Mutations in the transcriptional regulator MeCP2 severely impact key cellular and molecular signatures of human astrocytes during maturation.

Mutations in the MECP2 gene underlie a spectrum of neurodevelopmental disorders, most commonly Rett syndrome (RTT). We ask whether MECP2 mutations interfere with human astrocyte developmental maturation, thereby affecting their ability to support neurons. Using human-based models, we show that RTT-causing MECP2 mutations greatly impact the key role of astrocytes in regulating overall brain bioenergetics and that these metabolic aberrations are likely mediated by dysfunctional mitochondria. During post-natal maturation, astrocytes rely on neurons to induce their complex stellate morphology and transcriptional changes. While MECP2 mutations cause cell-intrinsic aberrations in the astrocyte transcriptional landscape, surprisingly, they do not affect the neuron-induced astrocyte gene expression. Notably, however, astrocytes are unable to develop complex mature morphology due to cell- and non-cell-autonomous aberrations caused by MECP2 mutations. Thus, MECP2 mutations critically impact key cellular and molecular features of human astrocytes and, hence, their ability to interact and support the structural and functional maturation of neurons.

Activity-dependent aberrations in gene expression and alternative splicing in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that affects about 1 in 10,000 female live births. The underlying cause of RTT is mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2); however, the molecular mechanism by which these mutations mediate the RTT neuropathology remains enigmatic. Specifically, although MeCP2 is known to act as a transcriptional repressor, analyses of the RTT brain at steady-state conditions detected numerous differentially expressed genes, while the changes in transcript levels were mostly subtle. Here we reveal an aberrant global pattern of gene expression, characterized predominantly by higher levels of expression of activity-dependent genes, and anomalous alternative splicing events, specifically in response to neuronal activity in a mouse model for RTT. Notably, the specific splicing modalities of intron retention and exon skipping displayed a significant bias toward increased retained introns and skipped exons, respectively, in the RTT brain compared with the WT brain. Furthermore, these aberrations occur in conjunction with higher seizure susceptibility in response to neuronal activity in RTT mice. Our findings advance the concept that normal MeCP2 functioning is required for fine-tuning the robust and immediate changes in gene transcription and for proper regulation of alternative splicing induced in response to neuronal stimulation.

Acute and crucial requirement for MeCP2 function upon transition from early to late adult stages of brain maturation.

Germline mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2), underlie most cases of Rett syndrome (RTT), an autism spectrum disorder affecting approximately one in 10 000 female live births. The disease is characterized in affected girls by a latent appearance of symptoms between 12 and 18 months of age while boys usually die before the age of two. The nature of the latency is not known, but RTT-like phenotypes are recapitulated in mouse models, even when MeCP2 is removed at different postnatal stages, including juvenile and adolescent stages. Unexpectedly, here, we show that within a very brief developmental window, between 10 (adolescent) and 15 (adult) weeks after birth, symptom initiation and progression upon removal of MeCP2 in male mice transitions from 3 to 4 months to only several days, followed by lethality. We further show that this accelerated development of RTT phenotype and lethality occur at the transition to adult stage (15 weeks of age) and persists thereafter. Importantly, within this abbreviated time frame of days, the brain acquires dramatic anatomical, cellular and molecular abnormalities, typical of classical RTT. This study reveals a new postnatal developmental stage, which coincides with full-brain maturation, where the structure/function of the brain is extremely sensitive to levels of MeCP2 and loss of MeCP2 leads to precipitous collapse of the neuronal networks and incompatibility with life within days.

An RNA binding protein promotes axonal integrity in peripheral neurons by destabilizing REST.

The RE1 Silencing Transcription Factor (REST) acts as a governor of the mature neuronal phenotype by repressing a large consortium of neuronal genes in non-neuronal cells. In the developing nervous system, REST is present in progenitors and downregulated at terminal differentiation to promote acquisition of mature neuronal phenotypes. Paradoxically, REST is still detected in some regions of the adult nervous system, but how REST levels are regulated, and whether REST can still repress neuronal genes, is not known. Here, we report that homeostatic levels of REST are maintained in mature peripheral neurons by a constitutive post-transcriptional mechanism. Specifically, using a three-hybrid genetic screen, we identify the RNA binding protein, ZFP36L2, associated previously only with female fertility and hematopoiesis, and show that it regulates REST mRNA stability. Dorsal root ganglia in Zfp36l2 knock-out mice, or wild-type ganglia expressing ZFP36L2 shRNA, show higher steady-state levels of Rest mRNA and protein, and extend thin and disintegrating axons. This phenotype is due, at least in part, to abnormally elevated REST levels in the ganglia because the axonal phenotype is attenuated by acute knockdown of REST in Zfp36l2 KO DRG explants. The higher REST levels result in lower levels of target genes, indicating that REST can still fine-tune gene expression through repression. Thus, REST levels are titrated in mature peripheral neurons, in part through a ZFP36L2-mediated post-transcriptional mechanism, with consequences for axonal integrity.

Oligodendrocyte lineage cells contribute unique features to Rett syndrome neuropathology.

Mutations in the methyl-CpG binding protein 2 gene, Mecp2, affect primarily the brain and lead to a wide range of neuropsychiatric disorders, most commonly Rett syndrome (RTT). Although the neuropathology of RTT is well understood, the cellular and molecular mechanism(s), which lead to the disease initiation and progression, has yet to be elucidated. RTT was initially attributed only to neuronal dysfunction, but our recent studies and those of others show that RTT is not exclusively neuronal but rather also involves interactions between neurons and glia. Importantly, studies have shown that MeCP2-restored astrocytes and microglia are able to attenuate the disease progression in otherwise MeCP2-null mice. Here we show that another type of glia, oligodendrocytes, and their progenitors are also involved in manifestation of specific RTT symptoms. Mice that lost MeCP2 specifically in the oligodendrocyte lineage cells, although overall normal, were more active and developed severe hindlimb clasping phenotypes. Inversely, restoration of MeCP2 in oligodendrocyte lineage cells, in otherwise MeCP2-null mice, although only mildly prolonging their lifespan, significantly improved the locomotor deficits and hindlimb clasping phenotype, both in male and female mice, and fully restored the body weight in male mice. Finally, we found that the level of some myelin-related proteins was impaired in the MeCP2-null mice. Expression of MeCP2 in oligodendrocytes of these mice only partially restored their expression, suggesting that there is a non-cell-autonomous effect by other cell types in the brains on the expression of myelin-related proteins in oligodendrocytes.

MeCP2 is critical for maintaining mature neuronal networks and global brain anatomy during late stages of postnatal brain development and in the mature adult brain.

Mutations in the X-linked gene, methyl-CpG binding protein 2 (Mecp2), underlie a wide range of neuropsychiatric disorders, most commonly, Rett Syndrome (RTT), a severe autism spectrum disorder that affects approximately one in 10,000 female live births. Because mutations in the Mecp2 gene occur in the germ cells with onset of neurological symptoms occurring in early childhood, the role of MeCP2 has been ascribed to brain maturation at a specific developmental window. Here, we show similar kinetics of onset and progression of RTT-like symptoms in mice, including lethality, if MeCP2 is removed postnatally during the developmental stage that coincides with RTT onset, or adult stage. For the first time, we show that brains that lose MeCP2 at these two different stages are actively shrinking, resulting in higher than normal neuronal cell density. Furthermore, we show that mature dendritic arbors of pyramidal neurons are severely retracted and dendritic spine density is dramatically reduced. In addition, hippocampal astrocytes have significantly less complex ramified processes. These changes accompany a striking reduction in the levels of several synaptic proteins, including CaMKII α/ß, AMPA, and NMDA receptors, and the synaptic vesicle proteins Vglut and Synapsin, which represent critical modifiers of synaptic function and dendritic arbor structure. Importantly, the mRNA levels of these synaptic proteins remains unchanged, suggesting that MeCP2 likely regulates these synaptic proteins post-transcriptionally, directly or indirectly. Our data suggest a crucial role for MeCP2 in post-transcriptional regulation of critical synaptic proteins involved in maintaining mature neuronal networks during late stages of postnatal brain development.

REST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells.

REST is a master repressor of neuronal genes; however, whether it has any role during nervous system development remains largely unknown. Here, we analyzed systematically the role of REST in embryonic stem cells and multipotent neural stem/progenitor (NS/P) cells, including neurogenic and gliogenic NS/P cells derived from embryonic stem (ES) cells or developing mouse embryos. We showed that REST-null ES cells remained pluripotent and generated teratomas consisting of the three germ layers. By contrast, multipotent NS/P cells lacking REST displayed significantly reduced self-renewal capacity owing to reduced cell cycle kinetics and precocious neuronal differentiation. Importantly, although early-born neurogenic NS/P cells that lack REST were capable of differentiating to neurons and glia, the neuronal and oligodendrocytic pools were significantly enlarged and the astrocytic pool was shrunken. However, gliogenic NS/P cells lacking REST were able to generate a normal astrocytic pool size, suggesting that the shrinkage of the astrocytic pool generated from neurogenic NS/P cells lacking REST probably occurs by default. Microarray profiling of early-born NS/P cells lacking REST showed upregulation of neuronal as well as oligodendrocytic genes, specifically those involved in myelination. Furthermore, chromatin immunoprecipitation analyses showed that some of the upregulated oligodendrocytic genes contain an RE1 motif and are direct REST targets. Together, our data support a central role for REST during neural development in promoting NS/P cell self-renewal while restricting the generation and maturation of neurons and oligodendrocytes.

Repressor element 1 silencing transcription factor (REST) controls radial migration and temporal neuronal specification during neocortical development.

Neurogenesis requires mechanisms that coordinate early cell-fate decisions, migration, and terminal differentiation. Here, we show that the transcriptional repressor, repressor element 1 silencing transcription factor (REST), regulates radial migration and the timing of neural progenitor differentiation during neocortical development, and that the regulation is contingent upon differential REST levels. Specifically, a sustained presence of REST blocks migration and greatly delays--but does not prevent--neuronal differentiation, resulting in a subcortical band heterotopia-like phenotype, reminiscent of loss of doublecortin. We further show that doublecortin is a direct gene target of REST, and that its overexpression rescues, at least in part, the aberrant phenotype caused by persistent presence of REST. Our studies support the view that the targeted down-regulation of REST to low levels in neural progenitors, and its subsequent disappearance during neurogenesis, is critical for timing the spatiotemporal transition of neural progenitor cells to neurons.

A role for glia in the progression of Rett's syndrome.

Rett's syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed in most tissues, loss of MeCP2 expression results primarily in neurological symptoms. Earlier studies suggested the idea that RTT is due exclusively to loss of MeCP2 function in neurons. Although defective neurons clearly underlie the aberrant behaviours, we and others showed recently that the loss of MECP2 from glia negatively influences neurons in a non-cell-autonomous fashion. Here we show that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter VGLUT1. Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports the targeting of glia as a strategy for improving the associated symptoms.

Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology.

The neurodevelopmental disorder Rett syndrome (RTT) is caused by sporadic mutations in the transcriptional factor methyl-CpG-binding protein 2 (MeCP2). Although it is thought that the primary cause of RTT is cell autonomous, resulting from a lack of functional MeCP2 in neurons, whether non-cell autonomous factors contribute to the disease is unknown. We found that the loss of MeCP2 occurs not only in neurons but also in glial cells of RTT brains. Using an in vitro co-culture system, we found that mutant astrocytes from a RTT mouse model, and their conditioned medium, failed to support normal dendritic morphology of either wild-type or mutant hippocampal neurons. Our studies suggest that astrocytes in the RTT brain carrying MeCP2 mutations have a non-cell autonomous effect on neuronal properties, probably as a result of aberrant secretion of soluble factor(s).

The many faces of REST oversee epigenetic programming of neuronal genes.

Nervous system development relies on a complex signaling network to engineer the orderly transitions that lead to the acquisition of a neural cell fate. Progression from the non-neuronal pluripotent stem cell to a restricted neural lineage is characterized by distinct patterns of gene expression, particularly the restriction of neuronal gene expression to neurons. Concurrently, cells outside the nervous system acquire and maintain a non-neuronal fate that permanently excludes expression of neuronal genes. Studies of the transcriptional repressor REST, which regulates a large network of neuronal genes, provide a paradigm for elucidating the link between epigenetic mechanisms and neurogenesis. REST orchestrates a set of epigenetic modifications that are distinct between non-neuronal cells that give rise to neurons and those that are destined to remain as nervous system outsiders.

REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis.

Regulation of neuronal gene expression is critical to central nervous system development. Here, we show that REST regulates the transitions from pluripotent to neural stem/progenitor cell and from progenitor to mature neuron. In the transition to progenitor cell, REST is degraded to levels just sufficient to maintain neuronal gene chromatin in an inactive state that is nonetheless poised for expression. As progenitors differentiate into neurons, REST and its co-repressors dissociate from the RE1 site, triggering activation of neuronal genes. In some genes, the level of expression is adjusted further in neurons by CoREST/MeCP2 repressor complexes that remain bound to a site of methylated DNA distinct from the RE1 site. Expression profiling based on this mechanism indicates that REST defines a gene set subject to plasticity in mature neurons. Thus, a multistage repressor mechanism controls the orderly expression of genes during development while still permitting fine tuning in response to specific stimuli.