Takeshi Kameyama, Muneaki Miyata, Hajime Shiotani, Jun Adachi, Soichiro Kakuta, Yasuo Uchiyama, Kiyohito Mizutani and Yoshimi Takai : Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain., iScience, Vol.26, No.10, 2023.
(Summary)
Astrocytes interact with not only synapses but also brain blood vessels through perivascular astrocyte endfeet (PV-AEF) to form the neurovascular unit (NVU). However, PV-AEF components have not been fully identified. Here, we biochemically isolated blood vessels from mouse brain homogenates and purified PV-AEF. The purified PV-AEF were observed in different sizes, similar to PV-AEF on brain blood vessels. Mass spectrometry analysis identified 9,762 proteins in the purified PV-AEF, including cell adhesion molecules, nectin-2δ, Kirrel2, and podoplanin. Immunofluorescence microscopic analysis revealed that nectin-2δ and podoplanin were concentrated mainly in arteries/arterioles and veins/venules of the mouse brain, whereas Kirrel2 was mainly in arteries/arterioles. Nectin-2α/δ, Kirrel2, and podoplanin were preferentially observed in large sizes of the purified PV-AEF. Furthermore, Kirrel2 potentially has cell adhesion activity of cultured astrocytes. Collectively, these results indicate that PV-AEF have heterogeneity in sizes and molecular components, implying different roles of PV-AEF in NVU function depending on vascular regions.
Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.
Tomohiko Maruo, Kiyohito Mizutani, Muneaki Miyata, Toshihiko Kuriu, Shotaro Sakakibara, Hatena Takahashi, Daichi Kida, Kouki Maesaka, Tsukiko Sugaya, Ayuko Sakane, Takuya Sasaki, Yoshimi Takai and Kenji Mandai : s-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons., The Journal of Biological Chemistry, Vol.299, No.4, 2023.
(Summary)
A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin in vivo and in vitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.
Hajime Shiotani, Muneaki Miyata, Takeshi Kameyama, Kenji Mandai, Miwako Yamasaki, Masahiko Watanabe, Kiyohito Mizutani and Yoshimi Takai : Nectin-2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula., The Journal of Comparative Neurology, Vol.529, No.2, 450-477, 2020.
(Summary)
The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and β-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, β-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.
Hajime Shiotani, Muneaki Miyata, Kiyohito Mizutani, Shujie Wang, Akira Mizoguchi, Hideki Mochizuki, Kenji Mandai and Yoshimi Takai : Interaction of nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula, Molecular and Cellular Neuroscience, Vol.94, 32-40, 2018.
(Summary)
The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.
Hajime Shiotani, Muneaki Miyata, Yu Itoh, Shujie Wang, Aika Kaito, Akira Mizoguchi, Miwako Yamasaki, Masahiko Watanabe, Kenji Mandai, Hideki Mochizuki and Yoshimi Takai : Localization of nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage-gated A-type K+ channel Kv4.2 in the medial habenula, The Journal of Comparative Neurology, Vol.526, No.9, 1527-1549, 2018.
(Summary)
The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin-like cell adhesion molecules (CAMs) nectin-2α and nectin-2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin-2α, but not nectin-2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin-2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin-2α at this boundary was not colocalized with the nectin-2α-binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin-2α forms a cell adhesion apparatus different from the Kv4.2-associated MSs. Genetic ablation of nectin-2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin-2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.
Hajime Shiotani, Tomohiko Maruo, Shotaro Sakakibara, Muneaki Miyata, Kenji Mandai, Hideki Mochizuki and Yoshimi Takai : Aging-dependent expression of synapse-related proteins in the mouse brain., Genes to Cells, Vol.22, No.5, 472-484, 2017.
(Summary)
A synapse is a cell adhesion structure that permits a neuron to pass a chemical or electrical signal to another neuron. They connect neurons and form neural networks that are essential for brain functions, such as learning and memory. At a chemical synapse, the presynapse and the postsynapse are connected by cell adhesion molecules. The presynapse contains synaptic vesicles and their release machinery, whereas the postsynapse contains postsynaptic densities and receptors for the neurotransmitters. Many proteins constituting a synapse have been identified, but their life-span expression profiles remain elusive. Here, we investigated the expression levels of representative synapse-related proteins by Western blot using the extranuclear supernatant fraction of the brains of mice at various ages. These proteins were classified into seven groups depending on their expression profiles during the embryonic stage, those from postnatal day 6 (P6) to P30, and those after P90. The expression levels of the majority of the proteins were gradually increased from the embryonic stage and then decreased at P14 or P30. After P90, the expression levels were not markedly changed or, in some proteins, increased. These results indicate that the expression levels of the synapse-related proteins are regulated orderly in an aging-dependent manner.
Muneaki Miyata, Kenji Mandai, Tomohiko Maruo, Junya Sato, Hajime Shiotani, Aika Kaito, Yu Itoh, Shujie Wang, Takeshi Fujiwara, Akira Mizoguchi, Yoshimi Takai and Yoshiyuki Rikitake : Localization of nectin-2δ at perivascular astrocytic endfoot processes and degeneration of astrocytes and neurons in nectin-2 knockout mouse brain., Brain Research, Vol.1649, No.Pt A, 90-101, 2016.
(Summary)
Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules. In the nervous system, among four members (nectin-1, -2, -3, and -4), nectin-1 and -3 are asymmetrically localized at puncta adherentia junctions formed between the mossy fiber terminals and the dendrites of CA3 pyramidal neurons in the mouse hippocampus and heterophilic trans-interactions between nectin-1 and nectin-3 are involved in the selective interaction of axons and dendrites of cultured neurons. By contrast, nectin-2, which has two splicing variants, nectin-2α and -2δ, has not been well characterized in the brain. We showed here that nectin-2α was expressed in both cultured mouse neurons and astrocytes whereas nectin-2δ was selectively expressed in the astrocytes. Nectin-2δ was localized at the adhesion sites between adjacent cultured astrocytes, but in the brain it was localized on the plasma membranes of astrocytic perivascular endfoot processes facing the basement membrane of blood vessels. Genetic ablation of nectin-2 caused degeneration of astrocytic perivascular endfoot processes and neurons in the cerebral cortex. These results uncovered for the first time the localization and critical functions of nectin-2 in the brain.
Kiyohito Mizutani, Muneaki Miyata, Hajime Shiotani, Takeshi Kameyama and Yoshimi Takai : Nectin-2 in general and in the brain., Molecular and Cellular Biochemistry, Vol.477, No.1, 167-180, Oct. 2021.
(Summary)
Nectins are immunoglobulin-like cell adhesion molecules constituting a family with four members, nectin-1, nectin-2, nectin-3, and nectin-4. In the brain, nectin-2 as well as nectin-1 and nectin-3 are expressed whereas nectin-4 is hardly expressed. In the nervous system, physiological functions of nectin-1 and nectin-3, such as synapse formation, mossy fiber trajectory regulation, interneurite affinity, contextual fear memory formation, and stress-related mental disorders, have been revealed. Nectin-2 is ubiquitously expressed in non-neuronal tissues and various nectin-2 functions in non-nervous systems have been extensively investigated, but nectin-2 functions in the brain have not been revealed until recently. Recent findings have revealed that nectin-2 is expressed in the specific areas of the brain and plays important roles, such as homeostasis of astrocytes and neurons and the formation of synapses. Moreover, a single nucleotide polymorphism in the human NECTIN2 gene is associated with Alzheimer's disease. We here summarize recent progress in our understanding of nectin-2 functions in the brain.
Kiyohito Mizutani, Muneaki Miyata, Hajime Shiotani, Takeshi Kameyama and Yoshimi Takai : Nectins and Nectin-like molecules in synapse formation and involvement in neurological diseases., Molecular and Cellular Neuroscience, Vol.115, Jul. 2021.
(Summary)
Synapses are interneuronal junctions which form neuronal networks and play roles in a variety of functions, including learning and memory. Two types of junctions, synaptic junctions (SJs) and puncta adherentia junctions (PAJs), have been identified. SJs are found at all excitatory and inhibitory synapses whereas PAJs are found at excitatory synapses, but not inhibitory synapses, and particularly well developed at hippocampal mossy fiber giant excitatory synapses. Both SJs and PAJs are mediated by cell adhesion molecules (CAMs). Major CAMs at SJs are neuroligins-neurexins and Nectin-like molecules (Necls)/CADMs/SynCAMs whereas those at PAJs are nectins and cadherins. In addition to synaptic PAJs, extrasynaptic PAJs have been identified at contact sites between neighboring dendrites near synapses and regulate synapse formation. In addition to SJs and PAJs, a new type of cell adhesion apparatus different from these junctional apparatuses has been identified and named nectin/Necl spots. One nectin spot at contact sites between neighboring dendrites at extrasynaptic regions near synapses regulates synapse formation. Several members of nectins and Necls had been identified as viral receptors before finding their physiological functions as CAMs and evidence is accumulating that many nectins and Necls are related to onset and progression of neurological diseases. We review here nectin and Necls in synapse formation and involvement in neurological diseases.