Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies DOI Creative Commons
Sigurbjörn Markússon, Arne Raasakka, Marcel Schröder

et al.

Journal of Neurochemistry, Journal Year: 2024, Volume and Issue: 169(1)

Published: Dec. 10, 2024

Abstract 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNPase) is an abundant constituent of central nervous system non‐compact myelin, and its loss in mice humans causes neurodegeneration. Additionally, CNPase frequently used as a marker antigen for myelinating cells. The catalytic activity CNPase, the 3′‐hydrolysis 2′,3′‐cyclic nucleotides, well characterised vitro, but vivo function remains unclear. interacts with actin cytoskeleton to counteract developmental closure cytoplasmic channels that travel through compact myelin; enzymatic may be involved adenosine metabolism RNA degradation. We developed set high‐affinity nanobodies recognising phosphodiesterase domain crystal structures each complex show five have distinct epitopes. One bound deep into active site acted inhibitor. Moreover, were imaging applications intrabodies, expressed mammalian cells, such primary oligodendrocytes. Fluorescently labelled functioned teased nerve fibres whole brain tissue sections, super‐resolution microscopy. These anti‐CNPase provide new tools structural functional studies on myelin formation, dynamics, disease, including high‐resolution tissue. image

Language: Английский

ARC/ARG3.1 binds the nuclear polyadenylate-binding protein RRM and regulates neuronal activity-dependent formation of nuclear speckles DOI Creative Commons
Tambudzai Kanhema,

Kamil Parobczak,

Sudarshan Patil

et al.

Cell Reports, Journal Year: 2025, Volume and Issue: 44(4), P. 115525 - 115525

Published: April 1, 2025

ARC is a neuronal activity-induced protein interaction hub with critical roles in synaptic plasticity and memory. localizes to synapses the nucleus, but its nuclear functions are little known. Following vivo long-term potentiation (LTP) induction dentate gyrus, we show that accumulates nucleosol fraction interchromatin space of granule cells. Proteomic analysis immunoprecipitated complexes identifies proteins involved pre-mRNA processing. We demonstrate endogenous protein-protein polyadenylate-binding 1 (PABPN1) paraspeckles polypyrimidine tract-binding (PTB)-associated splicing factor (PSF). In vitro peptide binding arrays direct purified PABPN1 poly(A)-RNA recognition motif. 3D morphometric imaging reveals structural changes foci corresponding classical speckles following LTP. Depletion disrupts maintenance activity-dependent formation speckles, thus implicating regulation speckle dynamics

Language: Английский

Citations

0
Correction: Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc DOI Creative Commons
José M. Godoy Muñoz, Lasse Neset, Sigurbjörn Markússon

et al.

PLoS ONE, Journal Year: 2025, Volume and Issue: 20(3), P. e0321004 - e0321004

Published: March 25, 2025

[This corrects the article DOI: 10.1371/journal.pone.0300453.].

Language: Английский

Citations

0
A Small Interfering Peptide Potentiates AMPA Receptor Diffusional Trapping and Prevents Social-Isolation-Induced Forgetting of Fear Memory DOI Open Access

Jens Edvard Trygstad Gundersen,

Xinrong Wu, Jiaxing Song

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 7, 2024

Abstract Regulation and dysregulation of AMPA receptor (AMPAR) diffusional trapping at synapses are critical for synaptic efficacy implicated in various neurological neuropsychiatric disorders. However, the limited availability reliable tools to modulate this process hinders our ability explore its role both physiological conditions disease, as well drug development. In study, we designed characterized a 21-amino-acid trans-activator transcription (TAT)-fused peptide that mimics binding region transmembrane AMPAR regulatory proteins (TARPs) C-tail activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) N-lobe. Acute intrahippocampal infusion enhanced perforant path-evoked transmission rat dentate gyrus (DG) vivo, likely by strengthening interaction between TARP postsynaptic density 95 (PSD-95), mechanism crucial anchoring AMPARs. Additionally, also slowed lateral diffusion blocked KCl-induced endocytosis, enhancing AMPARs synapse. Remarkably, 7-day prevented social-isolation-induced forgetting fear memory. Our findings suggest targeting trapping, particularly through disrupting TARP-Arc N-lobe interaction, could hold promise therapeutic approach disorders associated with impaired retention.

Language: Английский

Citations

1
A Nanobody-Based Proximity Ligation Assay Detects Constitutive and Stimulus-Regulated Native Arc/Arg3.1 Oligomers in Hippocampal Neuronal Dendrites DOI Creative Commons
Rodolfo Baldinotti, François Philippe Pauzin, Hauk Fevang

et al.

Molecular Neurobiology, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 5, 2024

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

Language: Английский

Citations

1
A nanobody-based proximity ligation assay detects constitutive and stimulus-regulated native Arc/Arg3.1 oligomers in hippocampal neuronal dendrites DOI Creative Commons
Rodolfo Baldinotti, François Philippe Pauzin, Hauk Fevang

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 17, 2024

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

Language: Английский

Citations

0
Arc/Arg3.1 binds the nuclear polyadenylate-binding protein RRM and regulates neuronal activity-dependent formation of nuclear speckles DOI Creative Commons
Tambudzai Kanhema,

Kamil Parobczak,

Sudarshan Patil

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 5, 2024

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

Language: Английский

Citations

0
Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies DOI Creative Commons
Sigurbjörn Markússon, Arne Raasakka, Marcel Schröder

et al.

Journal of Neurochemistry, Journal Year: 2024, Volume and Issue: 169(1)

Published: Dec. 10, 2024

Abstract 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNPase) is an abundant constituent of central nervous system non‐compact myelin, and its loss in mice humans causes neurodegeneration. Additionally, CNPase frequently used as a marker antigen for myelinating cells. The catalytic activity CNPase, the 3′‐hydrolysis 2′,3′‐cyclic nucleotides, well characterised vitro, but vivo function remains unclear. interacts with actin cytoskeleton to counteract developmental closure cytoplasmic channels that travel through compact myelin; enzymatic may be involved adenosine metabolism RNA degradation. We developed set high‐affinity nanobodies recognising phosphodiesterase domain crystal structures each complex show five have distinct epitopes. One bound deep into active site acted inhibitor. Moreover, were imaging applications intrabodies, expressed mammalian cells, such primary oligodendrocytes. Fluorescently labelled functioned teased nerve fibres whole brain tissue sections, super‐resolution microscopy. These anti‐CNPase provide new tools structural functional studies on myelin formation, dynamics, disease, including high‐resolution tissue. image

Language: Английский

Citations

0