Biology subjects

Kaneko, R.

Publications and source records attributed to Kaneko, R..

8 recordsLinked to original sources

bioRxiv2025

A compact glutamic acid decarboxylase 67 promoter enables inhibitory neuron-targeted AAV gene therapy for treatment-resistant epilepsy

Epilepsy often becomes treatment-resistant, partly due to impaired inhibitory neurotransmission and reduced {gamma}-aminobutyric acid (GABA) function. Enhancing inhibitory neuron activity via gene therapy may restore excitation-inhibition (E/I) balance. We developed a compact 410-bp glutamic acid decarboxylase 67 promoter (cmGAD67) that enables strong, selective transgene expression in inhibitory neurons while preserving AAV packaging capacity. When delivered systemically, AAV vectors carrying cmGAD67 preferentially targeted parvalbumin interneurons and supported effective circuit manipulation. To evaluate therapeutic potential, we expressed glutamic acid decarboxylase 65 (GAD65) under cmGAD67 (AAV-GAD65) in pentylenetetrazole (PTZ) epilepsy models. Systemic AAV-GAD65 suppressed abnormal delta oscillations, reduced seizure-like events, normalized anxiety-like behavior, and improved survival in a severe PTZ paradigm. Biochemical analyses confirmed increased cortical and hippocampal GABA levels, linking behavioral and electrophysiological improvements to enhanced inhibitory neurotransmitter synthesis. Prior clinical evidence indicates that AAV-GAD65 delivery to the subthalamic nucleus is safe and effective in Parkinsons disease. Building on this foundation, our findings establish the cmGAD67 promoter as a powerful platform for inhibitory neuron-targeted AAV gene therapy and highlight AAV-cmGAD67-GAD65 as a promising approach for treatment-resistant epilepsy and other disorders involving disrupted E/I balance.

neuroscience↗

bioRxiv2025

Reward-reset interval timing drives patch foraging decisions through neural state transitions in dorsomedial striatum

Activities with temporally distributed returns require deciding not only what to do, but when to stop. Although optimal foraging theory predicts that patch departure should depend on the total time invested in a patch, behavior across species often reflects sensitivity to recent rewards. The dorsomedial striatum (DMS) mediates both goal-directed decision-making and interval timing, two functions that converge during patch foraging, where animals must decide when to exit patches to maximize reward rates. We recorded extracellular activity from neurons in DMS while freely moving mice performed a patch-foraging task. Mice employed a reward-reset strategy, primarily basing their exit decisions on the time since the last reward, with systematic adjustments for patch residence time and environmental reward-rate context. Individual neurons in DMS underwent discrete firing rate transitions at characteristic delays following each reward. These transition times tiled the post-reward interval across the population, producing an accumulation-to-threshold signal that reset with each reward and whose rate was sensitive to the cost of time, increasing with higher environmental reward rates and as rewards occurred later in the patch. This accumulation reached a consistent threshold at the moment of patch exit, encoding the animals intended stopping time. Together, these results identify a time-cost-variable, reward-reset, accumulation-to-threshold computation in DMS that integrates environmental reward rate, elapsed time from pursuit engagement, and elapsed time from reward occurrence to determine when to abandon a depleting pursuit.

neuroscience↗

bioRxiv2025

Distribution of myogenic stem cell activator, hepatocyte growth factor, in skeletal muscle extracellular matrix and effect of short-term disuse and reloading

Hepatocyte growth factor (HGF) is a key myogenic stem cells (satellite cells) activator, that resides in the extracellular matrix (ECM). However, HGF distribution in the ECM varies depending on the muscle fiber type. Furthermore, aging impedes the binding of HGF to its receptors due to nitration by peroxynitrite (ONOO-). Though oxidative stress increases rapidly during muscle disuse atrophy, satellite cells are rapidly activated upon reloading. In this study, we investigated the distribution of HGF in the ECM in various muscle fiber types, and examined nitration of HGF in disuse and reloading models. Immunofluorescence staining was performed on the soleus (Sol), plantaris (Pla), and gastrocnemius (Gas) muscles of 10-week-old mice. Three mice were used to assess HGF distribution, while 12 mice, divided into control, disuse, and reloading groups were used for qualitative evaluation of nitrated HGF (nitroHGF). In Sol muscle, type IIa and IIx muscle fibers exhibited higher HGF distribution in the ECM (70.7{+/-}1.9% and 63.9{+/-}2.6%, respectively) than type I fibers (28.0{+/-}1.7%; p<0.001). In Pla and Gas muscle, type IIa (65.4{+/-}2.3% and 70.3{+/-}2.2%, respectively) and type IIx fibers (55.3{+/-}1.8% and 61.4{+/-}3.2%, respectively) had significantly higher HGF distribution in the ECM than type IIb fibers (13.1{+/-}1.8% and 5.9{+/-}1.6%; p<0.001, respectively). The amount of nitroHGF increased in the disuse group compared to that in the control group but decreased in the reloading group compared to that in the disuse group. This preferential HGF distribution around type IIa and IIx muscle fibers indicates a distinct mechanism for satellite cell activation, differing from the satellite cell-rich environment associated with type I fibers and the lower HGF association with type IIb fibers. Disuse-induced HGF nitration may inhibit satellite cell activation. Reloading likely triggers mechanisms that counteract nitration, enabling satellite cell reactivation in young muscle.

biochemistry↗

bioRxiv2025

Visualization of stochastic expression of clustered protocadherin β isoforms in vivo

The stochastic expression of clustered protocadherin (cPcdh) establishes a single-cell identity fundamental to cellular self/non-self-discrimination. However, it has been challenging to reveal the spatiotemporal patterning of the stochastic cPcdh expression in vivo. We developed XFP (tdTomato or GFP) knock-in mice using a new strategy to enhance XFP expression, which allows us to visualize cPcdh{beta}3 or 19-positive cells throughout the brain. These mouse lines demonstrate the cell-type selectivity, spatial biases, inter-individual differences, left-right asymmetry, developmental regulation, alteration in pathological or aging brain, and monoallelic expression of stochastic cPcdh{beta} expression in vivo. Our findings further demonstrate that the cPcdh{beta}3 expression undergoes significant changes in the mature brain over time. These results demonstrate the potential of these reporter mice to advance our understanding of cPcdh cellular barcode in vivo.\n\nOne Sentence SummaryNovel established cPcdh{beta} reporter mouse lines reveal stochastic expression of cPcdh{beta} isoforms in vivo.

neuroscience↗

bioRxiv2024

Impact of Temporal Uncertainty on Sign-tracking Behavior

Sign-tracking behavior, also known as "autoshaping", is defined as approach and interaction with reward-predictive cues. It is associated with addiction-related phenotypes and compulsive behavior. Several previous studies have demonstrated that when there is uncertainty about reward properties (e.g. probability, size), sign-tracking is increased. However, the effect of cue-uncertainty on sign-tracking behavior is not known. Here, using a Pavlovian conditioning paradigm, we manipulated the temporal uncertainty about the appearance of cues by implementing either fixed or variable inter-trial intervals (ITIs) of different durations across groups of mice. We found that temporal uncertainty during acquisition significantly enhances sign-tracking, which persists during extinction, even when ITI variability was different in the extinction session than in the acquisition session. This suggests that the effects of temporal uncertainty are learned and retained, rather than performance-based. Our results demonstrate that sign-tracking behavior is not only modified by the characteristic of the reward, but it can also be modified by the uncertainty regarding cues. These findings highlight how temporal predictability shapes cue-directed behaviors and has implications for understanding reward-related behavioral responses including sign-tracking behaviors.

animal behavior and cognition↗

bioRxiv2023

Visualization of trans homophilic interaction of clustered protocadherin in neurons

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in CNS. However, its interactions have yet not been visualized in neurons. We previously reported Pcdh{gamma}B2-FRET probes to be applicable only for cell lines. Herein, we newly designed Pcdh{gamma}B2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single Pcdh{gamma}B2 molecule and succeeded in visualizing Pcdh{gamma}B2 homophilic interaction in cultured hippocampal neurons. The {gamma}B2-FRET probe localized in the soma and neurites, and FRET signals were observed at contact sites between neurites and eliminated by EGTA addition. Live imaging revealed that the FRET-negative {gamma}B2 signals were rapidly moving along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the {gamma}B2 proteins at synapses rarely interact homophilically. The {gamma}B2-FRET probe would allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism. Significance StatementWe visualize the Pcdh homophilic interaction using a novel FRET-based probe, and reveal that the homophilically interacting Pcdh proteins are found at contact sites between the neurites and roots of neurites from the soma, and are stable at a location. Additionally, in neurons, Pcdh proteins are located at synapses but rarely interact homophilically.

neuroscience↗

bioRxiv2022

Teneurin-2 at the Synapse Construction Site is a Signpost for Cargo Unloading from Motor Proteins

In mature neurons, excitatory synapses are formed on the dendritic spine, whereas inhibitory synapses are formed on the dendritic shaft. Thus, it is primarily the accumulation of synaptic proteins that characterizes inhibitory synapses as distinct from non-synaptic regions. Protein accumulation is achieved by a combination of microtubule (MT)-based transport by kinesins and lateral diffusion across the plasma membrane; however, how and when proteins are released from kinesins remains unclear. Using primary cultured hippocampal neurons, we found that Teneurin-2 (TEN2) promotes synaptic protein accumulation by recruiting MTs via the representative MT plus end-tracking protein, EB1. MTs recruitment was enhanced when the extracellular domain of TEN2 successfully chose partners, and the lateral diffusion of TEN2 was inhibited. Conversely, if TEN2 partner choice is not achieved, MTs are not recruited, and thus synaptogenesis is not followed. Our study revealed that cargo release from kinesins through TEN2-MTs interactions supports the continuity from partner choice to synaptogenesis, which is a critical step in synaptic maturation.

cell biology↗

bioRxiv2022

In vitro generation of human embryonic stem cell-derived heart organoids possessing physiological ion currents

Recently, methods for in vitro organogenesis have been broadly developed due to their strong potential for human applications in medicine. In the present study, we optimized the culturing method of human heart organoids (hHOs) from embryonic stem cells (ESCs) in the presence of the highly concentrated laminin-entactin and fibroblast growth factor 4. The resulting hHOs showed distinctive cardiac morphology with atrium- and ventricle-like chambers composed of cardiac cells as well as expressed the integral proteins of gap junctions and ion channels. In fact, isolated cardiomyocytes from these hHOs exhibited Na and Ca currents by patch clamp analysis. These results indicated that the present method will provide a powerful tool for cardiac safety assessment of newly developed drugs as an in vitro human ESC-derived test system. One-Sentence SummaryFGF4 and ECM contribute to the generation of human HOs with heart compartments and electrophysiological properties.

bioengineering↗