Vollum Institute

The cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway regulates diverse cellular processes through precise spatiotemporal control across subcellular compartments. Förster resonance energy transfer (FRET)-based A-kinase activity reporters (AKARs) have enabled live-cell visualization of PKA activity, but their limited dynamic range constrains detection of subtle or compartment-specific signaling events. Here, we present a suite of sixth-generation cyan/yellow FRET-based PKA sensors (the AKAR6 series) with substantially enhanced sensitivity and improved selectivity. Systematic optimization of the FRET donor-acceptor pair and sensor backbone yields superior performance versus previous best-in-class FRET-based AKARs, enabling robust detection of subtle PKA activity changes across diverse experimental modalities, including flow cytometry, fluorescence lifetime-based FRET, and two-photon imaging in brain slices. We further leverage kinome atlas data to engineer a variant with improved selectivity for more accurate visualization of nuclear PKA activity. Using the AKAR6 toolkit, we showed that in contrast to strong GPCR-induced PKA activities across all tested compartments in PC12 cells, growth factors stimulated a significant PKA activity at the trans-Golgi network but no detectable activity at cis-Golgi, signifying highly compartmentalized PKA signaling at the sub-organelle level. Furthermore, NGF and EGF induced sustained and transient PKA activity, respectively, across various intracellular compartments, including the nucleus, suggesting that growth factor-specific temporal controls are maintained across subcellular compartments. Together, the AKAR6 toolkit provides a sensitive, selective, and versatile platform for dissecting compartmentalized PKA signaling across cells and tissues.

Although adenosine was identified in the brain many decades ago, our understanding of when, where, and how it functions has expanded rapidly in recent years, driven in part by innovative technological advances. Adenosine is now increasingly recognized as a key neuromodulator that dynamically regulates brain circuits important for sleep/wakefulness, movement, cognition, and homeostasis. In addition, growing attention has been directed toward the molecular mechanisms governing adenosine production and its downstream signaling pathways, both of which hold great promise as therapeutic targets for neuropsychiatric disorders and neurodegenerative diseases. This review highlights recent progress in detecting adenosine, unraveling its signaling pathways in vitro and in vivo, and understanding how it regulates brain function under physiological and pathological conditions.

Peripheral somatosensory neurons in the dorsal root ganglia (DRG) transduce mechanical force in the skin and other organs into electrical signals using specialized mechanically activated (MA) ion channels that initiate neuronal activation in response to force. Increasing evidence highlights PIEZO2 as the primary transducer of low-threshold mechanical force in DRG neurons. However, in the absence of Piezo2, mice and humans still respond to noxious painful stimuli like pinch, suggesting that additional MA channel(s) likely exist in DRG neurons. Developing strategies to identify Cre lines and DRG subpopulations that select for non-PIEZO2-expressing neurons is therefore an ongoing effort in the field to discover unknown mechanosensors. Here, we investigated a Vglut3-labeled mouse line as a candidate to identify non-PIEZO2 MA channels in a subtype of DRG neurons called C-fiber low-threshold mechanoreceptors (C-LTMRs). Our study carefully demonstrates that the Vglut3-IRES-Cre mouse line specifically and efficiently labels C-LTMR neurons of the DRG. Electrophysiological recordings using two different in vitro mechanical stimulation assays show that the genetically labeled Vglut3 neurons have robust indentation- and stretch-activated MA currents that are exclusively slowly or ultra-slowly adapting. To determine whether the Vglut3-IRES-Cre mouse line can be used to delete genes of interest and identify the underlying MA ion channels in C-LTMRs, we attempted to generate a Tmem63b conditional knockout using this Cre line but detected incomplete loss of Tmem63b transcript and lack of TMEM63B-dependent effect on C-LTMR MA currents. Together, our results emphasize that although the Vglut3-IRES-Cre line is robust in driving expression of a conditional reporter gene, it is inefficient in deleting genes like Tmem63b as well as Piezo2.

Canonical N-methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels with critical roles in the development and function of the nervous system. The excitatory currents they produce reflect stochastic transitions between multiple agonist-bound closed- and open-pore states. We leveraged the intrinsically high open probability (Po) of NMDARs composed of GluN1 and GluN2A subunits, together with judiciously chosen mutants and ligands, to achieve conditions in which receptors had a Po near unity. Using single-particle cryo–electron microscopy (cryo-EM), we captured three activated receptor states, each with distinct conformations of the gate-forming M3 helices. Separately, we carried out single-channel electrophysiology, together with statistical modeling, to relate the cryo-EM structures to the gating reaction. NMDAR channel opening involves bending of the pore-forming M3 helices to produce a transient open-channel conformation, subsequently stabilized by new interactions between the D2-M3 linkers with the pre-M1 helices and the pre-M4 loops, to yield the stable open channel.

Background: Hearing loss is known to cause structural and functional abnormalities in the central auditory pathways. Interventions with hearing aids that amplify acoustic signals have been developed to combat hearing loss. However, little is known about how such devices may affect the brain and mitigate the progression of hearing loss. We hypothesized that timely intervention that amplifies acoustic signals would delay further progression of hearing loss by maintaining central auditory activity and neural structure. Method: To that end, we provided eight weeks of acoustic stimulation tailored to compensate for subject-specific patterns of frequency loss in two mouse models of progressive hearing loss. We evaluated the effects of sound amplification on endbulb of Held anatomy at different ages of intervention in mice with early-onset (DBA/2) and late-onset (C57Bl/6) hearing loss. Results: We observed in both strains that endbulbs undergo rapid and progressive atrophy in untreated control subjects exposed to a baseline, unamplified, sound environment. In contrast, endbulb atrophy was significantly slowed in treated mice (p < 0.05). Conclusions: These data provide a possible explanation for how the brain benefits from sound amplification via hearing aid devices.

Stereocilia, the actin-based mechanosensory protrusions of inner ear sensory hair cells, require precise dimensional control for proper mechanotransduction, yet the mechanisms governing actin assembly during development remain unclear. Their size and shape are determined by a stable core of long, parallel, unbranched filamentous (F-) actin. We find that during stereocilia widening, which is a key process for function and stability, newly expressed actin first integrates at the tip, then along the periphery of the core. To understand how actin assembles, we probe for globular (G-) actin, F-actin barbed ends, and pointed ends, and identify a tip-enriched population of short actin filaments. Overexpressing actin increases these filaments at the stereocilia periphery, suggesting a role in widening. In addition, the tip-localized myosins MYO3A/B and MYO15A, essential for normal growth, generate or stabilize short filaments. We propose that these short filaments are intermediates that mature into the long F-actin known to comprise the stereocilia core.

Oligodendrocyte progenitor cells (OPCs) are highly dynamic, widely distributed glial cells of the central nervous system responsible for generating myelinating oligodendrocytes throughout life. However, the rates of OPC proliferation and differentiation decline dramatically with aging, which may impair homeostasis, remyelination and adaptive myelination during learning. To determine how aging influences OPCs, we generated a transgenic mouse line (Matn4-mEGFP) and performed single-cell RNA sequencing, providing enhanced resolution of transcriptional changes during key transitions from quiescence to proliferation and differentiation across the lifespan. We found that aging induces distinct transcriptomic changes in OPCs in different states, including enhanced activation of HIF-1α and WNT pathways. Pharmacological inhibition of these pathways in aged OPCs was sufficient to increase their ability to differentiate in vitro. Ultimately, Matn4-mEGFP mouse line and the sequencing dataset of cortical OPCs across ages will help to define the molecular changes guiding OPC behavior in various physiological and pathological contexts.

AnnSQL is a pip installable package that can be found at https://github.com/ArpiarSaundersLab/annsql along with documentation at https://docs.annsql.com .