Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation.

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

A comparative overview of DSCAM and its multifunctional roles in Drosophila and vertebrates.

DSCAM (Down syndrome cell adhesion molecule) is a unique neuronal adhesion protein with extensively documented multifaceted functionalities. DSCAM also has interesting properties in vertebrates and invertebrates, respectively. In Drosophila species, particularly, Dscam exhibits remarkable genetic diversity, with tens of thousands of splicing isoforms that modulate the specificity of neuronal wiring. Interestingly, this splice variant diversity of Dscam is absent in vertebrates. DSCAM plays a pivotal role in mitigating excessive adhesion between identical cell types, thereby maintaining the structural and functional coherence of neural networks. DSCAM contributes to the oversight of selective intercellular interactions such as synaptogenesis; however, the precise regulatory mechanisms underlying the promotion and inhibition of cell adhesion involved remain unclear. In this review, we aim to delineate the distinct molecules that interact with DSCAM and their specific roles within the biological landscapes of Drosophila and vertebrates. By integrating these comparative insights, we aim to elucidate the multifunctional nature of DSCAM, particularly its capacity to facilitate or deter intercellular adhesion.

Novel FABP3 ligand, HY-11-9, ameliorates neuropathological deficits in MPTP-induced Parkinsonism in mice.

Parkinson's disease (PD) is characterized by dopaminergic (DAergic) neuronal loss in the substantia nigra pars compacta (SNpc), resulting from α-synuclein (αSyn) toxicity. We previously reported that αSyn oligomerization and toxicity are regulated by the fatty-acid binding protein 3 (FABP3), and the therapeutic effects of the FABP3 ligand, MF1, was successfully demonstrated in PD models. Here, we developed a novel and potent ligand, HY-11-9, which has a higher affinity for FABP3 (Kd = 11.7 ± 8.8) than MF1 (Kd = 302.8 ± 130.3). We also investigated whether the FABP3 ligand can ameliorate neuropathological deterioration after the onset of disease in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism. Motor deficits were observed two weeks after MPTP treatment. Notably, oral administration of HY-11-9 (0.03 mg/kg) improved motor deficits in both beam-walking and rotarod tasks, whereas MF1 failed to improve the motor deficits in both tasks. Consistent with the behavioral tasks, HY-11-9 recovered dopamine neurons from MPTP toxicity in the substantia nigra and ventral tegmental areas. Furthermore, HY-11-9 reduced the accumulation of phosphorylated-serine129-α-synuclein (pS129-αSyn) and colocalization with FABP3 in tyrosine hydroxylase (TH)-positive DA neurons in the PD mouse model. Overall, HY-11-9 significantly improved MPTP-induced behavioral and neuropathological deterioration, suggesting that it may be a potential candidate for PD therapy.

Neuronal and astrocytic protein connections and associated adhesion molecules.

Astrocytes are morphologically complex, with a myriad of processes which allow contact with other astrocytes, blood vessels, and neurons. Adhesion molecules expressed by these cells regulate this connectivity. Adhesion molecules are required to form and maintain functional neural circuits, but their importance and mechanisms of action, particularly in astrocyte-neuron contact, remain unresolved. Several studies of neuron-astrocyte connections have demonstrated the vital functions of adhesion molecules, including neuron-glia cell adhesion molecules, astrotactins, and protocadherins. In this review, we provide an overview and perspective of astrocyte-neuron contacts mediated by adhesion molecules in developing neural circuits and synapse formation, especially in the cerebellum. We also outline a novel mechanism of interaction between neurons and astrocytes in the tripartite synapses that has been recently found by our group.

AUTS2 Governs Cerebellar Development, Purkinje Cell Maturation, Motor Function and Social Communication.

Autism susceptibility candidate 2 (AUTS2), a risk gene for autism spectrum disorders (ASDs), is implicated in telencephalon development. Because AUTS2 is also expressed in the cerebellum where defects have been linked to ASDs, we investigated AUTS2 functions in the cerebellum. AUTS2 is specifically localized in Purkinje cells (PCs) and Golgi cells during postnatal development. Auts2 conditional knockout (cKO) mice exhibited smaller and deformed cerebella containing immature-shaped PCs with reduced expression of Cacna1a. Auts2 cKO and knock-down experiments implicated AUTS2 participation in elimination and translocation of climbing fiber synapses and restriction of parallel fiber synapse numbers. Auts2 cKO mice exhibited behavioral impairments in motor learning and vocal communications. Because Cacna1a is known to regulate synapse development in PCs, it suggests that AUTS2 is required for PC maturation to elicit normal development of PC synapses and thus the impairment of AUTS2 may cause cerebellar dysfunction related to psychiatric illnesses such as ASDs.

DSCAM regulates delamination of neurons in the developing midbrain.

For normal neurogenesis and circuit formation, delamination of differentiating neurons from the proliferative zone must be precisely controlled; however, the regulatory mechanisms underlying cell attachment are poorly understood. Here, we show that Down syndrome cell adhesion molecule (DSCAM) controls neuronal delamination by local suppression of the RapGEF2-Rap1-N-cadherin cascade at the apical endfeet in the dorsal midbrain. Dscam transcripts were expressed in differentiating neurons, and DSCAM protein accumulated at the distal part of the apical endfeet. Cre-loxP-based neuronal labeling revealed that Dscam knockdown impaired endfeet detachment from ventricles. DSCAM associated with RapGEF2 to inactivate Rap1, whose activity is required for membrane localization of N-cadherin. Correspondingly, Dscam knockdown increased N-cadherin localization and ventricular attachment area at the endfeet. Furthermore, excessive endfeet attachment by Dscam knockdown was restored by co-knockdown of RapGEF2 or N-cadherin Our findings shed light on the molecular mechanism that regulates a critical step in early neuronal development.

Comprehensive and cell-type-based characterization of the dorsal midbrain during development.

The layer structure has been intensively characterized in the developing neocortex and cerebellum based on the various molecular markers. However, as to the developing dorsal midbrain, comprehensive analyses have not been intensely carried out, and thus, the name as well as the definition of each layer is not commonly shared. Here, we redefined the three layers, such as the ventricular zone, intermediate zone and marginal zone, based on various markers for proliferation and differentiation in embryonic dorsal midbrain. Biphasic Ki67 expression defines the classical VZ, in which there is clear separation of the mitotic and interphase zones. Next, we mapped the distribution of immature neurons to the defined layers, based on markers for glutamatergic and GABAergic lineage. Interestingly, Tbr2 and Neurog2 were expressed in the postmitotic neurons. We also report that active (phosphorylated) JNK is a useful marker to demarcate layers during the embryonic stage. Finally, we validated the final arrival layers of the migratory glutamatergic and GABAergic neurons. These results form a foundation for analyses of brain development, especially in the proliferation and migration of excitatory and inhibitory neurons in the dorsal midbrain.

TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.

Involvement of the globus pallidus in behavioral goal determination and action specification.

Multiple loop circuits interconnect the basal ganglia and the frontal cortex, and each part of the cortico-basal ganglia loops plays an essential role in neuronal computational processes underlying motor behavior. To gain deeper insight into specific functions played by each component of the loops, we compared response properties of neurons in the globus pallidus (GP) with those in the dorsal premotor cortex (PMd) and the ventrolateral and dorsolateral prefrontal cortex (vlPFC and dlPFC) while monkeys performed a behavioral task designed to include separate processes for behavioral goal determination and action selection. Initially, visual signals instructed an abstract behavioral goal, and seconds later, a choice cue to select an action was presented. When the instruction cue appeared, GP neurons started to reflect visual features as early as vlPFC neurons. Subsequently, GP neurons began to reflect goals informed by the visual signals no later than neurons in the PMd, vlPFC, and dlPFC, indicating that the GP is involved in the early determination of behavioral goals. In contrast, action specification occurred later in the GP than in the cortical areas, and the GP was not as involved in the process by which a behavioral goal was transformed into an action. Furthermore, the length of time representing behavioral goal and action was shorter in the GP than in the PMd and dlPFC, indicating that the GP may play an important role in detecting individual behavioral events. These observations elucidate the involvement of the GP in goal-directed behavior.

Local application of neurotrophins specifies axons through inositol 1,4,5-trisphosphate, calcium, and Ca2+/calmodulin-dependent protein kinases.

Neurons are highly polarized cells that have structurally distinct processes-the axons and dendrites-that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca(2+)) in an inositol 1,4,5-trisphosphate (IP(3))-dependent fashion as well as local activation of the Ca(2+) effector Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP(3)-Ca(2+)-CaMKK signaling in axon specification.

[Neural mechanisms underlying the integration of perception and action].

The hallmark of higher-order brain functions is the ability to integrate and associate diverse sets of information in a flexible manner. Thus, fundamental knowledge about the mechanisms underlying of information in the brain can be obtained by examining the neural mechanisms involved in the generation of an appropriate motor command based on perceived sensory signals. In this review article, we have focused on the involvement of the neuronal networks centered at the lateral aspect of the frontal cortex in the process of motor selection and motor planning based on visual signals. We have initially discussed the role of the lateral prefrontal cortex in integrating multiple sets of visual signals to select a reach target and the participation of the premotor cortex in retrieving and integrating diverse sets of motor information, such as where should one reach out or which arm is to be used. Next, based on the results of the studies on ideomotor apraxia, we have hypothesized that there are at least 2 distinct levels of neural representation (virtual level and physical level). We have reviewed the evidence supporting the operation of 2 distinct classes of neuronal activities corresponding to these 2 levels. In conclusion, we propose that the frontal cortex initially processes information across sensory and motor domains at the virtual level to generate information about a forthcoming motor action (virtual action plan) and that this information is subsequently transformed into a motor command, such as muscle activity or movement direction, for an actual body movement at the physical level (physical motor plan). This proposed framework may be useful for explaining the diverse clinical conditions caused by brain lesions as well as for clarifying the neural mechanisms underlying the integration of perception and action.

CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity.

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long-distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi-directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein-2 (CRMP-2) played key roles in axon elongation and neuronal polarization. CRMP-2 was also found to associate with the anterograde motor protein Kinesin-1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP-2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP-2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N-terminus of CRMP-2, which is the distinct side of CRMP-2's kinesin light chain-binding region. Furthermore, over-expression of the dynein-binding fragments of CRMP-2 prevented dynein-driven microtubule transport in COS-7 cells. Given that CRMP-2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP-2 might have an important role in axon formation, and neuronal development.

TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases.

The 43-kDa TAR DNA-binding protein (TDP-43) is known to be a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation, a key regulating factor of Rho family activity and intracellular localization. In contrast, overexpression of TDP-43 mitigated the cellular damage caused by pharmacological inhibition of geranylgeranylation. Furthermore administration of geranylgeranyl pyrophosphate partially restored cell viability and neurite outgrowth in TDP-43 knockdown cells. In summary, our data suggest that TDP-43 plays a key role in the maintenance of neuronal cell morphology and survival possibly through protein geranylgeranylation of Rho family GTPases.

Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27.

The neurotrophin receptors TrkA, TrkB, and TrkC are localized at the surface of the axon terminus and transmit key signals from brain-derived neurotrophic factor (BDNF) for diverse effects on neuronal survival, differentiation, and axon formation. Trk receptors are sorted into axons via the anterograde transport of vesicles and are then inserted into axonal plasma membranes. However, the transport mechanism remains largely unknown. Here, we show that the Slp1/Rab27B/CRMP-2 complex directly links TrkB to Kinesin-1, and that this association is required for the anterograde transport of TrkB-containing vesicles. The cytoplasmic tail of TrkB binds to Slp1 in a Rab27B-dependent manner, and CRMP-2 connects Slp1 to Kinesin-1. Knockdown of these molecules by siRNA reduces the anterograde transport and membrane targeting of TrkB, thereby inhibiting BDNF-induced ERK1/2 phosphorylation in axons. Our data reveal a molecular mechanism for the selective anterograde transport of TrkB in axons and show how the transport is coupled to BDNF signaling.

Neurofibromatosis type 1 (NF1) tumor suppressor, neurofibromin, regulates the neuronal differentiation of PC12 cells via its associating protein, CRMP-2.

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.

Neuronal polarity: from extracellular signals to intracellular mechanisms.

After they are born and differentiate, neurons break their previous symmetry, dramatically change their shape, and establish two structurally and functionally distinct compartments - axons and dendrites - within one cell. How do neurons develop their morphologically and molecularly distinct compartments? Recent studies have implicated several signalling pathways evoked by extracellular signals as having essential roles in a number of aspects of neuronal polarization.

Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation.

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.

IQGAP3, a novel effector of Rac1 and Cdc42, regulates neurite outgrowth.

Rac1 and Cdc42, members of the Rho family GTPases, control diverse cellular processes such as cell migration and morphogenesis through their effectors. Among the effectors, IQGAP1 plays pivotal roles in the establishment of cytoskeletal architecture and intercellular adhesions in various cells. However, its roles remain to be clarified, especially in neuronal cells. We have identified IQGAP3 as a novel member of the IQGAP family, which is highly expressed in brain. We found that IQGAP3, an effector of Rac1 and Cdc42, associates directly with actin filaments and accumulates asymmetrically at the distal region of axons in hippocampal neurons. The depletion of IQGAP3 impairs neurite or axon outgrowth in neuronal cells with the disorganized cytoskeleton, but depletion of IQGAP1 does not. Furthermore, IQGAP3 is indispensable for Rac1/Cdc42-promoted neurite outgrowth in PC12 cells. Taken together, these results indicate that IQGAP3 can link the activation of Rac1 and Cdc42 with the cytoskeletal architectures during neuronal morphogenesis.