In silico, in vitro, and in vivo approaches to identify molecular players in fragile X tremor and Ataxia syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative monogenetic disorder affecting carriers of premutation (PM) forms of the FMR1 gene, resulting in a progressive development of tremors, ataxia, and neuropsychological problems. This highly disabling disease is quite common in the general population with an estimation of about 20 million PM carriers worldwide. The chances of developing FXTAS increase dramatically with age, with about 45% of male carriers over the age of 50 being affected. Both the gene and pathogenic trigger, a mutant expansion of CGG RNA, causing FXTAS are known. This makes it an interesting disease to develop targeted therapeutic interventions for. Yet, no such interventions are available at this moment. Here we discuss in silico, in vitro, and in vivo approaches and how they have been used to identify the molecular determinants of FXTAS pathology. These approaches have yielded substantial information about FXTAS pathology and, consequently, many markers have emerged to play a key role in understanding the disease mechanism. Integration of the different approaches is expected to provide crucial information about the value of these markers as either therapeutic target or biomarker, essential to monitor therapeutic interventions in the future

Use of model systems to understand the etiology of fragile X-associated primary ovarian insufficiency (FXPOI)

Fragile X-associated primary ovarian insufficiency (FXPOI) is among the family of disorders caused by the expansion of a CGG repeat sequence in the 5' untranslated region of the X-linked gene FMR1. About 20% of women who carry the premutation allele (55 to 200 unmethylated CGG repeats) develop hypergonadotropic hypogonadism and cease menstruating before age 40. Some proportion of those who are still cycling show hormonal profiles indicative of ovarian dysfunction. FXPOI leads to subfertility and an increased risk of medical conditions associated with early estrogen deficiency. Little progress has been made in understanding the etiology of this clinically significant disorder. Understanding the molecular mechanisms of FXPOI requires a detailed knowledge of ovarian FMR1 mRNA and FMRP's function. In humans, non-invasive methods to discriminate the mechanisms of the premutation on ovarian function are not available, thus necessitating the development of model systems. Vertebrate (mouse and rat) and invertebrate (Drosophila melanogaster) animal studies for the FMR1 premutation and ovarian function exist and have been instrumental in advancing our understanding of the disease phenotype. For example, rodent models have shown that FMRP is highly expressed in oocytes where it is important for folliculogenesis. The two premutation mouse models studied to date show evidence of ovarian dysfunction and, together, suggest that the long repeat in the transcript itself may have some pathological effect quite apart from any effect of the toxic protein. Further, ovarian morphology in young animals appears normal and the primordial follicle pool size does not differ from that of wild-type animals. However, there is a progressive premature decline in the levels of most follicle classes. Observations also include granulosa cell abnormalities and altered gene expression patterns. Further comparisons of these models are now needed to gain insight into the etiology of the ovarian dysfunction. Premutation model systems in non-human primates and those based on induced pluripotent stem cells show particular promise and will complement current models. Here, we review the characterization of the current models and describe the development and potential of the new models. Finally, we will discuss some of the molecular mechanisms that might be responsible for FXPOI

Astroglial-targeted expression of the fragile X CGG repeat premutation in mice yields RAN translation, motor deficits and possible evidence for cell-to-cell propagation of FXTAS pathology

The fragile X premutation is a CGG trinucleotide repeat expansion between 55 and 200 repeats in the 5′- untranslated region of the fragile X mental retardation 1 (FMR1) gene. Human carriers of the premutation allele are at risk of developing the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Characteristic neuropathology associated with FXTAS includes intranuclear inclusions in neurons and astroglia. Previous studies recapitulated these histopathological features in neurons in a knock-in mouse model, but without significant astroglial pathology. To determine the role of astroglia in FXTAS, we generated a transgenic mouse line (Gfa2-CGG99-eGFP) that selectively expresses a 99-CGG repeat expansion linked to an enhanced green fluorescent protein (eGFP) reporter in astroglia throughout the brain, including cerebellar Bergmann glia. Behaviorally these mice displayed impaired motor performance on the ladder-rung test, but paradoxically better performance on the rotarod. Immunocytochemical analysis revealed that CGG99- eGFP co-localized with GFAP and S-100ß, but not with NeuN, Iba1, or MBP, indicating that CGG99-eGFP expression is specific to astroglia. Ubiquitin-positive intranuclear inclusions were found in eGFP-expressing glia throughout the brain. In addition, intracytoplasmic ubiquitin-positive inclusions were found outside the nucleus in distal astrocyte processes. Intriguingly, intranuclear inclusions, in the absence of eGFP mRNA and eGFP fluorescence, were present in neurons of the hypothalamus and neocortex. Furthermore, intranuclear inclusions in both neurons and astrocytes displayed immunofluorescent labeling for the polyglycine peptide FMRpolyG, implicating FMRpolyG in the pathology found in Gfa2-CGG99 mice. Considered together, these results show that Gfa2-CGG99 expression in mice is sufficient to induce key features of FXTAS pathology, including formation of intranuclear inclusions, translation of FMRpolyG, and deficits in motor function

Ammonium-Acetate Is Sensed by Gustatory and Olfactory Neurons in Caenorhabditis elegans

Background: Caenorhabditis elegans chemosensation has been successfully studied using behavioral assays that treat detection of volatile and water soluble chemicals as separate senses, analogous to smell and taste. However, considerable ambiguity has been associated with the attractive properties of the compound ammonium-acetate (NH 4Ac). NH 4Ac has been used in behavioral assays both as a chemosensory neutral compound and as an attractant. Methodology/Main Findings: Here we show that over a range of concentrations NH4Ac can be detected both as a water soluble attractant and as an odorant, and that ammonia and acetic acid individually act as olfactory attractants. We use genetic analysis to show that NaCl and NH4Ac sensation are mediated by separate pathways and that ammonium sensation depends on the cyclic nucleotide gated ion channel TAX-2/TAX-4, but acetate sensation does not. Furthermore we show that sodium-acetate (NaAc) and ammonium-chloride (NH4Cl) are not detected as Na + and Cl 2 specific stimuli, respectively. Conclusions/Significance: These findings clarify the behavioral response of C. elegans to NH4Ac. The results should have an impact on the design and interpretation of chemosensory experiments studying detection and adaptation to soluble compounds in the nematode Caenorhabditis elegans

Using C. elegans to decipher the cellular and molecular mechanisms underlying neurodevelopmental disorders

Prova tipográfica (uncorrected proof)Neurodevelopmental disorders such as epilepsy, intellectual disability (ID), and autism spectrum disorders (ASDs) occur in over 2 % of the population, as the result of genetic mutations, environmental factors, or combination of both. In the last years, use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. Nevertheless, following association of novel genes with a given disease, interpretation of findings is often difficult due to lack of information on gene function and effect of a given mutation in the corresponding protein. This brings the need to validate genetic associations from a functional perspective in model systems in a relatively fast but effective manner. In this context, the small nematode, Caenorhabditis elegans, presents a good compromise between the simplicity of cell models and the complexity of rodent nervous systems. In this article, we review the features that make C. elegans a good model for the study of neurodevelopmental diseases. We discuss its nervous system architecture and function as well as the molecular basis of behaviors that seem important in the context of different neurodevelopmental disorders. We review methodologies used to assess memory, learning, and social behavior as well as susceptibility to seizures in this organism. We will also discuss technological progresses applied in C. elegans neurobiology research, such as use of microfluidics and optogenetic tools. Finally, we will present some interesting examples of the functional analysis of genes associated with human neurodevelopmental disorders and how we can move from genes to therapies using this simple model organism.The authors would like to acknowledge Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-GMG/112577/2009). AJR and CB are recipients of FCT fellowships: SFRH/BPD/33611/2009 and SFRH/BPD/74452/2010, respectively

Use of model systems to understand the etiology of fragile X-associated primary ovarian insufficiency (FXPOI)

Fragile X-associated primary ovarian insufficiency (FXPOI) is among the family of disorders caused by the expansion of a CGG repeat sequence in the 5' untranslated region of the X-linked gene FMR1. About 20% of women who carry the premutation allele (55 to 200 unmethylated CGG repeats) develop hypergonadotropic hypogonadism and cease menstruating before age 40. Some proportion of those who are still cycling show hormonal profiles indicative of ovarian dysfunction. FXPOI leads to subfertility and an increased risk of medical conditions associated with early estrogen deficiency. Little progress has been made in understanding the etiology of this clinically significant disorder. Understanding the molecular mechanisms of FXPOI requires a detailed knowledge of ovarian FMR1 mRNA and FMRP’s function. In humans, non-invasive methods to discriminate the mechanisms of the premutation on ovarian function are not available, thus necessitating the development of model systems. Vertebrate (mouse and rat) and invertebrate (Drosophila melanogaster) animal studies for the FMR1 premutation and ovarian function exist and have been instrumental in advancing our understanding of the disease phenotype. For example, rodent models have shown that FMRP is highly expressed in oocytes where it is important for folliculogenesis. The two premutation mouse models studied to date show evidence of ovarian dysfunction and, together, suggest that the long repeat in the transcript itself may have some pathological effect quite apart from any effect of the toxic protein. Further, ovarian morphology in young animals appears normal and the primordial follicle pool size does not differ from that of wild-type animals. However, there is a progressive premature decline in the levels of most follicle classes. Observations also include granulosa cell abnormalities and altered gene expression patterns. Further comparisons of these models are now needed to gain insight into the etiology of the ovarian dysfunction. Premutation model systems in non-human primates and those based on induced pluripotent stem cells show particular promise and will complement current models. Here, we review the characterization of the current models and describe the development and potential of the new models. Finally, we will discuss some of the molecular mechanisms that might be responsible for FXPOI

Signaling proteins that regulate NaCL chemotaxis responses modulate longevity in C. elegans

The lifespan of the nematode Caenorhabditis elegans is regulated by sensory signals detected by the amphid neurons. In these neurons, C. elegans expresses at least 14 Gα subunits and a Gγ subunit. We have identified seven sensory Gα subunits that modulate lifespan. Genetic experiments suggest that multiple sensory signaling pathways exist that modulate lifespan and that some G proteins function in multiple pathways, most of which, but probably not all, involve insulinIGF-1 like signaling. Interestingly, of the sensory G proteins involved in regulating lifespan, only one Gα probably functions directly in the detection of sensory cues. The other G proteins seem to function in modulating the sensitivity of the sensory neurons. We hypothesize that in addition to the mere detection of sensory cues, regulation of the sensitivity of sensory neurons also plays a role in the regulation of lifespan

表紙・目次

Carriers of the fragile X premutation (FPM) have CGG trinucleotide repeat expansions of between 55 and 200 in the 5'-UTR of FMR1, compared to a CGG repeat length of between 5 and 54 for the general population. Carriers were once thought to be without symptoms, but it is now recognized that they can develop a variety of early neurological symptoms as well as being at risk for developing the late onset neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS). Several mouse models have contributed to our understanding of FPM and FXTAS, and findings from studies using these models are summarized here. This review also discusses how this information is improving our understanding of the molecular and cellular abnormalities that contribute to neurobehavioral features seen in some FPM carriers and in patients with FXTAS. Mouse models show much of the pathology seen in FPM carriers and in individuals with FXTAS, including the presence of elevated levels of Fmr1 mRNA, decreased levels of fragile X mental retardation protein, and ubiquitin-positive intranuclear inclusions. Abnormalities in dendritic spine morphology in several brain regions are associated with neurocognitive deficits in spatial and temporal memory processes, impaired motor performance, and altered anxiety. In vitro studies have identified altered dendritic and synaptic architecture associated with abnormal Ca(2+) dynamics and electrical network activity. FPM mice have been particularly useful in understanding the roles of Fmr1 mRNA, fragile X mental retardation protein, and translation of a potentially toxic polyglycine peptide in pathology. Finally, the potential for using these and emerging mouse models for preclinical development of therapies to improve neurological function in FXTAS is considered

Neuronal and molecular substrates for optimal foraging in Caenorhabditis elegans

Variation in food quality and abundance requires animals to decide whether to stay on a poor food patch or leave in search of better food. An important question in behavioral ecology asks when is it optimal for an animal to leave a food patch it is depleting. Although optimal foraging is central to evolutionary success, the neural and molecular mechanisms underlying it are poorly understood. Here we investigate the neuronal basis for adaptive food-leaving behavior in response to resource depletion in Caenorhabditis elegans, and identify several of the signaling pathways involved. The ASE neurons, previously implicated in salt chemoattraction, promote food-leaving behavior via a cGMP pathway as food becomes limited. High ambient O2 promotes food-leaving via the O2-sensing neurons AQR, PQR, and URX. Ectopic activation of these neurons using channelrhodopsin is sufficient to induce high food-leaving behavior. In contrast, the neuropeptide receptor NPR-1, which regulates social behavior on food, acts in the ASE neurons, the nociceptive ASH neurons, and in the RMG interneuron to repress food-leaving. Finally, we show that neuroendocrine signaling by TGF-β/DAF-7 and neuronal insulin signaling are necessary for adaptive food-leaving behavior. We suggest that animals integrate information about their nutritional state with ambient oxygen and gustatory stimuli to formulate optimal foraging strategies