing in-home feedback to parents: the use of spousal feedback.

We evaluated the contribution of spousal feedback to a parent education curriculum designed for parents of children with autism. A modified multiple baseline design across 3 husband-and-wife dyads was used to examine the effects of teaching parents to give each other feedback on their teaching performance. For 5 of 6 participants, improvement in teaching performance occurred following didactic presentations. However, additional improvement was observed for 5 participants when the spousal feedback component was implemented.

Leukocytes modulate 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in human granulosa-lutein cell cultures.

It is well established that there are interactions between the immune and reproductive systems. The ovary contains indigenous macrophages, as well as other classes of leukocytes in smaller numbers. Cytokines secreted by these cells have been shown to have the ability to regulate ovarian steroidogenesis. In the present study, the effect of leukocytes on 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in human granulosa-lutein cells was examined. In addition, individual cytokines were also tested for their ability to regulate this enzyme. The follicular aspirates of patients undergoing IVF treatment were used as a source of granulosa cells. Cells isolated from these aspirates were found to contain between 15 and 60% leukocytes as assessed by flow cytometry (FACS). Leukocytes were removed from the sample preparations by the use of immunomagnetic beads coated with CD45 antibody, which recognises a surface antigen on all classes of leukocyte. Removal of leukocytes significantly decreased the 11beta-HSD activity in the granulosa cells, assayed after 3 days of culture, from 7.3 (2-20) to 3.5 (1-10) pmol cortisone formed/50000 cells/4 h (medians and ranges, n = 15). Addition of IL-5 and IL-6 significantly increased the 11beta-HSD activity in granulosa cell cultures both in the presence and absence of leukocytes. Addition of IL-4 and IFN-gamma increased 11beta-HSD activity only in the leukocyte-depleted granulosa cell cultures, whereas IL-2 had no effect on either of the cultures. The data suggests that leukocytes interact with the ovarian cells through cytokine secretion and/or cell-cell contact to increase the 11beta-HSD activity in human granulosa cells.

The effect on maximal electroshock seizures induced by GABA agents and antiepileptic drugs microinfused into the nucleus reticularis pontis oralis.

The nucleus reticularis pontis oralis (RPO) is necessary for the expression of tonic hindlimb extension (THE) in maximal electroshock seizures (MES) of rats. Previous work in this laboratory has demonstrated that focal RPO microinfusion of NMDA antagonists inhibited THE while focal RPO microinfusion of NMDA induced convulsive activity similar to the audiogenic seizure response of rats. The purpose of the present study was to identify other receptors in the RPO that influence THE or induce convulsive activity. Bilateral microinfusion of bicuculline had no effect on the THE component of MES except when the bicuculline induced wild-running convulsions in which case the subsequent THE response to the MES stimulus was inhibited. The GABAergic agents muscimol, baclofen or 2-hydroxysaclofen neither altered the THE response nor induced convulsions. In addition, bilateral RPO microinfusion of the clinically effective antiepileptic drugs phenytoin, phenobarbital, valproate, ethosuximide or felbamate had no effect on the THE component of MES. These results indicate that the role of GABAergic receptors in the anticonvulsant activity mediated by the RPO is not prominent and that the RPO is unlikely to be the site of antiepileptic drug action in MES.

Postcaval thrombosis and delayed shunt migration after pleuro-peritoneal venous shunting for concurrent chylothorax and chylous ascites in a dog.

Simultaneous chylothorax and chylous ascites related to intestinal lymphangiectasia was diagnosed in a 4-year-old spayed female dog. Palliative pleural and peritoneal drainage was accomplished by placement of fenestrated silastic sheeting into surgically created diaphragmatic defects, and implantation of a pleuro-peritoneal venous shunt. The immediate postoperative period was complicated by acute renal failure secondary to postcaval thrombosis originating at the site of placement of the efferent pump catheter and extending to the level of the renal veins. Rapid resolution of this complication was accomplished with systemic anticoagulation. Clinical signs related to fluid accumulation resolved for 10 weeks after which acute decompensation occurred and the dog was euthanatized. Postmortem examination showed that reaccumulation of fluid was associated with migration of the efferent limb of the shunt from the caudal vena cava.

Infusion of NMDA antagonists into the nucleus reticularis pontis oralis inhibits the maximal electroshock seizure response.

The nucleus reticularis pontis oralis (RPO) is necessary for the expression of tonic hindlimb extension (THE) in maximal electroshock (MES) seizures of rats. Previous work in this laboratory has demonstrated that both systemic administration and focal RPO microinfusion of D-cycloserine inhibits THE. The purpose of the present study was to characterize specific components of the NMDA receptor/ionophore complex that regulate the anticonvulsant activity mediated by the RPO. Bilateral RPO microinfusion of the competitive NMDA antagonists (-)AP7 and D-CPP as well as the uncompetitive antagonist dizocilpine ((+)MK-801) inhibited THE in a dose-related fashion. Bilateral RPO microinfusion of NMDA did not affect the THE response to MES but did induce convulsions resembling audiogenic seizures in genetically epilepsy prone rats. Bilateral RPO microinfusion of the strychnine-insensitive glycine site partial agonist D-cycloserine and the antagonist 5,7-dichlorokynurenic acid inhibited THE. The strychnine-insensitive glycine partial agonists (+)HA-966 and ACPC, as well as the agonists glycine and D-serine, did not significantly affect the THE response. Strychnine microinfusions in the RPO had no effect on THE. The results support a hypothesis that the RPO is a site of anticonvulsant drug action in MES and indicate that either competitive or uncompetitive NMDA antagonist action regulates the anticonvulsant activity mediated by the RPO. The role of the strychnine-insensitive glycine site in the regulation of the anticonvulsant activity medicated by the RPO is uncertain.