CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance

Abstract

Objective Arcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17β-estradiol increases the M-current by regulating the mRNA expression of Kcnq2 , 3 , and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons. Methods We used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance. Results The virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5 . The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals. Conclusions The M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.

Integrating genome-wide association studies and population genomics analysis reveals the genetic architecture of growth and backfat traits in pigs

“…ANO1 gene defects or its expression in pancreatic islets might influence cytokine expression and elicit an immune response that could result in death of β cell (Xu et al, 2014). CRISPR-edited animals study demonstrated KCNQ3 expression was sensitive to the energy state of animals (Stincic et al, 2021). EYA2 had been documented to be closely associated with the biological processes of striated muscle tissue development, muscle cell and skeletal muscle cell differentiation (Li et al, 2019).…”

CRISPR/SaCas9 mutagenesis of stromal interaction molecule 1 in proopiomelanocortin neurons increases glutamatergic excitability and protects against diet-induced obesity

“…Our attempts to conditionally knockout Stim1 in POMC cells failed perhaps due to the fact POMC is not only expressed in neurons but pituitary corticotrophs [ 78 ]. Therefore, we took advantage of CRISPR technology [ 17 , 18 ] utilizing a sgRNA to selectively mutagenize Stim1 in adult ARH-POMC neurons in male mice. Importantly, there is not a pharmacological approach to inhibit STIM1 signaling in adult animals ( e.g.…”

“…However after extensive breeding over some twenty litters, we were unable to generate viable Pomc Cre ::Stim1 −/− offspring, even though we were able to produce Kiss1 Cre ::Stim1 −/− offspring that survived well into adulthood [ 16 ]. Therefore, we took advantage of a newly developed CRISPR technology [ 17 , 18 ] in which we utilized a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas) and a single guide RNA (sgRNA) to selectively mutagenize Stim1 in POMC neurons in male mice and measure changes in both cellular excitability and the whole animal physiological responses to high fat dieting.…”

<i>KCNQ2</i>- and <i>KCNQ3</i>-Associated Epilepsy

“…Importantly, previous studies have shown that fasting, which leads to increased firing activity of NPY/AgRP neurons, is associated with lower K V 7.2 and K V 7.3 expression and lower M-current [69]. Indeed, several of the effects of fasting on NPY/AgRP neuron excitability are recapitulated by blocking K V 7.2/K V 7.3 channels or by knocking down Kcnq3 [69,70]. Similar to NPY/AgRP neurons, corticotrophin-releasing hormone (CRH) neurons found in the paraventricular nucleus of the hypothalamus increase their activity depending on the behavioral state of the animal, in this case, following stress.…”