Active Transport by Cytoplasmic Dynein Maintains the Localization of MAP2 in Developing Neurons.

MAP2 has been widely used as a marker of neuronal dendrites because of its extensive restriction in the somatodendritic region of neurons. Despite that, how the precise localization of such a soluble protein is established and maintained against thermal forces and diffusion has been elusive and long remained a mystery in neuroscience. In this study, we aimed to uncover the mechanism behind how MAP2 is retained in the somatodendritic region. Using GFP-tagged MAP2 expressed in cultured hippocampal neurons, we discovered a crucial protein region responsible for the localization of MAP2, the serine/proline-rich (S/P) region. Our pulse-chase live-cell imaging revealed the slow but steady migration of MAP2 toward distal dendrites, which was not observed in a MAP2 mutant lacking the S/P region, indicating that S/P-dependent transport is vital for the proper localization of MAP2. Furthermore, our experiments using an inhibitor of cytoplasmic Dynein, ciliobrevin D, as well as Dynein knockdown, showed that cytoplasmic Dynein is involved in the transport of MAP2 in dendrites. We also found that Dynein complex binds to MAP2 through the S/P region in heterologous cells. Using mathematical modeling based on experimental data, we confirmed that an intermittent active transport mechanism is essential. Thus, we propose that the cytoplasmic Dynein recruits and transports free MAP2 toward distal dendrites, thereby maintaining the precise dendritic localization of MAP2 in neurons. Our findings shed light on the previously unknown mechanism behind MAP2 localization and provide a new direction for soluble protein trafficking research in the field of cell biology of neurons.

Identification of Tau protein as a novel marker for maturation and pathological changes of oligodendrocytes.

Microtubule-associated protein Tau is primarily expressed in axons of neurons, but also in Olig2-positive oligodendrocytes in adult rodent and monkey brains. In this study, we sought to determine at what cell stage Tau becomes expressed in the oligodendrocyte lineage. We performed immunostaining of adult mouse brain sections using well-known markers of oligodendrocyte lineage and found that Tau is expressed in mature oligodendrocytes, but not in oligodendrocyte progenitors and immature pre-oligodendrocytes. We also investigated Tau expression in developing mouse brain. Surprisingly, Tau expression occurred after the peak of myelination and even exceeded GSTπ expression, which has been considered as a marker of myelinating oligodendrocytes. These results suggest Tau as a novel marker of oligodendrocyte maturation. We then investigated whether Tau is important for oligodendrocyte development and/or myelination and how Tau changes in demyelination. First, we found no changes in myelination and oligodendrocyte markers in Tau knockout mice, suggesting that Tau is dispensable. Next, we analyzed the proteolipid protein 1 transgenic model of Pelizaeus-Merzbacher disease, which is a rare leukodystrophy. In hemizygous transgenic mice, the number of Tau-positive cells were significantly increased as compared with wild type mice. These cells were also positive for Olig2, CC1, and GSTπ, but not PDGFRα and GPR17. In stark contrast, the expression level of Tau, as well as GSTπ, was dramatically decreased in the cuprizone-induced model of multiple sclerosis. Taken together, we propose Tau as a new marker of oligodendrocyte lineage and for investigating demyelination lesions.

The organization of microtubules and Tau in oligodendrocytes: Tau pathology in damaged oligodendrocytes.

Tau is abundantly expressed in neurons, however previous reports and our recent study showed tau also exist in oligodendrocytes. Also the expression levels of tau are dramatical changed in hypomyelination model rat and in demyelination region of stroke model mice. The review demonstrate microtubule and its binding partner Tau might be necessary for oligodendrocyte function based on previous reports.

Inhibition of microtubule assembly competent tubulin synthesis leads to accumulation of phosphorylated tau in neuronal cell bodies.

Neurofibrillary tangles, a pathological hallmark of Alzheimer's disease (AD), are somatodendritic filamentous inclusions composed of hyperphosphorylated tau. Microtubule loss is also a common feature of affected neurons in AD. However, whether and how the disruptions of microtubules and the microtubule-associated proteins occur in the pathogenesis of AD remain unclear. Recent evidence indicates that reduced expression of tubulin by knocking down a tubulin chaperon can cause tau neurotoxicity. Thus, the disruption of tubulin homeostasis may result in the acquisition of tau pathogenesis and ultimately cause tauopathy. To investigate whether the disruption of tubulin maintenance induces tau abnormalities in mammalian neurons, we developed a miRNA-mediated knockdown system of tubulin-specific chaperon E (Tbce), which is a factor required for the de novo synthesis of tubulin. Tbce knockdown in mouse primary cultured neurons induced an increase in tubulin in the cell body at 14 days in vitro. Accumulated tubulin was not acetylated or incorporated in microtubules, indicating that they were functionally inert. Concomitantly, tau also accumulated in neuronal cell bodies. The mis-localized tau was phosphorylated at Ser202/Thr205 and Ser396/Ser404. These results indicate that Tbce knockdown in mammalian neurons induces not only a reduction in properly folded tubulins, which are microtubule assembly competent, but also an accumulation of phosphorylated tau in the cell body of mammalian neurons. These findings suggest that disruption of the homeostatic mechanism for maintaining tubulin biosynthesis and/or microtubules can cause tau accumulation in the cell body, which is commonly observed in tauopathies.

Regulatory mechanisms for the axonal localization of tau protein in neurons.

Tau is a microtubule (MT)-associated protein that is thought to be localized to the axon. However, its precise localization in developing neurons and mechanisms for the axonal localization have not been fully addressed. In this study, we found that the axonal localization of tau in cultured rat hippocampal neurons mainly occur during early neuronal development. Interestingly, transient expression of human tau in very immature neurons, but not in mature neurons, mimicked the developmental localization of endogenous tau to the axon. We therefore were able to establish an experimental model, in which exogenously expressed tau can be properly localized to the axon. Using this model, we obtained a surprising finding that the axonal localization of tau did not require stable MT binding. Tau lacking the MT-binding domain (MTBD) exhibited high diffusivity but localized properly to the axon. In contrast, a dephosphorylation-mimetic mutant of the proline-rich region 2 showed reinforced MT binding and mislocalization. Our results suggest that tight binding to MTs prevents tau from entering the axon and results in mislocalization in the soma and dendrites when expressed in mature neurons. This study therefore provides a novel mechanism independent of MTBD for the axonal localization of tau.

Ectopic Expression Induces Abnormal Somatodendritic Distribution of Tau in the Mouse Brain.

Tau is a microtubule (MT)-associated protein that is localized to the axon. In Alzheimer's disease, the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. To investigate how this mislocalization occurs, we recently developed immunohistochemical tools that can separately detect endogenous mouse and exogenous human tau with high sensitivity, which allows us to visualize not only the pathological but also the pre-aggregated tau in mouse brain tissues of both sexes. Using these antibodies, we found that in tau-transgenic mouse brains, exogenous human tau was abundant in dendrites and somata even in the presymptomatic period, whereas the axonal localization of endogenous mouse tau was unaffected. In stark contrast, exogenous tau was properly localized to the axon in human tau knock-in mice. We tracked this difference to the temporal expression patterns of tau. Endogenous mouse tau and exogenous human tau in human tau knock-in mice exhibited high expression levels during the neonatal period and strong suppression into the adulthood. However, human tau in transgenic mice was expressed continuously and at high levels in adult animals. These results indicated the uncontrolled expression of exogenous tau beyond the developmental period as a cause of mislocalization in the transgenic mice. Superresolution microscopic and biochemical analyses also indicated that the interaction between MTs and exogenous tau was impaired only in the tau-transgenic mice, but not in knock-in mice. Thus, the ectopic expression of tau may be critical for its somatodendritic mislocalization, a key step of the tauopathy.SIGNIFICANCE STATEMENT Somatodendritic localization of tau may be an early step leading to the neuronal degeneration in tauopathies. However, the mechanisms of the normal axonal distribution of tau and the mislocalization of pathological tau remain obscure. Our immunohistochemical and biochemical analyses demonstrated that the endogenous mouse tau is transiently expressed in neonatal brains, that exogenous human tau expressed corresponding to such tau expression profile can distribute into the axon, and that the constitutive expression of tau into adulthood (e.g., human tau in transgenic mice) results in abnormal somatodendritic localization. Thus, the expression profile of tau is tightly associated with the localization of tau, and the ectopic expression of tau in matured neurons may be involved in the pathogenesis of tauopathy.

Distribution of endogenous normal tau in the mouse brain.

Tau is a microtubule-associated protein (MAP) that is localized to the axon. In Alzheimer's disease (AD), the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. While the abnormal aggregated tau has been extensively studied in human patient tissues and animal models of AD, how normal tau localizes to the axon, which would be the foundation to understand how the mis-localization occurs, has not been well studied due to the poor detectability of normal unaggregated tau in vivo. Therefore, we developed immunohistochemical techniques that can detect normal mouse and human tau in brain tissues with high sensitivity. Using these techniques, we demonstrate the global distribution of tau in the mouse brain and confirmed that normal tau is exclusively localized to the axonal compartment in vivo. Interestingly, tau antibodies strongly labeled nonmyelinated axons such as hippocampal mossy fibers, while white matters generally exhibited low levels of immunoreactivity. Furthermore, mouse tau is highly expressed not only in neurons but also in oligodendrocytes. With super resolution imaging using the stimulated-depletion microscopy, axonal tau appeared punctate rather than fibrous, indicating that tau decorates microtubules sparsely. Co-labeling with presynaptic and postsynaptic markers revealed that normal tau is not localized to synapses but sparsely distributes in the axon. Taken together, this study reports novel antibodies to investigate the localization and mis-localization of tau in vivo and novel findings of normal tau localization in the mouse brain.

Perineuronal Nets in the Deep Cerebellar Nuclei Regulate GABAergic Transmission and Delay Eyeblink Conditioning.

Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum.SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions.

Precise localizations of voltage-gated sodium and potassium channels in neurons.

Neurons are extremely large and complex cells, and they regulate membrane potentials in multiple subcellular compartments using a variety of ion channels. Voltage-gated sodium (Nav) and potassium (Kv) channels are crucial in regulating neuronal membrane excitability owing to their diversity in subtypes, biophysical properties, and localizations. In particular, specific localizations of Nav and Kv channels in specific membrane compartments are essential to achieve a precise control of local membrane excitability. Recent advancement in super-resolution microscopy further substantiated nanoscale localizations of different ion channels in neuronal membranes. New questions arise from these new lines of evidence regarding how Nav and Kv channels are trafficked to a specific location and maintained against lateral diffusion. In this review, the aim is to summarize current information about ion channel localizations at nanoscopic levels and discuss what we can infer regarding the mechanisms. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 271-282, 2018.

Trafficking of Kv2.1 Channels to the Axon Initial Segment by a Novel Nonconventional Secretory Pathway.

Kv2.1 is a major delayed-rectifier voltage-gated potassium channel widely expressed in neurons of the CNS. Kv2.1 localizes in high-density cell-surface clusters in the soma and proximal dendrites as well as in the axon initial segment (AIS). Given the crucial roles of both of these compartments in integrating signal input and then generating output, this localization of Kv2.1 is ideal for regulating the overall excitability of neurons. Here we used fluorescence recovery after photobleaching imaging, mutagenesis, and pharmacological interventions to investigate the molecular mechanisms that control the localization of Kv2.1 in these two different membrane compartments in cultured rat hippocampal neurons of mixed sex. Our data uncover a unique ability of Kv2.1 channels to use two molecularly distinct trafficking pathways to accomplish this. Somatodendritic Kv2.1 channels are targeted by the conventional secretory pathway, whereas axonal Kv2.1 channels are targeted by a nonconventional trafficking pathway independent of the Golgi apparatus. We further identified a new AIS trafficking motif in the C-terminus of Kv2.1, and show that putative phosphorylation sites in this region are critical for the restricted and clustered localization in the AIS. These results indicate that neurons can regulate the expression and clustering of Kv2.1 in different membrane domains independently by using two distinct localization mechanisms, which would allow neurons to precisely control local membrane excitability.SIGNIFICANCE STATEMENT Our study uncovered a novel mechanism that targets the Kv2.1 voltage-gated potassium channel to two distinct trafficking pathways and two distinct subcellular destinations: the somatodendritic plasma membrane and that of the axon initial segment. We also identified a distinct motif, including putative phosphorylation sites, that is important for the AIS localization. This raises the possibility that the destination of a channel protein can be dynamically regulated via changes in post-translational modification, which would impact the excitability of specific membrane compartments.

Distinct Cell- and Layer-Specific Expression Patterns and Independent Regulation of Kv2 Channel Subtypes in Cortical Pyramidal Neurons.

The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K(+) current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT: Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.

BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells.

In myelinated axons, K(+) channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na(+) channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K(+) channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K(+) channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni(2+) elicited a similar effect on APs, indicating the involvement of Ni(2+)-sensitive Ca(2+) channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex.

Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility.

BACKGROUND: Identifying cellular signaling pathways that become corrupted in the presence of androgens that increase the metastatic potential of organ-confined tumor cells is critical to devising strategies capable of attenuating the metastatic progression of hormone-naïve, organ-confined tumors. In localized prostate cancers, gene fusions that place ETS-family transcription factors under the control of androgens drive gene expression programs that increase the invasiveness of organ-confined tumor cells. C-X-C chemokine receptor type 4 (CXCR4) is a downstream target of ERG, whose upregulation in prostate-tumor cells contributes to their migration from the prostate gland. Recent evidence suggests that CXCR4-mediated proliferation and metastasis of tumor cells is regulated by CXCR7 through its scavenging of chemokine CXCL12. However, the role of androgens in regulating CXCR4-mediated motility with respect to CXCR7 function in prostate-cancer cells remains unclear. METHODS: Immunocytochemistry, western blot, and affinity-purification analyses were used to study how androgens influenced the expression, subcellular localization, and function of CXCR7, CXCR4, and androgen receptor (AR) in LNCaP prostate-tumor cells. Moreover, luciferase assays and quantitative polymerase chain reaction (qPCR) were used to study how chemokines CXCL11 and CXCL12 regulate androgen-regulated genes (ARGs) in LNCaP prostate-tumor cells. Lastly, cell motility assays were carried out to determine how androgens influenced CXCR4-dependent motility through CXCL12. RESULTS: Here we show that, in the LNCaP prostate-tumor cell line, androgens coordinate the expression of CXCR4 and CXCR7, thereby promoting CXCL12/CXCR4-mediated cell motility. RNA interference experiments revealed functional interactions between AR and CXCR7 in these cells. Co-localization and affinity-purification experiments support a physical interaction between AR and CXCR7 in LNCaP cells. Unexpectedly, CXCR7 resided in the nuclear compartment and modulated AR-mediated transcription. Moreover, androgen-mediated cell motility correlated positively with the co-localization of CXCR4 and CXCR7 receptors, suggesting that cell migration may be linked to functional CXCR4/CXCR7 heterodimers. Lastly, CXCL12-mediated cell motility was CXCR7-dependent, with CXCR7 expression required for optimal expression of CXCR4 protein. CONCLUSIONS: Overall, our results suggest that inhibition of CXCR7 function might decrease the metastatic potential of organ-confined prostate cancers.

Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons.

The subcellular localization of neuronal membrane signaling molecules such as receptors and ion channels depends on intracellular trafficking mechanisms. Essentially, vesicular trafficking mechanisms ensure that a large number of membrane proteins are correctly targeted to different subcellular compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish the several different routes that neurons use for protein trafficking. Here we provide a novel protocol to selectively visualize post-Golgi transport vesicles carrying fluorescent-labeled ion channel proteins in living neurons. Further, we provide a number of analytical tools we developed to quantify characteristics of different types of transport vesicles. We demonstrate the application of our protocol to investigate whether ion channels are sorted into distinct vesicular populations at the Golgi apparatus. We also demonstrate how these techniques are suitable for pharmacological dissection of the transport mechanisms by which post-Golgi vesicles are trafficked in neurons. Our protocol uniquely combines the classic temperature-block with close monitoring of the transient expression of transfected protein tagged with fluorescent proteins, and provides a quick and easy way to study protein trafficking in living neurons. We believe that the procedures described here are useful for researchers who are interested in studying molecular mechanisms of protein trafficking in neurons.

Specific sorting and post-Golgi trafficking of dendritic potassium channels in living neurons.

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Kv2.2: a novel molecular target to study the role of basal forebrain GABAergic neurons in the sleep-wake cycle.

STUDY OBJECTIVES: The basal forebrain (BF) has been implicated as an important brain region that regulates the sleep-wake cycle of animals. Gamma-aminobutyric acidergic (GABAergic) neurons are the most predominant neuronal population within this region. However, due to the lack of specific molecular tools, the roles of the BF GABAergic neurons have not been fully elucidated. Previously, we have found high expression levels of the Kv2.2 voltage-gated potassium channel on approximately 60% of GABAergic neurons in the magnocellular preoptic area and horizontal limb of the diagonal band of Broca of the BF and therefore proposed it as a potential molecular target to study this neuronal population. In this study, we sought to determine the functional roles of the Kv2.2-expressing neurons in the regulation of the sleep-wake cycle. DESIGN: Sleep analysis between two genotypes and within each genotype before and after sleep deprivation. SETTING: Animal sleep research laboratory. PARTICIPANTS: Adult mice. Wild-type and Kv2.2 knockout mice with C57/BL6 background. INTERVENTIONS: EEG/EMG recordings from the basal state and after sleep-deprivation which was induced by mild agitation for 6 h. RESULTS: Immunostaining of a marker of neuronal activity indicates that these Kv2.2-expressing neurons appear to be preferentially active during the wake state. Therefore, we tested whether Kv2.2-expressing neurons in the BF are involved in arousal using Kv2.2-deficient mice. BF GABAergic neurons exhibited augmented expression of c-Fos. These knockout mice exhibited longer consolidated wake bouts than wild-type littermates, and that phenotype was further exacerbated by sleep deprivation. Moreover, in-depth analyses of their cortical electroencephalogram revealed a significant decrease in the delta-frequency activity during the nonrapid eye movement sleep state. CONCLUSIONS: These results revealed the significance of Kv2.2-expressing neurons in the regulation of the sleep-wake cycle.

Neuronal trafficking of voltage-gated potassium channels.

The computational ability of CNS neurons depends critically on the specific localization of ion channels in the somatodendritic and axonal membranes. Neuronal dendrites receive synaptic inputs at numerous spines and integrate them in time and space. The integration of synaptic potentials is regulated by voltage-gated potassium (Kv) channels, such as Kv4.2, which are specifically localized in the dendritic membrane. The synaptic potentials eventually depolarize the membrane of the axon initial segment, thereby activating voltage-gated sodium channels to generate action potentials. Specific Kv channels localized in the axon initial segment, such as Kv1 and Kv7 channels, determine the shape and the rate of action potentials. Kv1 and Kv7 channels present at or near nodes of Ranvier and in presynaptic terminals also influence the propagation of action potentials and neurotransmitter release. The physiological significance of proper Kv channel localization is emphasized by the fact that defects in the trafficking of Kv channels are observed in several neurological disorders including epilepsy. In this review, we will summarize the current understanding of the mechanisms of Kv channel trafficking and discuss how they contribute to the establishment and maintenance of the specific localization of Kv channels in neurons.

Immunolocalization of the voltage-gated potassium channel Kv2.2 in GABAergic neurons in the basal forebrain of rats and mice.

The Kv2 voltage-gated potassium channels, Kv2.1 and Kv2.2, are important regulators of neuronal excitability in mammalian brain. It has been shown that Kv2.1 channels are expressed in virtually all neurons in the brain. However, the cellular localization of Kv2.2 has not been fully elucidated. In this article we report that Kv2.2 is highly expressed in a subset of neurons in the magnocellular preoptic nucleus (MCPO) and the horizontal limb of the diagonal band of Broca (HDB) of the basal forebrain complex, which are areas highly implicated in the regulation of cortical activity and the sleep/wake cycle. It has been shown that MCPO and HDB contain distinct populations of neurons that differ in their neurochemicals, cholinergic, glutamatergic, and gamma-aminobutyric acid (GABA)ergic neurons. Using specific immunolabeling and knockin mice in which green fluorescent protein (GFP) is expressed in GABAergic neurons, we found that Kv2.2 is abundantly expressed in a large subpopulation of the GABAergic neurons in the MCPO and HDB. These data offer Kv2.2 as a molecular target to study the role of the specific subpopulation of basal forebrain GABAergic neurons.

Formation of heteromeric Kv2 channels in mammalian brain neurons.

The formation of heteromeric tetramers is a common feature of voltage-gated potassium (Kv) channels. This results in the generation of a variety of tetrameric Kv channels that exhibit distinct biophysical and biochemical characteristics. Kv2 delayed rectifier channels are, however, unique exceptions. It has been previously shown that mammalian Kv2.1 and Kv2.2 are localized in distinct domains of neuronal membranes and are not capable of forming heteromeric channels with each other (Hwang, P. M., Glatt, C. E., Bredt, D. S., Yellen, G., and Snyder, S. H. (1992) Neuron 8, 473-481). In this study, we report a novel form of rat Kv2.2, Kv2.2(long), which has not been previously recognized. Our data indicate that Kv2.2(long) is the predominant form of Kv2.2 expressed in cortical pyramidal neurons. In contrast to the previous findings, we also found that rat Kv2.1 and Kv2.2(long) are colocalized in the somata and proximal dendrites of cortical pyramidal neurons and are capable of forming functional heteromeric delayed rectifier channels. Our results suggest that the delayed rectifier currents, which regulate action potential firing, are encoded by heteromeric Kv2 channels in cortical neurons.

Homeostatic regulation of neuronal excitability by K(+) channels in normal and diseased brains.

K(+)-selective ion channels are critical determinants of membrane excitability in neuronal cells. Like many other cells in our body, neuronal cells have a propensity to maintain their homeostasis. Action potential firing is the most important function to maintain in brain neurons, as they are the elements of neural networks. If one element fires action potentials at an abnormally high rate, the entire network could become epileptic. Therefore, brain neurons adjust their intrinsic membrane excitability to maintain the firing rate within their own optimal operational range. When a neuron receives an enormous input, it will reduce the membrane excitability to prevent overshooting. When it is deprived of stimulus, the membrane becomes more excitable to avoid total quiescence. The homeostatic regulation of intrinsic excitability provides stability to the neural network in the face of dynamic and plastic synaptic inputs. In the past decade, we have learned that neurons achieve this type of homeostatic regulation through a variety of ion channels, including K(+) channels. It has also become clear that under certain pathological conditions, these homeostatic mechanisms provide neuroprotection. In this article, I will review recent advances in our understanding of K(+) channel-mediated homeostatic regulation of neuronal excitability and discuss involvement of these channels in hyperexcitable diseases where they provide neuroprotection.