Objective: Iaccarino et al. (2016) [1] exposed 1 h of light flickering at 40 Hz to awake 5XFAD Alzheimer's Disease (AD) mouse models, generating action potentials at 40 Hz, activating~54% of microglia to colocalize with Ab plaque, acutely, and clearing~50% of Ab plaque after seven days, but only in the visual cortex. Hypothesis: Transcranially delivered, focused ultrasound (tFUS) can replicate the results of Iaccarino et al. (2016) [1] but throughout its area of application. Methods: We exposed sedated 5XFAD mice to tFUS (2.0 MHz carrier frequency, 40 Hz pulse repetition frequency, 400 ms-long pulses, spatial peak pulse average value of 190 W/cm 2 ). Acute studies targeted tFUS into one hemisphere of brain centered on its hippocampus for 1 h. Chronic studies targeted comparable brain in each hemisphere for 1 h/day for five days. Results: Acute application of tFUS activated more microglia that colocalized with Ab plaque relative to sham ultrasound (36.0 ± 4.6% versus 14.2 ± 2.6% [mean ± standard error], z ¼ 2.45, p < 0.014) and relative to the contralateral hemisphere of treated brain (36.0 ± 4.6% versus 14.3 ± 4.0%, z ¼ 2.61, p < 0.009). Chronic application over five days reduced their Ab plaque burden by nearly half relative to paired sham animals (47.4 ± 5.8%, z ¼ -2.79, p < 0.005). Conclusion:Our results compare to those of Iaccarino et al. (2016) [1] but throughout the area of ultrasound-exposed brain. Our results also compare to those achieved by medications that target Ab, but over a substantially shorter period of time. The proximity of our ultrasound protocol to those shown safe for non-human primates and humans may motivate its rapid translation to human studies.

G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by posttranslational modifications, including N-glycosylation of asparagine residues. α 1D-adrenergic receptors (α 1D-ARs)-key regulators of central and autonomic nervous system function-contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α 1D-AR between transmembrane domain 2 and 3. Labelfree dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α 1D-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α 1D-AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α 1D-AR n-terminal glycosylation is required for complete translation of nascent, functional receptor. G protein-coupled receptors (GPCRs) are essential membrane proteins that regulate the vast majority of physiological functions in the human body. As a result, GPCRs have been estimated to be targeted by approximately one third of all currently approved medications 1. Adrenergic receptors (ARs) are a clinically relevant subfamily of GPCRs. Activated by the endogenous sympathetic neurotransmitters epinephrine and norepinephrine, adrenergic GPCRs consist of three major subtypes: α 1 , α 2 , and β. The α 1 sub-family-containing α 1A , α 1B , and α 1D subtypes 2-are targets for medications that regulate blood pressure 3,4 , bladder 5,6 , prostate 7,8 , and central nervous system function 9-11. Thus, understanding the molecular and cellular mechanisms regulating α 1-AR function will help spur the development of new medications associated with aberrant α 1-AR signaling, such as hypertension, PTSD, schizophrenia, and benign prostatic hypertrophy 12-15. Among the three α 1 subtypes, the α 1D-AR remains poorly understood due to technical challenges. Relative to the closely related α 1A and α 1B-AR subtypes, α 1D-AR displays limited functional responses and minimal plasma membrane expression when expressed in heterologous cell culture 16-18. Although pharmacologically detectable in intact isolated aortae in organ-tissue bath assays 19 , α 1D-AR functional expression rapidly disappears in primary vascular smooth muscle cell cultures within 24-48 hours 20. Also, immortalized cell lines that endogenously express α 1D-ARs have yet to be discovered. Combined, these experimental clues indicate the molecular and cellular ...