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Parkinson’s disease is one of the most common movement disorders and is characterized by dopaminergic cell loss and the accumulation of pathological α-synuclein, but its precise pathogenetic mechanisms remain elusive. To develop disease-modifying therapies for Parkinson’s disease, an animal model that recapitulates the pathology and symptoms of the disease, especially in the prodromal stage, is indispensable. As subjects with α-synuclein gene (SNCA) multiplication as well as point mutations develop familial Parkinson’s disease and a genome-wide association study in Parkinson’s disease has identified SNCA as a risk gene for Parkinson’s disease, the increased expression of α-synuclein is closely associated with the aetiology of Parkinson’s disease. In this study we generated bacterial artificial chromosome transgenic mice harbouring SNCA and its gene expression regulatory regions in order to maintain the native expression pattern of α-synuclein. Furthermore, to enhance the pathological properties of α-synuclein, we inserted into SNCA an A53T mutation, two single-nucleotide polymorphisms identified in a genome-wide association study in Parkinson’s disease and a Rep1 polymorphism, all of which are causal of familial Parkinson’s disease or increase the risk of sporadic Parkinson’s disease. These A53T SNCA bacterial artificial chromosome transgenic mice showed an expression pattern of human α-synuclein very similar to that of endogenous mouse α-synuclein. They expressed truncated, oligomeric and proteinase K-resistant phosphorylated forms of α-synuclein in the regions that are specifically affected in Parkinson’s disease and/or dementia with Lewy bodies, including the olfactory bulb, cerebral cortex, striatum and substantia nigra. Surprisingly, these mice exhibited rapid eye movement (REM) sleep without atonia, which is a key feature of REM sleep behaviour disorder, at as early as 5 months of age. Consistent with this observation, the REM sleep-regulating neuronal populations in the lower brainstem, including the sublaterodorsal tegmental nucleus, nuclei in the ventromedial medullary reticular formation and the pedunculopontine nuclei, expressed phosphorylated α-synuclein. In addition, they also showed hyposmia at 9 months of age, which is consistent with the significant accumulation of phosphorylated α-synuclein in the olfactory bulb. The dopaminergic neurons in the substantia nigra pars compacta degenerated, and their number was decreased in an age-dependent manner by up to 17.1% at 18 months of age compared to wild-type, although the mice did not show any related locomotor dysfunction. In conclusion, we created a novel mouse model of prodromal Parkinson’s disease that showed RBD-like behaviour and hyposmia without motor symptoms.

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

Parkinson's disease (PD) is characterized by dopaminergic (DA) cell loss and the accumulation of pathological alpha synuclein (asyn), but its precise pathomechanism remains unclear, and no appropriate animal model has yet been established. Recent studies have shown that a heterozygous mutation of glucocerebrosidase (gba) is one of the most important genetic risk factors in PD. To create mouse model for PD, we crossed asyn Bacterial Artificial Chromosome transgenic mice with gba heterozygous knockout mice. These double-mutant (dm) mice express human asyn in a physiological manner through its native promoter and showed an increase in phosphorylated asyn in the regions vulnerable to PD, such as the olfactory bulb and dorsal motor nucleus of the vagus nerve. Only dm mice showed a significant reduction in DA cells in the substantia nigra pars compacta, suggesting these animals were suitable for a prodromal model of PD. Next, we investigated the in vivo mechanism by which GBA insufficiency accelerates PD pathology, focusing on lipid metabolism. Dm mice showed an increased level of glucosylsphingosine without any noticeable accumulation of glucosylceramide, a direct substrate of GBA. In addition, the overexpression of asyn resulted in decreased GBA activity in mice, while dm mice tended to show an even further decreased level of GBA activity. In conclusion, we created a novel prodromal mouse model to study the disease pathogenesis and develop novel therapeutics for PD and also revealed the mechanism by which heterozygous gba deficiency contributes to PD through abnormal lipid metabolism under conditions of an altered asyn expression in vivo.

The aging process is accompanied by various neurophysiological changes, and the severity of neurodegenerative disorders such as Parkinson’s disease increases with aging. However, the precise neuroanatomical changes that accompany the aging process in both normal and pathological conditions remain unknown. This is in part because there is a lack of high-resolution imaging tool that has the capacity to image a desired volume of neurons in a high-throughput and automated manner. In the present study, focused ion beam/scanning electron microscopy (FIB/SEM) was used to image striatal neuropil in both wild-type (WT) mice and an A53T bacterial artificial chromosome human α-synuclein (A53T-BAC-SNCA) transgenic mouse model of Parkinson’s disease, at 1 month, 3 months, 6 months, and 22 months of age. We demonstrated that spine density gradually decreases, and average spine head volume gradually increases with age in WT mice; suggesting a homeostatic balance between spine head volume and spine density. However, this inverse relationship between spine head volume and spine density was not observed in A53T-BAC-SNCA transgenic mice. Taken together, our data suggest that Parkinson’s disease is accompanied by an abnormality in the mechanisms that control synapse growth and maturity. Significance statement Currently, the clinical diagnosis of Parkinson’s disease is based on the presence of motor symptoms. However, these symptoms only manifest after a significant proportion of dopaminergic cells have degenerated. The latent, prodromal phase is therefore of particular interest for the development of disease-modifying therapies to slow down or reverse the course of neurodegeneration. Although clinical markers to diagnose patients in the prodromal phase are gradually emerging, a structural marker that defines this phase has not yet been identified. Structural changes likely occur in the synapse before the onset of neurodegeneration. Thus, this study performed an ultrastructural analysis of synapses in a mouse model of prodromal Parkinson’s disease and revealed distinct, age-dependent structural changes.

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in labeling intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to labeling intensity. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. This imaging method enabled identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

Precise information on synapse organization in a dendrite is crucial to understanding the mechanisms underlying voltage integration and the variability in the strength of synaptic inputs across dendrites of different complex morphologies. Here, we used focused ion beam/scanning electron microscope (FIB/SEM) to image the dendritic spines of mice in the hippocampal CA1 region, CA3 region, somatosensory cortex, striatum, and cerebellum (CB). Our results show that the spine geometry and dimensions differ across neuronal cell types. Despite this difference, dendritic spines were organized in an orchestrated manner such that the postsynaptic density (PSD) area per unit length of dendrite scaled positively with the dendritic diameter in CA1 proximal stratum radiatum (PSR), cortex and CB. The ratio of the PSD area to neck length was kept relatively uniform across dendrites of different diameters in CA1 PSR. Computer simulation suggests that a similar level of synaptic strength across different dendrites in CA1 PSR enables the effective transfer of synaptic inputs from the dendrites towards soma. Excitatory postsynaptic potentials (EPSPs), evoked at single spines by glutamate uncaging and recorded at the soma, show that the neck length is more influential than head width in regulating the EPSP magnitude at the soma. Our study describes thorough morphological features and the organizational principles of dendritic spines in different brain regions. Significance statement Little is known about the characteristic anatomical features underlying the organization of spine synapses in a dendrite. This study used volume electron microscopy to make an extensive characterization of dendritic spine synapses in multiple regions of the mouse brain to uncover the principles underlying their placement along a dendritic shaft. By using a combination of approaches such as two-photon imaging, glutamate uncaging, electrophysiology, and computer simulation, we reveal the functional importance of regulated spine placement along a dendritic trunk. Our research presents a crucial step in understanding the synaptic computational principle in dendrites by highlighting the generalizable features of dendritic spine organization in a neuron.

The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Although it is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity, morphological and functional abnormalities at the neuronal circuit level because of perturbations in Golgi pH are not fully understood. In addition, morphological alteration of the Golgi apparatus is associated with several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. Here, we used anatomical and electrophysiological approaches to characterize morphological and functional abnormalities of neuronal circuits in Golgi pH regulator (GPHR) conditional knock-out mice. Purkinje cells (PCs) from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of basket cell (BC) terminals around PC soma, and electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber (CF) terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry.

SummaryCompetition between synapses contributes to activity-dependent refinement of the nervous system during development. Does local competition between neighboring synapses drive circuit remodeling during experience-dependent plasticity in the cerebral cortex? Here, we examined the role of activity-mediated competitive interactions in regulating dendritic spine structure and function on hippocampal CA1 neurons. We found that high-frequency glutamatergic stimulation at individual spines, which leads to input-specific synaptic potentiation, induces shrinkage and weakening of nearby unstimulated synapses. This heterosynaptic plasticity requires potentiation of multiple neighboring spines, suggesting that a local threshold of neural activity exists beyond which inactive synapses are punished. Notably, inhibition of calcineurin, IP3Rs, or group I metabotropic glutamate receptors (mGluRs) blocked heterosynaptic shrinkage without blocking structural potentiation, and inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) blocked structural potentiation without blocking heterosynaptic shrinkage. Our results support a model in which activity-induced shrinkage signal, and not competition for limited structural resources, drives heterosynaptic structural and functional depression during neural circuit refinement.

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.

The Kv2.1 voltage-gated K+ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity-dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+ -release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy-immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR-mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca2+ /calcineurin-dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell- and circuit-specific mechanism for coupling intracellular Ca2+ release to phosphorylation-dependent regulation of Kv2.1 to dynamically impact intrinsic excitability.

R-type calcium channels (RTCCs) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, we characterized the regional and subcellular localization of Cav2.3 in mice and rats at both light and electron microscopic levels. Cav2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique, or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained 3spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Cav2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites.

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 neuronsin situand 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 STATEMENTNeurons 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 neuronsin situand in heterologous cellsin vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.

My thesis deals with the localization of T-type calcium channel are R-type calcium channel in the brain. T-type calcium channels play a pivotal role in regulating neural membrane excitability. However, the precise subcellular distributions of T-type channel subunits are not well understood. Here, I investigated the subcellular distribution of the α1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the α1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the α1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited gradient such that the labeling density was the highest in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about three-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type specific expression levels and for uniform expression density of the α1G subunit over the plasma membrane of dLGN cells.R-type calcium channels (RTCC) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, I characterized the regional and subcellular localization of Cav2.3 at both light and electron microscopic levels. Cav2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained < 20 immunogold particles, with an average density of  260 immunoparticles per µm3 spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Cav2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites.