Epigenetic regulation of neuronal dendrite and dendrite spine development

Dendrites and the dendritic spines of neurons play key roles in the connectivity of the brain and have been recognized as the locus of long-term synaptic plasticity,which is correlated with learning and memory.The development of dendrites and spines in the mammalian central nervous system is a complex process that requires specific molecular events over a period of time.It has been shown that specific molecules are needed not only at the spine's point of contact,but also at a distance,providing signals that initiate a cascade of events leading to synapse formation.The specific molecules that act to signal neuronal differentiation,dendritic morphology,and synaptogenesis are tightly regulated by genetic and epigenetic programs.It has been shown that the dendritic spine structure and distribution are altered in many diseases,including many forms of mental retardation(MR),and can also be potentiated by neuronal activities and an enriched environment.Because dendritic spine pathologies are found in many types of MR,it has been proposed that an inability to form normal spines leads to the cognitive and motor deficits that are characteristic of MR.Epigenetic mechanisms,including DNA methylation,chromatin remodeling,and the noncoding RNA-mediated process,have profound regulatory roles in mammalian gene expression.The study of epigenetics focuses on cellular effects that result in a heritable pattern of gene expression without changes to genomic encoding.Despite extensive efforts to understand the molecular regulation of dendrite and spine development,epigenetic mechanisms have only recently been considered.In this review,we will focus on epigenetic mechanisms that regulate the development and maturation of dendrites and spines.We will discuss how epigenetic alterations could result in spine abnormalities that lead to MR,such as is seen in fragile X and Rett syndromes.We will also discuss both general methodology and recent technological advances in the study of neuronal dendrites and spines.     

Epigenetic regulation of neuronal dendrite and dendritic spine development

Dendrites and the dendritic spines of neurons play key roles in the connectivity of the brain and have been recognized as the locus of long-term synaptic plasticity, which is correlated with learning and memory. The development of dendrites and spines in the mammalian central nervous system is a complex process that requires specific molecular events over a period of time. It has been shown that specific molecules are needed not only at the spine’s point of contact, but also at a distance, providing signals that initiate a cascade of events leading to synapse formation. The specific molecules that act to signal neuronal differentiation, dendritic morphology, and synaptogenesis are tightly regulated by genetic and epigenetic programs. It has been shown that the dendritic spine structure and distribution are altered in many diseases, including many forms of mental retardation (MR), and can also be potentiated by neuronal activities and an enriched environment. Because dendritic spine pathologies are found in many types of MR, it has been proposed that an inability to form normal spines leads to the cognitive and motor deficits that are characteristic of MR. Epigenetic mechanisms, including DNA methylation, chromatin remodeling, and the noncoding RNA-mediated process, have profound regulatory roles in mammalian gene expression. The study of epigenetics focuses on cellular effects that result in a heritable pattern of gene expression without changes to genomic encoding. Despite extensive efforts to understand the molecular regulation of dendrite and spine development, epigenetic mechanisms have only recently been considered. In this review, we will focus on epigenetic mechanisms that regulate the development and maturation of dendrites and spines. We will discuss how epigenetic alterations could result in spine abnormalities that lead to MR, such as is seen in fragile X and Rett syndromes. We will also discuss both general methodology and recent technological advances in the study of neuronal dendrites and spines.     

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4–5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1–2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour. As evidence of the specificity of these NGF effects, epidermal growth factor and dibutyryl cAMP, agents that elicit responses in PC12 cells, but do not promote their neuronal differentiation, had no observable effect on NGF-deprived growth cones. These findings demonstrate that NGF exerts very rapid effects on growth cone shape and surface morphology. Such actions may play roles in regulation of growth cone movement and guidance by NGF.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Negative regulation of EphA2 receptor by Cbl

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.     

Dendritic spine geometry: functional implication and regulation

Dendritic spines are tiny protrusions on dendritic shafts where most excitatory synapses are located. Recent advances in imaging technologies have given us great insight into the function of spines as biochemical compartments. Here we review recent evidence suggesting that the geometry of dendritic spines controls postsynaptic calcium signaling and is bidirectionally regulated during synaptic plasticity.     

Cdc42: an important regulator of neuronal morphology

Regulation of neuronal morphology and activity-dependent synaptic modifications involves reorganization of the actin cytoskeleton. Dynamic changes of the actin cytoskeleton in many cell types are controlled by small GTPases of the Rho family, such as RhoA, Rac1 and Cdc42. As key regulators of both actin and microtubule cytoskeleton, Rho GTPases have also emerged as important regulators of dendrite and spine structural plasticity. Multiple studies suggest that Rac1 and Cdc42 are positive regulators promoting neurite outgrowth and growth cone protrusion, while the activation of RhoA induces stress fiber formation, leading to growth cone collapse and neurite retraction. This review focuses on recent advances in our understanding of the molecular mechanisms underlying physiological and pathological functions of Cdc42 in the nervous system. We also discuss application of different FRET-based biosensors as a powerful approach to examine the dynamics of Cdc42 activity in living cells.     

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development

Background

Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.

Methodology/Principal Findings

Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca²⁺ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.

Conclusions/Significance

These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.     

Synaptic activity prompts γ-secretase–mediated cleavage of EphA4 and dendritic spine formation

Alzheimer''s disease is an age-dependent neurodegenerative disorder that is characterized by a progressive decline in cognitive function. γ-secretase dysfunction is evident in many cases of early onset familial Alzheimer''s disease. However, the mechanism by which γ-secretase dysfunction results in memory loss and neurodegeneration is not fully understood. Here, we demonstrate that γ-secretase is localized at synapses and regulates spine formation. We identify EphA4, one of the Ephrin receptor family members, as a substrate of γ-secretase, and find that EphA4 processing is enhanced by synaptic activity. Moreover, overexpression of EphA4 intracellular domain increases the number of dendritic spines by activating the Rac signaling pathway. These findings reveal a function for EphA4-mediated intracellular signaling in the morphogenesis of dendritic spines and suggest that the processing of EphA4 by γ-secretase affects the pathogenesis of Alzheimer''s disease.     

Mitochondrial trafficking and morphology in neuronal injury

Alterations in mitochondrial function may have a central role in the pathogenesis of many neurodegenerative diseases. The study of mitochondrial dysfunction has typically focused on ATP generation, calcium homeostasis and the production of reactive oxygen species. However, there is a growing appreciation of the dynamic nature of mitochondria within cells. Mitochondria are highly motile organelles, and also constantly undergo fission and fusion. This raises the possibility that impairment of mitochondrial dynamics could contribute to the pathogenesis of neuronal injury. In this review we describe the mechanisms that govern mitochondrial movement, fission and fusion. The key proteins that are involved in mitochondrial fission and fusion have also been linked to some inherited neurological diseases, including autosomal dominant optic atrophy and Charcot–Marie–Tooth disease 2A. We will discuss the evidence that altered movement, fission and fusion are associated with impaired neuronal viability. There is a growing collection of literature that links impaired mitochondrial dynamics to a number of disease models. Additionally, the concept that the failure to deliver a functional mitochondrion to the appropriate site within a neuron could contribute to neuronal dysfunction provides an attractive framework for understanding the mechanisms underlying neurologic disease. However, it remains difficult to clearly establish that altered mitochondrial dynamics clearly represent a cause of neuronal dysfunction.     

NESH regulates dendritic spine morphology and synapse formation

Background

Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown.

Methodology/Principal Findings

NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines.

Conclusions/Significance

NESH is a novel F-actin binding protein that likely plays important roles in the regulation of dendritic spine morphogenesis and synapse formation.     

Human morphology and temperature regulation

For nearly a century individuals have believed that there is a link between human morphology and one’s thermoregulatory response in adverse environments. Most early research was focussed on the rate of core cooling in a male adult population and the role of subcutaneous adipose tissue, surface area and the surface-area-to-mass ratio in one’s ability to withstand varying degrees of cold stress. More recently research has addressed heat tolerance in various populations, exploring the role of subcutaneous adipose tissue, surface area and the surface-area-to-mass ratio in one’s ability to maintain thermal equilibrium in warm and hot, dry and humid environments. Since the late 1970s an emphasis has been placed on the role of muscle and muscle perfusion in total-body thermal insulation. Yet, despite the history of research pertaining to human morphology and temperature regulation there is little consensus as to the impact of variations in human morphology on thermoregulatory responses. Individuals differing in body size, shape and composition appear to respond quantitatively differently to variations in both ambient and core temperatures but the interrelations between morphological components and temperature regulation are complex. It is the purpose of this paper to examine the literature pertaining to the impact of variations in muscularity, adipose tissue thickness and patterning, surface area and the surface-area-to-mass ratio on thermoregulation and thermal stability in response to both heat and cold stress. Received: 6 May 1999 / Accepted: 14 July 1999     

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.     

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.     

Limonene inhibits methamphetamine-induced locomotor activity via regulation of 5-HT neuronal function and dopamine release

Methamphetamine is a psychomotor stimulant that produces hyperlocomotion in rodents. Limonene (a cyclic terpene from citrus essential oils) has been reported to induce sedative effects. In this study, we demonstrated that limonene administration significantly inhibited serotonin (5-hydroxytryptamine, 5-HT)-induced head twitch response in mice. In rats, pretreatment with limonene decreased hyperlocomotion induced by methamphetamine injection. In addition, limonene reversed the increase in dopamine levels in the nucleus accumbens of rats given methamphetamine. These results suggest that limonene may inhibit stimulant-induced behavioral changes via regulating dopamine levels and 5-HT receptor function.     

Synaptic connectivity and neuronal morphology: two sides of the same coin

Neurons often possess elaborate axonal and dendritic arbors. Why do these arbors exist and what determines their form and dimensions? To answer these questions, I consider the wiring up of a large highly interconnected neuronal network, such as the cortical column. Implementation of such a network in the allotted volume requires all the salient features of neuronal morphology: the existence of branching dendrites and axons and the presence of dendritic spines. Therefore, the requirement of high interconnectivity is, in itself, sufficient to account for the existence of these features. Moreover, the actual lengths of axons and dendrites are close to the smallest possible length for a given interconnectivity, arguing that high interconnectivity is essential for cortical function.     

Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology

The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.     

Wavelet packet fractal analysis of neuronal morphology

An image analysis method called two-dimensional wavelet packet analysis (2D WPA) is introduced to quantify branching complexity of neurons. Both binary silhouettes and contour profiles of neurons were analyzed to determine accuracy and precision of the fractal dimension in cell classification tasks. Two-dimensional WPA plotted the slope of decay for a sorted list of discrete wavelet packet coefficients belonging to the adapted wavelet best basis to obtain the fractal dimension for test images and binary representations of neurons. Two-dimensional WPA was compared with box counting and mass-radius algorithms. The results for 2D WPA showed that it could differentiate between neural branching complexity in cells of different type in agreement with accepted methods. The importance of the 2D WPA method is that it performs multiresolution decomposition in the horizontal, vertical, and diagonal orientations.     

Molecular regulation of dendritic spine shape and function

Dendritic spines are discrete membrane protrusions from dendritic shafts where the large majority of excitatory synapses are located. Their highly heterogeneous morphology is thought to be the morphological basis for synaptic plasticity. Electron microscopy and time-lapse imaging studies have suggested that the shape and number of spines can change after long-term potentiation (LTP), although there is no evidence that morphological changes are necessary for LTP induction and maintenance. An increasing number of proteins have been found to be morphogens for dendritic spines and provide new insights into the molecular mechanisms regulating spine formation and morphology.     

The development of neuronal morphology in insects

Neurons are highly polarized cells with some regions specified for information input--typically the dendrites--and others specialized for information output--the axons. By extending to a specific location and branching in a specific manner, the processes of neurons determine at a fundamental level how the nervous system is wired to produce behavior. Recent studies suggest that relatively small changes in neuronal morphology could conceivably contribute to striking behavioral distinctions between invertebrate species. We review recent data that begin to shed light on how neurons extend dendrites to their targets and acquire their particular branching morphologies, drawing primarily on data from genetic model organisms. We speculate about how and why the actions of these genes might facilitate the diversification of dendritic morphology.