No abstract

Endogenous biological clocks allow organisms to anticipate daily environmental cycles. The ability to achieve time-place associations is key to the survival and reproductive success of animals. The ability to link the location of a stimulus (usually food) with time of day has been coined time-place learning, but its circadian nature was only shown in honeybees and birds. So far, an unambiguous circadian time-place-learning paradigm for mammals is lacking. We studied whether expression of the clock gene Cryptochrome (Cry), crucial for circadian timing, is a prerequisite for time-place learning. Time-place learning in mice was achieved by developing a novel paradigm in which food reward at specific times of day was counterbalanced by the penalty of receiving a mild footshock. Mice lacking the core clock genes Cry1 and Cry2 (Cry double knockout mice; Cry1(-/-)Cry2(-/-)) learned to avoid unpleasant sensory experiences (mild footshock) and could locate a food reward in a spatial learning task (place preference). These mice failed, however, to learn time-place associations. This specific learning and memory deficit shows that a Cry-gene dependent circadian timing system underlies the utilization of time of day information. These results reveal a new functional role of the mammalian circadian timing system.

IntroductionGlycogenosis type II (Pompe disease, acid maltase deficiency; Online Mendelian Inheritance in Man no. 232300) is an autosomal recessive lysosomal storage disorder caused by acid ␣-glucosidase (GAA) deficiency. The disease is characterized by glycogen storage in liver, spleen, kidney, brain, and endothelial cells and most prominently in skeletal, heart, and smooth muscles. Symptoms arise from muscular weakness and wasting. Infants with complete enzyme deficiency present shortly after birth, lose all muscle strength within 8 months, and succumb to hypertrophic cardiomyopathy and respiratory failure in the first year of life. 1 Children and adults with residual GAA activity show a more protracted course and may become wheelchair bound, dependent on artificial ventilation, and have a shortened life expectancy. Presently, enzyme replacement therapy (ERT) based on intravenous infusion of recombinant human ␣-glucosidase, taken up by mannose 6-phosphate receptor mediated endocytosis, 2,3 is a major therapeutic advance that prolongs the life of affected infants but does not guarantee long-term symptom-free survival, requires biweekly administration, and may induce immune responses to the recombinant protein.As an alternative to ERT, in vivo gene therapy mediated by adenoviral vectors and adeno-associated virus vectors (AAVs) has been investigated in a mouse model of Pompe disease. [4][5][6] However, long-term efficacy can be significantly hampered by antibody formation, 7,8 and adverse immune responses to the vector has been observed after adenoviral and AAV gene therapy in patients. 9,10 For treatment of patients with other lysosomal enzyme deficiencies, allogeneic hematopoietic stem cell (HSC) transplantation has been proposed. 11 HSC transplantation proved effective in ameliorating the neurologic symptoms in murine globoid cell leukodystrophy and human patients 12,13 as well as in mucopolysaccharidosis I (Hurler syndrome). 14,15 Other lysosomal storage disorders such as metachromatic leukodystrophy (MLD) may require higher enzyme levels than provided by HSC transplantation; lentiviral (LV) vector-mediated overexpression of aryl-sulfatase A in HSCs effectively reversed the neuropathologic phenotype in the mouse model. 16 In addition, LV-mediated clinical gene therapy in trial phase of X-linked adrenoleukodystrophy halted progressive cerebral demyelination in 2 patients. 17 Recently, HSC transplantation was shown to promote immune tolerance to ERT in the Pompe mouse model. 18 The use of gene-modified autologous HSCs also overcomes the profound conditioning and immune barriers associated with allogeneic transplantation.The few attempts of HSC transplantation for Pompe disease have not met with success. 19 GAA levels, if any, are low in hematopoietic cells in mice, 18 and allogeneic transplantation is not an obvious treatment. Therefore, high-level vector-driven ectopic expression of the enzyme in hematopoietic cells would be required to accomplish efficacy. We tested the hypothesis that ex vivo LV For persona...

Routine diagnostic screening of inborn errors of metabolism (IEM) is currently performed by different targeted analyses of known biomarkers. This approach is time-consuming, targets a limited number of biomarkers and will not identify new biomarkers. Untargeted metabolomics generates a global metabolic phenotype and has the potential to overcome these issues. We describe a novel, single platform, untargeted metabolomics method for screening IEM, combining semi-automatic sample preparation with pentafluorophenylpropyl phase (PFPP)-based UHPLC- Orbitrap-MS. We evaluated analytical performance and diagnostic capability of the method by analysing plasma samples of 260 controls and 53 patients with 33 distinct IEM. Analytical reproducibility was excellent, with peak area variation coefficients below 20% for the majority of the metabolites. We illustrate that PFPP-based chromatography enhances identification of isomeric compounds. Ranked z-score plots of metabolites annotated in IEM samples were reviewed by two laboratory specialists experienced in biochemical genetics, resulting in the correct diagnosis in 90% of cases. Thus, our untargeted metabolomics platform is robust and differentiates metabolite patterns of different IEMs from those of controls. We envision that the current approach to diagnose IEM, using numerous tests, will eventually be replaced by untargeted metabolomics methods, which also have the potential to discover novel biomarkers and assist in interpretation of genetic data.

Long-term drug-induced alterations in gene expression underlying neuroplasticity in the nucleus accumbens (NAc) may play a crucial role in relapse behavior in abstinent drug addicts. In this respect, stimulus-induced relapse behavior is considered as the retrieval of stored drug-related information. Because the NAc shell may determine the impact of external and internal stimuli on goal-directed behavior, we compared long-term gene expression in this brain region after active and passive administration of different drugs of abuse. We made use of a preselected set of transcripts that were down-regulated 3 wk after active i.v. heroin self-administration. We found that most of these transcripts were not down-regulated long after passive exposure to the opiate. Most of the active heroin administration-regulated transcripts were also down-regulated in the NAc shell following active cocaine administration (common denominators). As observed with passive administration of heroin, passive exposure to cocaine was found to be relatively ineffective in reducing the expression of these transcripts. This work reveals that active drug consumption during self-administration (instrumental learning) is a crucial psychological factor directing long-term genomic responses in the brain.

Repeated exposure to addictive drugs results in long-lasting neuroadaptations in the brain, especially in the mesocorticolimbic system. Within this system, the nucleus accumbens (NAc) plays a major integrative role. As such, the NAc has been shown to be a target of short- and long-lasting drug-induced neuroadaptations at the levels of neurotransmission and cellular morphology. The long-lasting neuroadaptations might depend critically on alterations in gene expression. Recently, we obtained a set of transcripts by means of subtractive hybridization, of which the expression was decreased in the rat NAc shell after long-term extinction of intravenous heroin self-administration. Interestingly, the majority of these transcripts were also down-regulated upon long-term extinction of cocaine self-administration. Using the yoked-control operant paradigm, it was shown that non-contingent administration of these drugs resulted in a totally different gene expression profile. However, in the rat NAc core, both self-administration and non-contingent heroin administration induced a qualitatively similar expression profile. Hence, cognitive processes associated with drug self-administration seem to direct the long-term genomic responses in the NAc shell, whereas the NAc core might primarily mediate the persistent pharmacological effects of addictive drugs (including Pavlovian conditioning).

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid a-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.

Long-term drug-induced alterations in neurotransmission within the nucleus accumbens (NAc) shell and core may underlie relapse to drug-seeking behavior and drug-taking upon re-exposure to drugs and drug-associated stimuli (cues) during abstinence. Using an open screening strategy, we recently identified 25 gene transcripts, encoding for proteins involved in neuronal functioning and structure that are down-regulated in rat NAc shell after contingent (active), but not after non-contingent (passive), heroin administration. Studying the expression of the same transcripts in the NAc core by means of quantitative PCR, we now demonstrate that most of these transcripts are up-regulated in that NAc subregion long (3 weeks) after heroin self-administration in rats. A similar up-regulation in gene expression was also apparent in the NAc core of animals with a history of non-contingent heroin administration (yoked controls). These data indicate that heroin self-administration differentially regulates genes in the NAc core as compared with the shell. Moreover, whereas cognitive processes involved in active drug self-administration (e.g., instrumental learning) seems to direct gene expression in the NAc shell, neuroplasticity in the NAc core may be due to the pharmacological effects of heroin (including Pavlovian conditioning), as expressed in rats upon contingent as well as non-contingent administration of heroin.