Dysfunction of specific auditory fibers impacts cortical oscillations, driving an autism phenotype despite near-normal hearing.

Autism spectrum disorder is discussed in the context of altered neural oscillations and imbalanced cortical excitation-inhibition of cortical origin. We studied here whether developmental changes in peripheral auditory processing, while preserving basic hearing function, lead to altered cortical oscillations. Local field potentials (LFPs) were recorded from auditory, visual, and prefrontal cortices and the hippocampus of BdnfPax2 KO mice. These mice develop an autism-like behavioral phenotype through deletion of BDNF in Pax2+ interneuron precursors, affecting lower brainstem functions, but not frontal brain regions directly. Evoked LFP responses to behaviorally relevant auditory stimuli were weaker in the auditory cortex of BdnfPax2 KOs, connected to maturation deficits of high-spontaneous rate auditory nerve fibers. This was correlated with enhanced spontaneous and induced LFP power, excitation-inhibition imbalance, and dendritic spine immaturity, mirroring autistic phenotypes. Thus, impairments in peripheral high-spontaneous rate fibers alter spike synchrony and subsequently cortical processing relevant for normal communication and behavior.

Cortical Parvalbumin-Positive Interneuron Development and Function Are Altered in the APC Conditional Knockout Mouse Model of Infantile and Epileptic Spasms Syndrome.

Infantile and epileptic spasms syndrome (IESS) is a childhood epilepsy syndrome characterized by infantile or late-onset spasms, abnormal neonatal EEG, and epilepsy. Few treatments exist for IESS, clinical outcomes are poor, and the molecular and circuit-level etiologies of IESS are not well understood. Multiple human IESS risk genes are linked to Wnt/ß-catenin signaling, a pathway that controls developmental transcriptional programs and promotes glutamatergic excitation via ß-catenin's role as a synaptic scaffold. We previously showed that deleting adenomatous polyposis coli (APC), a component of the ß-catenin destruction complex, in excitatory neurons (APC cKO mice, APCfl/fl x CaMKIIαCre) increased ß-catenin levels in developing glutamatergic neurons and led to infantile behavioral spasms, abnormal neonatal EEG, and adult epilepsy. Here, we tested the hypothesis that the development of GABAergic interneurons (INs) is disrupted in APC cKO male and female mice. IN dysfunction is implicated in human IESS, is a feature of other rodent models of IESS, and may contribute to the manifestation of spasms and seizures. We found that parvalbumin-positive INs (PV+ INs), an important source of cortical inhibition, were decreased in number, underwent disproportionate developmental apoptosis, and had altered dendrite morphology at P9, the peak of behavioral spasms. PV+ INs received excessive excitatory input, and their intrinsic ability to fire action potentials was reduced at all time points examined (P9, P14, P60). Subsequently, GABAergic transmission onto pyramidal neurons was uniquely altered in the somatosensory cortex of APC cKO mice at all ages, with both decreased IPSC input at P14 and enhanced IPSC input at P9 and P60. These results indicate that inhibitory circuit dysfunction occurs in APC cKOs and, along with known changes in excitation, may contribute to IESS-related phenotypes.SIGNIFICANCE STATEMENT Infantile and epileptic spasms syndrome (IESS) is a devastating epilepsy with limited treatment options and poor clinical outcomes. The molecular, cellular, and circuit disruptions that cause infantile spasms and seizures are largely unknown, but inhibitory GABAergic interneuron dysfunction has been implicated in rodent models of IESS and may contribute to human IESS. Here, we use a rodent model of IESS, the APC cKO mouse, in which ß-catenin signaling is increased in excitatory neurons. This results in altered parvalbumin-positive GABAergic interneuron development and GABAergic synaptic dysfunction throughout life, showing that pathology arising in excitatory neurons can initiate long-term interneuron dysfunction. Our findings further implicate GABAergic dysfunction in IESS, even when pathology is initiated in other neuronal types.

Deletion of BDNF in Pax2 Lineage-Derived Interneuron Precursors in the Hindbrain Hampers the Proportion of Excitation/Inhibition, Learning, and Behavior.

Numerous studies indicate that deficits in the proper integration or migration of specific GABAergic precursor cells from the subpallium to the cortex can lead to severe cognitive dysfunctions and neurodevelopmental pathogenesis linked to intellectual disabilities. A different set of GABAergic precursors cells that express Pax2 migrate to hindbrain regions, targeting, for example auditory or somatosensory brainstem regions. We demonstrate that the absence of BDNF in Pax2-lineage descendants of Bdnf Pax2 KOs causes severe cognitive disabilities. In Bdnf Pax2 KOs, a normal number of parvalbumin-positive interneurons (PV-INs) was found in the auditory cortex (AC) and hippocampal regions, which went hand in hand with reduced PV-labeling in neuropil domains and elevated activity-regulated cytoskeleton-associated protein (Arc/Arg3.1; here: Arc) levels in pyramidal neurons in these same regions. This immaturity in the inhibitory/excitatory balance of the AC and hippocampus was accompanied by elevated LTP, reduced (sound-induced) LTP/LTD adjustment, impaired learning, elevated anxiety, and deficits in social behavior, overall representing an autistic-like phenotype. Reduced tonic inhibitory strength and elevated spontaneous firing rates in dorsal cochlear nucleus (DCN) brainstem neurons in otherwise nearly normal hearing Bdnf Pax2 KOs suggests that diminished fine-grained auditory-specific brainstem activity has hampered activity-driven integration of inhibitory networks of the AC in functional (hippocampal) circuits. This leads to an inability to scale hippocampal post-synapses during LTP/LTD plasticity. BDNF in Pax2-lineage descendants in lower brain regions should thus be considered as a novel candidate for contributing to the development of brain disorders, including autism.

The Spine Lab: A Short-Duration, Fully-Remote Course-Based Undergraduate Research Experience.

Course-based undergraduate research experiences (CUREs) are increasingly common approaches to provide students with authentic laboratory experiences. Typically, CUREs are semester-long, in-person experiences that can be financially and time prohibitive for some institutions, faculty, and students. Here, we developed a short-duration, fully-online CURE, the Spine Lab, to provide an opportunity for students to conduct original research. In this CURE, we focused on synaptic spines in the mammalian brain; synapses are the unit structure that functions in rapid information processing. The students worked together in pairs and as a class to analyze cortical neuron spine density and structural morphology changes between a mouse line with learning impairments (forebrain-specific ß-catenin knockouts [ß-cat cKOs]) and control (Ctl) littermates. The students showed their results in an online poster presentation. Their findings show that spine density is significantly reduced, while spine structural maturation is unaltered in the ß-cat cKO. Defining pathophysiological changes caused by CTNNB1/ß-catenin loss-of-function provides important insights relevant to human disorders caused by disruptive mutations in this gene. To assess the benefits of this CURE, students completed a pre- and post-test assessment including a content quiz, STEM identity survey, and a standardized CURE survey. Participation in the Spine Lab correlated with improved content and STEM identity scores, and decreased negative attitudes about science. Moreover, direct comparison to the CURE database reveals that the Spine Lab produces comparable benefits to traditional CUREs. This work as a whole suggests that short-duration, fully-online CUREs can provide benefit to students and could be an inclusive tool to improve student outcomes.

Excessive ß-Catenin in Excitatory Neurons Results in Reduced Social and Increased Repetitive Behaviors and Altered Expression of Multiple Genes Linked to Human Autism.

Multiple human autism risk genes are predicted to converge on the ß-catenin (ß-cat)/Wnt pathway. However, direct tests to link ß-cat up- or down-regulation with autism are largely lacking, and the associated pathophysiological changes are poorly defined. Here we identify excessive ß-cat as a risk factor that causes expression changes in several genes relevant to human autism. Our studies utilize mouse lines with ß-cat dysregulation in forebrain excitatory neurons, identified as cell types with a convergent expression of autism-linked genes in both human and mouse brains. We show that mice expressing excessive ß-cat display behavioral and molecular changes, including decreased social interest, increased repetitive behaviors, reduced parvalbumin and altered expression levels of additional genes identified as potential risk factors for human autism. These behavioral and molecular phenotypes are averted by reducing ß-cat in neurons predisposed by gene mutations to express elevated ß-cat. Using next-generation sequencing of the prefrontal cortex (PFC), we identify 87 dysregulated genes that are shared between mouse lines with excessive ß-cat and autism-like behaviors, but not mouse lines with reduced ß-cat and normal social behavior. Our findings provide critical new insights into ß-cat, Wnt pathway dysregulation in the brain causing behavioral phenotypes relevant to the disease and the molecular etiology which includes several human autism risk genes.

Learning impairments and molecular changes in the brain caused by ß-catenin loss.

Intellectual disability (ID), defined as IQ<70, occurs in 2.5% of individuals. Elucidating the underlying molecular mechanisms is essential for developing therapeutic strategies. Several of the identified genes that link to ID in humans are predicted to cause malfunction of ß-catenin pathways, including mutations in CTNNB1 (ß-catenin) itself. To identify pathological changes caused by ß-catenin loss in the brain, we have generated a new ß-catenin conditional knockout mouse (ß-cat cKO) with targeted depletion of ß-catenin in forebrain neurons during the period of major synaptogenesis, a critical window for brain development and function. Compared with control littermates, ß-cat cKO mice display severe cognitive impairments. We tested for changes in two ß-catenin pathways essential for normal brain function, cadherin-based synaptic adhesion complexes and canonical Wnt (Wingless-related integration site) signal transduction. Relative to control littermates, ß-cat cKOs exhibit reduced levels of key synaptic adhesion and scaffold binding partners of ß-catenin, including N-cadherin, α-N-catenin, p120ctn and S-SCAM/Magi2. Unexpectedly, the expression levels of several canonical Wnt target genes were not altered in ß-cat cKOs. This lack of change led us to find that ß-catenin loss leads to upregulation of γ-catenin (plakoglobin), a partial functional homolog, whose neural-specific role is poorly defined. We show that γ-catenin interacts with several ß-catenin binding partners in neurons but is not able to fully substitute for ß-catenin loss, likely due to differences in the N-and C-termini between the catenins. Our findings identify severe learning impairments, upregulation of γ-catenin and reductions in synaptic adhesion and scaffold proteins as major consequences of ß-catenin loss.

Social Stimulus Causes Aberrant Activation of the Medial Prefrontal Cortex in a Mouse Model With Autism-Like Behaviors.

Autism spectrum disorder (ASD) is a highly prevalent and genetically heterogeneous brain disorder. Developing effective therapeutic interventions requires knowledge of the brain regions that malfunction and how they malfunction during ASD-relevant behaviors. Our study provides insights into brain regions activated by a novel social stimulus and how the activation pattern differs between mice that display autism-like disabilities and control littermates. Adenomatous polyposis coli (APC) conditional knockout (cKO) mice display reduced social interest, increased repetitive behaviors and dysfunction of the ß-catenin pathway, a convergent target of numerous ASD-linked human genes. Here, we exposed the mice to a novel social vs. non-social stimulus and measured neuronal activation by immunostaining for the protein c-Fos. We analyzed three brain regions known to play a role in social behavior. Compared with control littermates, APC cKOs display excessive activation, as evidenced by an increased number of excitatory pyramidal neurons stained for c-Fos in the medial prefrontal cortex (mPFC), selectively in the infralimbic sub-region. In contrast, two other social brain regions, the medial amygdala and piriform cortex show normal levels of neuron activation. Additionally, APC cKOs exhibit increased frequency of miniature excitatory postsynaptic currents (mEPSCs) in layer 5 pyramidal neurons of the infralimbic sub-region. Further, immunostaining is reduced for the inhibitory interneuron markers parvalbumin (PV) and somatostatin (SST) in the APC cKO mPFC. Our findings suggest aberrant excitatory-inhibitory balance and activation patterns. As ß-catenin is a core pathway in ASD, we identify the infralimbic sub-region of the mPFC as a critical brain region for autism-relevant social behavior.

Extracellular matrix directs phenotypic heterogeneity of activated fibroblasts.

Activated fibroblasts are key players in the injury response, tumorigenesis, fibrosis, and inflammation. Dichotomous outcomes in response to varied stroma-targeted therapies in cancer emphasize the need to disentangle the roles of heterogeneous fibroblast subsets in physiological and pathophysiological settings. In wound healing, fibrosis, and myriad tumor types, fibroblast activation protein (FAP) and alpha-smooth muscle actin (αSMA) identify distinct, yet overlapping, activated fibroblast subsets. Prior studies established that FAPHi reactive fibroblasts and αSMAHi myofibroblasts can exert opposing influences in tumorigenesis. However, the factors that drive this phenotypic heterogeneity and the unique functional roles of these subsets have not been defined. We demonstrate that a convergence of ECM composition, elasticity, and transforming growth factor beta (TGF-ß) signaling governs activated fibroblast phenotypic heterogeneity. Furthermore, FAPHi reactive fibroblasts and αSMAHi myofibroblasts exhibited distinct gene expression signatures and functionality in vitro, illuminating potentially unique roles of activated fibroblast subsets in tissue remodeling. These insights into activated fibroblast heterogeneity will inform the rational design of stroma-targeted therapies for cancer and fibrosis.

APC conditional knock-out mouse is a model of infantile spasms with elevated neuronal ß-catenin levels, neonatal spasms, and chronic seizures.

Infantile spasms (IS) are a catastrophic childhood epilepsy syndrome characterized by flexion-extension spasms during infancy that progress to chronic seizures and cognitive deficits in later life. The molecular causes of IS are poorly defined. Genetic screens of individuals with IS have identified multiple risk genes, several of which are predicted to alter ß-catenin pathways. However, evidence linking malfunction of ß-catenin pathways and IS is lacking. Here, we show that conditional deletion in mice of the adenomatous polyposis coli gene (APC cKO), the major negative regulator of ß-catenin, leads to excessive ß-catenin levels and multiple salient features of human IS. Compared with wild-type littermates, neonatal APC cKO mice exhibit flexion-extension motor spasms and abnormal high-amplitude electroencephalographic discharges. Additionally, the frequency of excitatory postsynaptic currents is increased in layer V pyramidal cells, the major output neurons of the cerebral cortex. At adult ages, APC cKOs display spontaneous electroclinical seizures. These data provide the first evidence that malfunctions of APC/ß-catenin pathways cause pathophysiological changes consistent with IS. Our findings demonstrate that the APC cKO is a new genetic model of IS, provide novel insights into molecular and functional alterations that can lead to IS, and suggest novel targets for therapeutic intervention.

Adenomatous Polyposis Coli Protein Deletion in Efferent Olivocochlear Neurons Perturbs Afferent Synaptic Maturation and Reduces the Dynamic Range of Hearing.

Normal hearing requires proper differentiation of afferent ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) that carry acoustic information to the brain. Within individual IHCs, presynaptic ribbons show a size gradient with larger ribbons on the modiolar face and smaller ribbons on the pillar face. This structural gradient is associated with a gradient of spontaneous rates and threshold sensitivity, which is essential for a wide dynamic range of hearing. Despite their importance for hearing, mechanisms that direct ribbon differentiation are poorly defined. We recently identified adenomatous polyposis coli protein (APC) as a key regulator of interneuronal synapse maturation. Here, we show that APC is required for ribbon size heterogeneity and normal cochlear function. Compared with wild-type littermates, APC conditional knock-out (cKO) mice exhibit decreased auditory brainstem responses. The IHC ribbon size gradient is also perturbed. Whereas the normal-developing IHCs display ribbon size gradients before hearing onset, ribbon sizes are aberrant in APC cKOs from neonatal ages on. Reporter expression studies show that the CaMKII-Cre used to delete the floxed APC gene is present in efferent olivocochlear (OC) neurons, not IHCs or SGNs. APC loss led to increased volumes and numbers of OC inhibitory dopaminergic boutons on neonatal SGN fibers. Our findings identify APC in efferent OC neurons as essential for regulating ribbon heterogeneity, dopaminergic terminal differentiation, and cochlear sensitivity. This APC effect on auditory epithelial cell synapses resembles interneuronal and nerve-muscle synapses, thereby defining a global role for APC in synaptic maturation in diverse cell types. SIGNIFICANCE STATEMENT: This study identifies novel molecules and cellular interactions that are essential for the proper maturation of afferent ribbon synapses in sensory cells of the inner ear, and for normal hearing.

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels.

Small conductance Ca(2+)-sensitive potassium (SK2) channels are voltage-independent, Ca(2+)-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca(2+) permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca(2+) and Ca(2+)-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.

The postsynaptic adenomatous polyposis coli (APC) multiprotein complex is required for localizing neuroligin and neurexin to neuronal nicotinic synapses in vivo.

Synaptic efficacy requires that presynaptic and postsynaptic specializations align precisely and mature coordinately. The underlying mechanisms are poorly understood, however. We propose that adenomatous polyposis coli protein (APC) is a key coordinator of presynaptic and postsynaptic maturation. APC organizes a multiprotein complex that directs nicotinic acetylcholine receptor (nAChR) localization at postsynaptic sites in avian ciliary ganglion neurons in vivo. We hypothesize that the APC complex also provides retrograde signals that direct presynaptic active zones to develop in register with postsynaptic nAChR clusters. In our model, the APC complex provides retrograde signals via postsynaptic neuroligin that interacts extracellularly with presynaptic neurexin. S-SCAM (synaptic cell adhesion molecule) and PSD-93 (postsynaptic density-93) are scaffold proteins that bind to neuroligin. We identify S-SCAM as a novel component of neuronal nicotinic synapses. We show that S-SCAM, PSD-93, neuroligin and neurexin are enriched at alpha3*-nAChR synapses. PSD-93 and S-SCAM bind to APC and its binding partner beta-catenin, respectively. Blockade of selected APC and beta-catenin interactions, in vivo, leads to decreased postsynaptic accumulation of S-SCAM, but not PSD-93. Importantly, neuroligin synaptic clusters are also decreased. On the presynaptic side, there are decreases in neurexin and active zone proteins. Further, presynaptic terminals are less mature structurally and functionally. We define a novel neural role for APC by showing that the postsynaptic APC multiprotein complex is required for anchoring neuroligin and neurexin at neuronal synapses in vivo. APC human gene mutations correlate with autism spectrum disorders, providing strong support for the importance of the association, demonstrated here, between APC, neuroligin and neurexin.

Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors.

The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.

Endogenous cAbl regulates receptor endocytosis.

There are two key processes underlying ligand-induced receptor endocytosis: receptor ubiquitylation and remodeling of the actin cytoskeleton. Tyrosine kinases play critical roles in both receptor endocytosis and actin reorganization. Interestingly, members of the Abl family are the only known tyrosine kinases that possess an actin-binding domain and thus have the potential to directly regulate the actin cytoskeleton. However, the role of non-transforming cAbl in receptor endocytosis remains undefined. We report that cAbl promotes ligand-induced antigen receptor endocytosis in B lymphocytes. We show that pharmacologic inhibition or genetic deletion of cAbl causes a defect in tyrosine phosphorylation of the cytoskeletal adapter CrkII. cAbl inhibition or ablation also impairs Rac activation downstream of CrkII, as well as antigen receptor capping and endocytosis. Although phosphorylation of CrkII has been suggested to maintain it in a closed inactive conformation, we demonstrate that it is in fact essential for the activation of Rac. On the other hand, association of CrkII with cCbl, a key mediator of receptor ubiquitylation, does not require CrkII phosphorylation and is cAbl-independent. Phosphorylation of cCbl itself is also cAbl-independent. Our results thus indicate that CrkII links receptor engagement to cytoskeletal remodeling by coupling cCbl- and cAbl-mediated signaling pathways that cooperatively regulate ligand-induced receptor endocytosis.

Dystrophin and utrophin isoforms are expressed in glia, but not neurons, of the avian parasympathetic ciliary ganglion.

Muscular dystrophy patients often show cognitive impairment, in addition to muscle degeneration caused by dystrophin gene defects. The cognitive impairments lead to speculation that the dystrophin protein family may play a key role at neuronal synapses. Dystrophin regulates the stability of selected GABA(A) receptor subtypes and alpha3-containing nicotinic acetylcholine receptors (nAChRs) at a subset of central GABAergic and peripheral sympathetic nicotinic neuron synapses. Similarly, utrophin, the autosomal homologue of dystrophin, is not required for clustering but indirectly stabilizes muscle-type nAChRs at the neuromuscular junction. We examined dystrophin and utrophin expression and localization in the avian parasympathetic ciliary ganglion (CG) to determine whether these proteins play a general role at neuronal nicotinic synapses. We have determined that full-length utrophin and dystrophin and the short dystrophin isoform Dp116 are the major isoforms expressed in the CG based on immunoblotting and immunolabeling. Unexpectedly, the cytoskeletal proteins were not detected at nicotinic synapses or in CG neurons. They are expressed in myelinating and non-myelinating Schwann cells. Further, utrophin expression developmentally precedes that of dystrophin. The proteins show partially overlapping distributions, but also differential accumulation along the surface membrane of Schwann cells adjacent to neuronal somata versus axonal processes. Our findings are consistent with reports that dystrophin protein family members function in the maintenance of cell-cell interactions and myelination by anchoring the Schwann cell surface membrane to the basal lamina. In contrast, our results differ from those in skeletal muscle and a subset of sympathetic neurons where utrophin and dystrophin localize at nicotinic synapses.

Fibroblast migration is mediated by CD44-dependent TGF beta activation.

CD44 contributes to inflammation and fibrosis in response to injury. As fibroblast recruitment is critical to wound healing, we compared cytoskeletal architecture and migration of wild-type (CD44WT) and CD44-deficient (CD44KO) fibroblasts. CD44KO fibroblasts exhibited fewer stress fibers and focal adhesion complexes, and their migration was characterized by increased velocity but loss of directionality, compared with CD44WT fibroblasts. Mechanistically, we demonstrate that CD44WT cells generated more active TGFbeta than CD44KO cells and that CD44 promotes the activation of TGFbeta via an MMP-dependent mechanism. Reconstitution of CD44 expression completely rescued the phenotype of CD44KO cells whereas exposure of CD44KO cells to exogenous active TGFbeta rescued the defect in stress fibers and migrational velocity, but was not sufficient to restore directionality of migration. These results resolve the TGFbeta-mediated and TGFbeta-independent effects of CD44 on fibroblast migration and suggest that CD44 may be critical for the recruitment of fibroblasts to sites of injury and the function of fibroblasts in tissue remodeling and fibrosis.

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex.

The neuronal nicotinic synapse plays a central role in normal cognitive and autonomic function. Molecular mechanisms that direct the assembly of this synapse remain poorly defined, however. We show here that adenomatous polyposis coli (APC) organizes a multi-molecular complex that is essential for targeting alpha3(*)nAChRs to synapses. APC interaction with microtubule plus-end binding protein EB1 is required for alpha3(*)nAChR surface membrane insertion and stabilization. APC brings together EB1, the key cytoskeletal regulators macrophin and IQGAP1, and 14-3-3 adapter protein at nicotinic synapses. 14-3-3, in turn, links the alpha3-subunit to APC. This multi-molecular APC complex stabilizes the local microtubule and F-actin cytoskeleton and links postsynaptic components to the cytoskeleton--essential functions for controlling the molecular composition and stability of synapses. This work identifies macrophin, IQGAP1 and 14-3-3 as novel nicotinic synapse components and defines a new role for APC as an in vivo coordinator of nicotinic postsynaptic assembly in vertebrate neurons.

Dual role of Cbl links critical events in BCR endocytosis.

Receptor endocytosis down-regulates ligand-induced signaling in a timely manner and depends on cytoskeletal remodeling. In B lymphocytes, internalization of B cell receptors (BCRs) is also critical to antigen presentation. However, the mechanisms underlying BCR endocytosis are not fully understood. Similarly, the molecular mechanisms linking endocytosis to cytoskeletal remodeling remain poorly defined. We used flow cytometry, pull-down assays, immunochemistry and fluorescence microscopy to investigate BCR internalization in the DT40 B cell line. We demonstrate that ablation of Cbl impacts BCR endocytosis and the underlying cytoskeletal dynamics. Specifically, we demonstrate that ligand-induced endocytosis is impaired in Cbl-/- cells and that the ubiquitin ligase activity is required for Cbl to promote endocytosis. We also show that phosphorylation of CrkII, activation of Rac downstream of CrkII and BCR capping require Cbl. Furthermore, we show that the association of Cbl and CrkII requires phosphorylation of Cbl, but not its ubiquitin ligase activity. Our data indicate that Cbl promotes BCR endocytosis and attenuates ligand-induced signaling by virtue of its ability to orchestrate receptor ubiquitylation and cytoskeletal dynamics.

Kainate receptors and RNA editing in cholinergic neurons.

Parasympathetic ganglia are considered simple relay systems that have cholinergic input and output, with modulation occurring centrally. Greater complexity is suggested, however, by our showing here that avian ciliary ganglion (CG) neurons also express a different excitatory receptor type--ionotropic glutamate receptors of the kainate subtype (KARs). This is the first report of glutamate receptor expression in the CG and KAR expression in any cholinergic neuron. We show that KARs form functional channels on CG neurons. KARs localize to CG neuron axons and somata as well as axons and terminals of pre-synaptic inputs to the CG. Glutamate transporters are expressed on Schwann cells that surround synapses on neuronal somata, and may provide a local source of glutamate. CG neurons express multiple KAR subunit mRNAs (GluR5, GluR7, and KA1), and their relative levels change dramatically during axon outgrowth and synaptic differentiation. The developmental role for KARs may depend upon their calcium permeability, a property regulated by mRNA editing. We show GluR5 editing increases predominantly at the time CG axons contact peripheral targets. Our data suggest that glutamatergic signaling may function as a local circuit mechanism to modulate excitability and calcium signaling during synapse formation and maturation in the CG in vivo.

Identification of 12/15-lipoxygenase as a suppressor of myeloproliferative disease.

Though Abl inhibitors are often successful therapies for the initial stages of chronic myelogenous leukemia (CML), refractory cases highlight the need for novel molecular insights. We demonstrate that mice deficient in the enzyme 12/15-lipoxygenase (12/15-LO) develop a myeloproliferative disorder (MPD) that progresses to transplantable leukemia. Although not associated with dysregulation of Abl, cells isolated from chronic stage 12/15-LO-deficient (Alox15) mice exhibit increased activation of the phosphatidylinositol 3-kinase (PI3-K) pathway, as indicated by enhanced phosphorylation of Akt. Furthermore, the transcription factor interferon consensus sequence binding protein (ICSBP) is hyperphosphorylated and displays decreased nuclear accumulation, translating into increased levels of expression of the oncoprotein Bcl-2. The ICSBP defect, exaggerated levels of Bcl-2, and prolonged leukemic cell survival associated with chronic stage Alox15 MPD are all reversible upon treatment with a PI3-K inhibitor. Remarkably, the evolution of Alox15 MPD to leukemia is associated with additional regulation of ICSBP on an RNA level, highlighting the potential usefulness of the Alox15 model for understanding the transition of CML to crisis. Finally, 12/15-LO expression suppresses the growth of a human CML-derived cell line. These data identify 12/15-LO as an important suppressor of MPD via its role as a critical upstream effector in the regulation of PI3-K-dependent ICSBP phosphorylation.