The autism-associated loss of δ-catenin functions disrupts social behavior.

δ-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the δ-catenin gene has been found in autism spectrum disorder (ASD) patients and results in loss of δ-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of δ-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we identify that the G34S mutation increases glycogen synthase kinase 3ß (GSK3ß)-dependent δ-catenin degradation to reduce δ-catenin levels, which likely contributes to the loss of δ-catenin functions. Synaptic δ-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the δ-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. δ-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, pharmacological inhibition of GSK3ß activity reverses the G34S-induced loss of δ-catenin function effects in cells and mice. Finally, using δ-catenin knockout mice, we confirm that δ-catenin is required for GSK3ß inhibition-induced restoration of normal social behavior in δ-catenin G34S mutant animals. Taken together, we reveal that the loss of δ-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3ß inhibition can reverse δ-catenin G34S-induced synaptic and behavioral deficits.

The autism-associated loss of δ-catenin functions disrupts social behaviors.

δ-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the δ-catenin gene is found in autism spectrum disorder (ASD) patients and induces loss of δ-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of δ-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we discover that the G34S mutation generates an additional phosphorylation site for glycogen synthase kinase 3ß (GSK3ß). This promotes δ-catenin degradation and causes the reduction of δ-catenin levels, which likely contributes to the loss of δ-catenin functions. Synaptic δ-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the δ-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. δ-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, inhibition of GSK3ß activity reverses the G34S-induced loss of δ-catenin function effects in cells and mice. Finally, using δ-catenin knockout mice, we confirm that δ-catenin is required for GSK3ß inhibition-induced restoration of normal social behaviors in δ-catenin G34S mutant animals. Taken together, we reveal that the loss of δ-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3ß inhibition can reverse δ-catenin G34S-induced synaptic and behavioral deficits. Significance Statement: δ-catenin is important for the localization and function of glutamatergic AMPA receptors at synapses in many brain regions. The glycine 34 to serine (G34S) mutation in the δ-catenin gene is found in autism patients and results in the loss of δ-catenin functions. δ-catenin expression is also closely linked to other autism-risk genes involved in synaptic structure and function, further implying that it is important for the autism pathophysiology. Importantly, social dysfunction is a key characteristic of autism. Nonetheless, the links between δ-catenin functions and social behaviors are largely unknown. The significance of the current research is thus predicated on filling this gap by discovering the molecular, cellular, and synaptic underpinnings of the role of δ-catenin in social behaviors.

Complement Component C3 Loss leads to Locomotor Deficits and Altered Cerebellar Internal Granule Cell In Vitro Synaptic Protein Expression in C57BL/6 Mice.

Complement component 3 (C3) expression is increased in the cerebellum of aging mice that demonstrate locomotor impairments and increased excitatory synapse density. However, C3 regulation of locomotion, as well as C3 roles in excitatory synapse function, remains poorly understood. Here, we demonstrate that constitutive loss of C3 function in mice evokes a locomotor phenotype characterized by decreased speed, increased active state locomotor probability, and gait ataxia. C3 loss does not alter metabolism or body mass composition. No evidence of significant muscle weakness or degenerative arthritis was found in C3 knockout mice to explain decreased gait speeds. In an enriched primary cerebellar granule cell culture model, loss of C3 protein results in increased excitatory synaptic density and increased response to KCl depolarization. Our analysis of excitatory synaptic density in the cerebellar internal granule cell and molecular layers did not demonstrate increased synaptic density in vivo, suggesting the presence of compensatory mechanisms regulating synaptic development. Functional deficits in C3 knockout mice are therefore more likely to result from altered synaptic function and/or connectivity than gross synaptic deficits. Our data demonstrate a novel role for complement proteins in cerebellar regulation of locomotor output and control.

Novel phospho-switch function of delta-catenin in dendrite development.

In neurons, dendrites form the major sites of information receipt and integration. It is thus vital that, during development, the dendritic arbor is adequately formed to enable proper neural circuit formation and function. While several known processes shape the arbor, little is known of those that govern dendrite branching versus extension. Here, we report a new mechanism instructing dendrites to branch versus extend. In it, glutamate signaling activates mGluR5 receptors to promote Ckd5-mediated phosphorylation of the C-terminal PDZ-binding motif of delta-catenin. The phosphorylation state of this motif determines delta-catenin's ability to bind either Pdlim5 or Magi1. Whereas the delta:Pdlim5 complex enhances dendrite branching at the expense of elongation, the delta:Magi1 complex instead promotes lengthening. Our data suggest that these complexes affect dendrite development by differentially regulating the small-GTPase RhoA and actin-associated protein Cortactin. We thus reveal a "phospho-switch" within delta-catenin, subject to a glutamate-mediated signaling pathway, that assists in balancing the branching versus extension of dendrites during neural development.

Mechanisms of axon polarization in pyramidal neurons.

Neurons are highly polarized cells that have specialized regions for synaptic input, the dendrites, and synaptic output, the axons. This polarity is critical for appropriate neural circuit formation and function. One of the central gaps in our knowledge is understanding how developing neurons initiate axon polarity. Given the critical nature of this polarity on neural circuit formation and function, neurons have evolved multiple mechanisms comprised of extracellular and intracellular cues that allow them to initiate and form axons. These mechanisms engage a variety of signaling cascades that provide positive and negative cues to ensure axon polarization. This review highlights our current knowledge of the molecular underpinnings of axon polarization in pyramidal neurons and their relevance to the development of the brain.

δ-Catenin engages the autophagy pathway to sculpt the developing dendritic arbor.

The development of the dendritic arbor in pyramidal neurons is critical for neural circuit function. Here, we uncovered a pathway in which δ-catenin, a component of the cadherin-catenin cell adhesion complex, promotes coordination of growth among individual dendrites and engages the autophagy mechanism to sculpt the developing dendritic arbor. Using a rat primary neuron model, time-lapse imaging, immunohistochemistry, and confocal microscopy, we found that apical and basolateral dendrites are coordinately sculpted during development. Loss or knockdown of δ-catenin uncoupled this coordination, leading to retraction of the apical dendrite without altering basolateral dendrite dynamics. Autophagy is a key cellular pathway that allows degradation of cellular components. We observed that the impairment of the dendritic arbor resulting from δ-catenin knockdown could be reversed by knockdown of autophagy-related 7 (ATG7), a component of the autophagy machinery. We propose that δ-catenin regulates the dendritic arbor by coordinating the dynamics of individual dendrites and that the autophagy mechanism may be leveraged by δ-catenin and other effectors to sculpt the developing dendritic arbor. Our findings have implications for the management of neurological disorders, such as autism and intellectual disability, that are characterized by dendritic aberrations.

A selective role for a component of the autophagy pathway in coupling the Golgi apparatus to dendrite polarity in pyramidal neurons.

Pyramidal neurons have a characteristic morphology that is critical to their ability to integrate into functional neural circuits. In addition to axon dendrite polarity, pyramidal neurons also exhibit dendritic polarity such that apical and basolateral dendrites differ in size, structure and inputs. Dendrite polarity in pyramidal neurons coincides with polarity of the Golgi apparatus, a key feature relevant to directed secretory trafficking, both in vitro and in vivo. We identify a novel autophagy based mechanism that uncouples the polarity of the Golgi apparatus from dendrite polarity. Autophagy is a universal cellular pathway that promotes cellular homeostasis via degradation of cellular components. Our data indicate that knockdown of ATG7, a key component of the autophagy mechanism, disrupts the polarity of the Golgi apparatus without impacting dendritic polarity in primary pyramidal neurons, providing the first evidence that dendrite polarity can be uncoupled from Golgi polarity. Interestingly, these effects are restricted to ATG7 knockdown and are not replicated by the knockdown of ATG16L1, another component of the autophagy mechanism. We propose that cellular mechanisms exist to couple Golgi polarity to dendrite polarity. Components of the autophagy mechanism are leveraged to actively couple Golgi polarity to dendrite polarity, thus impacting secretory trafficking into individual dendrites in pyramidal neurons.

In the Telencephalon, GluN2C NMDA Receptor Subunit mRNA is Predominately Expressed in Glial Cells and GluN2D mRNA in Interneurons.

N-methyl-D-aspartate receptors (NMDARs) are widely distributed in the brain with high concentrations in the telencephalon where they modulate synaptic plasticity, working memory, and other functions. While the actions of the predominate GluN2 NMDAR subunits, GluN2A and GluN2B are relatively well understood, the function of GluN2C and GluN2D subunits in the telencephalon is largely unknown. To better understand the possible role of GluN2C subunits, we used fluorescence in situ hybridization (FISH) together with multiple cell markers to define the distribution and type of cells expressing GluN2C mRNA. Using a GluN2C-KO mouse as a negative control, GluN2C mRNA expression was only found in non-neuronal cells (NeuN-negative cells) in the hippocampus, striatum, amygdala, and cerebral cortex. For these regions, a significant fraction of GFAP-positive cells also expressed GluN2C mRNA. Overall, for the telencephalon, the globus pallidus and olfactory bulb were the only regions where GluN2C was expressed in neurons. In contrast to GluN2C, GluN2D subunit mRNA colocalized with neuronal and not astrocyte markers or GluN2C mRNA in the telencephalon (except for the globus pallidus). GluN2C mRNA did, however, colocalize with GluN2D in the thalamus where neuronal GluN2C expression is found. These findings strongly suggest that GluN2C has a very distinct function in the telencephalon compared to its role in other brain regions and compared to other GluN2-containing NMDARs. NMDARs containing GluN2C may have a specific role in regulating L-glutamate or D-serine release from astrocytes in response to L-glutamate spillover from synaptic activity.

Neuron-Type Specific Loss of CDKL5 Leads to Alterations in mTOR Signaling and Synaptic Markers.

CDKL5 disorder is a devastating neurodevelopmental disorder associated with epilepsy, developmental retardation, autism, and related phenotypes. Mutations in the CDKL5 gene, encoding CDKL5, have been identified in this disorder. CDKL5 is a protein with homology to the serine-threonine kinases and incompletely characterized function. We generated and validated a murine model bearing a floxed allele of CDKL5 and polyclonal antibodies to CDKL5. CDKL5 is well expressed in the cortex, hippocampus, and striatum, localized to synaptosomes and nuclei and developmentally regulated in the hippocampus. Using Cre-mediated mechanisms, we deleted CDKL5 from excitatory CaMKIIα-positive neurons or inhibitory GABAergic neurons. Our data indicate that loss of CDKL5 in excitatory neurons of the cortex or inhibitory neurons of the striatum differentially alters expression of some components of the mechanistic target of rapamycin (mTOR) signaling pathway. Further loss of CDKL5 in excitatory neurons of the cortex or inhibitory neurons of the striatum leads to alterations in levels of synaptic markers in a neuron-type specific manner. Taken together, these data support a model in which loss of CDKL5 alters mTOR signaling and synaptic compositions in a neuron-type specific manner and suggest that CDKL5 may have distinct functional roles related to cellular signaling in excitatory and inhibitory neurons. Thus, these studies provide new insights into the biology of CDKL5 and suggest that the molecular pathology in CDKL5 disorder may have distinct neuron-type specific origins and effects.

A role for proteolytic regulation of δ-catenin in remodeling a subpopulation of dendritic spines in the rodent brain.

Neural wiring and activity are essential for proper brain function and behavioral outputs and rely on mechanisms that guide the formation, elimination, and remodeling of synapses. During development, it is therefore vital that synaptic densities and architecture are tightly regulated to allow for appropriate neural circuit formation and function. δ-Catenin, a component of the cadherin-catenin cell adhesion complex, has been demonstrated to be a critical regulator of synaptic density and function in the developing central neurons. In this study, we identified forms of δ-catenin that include only the N-terminal (DcatNT) or the C-terminal (DcatCT) regions. We found that these δ-catenin forms are differentially expressed in different regions of the male mouse brain. Our results also indicated that in rat primary cortical culture, these forms are generated in an activity-dependent manner by Ca2+-dependent and calpain-mediated cleavage of δ-catenin or in an activity-independent but lysosome-dependent manner. Functionally, loss of the domain containing the calpain-cleavage sites allowing for generation of DcatCT and DcatNT perturbed the density of a subpopulation of dendritic protrusions in rat hippocampal neurons. This subpopulation likely included protrusions that are either in transition toward becoming mature mushroom spines or in the process of being eliminated. By influencing this subpopulation of spines, proteolytic processing of δ-catenin can likely regulate the balance between mature and immature dendritic protrusions in coordination with neural activity. We conclude that by undergoing cleavage, δ-catenin differentially regulates the densities of subpopulations of dendritic spines and contributes to proper neural circuit wiring in the developing brain.

Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis.

Alzheimer's disease (AD) is a neurodegenerative syndrome classically depicted by the parenchymal accumulation of extracellular amyloid beta plaques. However, recent findings suggest intraneuronal amyloid beta (iAß1-42) accumulation precedes extracellular deposition. Furthermore, the pathologic increase in iAß1-42 has been implicated in dysregulation of cellular mechanisms critically important in axonal transport. Owing to neuronal cell polarity, retrograde and anterograde axonal transport are essential trafficking mechanism necessary to convey membrane bound neurotransmitters, neurotrophins, and endosomes between soma and synaptic interfaces. Although iAß1-42 disruption of axonal transport has been implicated in dysregulation of neuronal synaptic transmission, the role of iAß1-42 and its influence on signal transduction involving the mitogen-activated protein kinase (MAPK) and morphogenetic signaling axis are unknown. Our biochemical characterization of intracellular amyloid beta accumulation on MAPK and morphogenetic signaling have revealed increased iAß1-42 expression leads to significant reduction in ERK 1/2 phosphorylation and increased bone morphogenetic protein 2 dependent Smad 1/5/8 phosphorylation. Furthermore, rescue of iAß1-42 mediated attenuation of MAPK signaling can be accomplished with the small molecule PLX4032 as a downstream enhancer of the MAPK pathway. Consequently, our observations regarding the dysregulation of these gatekeepers of neuronal viability may have important implications in understanding the iAß1-42 mediated effects observed in AD.

Functional roles of p120ctn family of proteins in central neurons.

The cadherin-catenin complex in central neurons is associated with a variety of cytosolic partners, collectively called catenins. The p120ctn members are a family of catenins that are distinct from the more ubiquitously expressed α- and ß-catenins. It is becoming increasingly clear that the functional roles of the p120ctn family of catenins in central neurons extend well beyond their functional roles in non-neuronal cells in partnering with cadherin to regulate adhesion. In this review, we will provide an overview of the p120ctn family in neurons and their varied functional roles in central neurons. Finally, we will examine the emerging roles of this family of proteins in neurodevelopmental disorders.

Quantitative assessment of neural outgrowth using spatial light interference microscopy.

Optimal growth as well as branching of axons and dendrites is critical for the nervous system function. Neuritic length, arborization, and growth rate determine the innervation properties of neurons and define each cell's computational capability. Thus, to investigate the nervous system function, we need to develop methods and instrumentation techniques capable of quantifying various aspects of neural network formation: neuron process extension, retraction, stability, and branching. During the last three decades, fluorescence microscopy has yielded enormous advances in our understanding of neurobiology. While fluorescent markers provide valuable specificity to imaging, photobleaching, and photoxicity often limit the duration of the investigation. Here, we used spatial light interference microscopy (SLIM) to measure quantitatively neurite outgrowth as a function of cell confluence. Because it is label-free and nondestructive, SLIM allows for long-term investigation over many hours. We found that neurons exhibit a higher growth rate of neurite length in low-confluence versus medium- and high-confluence conditions. We believe this methodology will aid investigators in performing unbiased, nondestructive analysis of morphometric neuronal parameters.

Cell density modulates intracellular mass transport in neural networks.

In order to fully understand brain connectivity and elucidate the mechanisms involved in central nervous system disease, the field of neuroscience depends on quantitative studies of neuronal structure and function. Cell morphology and neurite (axonal and dendritic) arborization are typically studied by immunohistochemical and fluorescence techniques. However, dry mass content and intracellular mass transport rates have largely been under-investigated given the inherent difficulties in their measurement. Here, spatial light interference microscopy (SLIM) and dispersion-relation phase spectroscopy (DPS) were used to measure pathlength fluctuations that report on the dry mass and transport within cultured primary neurons across low, medium, and high cell density conditions. It was found that cell density (confluence) affects significantly both the growth rate and mass transport. The analysis method is label-free and does not require neuronal tracing, particle tracking, or neuron reconstruction. Since SLIM can upgrade any existing phase contrast microscope and the imaging and analysis are high-throughput, we anticipate that this approach will be embraced by neuroscientists for broad scale studies. © 2017 International Society for Advancement of Cytometry.

Halo-free Phase Contrast Microscopy.

We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric module, which generates sufficient data for reversing the halo artifact. Acquiring four independent intensity images, our approach first measures haloed phase maps of the sample. We solve for the halo-free sample transmission function by using a physical model of the image formation under partial spatial coherence. Using this halo-free sample transmission, we can numerically generate artifact-free PC images. Furthermore, this transmission can be further used to obtain quantitative information about the sample, e.g., the thickness with known refractive indices, dry mass of live cells during their cycles. We tested our hfPC method on various control samples, e.g., beads, pillars and validated its potential for biological investigation by imaging live HeLa cells, red blood cells, and neurons.

Regulation of morphine-induced synaptic alterations: Role of oxidative stress, ER stress, and autophagy.

Our findings suggest that morphine dysregulates synaptic balance in the hippocampus, a key center for learning and memory, via a novel signaling pathway involving reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy. We demonstrate in this study that exposure of morphine to hippocampal neurons leads to a reduction in excitatory synapse densities with a concomitant enhancement of inhibitory synapse densities via activation of the µ opioid receptor. Furthermore, these effects of morphine are mediated by up-regulation of intracellular ROS from NADPH oxidase, leading, in turn, to sequential induction of ER stress and autophagy. The detrimental effects of morphine on synaptic densities were shown to be reversed by platelet-derived growth factor (PDGF), a pleiotropic growth factor that has been implicated in neuroprotection. These results identify a novel cellular mechanism involved in morphine-mediated synaptic alterations with implications for therapeutic interventions by PDGF.

Age-related changes in cerebellar and hypothalamic function accompany non-microglial immune gene expression, altered synapse organization, and excitatory amino acid neurotransmission deficits.

We describe age-related molecular and neuronal changes that disrupt mobility or energy balance based on brain region and genetic background. Compared to young mice, aged C57BL/6 mice exhibit marked locomotor (but not energy balance) impairments. In contrast, aged BALB mice exhibit marked energy balance (but not locomotor) impairments. Age-related changes in cerebellar or hypothalamic gene expression accompany these phenotypes. Aging evokes upregulation of immune pattern recognition receptors and cell adhesion molecules. However, these changes do not localize to microglia, the major CNS immunocyte. Consistent with a neuronal role, there is a marked age-related increase in excitatory synapses over the cerebellum and hypothalamus. Functional imaging of these regions is consistent with age-related synaptic impairments. These studies suggest that aging reactivates a developmental program employed during embryogenesis where immune molecules guide synapse formation and pruning. Renewed activity in this program may disrupt excitatory neurotransmission, causing significant behavioral deficits.

Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders.

The common underlying feature of most neurodegenerative diseases such as Alzheimer disease (AD), prion diseases, Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) involves accumulation of misfolded proteins leading to initiation of endoplasmic reticulum (ER) stress and stimulation of the unfolded protein response (UPR). Additionally, ER stress more recently has been implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Autophagy plays an essential role in the clearance of aggregated toxic proteins and degradation of the damaged organelles. There is evidence that autophagy ameliorates ER stress by eliminating accumulated misfolded proteins. Both abnormal UPR and impaired autophagy have been implicated as a causative mechanism in the development of various neurodegenerative diseases. This review highlights recent advances in the field on the role of ER stress and autophagy in AD, prion diseases, PD, ALS and HAND with the involvement of key signaling pathways in these processes and implications for future development of therapeutic strategies.

Differential regulation of apical-basolateral dendrite outgrowth by activity in hippocampal neurons.

Hippocampal pyramidal neurons have characteristic dendrite asymmetry, characterized by structurally and functionally distinct apical and basolateral dendrites. The ability of the neuron to generate and maintain dendrite asymmetry is vital, since synaptic inputs received are critically dependent on dendrite architecture. Little is known about the role of neuronal activity in guiding maintenance of dendrite asymmetry. Our data indicate that dendrite asymmetry is established and maintained early during development. Further, our results indicate that cell intrinsic and global alterations of neuronal activity have differential effects on net extension of apical and basolateral dendrites. Thus, apical and basolateral dendrite extension may be independently regulated by cell intrinsic and network neuronal activity during development, suggesting that individual dendrites may have autonomous control over net extension. We propose that regulated individual dendrite extension in response to cell intrinsic and neuronal network activity may allow temporal control of synapse specificity in the developing hippocampus.