Self-assembly of VPS41 promotes sorting required for biogenesis of the regulated secretory pathway.

The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 in mammals has been considered to function in the endolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.

Influence of electron dose rate on electron counting images recorded with the K2 camera.

A recent technological breakthrough in electron cryomicroscopy (cryoEM) is the development of direct electron detection cameras for data acquisition. By bypassing the traditional phosphor scintillator and fiber optic coupling, these cameras have greatly enhanced sensitivity and detective quantum efficiency (DQE). Of the three currently available commercial cameras, the Gatan K2 Summit was designed specifically for counting individual electron events. Counting further enhances the DQE, allows for practical doubling of detector resolution and eliminates noise arising from the variable deposition of energy by each primary electron. While counting has many advantages, undercounting of electrons happens when more than one electron strikes the same area of the detector within the analog readout period (coincidence loss), which influences image quality. In this work, we characterized the K2 Summit in electron counting mode, and studied the relationship of dose rate and coincidence loss and its influence on the quality of counted images. We found that coincidence loss reduces low frequency amplitudes but has no significant influence on the signal-to-noise ratio of the recorded image. It also has little influence on high frequency signals. Images of frozen hydrated archaeal 20S proteasome (~700 kDa, D7 symmetry) recorded at the optimal dose rate retained both high-resolution signal and low-resolution contrast and enabled calculating a 3.6 Å three-dimensional reconstruction from only 10,000 particles.

Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein alpha-synuclein.

The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than ß- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease.

Neuroanatomical substrates for paroxysmal dyskinesia in lethargic mice.

The paroxysmal dyskinesias are a group of neurological disorders described by intermittent attacks of involuntary abnormal movements superimposed on a relatively normal baseline. The neuroanatomical substrates for these attacks are not fully understood, though available evidence from studies of affected people and animal models points to dysfunction in the basal ganglia or cerebellum. In the current studies, the anatomical basis for paroxysmal dyskinesias in lethargic mice was determined via histochemical methods sensitive to changes in regional brain activity followed by surgical elimination of the suspected source. Cytochrome oxidase histochemistry revealed increased activity in the red nucleus. Surgical removal of the cerebellum worsened ataxia but eliminated paroxysmal dyskinesias. These studies support the hypothesis that abnormal cerebellar output contributes to paroxysmal dyskinesias.

A human neuronal tissue culture model for Lesch-Nyhan disease.

Mutations in the gene encoding the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease, a neurodevelopmental disorder characterized by cognitive, neurological, and behavioral abnormalities. Despite detailed knowledge of the enzyme's function, the key pathophysiological changes that accompany loss of purine recycling are unclear. To facilitate delineating the consequences of HPRT deficiency, four independent HPRT-deficient sublines of the human dopaminergic neuroblastoma, SK-N-BE(2) M17, were isolated by targeted mutagenesis with triple helix-forming oligonucleotides. As a group, these HPRT-deficient cells showed several significant abnormalities: (i) impaired purine recycling with accumulation of hypoxanthine, guanine, and xanthine, (ii) reduced guanylate energy charge and GTP:GDP ratio, but normal adenylate energy charge and no changes in any adenine nucleotide ratios, (iii) increased levels of UTP and NADP+, (iv) reduced DOPA decarboxylase, but normal monoamines, and (v) reduction in cell soma size. These cells combine the analytical power of multiple lines and a human, neuronal origin to provide an important tool to investigate the pathophysiology of HPRT deficiency.

Basal ganglia dopamine loss due to defect in purine recycling.

Several rare inherited disorders have provided valuable experiments of nature highlighting specific biological processes of particular importance to the survival or function of midbrain dopamine neurons. In both humans and mice, deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) is associated with profound loss of striatal dopamine, with relative preservation of other neurotransmitters. In the current studies of knockout mice, no morphological signs of abnormal development or degeneration were found in an exhaustive battery that included stereological and morphometric measures of midbrain dopamine neurons, electron microscopic studies of striatal axons and terminals, and stains for degeneration or gliosis. A novel culture model involving HPRT-deficient dopaminergic neurons also exhibited significant loss of dopamine without a morphological correlate. These results suggest that dopamine loss in HPRT deficiency has a biochemical rather than anatomical basis and imply that purine recycling to be a biochemical process of particular importance to the function of dopaminergic neurons.

Nifedipine suppresses self-injurious behaviors in animals.

Self-injurious behavior is a common problem in many developmental disorders. The neurobiology of this behavior is not well understood, but the differing behavioral manifestations and associations with different disorders suggest that the underlying biological mechanisms are heterogeneous. The behavioral and biological heterogeneity is also evident in several animal models, where different manifestations can be provoked under different experimental conditions. Identifying commonalities among the different mechanisms is likely to be helpful in the design of treatments useful for the broadest populations of patients. The current studies reveal that nifedipine suppresses self-injurious behavior in 4 unrelated animal models: acute administration of high doses of +/-BayK 8644 or methamphetamine in mice, dopamine agonist treatment in rats with lesions of dopamine pathways during early development and repeated administration of pemoline in rats. The effect of nifedipine does not appear to be due to nonspecific mechanisms, such as sedation, since other classes of behaviors are unaffected or exaggerated. These results suggest that nifedipine may target a common biological mechanism in the expression of self-injurious behavior, and they suggest it should be considered in the treatment of self-injury in humans.

Abnormal latent inhibition and impulsivity in coloboma mice, a model of ADHD.

Attention deficit hyperactivity disorder (ADHD) is characterized by hyperactivity, inattention, and impulsivity. The coloboma mouse model of ADHD exhibits profound hyperactivity. To determine whether coloboma mice exhibit other signs of ADHD, we assessed latent inhibition as a test of attention, and impulsivity in a delayed reinforcement paradigm. Latent inhibition was present in control mice but was disrupted in coloboma mice. Coloboma mice also exhibited impaired performance on the delayed reinforcement task and were not able to wait as long as control mice to obtain the greater reinforcer. Because norepinephrine mediates hyperactivity in coloboma mice, we examined the role of norepinephrine in disrupted latent inhibition and impulsivity. Reduction of norepinephrine with DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride) restored latent inhibition but did not ameliorate impulsivity. In summary, coloboma mice exhibit hyperactivity, inattention as determined by latent inhibition, and impulsivity, and norepinephrine mediates hyperactivity and inattention but not impulsivity in these mice.

The role of dopamine receptors in the neurobehavioral syndrome provoked by activation of L-type calcium channels in rodents.

In rodents, activation of L-type calcium channels with +/-BayK 8644 causes an unusual behavioral syndrome that includes dystonia and self-biting. Prior studies have linked both of these behaviors to dysfunction of dopaminergic transmission in the striatum. The current studies were designed to further elucidate the relationship between +/-BayK 8644 and dopaminergic transmission in the expression of the behavioral syndrome. The drug does not appear to release presynaptic dopamine stores, since microdialysis of the striatum revealed dopamine release was unaltered by +/-BayK 8644. In addition, the behaviors were preserved or even exaggerated in mice or rats with virtually complete dopamine depletion. On the other hand, pretreatment of mice with D(3) or D(1/5) dopamine receptor antagonists attenuated the behavioral effects of +/-BayK 8644, while pretreatment with D(2) or D(4) antagonists had no effect. In D(3) receptor knockout mice, +/-BayK 8644 elicited both dystonia and self-biting, but these behaviors were less severe than in matched controls. In D(1) receptor knockout mice, behavioral responses to +/-BayK 8644 appeared exaggerated. These results argue that the behavioral effects of +/-BayK 8644 are not mediated by a presynaptic influence. Instead, the behaviors appear to result from a postsynaptic activation of the drug, which does not require but can be modified by D(3) or D(1/5) receptors.

Expression of c-FOS in the brain after activation of L-type calcium channels.

In rodents, administration of the L-type calcium channel activators, +/-Bay K 8644 and FPL 64176, causes an unusual neurobehavioral syndrome that includes dystonia and self-injurious biting. To determine the regional influence of these drugs in the brain, the induction of c-FOS was mapped after administration of these drugs to mice. In situ hybridization with an antisense riboprobe directed to c-FOS mRNA revealed widespread induction, with the highest levels in the striatum, cortex, hippocampus, locus coeruleus, and cerebellum. The induction of c-FOS mRNA was dose dependent, reached maximal expression approximately 60 min after drug treatment, and could be blocked by pretreatment with the L-type calcium channel antagonist, nifedipine. Immunohistochemical stains with an antibody directed to c-FOS protein revealed a pattern of induction similar to that obtained with in situ hybridization in most brain regions. These results demonstrate a very heterogeneous influence of L-type calcium channel activation in different brain regions, despite the nearly universal expression of these channels implied by more classical anatomical methods.

Self-biting induced by activation of L-type calcium channels in mice: serotonergic influences.

The L-type calcium channel activator +/-Bay K 8644 has recently been shown to provoke self-injurious biting in young mice. Since the serotonergic systems have been implicated in the expression of self-injurious behavior in both humans and animals, the present studies tested whether drugs influencing serotonin systems could modify the ability of +/-Bay K 8644 to cause this behavior. The ability of +/-Bay K 8644 to provoke self-biting behavior was increased by the serotonin uptake inhibitor fluoxetine or the monoamine oxidase inhibitor clorgyline. On the other hand, the ability of +/-Bay K 8644 to provoke self-biting was decreased by depletion of serotonin with p-chlorophenylalanine or 5,7-dihyroxytryptamine. These results suggest that the ability of +/-Bay K 8644 to provoke self-injurious behaviors may be mediated by serotonergic influences.