One-carbon metabolism in neurodevelopmental disorders: using broad-based nutraceutics to treat cognitive deficits in complex spectrum disorders.

Folate and choline, two nutrients involved in the one-carbon metabolic cycle, are intimately involved in regulating DNA integrity, synthesis, biogenic amine synthesis, and methylation. In this review, we discuss evidence that folate and choline play an important role in normal cognitive development, and that altered levels of these nutrients during periods of high neuronal proliferation and synaptogenesis can result in diminished cognitive function. We also discuss the use of these nutrients as therapeutic agents in a spectrum of developmental disorders in which intellectual disability is a prominent feature, such as in Fragile-X, Rett syndrome, Down syndrome, and Autism spectrum disorders. A survey of recent literature suggests that nutritional supplements have mild, but generally consistent, effects on improving cognition. Intervening with supplements earlier rather than later during development is more effective in improving cognitive outcomes. Given the mild improvements seen after treatments using nutrients alone, and the importance of the genetic profile of parents and offspring, we suggest that using nutraceutics early in development and in combination with other therapeutics are likely to have positive impacts on cognitive outcomes in a broad spectrum of complex neurodevelopmental disorders.

Acetyl-L-carnitine improves behavior and dendritic morphology in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder affecting 1 in 10,000 girls. Approximately 90% of cases are caused by spontaneous mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Girls with RTT suffer from severe motor, respiratory, cognitive and social abnormalities attributed to early deficits in synaptic connectivity which manifest in the adult as a myriad of physiological and anatomical abnormalities including, but not limited to, dimished dendritic complexity. Supplementation with acetyl-L-carnitine (ALC), an acetyl group donor, ameliorates motor and cognitive deficits in other disease models through a variety of mechanisms including altering patterns of histone acetylation resulting in changes in gene expression, and stimulating biosynthetic pathways such as acetylcholine. We hypothesized ALC treatment during critical periods in cortical development would promote normal synaptic maturation, and continuing treatment would improve behavioral deficits in the Mecp2(1lox) mouse model of RTT. In this study, wildtype and Mecp2(1lox) mutant mice received daily injections of ALC from birth until death (postnatal day 47). General health, motor, respiratory, and cognitive functions were assessed at several time points during symptom progression. ALC improved weight gain, grip strength, activity levels, prevented metabolic abnormalities and modestly improved cognitive function in Mecp2 null mice early in the course of treatment, but did not significantly improve motor or cognitive functions assessed later in life. ALC treatment from birth was associated with an almost complete rescue of hippocampal dendritic morphology abnormalities with no discernable side effects in the mutant mice. Therefore, ALC appears to be a promising therapeutic approach to treating early RTT symptoms and may be useful in combination with other therapies.

Preclinical research in Rett syndrome: setting the foundation for translational success.

In September of 2011, the National Institute of Neurological Disorders and Stroke (NINDS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the International Rett Syndrome Foundation (IRSF) and the Rett Syndrome Research Trust (RSRT) convened a workshop involving a broad cross-section of basic scientists, clinicians and representatives from the National Institutes of Health (NIH), the US Food and Drug Administration (FDA), the pharmaceutical industry and private foundations to assess the state of the art in animal studies of Rett syndrome (RTT). The aim of the workshop was to identify crucial knowledge gaps and to suggest scientific priorities and best practices for the use of animal models in preclinical evaluation of potential new RTT therapeutics. This review summarizes outcomes from the workshop and extensive follow-up discussions among participants, and includes: (1) a comprehensive summary of the physiological and behavioral phenotypes of RTT mouse models to date, and areas in which further phenotypic analyses are required to enhance the utility of these models for translational studies; (2) discussion of the impact of genetic differences among mouse models, and methodological differences among laboratories, on the expression and analysis, respectively, of phenotypic traits; and (3) definitions of the standards that the community of RTT researchers can implement for rigorous preclinical study design and transparent reporting to ensure that decisions to initiate costly clinical trials are grounded in reliable preclinical data.

Gene-environment interactions and epigenetic pathways in autism: the importance of one-carbon metabolism.

Both genetic and epigenetic factors play important roles in the rate and severity of classic autism and autism spectrum disorders (ASDs). This review focuses on DNA methylation as a key epigenetic mechanism in autism. The critical role that one-carbon (C1) metabolism plays in establishing and maintaining DNA methylation patterns makes it a likely candidate pathway to regulate epigenetic processes in ASDs. This review is the first, to our knowledge, to examine how altering C1 metabolic function through genetic and environmental factors (focusing on diet) may lead to aberrant DNA methylation and increase susceptibility to ASDs. Additionally, the critical time windows for sensitivity to genetic and dietary factors both during the development of cortical networks implicated in ASDs and in regard to potential treatments are discussed. One thing is clear, if C1 metabolism plays a critical role in ASDs, it provides a potential avenue for treatment and perhaps, ultimately, prevention.

Glutamate carboxypeptidase II and folate deficiencies result in reciprocal protection against cognitive and social deficits in mice: implications for neurodevelopmental disorders.

Interactions between genetic and environmental risk factors underlie a number of neuropsychiatric disorders, including schizophrenia (SZ) and autism (AD). Due to the complexity and multitude of the genetic and environmental factors attributed to these disorders, recent research strategies focus on elucidating the common molecular pathways through which these multiple risk factors may function. In this study, we examine the combined effects of a haplo-insufficiency of glutamate carboxypeptidase II (GCPII) and dietary folic acid deficiency. In addition to serving as a neuropeptidase, GCPII catalyzes the absorption of folate. GCPII and folate depletion interact within the one-carbon metabolic pathway and/or of modulate the glutamatergic system. Four groups of mice were tested: wild-type, GCPII hypomorphs, and wild-types and GCPII hypomorphs both fed a folate deficient diet. Due to sex differences in the prevalence of SZ and AD, both male and female mice were assessed on a number of behavioral tasks including locomotor activity, rotorod, social interaction, prepulse inhibition, and spatial memory. Wild-type mice of both sexes fed a folic acid deficient diet showed motor coordination impairments and cognitive deficits, while social interactions were decreased only in males. GCPII mutant mice of both sexes also exhibited reduced social propensities. In contrast, all folate-depleted GCPII hypomorphs performed similarly to untreated wild-type mice, suggesting that reduced GCPII expression and folate deficiency are mutually protective. Analyses of folate and neurometabolite levels associated with glutamatergic function suggest several potential mechanisms through which GCPII and folate may be interacting to create this protective effect.

Cognitive deficits in Rett syndrome: what we know and what we need to know to treat them.

Rett syndrome is an autism spectrum disorder and a leading cause of severe mental retardation in girls. The nature of the cognitive abnormalities in Rett, as described in humans and other animal models, and its potential reversibility and treatment are the subject of this review. Rett syndrome is associated with severe mental retardation and a host of impairments that include social and motor deficits, and respiratory and bone abnormalities. More than 80% of Rett girls have mutations in the gene that encodes MeCP2, which is a protein with a complex set of functions that include transcriptional repression and activation. The complex phenotype associated with Rett and the knowledge of the causal genetic mutation provide a unique opportunity within the autism spectrum to explore the relationship between transcriptional control, brain abnormalities and specific behavioral functions, importantly the elusive cognitive dysfunctions associated with mental retardation. The nature of the cognitive abnormalities related to Rett and the potential reversibility and treatment of these abnormalities have not been studied as extensively as some of the other aspects of the Rett phenotype. The cognitive phenotype associated with Rett is also less well studied relative to that in other well known developmental disorders, such as Down syndrome and Fragile X. Nevertheless, some recent studies provide hope that the cognitive impairments, as well as other symptoms of Rett, can be rescued.

Cognitive and social functions and growth factors in a mouse model of Rett syndrome.

Rett syndrome (RTT) is an autism-spectrum disorder caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Abnormalities in social behavior, stereotyped movements, and restricted interests are common features in both RTT and classic autism. While mouse models of both RTT and autism exist, social behaviors have not been explored extensively in mouse models of RTT. Here, we report cognitive and social abnormalities in Mecp2(1lox) null mice, an animal model of RTT. The null mice show severe deficits in short- and long-term object recognition memories, reminiscent of the severe cognitive deficits seen in RTT girls. Social behavior, however, is abnormal in that the null mice spend more time in contact with stranger mice than do wildtype controls. These findings are consistent with reports of increased reciprocal social interaction in RTT girls relative to classic autism. We also report here that the levels of the neurotrophins brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), and nerve growth factor (NGF) are decreased in the hippocampus of the null mice, and discuss how this may provide an underlying mechanism for both the cognitive deficits and the increased motivation for social contact observed in the Mecp2(1lox) null mice. These studies support a differential etiology between RTT and autism, particularly with respect to sociability deficits.

Phenotypic characterization of mice heterozygous for a null mutation of glutamate carboxypeptidase II.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of N-methyl-D-aspartate receptors (NMDAR) has been implicated in the pathophysiology of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N-acetyl-alpha L-aspartyl-L-glutamate (NAAG) into glutamate and N-acetyl-aspartate. NAAG is a neuropeptide that is an NMDAR antagonist as well as an agonist for the metabotropic glutamate receptor-3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDAR activation. To manipulate the expression of GCP II, LoxP sites were inserted flanking exons 1 and 2, which were excised by crossing with a Cre-expressing mouse. The mice heterozygous for this deletion showed a 50% reduction in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N > 200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm our hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory.

Efficacy of a murine-p75-saporin immunotoxin for selective lesions of basal forebrain cholinergic neurons in mice.

Selective lesioning of cholinergic neurons in the basal forebrain provides a tool for examining the functional significance of cholinergic loss, which is associated with a number of developmental and neurodegenerative disorders. A new version of an immunotoxin (murine-p75NTR-saporin) was used to produce a selective loss of cholinergic neurons in the adult basal forebrain of the mouse. This new version of the toxin is significantly more potent and selective than a previously developed version. C57Bl/6J mice (n=30) were given 1 microL of either saline or murine-p75NTR-saporin (0.65 microg/microL or 1.3 microg/microL) into the lateral ventricles, and then sacrificed 10-12 days post-surgery for histological analysis. In contrast to results from the previous version of the toxin, survival of the toxin-treated mice was 100% at both doses. A complete loss of cholinergic neurons was seen in the medial septum (MS) with both doses, while a dose-dependent loss of cholinergic neurons was observed in the nucleus basalis magnocellularis (nBM). The lesions were associated with locomotor hypoactivity and anxiolytic-type behavioral effects. These studies describe the efficacy and selectivity of this new version of murine-p75NTR-saporin, which may be used to provide insight into functional deficits that result from the loss of cholinergic neurons in the mouse basal forebrain.

Nutritional factors in a mouse model of Rett syndrome.

Environmental factors such as nutrition and housing can influence behavioral and anatomical characteristics of several neurological disorders, including Rett syndrome (RTT). RTT is associated with mutations in the X-linked gene encoding MeCP2, a transcriptional repressor that binds methylated DNA. While direct genetic intervention in humans is impossible at this time, motor and cognitive deficits in RTT may be ameliorated through manipulations of epigenetic/environmental factors. For example, studies in rodents suggest that choline nutrient supplementation during critical periods of brain development enhances cholinergic neurotransmission, alters neuronal size and distribution, and facilitates performance of memory and motoric tasks. Recent work in a mouse model of RTT shows that enhancing maternal nutrition through choline supplementation improves both anatomical and behavioral symptoms in the mutant offspring. We describe here cellular and molecular mechanisms that may underlie this specific enhancement and may provide more general insights into mechanisms underlying gene-environment interactions in neurological disorders.

Environmental enrichment alters locomotor behaviour and ventricular volume in Mecp2 1lox mice.

Rett syndrome (RTT) is an autistic spectrum developmental disorder associated with mutations in the X-linked Mecp2 gene, and severe behavioural and neuropathological deficits. In a mouse model of RTT (Mecp2(1lox)), we examined whether environmental enrichment (EE) alters behavioural performance and regional brain volume. At weaning, Mecp2(1lox) and control mice were assigned to enriched or standard housing. From postnatal day 29 to 43, mice were subjected to behavioural tasks measuring motor and cognitive performance. At postnatal day 44, volumes of whole brain, cerebellum, ventricles, and motor cortex were measured using magnetic resonance imaging. EE provided subtle improvements to locomotor activity and contextual fear conditioning in Mecp2(1lox) mice. Additionally, EE reduced ventricular volumes, which correlated with improved locomotor activity, suggesting that neuroanatomical changes contribute to improved behaviour. Our results suggest that post-weaning EE may provide a non-invasive palliative treatment for RTT.

Neurochemical changes in a mouse model of Rett syndrome: changes over time and in response to perinatal choline nutritional supplementation.

Rett syndrome (RTT), the second leading cause of mental retardation in girls, is caused by mutations in the X-linked gene for methyl-CpG-binding protein 2 (MeCP2), a transcriptional repressor. In addition to well-documented neuroanatomical and behavioral deficits, RTT is characterized by reduced markers of cholinergic activity and general neuronal health. Previously, we have shown that early postnatal choline (Cho) supplementation improves behavioral and neuroanatomical symptoms in a mouse model of RTT (Mecp2(1lox) mice). In this study, we use NMR spectroscopy to quantify the relative amounts of Cho, Glutamate (Glu), Glutamine (Gln), and N-acetyl aspartate (NAA) in the brains of wild type and mutant mice at 21, 35, and 42 days of age and in mice receiving postnatal Cho supplementation. We find that the mutant mice have reduced levels of Cho, Glu, and NAA, but elevated Gln levels, compared with their wild type littermates. These differences emerge at different developmental ages. Cho supplementation increases NAA levels, a marker of neuronal integrity, but has no effect on Cho, Glu, or Gln. These data suggest that postnatal nutritional supplementation may improve neuronal function and could serve as a therapeutic agent for human RTT patients.

Effects of postnatal dietary choline supplementation on motor regional brain volume and growth factor expression in a mouse model of Rett syndrome.

Nutritional status during pregnancy and lactation can influence behavioral and anatomical characteristics of several neurological disorders in the offspring, including Rett syndrome (RTT). RTT is associated with mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCp2), a transcriptional repressor that binds methylated DNA. In Mecp2(1lox) mice, a model of RTT, enhancing maternal nutrition through choline supplementation attenuates motor coordination deficits in the mutant offspring. Here, we examine alterations in brain volume and growth factor expression in the cerebellum and striatum, motor regions that may contribute to the improved behavioral performance seen with choline supplementation. Mecp2(1lox) dams were given choline in drinking water, and pups nursed from birth to weaning. Brains of male offspring were collected at postnatal day 42 for volumetric and growth factor expression analyses. Compared to wild-type mice, Mecp2(1lox) null mice had decreased whole brain, cerebellar and striatal volume. Choline supplementation had no effect on brain volume. Nerve growth factor and insulin-like growth factor-1 expression was similar between wild-type and Mecp2(1lox) mice while brain derived neurotrophic factor was reduced in Mecp2(1lox) mice. Choline supplementation increased striatal nerve growth factor expression in wild-type and Mecp2(1lox) mice, suggesting that neuronal proliferation and survival may contribute to improved motor performance in this model of RTT.

Sectioning of brain tissues.

INTRODUCTIONA fixed brain, although harder than the brain in its natural state, is still not hard enough to cut into very thin slices. To make uniform, thin sections, the brain must be frozen. A wide variety of sectioning devices are available, each with their own requirements. With a microtome, for example, a very sharp knife is drawn manually across a brain that has been frozen to a chilled platform using dry ice. After each cut, the knife is lowered mechanically by a preset amount, giving consistent sections of a required thickness. A drawback of this instrument is that the temperature of the platform can fluctuate, thus causing changes in the quality of the sections. Other instruments, such as a cryostat, maintain the platform-mounted brain and the knife inside an enclosed refrigerated compartment. The temperature is kept at -20°C, and electronics are used to control the speed of the knife and the thickness of the sections. Some models can be set up to make a series of sections automatically.

Perfusion of brain tissues with fixative.

INTRODUCTIONIn many experiments it is necessary to section the brain to determine the location of a treatment (lesion or electrode) or to look at the histology of the brain using various staining techniques. Because the texture of the brain is so soft (often likened to soft cheese), it must be "fixed" before it can be removed from the skull. A fixative is a chemical that cross-links the molecules of the tissue, rendering it hard and preserving the tissue. This protocol describes a method for perfusing the brain with fixative (specifically, it describes how to perfuse a rat brain; slight modifications may be needed for different animals). A relatively simple gravity feed and the pumping mechanism of the heart is used to get fixative into the brain. A cannula is placed in the heart, or directly in the ascending aorta, of a deeply anesthetized animal. Blood is flushed out with saline first, and then with a fixative. The choice of fixative is often important if a specific staining technique is to be used, especially in immunocytochemistry, because the fixative can interfere with the staining sensitivity.

Dissection of rat brains.

INTRODUCTIONIn this protocol, the perfused brain of a rat is separated from the surrounding tissue and post-fixed in a formalin/sucrose solution in preparation for freezing and sectioning. Dissection should be done as soon as possible after perfusion to prevent desiccation of the brain. This procedure can also be used to dissect fresh (non-perfused) brains, for example, for Golgi-Cox stain or neurochemical assays.

Subbing slides.

INTRODUCTIONWhen mounting sections on a slide, it is essential that the slide be ultraclean. To ensure that the section sticks to the slide, slides can be covered with a thin layer of gelatin. The process of cleaning and gelatin-coating is called "subbing." Several boxes of slides should be subbed at a time and stored, covered, in the refrigerator until they are needed. Subbed slides can also be purchased commercially.