Weltzin, M. M. | bioRxiv Science

bioRxiv2026

Serotype-dependent differences in AAV cellular transduction rates in the hypothalamus of Arctic ground squirrels

Adeno-associated viral (AAV) vectors are foundational tools for dissecting brain structure-function relationships, but AAV serotype tropism varies across brain regions and species, requiring empirical validation to inform experimental design. This need is especially important in non-model organisms, where molecular neuroscience tools remain underdeveloped and access to research subjects is often limited. The Arctic ground squirrel (AGS, Urocitellus parryii) is a valuable model for studying extreme physiology, including metabolic suppression during hibernation and resistance to cerebral ischemia/reperfusion, yet no studies have evaluated AAV performance in the AGS brain. Here, we investigated the ability of AAV serotypes 1, 8, 9, and DJ to transduce the AGS hypothalamus using the human synapsin (hSyn) promoter and directly compared cellular transduction rates in a region implicated in thermoregulation and hibernation. To maximize data collection from a limited experimental population, we used a within-animal, contralateral stereotaxic injection design. Recombinant AAV vectors expressing enhanced green fluorescent protein or mCherry were delivered bilaterally, and reporter expression was analyzed four weeks later. All tested serotypes produced clear and reproducible reporter expression, establishing AAV as a viable molecular tool in the AGS hypothalamus. AAV1 produced significantly greater cellular transduction rates than AAV-DJ (17.2% {+/-} 3.5% vs 8.4% {+/-} 2.9%, paired t-test, p = 0.032). AAV8 and AAV9 showed transduction rates of 22.8% {+/-} 0.6% and 20.1% {+/-} 1.5%, respectively; however, with only two biological replicates per serotype, formal statistical comparison was not performed. These findings provide the first direct characterization of AAV-mediated gene delivery in the AGS brain and establish a foundation for future molecular interrogation of hypothalamic circuits in this extreme mammalian hibernator.

neuroscience↗

bioRxiv2025

Development of a novel alpha7-nicotinic acetylcholine receptor-selective cell-penetrating peptide for intracellular cargo transport

Drug delivery into the brain remains a critical barrier for the treatment of neurological diseases. While the brain is shielded from many toxins and viruses by the blood-brain barrier (BBB), therapeutics are being created that exploit natural bypass mechanisms by forming complexes with cell-penetrating peptides (CPPs) derived from viruses. Neurological diseases often impact specific proteins in the brain; however, current CPPs lack the ability to selectively target precise cellular macromolecules. As a result, they are distributed broadly throughout the brain and cause off-target side effects. Neurotropic CPPs derived from the rabies virus glycoprotein (RVG) can access the brain by binding to plasma membrane targets, including, but not exclusively, nicotinic acetylcholine receptors (nAChRs). To overcome this barrier of minimal target selectivity, we designed several chimeric peptides composed of regions from the RVG and -bungarotoxin, an 7 subtype-selective protein. Using human nAChRs expressed in Xenopus laevis oocytes, we screened the selectivity of our peptides using two-electrode voltage clamp electrophysiology. We identified a peptide with improved 7 nAChR subtype selectivity and apparent potency compared to the control RVG peptide. Using mammalian Neuro-2a cells, we demonstrated that our peptide depends on 7 nAChR plasma membrane expression to internalize and carry small-molecule payloads into the neuronal-like cells, without significant cytotoxic effects. Our novel 7 nAChR subtype-selective CPP may be useful in research applications requiring cargo delivery. Translationally, our 7 nAChR selective CPP holds potential to be a dual drug delivery system to transport cargo into the brain for the treatment of neurological diseases.

bioengineering↗