Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function
Tae Jun Lee, … , Andrea Santeford, Rajendra S. Apte
Tae Jun Lee, … , Andrea Santeford, Rajendra S. Apte
Published January 16, 2024
Citation Information: JCI Insight. 2024;9(4):e173707. https://doi.org/10.1172/jci.insight.173707.
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Research Article Metabolism Ophthalmology

Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

Abstract

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, β, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2-deficient mice displayed visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor–knockout mice. These findings highlight a therapeutically targetable PRKAA2 isoform–specific function of AMPK in regulating photoreceptor metabolism and function through a potentially previously uncharacterized mechanism affecting IMPDH activity.

Authors

Tae Jun Lee, Yo Sasaki, Philip A. Ruzycki, Norimitsu Ban, Joseph B. Lin, Hung-Ting Wu, Andrea Santeford, Rajendra S. Apte

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Figure 5

Dysfunctional PRKAA2 cannot regulate IMPDH, leading to IMPDH hyperactivity.

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Dysfunctional PRKAA2 cannot regulate IMPDH, leading to IMPDH hyperactivi...
(A) Schematic representation of IMPDH function in rod photoreceptors according to light exposure. Previous work has shown GTP allosterically inhibits IMPDH in dark-adapted conditions while IMPDH is active to produce GMP in light-adapted conditions. (B) Ambient light–adapted retinas from Prkaa2-Rhod/-Rhod were assessed for IMPDH activity (n = 4). No significant changes were detected. (C) Dark-adapted retinas from Prkaa2-Rhod/-Rhod showed significantly increased IMPDH activity (n = 6; **P < 0.01 by 2-way ANOVA). (D) Ambient light–adapted wild-type retinas were assessed for IMPDH activity after treatment with 5 mM metformin, an AMPK activator. Metformin is a slower activator of AMPK, and metformin treatment inhibits IMPDH activity compared with that of control retinas (n = 6, P < 0.0001 by 2-way ANOVA). (E) Dark-adapted wild-type retinas were assessed for IMPDH activity after treatment with 40 μM compound C, an AMPK inhibitor. Lysates treated with compound C exhibited significantly higher IMPDH activity compared with controls (n = 4, **P < 0.01 by 2-way ANOVA). (F and G) Recombinant human AMPK (α2β1γ1) was incubated with recombinant human (F) IMPDH1 or (G) IMPDH2 to assess the effect of AMPK on IMPDH activity. Both IMPDH1 and IMPDH2 when incubated with AMPK had significantly attenuated IMPDH activity compared with IMPDH incubated alone (n = 4, P < 0.0001 by 2-way ANOVA). (H) Schematic workflow of the co-immunoprecipitation protocol of IMPDH1. Six dark-adapted retinas from wild-type mice were dissected and lysed in extraction buffer. Dynabeads coated with IMPDH1 antibody were added to the suspension and allowed to bind to IMPDH1. Attached IMPDH1 along with bound proteins were isolated. The resulting eluant was then used for Western blots to detect IMPDH1 and bound protein. (I) The results of Western blot detection of co-immunoprecipitation samples are depicted. Clear bands representing IMPDH1 along with PRKAA2 demonstrate PRKAA2 was bound to IMPDH1 in wild-type retinas. Values are mean ± SEM.