A comparison in the brain levels of cholinergic transmission components between genotype and age groups. a AChE enzymatic activity was significantly reduced in both young (10–11 months old) and old (30–31 months old) ache mutants as compared to wildtype animals, and a significant overall age-related decline in AChE activity was observed. b A significant effect of genotype was revealed in brain ACh levels with ache mutants having reduced levels of ACh. c An age-dependent decline that was more robust in the wildtype group was revealed in the AChE/ACh ratio. d No significant change was observed in the protein expression levels of the cholinergic receptor subunit nAChR-a7. The group means + standard error (SE) are represented. *: p <0.05, ***: p <0.001.

Representative Western blot images for cellular, synaptic, and inflammatory proteins that were used in the current study to examine the effects of genotype and age. All antibodies gave bands at their expected molecular weights.

Relative levels of neuronal, glial, and proliferation markers between genotype and age groups. a HuC protein expression levels were altered significantly depending on the genotype of the animal with ache mutants having higher levels of HuC at both young and old ages. b No significant changes were observed in the whole brain levels of the post-mitotic neuronal marker DCAMKL1. c Glial marker GFAP showed no significant changes. d Global proliferation marker PCNA was stable among age and genotype groups. The group means + SE are represented. *: p <0.05, **: p <0.01.

Relative levels of synaptic protein between genotype and age groups. a No significant effect was observed in PSD95, a clustering protein in the excitatory neurotransmission system. b The levels of AMPA receptor subunits GluR2/3 were altered significantly depending on the genotype, and ache animals were characterized with lower levels of GluR2/3 at an old age. c Genotype effect was also significant on the levels of NR2B, an NMDA receptor subunit, and ache animals had significantly lower NR2B levels at both young and old ages. d The elements of the inhibitory neurotransmission system including GEP and GABA-a1 (e) were not significantly altered depending on the genotype and age of the animals. f The levels of SYP, a presynaptic integrity marker, showed an increase at older ages but this age effect was significantly driven by ache mutants, not the wildtype zebrafish. The group means + SE are represented. *: p <0.05, **: p <0.01.

Alterations in the levels of inflammatory and oxidative stress markers. a TNF-a, a pro-inflammatory protein marker, was not altered significantly by the factors of genotype or age. b No significant age- or genotype-dependent changes were observed in ROS activity levels, an indicator of free radical content. The group means + SE are represented.

Summary figure indicating alterations in response to long-term reduction of AChE activity and aging. Overall, brain levels of AChE activity and ACh content decrease significantly in the ache mutants. On the other hand, mutants as compared to wildtype controls are characterized by elevated levels of the immature neuronal marker, HuC, and reduced levels of the glial marker, GFAP, as well as excitatory synaptic proteins GluR2/3 and NR2B. In wildtype animals, the cholinergic ratio AChE/ACh declines with increasing age, while this index is stable in the mutants across the lifespan. Additionally, with aging, the levels of presynaptic protein SYP are elevated in the ache zebrafish. The figure was created with BioRender.