Effect of recurrent yohimbine on immediate and post-hoc behaviors, stress hormones, and energy homeostatic parameters.

Evidence from experimental models has suggested that acute activation of brain stress and anxiety pathways impacts subsequent behaviors that are mediated or modulated by limbic circuitry. There have been limited investigations of prior or chronic activation of these pathways on subsequent limbic-mediated behaviors. In this study, we tested whether recurrent administration of the anxiogenic compound yohimbine (YOH) could have post-injection effects on brain activation, stress hormones, and performance in sucrose self-administration and startle response paradigms. Rats received six injections across two weeks of either 2mg/kg YOH or saline. Behavioral evaluation confirmed the continued efficacy of the YOH regimen, and increased adrenal corticosterone (CORT) was observed. Several days following YOH or SAL administration, cFos, CORT and adrenocorticotropin hormone (ACTH), and behavioral performance were measured. cFos was elevated post-YOH in the hippocampus; ventral tegmental area/zona inserta; and central and medial nuclei of the amygdala. This activation is consistent with a sustained effect of YOH to activate fear and anxiety circuitries in the CNS. CORT but not ACTH was elevated in the YOH-rats following startle testing. Self-administration and startle tests suggested an increase of non-specific activity in the post-YOH rats; there was no increase in sucrose self-administration or startle response per se. Our findings suggest that recurrent YOH administration may prove a useful and reliable model for simulating recurrent stress/anxiety, and that enhancements to the paradigm such as higher or more frequent dosing of YOH could yield stronger or more extensive behavioral effects.

Establishing a Canadian registry of patients with amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating cause of progressive weakness, respiratory failure and death. To date there is no effective therapy to meaningfully extend survival but continuously emerging targets and putative treatments are studied in clinical trials. Canadian epidemiological data on ALS is scarce and the socioeconomic impact of ALS on Canadian society is unclear. The Canadian Neuromuscular Disease Registry (CNDR) is a national clinic-based registry of patients with neuromuscular diseases with the goal of facilitating the design and execution of clinical research. METHODS: We conducted a national stakeholder survey to assess interest for a Canadian ALS registry and an assessment of expected case ascertainment. A dataset derivation meeting was held to establish the registry medical dataset. RESULTS: We report the results of the national stakeholder survey, case ascertainment assessment, and the derived dataset that have resulted in the current implementation of a Canadian registry of patients with ALS. CONCLUSIONS: The development of this long sought-after resource is a significant step forward for the Canadian ALS patient and research communities that will result in more efficient clinical trial recruitment and advancements in our understanding of ALS in Canada.

Loss of islet sympathetic nerves and impairment of glucagon secretion in the NOD mouse: relationship to invasive insulitis.

AIMS/HYPOTHESIS: We hypothesised that non-obese diabetic mice (NOD) mice have an autoimmune-mediated loss of islet sympathetic nerves and an impairment of sympathetically mediated glucagon responses. We aimed: (1) to determine whether diabetic NOD mice have an early impairment of the glucagon response to insulin-induced hypoglycaemia (IIH) and a coincident loss of islet sympathetic nerves; (2) to determine whether invasive insulitis is required for this nerve loss; and (3) to determine whether sympathetically mediated glucagon responses are also impaired. METHODS: We measured glucagon responses to both IIH and tyramine in anaesthetised mice. We used immunohistochemistry to quantify islet sympathetic nerves and invasive insulitis. RESULTS: The glucagon response to IIH was markedly impaired in NOD mice after only 3 weeks of diabetes (change, -70%). Sympathetic nerve area within the islet was also markedly reduced at this time (change, -66%). This islet nerve loss was proportional to the degree of invasive insulitis. More importantly, blocking the infiltration prevented the nerve loss. Mice with autoimmune diabetes had an impaired glucagon response to sympathetic nerve activation, whereas those with non-autoimmune diabetes did not. CONCLUSIONS/INTERPRETATION: The invasive insulitis seen in diabetic NOD mice causes early sympathetic islet neuropathy. Further studies are needed to confirm that early sympathetic islet neuropathy is responsible for the impaired glucagon response to tyramine.

Effect of moderate intake of sweeteners on metabolic health in the rat.

The rise in prevalence of obesity, diabetes, metabolic syndrome, and fatty liver disease has been linked to increased consumption of fructose-containing foods or beverages. Our aim was to compare the effects of moderate consumption of fructose-containing and non-caloric sweetened beverages on feeding behavior, metabolic and serum lipid profiles, and hepatic histology and serum liver enzymes, in rats. Behavioral tests determined preferred (12.5-15%) concentrations of solutions of agave, fructose, high fructose corn syrup (HFCS), a combination of HFCS and Hoodia (a putative appetite suppressant), or the non-caloric sweetener Stevia (n=5/gp). HFCS intake was highest, in preference and self-administration tests. Groups (n=10/gp) were then assigned to one of the sweetened beverages or water as the sole source of liquid at night (3 nights/wk, 10wks). Although within the normal range, serum cholesterol was higher in the fructose and HFCS groups, and serum triglycerides were higher in the Agave, HFCS, and HFCS/Hoodia groups (vs. water-controls, p<0.05). Liver histology was normal in all groups with no evidence of steatosis, inflammation, or fibrosis; however serum alanine aminotransferase was higher in the fructose and HFCS groups (vs. water-controls, p<0.05). Serum inflammatory marker levels were comparable among Stevia, agave, fructose, HFCS, and water-consuming groups, however levels of IL-6 were significantly lower in association with the ingestion of Hoodia. There were no differences in terminal body weights, or glucose tolerance assessed by 120-min IVGTTs performed at the end of the 10-week regimen. We conclude that even moderate consumption of fructose-containing liquids may lead to the onset of unfavorable changes in the plasma lipid profile and one marker of liver health, independent of significant effects of sweetener consumption on body weight.

A large-scale international meta-analysis of paraoxonase gene polymorphisms in sporadic ALS.

BACKGROUND: Six candidate gene studies report a genetic association of DNA variants within the paraoxonase locus with sporadic amyotrophic lateral sclerosis (ALS). However, several other large studies, including five genome-wide association studies, have not duplicated this finding. METHODS: We conducted a meta-analysis of 10 published studies and one unpublished study of the paraoxonase locus, encompassing 4,037 ALS cases and 4,609 controls, including genome-wide association data from 2,018 ALS cases and 2,425 controls. RESULTS: The combined fixed effects odds ratio (OR) for rs662 (PON1 Q192R) was 1.09 (95% confidence interval [CI], 1.02-1.16, p = 0.01); the genotypic OR for RR homozygotes at Q192R was 1.25 (95% CI, 1.07-1.45, p = 0.0004); the combined OR for rs854560 (PON1 L55M) was 0.97 (95% CI, 0.86-1.10, p = 0.62); the OR for rs10487132 (PON2) was 1.08 (95% CI, 0.92-1.27, p = 0.35). Although the rs662 polymorphism reached a nominal level of significance, no polymorphism was significant after multiple testing correction. In the subanalysis of samples with genome-wide data from which population outliers were removed, rs662 had an OR of 1.06 (95% CI, 0.97-1.16, p = 0.22). CONCLUSIONS: In contrast to previous positive smaller studies, our genetic meta-analysis showed no significant association of amyotrophic lateral sclerosis (ALS) with the PON locus. This is the largest meta-analysis of a candidate gene in ALS to date and the first ALS meta-analysis to include data from whole genome association studies. The findings reinforce the need for much larger and more collaborative investigations of the genetic determinants of ALS.

Paraoxonase gene polymorphisms and sporadic ALS.

BACKGROUND: The human paraoxonase (PON) gene family consists of three members, PON1, PON2, and PON3, located adjacent to each other on chromosome 7. PON catalytic activity may be influenced by frequent amino acid variants. Chronic exposure to certain chemicals or to environmental factors causing enhanced lipid peroxidation metabolized by paraoxonases may be a risk factor for sporadic ALS (sALS). OBJECTIVE: The aim of this study was to examine the association between PON1 Q192R, PON1 L55M, and PON2 C311S functional polymorphisms and the risk of sALS in a Polish population. METHODS: The authors included 185 patients with a definite or probable diagnosis of sALS (El Escorial Criteria) and 437 healthy controls of similar age and gender. The paraoxonase polymorphisms were studied by PCR and restriction enzyme digestion. RESULTS: Using logistic regression analyses, the C allele of the C311S polymorphism was associated with sALS in dominant and additive models, whereas the R allele of the Q192R polymorphism was associated with sALS in recessive, additive, and dominant models. The authors compared the distribution of haplotypes between cases and controls. The R-C haplotype was overrepresented among cases (odds ratio 3.44, 95% CI: 1.55 to 7.62, p = 0.002). CONCLUSIONS: Frequent amino acid variants in the paraoxonase 1 and paraoxonase 2 genes are associated with sporadic ALS in a Polish population.

FRG2, an FSHD candidate gene, is transcriptionally upregulated in differentiating primary myoblast cultures of FSHD patients.

BACKGROUND: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. METHODS AND RESULTS: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. CONCLUSION: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.

DD genotype of ACE gene is a risk factor for intracerebral hemorrhage.

Genetic factors may play a role in susceptibility to stroke. The angiotensin converting enzyme (ACE) gene is a candidate gene for two phenotypically different types of stroke affecting small perforating arteries: spontaneous intracerebral hemorrhage (SIH) and ischemic stroke due to small vessel disease (SVD). The authors report evidence that ACE gene DD homozygosity of the I/D polymorphism in intron 16 is an independent risk factor for SIH, and not for SVD stroke, in a Polish population.

Expression of receptors for insulin and leptin in the ventral tegmental area/substantia nigra (VTA/SN) of the rat.

Recent studies have demonstrated that the metabolic hormones insulin and leptin can modulate behavioral performance in reward-related paradigms. However, specific anatomical substrate(s) within the CNS for these effects remain to be identified. We hypothesize that midbrain dopamine neurons, which have been implicated to be critical in the mediation of motivational and reward aspects of stimuli, contribute to these behavioral effects of insulin and leptin. As one approach to evaluate this hypothesis, we used double-labeling fluorescence immunohistochemistry to determine whether the midbrain dopamine neurons express insulin receptors or leptin receptors. Extensive co-expression of tyrosine hydroxylase (a marker for dopamine neurons) with both the insulin receptor and the leptin receptor was observed in the ventral tegmentum and substantia nigra. These findings suggest that midbrain dopamine neurons are direct targets of insulin and leptin, and that they participate in mediating the effects of these hormones on reward-seeking behavior.

Effects of repetitive hypoglycemia on neuroendocrine response and brain tyrosine hydroxylase activity in the rat.

Hypoglycemia-associated autonomic failure (HAAF) is a syndrome of acute adaptation to a metabolic stressor, in which neuroendocrine responses to repetitive hypoglycemic bouts are blunted. The CNS mechanisms that contribute to HAAF are unknown. In the present study, we modeled HAAF in the rat and measured the activity of tyrosine hydroxylase (TH) as an index of acute noradrenergic activation, to test the hypothesis that noradrenergic activation of the hypothalamus might be impaired. In association with a significant counter-regulatory response to a single bout of hypoglycemia (elevated corticosterone, catecholamines, and glucagon), TH activity was elevated overall in brainstem NE cell body areas and hypothalamus. With multiple hypoglycemic episodes in a 24 h period, the counter-regulatory response was blunted, and hypothalamic TH activity was comparable to that of saline-infused controls. In a similar paradigm, multiple bouts of CNS neuroglucopenia did not blunt the hyperglycemic or corticosterone responses, and were required for elevation of TH activity. This alternate response pattern suggests that insulin-induced hypoglycemia and cerebral neuroglucopenia represent somewhat different metabolic stressors at the CNS.

Intraventricular insulin decreases kappa opioid-mediated sucrose intake in rats.

The hormone insulin acts in the central nervous system (CNS) as a regulator of body adiposity and food intake. Recent work from our laboratory has provided evidence that one way by which insulin may decrease food intake is by decreasing the rewarding properties of food. Evidence from others suggests that endogenous opioids may mediate the palatable properties of foods, and insulin may decrease nonfood-related reward via interaction with some CNS kappa opioid systems. In the present study we examined the ability of insulin to interact with exogenous or endogenous kappa opioids to modulate feeding of palatable sucrose pellets by nondeprived rats. Insulin (5 mU intracerebroventricular (i.c.v.), t=-3h) completely reversed the ability of the exogenous kappa agonist U50,488 (26 microg, i.c.v., t=-15 min) to stimulate 90-min sucrose feeding (211+/-32% reduced to 125+/-23% of 90-min baseline intake). Further, i.c.v. insulin (5 mU, t=-3h) interacted with a subthreshold dose of the kappa receptor antagonist norbinaltorphimine (5 microg, i.c.v., t=-15 min) to decrease the 90-min sucrose intake baseline (77+/-11% versus 109+/-10% of 90 min baseline intake, insulin/norbinaltorphimine versus norbinaltorphimine). Together these studies provide new evidence that insulin in the CNS may decrease the action of CNS kappa opioid system(s) that mediate palatable feeding.

Methylation of the FSHD syndrome-linked subtelomeric repeat in normal and FSHD cell cultures and tissues.

Facioscapulohumeral muscular dystrophy (FSHD) has an unusual molecular etiology. In a putatively heterochromatic subtelomeric region of each chromosome 4 homologue (4q35), unaffected individuals have 11 to about 95 tandem copies of a complex 3.3-kb repeat (D4Z4). Most FSHD patients have less than 10 copies at one allelic 4q35. This has been proposed to lead to the loss of heterochromatinization and, thereby, inappropriate gene expression by position effects, explaining the dominant nature of FSHD and the role of a decreased number of copies of D4Z4 at 4q35 but not at 10q26. Consistent with the proposed heterochromatinization of this repeat, by Southern blot analysis, we found that SmaI, MluI, SacII, and EagI sites in D4Z4 are highly methylated in normal and FSHD cell lines and somatic tissues, including skeletal muscle. Like repeated DNA sequences in the juxtacentromeric heterochromatin of chromosomes 1, 9, and 16, D4Z4 was hypomethylated at numerous CpGs in sperm and in cell lines from patients with an unrelated DNA methyltransferase deficiency syndrome (ICF; immunodeficiency, centromeric region instability, facial anomalies) in contrast to its hypermethylation in non-ICF postnatal somatic tissues. Our data on FSHD samples suggest that the disease-associated 4q35 D4Z4 repeats, which constitute a small percentage of the total D4Z4 repeats, are not generally hypomethylated relative to the other repeats of this sequence. However, in individuals not affected with FSHD, the hypermethylation of tandem, high-copy-number D4Z4 repeats might help stabilize heterochromatinization at allelic 4q35 regions just as hypermethylation elsewhere in the genome has been linked to chromatin compaction.

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

PVN activation is suppressed by repeated hypoglycemia but not antecedent corticosterone in the rat.

The mechanism(s) underlying hypoglycemia-associated autonomic failure (HAAF) are unknown. To test the hypothesis that the activation of brain regions involved in the counterregulatory response to hypoglycemia is blunted with HAAF, rats were studied in a 2-day protocol. Neuroendocrine responses and brain activation (c-Fos immunoreactivity) were measured during day 2 insulin-induced hypoglycemia (0.5 U insulin x 100 g body x wt(-1) x h(-1) iv for 2 h) after day 1 hypoglycemia (Hypo-Hypo) or vehicle. Hypo-Hypo animals demonstrated HAAF with blunted epinephrine, glucagon, and corticosterone (Cort) responses and decreased activation of the medial hypothalamus [the paraventricular (PVN), dorsomedial (DMH), and arcuate (Arc) nuclei]. To evaluate whether increases in day 1 Cort were responsible for the decreased hypothalamic activation, Cort was infused intracerebroventricularly (72 microg) on day 1 and the response to day 2 hypoglycemia was measured. Intracerebroventricular Cort infusion failed to alter the neuroendocrine response to day 2 hypoglycemia, despite elevating both central nervous system and peripheral Cort levels. However, day 1 Cort blunted responses in two of the same hypothalamic regions as Hypo-Hypo (the DMH and Arc) but not in the PVN. These results suggest that decreased activation of the PVN may be important in the development of HAAF and that antecedent exposure to elevated levels of Cort is not always sufficient to produce HAAF.

Superoxide-induced nitric oxide release from cultured glial cells.

Nitric oxide (NO) has been implicated as a potential contributor to neural cell death in a variety of neurological conditions. Cultured glial cells were exposed to extracellular superoxide generated by the action of xanthine oxidase on xanthine. In this experimental paradigm, both C6 glioma cells and primary astrocytes from rat cerebral cortex produced a rapid release of nitric oxide, measured using an NO specific electrode, in response to the applied superoxide stimulus. Application of a superoxide scavenger, or over-expression of Cu/Zn superoxide dismutase decreased the observed NO release. Authenticity of the NO signal was confirmed by the addition of the NO scavenger 2-(carboxyphenyl)-4,4,5,5-tetramethyllimidazoline-1-oxyl 3-oxide (carboxy-PTIO), which abolished the observed NO release without affecting simultaneously measured superoxide. Therefore, we suggest that glial cells may produce NO under free radical stimulation, which may be relevant to several neurological disorders where superoxide radicals are generated in the vicinity of glia. This would be predicted to result in the release of NO, which may exert toxic effects on neighbouring cells.

Clinical implications of the genetics of ALS and other motor neuron diseases.

Genetic mutations have been identified in the major motor neuron diseases, including ALS, spinal muscular atrophy, bulbospinal muscular atrophy (Kennedy's disease), the hereditary spastic paraplegias, and rarer conditions such as GM2 gangliosidosis (hexosaminidase A deficiency). These include mutations in the SOD1 gene, deletions of the telomeric copy of the SMN gene, expansions of the trinucleotide repeat region in the first exon of the androgen receptor gene, other rare mutations, and diseases where linkage has been established but the gene not identified. Identification of one of these genetic abnormalities will allow specific diagnosis in patients. Because cure is not yet available, presymptomatic testing is seldom indicated; in such cases, careful counseling is appropriate.

Leptin reverses sucrose-conditioned place preference in food-restricted rats.

Previous studies have suggested that food restriction can modify performance in the conditioned place preference (CPP) paradigm. In the present study, we tested the hypotheses that food restriction would enhance the development of a CPP to low-calorie sucrose pellets and that peripheral leptin replacement in food-restricted animals would reverse this effect. Using a range of 45-mg sucrose pellets (0-15 pellets) as a reward, we observed that a significant place preference was conditioned in food-restricted, but not ad libitum-fed rats. This CPP was reversed either by treatment of food-restricted rats with the dopamine receptor antagonist alpha-flupenthixol (200 microg/kg ip) during the training protocol or by chronic subcutaneous replacement of leptin (125 microg/kg/day) that attenuated the food restriction-induced decrease of circulating leptin. We conclude that dopaminergic signaling and the fall of plasma leptin concentrations contribute to the CPP of food-restricted rats. This finding suggests that in addition to metabolic adaptations, hypoleptinemia results in behavioral adaptations during states of energy deprivation.

Nitrotyrosination contributes minimally to toxicity of mutant SOD1 associated with ALS.

Enhanced production of nitrotyrosine and subsequent protein nitration has been proposed as the mechanism by which mutant SOD1 causes death of motor neurons in a familial form of amyotrophic lateral sclerosis (FALS-1). We have tested this hypothesis in a primary culture model in which mutant human SOD1 was expressed in motor neurons of dissociated spinal cord cultures. Preventing formation of nitrotyrosine by inhibiting nitric oxide synthase rescued cultured motor neurons from excitotoxic death induced by adding glutamate to the culture medium, but failed to significantly delay death of motor neurons expressing the G93A mutant SOD1. The results do not support generation of nitrotyrosine being the predominant lethal gain of function conferred by mutations in SOD1.