Search

Cocaine abuse is highly disruptive to circadian physiological and behavioral rhythms. The present study was undertaken to determine whether such effects are manifest through actions on critical photic and nonphotic regulatory pathways in the master circadian clock of the mouse suprachiasmatic nucleus (SCN). Impairment of SCN photic signaling by systemic (intraperitoneal) cocaine injection was evidenced by strong (60%) attenuation of light-induced phase-delay shifts of circadian locomotor activity during the early night. A nonphotic action of cocaine was apparent from its induction of 1-h circadian phase-advance shifts at midday. The serotonin receptor antagonist, metergoline, blocked shifting by 80%, implicating a serotonergic mechanism. Reverse microdialysis perfusion of the SCN with cocaine at midday induced 3.7 h phase-advance shifts. Control perfusions with lidocaine and artificial cerebrospinal fluid had little shifting effect. In complementary in vitro experiments, photic-like phase-delay shifts of the SCN circadian neuronal activity rhythm induced by glutamate application to the SCN were completely blocked by cocaine. Cocaine treatment of SCN slices alone at subjective midday, but not the subjective night, induced 3-h phase-advance shifts. Lidocaine had no shifting effect. Cocaine-induced phase shifts were completely blocked by metergoline, but not by the dopamine receptor antagonist, fluphenazine. Finally, pretreatment of SCN slices for 2 h with a low concentration of serotonin agonist (to block subsequent serotonergic phase resetting) abolished cocaine-induced phase shifts at subjective midday. These results reveal multiple effects of cocaine on adult circadian clock regulation that are registered within the SCN and involve enhanced serotonergic transmission.

People with physical disabilities have ranked object retrieval as a high-priority task for assistive robots. We have developed Dusty, a teleoperated mobile manipulator that fetches objects from the floor and delivers them to users at a comfortable height. In this paper, we first demonstrate the robot's high success rate (98.4%) when autonomously grasping 25 objects considered being important by people with amyotrophic lateral sclerosis (ALS). We tested the robot with each object in five different configurations on five types of flooring. We then present the results of an experiment in which 20 people with ALS operated Dusty. Participants teleoperated Dusty to move around an obstacle, pick up an object and deliver the object to themselves. They successfully completed this task in 59 out of 60 trials (3 trials each) with a mean completion time of 61.4 SD = 20.5 seconds), and reported high overall satisfaction using Dusty (7-point Likert scale; 6.8 SD = 0.6). Participants rated Dusty to be significantly easier to use than their own hands, asking family members, and using mechanical reachers (p < 0.03, paired t-tests). Fourteen of the 20 participants reported that they would prefer using Dusty over their current methods. [Box: see text].

Excitotoxicity is thought to play a pathogenic role in amyotrophic lateral sclerosis (ALS). Excitotoxic motor neuron death is mediated through the Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type of glutamate receptors and Ca(2+) permeability is determined by the GluR2 subunit. We investigated whether polymorphisms or mutations in the GluR2 gene (GRIA2) predispose patients to ALS. Upon sequencing 24 patients and 24 controls no nonsynonymous coding variants were observed but 24 polymorphisms were identified, 9 of which were novel. In a screening set of 310 Belgian ALS cases and 794 healthy controls and a replication set of 3157 cases and 5397 controls from 6 additional populations no association with susceptibility, age at onset, or disease duration was observed. We conclude that polymorphisms in the GluR2 gene (GRIA2) are not a major contributory factor in the pathogenesis of ALS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oxidative stress is commonly implicated in the pathogenesis of motor neuron disease. However, the cause and effect relationship between oxidative stress and motor neuron degeneration is poorly defined. We recently identified denervation at the neuromuscular junction in mice lacking the antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD1) (Fischer et al., 2011). These mice show a phenotype of progressive muscle atrophy and weakness in the setting of chronic oxidative stress. Here, we investigated further the extent of motor neuron pathology in this model, and the relationship between motor pathology and oxidative stress. We report preferential denervation of fast-twitch muscles beginning between 1 and 4 months of age, with relative sparing of slow-twitch muscle. Motor axon terminals in affected muscles show widespread sprouting and formation of large axonal swellings. We confirmed, as was previously reported, that spinal motor neurons and motor and sensory nerve roots in these mice are preserved, even out to 18 months of age. We also found preservation of distal sensory fibers in the epidermis, illustrating the specificity of pathology in this model for distal motor axons. Using HPLC measurement of the glutathione redox potential, we quantified oxidative stress in peripheral nerve and muscle at the onset of denervation. SOD1 knockout tibial nerve, but not gastrocnemius muscle, showed significant oxidation of the glutathione pool, suggesting that axonal degeneration is a consequence of impaired redox homeostasis in peripheral nerve. We conclude that the SOD1 knockout mouse is a model of oxidative stress-mediated motor axonopathy. Pathology in this model primarily affects motor axon terminals at the neuromuscular junction, demonstrating the vulnerability of this synapse to oxidative injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Detergent-insoluble protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases. Here, we describe the identification of septin 11 (SEPT11), an enriched component of detergent-resistant fractions in frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U), using large-scale unbiased proteomics approaches., Results: We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U. Using these approaches, we proteomically profiled detergent-insoluble protein extracts prepared from frontal cortex of FTLD-U cases, unaffected controls, or neurologic controls (i.e. Alzheimer's disease; AD). Among the proteins altered specifically in FTLD-U, we identified TAR DNA binding protein-43 (TDP-43), a known component of ubiquitinated inclusions. Moreover, we identified additional proteins enriched in detergent-resistant fractions in FTLD-U, and characterized one of them, SEPT11, in detail. Using independent highly sensitive targeted proteomics approaches, we confirmed the enrichment of SEPT11 in FTLD-U extracts. We further showed that SEPT11 is proteolytically cleaved into N-terminal fragments and, in addition to its prominent glial localization in normal brain, accumulates in thread-like pathology in affected cortex of FTLD-U patients., Conclusions: The proteomic discovery of insoluble SEPT11 accumulation in FTLD-U, along with novel pathological associations, highlights a role for this cytoskeleton-associated protein in the pathogenesis of this complex disorder.

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes a complex array of pathological events, through toxic gain of function mechanisms, leading to selective motor neuron degeneration. Mitochondrial dysfunction is among the well established toxic effects of mutant SOD1, but its mechanisms are just starting to be elucidated. A portion of mutant SOD1 is localized in mitochondria, where it accumulates mostly on the outer membrane and inside the intermembrane space (IMS). Evidence in cultured cells suggests that mutant SOD1 in the IMS causes mitochondrial dysfunction and compromises cell viability. Therefore, to test its pathogenic role in vivo we generated transgenic mice expressing G93A mutant or wild-type (WT) human SOD1 targeted selectively to the mitochondrial IMS (mito-SOD1). We show that mito-SOD1 is correctly localized in the IMS, where it oligomerizes and acquires enzymatic activity. Mito-G93ASOD1 mice, but not mito-WTSOD1 mice, develop a progressive disease characterized by body weight loss, muscle weakness, brain atrophy, and motor impairment, which is more severe in females. These symptoms are associated with reduced spinal motor neuron counts and impaired mitochondrial bioenergetics, characterized by decreased cytochrome oxidase activity and defective calcium handling. However, there is no evidence of muscle denervation, a cardinal pathological feature of ALS. Together, our findings indicate that mutant SOD1 in the mitochondrial IMS causes mitochondrial dysfunction and neurodegeneration, but per se it is not sufficient to cause a full-fledged ALS phenotype, which requires the participation of mutant SOD1 localized in other cellular compartments.

The PER2 clock gene modulates ethanol consumption, such that mutant mice not expressing functional mPer2 have altered circadian behavior that promotes higher ethanol intake and preference. Experiments were undertaken to characterize circadian-related behavioral effects of mPer2 deletion on ethanol intake and to explore how acamprosate (used to reduce alcohol dependence) alters diurnal patterns of ethanol intake. Male mPer2 mutant and WT (wild-type) mice were entrained to a 12:12 h light-dark (12L:12D) photocycle, and their locomotor and drinking activities were recorded. Circadian locomotor measurements confirmed that mPer2 mutants had an advanced onset of nocturnal activity of about 2 h relative to WTs, and an increased duration of nocturnal activity (p < .01). Also, mPer2 mutants preferred and consumed more ethanol and had more daily ethanol drinking episodes vs. WTs. Measurements of systemic ethanol using subcutaneous microdialysis confirmed the advanced rise in ethanol intake in the mPer2 mutants, with 24-h averages being ∼60 vs. ∼25 mM for WTs (p < .01). A 6-day regimen of single intraperitoneal (i.p.) acamprosate injections (300 mg/kg) at zeitgeber time (ZT) 10 did not alter the earlier onset of nocturnal ethanol drinking in the mPer2 mutants, but reduced the overall amplitude of drinking and preference (both p < .01). Acamprosate also reduced these parameters in WTs. These results suggest that elevated ethanol intake in mPer2 mutants may be a partial consequence of an earlier nighttime activity onset and increase in nocturnal drinking activity. The suppressive action of acamprosate on ethanol intake is not due to an altered diurnal pattern of drinking, but rather a decrease in the number of daily drinking bouts and amount of drinking per bout.

Acamprosate suppresses alcohol intake and craving in recovering alcoholics; however, the central sites of its action are unclear. To approach this question, brain regions responsive to acamprosate were mapped using acamprosate microimplants targeted to brain reward and circadian areas implicated in alcohol dependence. mPer2 mutant mice with nonfunctional mPer2, a circadian clock gene that gates endogenous timekeeping, were included, owing to their high levels of ethanol intake and preference. Male wild-type (WT) and mPer2 mutant mice received free-choice (15%) ethanol/water for 3 wk. The ethanol was withdrawn for 3 wk and then reintroduced to facilitate relapse. Four days before ethanol reintroduction, mice received bilateral blank or acamprosate-containing microimplants releasing ∼50 ng/day into reward [ventral tegmental (VTA), peduculopontine tegmentum (PPT), and nucleus accumbens (NA)] and circadian [intergeniculate leaflet (IGL) and suprachiasmatic nucleus (SCN)] areas. The hippocampus was also targeted. Circadian locomotor activity was measured throughout. Ethanol intake and preference were greater in mPer2 mutants than in wild-type (WT) mice (27 g·kg(-1)·day(-1) vs. 13 g·kg(-1)·day(-1) and 70% vs. 50%, respectively; both, P < 0.05). In WTs, acamprosate in all areas, except hippocampus, suppressed ethanol intake and preference (by 40-60%) during ethanol reintroduction. In mPer2 mutants, acamprosate in the VTA, PPT, and SCN suppressed ethanol intake and preference by 20-30%. These data are evidence that acamprosate's suppression of ethanol intake and preference are manifest through actions within major reward and circadian sites.

Background: Alcohol dependence is associated with impaired circadian rhythms and sleep. Ethanol administration disrupts circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol dependence on the circadian timing system. In this study, we extend previous work in C57BL/6J mice to: (i) characterize the suprachiasmatic nucleus (SCN) pharmacokinetics of acute systemic ethanol administration, (ii) explore the effects of acute ethanol on photic and nonphotic phase-resetting, and (iii) determine if the SCN is a direct target for photic effects., Methods: First, microdialysis was used to characterize the pharmacokinetics of acute intraperitoneal (i.p.) injections of 3 doses of ethanol (0.5, 1.0, and 2.0 g/kg) in the mouse SCN circadian clock. Second, the effects of acute i.p. ethanol administration on photic phase delays and serotonergic ([+]8-OH-DPAT-induced) phase advances of the circadian activity rhythm were assessed. Third, the effects of reverse-microdialysis ethanol perfusion of the SCN on photic phase-resetting were characterized., Results: Peak ethanol levels from the 3 doses of ethanol in the SCN occurred within 20 to 40 minutes postinjection with half-lives for clearance ranging from 0.6 to 1.8 hours. Systemic ethanol treatment dose-dependently attenuated photic and serotonergic phase-resetting. This treatment also did not affect basal SCN neuronal activity as assessed by Fos expression. Intra-SCN perfusion with ethanol markedly reduced photic phase delays., Conclusions: These results confirm that acute ethanol attenuates photic phase-delay shifts and serotonergic phase-advance shifts in the mouse. This dual effect could disrupt photic and nonphotic entrainment mechanisms governing circadian clock timing. It is also significant that the SCN clock is a direct target for disruptive effects of ethanol on photic shifting. Such actions by ethanol could underlie the disruptive effects of alcohol abuse on behavioral, physiological, and endocrine rhythms associated with alcoholism., (Copyright © 2011 by the Research Society on Alcoholism.)

In this report we describe an ALS family with a novel missense SOD1 mutation with substitution of serine for cysteine at the sixth amino acid (C6S). This mutation has interesting implications for the role of disulfides in causing disease. After identification of the ALS proband, we examined 17 members of an extended family and performed DNA mutation analysis on 21 family members. The level and activity of SOD1 in C6S carriers and wild-type family members was analyzed in erythrocytes. We found that the C6S mutation results in disease with an autosomal dominant mode of inheritance and markedly reduced penetrance. The S6 mutated protein demonstrates high stability relative to the C6 wild-type protein. The specific dismutation activity of S6 SOD1 is normal. In conclusion, C6S is a novel FALS associated mutation with reduced disease penetrance, long survival time and a phenotype very different from the other SOD1 mutations reported in codon C6. This mutation may provide insight into the role of SOD1 structural changes in disease.

The Alzheimer Disease Genetics Consortium (ADGC) performed a genome-wide association study of late-onset Alzheimer disease using a three-stage design consisting of a discovery stage (stage 1) and two replication stages (stages 2 and 3). Both joint analysis and meta-analysis approaches were used. We obtained genome-wide significant results at MS4A4A (rs4938933; stages 1 and 2, meta-analysis P (P(M)) = 1.7 × 10(-9), joint analysis P (P(J)) = 1.7 × 10(-9); stages 1, 2 and 3, P(M) = 8.2 × 10(-12)), CD2AP (rs9349407; stages 1, 2 and 3, P(M) = 8.6 × 10(-9)), EPHA1 (rs11767557; stages 1, 2 and 3, P(M) = 6.0 × 10(-10)) and CD33 (rs3865444; stages 1, 2 and 3, P(M) = 1.6 × 10(-9)). We also replicated previous associations at CR1 (rs6701713; P(M) = 4.6 × 10(-10), P(J) = 5.2 × 10(-11)), CLU (rs1532278; P(M) = 8.3 × 10(-8), P(J) = 1.9 × 10(-8)), BIN1 (rs7561528; P(M) = 4.0 × 10(-14), P(J) = 5.2 × 10(-14)) and PICALM (rs561655; P(M) = 7.0 × 10(-11), P(J) = 1.0 × 10(-10)), but not at EXOC3L2, to late-onset Alzheimer's disease susceptibility.

FUS, EWS, and TAF15 belong to the TET family of structurally similar DNA/RNA-binding proteins. Mutations in the FUS gene have recently been discovered as a cause of familial amyotrophic lateral sclerosis (FALS). Given the structural and functional similarities between the three genes, we screened TAF15 and EWS in 263 and 94 index FALS cases, respectively. No coding variants were found in EWS, while we identified six novel changes in TAF15. Of these, two 24 bp deletions and a R388H missense variant were also found in healthy controls. A D386N substitution was shown not to segregate with the disease in the affected pedigree. A single A31T and two R395Q changes were identified in FALS cases but not in over 1,100 controls. Interestingly, one of the R395Q FALS cases also harbors a TARDBP mutation (G384R). Altogether, these results suggest that additional studies are needed to determine whether mutations in the TAF15 gene represent a cause of FALS., (Copyright © 2011 Wiley-Liss, Inc.)

Amyotrophic lateral sclerosis and spinal muscular atrophy are devastating neurodegenerative diseases that lead to the specific loss of motor neurons. Recently, stem cell technologies have been developed for the investigation and treatment of both diseases. Here we discuss the different stem cells currently being studied for mechanistic discovery and therapeutic development, including embryonic, adult and induced pluripotent stem cells. We also present supporting evidence for the utilization of stem cell technology in the treatment of amyotrophic lateral sclerosis and spinal muscular atrophy, and describe key issues that must be considered for the transition of stem cell therapies for motor neuron diseases from bench to bedside. Finally, we discuss the first-in-human Phase I trial currently underway examining the safety and feasibility of intraspinal stem cell injections in amyotrophic lateral sclerosis patients as a foundation for translating stem cell therapies for various neurological diseases.

Motor axon degeneration is a critical but poorly understood event leading to weakness and muscle atrophy in motor neuron diseases. Here, we investigated oxidative stress-mediated axonal degeneration in mice lacking the antioxidant enzyme, Cu,Zn superoxide dismutase (SOD1). We demonstrate a progressive motor axonopathy in these mice and show that Sod1(-/-) primary motor neurons extend short axons in vitro with reduced mitochondrial density. Sod1(-/-) neurons also show oxidation of mitochondrial--but not cytosolic--thioredoxin, suggesting that loss of SOD1 causes preferential oxidative stress in mitochondria, a primary source of superoxide in cells. SOD1 is widely regarded as the cytosolic isoform of superoxide dismutase, but is also found in the mitochondrial intermembrane space. The functional significance of SOD1 in the intermembrane space is unknown. We used a transgenic approach to express SOD1 exclusively in the intermembrane space and found that mitochondrial SOD1 is sufficient to prevent biochemical and morphological defects in the Sod1(-/-) model, and to rescue the motor phenotype of these mice when followed to 12 months of age. These results suggest that SOD1 in the mitochondrial intermembrane space is fundamental for motor axon maintenance, and implicate oxidative damage initiated at mitochondrial sites in the pathogenesis of motor axon degeneration.

Background: The genetic basis of amyotrophic lateral sclerosis (ALS) is not entirely clear. While there are families with rare highly penetrant mutations in Cu/Zn superoxide dismutase 1 and several other genes that cause apparent Mendelian inheritance of the disease, most ALS occurs in families without another affected individual. However, twin studies suggest that all ALS has a substantial genetic basis. Herein, we estimate the genetic contribution to ALS in a clinically ascertained case series from the United States., Methodology/principal Findings: We used the database of the Emory ALS Center to ascertain individuals with ALS along with their family histories to determine the concordance among parents and offspring for the disease. We found that concordance for all parent-offspring pairs was low (<2%). With this concordance we found that ALS heritability, or the proportion of the disease explained by genetic factors, is between 40 and 45% for all likely estimates of ALS lifetime prevalence., Conclusions/significance: We found the lifetime risk of ALS is 1.1% in first-degree relatives of those with ALS. Environmental and genetic factors appear nearly equally important for the development of ALS.

Timing of the mammalian circadian clock of the suprachiasmatic nucleus (SCN) is regulated by photic input from the retina. Retinorecipient units entrain rhythmicity of SCN pacemaker cells in part through their release of vasoactive intestinal polypeptide (VIP). The underlying nature of this process is conjectural, however, as in-vivo SCN VIP release has never been measured. Here, SCN microdialysis was used to investigate mechanisms regulating VIP. Hamsters under light-dark cycle of 14:10 exhibited a daily peak in synaptic VIP release near midday. Under constant darkness, this output was arrhythmic. Light and the glutamatergic agonist, N-methyl-D-aspartate, stimulated VIP release at night, whereas the serotonin (1A,7) agonist, (±)8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide, suppressed release at midday. Hence, SCN VIP activity is stimulated by photic input and inhibited by serotonin.

Jonathan D. Glass, M.D., is leading a clinical trial testing the safety of using adult stem cells to treat patients with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that remains untreatable. This trial, along with others like it, is just the beginning of a time-intensive process necessary to determine whether stem-cell treatments are safe and effective, meaning that the benefits-if there prove to be any-outweigh the risks. But even as FDA-approved trials are underway, some people with neurodegenerative disorders are turning to dangerous, unapproved stem-cell treatments out of desperation. Dr. Glass warns that researchers must strictly adhere to the scientific process in order to convert the hope of stem-cell treatments into reality.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons that results in progressive weakness and death from respiratory failure, commonly within about 3 years. Previous studies have shown association of a locus on chromosome 9p with ALS and linkage with ALS-frontotemporal dementia. We aimed to test whether this genomic region is also associated with ALS in an independent set of UK samples, and to identify risk factors associated with ALS in a further genome-wide association study that combined data from the independent analysis with those from other countries., Methods: We collected samples from patients with sporadic ALS from 20 UK hospitals and obtained UK control samples from the control groups of the Depression Case Control study, the Bipolar Affective Case Control Study, and the British 1958 birth cohort DNA collection. Genotyping of DNA in this independent analysis was done with Illumina HumanHap550 BeadChips. We then undertook a joint genome-wide analysis that combined data from the independent set with published data from the UK, USA, Netherlands, Ireland, Italy, France, Sweden, and Belgium. The threshold for significance was p=0·05 in the independent analysis, because we were interested in replicating a small number of previously reported associations, whereas the Bonferroni-corrected threshold for significance in the joint analysis was p=2·20×10(-7), Findings: After quality control, samples were available from 599 patients and 4144 control individuals in the independent set. In this analysis, two single nucleotide polymorphisms in a locus on chromosome 9p21.2 were associated with ALS: rs3849942 (p=2·22×10(-6); odds ratio [OR] 1·39, 95% CI 1·21-1·59) and rs2814707 (p=3·32×10(-6); 1·38, 1·20-1·58). In the joint analysis, which included samples from 4312 patients with ALS and 8425 control individuals, rs3849942 (p=4·64×10(-10); OR 1·22, 95% CI 1·15-1·30) and rs2814707 (p=4·72×10(-10); 1·22, 1·15-1·30) were associated with ALS., Interpretation: We have found strong evidence of a genetic association of two single nucleotide polymorphisms on chromosome 9 with sporadic ALS, in line with findings from previous independent GWAS of ALS and linkage studies of ALS-frontotemporal dementia. Our findings together with these earlier findings suggest that genetic variation at this locus on chromosome 9 causes sporadic ALS and familial ALS-frontotemporal dementia. Resequencing studies and then functional analysis should be done to identify the defective gene., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The discovery of biomarkers for neurodegenerative diseases will have a major impact on the efficiency of therapeutic clinical trials and may be important for understanding basic pathogenic mechanisms. We have approached the discovery of protein biomarkers for amyotrophic lateral sclerosis (ALS) by profiling affected tissues in a relevant animal model and then validating the findings in human tissues. Ventral roots from SOD1(G93A) "ALS" mice were analyzed by label-free quantitative mass spectrometry, and the resulting data were compared with data for matched samples from nontransgenic littermates and transgenic mice carrying wild-type human SOD1 (SOD1(WT)). Of 1299 proteins, statistical inference of the data in the three groups identified 14 proteins that were dramatically altered in the ALS mice compared with the two control groups. The protein galectin-3 emerged as a lead biomarker candidate on the basis of its differential expression as assessed by immunoblot and immunocytochemistry in SOD1(G93A) mice as compared to controls and because it is a secreted protein that could potentially be measured in human biofluids. Spinal cord tissue from ALS patients also exhibited increased levels of galectin-3 when compared to controls. Further measurement of galectin-3 in cerebrospinal fluid samples showed that ALS patients had approximately twice as much galectin-3 as normal and disease controls. These results provide the proof of principle that biomarker identification in relevant and well-controlled animal models can be translated to human disease. The challenge is to validate our biomarker candidate proteins as true biomarkers for ALS that will be useful for diagnosis and/or monitoring disease activity in future clinical trials.

Daily timing of the mammalian circadian clock of the suprachiasmatic nucleus (SCN) is regulated by photic input from the retina via the retinohypothalamic tract. This signaling is mediated by glutamate, which activates SCN retinorecipient units communicating to pacemaker cells in part through the release of gastrin-releasing peptide (GRP). Efferent signaling from the SCN involves another SCN-containing peptide, arginine vasopressin (AVP). Little is known regarding the mechanisms regulating these peptides, as literature on in vivo peptide release in the SCN is sparse. Here, microdialysis-radioimmunoassay procedures were used to characterize mechanisms controlling GRP and AVP release in the hamster SCN. In animals housed under a 14/10-h light-dark cycle both peptides exhibited daily fluctuations of release, with levels increasing during the morning to peak around midday. Under constant darkness, this pattern persisted for AVP, but rhythmicity was altered for GRP, characterized by a broad plateau throughout the subjective night and early subjective day. Neuronal release of the peptides was confirmed by their suppression with reverse-microdialysis perfusion of calcium blockers and stimulation with depolarizing agents. Reverse-microdialysis perfusion with the 5-HT(1A,7) agonist 8-OH-DPAT ((±)-8-hydroxydipropylaminotetralin hydrobromide) during the day significantly suppressed GRP but had little effect on AVP. Also, perfusion with the glutamate agonist NMDA, or exposure to light at night, increased GRP but did not affect AVP. These analyses reveal distinct daily rhythms of SCN peptidergic activity, with GRP but not AVP release attenuated by serotonergic activation that inhibits photic phase-resetting, and activated by glutamatergic and photic stimulation that mediate this phase-resetting., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

A series of peptidyl alpha-ketoamides with the general structure Cbz-L-Leu-D,L-AA-CONH-R were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. Nucleobases, methylpiperazine, and dimethylaminoalkyl groups were incorporated into the primed region of the inhibitors to generate compounds that potentially cross the blood-brain barrier. Two of these compounds (Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-adenin-9-yl and Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-(4-methylpiperazin-1-yl) have been shown to have useful concentrations in the brain in animals. The best inhibitor for calpain I was Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-2-methoxyadenin-9-yl (K(i) = 23 nM), and the best inhibitor for calpain II was Cbz-Leu-D,L-Phe-CONH-(CH(2))(3)-adenin-9-yl (K(i) = 68 nM). On the basis of the crystal structure obtained with heterocyclic peptidyl alpha-ketoamides, we have improved inhibitor potency by introducing a small hydrophobic group on the adenine ring. These inhibitors have good potential to be used in the treatment of neurodegenerative diseases.

Background: Alcohol abuse leads to marked disruptions of circadian rhythms, and these disturbances in themselves can increase the drive to drink. Circadian clock timing is regulated by light, as well as by nonphotic influences such as food, social interactions, and wheel running. We previously reported that alcohol markedly disrupts photic and nonphotic modes of circadian rhythm regulation in Syrian hamsters. As an extension of this work, we characterize the hedonic interrelationship between wheel running and ethanol (EtOH) intake and the effects of environmental circadian disruption (long-term exposure to constant light [LL]) on the drive to drink., Methods: First, we tested the effect of wheel running on chronic free-choice consumption of a 20% (v/v) EtOH solution and water. Second, the effect of this alcohol drinking on wheel running in alcohol-naive animals was investigated. Third, we assessed the influence of LL, known to suppress locomotor activity and cause circadian rhythm disruption, on EtOH consumption and wheel-running behavior., Results: Inhibitory effects of wheel running on EtOH intake and vice versa were observed. Exposure to LL, while not affecting EtOH intake, induced rhythm splitting in 75% of the animals. Notably, the splitting phenotype was associated with lower levels of EtOH consumption and preference prior to, and throughout, the period of LL exposure., Conclusions: These results are evidence that exercise may offer an efficacious clinical approach to reducing EtOH intake. Also, predisposition for light-induced (or other) forms of circadian disruption may modulate the drive to drink.

Three clustered, homologous paraoxonase genes (PON1, PON2, and PON3) have roles in preventing lipid oxidation and detoxifying organophosphates. Recent reports describe a genetic association between the PON genes and sporadic amyotrophic lateral sclerosis (ALS). We now report that in genomic DNA from individuals with familial and sporadic ALS, we have identified at least 7 PON gene mutations that are predicted to alter PON function.

Background: Chronic ethanol abuse is associated with disrupted circadian rhythms and sleep. Ethanol administration impairs circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol abuse on circadian timing. Here, we extend previous studies to explore the effects of chronic forced ethanol on photic phase-resetting, photic entrainment, and daily locomotor activity patterns in C57BL/6J mice., Methods: First, microdialysis was used to characterize the circadian patterns of ethanol uptake in the suprachiasmatic (SCN) circadian clock and correlate this with systemic ethanol levels and episodic drinking of 10 or 15% ethanol. Second, the effects of chronic forced ethanol drinking and withdrawal on photic phase-delays of the circadian activity rhythm were assessed. Third, the effects of chronic ethanol drinking on entrainment to a weak photic zeitgeber (1 minute of 25 lux intensity light per day) were assessed. This method was used to minimize any masking actions of light that could mask ethanol effects on clock entrainment., Results: Peak ethanol levels in the SCN and periphery occurred during the dark phase and coincided with the time when light normally induces phase-delays in mice. These delays were dose-dependently inhibited by chronic ethanol and its withdrawal. Chronic ethanol did not impede re-entrainment to a shifted light cycle but affected entrainment under the weak photic zeitgeber and disrupted the daily pattern of locomotor activity., Conclusions: These results confirm that chronic ethanol consumption and withdrawal markedly impair circadian clock photic phase-resetting. Ethanol also disturbs the temporal structure of nighttime locomotor activity and photic entrainment. Collectively, these results suggest a direct action of ethanol on the SCN clock.

More than 30 phase II or III clinical trials have been carried out in amyotrophic lateral sclerosis (ALS). Only riluzole, however, has been shown to extend survival and/or time to tracheostomy. Many early ALS trials lacked solid pharmacodynamic and pharmacokinetic data for the treatment being tested, challenging the interpretation of the efficacy and pathway relevance. Understanding of the genetics and pathophysiology of ALS has improved considerably in the past decade, but biomarkers of disease activity remain lacking. A more efficient approach to early phase clinical trials is needed to accelerate the identification of useful agents for ALS. Here we summarize our current thinking about phase II design options and the potential benefits of a clinical trial network for phase II trials in ALS.

Background: Peripheral neuropathy is the dose-limiting toxicity of stavudine and didanosine (nucleoside analogs used in HIV treatment) and is attributed to mitochondrial toxicity from these drugs. Acetyl L-carnitine (ALC) and co-enzyme Q(10) are proposed as neuropathy treatments, but evidence to support these is limited., Methods: We examined ALC and a water-soluble formulation of co-enzyme Q(10) (H(Q)O) for the prevention of d4T and ddI neurotoxicity using cultured fetal rat DRG as an in vitro model., Results: DdI (33microM) and d4T (50microM) caused clear toxicity (impaired neurite growth) by day 8 of DRG culture. H(Q)O at concentrations 1-100microM completely prevented the toxicity of 33microM ddI in vitro and ALC at concentrations 1-100 microM substantially (but incompletely) prevented ddI toxicity in this model. In contrast, ALC was ineffective at all concentrations tested for preventing the toxicity of 50microM d4T. H(Q)O showed dose-dependent efficacy for preventing d4T toxicity. H(Q)O (1microM) partially prevented d4T toxicity while 10 and 100microM H(Q)O completely prevented d4T toxicity in this model., Conclusions: We find H(Q)O is superior to ALC for preventing the neurotoxicity of d4T (the HIV treatment most associated with neuropathy) and ddI in vitro. Further study is needed to clarify any clinical role for co-enzyme Q(10) co-administration with d4T and ddI and to assess whether this compound may have a role in treating established cases of neuropathy.

Background: Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinson's disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed., Methods and Findings: Here we present the neurobiological actions of dopamine neuron stimulating peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of fetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth. In MN9D cells, DNSP-11 protects against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced cell death, significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the normal adult rat substantia nigra is taken up rapidly into neurons and increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotational behavior, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Unlike GDNF, DNSP-11 was found to block staurosporine- and gramicidin-induced cytotoxicity in nutrient-deprived dopaminergic B65 cells, and its neuroprotective effects included preventing the release of cytochrome c from mitochondria., Conclusions: Collectively, these data support that DNSP-11 exhibits potent neurotrophic actions analogous to GDNF, making it a viable candidate for a PD therapeutic. However, it likely signals through pathways that do not directly involve the GFRalpha1 receptor.

Timing of the circadian clock of the suprachiasmatic nucleus (SCN) is regulated by photic and non-photic inputs. Of these, neuropeptide Y (NPY) signaling from the intergeniculate leaflet (IGL) to the SCN plays a prominent role. Although NPY is critical to clock regulation, neither the mechanisms modulating IGL NPY neuronal activity nor the nature of regulatory NPY signaling in the SCN clock are understood, as NPY release in the SCN has never been measured. Here, microdialysis procedures for in vivo measurement of NPY were used in complementary experiments to address these questions. First, neuronal release of NPY in the hamster SCN was rhythmic under a 14L : 10D photocycle, with the acrophase soon after lights-on and the nadir at midday. No rhythmic fluctuation in NPY occurred under constant darkness. Second, a behavioral phase-resetting stimulus (wheel-running at midday that induces IGL serotonin release) acutely stimulated SCN NPY release. Third, bilateral IGL microinjection of the serotonin agonist, (+/-)-2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT) (another non-photic phase-resetting stimulant), at midday enhanced SCN NPY release. Conversely, similar application of the serotonin antagonist, metergoline, abolished wheel-running-induced SCN NPY release. IGL microinjection of the GABA agonist, muscimol, suppressed SCN NPY release. These results support an intra-IGL mechanism whereby behavior-induced serotonergic activity suppresses inhibitory GABAergic transmission, promoting NPY activity and subsequent phase resetting. Collectively, these results confirm IGL-mediated NPY release in the SCN and verify that its daily rhythm of release is dependent upon the 14L : 10D photocycle, and that it is modulated by appropriately-timed phase-resetting behavior, probably mediated by serotonergic activation of NPY units in the IGL.

Axonal degeneration is a common pathologic feature in peripheral neuropathy, neurodegenerative disease, and normal aging. Oxidative stress may be an important mechanism of axonal degeneration, but is underrepresented among current experimental models. To test the effects of loss of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) on axon survival, we cultured dorsal root ganglion (DRG) neurons from SOD1 knockout mice. Beginning as early as 48-72 h, we observed striking degeneration of Sod1-/- axons that was prevented by introduction of human SOD1 and was attenuated by antioxidant treatment. To test susceptibility to increased superoxide production, we exposed wild-type DRGs to the redox-cycling herbicides paraquat and diquat (DQ). Dose-dependent axon degeneration was observed, and toxicity of DQ was exacerbated by SOD1 deficiency. MTT staining suggested that DRG axons are more susceptible to injury than their parent cell bodies in both paradigms. Taken together, these data demonstrate susceptibility of DRG axons to oxidative stress-mediated injury due to loss of SOD1 or excess superoxide production. These in vitro models provide a novel means of investigating oxidative stress-mediated injury to axons, to improve our understanding of axonal redox control and dysfunction in peripheral neuropathy.

(S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (EM011) is a tubulin-binding agent with significant anticancer activity. Here we show that EM011 modulates microtubule dynamics at concentrations that do not alter the total polymer mass of tubulin. In particular, EM011 decreases the transition frequencies between growth and shortening phases and increases the duration microtubules spend in an idle 'pause' state. Using B16LS9 murine melanoma cells, we show that EM011 briefly arrests cell-cycle progression at the G2/M phase by formation of multiple aster spindles. An aberrant mitotic exit without cytokinesis then occurs, leading to the accumulation of abnormal multinucleated cells prior to apoptosis. Our pharmacokinetic studies conformed to a linear dose-response relationship upto 150 mg/kg. However, non-linearity was observed at 300 mg/kg. In a syngeneic murine model of subcutaneous melanoma, better antitumor responses were seen at 150 mg/kg compared to 300 mg/kg of EM011. Unlike currently available chemotherapeutics, EM011 is non-toxic to normal tissues and most importantly, does not cause any immunosuppression and neurotoxicity. Our data thus warrant a clinical evaluation of EM011 for melanoma therapy.

Ethanol modulates the actions of multiple neurotransmitter systems, including GABA. However, its enhancing effects on GABA signaling typically are seen only at high concentrations. In contrast, although GABA is a prominent neurotransmitter in the circadian clock of the suprachiasmatic nucleus (SCN), we see ethanol modulation of clock phase resetting at low concentrations (<50 mM). A possible explanation is that ethanol enhances GABAergic signaling in the SCN through activating GABA(A) receptors that contain the delta subunit (GABA(Adelta) receptors), which are sensitive to low ethanol concentrations. Therefore, we investigated whether ethanol acts on GABA(Adelta) receptors in the SCN. Here we show that acute application of the GABA(Adelta) receptor antagonist, RO15-4513, to mouse hypothalamic slices containing the SCN prevents ethanol inhibition of nighttime glutamate-induced (photic-like) phase delays of the circadian clock. Diazepam, which enhances activity of GABA(A) receptors containing the gamma subunit (GABA(Agamma) receptors), does not modulate these phase shifts. Moreover, we find that RO15-4513 prevents ethanol enhancement of daytime serotonergic (non-photic) phase advances of the circadian clock. Furthermore, diazepam phase-advances the SCN circadian clock when applied alone in the daytime, while ethanol has no effect by itself at that time. These data support the hypothesis that ethanol acts on GABA(Adelta) receptors in the SCN to modulate photic and non-photic circadian clock phase resetting. They also reveal distinct modulatory roles of different GABA(A) receptor subtypes in circadian clock phase regulation.

Background: Tolerance to ethanol is observed over a variety of time courses, from minutes to days. Acute tolerance, which develops over 5 to 60 minutes, has been observed for both behavioral and neurophysiological variables and may involve changes in signaling through NMDA, GABA, or other receptors. Previous work has shown that both acute and chronic ethanol treatments modulate photic and nonphotic phase resetting of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Although not specifically tested, the data thus far do not point to the development of chronic tolerance to the modulatory effects of ethanol. Here we investigated whether acute tolerance the ethanol occurs with respect to in vitro phase modulation of the SCN clock., Methods: Mouse brain slices containing the SCN were pretreated with ethanol for varying lengths of time, followed by treatment concurrent with either glutamate or the serotonin agonist, 8-hydroxy-DPAT (DPAT). The phase of the SCN circadian clock was assessed the following day through extracellular recordings of SCN neuronal activity. SCN neuronal activity normally peaks during mid-day, and this rhythm can be shifted by treatment with either glutamate or DPAT., Results: While concurrent treatment of SCN-containing brain slices with ethanol and glutamate blocks glutamate-induced phase delays of the SCN clock, pretreating the slices with ethanol for > or =15 minutes prevents this inhibition. Likewise, while concurrent treatment with ethanol and DPAT enhances DPAT-induced phase advances of the SCN clock, pretreating the slices with ethanol for > or =30 minutes prevents this enhancement., Conclusions: Both the inhibiting and enhancing effects of ethanol on in vitro SCN clock phase resetting show acute tolerance. Additional experiments are needed to determine whether more slowly developing forms of tolerance also occur with respect to the SCN circadian clock.

Mutations in the enzyme superoxide dismutase 1 (SOD1) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this issue of the JCI, Zhong et al. report that the endogenous anticoagulant activated protein C (APC) is able to cross the blood-spinal cord barrier in mice and signal to both neuronal and non-neuronal cells (see the related article beginning on page 3437). This signaling resulted in the suppression of mutant SOD1 synthesis and retarded disease progression in a murine model of ALS. Here we discuss the potential importance of these data and possible relevance to human neurodegenerative diseases.

We conducted a genome-wide association study among 2,323 individuals with sporadic amyotrophic lateral sclerosis (ALS) and 9,013 control subjects and evaluated all SNPs with P < 1.0 x 10(-4) in a second, independent cohort of 2,532 affected individuals and 5,940 controls. Analysis of the genome-wide data revealed genome-wide significance for one SNP, rs12608932, with P = 1.30 x 10(-9). This SNP showed robust replication in the second cohort (P = 1.86 x 10(-6)), and a combined analysis over the two stages yielded P = 2.53 x 10(-14). The rs12608932 SNP is located at 19p13.3 and maps to a haplotype block within the boundaries of UNC13A, which regulates the release of neurotransmitters such as glutamate at neuromuscular synapses. Follow-up of additional SNPs showed genome-wide significance for two further SNPs (rs2814707, with P = 7.45 x 10(-9), and rs3849942, with P = 1.01 x 10(-8)) in the combined analysis of both stages. These SNPs are located at chromosome 9p21.2, in a linkage region for familial ALS with frontotemporal dementia found previously in several large pedigrees.

Acute ethanol (EtOH) administration impairs circadian clock phase resetting, suggesting a mode for the disruptive effect of alcohol abuse on human circadian rhythms. Here, we extend this research by characterizing the chronobiological effects of chronic alcohol consumption. First, daily profiles of EtOH were measured in the suprachiasmatic nucleus (SCN) and subcutaneously using microdialysis in hamsters drinking EtOH. In both cases, EtOH peaked near lights-off and declined throughout the dark-phase to low day-time levels. Drinking bouts preceded EtOH peaks by approximately 20 min. Second, hamsters chronically drinking EtOH received a light pulse during the late dark phase [Zeitgeber time (ZT) 18.5] to induce photic phase advances. Water controls had shifts of 1.2 +/- 0.2 h, whereas those drinking 10% and 20% EtOH had much reduced shifts (0.5 +/- 0.1 and 0.3 +/- 0.1 h, respectively; P < 0.001 vs. controls). Third, incremental decreases in light intensity (270 lux to 0.5 lux) were used to explore chronic EtOH effects on photic entrainment and rhythm stability. Activity onset was unaffected by 20% EtOH at all light intensities. Conversely, the 24-h pattern of activity bouts was disrupted by EtOH under all light intensities. Finally, replacement of chronic EtOH with water was used to examine withdrawal effects. Water controls had photic phase advances of 1.1 +/- 0.3 h, while hamsters deprived of EtOH for 2-3 days showed enhanced shifts (2.1 +/- 0.3 h; P < 0.05 vs. controls). Thus, in chronically drinking hamsters, brain EtOH levels are sufficient to inhibit photic phase resetting and disrupt circadian activity. Chronic EtOH did not impair photic entrainment; however, its replacement with water potentiated photic phase resetting.

Amyotrophic lateral sclerosis is a degenerative disorder of motor neurons that typically develops in the 6th decade and is uniformly fatal, usually within 5 years. To identify genetic variants associated with susceptibility and phenotypes in sporadic ALS, we performed a genome-wide SNP analysis in sporadic ALS cases and controls. A total of 288,357 SNPs were screened in a set of 1,821 sporadic ALS cases and 2,258 controls from the U.S. and Europe. Survival analysis was performed using 1,014 deceased sporadic cases. Top results for susceptibility were further screened in an independent sample set of 538 ALS cases and 556 controls. SNP rs1541160 within the KIFAP3 gene (encoding a kinesin-associated protein) yielded a genome-wide significant result (P = 1.84 x 10(-8)) that withstood Bonferroni correction for association with survival. Homozygosity for the favorable allele (CC) conferred a 14.0 months survival advantage. Sequence, genotypic and functional analyses revealed that there is linkage disequilibrium between rs1541160 and SNP rs522444 within the KIFAP3 promoter and that the favorable alleles of rs1541160 and rs522444 correlate with reduced KIFAP3 expression. No SNPs were associated with risk of sporadic ALS, site of onset, or age of onset. We have identified a variant within the KIFAP3 gene that is associated with decreased KIFAP3 expression and increased survival in sporadic ALS. These findings support the view that genetic factors modify phenotypes in this disease and that cellular motor proteins are determinants of motor neuron viability.

Neuropathy and myopathy can cause weakness during critical illness. To determine whether reduced excitability of peripheral nerves, rather than degeneration, is the mechanism underlying acute neuropathy in critically ill patients, we prospectively followed patients during the acute phase of critical illness and early recovery and assessed nerve conduction. During the period of early recovery from critical illness, patients recovered from neuropathy within days. This rapidly reversible neuropathy has not to our knowledge been previously described in critically ill patients and may be a novel type of neuropathy. In vivo intracellular recordings from dorsal root axons in septic rats revealed reduced action potential amplitude, demonstrating that reduced excitability of nerve was the mechanism underlying neuropathy. When action potentials were triggered by hyperpolarizing pulses, their amplitudes largely recovered, indicating that inactivation of sodium channels was an important contributor to reduced excitability. There was no depolarization of axon resting potential in septic rats, which ruled out a contribution of resting potential to the increased inactivation of sodium channels. Our data suggest that a hyperpolarized shift in the voltage dependence of sodium channel inactivation causes increased sodium inactivation and reduced excitability. Acquired sodium channelopathy may be the mechanism underlying acute neuropathy in critically ill patients.

Brief ( approximately 2 day) constant light exposure (LL(b)) in hamsters dramatically enhances circadian phase-resetting induced by the 5-HT receptor agonist, (+/-)-2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT) and other nonphotic stimuli. The present study was undertaken to determine if LL(b) can also amplify phase-resetting responses to endogenous 5-HT and accelerate re-entrainment to large-magnitude advance and delay shifts of the light/dark (LD) cycle. First, central serotonergic activity was increased by i.p. injection of L-tryptophan+/-the 5-HT reuptake inhibitor fluoxetine. Hamsters under LD or exposed to LL(b) received vehicle or drugs during the early morning, and phase-shifts of the locomotor activity rhythm were measured after release to constant darkness. Neither drug phase-shifted animals not exposed to LL(b) (P>0.5 vs. vehicle); however in animals receiving LL(b,)L-tryptophan with and without fluoxetine produced large phase-advance shifts (means=2.5+/-0.4 h and 2.6+/-0.2 h, respectively; both P<0.035 vs. vehicle). Next, the effects of LL(b) combined with 8-OH-DPAT or L-tryptophan+fluoxetine on serotonergic re-entrainment to 10 h phase-advance and phase-delay shifts of the LD cycle were assessed. In groups not exposed to LL(b), vehicle controls re-entrained slowly to the advance and delay shifts (means=16+/-1 and 24+/-4 days, respectively), but those treated with 8-OH-DPAT re-entrained faster (means=11+/-2 and 9+/-2 days, respectively; both P<0.05 vs. vehicle). In groups exposed to LL(b), vehicle controls re-entrained slowly to the advance and delay shifts (means=15+/-2 and 25+/-3 days, respectively); however those receiving 8-OH-DPAT rapidly re-entrained to the delay and advance shifts, with the majority (75%) requiring only 1-2 days (means=2+/-1 and 4+/-2 days, respectively; both P<0.05 vs. vehicle). Animals exposed to LL(b) and treated with L-tryptophan+fluoxetine also exhibited accelerated re-entrainment to a 10 h advance shift (mean=5+/-2 days; P<0.05 vs. vehicle). Thus through enhancing serotonergic phase-resetting, LL(b) facilitates rapid re-entrainment to large shifts of the LD cycle which offers a potential approach for treating circadian-related desynchronies.

Disrupted circadian rhythmicity is associated with ethanol (EtOH) abuse, yet little is known about how EtOH affects the mammalian circadian clock of the suprachiasmatic nucleus (SCN). Clock timing is regulated by photic and nonphotic inputs to the SCN involving glutamate release from the retinohypothalamic tract and serotonin (5-HT) from the midbrain raphe, respectively. Our recent in vitro studies in the SCN slice revealed that EtOH blocks photic phase-resetting action of glutamate and enhances the nonphotic phase-resetting action of the 5-HT1A,7 agonist, 8-OH-DPAT. To explore the basis of these effects in the whole animal, we used microdialysis to characterize the pharmacokinetics of intraperitoneal injection of EtOH in the hamster SCN extracellular fluid compartment and then studied the effects of such EtOH treatment on photic and serotonergic phase resetting of the circadian locomotor activity rhythm. Peak EtOH levels (approximately 50 mM) from a 2 g/kg injection occurred within 20-40 min with a half-life of approximately 3 h. EtOH treatment dose-dependently attenuated photic phase advances but had no effect on phase delays and, contrary to in vitro findings, markedly attenuated 8-OH-DPAT-induced phase advances. In a complementary experiment using reverse microdialysis to deliver a timed SCN perfusion of EtOH during a phase-advancing light pulse, the phase advances were blocked, similar to systemic EtOH treatment. These results are evidence that acute EtOH significantly affects photic and nonphotic phase-resetting responses critical to circadian clock regulation. Notably, EtOH inhibition of photic signaling is manifest through direct action in the SCN. Such actions could underlie the disruption of circadian rhythmicity associated with alcohol abuse.

Computerized treadmill gait analysis in models of toxicant exposure and neurodegenerative disorders holds much potential for detection and therapeutic intervention in these models, and researchers must validate the technology that assists in that data collection and analysis. The present authors used a commercially available computerized gait analysis system that used (a) a motorized treadmill on retired breeder male C57BL/6J mice, (b) the toxicant-induced (1-methyl-1-, 2-, 3-, 6-tetrahydropyridine) MPTP mouse model of Parkinson's disease (PD), and (c) the superoxide dismutase 1 (SOD1) G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS). The authors compared the detection of deficits by computerized treadmill gait analysis in MPTP-treated mice with inked-paw stride length and correlated these measures to dopamine (DA) loss. The authors found that the computerized treadmill gait analysis system did not distinguish MPTP-treated mice from vehicle controls, despite a nearly 90% deficit of striatal DA. In contrast, decreases in inked-paw stride length correlated strongly with DA losses in these same animals. Computerized treadmill gait analysis could neither reliably distinguish SOD1 G93A mutant mice from controls from 6 to 12 weeks of age nor detect any consistent early motor deficits in these mice. On the basis of the authors' findings, they inferred that computerized gait analysis on a motorized treadmill is not suited to measuring motor deficits in either the MPTP mouse model of PD or the SOD1 G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disorder of upper and lower motor neurons. Genetic variants in the paraoxonase gene cluster have been associated with susceptibility to sporadic ALS. Because these studies have yielded conflicting results, we have further investigated this association in a larger data set. Twenty SNPs spanning the paraoxonase gene cluster were genotyped on a panel of 597 case and 692 control samples and tested for association with risk of sporadic ALS and with ALS sub-phenotypes. Our study revealed two SNPs, rs987539 and rs2074351, within the paraoxonase gene cluster that are associated with susceptibility to sporadic ALS (uncorrected p=6.47E-04 and 7.87E-04, respectively). None of the 20 SNPs displayed significant associations with age of onset, site of onset or disease survival. Using a sliding window approach, we have also identified a 5-SNP haplotype that is significantly associated with risk of sporadic ALS (p=2.75E-05). We conclude that a common haplotype within the PON1 promoter region is associated with susceptibility to sporadic ALS.

The objective was to test the hypothesis that a described association between homozygosity for a 50bp deletion in the SOD1 promoter 1684bp upstream of the SOD1 ATG and an increased age of onset in SALS can be replicated in additional SALS and control sample sets from other populations. Our second objective was to examine whether this deletion attenuates expression of the SOD1 gene. Genomic DNA from more than 1200 SALS cases from Ireland, Scotland, Quebec and the USA was genotyped for the 50bp SOD1 promoter deletion. Reporter gene expression analysis, electrophoretic mobility shift assays and chromatin immunoprecipitation studies were utilized to examine the functional effects of the deletion. The genetic association for homozygosity for the promoter deletion with an increased age of symptom onset was confirmed overall in this further study (p=0.032), although it was only statistically significant in the Irish subset, and remained highly significant in the combined set of all cohorts (p=0.001). Functional studies demonstrated that this polymorphism reduces the activity of the SOD1 promoter by approximately 50%. In addition we revealed that the transcription factor SP1 binds within the 50bp deletion region in vitro and in vivo. Our findings suggest the hypothesis that this deletion reduces expression of the SOD1 gene and that levels of the SOD1 protein may modify the phenotype of SALS within selected populations.

Objective: Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disorder involving upper and lower motor neurons. The vesicle-associated membrane protein B (VAPB) gene has been genetically linked to ALS in several large Brazilian families in which the disorder is caused by a proline to serine mutation at codon 56 (P56S). No additional mutations have been identified., Methods: To establish the prevalence of VAPB mutations, we screened 80 familial ALS samples by DNA sequencing., Results: Our study failed to identify any novel VAPB gene mutations but identified a single Brazilian family harboring the P56S mutation. In a second familial ALS case, we identified a three-base pair deletion within exon 5 of the VAPB gene that deleted the serine residue at position 160 (Delta S160). This variant is detected in a normal population at low frequency (0.45%). Analyses of homology alignment and secondary structure predict that this deletion significantly alters the structure of VAPB, although a GFP-Delta S160 VAPB fusion protein demonstrates a wild-type subcellular localization. This contrasts the aberrant localization observed in a GFP-P56S VAPB fusion protein. The allele frequency of Delta S160 in patients with sporadic ALS does not differ significantly from that in the normal population., Conclusions: Mutations in the VAPB gene are rare and the Delta S160 variant does not contribute to the development of amyotrophic lateral sclerosis.

Alcohol abuse is associated with sleep problems, which are often linked to circadian rhythm disturbances. However, there is no information on the direct effects of ethanol on the mammalian circadian clock. Acute ethanol inhibits glutamate signaling, which is the primary mechanism through which light resets the mammalian clock in the suprachiasmatic nucleus (SCN). Glutamate and light also inhibit circadian clock resetting induced by nonphotic signals, including 5-HT. Thus, we investigated the effects of acute ethanol on both glutamatergic and serotoninergic resetting of the mouse SCN clock in vitro. We show that ethanol dose-dependently inhibits glutamate-induced phase shifts and enhances serotonergic phase shifts. The inhibition of glutamate-induced phase shifts is not affected by excess glutamate, glycine or d-serine, but is prevented by excess brain-derived neurotrophic factor (BDNF). BDNF is known to augment glutamate signaling in the SCN and to be necessary for glutamate/light-induced phase shifts. Thus, ethanol may inhibit glutamate-induced clock resetting at least in part by blocking BDNF enhancement of glutamate signaling. Ethanol enhancement of serotonergic phase shifts is mimicked by treatments that suppress glutamate signaling in the SCN, including antagonists of glutamate receptors, BDNF signaling and nitric oxide synthase. The combined effect of ethanol with these treatments is not additive, suggesting they act through a common pathway. Our data indicate further that the interaction between 5-HT and glutamate in the SCN may occur downstream from nitric oxide synthase activation. Thus, acute ethanol disrupts normal circadian clock phase regulation, which could contribute to the physiological and psychological problems associated with alcohol abuse.

The distal to proximal degeneration of axons, or "dying back" is a common pattern of neuropathology in many diseases of the PNS and CNS. A long-standing debate has centered on whether this pattern of neurodegeneration is due to an insult to the cell body or to the axon itself, although it is likely that mechanisms are different for specific disease entities. We have addressed this question in a model system of vincristine-induced axonal degeneration. Here, we created a novel experimental apparatus combining a microfluidic divider with a multielectrode array substrate to allow for independent monitoring of injury-induced electrical activity from dorsal root ganglion (DRG) cell bodies and axons while isolating them into their own culture microenvironments. At specified doses, exposure of the cell body to vincristine caused neither morphological neurodegeneration nor persistent hyperexcitability. In comparison, exposure of the distal axon to the same dose of vincristine first caused a decrease in the excitability of the axon and then axonal degeneration in a dying back pattern. Additionally, exposure of axons to vincristine caused an initial period of hyperexcitability in the cell bodies, suggesting that a signal is transmitted from the distal axon to the soma during the progression of vincristine-induced axonal degeneration. These data support the proposition that vincristine has a direct neurotoxic effect on the axon.

This paper describes a microfabricated compartmented culture system (mu-CCS) for studying the effects of drugs on cultured neurons. We describe the fabrication of the microsystem and show the ability to culture DRG neurons in the microsystem. Furthermore, we demonstrate the ability to culture neurons with extensions growing into adjoining compartments while maintaining fluid isolation between compartments. The axonal growth pattern was controlled along the surface of the glass microelectrode substrate using a micropatterned collagen film. The ability to isolate fluids to selected compartments while simultaneously allowing intercompartmental growth of the axons enables various studies in which selected segments of neurons or populations of neurons can be selectively exposed to biochemical treatment. The neurotoxin vincristine was used as the test vehicle to demonstrate the functionality of the mu-CCS. Vincristine was applied to the axonal compartment to show that the interaction of drugs with DRG neurons progresses in a way similar to neurons cultured/exposed using conventional techniques.

The slow Wallerian degeneration protein (Wld(S)), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld(S) mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld(S) mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld(S), and lentiviral overexpressed enzyme-dead Wld(S) still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld(S) at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld(S) requires more N-terminal sequences of the protein.

Growing evidence from animal models and patients with amyotrophic lateral sclerosis (ALS) suggests that distal axonal degeneration begins very early in this disease, long before symptom onset and motor neuron death. The cause of axonal degeneration is unknown, and may involve local axonal damage, withdrawal of trophic support from a diseased cell body, or both. It is increasingly clear that axons are not passive extensions of their parent cell bodies, and may die by mechanisms independent of cell death. This is supported by studies in which protection of motor neurons in models of ALS did not significantly improve symptoms or prolong lifespan, likely due to a failure to protect axons. Here, we will review the evidence for early axonal degeneration in ALS, and discuss possible mechanisms by which it might occur, with a focus on oxidative stress. We contend that axonal degeneration may be a primary feature in the pathogenesis of motor neuron disease, and that preventing axonal degeneration represents an important therapeutic target that deserves increased attention., (Copyright (c) 2007 S. Karger AG, Basel.)