Biology subjects

Ishizuka, Y.

Publications and source records attributed to Ishizuka, Y..

5 recordsLinked to original sources

bioRxiv2026

Validation of a Commercially Available Non-Woven Fabric Electrode for Mouse Electroretinography

Purpose: To evaluate the performance and usability of a commercially available nonwoven fabric electrode as a cost-effective and user-friendly alternative to standard corneal contact lens electrodes for recording murine electroretinography (ERG). Methods: Full-field ERGs were recorded in the same individual mice using both the commercially available non-woven fabric electrode and a conventional contact lens electrode in a paired design. Key parameters evaluated included (1) baseline noise stability, (2) dark-adapted intensity-response curves (0.01-100 cd{middle dot}s{middle dot}m-2), (3) light-adapted intensity-response curves (3-100 cd{middle dot}s{middle dot}m-2), (4) 20 and 30 Hz flicker responses, (5) extended recordings targeting slow c-wave kinetics under dark-adapted conditions, and (6) inter-eye consistency via bilateral simultaneous recordings. Results: Eliminating the need for viscous lubricants, the non-woven fabric electrode simplified corneal placement, alignment, and re-application. It yielded a- and b-wave amplitudes and peak times comparable to those obtained with standard contact lens electrodes. Notably, during prolonged recordings, the non-woven fabric electrode yielded significantly larger c-wave amplitudes while maintaining equivalent baseline noise stability. Bilateral simultaneous recordings demonstrated high inter-eye correlation and low measurement variability between contralateral eyes. Conclusions: The non-woven fabric electrode provides high-quality, reproducible ERG recordings comparable to, and for c-waves superior to, standard corneal contact lens electrodes. This accessible method facilitates standardized data acquisition and enhances inter-laboratory reproducibility in murine ERG. Translational Relevance: By reducing financial and technical barriers, this non-woven fabric electrode offers an alternative to specialized recording setups, improving experimental throughput and efficiency in ophthalmic research.

neuroscience↗

bioRxiv2024

Arc/Arg3.1 binds the nuclear polyadenylate-binding protein RRM and regulates neuronal activity-dependent formation of nuclear speckles

Arc is a neuronal activity-induced protein interaction hub with critical roles in synaptic plasticity and memory. Arc localizes to synapses and the nucleus, but its nuclear functions are little known. We show that Arc accumulates in the interchromatin space of dentate granule cell nuclei and the nucleosol subcellular fraction following seizure activity and in vivo dentate gyrus LTP. Proteomic analysis of affinity-purified Arc complexes identified proteins with functions in post-transcriptional mRNA processing. During LTP, Arc undergoes enhanced complex formation with polyadenylate binding protein nuclear 1 (PABPN1) and paraspeckle splicing factor (PSF) in the nucleosol. In vitro peptide binding arrays show selective binding of Arc to the PABPN1 polyA RNA recognition motif. In hippocampal neuronal cultures, Arc knockdown increases formation of PABPN1 nuclear speckles and blocks chemical-LTP associated increases in small PABPN1 foci. These results implicate Arc in basal and neuronal activity-dependent regulation of PABPN1 speckles involved in mRNA processing and polyadenylation.

neuroscience↗

bioRxiv2024

A nanobody-based proximity ligation assay detects constitutive and stimulus-regulated native Arc/Arg3.1 oligomers in hippocampal neuronal dendrites

Activity-regulated cytoskeleton-associated (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state, however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody.

neuroscience↗

bioRxiv2022

Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function

Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N-and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc and analysis of capsid domain specific functions.

neuroscience↗

bioRxiv2020

Herpes Simplex Virus type 1 neuronal infection triggers disassembly of key structural components of dendritic spines

Herpes simplex virus type 1 (HSV-1) is a widespread neurotropic virus. The primary infection in facial epithelium leads to retrograde axonal transport to the central nervous system (CNS) where it establishes latency. Under stressful conditions, the virus reactivates, and new progeny is transported anterogradely to the primary site of infection. In late stages of neuronal infection, axonal damage is known to occur. However, the impact of HSV-1 infection on morphology and functional integrity at earlier stages of infection in neuronal dendrites is unknown. Previously, we demonstrated that acute HSV-1 infection in neuronal cell lines selectively enhances the expression of Arc protein - a major regulator of long-term synaptic plasticity and memory consolidation, known for being a protein-interaction hub in the postsynaptic dendritic compartment. Thus, HSV-1 induced Arc may alter the functionality of the infected neurons having an impact on dendritic spine dynamics. In this study we demonstrated that HSV-1 infection causes structural disassembly and functional deregulation in cultured cortical neurons, through protein homeostasis alteration with intracellular accumulation of Arc, and decreased expression of spine scaffolding-like proteins such as PSD-95, Drebrin and CaMKII{beta}. Our findings reveal progressive deleterious effects of HSV-1 infection on excitatory neuronal synapse function and dendritic morphology, supporting the thesis of the infectious origin of neurodegenerative processes.

neuroscience↗