RETRACTED ARTICLE: Alteration of astrocytes and Wnt/β-catenin signaling in the frontal cortex of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. But the etiology of this disorder is poorly understood. Animal models offer opportunities for conducting biological studies to understand the mechanisms responsible for the phenotypes. The BTBR T + tfJ (BTBR) mice are currently used as a mouse model for understanding mechanisms that may be responsible for the pathogenesis of autism since they demonstrate the three core autistic symptoms [1–4]. In addition, the Neuroligin-3(NL3) knockdown mouse may be a useful model for studying autistic-related behaviors since this mouse model mimics autistic core symptoms [5].

The brain consists of two major cell types - neurons and glial cells. In the past, neurons have been extensively studied. However, research on glial cells has increased in the last decade. The development of the nervous system requires choreographed neuronal migration, axon guidance, target selection, dendritic growth and synapse formation.

Proper orchestration of each of these stages of neuronal development requires glial-derived factors. Glial cells in the central neural system (CNS) are categorized into four types, which include astrocytes, oligodendrocytes, microglia, and chondroitin sulfate proteoglycan NG2-positive cells. Astrocytes are a very heterogeneous population of cells, which interact with neurons and blood vessels. These cells detect neuronal activity and can modulate neuronal networks. Oligodendrocytes (or Schwann cells in the peripheral nervous system) form myelin and thus are prerequisites for the high conduction velocity of axons in vertebrates. Microglia cells are the immune cells of the CNS and respond to changes in the environment [6]. Together these cells play essential roles in nervous system development and function, from simple trophic support of neurons, and wrapping axons and allowing for rapid nerve impulse conduction, to modulating synaptic connectivity and efficacy. During nervous system development, the neural progenitor cells (NPCs) generate neurons first, followed by glia. The switch from neurontoglia at the proper time is critical for the establishment of normal brain function. The mechanisms regulating this transition and development of glia are complex, and currently are poorly understood [7]. However, a number of recent studies indicate that the fate switch is governed by both extrinsic environmental cues that promote astrogenesis in NPCs and NPC-intrinsic mechanisms that decrease neurogenic and increase astrogenic competence over developmental time. The Wnt(wingless-type MMTV integration site1) pathway has been shown to be required for the activation of the proneural genes neurogenin1 (ngn1) and neurogenin2 (ngn2) in NPCs, where, at an early stage, they act to promote neuronal differentiation [8–10]. Recently, Episcopo et al. demonstrated that Wnt1-regulated Frizzled-1/β-Catenin signaling pathway can also act as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk [11]. In addition, it was shown that Wnt/β-Catenin signaling increases in proliferating NG2+ progenitors and astrocytes during post-traumatic gliogenesis in the adult brain [12].

Recently, a number of studies have suggested that abnormal functioning of glia/astrocytes may play a role in the development of autism. Laurence and Fatemi reported that the glial fibrillary acidic protein (GFAP), a marker for astrocytes, is elevated in the superior frontal, parietal and cerebellar cortices of autistic subjects [13]. GFAP was also reported to be three times higher in the cerebrospinal fluid of autistic and autistic-like conditions as compared with a control group [14]. In addition, a study reported that the expression of the astrocytic markers aquaporin4 and connexin43 are altered in the brains of subjects with autism. Most recently, a role for glia in the progression of Rett syndrome (RTT), an X-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2), has been reported [15–17]. In these studies, it was found that mutant astrocytes from anRTT mouse model and their conditioned medium, fail to support normal dendritic morphology of either wild-type or mutant hippocampal neurons. It was also shown 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, a recent study demonstrated that astrocytes in the fragile X mouse model induced developmental delays in normal dendrites including maturation and synaptic protein expression, and implicated a role for astrocytes in the development of the fragile X syndrome [18]. Taken together, the evidence suggests that glia/astrocytes could develop or be regulated abnormally in the autistic brain and that alterations of glia/astrocytes could be critically involved in the pathogenesis of autism. However, as yet there is no study directly investigating how astrocytes develop in the brain of autistic individuals. The aim of this study was to examine the development and morphology of astrocytes in the brains of autistic subjects, as well as in the brains of BTBR mice and NL3 knockout murine models of autism.

Previously, a number of studies have suggested that abnormal functioning of glia and astrocytes may play a role in the development of autism. GFAP expression, a marker for astrocytes, has been reported to be significantly elevated in the cortex and cerebrospinal fluid of autistic subjects [13, 14]. Other astrocyte markers such as aquaporin 4 and connexin 43 have also been shown to be altered in the brains of autistic individuals [20]. In particular, several recent studies have demonstrated that the abnormal functions of glia may also contribute to the progression of RTT, an X- linked autism spectrum disorder, and to the fragile X syndrome [15–18]. However, to date the information about glia/astrocyte development and function in the autistic brain is very limited. In this study, by employing western blotting and immunohistochemical approaches, we found that the morphology of astrocytes in the frontal cortex of autistic subjects was markedly altered compared with controls. The astrocytes in the autistic cortex exhibited significantly reduced branching processes, and the total branch length as well as the cell body size were significantly decreased. Further, the number of astrocytes was markedly increased compared with the controls. These results indicate that there is an astrocytosis in the autistic brain, and the structures of the astrocytes are altered.

Astrocytes are the most abundant cells in the CNS and have been suggested to detect neuronal activity and modulate neuronal networks. Thus their structural integrity and sustained function are essential for neuronal viability [21–23]. The astrocyte branching processes are important structures, which can interdigitate between and closely approximate adjacent neuronal elements, thereby facilitating the local homeostasis of a range of molecules, including glutamate [24–26]. Studies have shown that the neurons depend upon the physical proximity of the astrocyte processes for normal function [23]. Torres-Plataset al. also reported that changes in astrocyte structures including branching processes, and cell body sizes may be significantly involved in mood disorders [27]. Thus, we suggest that the interruption of the astrocyte structures in the autistic cortex could critically impair neuronal function and the homeostasis of certain molecules such as glutamate, which may lead to the development of autistic-like behaviors.

Recently, studies have also shown that astrocytes have a complex, dual role in the local regulation of immune reactivity. They form the glia limitans around blood vessels restricting the access of immune cells to the CNS parenchyma [28]. Astrocytes have also been shown to be important regulators of neuroinflammation. Previous studies have demonstrated that astrocytes carry a series of germline-encoded pattern-recognition receptors (PRRs), which are important for the primary recognition of infectious agents [29]. Several cytokines, including IL-1 and IL-6, have been implicated in the induction and modulation of reactive astrogliosis and pathological inflammatory responses [30–34]. In addition, astrocytes have been reported to secrete inflammatory cytokine IL-6 [35]. Recently, various studies have suggested that abnormal immunity and localized inflammation of the central nervous system may contribute to the pathogenesis of autism. A number of studies including ours have demonstrated that cytokines including IL-6, IL-1β TNF-α and IFN-γ are elevated in the serum and brain tissue of autistic individuals [35–41]. We reckon that the astrocytic changes could result from an inflammatory process.

It will be important to determine whether the observed changes in astrocyte structure, as well as the astrocytosis found in the autistic brain are associated with elevated inflammatory cytokines such as IL-6. In this study, we did not determine the IL6 concentration in the same sample used for examining the astrocytes. Further studies can be conducted to examine cytokines including IL-6 and astrocytes in the same brain region at the same time. We suggest that it is also possible that the increased cytokines, in particular IL-6 in the autistic brain, could result from the astrocytosis.

We next undertook to determine whether the alterations in the structure and density of astrocytes in the autistic brain also occurred in murine models of autism, including NL3 knockdown mice and BTBR mice. We found that the morphology of astrocytes in the NL3 knockdown mouse exhibited similar changes to that found in the autistic brain. They exhibit significantly reduced branching processes and total branch lengths, and as well the astrocytic cell body sizes were significantly decreased in comparison with the controls. Neuroligins are cell adhesion molecules localized postsynaptically in glutamatergic synapses, and interact with presynaptic neurexins to form heterophilic complexes, which likely play critical roles in synaptic transmission and differentiation of synaptic contacts [42–45]. A role of neuroligins in autism was implied by the discovery of deletions at Xp22.1 containing the NL4X gene in three female autistic individuals and a missense mutation (R451C) in NL3 in two Swedish families with autism [46, 47]. NL3 knockdown mice have been shown to mimic certain human autistic behaviors [5]. Recent studies have demonstrated that NL3 is expressed in many types of glia during the development of the nervous system. In particular, NL3 is expressed in the olfactory ensheathing glia, retinal astrocytes, Schwann cells, and spinal cord astrocytes in the developing embryo [48]. The NL3 knock-down mouse in the current study was shown to exhibit autistic-like behaviors including increased anxiety, impaired cognition, vocal communication deficits and decreased social interactions (unpublished data). Thus, there is a possibility that alteration in astrocyte structure could be partially responsible for the development of autistic-like behavior in NL-3 knockdown mice. The mechanisms through which structural change in astrocytes could lead to behavioral changes remains to be further investigated. A limitation of this study was that we only had one NL3 knockdown mouse that could be analyzed. More studies are needed to further confirm our observations.

We did not detect a significant change in the morphology of astrocytes in either the cortex or cerebellum of the BTBR mice, another murine model of autism. There were no significant differences in the number of astrocyte branching processes, the total length of processes orcell body size between the BTBR and control B6 mice. Nor did we find that there was an astrocytosis in the brain of BTBR mice similar to that found in the autistic brain. The density of astrocytes remained unchanged compared with the control mouse. However, we have not examined the orientation of the glial fibers. Recently, it was reported that there is a misorientation of selected glial fibers present in the BTBR forebrain [49]. This study found that the astrocytic processes were oriented dorsoventrally rather than mediolaterally in the cingulum and alveus at the levels of the striatum and hippocampus. The misorientation of glial processes was only found in brain regions that normally receive corpus callosal innervations, indicating that these findings are likely to be a consequence of callosal agenesis [49]. We therefore reason that although there are no changes observed in the astrocyte density, as well as in the number of branching processes and cell body sizes in BTBR mice, a misorientation of glial processes could lead to impairments in the functions of astrocytes, and consequentially impair synaptic plasticity and various neural functions and might contribute to the development of autistic-like behaviors. It has been demonstrated that astrocyte secreted proteins selectively increase hippocampal GABAergic axon length, branching, and synaptogenesis [50]. Whether the change in astrocytes in autistic subjects, or NL3 knockdown and BTBR mice could impair the development of GABAergicaxons, remains to be further studied.

Both NL3 knockdown and BTBR mice have been demonstrated to exhibit core autistic-like behaviors. Alterations found in the astrocytes of autistic subjects and the mice models imply that NL3 knockdown mice and BTBR mice could offer opportunities for conducting biological studies to understand the mechanisms responsible for autism.

More and more evidence suggests that astrocytes are intimately associated with synapses and govern key steps in synapse formation and plasticity. However, we understand little about the molecular underpinnings of astrocyte development. It is unclear how astrocytes are specified at the appropriate developmental time from NPCs and how their development and maturation are regulated. The Wnt/β-catenin signaling pathway has been intensely studied as a key regulator of cell proliferation and cell fate during development, including neural development [10, 23, 51–53]. Recently, several studies have reported a role of Wnt/β-catenin signaling in the development of astrocytes [12]. It has been shown that Wnt/β-catenin pathway signaling regulates post-traumatic gliogenesis. Wnt/β-catenin pathway has also been demonstrated to act as a candidate regulatory circuit that controls mesencephalic dopaminergic neuron-astrocyte crosstalk [11]. In this study, we found that both Wnt and β-catenin protein expression were decreased in the brains of autistic subjects, suggesting that Wnt/β-catenin signaling activities are down-regulated. There is some evidence for a direct genetic link between Wnt2 and autism spectrum disorders. Two studies have found correlations between mutations of the WNT2 locus and the incidence of autism in different populations [54, 55]. Wnt2 has also been found to be expressed at lower levels in a mouse model of fragile X syndrome, a human disease strongly associated with autism [56]. Our findings imply that the decreased expression of Wnt and β-catenin may be associated with changes in astrocytes in the frontal cortex of autistic subjects. Further studies will be carried out to determine whether down-regulation of Wnt/β-catenin impairs the structure and density of astrocytes.

In summary, our study demonstrated that the morphology of astrocytes in the frontal cortex of autistic subjects was significantly altered compared with age- and sex-matched controls. The astrocytes in autisitc subjects exhibited significantly reduced branching processes, reduced total branching lengths and decreased cell body sizes. In addition, we detected astrocytosis present in the frontal cortex of autistic subjects. However these alterations of astrocytes are not detected in the autistic cerebellum. Interestingly, we found that astrocytes in the frontal cortex of the NL3 knockdown mouse also showed significantly reduced branching processes, reduced total branching length and decreased cell body sizes, which mimic the changes in the autistic brain. These findings imply that the defects in astrocytes could impair neuronal plasticity and functions, and may partially contribute to the development of autistic-like behaviors in both humans and mice. We did not detect significant alterations in the density and morphology of astrocytes from the frontal cortex or cerebellum of BTBR mice. Recently, one study reported that there is a misorientation of selected glial fibers present in the BTBR forebrain. We suggest that even small changes like misorientation of glial fibers could affect astrocyte functions and consequently neuronal networks and lead to behavioral changes. Finally, we found that both Wnt and β-catenin protein expression was decreased in the brains of autistic subjects, which suggests that the Wnt/β-catenin signaling activities may be down-regulated. We suggest that the alteration of the Wnt/β-catenin pathway in the frontal cortex of autistic subjects could be one of the underlying mechanisms responsible for the observed changes of astrocytes. Our findings indicate that astrocytes may play an important role in the development of autism and may further suggest new potential therapeutic targets and strategies for intervention in autism.