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.

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.

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.

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.

A method to analyze gene expression profiles from hippocampal neurons electrophysiologically recorded in vivo.

Hippocampal pyramidal neurons exhibit diverse spike patterns and gene expression profiles. However, their relationships with single neurons are not fully understood. In this study, we designed an electrophysiology-based experimental procedure to identify gene expression profiles using RNA sequencing of single hippocampal pyramidal neurons whose spike patterns were recorded in living mice. This technique involves a sequence of experiments consisting of in vivo juxtacellular recording and labeling, brain slicing, cell collection, and transcriptome analysis. We demonstrated that the expression levels of a subset of genes in individual hippocampal pyramidal neurons were significantly correlated with their spike burstiness, submillisecond-level spike rise times or spike rates, directly measured by in vivo electrophysiological recordings. Because this methodological approach can be applied across a wide range of brain regions, it is expected to contribute to studies on various neuronal heterogeneities to understand how physiological spike patterns are associated with gene expression profiles.

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.

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.

Key regulators in neuronal polarity.

Neurons are highly polarized cells, most of which develop a single axon and several dendrites. These two compartments acquire specific characteristics that enable neurons to transmit intercellular signals from several dendrites to an axon. A wealth of recent studies has shown that PI 3-kinase, Rho family GTPases, the Par complex, and cytoskeleton-related proteins participate in the initial events of neuronal polarization. Here, we review the role of polarity-regulating molecules and the potential mechanisms underlying the specification of an axon and dendrites.

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.

Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones.

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.

Signaling networks in neuronal polarization.

A mature neuron is typically polarized both structurally and functionally, with a single long axon and several dendrites. Neuronal polarity is essential for unidirectional signal flow from somata or dendrites to axons. The initial event in establishing a polarized neuron is the specification of a single axon. Early in neuronal development, one immature neurite becomes differentiated from other neurites to form an axon. Although studies in the past two decades have yielded a catalog of structural, molecular, and functional differences between axons and dendrites, we are only now beginning to understand the molecular mechanisms involved in the establishment of neuronal polarity. In the last few years, neuronal polarity-regulating molecules have been revealed. There are two major signaling cascades in neuronal polarization. Several groups, including ours, reported that the phosphatidylinositol 3-kinase (PI3-kinase)/Akt/glycogen synthase kinase-3beta (GSK-3beta)/collapsin response mediator protein-2 pathway is important for axon specification and elongation. Recent studies have revealed that the positive feedback loop composed of Rho family small GTPases and the Par3/Par6/atypical protein kinase C complex plays a role in the initial events of neuronal polarization downstream of PI3-kinase. Here, we discuss the roles of signaling molecules for axon specification.

Molecular mechanisms of axon specification and neuronal disorders.

A cardinal feature of neurons is the morphological polarity of neurons with serious functional implications. Typically, a neuron has a single axon and several dendrites. Neuronal polarity is essential for the unidirectional signal flow from somata or dendrites to axons in neurons. The initial event in establishing a polarized neuron is the specification of a single axon. Although researchers are accumulating a catalog of structural, molecular, and functional differences between axons and dendrites, we are only now beginning to understand the molecular mechanisms involved in the establishment of neuronal polarity. We have described recent advances in the understanding of cellular events in the early development of an axon and dendrites. Several groups, including ours, reported that the phosphatidylinositol 3-kinase (PI3-kinase)/Akt (also called protein kinase B)/glycogen synthase kinase-3beta (GSK-3beta)/collapsin response mediator protein-2 (CRMP-2) pathway is important for axon specification and elongation. Recent studies have revealed the roles that Rho family small GTPases, the Par complex, and cytoskeleton-related proteins play in the initial events of neuronal polarization downstream of PI3-kinase. We discuss the roles of polarity-regulating molecules and the potential mechanisms underlying the specification of an axon and dendrites. Polarity-regulating molecules participate in various neuronal disorders. In this review, the signal transduction of GSK-3beta and CRMP-2 is introduced as a new target for the treatment of Alzheimer's disease (AD) and nerve injury. These findings may help clarify causes of and treatments aimed at reversing AD and nerve injury.

[Molecular mechanisms of neuronal polarity].

Neurons are one of the most highly polarized cells known and are comprised of two structurally and functionally distinct parts, an axon and dendrites. The specification of the axon is thought to depend on its length relative to the other minor processes, which are called immature neurites. Elongation of one of immature neurites is necessary for axon specification. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axons in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. We here reviewed the molecular mechanisms of the axon formation.

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.

[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.