Melatonin prevents the dynamin‐related protein 1‐dependent mitochondrial fission and oxidative insult in the cortical neurons after 1‐methyl‐4‐phenylpyridinium treatment

Mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of Parkinson's disease (PD). Mitochondrial morphology is dynamic and precisely regulated by the mitochondrial fission and fusion machinery. Aberrant mitochondrial fragmentation controlled by the mitochondrial fission protein, dynamin‐related protein 1 (Drp1), may result in cell death. Our previous results showed that melatonin protected neurons by inhibiting oxidative stress in a 1‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced PD model. However, the effect of melatonin on mitochondrial dynamics remains uncharacterized. Herein, we investigated the effect of melatonin and the role of Drp1 on MPP⁺‐induced mitochondrial fission in rat primary cortical neurons. We found that MPP⁺ induced a rapid increase in the ratio of GSSG:total glutathione (a marker of oxidative stress) and mitochondrial fragmentation, Drp1 upregulation within 4 hours, and finally resulted in neuron loss 48 hours after the treatment. Neurons overexpressing wild‐type Drp1 promoted mitochondrial and nuclear fragmentation; however, neurons overexpressing dominant‐negative Drp1^K38A or cotreated with melatonin exhibited significantly reduced MPP⁺‐induced mitochondrial fragmentation and neuron death. Moreover, melatonin cotreatment prevented an MPP⁺‐induced high ratio of GSSG and mitochondrial Drp1 upregulation. The prevention of mitochondrial fission by melatonin was not found in neurons transfected with wild‐type Drp1. These results provide a new insight that the neuroprotective effect of melatonin against MPP⁺ toxicity is mediated by inhibiting the oxidative stress and Drp1‐mediated mitochondrial fragmentation.     

Decreased sensitivity of palmitoyl protein thioesterase 1-deficient neurons to chemical anoxia

Infantile CLN1 disease, also known as infantile neuronal ceroid lipofuscinosis, is a fatal childhood neurodegenerative disorder caused by mutations in the CLN1 gene. CLN1 encodes a soluble lysosomal enzyme, palmitoyl protein thioesterase 1 (PPT1), and it is still unclear why neurons are selectively vulnerable to the loss of PPT1 enzyme activity in infantile CLN1 disease. To examine the effects of PPT1 deficiency on several well-defined neuronal signaling and cell death pathways, different toxic insults were applied in cerebellar granule neuron cultures prepared from wild type (WT) and palmitoyl protein thioesterase 1-deficient (Ppt1 ^?/?) mice, a model of infantile CLN1 disease. Glutamate uptake inhibition by t-PDC (L-trans-pyrrolidine-2,4-dicarboxylic acid) or Zn²⁺-induced general mitochondrial dysfunction caused similar toxicity in WT and Ppt1 ^?/? cultures. Ppt1 ^?/? neurons, however, were more sensitive to mitochondrial complex I inhibition by MPP⁺ (1-methyl-4-phenylpyridinium), and had significantly decreased sensitivity to chemical anoxia induced by the mitochondrial complex IV inhibitor, sodium azide. Our results indicate that PPT1 deficiency causes alterations in the mitochondrial respiratory chain.

     

Hepatitis C virus infection: a risk factor for Parkinson's disease

Recent studies found that hepatitis C virus (HCV) may invade the central nervous system, and both HCV and Parkinson's disease (PD) have in common the overexpression of inflammatory biomarkers. We analysed data from a community‐based integrated screening programme based on a total of 62 276 subjects. We used logistic regression models to investigate association between HCV infection and PD. The neurotoxicity of HCV was evaluated in the midbrain neuron–glia coculture system in rats. The cytokine/chemokine array was performed to measure the differences of amounts of cytokines released from midbrain in the presence and absence of HCV. The crude odds ratios (ORs) for having PD were 0.62 [95% confidence interval (CI), 0.48–0.81] and 1.91 (95% CI, 1.48–2.47) for hepatitis B virus (HBV) and HCV. After controlling for potential confounders, the association between HCV and PD remained statistically significant (adjusted OR = 1.39; 95% CI, 1.07–1.80), but not significantly different between HBV and PD. The HCV induced 60% dopaminergic neuron death in the midbrain neuron–glia coculture system in rats, similar to that of 1‐methyl‐4‐phenylpyridinium (MPP⁺) but not caused by HBV. This link was further supported by the finding that HCV infection may release the inflammatory cytokines, which may play a role in the pathogenesis of PD. In conclusion, our study demonstrated a significantly positive epidemiological association between HCV infection and PD and corroborated the dopaminergic toxicity of HCV similar to that of MPP⁺.     

Transgenic mice expressing human Bcl-2 in their neurons are resistant to 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine neurotoxicity

The protooncogene bcl-2 inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE-hbcl-2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE-hbcl-2 transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP⁺. Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE-hbcl-2 heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.     

Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways,accumulation of α-synuclein,and apoptotic cell death in aged mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson''s disease. LRRK2 is a large protein containing a small GTPase domain and a kinase domain, but its physiological role is unknown. To identify the normal function of LRRK2 in vivo, we generated two independent lines of germ-line deletion mice. The dopaminergic system of LRRK2^−/− mice appears normal, and numbers of dopaminergic neurons and levels of striatal dopamine are unchanged. However, LRRK2^−/− kidneys, which suffer the greatest loss of LRRK compared with other organs, develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins at 20 months of age. The autophagy–lysosomal pathway is also impaired in the absence of LRRK2, as indicated by accumulation of lipofuscin granules as well as altered levels of LC3-II and p62. Furthermore, loss of LRRK2 dramatically increases apoptotic cell death, inflammatory responses, and oxidative damage. Collectively, our findings show that LRRK2 plays an essential and unexpected role in the regulation of protein homeostasis during aging, and suggest that LRRK2 mutations may cause Parkinson''s disease and cell death via impairment of protein degradation pathways, leading to α-synuclein accumulation and aggregation over time.     

Cytochrome b5 reductase,a plasma membrane redox enzyme,protects neuronal cells against metabolic and oxidative stress through maintaining redox state and bioenergetics

The plasma membrane redox system (PMRS) containing NADH-dependent reductases is known to be involved in the maintenance of redox state and bioenergetics. Neuronal cells are very vulnerable to oxidative stress and altered energy metabolism linked to mitochondrial dysfunction. However, the role of the PMRS in these pathways is far from clear. In this study, in order to investigate how cytochrome b5 reductase (b5R), one of the PM redox enzymes, regulates cellular response under stressed conditions, human neuroblastoma cells transfected with b5R were used for viability and mitochondrial functional assays. Cells transfected with b5R exhibited significantly higher levels of the NAD⁺/NADH ratio, consistent with increased levels of b5R activity. Overexpression of b5R made cells more resistant to H₂O₂ (oxidative stress), 2-deoxyglucose (metabolic stress), rotenone and antimycin A (energetic stress), and lactacystin (proteotoxic stress), but did not protect cells against H₂O₂ and serum withdrawal. Overexpression of b5R induced higher mitochondrial functions such as ATP production rate, oxygen consumption rate, and activities of complexes I and II, without formation of further reactive oxygen species, consistent with lower levels of oxidative/nitrative damage and resistance to apoptotic cell death. In conclusion, higher NAD⁺/NADH ratio and consequent more efficient mitochondrial functions are induced by the PMRS, enabling them to maintain redox state and energy metabolism under conditions of some energetic stresses. This suggests that b5R can be a target for therapeutic intervention for aging and neurodegenerative diseases.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-015-9859-9) contains supplementary material, which is available to authorized users.     

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1^−/− mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1^−/−;Wnt5a^−/− mice revealed a greater loss of Nurr1⁺ cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson’s disease.     

From the Cover: Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome

To explore the lethal, ataxic phenotype of complex I deficiency in Ndufs4 knockout (KO) mice, we inactivated Ndufs4 selectively in neurons and glia (NesKO mice). NesKO mice manifested the same symptoms as KO mice including retarded growth, loss of motor ability, breathing abnormalities, and death by ~7 wk. Progressive neuronal deterioration and gliosis in specific brain areas corresponded to behavioral changes as the disease advanced, with early involvement of the olfactory bulb, cerebellum, and vestibular nuclei. Neurons, particularly in these brain regions, had aberrant mitochondrial morphology. Activation of caspase 8, but not caspase 9, in affected brain regions implicate the initiation of the extrinsic apoptotic pathway. Limited caspase 3 activation and the predominance of ultrastructural features of necrotic cell death suggest a switch from apoptosis to necrosis in affected neurons. These data suggest that dysfunctional complex I in specific brain regions results in progressive glial activation that promotes neuronal death that ultimately results in mortality.     

GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana

Higher plant species differ widely in their growth responses to ammonium (NH₄⁺). However, the molecular genetic mechanisms underlying NH₄⁺ sensitivity in plants remain unknown. Here, we report that mutations in the Arabidopsis gene encoding GDP-mannose pyrophosphorylase (GMPase) essential for synthesizing GDP-mannose confer hypersensitivity to NH₄⁺. The in planta activities of WT and mutant GMPases all were inhibited by NH₄⁺, but the magnitude of the inhibition was significantly larger in the mutant. Despite the involvement of GDP-mannose in both l-ascorbic acid (AsA) and N-glycoprotein biosynthesis, defective protein glycosylation in the roots, rather than decreased AsA content, was linked to the hypersensitivity of GMPase mutants to NH₄⁺. We conclude that NH₄⁺ inhibits GMPase activity and that the level of GMPase activity regulates Arabidopsis sensitivity to NH₄⁺. Further analysis showed that defective N-glycosylation of proteins, unfolded protein response, and cell death in the roots are likely important downstream molecular events involved in the growth inhibition of Arabidopsis by NH₄⁺.     

The nature of the transport ATPase-digitalis complex. II. Some species differences and ouabain "exchange" characteristics

[³H]ouabain, bound to Na⁺, K⁺-ATPase in the presence of ATP, Mg²⁺ + Na⁺ (complex I) or in the prescence of Mg²⁺ + inorganic phosphate (complex II), was more stable when beef heart rather than rabbit heart was the enzyme source. For both species, complex II was more stable than complex I. Unlabeled ouabain in the wash-off medium did not affect drug dissociation kinetics, but did cause inhibition of enzyme activity, suggesting ouabain exchange.     

Dopaminergic degeneration is enhanced by chronic brain hypoperfusion and inhibited by angiotensin receptor blockage

The possible interaction between brain hypoperfusion related to aging and/or vascular disease, vascular parkinsonism and Parkinson’s disease, as well as the possible contribution of aging-related chronic brain hypoperfusion in the development or severity of Parkinson’s disease are largely unknown. We used a rat model of chronic cerebral hypoperfusion to study the long-term effects of hypoperfusion on dopaminergic neurons and the possible synergistic effects between chronic hypoperfusion and factors that are deleterious to dopaminergic neurons, such as the dopaminergic neurotoxin 6-hydroxydopamine. Chronic hypoperfusion induced significant loss of dopaminergic neurons and striatal dopaminergic terminals and a reduction in striatal dopamine levels. Furthermore, intrastriatal administration of 6-hydroxydopamine in rats subjected to chronic hypoperfusion induced a significantly greater loss of dopaminergic neurons than in sham-operated control rats. The dopaminergic neuron loss was significantly reduced by oral treatment with angiotensin type 1 receptor antagonist candesartan (3 mg/kg/day). The levels of angiotensin type 2 receptors were lower and the levels of angiotensin type 1 receptors, interleukin-1 β and nicotinamide adenine dinucleotide phosphate oxidase activity were higher in the substantia nigra of rats subjected to chronic hypoperfusion than in control rats; this was significantly reduced by treatment with candesartan. The results suggest that early treatment of vascular disease should be considered in the treatment of aged Parkinson’s disease patients and Parkinson’s disease patients with cerebrovascular risk factors. The findings also suggest that inhibition of brain renin–angiotensin activity may be useful as a neuroprotective strategy.     

The silkworm Green b locus encodes a quercetin 5-O-glucosyltransferase that produces green cocoons with UV-shielding properties

In the silkworm Bombyx mori, dietary flavonoids are metabolized and accumulate in cocoons, thereby causing green coloration. Classical genetic studies suggest that more than seven independent loci are associated with this trait; however, because of the complex inheritance pattern, none of these loci have been characterized molecularly, and a plausible and comprehensive model for their action has not been proposed. Here, we report the identification of the gene responsible for the Green b (Gb) locus involving the green cocoon trait. In +^Gb animals, glucosylation at the 5-O position of dietary quercetin did not occur, and the total amount of flavonoids in tissues and cocoons was dramatically reduced. We performed positional cloning of Gb and found a 38-kb deletion in a UDP-glucosyltransferase (UGT) gene cluster associated with the +^Gb allele. RT-PCR and biochemical studies suggested that deletion of Bm-UGT10286 (UGT) is responsible for Gb and Bm-UGT10286 is virtually the sole source of UGT activity toward the 5-O position of quercetin. Our data show that the regiospecific glucosylation of flavonoids by the quercetin 5-O-glucosyltransferase can greatly affect the overall bioavailability of flavonoids in animals. Furthermore, we provide evidence that flavonoids increase the UV-shielding activity of cocoons and thus could confer an increased survival advantage to insects contained in these cocoons. This study will lead to greater understanding of mechanisms for metabolism, uptake, and transport of dietary flavonoids, which have a variety of biological activities in animals and beneficial effects on human health.     

Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin''s ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP⁺) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD.     

Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing

Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z⁺ agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z⁺ agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z⁺ agrin in motor neurons rescued AChR clustering. Surprisingly, however, these rescued mice remained paralyzed, while electrophysiologic studies demonstrated that the motor axon and synapse were functional-spontaneous and evoked recordings revealed synaptic transmission and muscle contraction. These results point to a proximal defect in motor neuron firing in the absence of Nova and reveal a previously unsuspected role for RNA regulation in the physiologic activation of motor neurons.     

Bidirectional influence of sodium channel activation on NMDA receptor–dependent cerebrocortical neuron structural plasticity

Neuronal activity regulates brain development and synaptic plasticity through N-methyl-d-aspartate receptors (NMDARs) and calcium-dependent signaling pathways. Intracellular sodium ([Na⁺]_i) also exerts a regulatory influence on NMDAR channel activity, and [Na⁺]_i may, therefore, function as a signaling molecule. In an attempt to mimic the influence of neuronal activity on synaptic plasticity, we used brevetoxin-2 (PbTx-2), a voltage-gated sodium channel (VGSC) gating modifier, to manipulate [Na⁺]_i in cerebrocortical neurons. The acute application of PbTx-2 produced concentration-dependent increments in both intracellular [Na⁺] and [Ca²⁺]. Pharmacological evaluation showed that PbTx-2–induced Ca²⁺ influx primarily involved VGSC activation and NMDAR-mediated entry. Additionally, PbTx-2 robustly potentiated NMDA-induced Ca²⁺ influx. PbTx-2–exposed neurons showed enhanced neurite outgrowth, increased dendritic arbor complexity, and increased dendritic filopodia density. The appearance of spontaneous calcium oscillations, reflecting synchronous neuronal activity, was accelerated by PbTx-2 treatment. Parallel to this response, PbTx-2 increased cerebrocortical neuron synaptic density. PbTx-2 stimulation of neurite outgrowth, dendritic arborization, and synaptogenesis all exhibited bidirectional concentration–response profiles. This profile paralleled that of NMDA, which also produced bidirectional concentration–response profiles for neurite outgrowth and synaptogenesis. These data are consistent with the hypothesis that PbTx-2–enhanced neuronal plasticity involves NMDAR-dependent signaling. Our results demonstrate that PbTx-2 mimics activity-dependent neuronal structural plasticity in cerebrocortical neurons through an increase in [Na⁺]_i, up-regulation of NMDAR function, and engagement of downstream Ca²⁺-dependent signaling pathways. These data suggest that VGSC gating modifiers may represent a pharmacologic strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.     

Hugin+ neurons provide a link between sleep homeostat and circadian clock neurons

Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin⁺ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin⁺ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin⁺ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin⁺ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin⁺ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide–dependent fashion. We propose that hugin⁺ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.

Sleep is regulated by two processes, circadian and homeostatic (1). The endogenous circadian clock, together with its downstream pathways, is synchronized to external day–night cycles and determines the timing of sleep to produce 24-h rhythms in sleep and wake. The homeostatic process tracks sleep:wake history and generates sleep drive, based on the extent of wakefulness. Overtly, sleep homeostasis can be seen as an increase in sleep duration and depth after prolonged wakefulness. Generally, circadian and homeostatic processes are studied as separate mechanisms that regulate sleep, but they clearly intersect and are coordinated in a daily cycle to promote the onset and maintenance of sleep at night. Following sleep deprivation, the homeostatic system can drive sleep at the wrong time of day, but even under these conditions, interactions between the two systems determine the timing and duration of sleep. However, the neuronal mechanisms by which circadian and homeostatic pathways signal to each other are largely unknown.The functions and regulation of sleep are extensively studied in model organisms, such as Drosophila melanogaster (2). In the Drosophila brain, the circadian clock is expressed in ∼150 clock neurons that are organized into neuroanatomical groups, of which the ventral and dorsal lateral neurons are the most important for driving rhythms of locomotor activity (3, 4). The small ventrolateral neurons (s-LNvs) link to other brain regions through different circuits. One of those circuits connects the s-LNvs to the site of the motor ganglion, the thoracic nerve cord: s-LNvs → DN1s → Dh44⁺ neurons → hugin⁺ neurons→ ventral nerve cord (5). Dh44-expressing neurons in the pars intercerebralis regulate locomotor activity rhythms in part through the signaling of DH44 neuropeptide to hugin-expressing neurons in the subesophageal zone (SEZ) (6, 7). Dh44⁺ and hugin⁺ circadian output neurons do not contain canonical molecular clocks, but display cycling in neuronal activity or peptide release, likely under control of upstream circadian signals (7–9). Links between these neurons and loci regulating sleep homeostasis have not been identified yet.Regulation of sleep homeostasis in flies involves the central complex and mushroom body (10–13). Of particular importance is a group of sleep-promoting neurons labeled by the 23E10-GAL4 driver that projects to the dorsal fan-shaped body (dFB) neuropil in the central complex. These dFB neurons promote sleep when activated (14, 15), and they are required for normal sleep rebound after deprivation (16). 23E10⁺ neurons receive input signals from R5 ellipsoid body neurons, which track sleep need (17).As hugin⁺ neurons are significantly downstream of central clock neurons and close to behavioral outputs, we asked whether they also have a role in sleep. We find that hugin⁺ neurons are dispensable for determining daily sleep amount, but they show decreases in activity following sleep deprivation. They also receive projections from the dFB and counter sleep-promoting effects of 23E10⁺ neurons, such that ablation of hugin⁺ neurons enhances sleep driven by 23E10⁺ cells. Further supporting a role in sleep, mutations in Hugin peptides affect recovery sleep after deprivation and enhance sleep driven by 23E10⁺ cells. hugin⁺ neurons target PDF⁺ s-LNv clock neurons, which also show decreases in intracellular Ca²⁺ levels following sleep deprivation. Thus hugin⁺ neurons serve as an integrations site of signals from both the sleep homeostat and the circadian clock.     

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants

We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.     

Neuroprotective efficacy of aminopropyl carbazoles in a mouse model of Parkinson disease

We previously reported the discovery of P7C3, an aminopropyl carbazole having proneurogenic and neuroprotective properties in newborn neural precursor cells of the dentate gyrus. Here, we provide evidence that P7C3 also protects mature neurons in brain regions outside of the hippocampus. P7C3 blocks 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated cell death of dopaminergic neurons in the substantia nigra of adult mice, a model of Parkinson disease (PD). Dose–response studies show that the P7C3 analog P7C3A20 blocks cell death with even greater potency and efficacy, which parallels the relative potency and efficacy of these agents in blocking apoptosis of newborn neural precursor cells of the dentate gyrus. P7C3 and P7C3A20 display similar relative effects in blocking 1-methyl-4-phenylpyridinium (MPP⁺)-mediated death of dopaminergic neurons in Caenorhabditis elegans, as well as in preserving C. elegans mobility following MPP⁺ exposure. Dimebon, an antihistaminergic drug that is weakly proneurogenic and neuroprotective in the dentate gyrus, confers no protection in either the mouse or the worm models of PD. We further demonstrate that the hippocampal proneurogenic efficacy of eight additional analogs of P7C3 correlates with their protective efficacy in MPTP-mediated neurotoxicity. In vivo screening of P7C3 analogs for proneurogenic efficacy in the hippocampus may thus provide a reliable means of predicting neuroprotective efficacy. We propose that the chemical scaffold represented by P7C3 and P7C3A20 provides a basis for optimizing and advancing pharmacologic agents for the treatment of patients with PD.Parkinson disease (PD) is an incurable and progressive neurodegenerative disorder of predominantly idiopathic origin that is characterized by the death of dopaminergic neurons in the substantia nigra pars compacta (SNc), a region of the brain that controls motor activity by projecting dopaminergic axons to the striatum (1). Early symptoms in PD are primarily movement related, including shaking, rigidity, brady- and hypokinesia, tremor, and difficulty walking. More advanced stages of PD are associated with cognitive and behavioral problems, including dementia. Current treatment strategies for PD consist primarily of partial management of early motor symptoms with drugs that enhance dopaminergic signaling, such as l-DOPA or dopamine receptor agonists. Unfortunately, as greater numbers of dopaminergic neurons in the SNc die, these drugs fail to alleviate symptoms and additionally produce dyskinesia. There is thus a significant unmet need for new pharmacologic strategies to slow the progression of PD, such as drugs capable of blocking the death of SNc dopaminergic neurons.We have previously reported the identification of an aminopropyl carbazole (P7C3) discovered via an unbiased, in vivo screen for small molecules capable of enhancing postnatal hippocampal neurogenesis. P7C3 displays enantiomeric-selective stabilization of mitochondrial membrane potential and enhances neurogenesis by blocking apoptosis of newborn neurons in the dentate gyrus (2). Prolonged oral or i.p. administration of P7C3 to rodents safely improves hippocampal functioning. For example, administration of P7C3 to mice suffering from pathologically high levels of neuronal apoptosis in the dentate gyrus, neuronal PAS domain protein 3 (NPAS3)-deficient mice (3), restored hippocampal structure and function with no obvious physiologic side effects (2). In addition, extended administration of P7C3 to aged rats safely impeded hippocampal cell death and preserved cognitive ability as a function of terminal aging (2).Through an in vivo structure–activity relationship (SAR) study, we have identified analogs of P7C3 displaying either increased or decreased activity. In particular, a chemical variant known as P7C3A20 was observed to have greater potency and efficacy than P7C3. P7C3A20 differs from P7C3 by virtue of replacing the hydroxyl group at the chiral center of the linker with a fluorine and the addition of a methoxy group to the aniline ring (Fig. 1). This analog displays a more favorable toxicity profile than P7C3, with no hERG channel binding, histamine receptor binding, or toxicity to HeLa cells (2, 4, 5). We have also found that Dimebon, an antihistaminergic drug long deployed in Russia that is claimed to have anti-apoptotic and mitochondrial protective properties (6, 7), displays modest efficacy in the same biologic assays used to discover and characterize P7C3 and P7C3A20 (2). Here, we report that the neuroprotective activity of these agents extends beyond promoting long-term survival of newborn cells in the adult hippocampus. Specifically, we show that the most active variants of P7C3 exhibit robust protection of mature dopaminergic neurons in both mouse and worm models of neurodegeneration and propose that substituted carbazoles may represent attractive chemical scaffolds for the optimization of therapeutic agents for the treatment of Parkinson disease.Open in a separate windowFig. 1.Neuroprotective efficacy of P7C3, P7C3A20, and Dimebon for newborn neurons in the adult hippocampus. Test compounds were evaluated by dose–response assay for their ability to block normal apoptotic cell death of newborn neural precursor cells in the adult dentate gyrus. P7C3A20 exhibits the greatest potency and ceiling of efficacy, and Dimebon the least. P7C3 is intermediate in both measures. Six animals were tested per group. Dosing is expressed as total milligrams per day, and compounds were administered intraperitoneally in divided doses twice daily. Data are expressed as mean ± SEM. Values for P7C3 and P7C3A20 were compared with those of vehicle (VEH), and values for Dimebon were compared with those of saline (SAL).     

Melatonin protects against rotenone-induced cell injury via inhibition of Omi and Bax-mediated autophagy in Hela cells

Parkinson's disease is the second most common neurodegenerative disease, and environmental toxins such as rotenone play an important role in causing degeneration of dopaminergic neurons. Melatonin, a major secretory product of pineal, is recently reported to protect against rotenone-induced cell death in animal models. Yet, the mechanism involved in this protection needs to be elucidated. Here, we report that rotenone treatment (0-100 μM) decreased cell survival of Hela cells in a dose-dependent manner. At concentrations ranging from 0.1 to 100 μM, rotenone induced a dose-dependent increase in the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, a protein associated with the autophagosomal membrane. Knockdown of Bax or Omi using shRNA inhibited 1 μM rotenone-induced autophagy. To determine whether melatonin would protect cells against rotenone-induced cell death and autophagy, we pretreated Hela cells with 250 μM melatonin for 24 hr in the presence of rotenone. Melatonin inhibited Bax expression and the release of the omi/HtrA2 into the cytoplasm induced by 1 μM rotenone. Melatonin 250 μM treatment also suppressed cell death induced by 0.1-100 μM rotenone and protected against the formation of LC3-II in cells exposed to 1 μM rotenone. This work demonstrates a novel role for melatonin as a neuroprotective agent against rotenone.