Dendritic spine dysgenesis in Rett syndrome

Spines are small cytoplasmic extensions of dendrites that form the postsynaptic compartment of the majority of excitatory synapses in the mammalian brain. Alterations in the numerical density, size, and shape of dendritic spines have been correlated with neuronal dysfunction in several neurological and neurodevelopmental disorders associated with intellectual disability, including Rett syndrome (RTT). RTT is a progressive neurodevelopmental disorder associated with intellectual disability that is caused by loss of function mutations in the transcriptional regulator methyl CpG-binding protein 2 (MECP2). Here, we review the evidence demonstrating that principal neurons in RTT individuals and Mecp2-based experimental models exhibit alterations in the number and morphology of dendritic spines. We also discuss the exciting possibility that signaling pathways downstream of brain-derived neurotrophic factor (BDNF), which is transcriptionally regulated by MeCP2, offer promising therapeutic options for modulating dendritic spine development and plasticity in RTT and other MECP2-associated neurodevelopmental disorders.


INTRODUCTION
Espinas dendríticas are small cytoplasmic extensions emerging from the dendrites of neurons that were first described in the cerebellum and cerebrum of birds and mammals by Santiago Ramón y Cajal at the end of the 19th century (Ramón y Cajal, 1888, 1891b, 1896; as cited in Yuste, 2010). Cajal had already envisioned that dendritic spines are contacted by axons at synapses (Ramón y Cajal, 1891a, 1894, and used this arrangement as the main example in support of his Neuronal Doctrine (Ramón y Cajal, 1933). With the aid of electron microscopy and confocal fluorescence microscopy, it is now well established that spines are the postsynaptic sites of most excitatory synapses in the brain, receiving inputs from glutamatergic axons (Bhatt et al., 2009;Yuste, 2010;Shirao and Gonzalez-Billault, 2013). Despite their small size (∼1 μm in diameter), proper dendritic spine formation is critical for brain function. Numerous proteins, including neurotransmitter and neuropeptide receptors, signaling kinases, and phosphatases, as well as ion channels are expressed in dendritic spines, where they participate in excitatory synaptic transmission and activity-dependent synaptic plasticity, and ultimately in learning and memory (Sala and Segal, 2014). During development and throughout adulthood, the numerical density and morphology of individual spines are critical for the fine-tuning of neuronal and synaptic excitability, allowing the initial establishment and activity-dependent remodeling of connectivity of neuronal circuits (Luebke et al., 2010).
The morphology of dendritic spines is highly variable, and by defining the biochemical and electrical properties of the postsynaptic compartment, it contributes to the strength and plasticity of excitatory synapses (Luebke et al., 2010). Spines have been broadly classified into three morphological types: stubby, mushroom and thin (Peters and Kaiserman-Abramof, 1969). Mushroom spines have a large head that is connected to the parent dendrite through a narrow neck. Stubby spines do not have a noticeable neck and are most common during postnatal development (Chapleau et al., 2009b;Rochefort and Konnerth, 2012). These two types of large spines are referred to as "memory spines," because they are stable, persist for longer periods of time, and are the postsynaptic side of strong excitatory synapses (Trachtenberg et al., 2002;Kasai et al., 2003). Conversely, thin spines have a thin, long neck, and a small bulbous head, are highly motile, unstable, and often short-lived, usually representing weak or silent synapses (Rochefort and Konnerth, 2012). Because thin spines are more plastic than large spines and have the potential to become stable spines, they have been dubbed "learning spines" (Grutzendler et al., 2002;Trachtenberg et al., 2002;Kasai et al., 2003;Holtmaat et al., 2005). It should be noted that thin protrusions longer than thin spines and without a noticeable head are called dendritic filopodia, and are more numerous than spines in developing neurons. Dendritic filopodia are transient and highly motile protrusions that can receive synaptic input and develop into mature spines, thus initiating synaptogenesis (Fiala et al., 1998;Luebke et al., 2010).
Following the well-known relationship between form and function in biological systems, recent in vitro and in vivo studies have demonstrated that the morphology of spines relates closely to the function and plasticity of the synapses they belong to (Yuste et al., 2000;Trachtenberg et al., 2002;Mizrahi et al., 2004;Segal, 2005;Kasai et al., 2010). For example, the volume of the spine head is directly proportional to the area of the postsynaptic density and the number of synaptic vesicles docked at the presynaptic active zone (Harris and Stevens, 1989;Schikorski and Stevens, 1999), the number of postsynaptic receptors (Nusser et al., 1998), and hence to the size of synaptic currents and synaptic strength (Yuste and Bonhoeffer, 2001;Luebke et al., 2010). Two-photon uncaging of glutamate on large spines evoked Frontiers in Neuroanatomy www.frontiersin.org larger postsynaptic currents mediated by AMPA receptors than uncaging on small spines (Matsuzaki et al., 2001;Kasai et al., 2010). Such a structure-function relationship is also evident in the intracellular Ca 2+ signals within spines triggered by afferent synaptic activity (Yuste et al., 2000;Nimchinsky et al., 2002;Bloodgood and Sabatini, 2007). Together with structural changes in response to afferent synaptic stimulation (Murphy and Segal, 1996;Srivastava and Penzes, 2011), all these findings support the long held view that dendritic spines are the morphological substrate of neuronal plasticity and learning and memory (Sala and Segal, 2014). In support of this notion, induction of long-term potentiation (LTP) leads to spine enlargement (Matsuzaki et al., 2004;Park et al., 2006), whereas long-term depression (LTD) causes spine shrinkage (Nagerl et al., 2004;Zhou et al., 2004;Hoogenraad and Akhmanova, 2010;Penzes et al., 2011b). The relationship between dendritic spines and cognitive abilities was noted in early studies, when the term "spine dysgenesis" was coined by Dominick Purpura (Huttenlocher, 1970;Marin-Padilla, 1972;Purpura, 1974). Such anomalies in the morphology -and likely function -of dendritic spines have been described in several neurological disorders associated with cognitive decline, including typical aging, Alzheimer's and Huntington diseases, schizophrenia, neurodevelopmental intellectual disabilities, and autism spectrum disorders (Fiala et al., 2002;Fukuda et al., 2005;Zhao et al., 2007;Bourgeron, 2009;Chapleau et al., 2009b;Garey, 2010;Penzes et al., 2011a;Levenga and Willemsen, 2012).

DENDRITIC SPINE DYSGENESIS IN RETT SYNDROME
Rett syndrome (RTT) is an X-linked progressive autism spectrum disorder associated with intellectual disability that affects girls during early childhood (∼1:15,000 birth worldwide; Neul and Zoghbi, 2004;Chapleau et al., 2009b). The disorder is characterized by a seemingly typical development for 6 to 18 months followed by regression and onset of a variety of neurological features, including motor impairments, loss of acquired language, intellectual disability, seizures, and anxiety (Chahrour and Zoghbi, 2007). The majority of RTT individuals carry loss-of-function mutations in MECP2, the gene encoding methyl CpG-binding protein 2 (MeCP2), a global transcriptional regulator that binds to methylated CpG sites in promoter regions of DNA (Amir et al., 1999;Chahrour et al., 2008). Emerging evidence indicates that RTT results from a deficit in synaptic maturation in the brain, and that MeCP2 plays a critical role in neuronal and synaptic maturation and pruning during development (Cohen et al., 2003;Calfa et al., 2011b), as well as in the function of established neuronal networks in adulthood (McGraw et al., 2011).
The spine dysgenesis phenotype in pyramidal neurons of the hippocampus in Mecp2 KO mice has revealed unexpected complexities. CA1 and CA3 pyramidal neurons have lower spine density only in neonatal (postnatal day-7) Mecp2 KOs, well before excitatory synapse expansion. Spine density reaches wildtype (WT) levels a week later (postnatal day-15), and is maintained at WT levels throughout the symptomatic stage (postnatal day-40 to 60). Quantitative electron microscopy confirmed that the density of asymmetric spine synapses in CA1 stratum radiatum of Mecp2 KOs is comparable to that of WT mice (Calfa et al., 2011a;Chapleau et al., 2012). This developmental progression of the spine density phenotype is also reflected in the density of excitatory synapses imaged as VGLUT1-PSD95 immunofluorescent puncta, which is lower in area CA1 of 2 week-old Mecp2 null mice, but comparable to WT levels at 5 weeks of age (Chao et al., 2007). Altogether, these data demonstrate that proper MeCP2 functioning is required for dendritic spine formation during early postnatal development, and that a secondary compensatory mechanism seems to take place during atypical development in Mecp2 KOs. A couple of possibilities exist as to the extent of the compensatory mechanisms necessary to bring spine density to WT levels in hippocampal neurons. One possibility is that enhanced hippocampal network activity in Mecp2 KOs promotes dendritic spine formation (Calfa et al., 2011a). A second possibility is that deranged homeostatic plasticity promotes spinogenesis, while still affecting pyramidal neuron function (Blackman et al., 2012;Qiu et al., 2012). Consistent with a model that tightly regulated MeCP2 levels are necessary during brain development and adulthood, overexpression of Mecp2 in vitro or in the MECP2 duplication mouse model (Mecp2 Tg1 ) either increased (Jugloff et al., 2005;Chao et al., 2007;Jiang et al., 2013) or decreased (Zhou et al., 2006;Chapleau et al., 2009a;Cheng et al., 2014) dendritic complexity, spine density, and the density of excitatory synapses. Table 1 sumarizes all the published work on dendritic spines in RTT and experimenal models based on MeCP2 loss-of-function.
Activity-dependent release of endogenously expressed (native) BDNF also modulates spine morphology in conjunction with spontaneous neurotransmitter release (Tyler et al., 2002b;Tyler and Pozzo-Miller, 2003;Tanaka et al., 2008). In addition, proper secretory trafficking of BDNF is essential for its actions on dendritic spine development and plasticity. The human BDNF gene has a single nucleotide polymorphism -a methionine (met) substitution for valine (val) at codon 66 -that impairs BDNF trafficking and its activity-dependent release, resulting in cognitive dysfunction in the general population (Egan et al., 2003;Chen et al., 2004), as well as more severe neurological symptoms in RTT individuals (Zeev et al., 2009). Consistently, dendritic complexity is reduced in dentate granule cells of Val66Met knock-in mice (Chen et al., 2006). Therefore, expression of this BDNF polymorphism might lead to deleterious effects on dendritic spine density and morphology.
The main limitation of BDNF-based therapies for neurological disorders, including RTT, is its poor blood-brain barrier permeability. Synthetic BDNF-loop mimetics with selective TrkB agonist activity are exciting alternatives (Massa et al., 2010;Kajiya et al., 2014). Indeed, systemic treatment with LM22A-4 rescues respiratory deficits in female Mecp2 heterozygous mice (Schmid et al., 2012), and prevents spine loss in striatal medium-spiny neurons in a mouse model of Huntington's, rescuing their motor deficits (Simmons et al., 2013).
Other intriguing substitutes for BDNF are insulin-like growth factor-1 (IGF-1) and its active tripeptide ([1-3]IGF-1, also known as glypromate, GPE), a hormone widely expressed in the CNS during brain development that promotes neuronal survival as well as synaptic maturation (D'Ercole, 1996;O'Kusky et al., 2000;Tropea et al., 2009). Indeed, systemic treatment of male Mecp2 KO mice with [1-3]IGF-1 significantly increased activity of signaling pathways downstream of TrkB and improved several RTT-like symptoms and increased dendritic spine density in cortical neurons (Tropea et al., 2009), effects that are all recapitulated by full-length IGF-1 (Castro et al., 2014). These effects are due to the activation of IGF-1 receptors directly by IGF-1, and indirectly by [1-3]IGF-1, which does not bind to the IGF-1 receptor but rather increases the expression of IGF-1 (Carlsson-Skwirut et al., 1989;Corvin et al., 2012). It should be noted that full-length IGF-1 worsened a metabolic syndrome in Mecp2 KO mice, and did not affect dendritic spine density in hippocampal neurons (Pitcher et al., 2013). The safety and efficacy of recombinant human fulllength IGF-1 (mecasermin) in a Phase-1 clinical trial in RTT individuals have been recently reported (Khwaja et al., 2014). The [1-3]IGF-1 analog glycyl-L-methylprolyl-L-glutamic acid (NNZ-2566; Neuren Pharmaceuticals) is in a Phase-2 clinical trial in RTT individuals.

CONCLUSION
Activity-dependent plasticity of dendritic spines includes both the formation of new spines and their maturation from thin, filipodia-like protrusions to "memory spines" that accompany excitatory synapse formation during brain development, as well as the structural remodeling of already existing spines. Alterations in neuronal circuitry are due to, or at least reflected by, deficits in dendritic spine structure and function. Dendritic spine anomalies have been identified in multiple brain regions in RTT and Mecp2-based mouse models. Since BDNF promotes the formation, maintenance, and activity-dependent sculpting of dendritic spines, and plays a critical role in neurological dysfunction in RTT, it emerges as one of the most exciting therapeutic agents for RTT. Thus, treatments that target the BDNF receptor TrkB and/or its downstream signaling pathways stand out as strong candidates to improve not only the spine dysgenesis phenotype, but also other synaptic plasticity deficits in RTT and other neurodevelopmental disorders caused by impaired BDNF availability.