Fig 1: Schematic for the proposed mechanisms of PRG-1 modulating RNS-induced hyperalgesia, learning, and memory defectsLeft: PRG-1/NSF interactions maintain PRG-1 function and neurotransmitter receptor stability under normal conditions. Right: Following neonatal RNS stress, the release of extracellular ATP is increased and further inhibits PRG-1 function and PRG-1/NSF interaction. Two pathways involving PRG-1 are initiated. On one pathway, inhibition of PRG-1 promotes the release of glutamate at the extracellular presynaptic membrane. Along the other pathway, PRG-1/NSF interaction interferes with the sorting, transport and interaction of neurotransmitter receptors, thus causing cell-autonomous AMPAR GluR2 trafficking deficiency, resulting in changes in synaptic activity, function, and structure. The two pathways modulate RNS-induced persistent hyperalgesia in a synergistic effect.
Fig 2: PRG-1/NSF relieves RNS-induced allodynia by reversing synaptic depression in the hippocampus(A–D) (A) Images of Golgi-Cox staining and (B–D) quantitative analysis showing that the spine density depression induced by RNS was reversed by ATP?S and PRG-1 overexpression (B, n = 12 CON, 11 RNS, 11 RNS + ATP?S and 13 RNS + PRG-1 OE; C, n = 12 CON, 9 RNS, 14 RNS+ ATP?S and 10 RNS + PRG-1 OE; D, n = 12 CON, 8 RNS, 12 RNS+ ATP?S and 15 RNS + PRG-1 OE; B–D, one-way ANOVA with Bonferroni post hoc; n represents analyzed dendritic segments; *: vs CON group; #: vs RNS group). stratum radiatum (sr, apical dendrites) and stratum oriens (so, basal dendrites) of the CA1 region, and the molecular layer of dentate gyri (moDG).Data are presented as mean ± SEM with *p < 0.05, **p < 0.01, ***p < 0.001; RNS, repetitive noxious stimuli.
Fig 3: PRG-1/NSF interaction relieves allodynia, learning, and memory impairments in RNS rats(A) Schematic diagram of hippocampal microinjection of adult rats (x = ±2.0 mm, y = −3.72 mm, and z = −3.0 mm).(B) Chronic ATPγS (100 μM per day, P60-62) administration in adult rats (at 9 weeks) attenuated RNS-induced TWL impairments on P61-63 (n = 6; repeated measures two-way ANOVA with Tukey’s post-hoc multiple comparisons test; ∗: vs CON group at the same time point; #: vs RNS group at the same time point; arrows indicate pharmacological treatment).(C) Multiple ATPγS (100 μM per day, P60-62) administration attenuated RNS-induced MWT impairments on P60-63. (n = 6; repeated measures two-way ANOVA with Tukey’s post-hoc multiple comparisons test; ∗: vs CON group at the same time point; #: vs RNS group at the same time point; arrows indicate pharmacological treatment).(D–F) ATPγS reversed the spatial learning and memory impairments induced by RNS, and showed shortened escape latency time to target (D), less escape path length to target (E) in spatial acquisition trials, and more time in the quadrant zone in probe trials (F) compared with RNS group (n = 6; D-E, repeated measures two-way ANOVA with Tukey’s post-hoc multiple comparisons test; F, one-way ANOVA with Bonferroni post hoc; ∗: vs CON group at the same time point; #: vs RNS group at the same time point).Data are presented as mean ± SEM with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; RNS, repetitive noxious stimuli; TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold.
Fig 4: PRG-1 interacts with NSF(A) CoIP using a PRG-1-specific antibody detected PRG-1/NSF interaction in the hippocampus.(B) PRG-1/NSF interaction was inhibited in RNS rats compared with CON rats.(C) CoIP using a PRG-1 antibody shows PRG-1 and NSF association in/HEK293 cells transfected with wild-type PRG-1, and that PRG-1-R346T mutation lost this interaction.(D) Interaction between PRG-1 and NSF is enhanced by ATPγS (100 μM, 2 h) while decreased by ATP (100 μM, 2 h).RNS, repetitive noxious stimuli; NSF, N-Ethylmaleimide sensitive fusion protein.
Fig 5: PRG-1/NSF relieves RNS-induced pain in rats via glutamine/GluR2 signaling(A) Quantitative ELISA showing that secreted glutamate content was increased in hippocampus of RNS rats at 9 weeks compared with age-matched CON group (n = 6, two-tailed unpaired student t-test, *: vs CON group).(B) Quantitative analysis showing that ATP?S or PRG-1 overexpression induced the secreted glutamate content decreased in hippocampus at 9 weeks in RNS rats (n = 6, one-way ANOVA with Bonferroni post hoc; *: vs CON group; #: vs RNS group).(C–E) (C) Western blot and (D and E) quantitative analysis of gray values (normalized to CON values) showing PRG-1 and GluR2 expression in CON, RNS, RNS+ virus or drug group at 9 weeks (n = 6, *: vs CON group, D–E: one-sample t test. #: vs RNS group, D: one-way ANOVA with Bonferroni post hoc, E: Kruskal-Wallis test with a Dunn’s multiple comparisons test). Data are presented as mean ± SEM with *p < 0.05, **p < 0.01, ***p < 0.001; RNS, repetitive noxious stimuli; OE, overexpression; SI, silencing.
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