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1.  Loss of MeCP2 in Parvalbumin-and Somatostatin-Expressing Neurons in Mice Leads to Distinct Rett Syndrome-like Phenotypes 
Neuron  2015;88(4):651-658.
SUMMARY
Inhibitory neurons are critical for proper brain function, and their dysfunction is implicated in several disorders, including autism, schizophrenia, and Rett syndrome. These neurons are heterogeneous, and it is unclear which subtypes contribute to specific neurological phenotypes. We deleted Mecp2, the mouse homolog of the gene that causes Rett syndrome, from the two most populous subtypes, parvalbumin-positive (PV+) and somatostatin-positive (SOM+) neurons. Loss of MeCP2 partially impairs the affected neuron, allowing us to assess the function of each subtype without profound disruption of neuronal circuitry. We found that mice lacking MeCP2 in either PV+ or SOM+ neurons have distinct, non-overlapping neurological features: mice lacking MeCP2 in PV+ neurons developed motor, sensory, memory, and social deficits, whereas those lacking MeCP2 in SOM+ neurons exhibited seizures and stereotypies. Our findings indicate that PV+ and SOM+ neurons contribute complementary aspects of the Rett phenotype and may have modular roles in regulating specific behaviors.
doi:10.1016/j.neuron.2015.10.029
PMCID: PMC4656196  PMID: 26590342
2.  TRIM28 regulates the nuclear accumulation and toxicity of both alpha-synuclein and tau 
eLife  null;5:e19809.
Several neurodegenerative diseases are driven by the toxic gain-of-function of specific proteins within the brain. Elevated levels of alpha-synuclein (α-Syn) appear to drive neurotoxicity in Parkinson's disease (PD); neuronal accumulation of tau is a hallmark of Alzheimer's disease (AD); and their increased levels cause neurodegeneration in humans and model organisms. Despite the clinical differences between AD and PD, several lines of evidence suggest that α-Syn and tau overlap pathologically. The connections between α-Syn and tau led us to ask whether these proteins might be regulated through a shared pathway. We therefore screened for genes that affect post-translational levels of α-Syn and tau. We found that TRIM28 regulates α-Syn and tau levels and that its reduction rescues toxicity in animal models of tau- and α-Syn-mediated degeneration. TRIM28 stabilizes and promotes the nuclear accumulation and toxicity of both proteins. Intersecting screens across comorbid proteinopathies thus reveal shared mechanisms and therapeutic entry points.
DOI: http://dx.doi.org/10.7554/eLife.19809.001
eLife digest
Behind many neurodegenerative diseases are specific proteins that abnormally accumulate inside neurons and damage the cells. In Parkinson’s disease, the protein alpha-synuclein accumulates; in Alzheimer’s disease, the protein tau is one of the toxic culprits; and in other neurodegenerative diseases, alpha-synuclein and tau both accumulate. Genetic studies suggest that accumulation of the two proteins may be linked, but little is known about the factors that regulate the levels of these proteins inside neurons.
Rousseaux et al. set out to identify how these proteins are regulated in the hope of finding new ways of targeting them and reducing their toxicity. Screening a subset of human genes led to one that encodes a protein called TRIM28, which regulates the levels of both alpha-synuclein and tau. When the TRIM28 protein was depleted in human and mouse cells, the levels of alpha-synuclein and tau also went down. This effect was specific because the levels of other proteins with the potential to cause neurodegeneration remained unaffected.
Models of neurodegenerative disease in fruit flies and mice were then used to explore how TRIM28 affects the levels of tau and alpha-synuclein in animals. In each case, the proteins’ levels dropped when TRIM28 was suppressed and this in turn protected the neurons from damage. Rousseaux et al. went on to show that TRIM28 affected how alpha-synuclein and tau were cleared in cells. Overexpressing TRIM28 revealed that it could encourage both alpha-synuclein and tau to accumulate in the nucleus of cells over time.
Finally, Rousseaux et al. compared post-mortem brain tissue from people who had neurodegenerative conditions that are driven by or associated with tau and alpha-synuclein with tissue from those who did not. The cell nuclei in the diseased tissue had much more TRIM28 associated with alpha-synuclein and tau than those in the healthy tissues.
Overall, the findings show that TRIM28 promotes the accumulation and damaging effects of both alpha-synuclein and tau. The next steps will be to understand how TRIM28 does this. It will also be important to determine if this effect can be targeted, whilst leaving others roles of TRIM28 intact, in order to explore it as a potential target to treat or prevent neurodegenerative diseases.
DOI: http://dx.doi.org/10.7554/eLife.19809.002
doi:10.7554/eLife.19809
PMCID: PMC5104516  PMID: 27779468
Parkinson's disease; Alzheimer's disease; neurodegeneration; synucleinopathies; tauopathies; D. melanogaster; Human; Mouse
3.  Rett syndrome: disruption of epigenetic control of postnatal neurological functions 
Human Molecular Genetics  2015;24(R1):R10-R16.
Loss-of-function mutations in the X-linked gene Methyl-CpG-binding protein 2 (MECP2) cause a devastating pediatric neurological disorder called Rett syndrome. In males, these mutations typically result in severe neonatal encephalopathy and early lethality. On the other hand, owing to expression of the normal allele in ∼50% of cells, females do not suffer encephalopathy but instead develop Rett syndrome. Typically females with Rett syndrome exhibit a delayed onset of neurologic dysfunction that manifests around the child's first birthday and progresses over the next few years. Features of this disorder include loss of acquired language and motor skills, intellectual impairment and hand stereotypies. The developmental regression observed in patients with Rett syndrome arises from altered neuronal function and is not the result of neurodegeneration. Maintenance of an appropriate level of MeCP2 appears integral to the function of healthy neurons as patients with increased levels of MeCP2, owing to duplication of the Xq28 region encompassing the MECP2 locus, also present with intellectual disability and progressive neurologic symptoms. Despite major efforts over the past two decades to elucidate the molecular functions of MeCP2, the mechanisms underlying the delayed appearance of symptoms remain unclear. In this review, we will highlight recent findings that have expanded our knowledge of MeCP2's functions, and we will discuss how epigenetic regulation, chromatin organization and circuit dynamics may contribute to the postnatal onset of Rett syndrome.
doi:10.1093/hmg/ddv217
PMCID: PMC4571996  PMID: 26060191
4.  MECP2 disorders: from the clinic to mice and back 
The Journal of Clinical Investigation  2015;125(8):2914-2923.
Two severe, progressive neurological disorders characterized by intellectual disability, autism, and developmental regression, Rett syndrome and MECP2 duplication syndrome, result from loss and gain of function, respectively, of the same critical gene, methyl-CpG–binding protein 2 (MECP2). Neurons acutely require the appropriate dose of MECP2 to function properly but do not die in its absence or overexpression. Instead, neuronal dysfunction can be reversed in a Rett syndrome mouse model if MeCP2 function is restored. Thus, MECP2 disorders provide a unique window into the delicate balance of neuronal health, the power of mouse models, and the importance of chromatin regulation in mature neurons. In this Review, we will discuss the clinical profiles of MECP2 disorders, the knowledge acquired from mouse models of the syndromes, and how that knowledge is informing current and future clinical studies.
doi:10.1172/JCI78167
PMCID: PMC4563741  PMID: 26237041
5.  Restoration of Mecp2 expression in GABAergic neurons is sufficient to rescue multiple disease features in a mouse model of Rett syndrome 
eLife  null;5:e14198.
The postnatal neurodevelopmental disorder Rett syndrome, caused by mutations in MECP2, produces a diverse array of symptoms, including loss of language, motor, and social skills and the development of hand stereotypies, anxiety, tremor, ataxia, respiratory dysrhythmias, and seizures. Surprisingly, despite the diversity of these features, we have found that deleting Mecp2 only from GABAergic inhibitory neurons in mice replicates most of this phenotype. Here we show that genetically restoring Mecp2 expression only in GABAergic neurons of male Mecp2 null mice enhanced inhibitory signaling, extended lifespan, and rescued ataxia, apraxia, and social abnormalities but did not rescue tremor or anxiety. Female Mecp2+/- mice showed a less dramatic but still substantial rescue. These findings highlight the critical regulatory role of GABAergic neurons in certain behaviors and suggest that modulating the excitatory/inhibitory balance through GABAergic neurons could prove a viable therapeutic option in Rett syndrome.
DOI: http://dx.doi.org/10.7554/eLife.14198.001
eLife digest
Rett syndrome is a childhood brain disorder that mainly affects girls and causes symptoms including anxiety, tremors, uncoordinated movements and breathing difficulties. Rett syndrome is caused by mutations in a gene called MECP2, which is found on the X chromosome. Males with MECP2 mutations are rare but have more severe symptoms and die young. Many researchers who study Rett syndrome use mice as a model of the disorder. In particular, male mice with the mouse equivalent of the human MECP2 gene switched off in every cell in the body (also known as Mecp2-null mice) show many of the features of Rett syndrome and die at a young age.
The MECP2 gene is important for healthy brain activity. The brain contains two major types of neurons: excitatory neurons, which encourage other neurons to be active; and inhibitory neurons, which stop or dampen the activity of other neurons. In 2010, researchers reported that mice lacking Mecp2 in only their inhibitory neurons develop most of the same problems as those mice with no Mecp2 at all. This discovery led Ure et al. – including a researcher involved in the 2010 study – to ask if activating Mecp2 in the same neurons in otherwise Mecp2-null mice was enough to prevent some of their Rett syndrome-like symptoms.
The experiments showed that male mice that only have Mecp2 activated in their inhibitory neurons lived several months longer than male Mecp2-null mice. These male “rescue mice” also moved normally and had a normal body weight, though they still experienced anxiety, tremors and breathing difficulties. Female mice represent a better model of human Rett syndrome patients, and Ure et al. found that female rescue mice showed smaller improvements than the males.
These data suggest that when a brain is missing Mecp2 everywhere, as in male Mecp2-null mice, turning on Mecp2 in inhibitory neurons can make the brain network nearly normal and prevent most Rett-syndrome-like symptoms. However, the brains of female rescue mice contain both normal cells and cells with mutated Mecp2. This mixture of normal and abnormal cells appears to cause abnormalities that cannot be overcome by rescuing just the activity of the inhibitory neurons. These findings also highlight the importance of doing future studies in female mice to better understand the development of Rett syndrome.
The next challenge is to test different ways of activating the inhibitory neurons in the female mouse brain, for example by using drugs that target these neurons. It is hoped these methods will help researchers to refine a path toward potential new treatments for Rett syndrome patients. Finally, in a related study, Meng et al. asked how deleting or activating Mecp2 only in the excitatory neurons of mice affected Rett-syndrome-like symptoms.
DOI: http://dx.doi.org/10.7554/eLife.14198.002
doi:10.7554/eLife.14198
PMCID: PMC4946897  PMID: 27328321
Rett syndrome; GABAergic neurons; inhibition; Mouse
6.  Manipulations of MeCP2 in glutamatergic neurons highlight their contributions to Rett and other neurological disorders 
eLife  null;5:e14199.
Many postnatal onset neurological disorders such as autism spectrum disorders (ASDs) and intellectual disability are thought to arise largely from disruption of excitatory/inhibitory homeostasis. Although mouse models of Rett syndrome (RTT), a postnatal neurological disorder caused by loss-of-function mutations in MECP2, display impaired excitatory neurotransmission, the RTT phenotype can be largely reproduced in mice simply by removing MeCP2 from inhibitory GABAergic neurons. To determine what role excitatory signaling impairment might play in RTT pathogenesis, we generated conditional mouse models with Mecp2 either removed from or expressed solely in glutamatergic neurons. MeCP2 deficiency in glutamatergic neurons leads to early lethality, obesity, tremor, altered anxiety-like behaviors, and impaired acoustic startle response, which is distinct from the phenotype of mice lacking MeCP2 only in inhibitory neurons. These findings reveal a role for excitatory signaling impairment in specific neurobehavioral abnormalities shared by RTT and other postnatal neurological disorders.
DOI: http://dx.doi.org/10.7554/eLife.14199.001
eLife digest
Rett syndrome is a childhood brain disorder that mainly affects girls and causes symptoms including anxiety, tremors, uncoordinated movements and breathing difficulties. Rett syndrome is caused by mutations in a gene called MECP2, which is found on the X chromosome. Males with MECP2 mutations are rare but have more severe symptoms and die young. Many researchers who study Rett syndrome use mice as a model of the disorder. In particular, male mice with the mouse equivalent of the human MECP2 gene switched off in every cell in the body (also known as Mecp2-null mice) show many of the features of Rett syndrome and die at a young age.
The MECP2 gene is important for healthy brain activity. The brain contains two major types of neurons: excitatory neurons, which encourage other neurons to be active; and inhibitory neurons, which stop or dampen the activity of other neurons. In 2010, researchers reported that mice lacking Mecp2 in only their inhibitory neurons develop most of the same problems as those mice with no Mecp2 at all.
Now, Meng et al. – who include two researchers involved in the 2010 study – have asked how deleting or activating Mecp2 only in excitatory neurons of mice affects Rett-syndrome-like symptoms. The experiments showed that male mice without Mecp2 in their excitatory neurons develop tremors, anxiety-like behaviors, abnormal seizure-like brain activity and severe obesity; these mice also die earlier than normal mice. Female mice lacking Mecp2 in half of their excitatory neurons (because the gene is on the X chromosome) were less affected than the males, and had normal life spans. These symptoms are different from those seen in mice missing Mecp2 only in inhibitory neurons.
Meng et al. also found that if Mecp2 was switched on only in excitatory neurons of female mice (which are a model of human Rett syndrome patients) the mice were almost completely normal. In male mice (which show more severe symptoms), activating Mecp2 in only the excitatory neurons reduced the anxiety and tremors. These findings suggest that impaired excitatory neurons may be responsible for specific symptoms such as anxiety and tremors amongst other Rett-syndrome-like features.
The next challenge is to explore how the loss of Mecp2 changes the activity of excitatory neurons in different brain regions. Further studies could also investigate if drugs that improve the activity of excitatory neurons can be used to treat Rett syndrome patients. Finally, in a related study, Ure et al. asked if activating Mecp2 in inhibitory neurons in otherwise Mecp2-null mice was enough to prevent some of their Rett syndrome-like symptoms.
DOI: http://dx.doi.org/10.7554/eLife.14199.002
doi:10.7554/eLife.14199
PMCID: PMC4946906  PMID: 27328325
glutamatergic neurons; neurological disorders; MeCP2; Rett syndrome; Mouse
7.  Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligos 
Nature  2015;528(7580):123-126.
Copy number variations have been frequently associated with developmental delay, intellectual disability, and autism spectrum disorders1. MECP2 duplication syndrome is one of the most common genomic rearrangements in males2 and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections, and early death3–5. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question, however, was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders6–8, including loss of MeCP2 in Rett syndrome9, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we hypothesized that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. Therefore, we first generated and characterized a conditional Mecp2-overexpressing mouse model and showed that correction of MeCP2 levels largely reversed the behavioral, molecular, and electrophysiological deficits. Next, we sought a translational strategy to reduce MeCP2 and turned to antisense oligonucleotides (ASOs). ASOs are small modified nucleic acids that can selectively hybridize with mRNA transcribed from a target gene and silence it10,11, and have been successfully used to correct deficits in different mouse models12–18. We found that ASO treatment induced a broad phenotypic rescue in adult symptomatic transgenic MECP2 duplication mice (MECP2-TG)19,20, and corrected MECP2 levels in lymphoblastoid cells from MECP2 duplication patients in a dose-dependent manner.
doi:10.1038/nature16159
PMCID: PMC4839300  PMID: 26605526
8.  Forniceal deep brain stimulation rescues hippocampal memory in Rett syndrome mice 
Nature  2015;526(7573):430-434.
Deep brain stimulation (DBS) has improved the prospects for many individuals with diseases affecting motor control, and recently it has shown promise for improving cognitive function as well. Several studies in individuals with Alzheimer disease and in amnestic rats have demonstrated that DBS targeted to the fimbria-fornix1-3, the region that appears to regulate hippocampal activity, can mitigate defects in hippocampus-dependent memory3-5. Despite these promising results, DBS has not been tested for its ability to improve cognition in any childhood intellectual disability disorder (IDD). IDDs are a pressing concern: they affect as much as 3% of the population and involve hundreds of different genes. We hypothesized that stimulating the neural circuits that underlie learning and memory might provide a more promising route to treating these otherwise intractable disorders than seeking to adjust levels of one molecule at a time. We therefore studied the effects of forniceal DBS in a well-characterized mouse model of Rett Syndrome (RTT), which is a leading cause of intellectual disability in females. Caused by mutations that impair the function of MeCP26, RTT appears by the second year of life, causing profound impairment in cognitive, motor, and social skills along with an array of neurological features7; RTT mice, which reproduce the broad phenotype of this disorder, also show clear deficits in hippocampus-dependent learning and memory and hippocampal synaptic plasticity8-11. Here we show that forniceal DBS in RTT mice rescued contextual fear memory as well as spatial learning and memory. In parallel, forniceal DBS restored in vivo hippocampal long-term potentiation (LTP) and hippocampal neurogenesis. These results indicate that forniceal DBS might mitigate cognitive dysfunction in RTT.
doi:10.1038/nature15694
PMCID: PMC4828032  PMID: 26469053
9.  Fragile X-like behaviors and abnormal cortical dendritic spines in Cytoplasmic FMR1-interacting protein 2-mutant mice 
Human Molecular Genetics  2014;24(7):1813-1823.
Silencing of fragile X mental retardation 1 (FMR1) gene and loss of fragile X mental retardation protein (FMRP) cause fragile X syndrome (FXS), a genetic disorder characterized by intellectual disability and autistic behaviors. FMRP is an mRNA-binding protein regulating neuronal translation of target mRNAs. Abnormalities in actin-rich dendritic spines are major neuronal features in FXS, but the molecular mechanism and identity of FMRP targets mediating this phenotype remain largely unknown. Cytoplasmic FMR1-interacting protein 2 (Cyfip2) was identified as an interactor of FMRP, and its mRNA is a highly ranked FMRP target in mouse brain. Importantly, Cyfip2 is a component of WAVE regulatory complex, a key regulator of actin cytoskeleton, suggesting that Cyfip2 could be implicated in the dendritic spine phenotype of FXS. Here, we generated and characterized Cyfip2-mutant (Cyfip2+/−) mice. We found that Cyfip2+/− mice exhibited behavioral phenotypes similar to Fmr1-null (Fmr1−/y) mice, an animal model of FXS. Synaptic plasticity and dendritic spines were normal in Cyfip2+/− hippocampus. However, dendritic spines were altered in Cyfip2+/− cortex, and the dendritic spine phenotype of Fmr1−/y cortex was aggravated in Fmr1−/y; Cyfip2+/− double-mutant mice. In addition to the spine changes at basal state, metabotropic glutamate receptor (mGluR)-induced dendritic spine regulation was impaired in both Fmr1−/y and Cyfip2+/− cortical neurons. Mechanistically, mGluR activation induced mRNA translation-dependent increase of Cyfip2 in wild-type cortical neurons, but not in Fmr1−/y or Cyfip2+/− neurons. These results suggest that misregulation of Cyfip2 function and its mGluR-induced expression contribute to the neurobehavioral phenotypes of FXS.
doi:10.1093/hmg/ddu595
PMCID: PMC4355018  PMID: 25432536
10.  Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels 
Cell  2015;160(6):1087-1098.
SUMMARY
Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1 this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but that it also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1+/− mice to SCA1 mice (Atxn1154Q/+) exacerbated disease progression, whereas breeding them to Atxn1+/− mice normalized Ataxin1 levels and largely rescued the Pum1+/− phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.
doi:10.1016/j.cell.2015.02.012
PMCID: PMC4383046  PMID: 25768905
11.  Ataxin-1 oligomers induce local spread of pathology and decreasing them by passive immunization slows Spinocerebellar ataxia type 1 phenotypes 
eLife  null;4:e10891.
Previously, we reported that ATXN1 oligomers are the primary drivers of toxicity in Spinocerebellar ataxia type 1 (SCA1; Lasagna-Reeves et al., 2015). Here we report that polyQ ATXN1 oligomers can propagate locally in vivo in mice predisposed to SCA1 following intracerebral oligomeric tissue inoculation. Our data also show that targeting these oligomers with passive immunotherapy leads to some improvement in motor coordination in SCA1 mice and to a modest increase in their life span. These findings provide evidence that oligomer propagation is regionally limited in SCA1 and that immunotherapy targeting extracellular oligomers can mildly modify disease phenotypes.
DOI: http://dx.doi.org/10.7554/eLife.10891.001
doi:10.7554/eLife.10891
PMCID: PMC4821792  PMID: 26673892
ataxin-1; propagation; immunotherapy; oligomers; Mouse
12.  Characterization of the Transcriptome of Nascent Hair Cells and Identification of Direct Targets of the Atoh1 Transcription Factor 
The Journal of Neuroscience  2015;35(14):5870-5883.
Hair cells are sensory receptors for the auditory and vestibular system in vertebrates. The transcription factor Atoh1 is both necessary and sufficient for the differentiation of hair cells, and is strongly upregulated during hair-cell regeneration in nonmammalian vertebrates. To identify genes involved in hair cell development and function, we performed RNA-seq profiling of purified Atoh1-expressing hair cells from the neonatal mouse cochlea. We identified >600 enriched transcripts in cochlear hair cells, of which 90% have not been previously shown to be expressed in hair cells. We identified 233 of these hair cell genes as candidates to be directly regulated by Atoh1 based on the presence of Atoh1 binding sites in their regulatory regions and by analyzing Atoh1 ChIP-seq datasets from the cerebellum and small intestine. We confirmed 10 of these genes as being direct Atoh1 targets in the cochlea by ChIP-PCR. The identification of candidate Atoh1 target genes is a first step in identifying gene regulatory networks for hair-cell development and may inform future studies on the potential role of Atoh1 in mammalian hair cell regeneration.
doi:10.1523/JNEUROSCI.5083-14.2015
PMCID: PMC4388939  PMID: 25855195
Atoh1; cochlea; hair cells; inner ear
13.  NUDT21-spanning CNVs lead to neuropsychiatric disease and altered MeCP2 abundance via alternative polyadenylation 
eLife  null;4:e10782.
The brain is sensitive to the dose of MeCP2 such that small fluctuations in protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2 levels to brain function, little is known about its regulation. In this study, we report eleven individuals with neuropsychiatric disease and copy-number variations spanning NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2 protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in the MECP2 3′ UTR, resulting in an enrichment of inefficiently translated long mRNA isoforms. Furthermore, normalization of NUDT21 via siRNA-mediated knockdown in duplication patient lymphoblasts restores MeCP2 to normal levels. Ultimately, we identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via altered alternative polyadenylation.
DOI: http://dx.doi.org/10.7554/eLife.10782.001
eLife digest
The X-chromosome carries a number of genes that are involved in a child's intellectual development. One of these genes encodes a protein called MeCP2, which is important for brain function after birth. Mutations in the MECP2 gene cause a disorder known as Rett syndrome. At around 18 months of age, affected children begin to lose the cognitive and motor skills that they had previously acquired. Individuals with extra copies of this gene also show cognitive impairments. For both diseases, individuals with levels of the MeCP2 protein that are the most different from those found in healthy individuals also show the most severe symptoms.
To produce the protein that is encoded by a particular gene, enzymes inside the cell must first make a copy of that gene using a molecule called messenger ribonucleic acid (or mRNA). This mRNA is then used as a template to assemble the protein itself. In the case of MECP2, two different mRNA templates are produced: a long version and a short version. A gene called NUDT21 makes a protein that regulates whether the long or short version of MECP2 mRNA is made.
Gennarino, Alcott et al. have now discovered that people with too many, or too few, copies of the NUDT21 gene have intellectual disabilities and altered levels of MeCP2 protein. Specifically, individuals with extra copies of NUDT21—and thus higher levels of the corresponding protein—produce more of the long MECP2 mRNA. The production of proteins from this long mRNA is less efficient than from the short mRNA; therefore, these individuals have lower levels of MeCP2 protein. The opposite is true for individuals who lack a copy of the NUDT21 gene.
To confirm these data, Gennarino, Alcott et al. grew cells in the laboratory from patients with extra copies of the NUDT21 gene and found that reducing the production of its protein returned the levels of the MeCP2 protein back to normal. These findings show that alterations in the NUDT21 gene cause changes in the level of MeCP2 protein in cells and leads to neuropsychiatric diseases.
DOI: http://dx.doi.org/10.7554/eLife.10782.002
doi:10.7554/eLife.10782
PMCID: PMC4586391  PMID: 26312503
alternative polyadenylation; neuropsychiatric disease; NUDT21; MeCP2; intellectual disability; human
14.  A native interactor scaffolds and stabilizes toxic ATAXIN-1 oligomers in SCA1 
eLife  null;4:e07558.
Recent studies indicate that soluble oligomers drive pathogenesis in several neurodegenerative proteinopathies, including Alzheimer and Parkinson disease. Curiously, the same conformational antibody recognizes different disease-related oligomers, despite the variations in clinical presentation and brain regions affected, suggesting that the oligomer structure might be responsible for toxicity. We investigated whether polyglutamine-expanded ATAXIN-1, the protein that underlies spinocerebellar ataxia type 1, forms toxic oligomers and, if so, what underlies their toxicity. We found that mutant ATXN1 does form oligomers and that oligomer levels correlate with disease progression in the Atxn1154Q/+ mice. Moreover, oligomeric toxicity, stabilization and seeding require interaction with Capicua, which is expressed at greater ratios with respect to ATXN1 in the cerebellum than in less vulnerable brain regions. Thus, specific interactors, not merely oligomeric structure, drive pathogenesis and contribute to regional vulnerability. Identifying interactors that stabilize toxic oligomeric complexes could answer longstanding questions about the pathogenesis of other proteinopathies.
DOI: http://dx.doi.org/10.7554/eLife.07558.001
eLife digest
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease in which damage to the brain regions that control movement results in the gradual loss of coordination and motor skills. The disease is a caused by a mutation in the gene that codes for a protein called ATAXIN-1. In healthy individuals this protein contains up to 39 copies of an amino acid called glutamine. However, the mutant gene can encode for 40 or more copies of glutamine, which results in a longer-than-usual ATAXIN-1 protein with toxic properties.
Within the brain, some of the toxic ATAXIN-1 proteins form insoluble deposits, while the rest remain soluble. At first it was assumed that the insoluble deposits were responsible for the neurodegeneration seen in SCA1. However, closer examination revealed that these deposits form mainly in brain regions that do not degenerate, which suggests that they might instead have a protective role. This is consistent with evidence from research into other brain disorders, including Alzheimer's disease, which suggests that the soluble form of the toxic proteins might be causing these diseases.
Lasagna-Reeves et al. now provide the first direct evidence that the soluble form of the toxic ATAXIN-1 proteins are indeed harmful in a mouse model of SCA1. Experiments reveal that these soluble proteins accumulate in the brain regions that undergo degeneration in SCA1, such as the cerebellum, but not in those regions that remain intact. Moreover, the motor skills and coordination of the mice get worse as the level of soluble toxic ATAXIN-1 increases.
Lasagna-Reeves et al. go on to show that a protein called capicua stabilizes the toxic ATAXIN-1 proteins, which keeps them from forming insoluble deposits. Since capicua is particularly abundant in the cerebellum, this could explain the high levels of toxic ATAXIN-1 in this region and why it is vulnerable to degeneration.
Future experiments are needed to investigate whether proteins equivalent to capicua might play a similar role in stabilizing toxic proteins in Alzheimer's, Parkinson's and Huntington's diseases, and whether preventing this stabilization could have therapeutic potential.
DOI: http://dx.doi.org/10.7554/eLife.07558.002
doi:10.7554/eLife.07558
PMCID: PMC4462648  PMID: 25988806
ataxin-1; amyloid oligomers; native interactors; proteinopathies; mouse
15.  MECP2 Mutations in People without Rett Syndrome 
Mutations in Methyl-CpG-Binding protein 2 (MECP2) are commonly associated with and the neurodevelopmental disorder Rett syndrome (RTT). However, some people with RTT do not have mutations in MECP2, and interestingly there have been people identified with MECP2 mutations that do not have the clinical features of RTT. In this report we present four people with neurodevelopmental abnormalities and clear RTT-disease causing MECP2 mutation but lacking the characteristic clinical features of RTT. One patient's symptoms suggest an extension of the known spectrum of MECP2 associated phenotypes to include Global Developmental Delay (GDD) with Obsessive Compulsive Disorder (OCD) and Attention Deficit Hyperactivity Disorder (ADHD). These results furthermore reemphasize that RTT should remain a clinical diagnosis, based on the recent refurbished consensus criteria.
doi:10.1007/s10803-013-1902-z
PMCID: PMC3880396  PMID: 23921973
Rett syndrome; autism; neurodevelopmental disorders; MECP2; epigenetics; neurogenetics
16.  Deficiency of Capicua disrupts bile acid homeostasis 
Scientific Reports  2015;5:8272.
Capicua (CIC) has been implicated in pathogenesis of spinocerebellar ataxia type 1 and cancer in mammals; however, the in vivo physiological functions of CIC remain largely unknown. Here we show that Cic hypomorphic (Cic-L-/-) mice have impaired bile acid (BA) homeostasis associated with induction of proinflammatory cytokines. We discovered that several drug metabolism and BA transporter genes were down-regulated in Cic-L-/- liver, and that BA was increased in the liver and serum whereas bile was decreased within the gallbladder of Cic-L-/- mice. We also found that levels of proinflammatory cytokine genes were up-regulated in Cic-L-/- liver. Consistent with this finding, levels of hepatic transcriptional regulators, such as hepatic nuclear factor 1 alpha (HNF1α), CCAAT/enhancer-binding protein beta (C/EBPβ), forkhead box protein A2 (FOXA2), and retinoid X receptor alpha (RXRα), were markedly decreased in Cic-L-/- mice. Moreover, induction of tumor necrosis factor alpha (Tnfα) expression and decrease in the levels of FOXA2, C/EBPβ, and RXRα were found in Cic-L-/- liver before BA was accumulated, suggesting that inflammation might be the cause for the cholestasis in Cic-L-/- mice. Our findings indicate that CIC is a critical regulator of BA homeostasis, and that its dysfunction might be associated with chronic liver disease and metabolic disorders.
doi:10.1038/srep08272
PMCID: PMC4317698  PMID: 25653040
17.  Mutations in NGLY1 Cause an Inherited Disorder of the Endoplasmic Reticulum-Associated Degradation (ERAD) Pathway 
Purpose
The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for the translocation of misfolded proteins across the ER membrane into the cytosol for subsequent degradation by the proteasome. In order to understand the spectrum of clinical and molecular findings in a complex neurological syndrome, we studied a series of eight patients with inherited deficiency of N-glycanase 1 (NGLY1), a novel disorder of cytosolic ERAD dysfunction.
Methods
Whole-genome, whole-exome or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data.
Results
All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypo- or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele.
Conclusions
NGLY1 deficiency is a novel autosomal recessive disorder of the ERAD pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a more broad range of mutations are detected.
doi:10.1038/gim.2014.22
PMCID: PMC4243708  PMID: 24651605
NGLY1; alacrima; choreoathetosis; seizures; liver disease
18.  Rett-causing mutations reveal two domains critical for MeCP2 function and for toxicity in MECP2 duplication syndrome mice 
eLife  2014;3:e02676.
Loss of function of the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2) causes the progressive neurological disorder Rett syndrome (RTT). Conversely, duplication or triplication of Xq28 causes an equally wide-ranging progressive neurological disorder, MECP2 duplication syndrome, whose features overlap somewhat with RTT. To understand which MeCP2 functions cause toxicity in the duplication syndrome, we generated mouse models expressing endogenous Mecp2 along with a RTT-causing mutation in either the methyl-CpG binding domain (MBD) or the transcriptional repression domain (TRD). We determined that both the MBD and TRD must function for doubling MeCP2 to be toxic. Mutating the MBD reproduces the null phenotype and expressing the TRD mutant produces milder RTT phenotypes, yet both mutations are harmless when expressed with endogenous Mecp2. Surprisingly, mutating the TRD is more detrimental than deleting the entire C-terminus, indicating a dominant-negative effect on MeCP2 function, likely due to the disruption of a basic cluster.
DOI: http://dx.doi.org/10.7554/eLife.02676.001
eLife digest
Rett syndrome is a disorder that affects the development of the brain after birth. Infants with this condition develop as normal until they are 6–18 months old, when the development of their language and motor skills stops, or even regresses. Most cases of Rett syndrome are caused by mutations in a gene called MECP2.
If an individual mistakenly inherits an extra copy of the MECP2 gene, it can cause another developmental disorder called MECP2 duplication syndrome. This condition, which also affects the brain, gets worse over time and shares many features with Rett syndrome. The extra copy of the MECP2 gene leads to the production of too much MeCP2 protein. However, how doubling the level of this protein causes the syndrome and, in particular, which parts of the protein are involved are unknown.
Previously, researchers engineered mice that expressed a copy of the human MECP2 gene alongside their own version of the gene. These mice developed a condition similar to MECP2 duplication syndrome and many of these mice suffered from seizures and died within their first year.
Heckman et al. have now engineered mice that also have an extra human MECP2 gene but with one of two mutations that cause Rett syndrome in humans. Some mice had a mutation in a part of the MeCP2 protein that binds to DNA that is marked with small chemical tags called methyl groups. Other mice had a mutation in a domain of the protein that works to switch off genes. Heckman et al. found that mice with extra MeCP2 protein with either of these two mutations were as healthy as normal mice and showed none of the signs of MECP2 duplication syndrome. This indicates that both of these domains must be intact for doubling the levels of the MeCP2 protein to be harmful. Furthermore, Heckman et al. discovered that the mutation in the part of MeCP2 that works to switch genes off also reduces the protein's ability to bind to DNA.
The next challenge is to understand the mechanism by which doubling the levels of this protein causes harm to the brain. Further work is also needed to uncover why having too much MeCP2 protein or none at all cause syndromes that share many features.
DOI: http://dx.doi.org/10.7554/eLife.02676.002
doi:10.7554/eLife.02676
PMCID: PMC4102243  PMID: 24970834
MeCP2; Rett syndrome; MECP2 duplication syndrome; neurobiology; genetics; mouse
19.  Dendritic Arborization and Spine Dynamics Are Abnormal in the Mouse Model of MECP2 Duplication Syndrome 
The Journal of Neuroscience  2013;33(50):19518-19533.
MECP2 duplication syndrome is a childhood neurological disorder characterized by intellectual disability, autism, motor abnormalities, and epilepsy. The disorder is caused by duplications spanning the gene encoding methyl-CpG-binding protein-2 (MeCP2), a protein involved in the modulation of chromatin and gene expression. MeCP2 is thought to play a role in maintaining the structural integrity of neuronal circuits. Loss of MeCP2 function causes Rett syndrome and results in abnormal dendritic spine morphology and decreased pyramidal dendritic arbor complexity and spine density. The consequences of MeCP2 overexpression on dendritic pathophysiology remain unclear. We used in vivo two-photon microscopy to characterize layer 5 pyramidal neuron spine turnover and dendritic arborization as a function of age in transgenic mice expressing the human MECP2 gene at twice the normal levels of MeCP2 (Tg1; Collins et al., 2004). We found that spine density in terminal dendritic branches is initially higher in young Tg1 mice but falls below control levels after postnatal week 12, approximately correlating with the onset of behavioral symptoms. Spontaneous spine turnover rates remain high in older Tg1 animals compared with controls, reflecting the persistence of an immature state. Both spine gain and loss rates are higher, with a net bias in favor of spine elimination. Apical dendritic arbors in both simple- and complex-tufted layer 5 Tg1 pyramidal neurons have more branches of higher order, indicating that MeCP2 overexpression induces dendritic overgrowth. P70S6K was hyperphosphorylated in Tg1 somatosensory cortex, suggesting that elevated mTOR signaling may underlie the observed increase in spine turnover and dendritic growth.
doi:10.1523/JNEUROSCI.1745-13.2013
PMCID: PMC3858623  PMID: 24336718
20.  Atoh1-dependent rhombic lip neurons are required for temporal delay between independent respiratory oscillators in embryonic mice 
eLife  2014;3:e02265.
All motor behaviors require precise temporal coordination of different muscle groups. Breathing, for example, involves the sequential activation of numerous muscles hypothesized to be driven by a primary respiratory oscillator, the preBötzinger Complex, and at least one other as-yet unidentified rhythmogenic population. We tested the roles of Atoh1-, Phox2b-, and Dbx1-derived neurons (three groups that have known roles in respiration) in the generation and coordination of respiratory output. We found that Dbx1-derived neurons are necessary for all respiratory behaviors, whereas independent but coupled respiratory rhythms persist from at least three different motor pools after eliminating or silencing Phox2b- or Atoh1-expressing hindbrain neurons. Without Atoh1 neurons, however, the motor pools become temporally disorganized and coupling between independent respiratory oscillators decreases. We propose Atoh1 neurons tune the sequential activation of independent oscillators essential for the fine control of different muscles during breathing.
DOI: http://dx.doi.org/10.7554/eLife.02265.001
eLife digest
A healthy adult at rest will breathe in and out around 20 times per minute. Each breath requires a complex series of coordinated muscle activity. Inhalation begins with the opening of the airway followed by the contraction of the diaphragm and the intercostal muscles between the ribs, causing the chest cavity to expand. As the lungs increase in volume, the pressure inside them drops and air is drawn in. Relaxation of the diaphragm and intercostal muscles compresses the lungs, causing us to exhale.
Breathing is driven by the brainstem and it cannot be suppressed indefinitely: holding your breath eventually triggers a reflex that forces breathing to resume. The region of the brainstem that controls breathing is called the preBötzinger Complex. However, there is increasing evidence that a second region in the brainstem is also involved. This region, which is called the retrotrapezoid nucleus/parafacial respiratory group, consists of three types of excitatory neurons—Dbx1 neurons, Phox2b neurons, and Atoh1 neurons—but their roles had not been clear. Now, using multiple lines of genetically modified mice, Tupal et al. have teased apart the roles of these three cell types.
These experiments showed that the Dbx1 neurons—which are also found in the preBötzinger Complex—have an essential role in sending the signals from the brain that drive the different muscle activities needed to breathe. The Phox2b neurons modulate breathing based on the level of carbon dioxide in the blood. Atoh1 neurons help control the sequence of respiratory muscle activity during a breath, probably by selectively inhibiting different populations of Dbx1 neurons.
The work of Tupal et al. indicates that distinct populations of neurons within the brainstem independently control two different aspects of breathing: the generation of breathing rhythms, and the coordination of these rhythms. Given that many other physiological processes involve rhythmic activity patterns, this model may help us to understand how the brain generates and controls complex behaviors more generally.
DOI: http://dx.doi.org/10.7554/eLife.02265.002
doi:10.7554/eLife.02265
PMCID: PMC4060005  PMID: 24842997
breathing; central pattern generator; PreBötzinger Complex; oscillator; transcription; mouse
21.  Genetic screens reveal RAS/MAPK/MSK1 modulate ataxin 1 protein levels and toxicity in SCA1 
Nature  2013;498(7454):325-331.
Many neurodegenerative disorders such as Alzheimer’s, Parkinson’s and polyglutamine diseases share a common pathogenic mechanism: the abnormal accumulation of disease-causing proteins, due to either the mutant protein’s resistance to degradation or overexpression of the wild-type protein. We developed a strategy to identify therapeutic entry points for such neurodegenerative disorders by screening for genetic networks that influence the levels of disease-driving proteins. We applied this approach, which integrates parallel cell-based and Drosophila genetic screens, to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of a polyglutamine tract in ataxin 1 (ATXN1). Our approach revealed that downregulation of several components of the RAS–MAPK–MSK1 pathway decreases ATXN1 levels and suppresses neurodegeneration in Drosophila and mice. Importantly, pharmacologic inhibitors of components of this pathway also decrease ATXN1 levels, suggesting that these components represent new therapeutic targets in mitigating SCA1. Collectively, these data reveal new therapeutic entry points for SCA1 and provide a proof-of-principle for tackling other classes of intractable neurodegenerative diseases.
doi:10.1038/nature12204
PMCID: PMC4020154  PMID: 23719381
22.  SHANK3 overexpression causes manic-like behavior with unique pharmacogenetic properties 
Nature  2013;503(7474):72-77.
Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, suggesting that proper SHANK3 dosage is critical for normal brain function. SHANK3 overexpression per se has not been established as a cause of human disorders, however, because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modeling a human SHANK3 duplication exhibit manic-like behavior and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings suggest SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behavior of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.
doi:10.1038/nature12630
PMCID: PMC3923348  PMID: 24153177
23.  Synaptic Dysfunction in Neurodevelopmental Disorders Associated with Autism and Intellectual Disabilities 
The discovery of the genetic causes of syndromic autism spectrum disorders and intellectual disabilities has greatly informed our understanding of the molecular pathways critical for normal synaptic function. The top-down approaches using human phenotypes and genetics helped identify causative genes and uncovered the broad spectrum of neuropsychiatric features that can result from various mutations in the same gene. Importantly, the human studies unveiled the exquisite sensitivity of cognitive function to precise levels of many diverse proteins. Bottom-up approaches applying molecular, biochemical, and neurophysiological studies to genetic models of these disorders revealed unsuspected pathogenic mechanisms and identified potential therapeutic targets. Moreover, studies in model organisms showed that symptoms of these devastating disorders can be reversed, which brings hope that affected individuals might benefit from interventions even after symptoms set in. Scientists predict that insights gained from studying these rare syndromic disorders will have an impact on the more common nonsyndromic autism and mild cognitive deficits.
Approximately 1% of the human population has an autism spectrum disorder (ASD). Key insight into synaptic function has been gained by studying the many genetic alterations that cause ASD.
doi:10.1101/cshperspect.a009886
PMCID: PMC3282414  PMID: 22258914
24.  Female Mecp2+/− mice display robust behavioral deficits on two different genetic backgrounds providing a framework for pre-clinical studies 
Human Molecular Genetics  2012;22(1):96-109.
Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the gene encoding the transcriptional modulator methyl-CpG-binding protein 2 (MeCP2). Typical RTT primarily affects girls and is characterized by a brief period of apparently normal development followed by the loss of purposeful hand skills and language, the onset of anxiety, hand stereotypies, autistic features, seizures and autonomic dysfunction. Mecp2 mouse models have extensively been studied to demonstrate the functional link between MeCP2 dysfunction and RTT pathogenesis. However, the majority of studies have focused primarily on the molecular and behavioral consequences of the complete absence of MeCP2 in male mice. Studies of female Mecp2+/− mice have been limited because of potential phenotypic variability due to X chromosome inactivation effects. To determine whether reproducible and reliable phenotypes can be detected Mecp2+/− mice, we analyzed Mecp2+/− mice of two different F1 hybrid isogenic backgrounds and at young and old ages using several neurobehavioral and physiological assays. Here, we report a multitude of phenotypes in female Mecp2+/− mice, some presenting as early as 5 weeks of life. We demonstrate that Mecp2+/− mice recapitulate several aspects of typical RTT and show that mosaic expression of MeCP2 does not preclude the use of female mice in behavioral and molecular studies. Importantly, we uncover several behavioral abnormalities that are present in two genetic backgrounds and report on phenotypes that are unique to one background. These findings provide a framework for pre-clinical studies aimed at improving the constellation of phenotypes in a mouse model of RTT.
doi:10.1093/hmg/dds406
PMCID: PMC3522402  PMID: 23026749
25.  Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1 
Polyglutamine diseases are dominantly inherited neurodegenerative diseases caused by an expansion of a CAG trinucleotide repeat encoding a glutamine tract in the respective disease-causing proteins. Extensive studies have been performed to unravel disease pathogenesis and to develop therapeutics. Here, we report on several lines of evidence demonstrating that Nemo-like kinase (NLK) is a key molecule modulating disease toxicity in spinocerebellar ataxia type 1 (SCA1), a disease caused by a polyglutamine expansion in the protein ATAXIN1 (ATXN1). Specifically, we show that NLK, a serine/threonine kinase that interacts with ATXN1, modulates disease phenotypes of polyglutamine-expanded ATXN1 in a Drosophila model of SCA1. Importantly, the effect of NLK on SCA1 pathology is dependent upon NLK’s enzymatic activity. Consistent with this, reduced Nlk expression suppresses the behavioral and neuropathological phenotypes in SCA1 knock-in mice. These data clearly indicate that either reducing NLK enzymatic activity or decreasing NLK expression levels can have beneficial effects against the toxicity induced by polyglutamine-expanded ATXN1.
doi:10.1523/JNEUROSCI.3465-12.2013
PMCID: PMC3710458  PMID: 23719801

Results 1-25 (78)