Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function
Tae Jun Lee, Yo Sasaki, Philip A. Ruzycki, Norimitsu Ban, Joseph B. Lin, Hung-Ting Wu, Andrea Santeford, Rajendra S. Apte
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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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 2

PRKAA2, but not PRKAA1, dysfunction leads to rod photoreceptor structural abnormalities and visual dysfunction.

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PRKAA2, but not PRKAA1, dysfunction leads to rod photoreceptor structura...
(A and B) Schematic representations of the protein sequences in the Prkaa1-Rhod/-Rhod and Prkaa2-Rhod/-Rhod models, respectively. Knockout domains are within the kinase domain to abrogate function but preserve overall expression of the proteins. (A) Schematic representation of the PRKAA1 and (B) PRKAA2 protein sequence. (C) Representative transmission electron microscopy images of magnification, 2,500×, of rod photoreceptors. (Left) Rod photoreceptors of control mice (Prkaa2fl/fl) demonstrate similar features to those of Prkaa1-Rhod/-Rhod with consistent membrane structure and organization. (Middle) Rod photoreceptors of Prkaa1-Rhod/-Rhod demonstrate intact connections between the outer and inner segments with consistent laminar organization of the outer segment membranes. (Right) Rod photoreceptors of Prkaa2-Rhod/-Rhod consistently demonstrate detachments between the outer and inner segments and occasional outer segment membrane dysmorphisms (red arrow). (D) Representative transmission electron microscopy images of magnification, 6,000×, of the outer segments of rod photoreceptors. (Left) Outer segments from control mice (Prkaa2fl/fl) show consistent striations and laminar organization resembling Prkaa1-Rhod/-Rhod. (Middle) Prkaa1-Rhod/-Rhod outer segments demonstrated organized and laminar structure indicative of normal wild-type structure. (Right) Prkaa2-Rhod/-Rhod outer segments exhibited disorganized membrane layers and occasional manifestation of granular debris (red arrows). (E and F) Scotopic electroretinography of Prkaa1-Rhod/-Rhod and respective wild-type littermates. (E) Representative traces of 0 dB intensity flashes demonstrate similar waveforms between Prkaa1fl/fl and Prkaa1-Rhod/-Rhod (n = 7). (F) Analyses of scotopic a (left) and scotopic b (right) amplitude measurements reveal no significant changes in Prkaa1-Rhod/-Rhod. (G and H) Scotopic electroretinography of Prkaa2-Rhod/-Rhod and respective wild-type littermates (n = 7). (G) Representative traces of 0 dB intensity flashes show a diminutive waveform from Prkaa2-Rhod/-Rhod. (H) Analyses of scotopic a (left) and scotopic b (right) amplitude measurements confirm significant attenuation in Prkaa2-Rhod/-Rhod measurements (**P < 0.01, ****P < 0.0001 by 2-way ANOVA with post hoc Bonferroni’s multiple comparisons test). Values are mean ± SEM. Scale bars represent 2 μm. Representative images selected from 40 images for each group.