Mitochondrial Morphogenesis, Dendrite Development, and Synapse Formation in Cerebellum Require both Bcl-w and the Glutamate Receptor δ2

Bcl-w belongs to the prosurvival group of the Bcl-2 family, while the glutamate receptor δ2 (Grid2) is an excitatory receptor that is specifically expressed in Purkinje cells, and required for Purkinje cell synapse formation. A recently published result as well as our own findings have shown that Bcl-w can physically interact with an autophagy protein, Beclin1, which in turn has been shown previously to form a protein complex with the intracellular domain of Grid2 and an adaptor protein, nPIST. This suggests that Bcl-w and Grid2 might interact genetically to regulate mitochondria, autophagy, and neuronal function. In this study, we investigated this genetic interaction of Bcl-w and Grid2 through analysis of single and double mutant mice of these two proteins using a combination of histological and behavior tests. It was found that Bcl-w does not control the cell number in mouse brain, but promotes what is likely to be the mitochondrial fission in Purkinje cell dendrites, and is required for synapse formation and motor learning in cerebellum, and that Grid2 has similar phenotypes. Mice carrying the double mutations of these two genes had synergistic effects including extremely long mitochondria in Purkinje cell dendrites, and strongly aberrant Purkinje cell dendrites, spines, and synapses, and severely ataxic behavior. Bcl-w and Grid2 mutations were not found to influence the basal autophagy that is required for Purkinje cell survival, thus resulting in these phenotypes. Our results demonstrate that Bcl-w and Grid2 are two critical proteins acting in distinct pathways to regulate mitochondrial morphogenesis and control Purkinje cell dendrite development and synapse formation. We propose that the mitochondrial fission occurring during neuronal growth might be critically important for dendrite development and synapse formation, and that it can be regulated coordinately by multiple pathways including Bcl-2 and glutamate receptor family members.     

Synaptic Competition Sculpts the Development of GABAergic Axo-Dendritic but Not Perisomatic Synapses

The neurotransmitter GABA regulates many aspects of inhibitory synapse development. We tested the hypothesis that GABA_A receptors (GABA_ARs) work together with the synaptic adhesion molecule neuroligin 2 (NL2) to regulate synapse formation in different subcellular compartments. We investigated mice (“γ2 knockdown mice”) with an engineered allele of the GABA_AR γ2 subunit gene which produced a mosaic expression of synaptic GABA_ARs in neighboring neurons, causing a strong imbalance in synaptic inhibition. Deletion of the γ2 subunit did not abolish synapse formation or the targeting of NL2 to distinct types of perisomatic and axo-dendritic contacts. Thus synaptic localization of NL2 does not require synaptic GABA_ARs. However, loss of the γ2 subunit caused a selective decrease in the number of axo-dendritic synapses on cerebellar Purkinje cells and cortical pyramidal neurons, whereas perisomatic synapses were not significantly affected. Notably, γ2-positive cells had increased axo-dendritic innervation compared with both γ2-negative and wild-type counterparts. Moreover heterologous synapses on spines, that are found after total deletion of GABA_ARs from all Purkinje cells, were rare in cerebella of γ2 knockdown mice. These findings reveal a selective role of γ2 subunit-containing GABA_ARs in regulating synapse development in distinct subcellular compartments, and support the hypothesis that the refinement of axo-dendritic synapses is regulated by activity-dependent competition between neighboring neurons.     

Persistent Posttetanic Depression at Cerebellar Parallel Fiber to Purkinje Cell Synapses

Plasticity at the cerebellar parallel fiber to Purkinje cell synapse may underlie information processing and motor learning. In vivo, parallel fibers appear to fire in short high frequency bursts likely to activate sparsely distributed synapses over the Purkinje cell dendritic tree. Here, we report that short parallel fiber tetanic stimulation evokes a ∼7–15% depression which develops over 2 min and lasts for at least 20 min. In contrast to the concomitantly evoked short-term endocannabinoid-mediated depression, this persistent posttetanic depression (PTD) does not exhibit a dependency on the spatial pattern of synapse activation and is not caused by any detectable change in presynaptic calcium signaling. This persistent PTD is however associated with increased paired-pulse facilitation and coefficient of variation of synaptic responses, suggesting that its expression is presynaptic. The chelation of postsynaptic calcium prevents its induction, suggesting that post- to presynaptic (retrograde) signaling is required. We rule out endocannabinoid signaling since the inhibition of type 1 cannabinoid receptors, monoacylglycerol lipase or vanilloid receptor 1, or incubation with anandamide had no detectable effect. The persistent PTD is maximal in pre-adolescent mice, abolished by adrenergic and dopaminergic receptors block, but unaffected by adrenergic and dopaminergic agonists. Our data unveils a novel form of plasticity at parallel fiber synapses: a persistent PTD induced by physiologically relevant input patterns, age-dependent, and strongly modulated by the monoaminergic system. We further provide evidence supporting that the plasticity mechanism involves retrograde signaling and presynaptic diacylglycerol.     

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase–related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.     

Quantitative Organization of GABAergic Synapses in the Molecular Layer of the Mouse Cerebellar Cortex

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABA_A receptor α1 subunit (GABA_ARα1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the α1 subunit resulted in a complete loss of GABA_AR aggregates from Purkinje cells, allowing us to determine the density of GABA_AR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using α-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex.     

Mitochondrial Morphogenesis,Dendrite Development,and Synapse Formation in Cerebellum Require both Bcl-w and the Glutamate Receptor δ2

Bcl-w belongs to the prosurvival group of the Bcl-2 family, while the glutamate receptor δ2 (Grid2) is an excitatory receptor that is specifically expressed in Purkinje cells, and required for Purkinje cell synapse formation. A recently published result as well as our own findings have shown that Bcl-w can physically interact with an autophagy protein, Beclin1, which in turn has been shown previously to form a protein complex with the intracellular domain of Grid2 and an adaptor protein, nPIST. This suggests that Bcl-w and Grid2 might interact genetically to regulate mitochondria, autophagy, and neuronal function. In this study, we investigated this genetic interaction of Bcl-w and Grid2 through analysis of single and double mutant mice of these two proteins using a combination of histological and behavior tests. It was found that Bcl-w does not control the cell number in mouse brain, but promotes what is likely to be the mitochondrial fission in Purkinje cell dendrites, and is required for synapse formation and motor learning in cerebellum, and that Grid2 has similar phenotypes. Mice carrying the double mutations of these two genes had synergistic effects including extremely long mitochondria in Purkinje cell dendrites, and strongly aberrant Purkinje cell dendrites, spines, and synapses, and severely ataxic behavior. Bcl-w and Grid2 mutations were not found to influence the basal autophagy that is required for Purkinje cell survival, thus resulting in these phenotypes. Our results demonstrate that Bcl-w and Grid2 are two critical proteins acting in distinct pathways to regulate mitochondrial morphogenesis and control Purkinje cell dendrite development and synapse formation. We propose that the mitochondrial fission occurring during neuronal growth might be critically important for dendrite development and synapse formation, and that it can be regulated coordinately by multiple pathways including Bcl-2 and glutamate receptor family members.     

δ-Glutamate Receptors Are Differentially Distributed at Parallel and Climbing Fiber Synapses on Purkinje Cells

Abstract: Neurons containing multiple excitatory inputs may sort and target glutamate receptor subtypes to subsets of synapses. A good model for testing this hypothesis is the Purkinje cell, which expresses significant levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, kainate, N -methyl- d -aspartate, δ-, and metabotropic glutamate receptors. Purkinje cells receive two excitatory inputs, the parallel and climbing fibers; the combined effect of stimulation of these two inputs is to produce long-term depression of parallel fiber/Purkinje cell neurotransmission. Distribution of glutamate receptors in these two synapse populations in rat cerebella was studied using preembedding immunocytochemistry with antibodies to GluR1, GluR2/3, GluR5-7, NR1, δ1/2, and mGluR1α. Moderate/dense postsynaptic staining was most frequent in postsynaptic densities and spines of both parallel and climbing fiber synapses with mGluR1α antibody, was intermediate in frequency with GluR2/3 and GluR5-7 antibodies, and was least frequent with GluR1 and NR1 antibodies. The most striking finding was the absence of significant postsynaptic staining with δ1/2 antibody in climbing fiber synapses in adult animals, even though postsynaptic staining was prevalent in parallel fiber synapses with this antibody. In contrast to adults, moderate/dense postsynaptic immunolabeling of climbing fiber synapses with δ1/2 antibody was common in rats at 10 days postnatal. This study provides direct morphological evidence that δ-glutamate receptors are differentially targeted to synapse populations. Our results support previous suggestions that δ2 is involved in development of parallel and climbing fiber synapses and in long-term depression of parallel fiber/Purkinje synaptic responses in adults.     

Long-term synaptic changes induced in the cerebellar cortex by fear conditioning

To better understand learning mechanisms, one needs to study synaptic plasticity induced by behavioral training. Recently, it has been demonstrated that the cerebellum is involved in the consolidation of fear memory. Nevertheless, how the cerebellum contributes to emotional behavior is far from known. In cerebellar slices at 10 min and 24 hr following fear conditioning, we found a long-lasting potentiation of the synapse between parallel fibers and Purkinje cells in vermal lobules V-VI, but not in the climbing fiber synapses. The mechanism is postsynaptic, due to an increased AMPA response. In addition, in hotfoot mice with a primary deficiency of the parallel fiber to Purkinje cell synapse, cued (but not contextual) fear conditioning is affected. We propose that this synapse plays an important role in the learned fear and that its long-term potentiation may represent a contribution to the neural substrate of fear memory.     

Nitric oxide regulates input specificity of long-term depression and context dependence of cerebellar learning

Recent studies have shown that multiple internal models are acquired in the cerebellum and that these can be switched under a given context of behavior. It has been proposed that long-term depression (LTD) of parallel fiber (PF)–Purkinje cell (PC) synapses forms the cellular basis of cerebellar learning, and that the presynaptically synthesized messenger nitric oxide (NO) is a crucial “gatekeeper” for LTD. Because NO diffuses freely to neighboring synapses, this volume learning is not input-specific and brings into question the biological significance of LTD as the basic mechanism for efficient supervised learning. To better characterize the role of NO in cerebellar learning, we simulated the sequence of electrophysiological and biochemical events in PF–PC LTD by combining established simulation models of the electrophysiology, calcium dynamics, and signaling pathways of the PC. The results demonstrate that the local NO concentration is critical for induction of LTD and for its input specificity. Pre- and postsynaptic coincident firing is not sufficient for a PF–PC synapse to undergo LTD, and LTD is induced only when a sufficient amount of NO is provided by activation of the surrounding PFs. On the other hand, above-adequate levels of activity in nearby PFs cause accumulation of NO, which also allows LTD in neighboring synapses that were not directly stimulated, ruining input specificity. These findings lead us to propose the hypothesis that NO represents the relevance of a given context and enables context-dependent selection of internal models to be updated. We also predict sparse PF activity in vivo because, otherwise, input specificity would be lost.     

Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase Acts as a Cdc42 Effector in Promoting Cytoskeletal Reorganization

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ~190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCKα and Cdc42^V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCKα resulted in actin and myosin reorganization. Expression of kinase-dead MRCKα blocked Cdc42^V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCKα mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCKα plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCKα mutants. These results suggest that MRCKα may act as a downstream effector of Cdc42 in cytoskeletal reorganization.     

Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2

The precise regulation of synapse maintenance is critical to the development and function of neuronal circuits. Using an in vivo RNAi screen targeting the Drosophila kinome and phosphatome, we identify 11 kinases and phosphatases controlling synapse stability by regulating cytoskeletal, phospholipid, or metabolic signaling. We focus on casein kinase 2 (CK2) and demonstrate that the regulatory (β) and catalytic (α) subunits of CK2 are essential for synapse maintenance. CK2α kinase activity is required in the presynaptic motoneuron, and its interaction with CK2β, mediated cooperatively by two N-terminal residues of CK2α, is essential for CK2 holoenzyme complex stability and function in vivo. Using genetic and biochemical approaches we identify Ankyrin2 as a key presynaptic target of CK2 to maintain synapse stability. In addition, CK2 activity controls the subcellular organization of individual synaptic release sites within the presynaptic nerve terminal. Our study identifies phosphorylation of structural synaptic components as a compelling mechanism to actively control the development and longevity of synaptic connections.     

N-cadherin and Neuroligins Cooperate to Regulate Synapse Formation in Hippocampal Cultures

Cadherins and neuroligins (NLs) represent two families of cell adhesion proteins that are essential for the establishment of synaptic connections in vitro; however, it remains unclear whether these proteins act in concert to regulate synapse density. Using a combination of overexpression and knockdown analyses in primary hippocampal neurons, we demonstrate that NL1 and N-cadherin promote the formation of glutamatergic synapses through a common functional pathway. Analysis of the spatial relationship between N-cadherin and NL1 indicates that in 14-day in vitro cultures, almost half of glutamatergic synapses are associated with both proteins, whereas only a subset of these synapses are associated with N-cadherin or NL1 alone. This suggests that NL1 and N-cadherin are spatially distributed in a manner that enables cooperation at synapses. In young cultures, N-cadherin clustering and its association with synaptic markers precede the clustering of NL1. Overexpression of N-cadherin at this time point enhances NL1 clustering and increases synapse density. Although N-cadherin is not sufficient to enhance NL1 clustering and synapse density in more mature cultures, knockdown of N-cadherin at later time points significantly attenuates the density of NL1 clusters and synapses. N-cadherin overexpression can partially rescue synapse loss in NL1 knockdown cells, possibly due to the ability of N-cadherin to recruit NL2 to glutamatergic synapses in these cells. We demonstrate that cadherins and NLs can act in concert to regulate synapse formation.     

Associative short-term synaptic plasticity mediated by endocannabinoids

Associative learning is important on rapid timescales, but no suitable form of short-term plasticity has been identified that is both associative and synapse specific. Here, we assess whether endocannabinoids can mediate such plasticity. In the cerebellum, bursts of parallel fiber (PF) activity evoke endocannabinoid release from Purkinje cell dendrites that results in retrograde synaptic inhibition lasting seconds. We find that the powerful climbing fiber (CF) to Purkinje cell synapse regulates this inhibition. Compared to PF stimulation alone, coactivation of PF and CF synapses greatly enhanced endocannabinoid-mediated inhibition of PF synapses. Retrograde inhibition was restricted to PFs activated within several hundred milliseconds of CF activation. This associative plasticity reflects two aspects of calcium-dependent endocannabinoid release. First, PF-mediated activation of metabotropic glutamate receptors locally reduced the dendritic calcium levels required for endocannabinoid release. Second, CF and PF coactivation evoked localized supralinear dendritic calcium signals. Thus, endocannabinoids mediate transient associative synaptic plasticity.     

Long-term depression of the cerebellar climbing fiber--Purkinje neuron synapse

In classic Marr-Albus-Ito models of cerebellar function, coactivation of the climbing fiber (CF) synapse, which provides massive, invariant excitation of Purkinje neurons (coding the unconditioned stimulus), together with a graded parallel fiber synaptic array (coding the conditioned stimulus) leads to long-term depression (LTD) of parallel fiber-Purkinje neuron synapses, underlying production of a conditioned response. Here, we show that the supposedly invariant CF synapse can also express LTD. Brief 5 Hz stimulation of the CF resulted in a sustained depression of CF EPSCs that did not spread to neighboring parallel fiber synapses. Like parallel fiber LTD, CF LTD required postsynaptic Ca2+ elevation, activation of group 1 mGluRs, and activation of PKC. CF LTD is potentially relevant for models of cerebellar motor control and learning and the developmental conversion from multiple to single CF innervation of Purkinje neurons.     

Developing T cells form an immunological synapse for passage through the β-selection checkpoint

The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T cell receptor β (TCRβ). Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-selection checkpoint establish an immunological synapse in in vitro and in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the β-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the β-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell to allow transition beyond the β-selection checkpoint.     

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency.     

Maternal Dietary Loads of Alpha-Tocopherol Increase Synapse Density and Glial Synaptic Coverage in the Hippocampus of Adult Offspring

An increased intake of the antioxidant α-Tocopherol (vitamin E) is recommended in complicated pregnancies, to prevent free radical damage to mother and fetus. However, the anti-PKC and antimitotic activity of α-Tocopherol raises concerns about its potential effects on brain development. Recently, we found that maternal dietary loads of α-Tocopherol through pregnancy and lactation cause developmental deficit in hippocampal synaptic plasticity in rat offspring. The defect persisted into adulthood, with behavioral alterations in hippocampus-dependent learning. Here, using the same rat model of maternal supplementation, ultrastructural morphometric studies were carried out to provide mechanistic interpretation to such a functional impairment in adult offspring by the occurrence of long-term changes in density and morphological features of hippocampal synapses. Higher density of axo-spinous synapses was found in CA1 stratum radiatum of α-Tocopherol-exposed rats compared to controls, pointing to a reduced synapse pruning. No morphometric changes were found in synaptic ultrastructural features, i.e., perimeter of axon terminals, length of synaptic specializations, extension of bouton-spine contact. Gliasynapse anatomical relationship was also affected. Heavier astrocytic coverage of synapses was observed in Tocopherol-treated offspring, notably surrounding axon terminals; moreover, the percentage of synapses contacted by astrocytic endfeet at bouton-spine interface (tripartite synapses) was increased.These findings indicate that gestational and neonatal exposure to supranutritional Tocopherol intake can result in anatomical changes of offspring hippocampus that last through adulthood. These include a surplus of axo-spinous synapses and an aberrant gliasynapse relationship, which may represent the morphological signature of previously described alterations in synaptic plasticity and hippocampus-dependent learning.Key words: Vitamin E, CA1 stratum radiatum, axo-spinous synapses, glia-synapse relationship, tripartite synapses, morphometry, electron microscopy