Faculty Bibliography

Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis in Drosophila, as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, suggesting that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, indicating that the Inc C-terminus has the properties of a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and support the notion that Inc-Cul3 complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Inc-Cul3 complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated with Cul3 mutations.

The immune system is a key player in the onset and progression of neurodegenerative disorders. While brain resident immune cell-mediated neuroinflammation and peripheral immune cell (eg, T cell) infiltration into the brain have been shown to significantly contribute to Alzheimer's disease (AD) pathology, the nature and extent of immune responses in the brain in the context of AD and related dementias (ADRD) remain unclear. Furthermore, the roles of the peripheral immune system in driving ADRD pathology remain incompletely elucidated. In March of 2023, the Alzheimer's Association convened the Alzheimer's Association International Conference (AAIC), Advancements: Immunity, to discuss the roles of the immune system in ADRD. A wide range of topics were discussed, such as animal models that replicate human pathology, immune-related biomarkers and clinical trials, and lessons from other fields describing immune responses in neurodegeneration. This manuscript presents highlights from the conference and outlines avenues for future research on the roles of immunity in neurodegenerative disorders. HIGHLIGHTS: The immune system plays a central role in the pathogenesis of Alzheimer's disease. The immune system exerts numerous effects throughout the brain on amyloid-beta, tau, and other pathways. The 2023 AAIC, Advancements: Immunity, encouraged discussions and collaborations on understanding the role of the immune system.

Glutamatergic dentate gyrus (DG) mossy cells (MCs) innervate the primary DG cell type, granule cells (GCs). Numerous MC synapses are on GC proximal dendrites in the inner molecular layer (IML). However, field recordings of the GC excitatory postsynaptic potential (fEPSPs) have not been used to study this pathway selectively. Here we describe methods to selectively activate MC axons in the IML using mice with Cre recombinase expressed in MCs. Slices were made after injecting adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) in the DG. In these slices, we show that fEPSPs could be recorded reliably in the IML in response to optogenetic stimulation of MC axons. Furthermore, fEPSPs were widespread across the septotemporal axis. However, fEPSPs were relatively weak because they were small in amplitude and did not elicit a significant population spike in GCs. They also showed little paired pulse facilitation. We confirmed the extracellular findings with patch clamp recordings of GCs despite different recording chambers and other differences in methods. Together the results provide a simple method for studying MC activation of GCs and add to the evidence that this input is normally weak but widespread across the GC population.

BACKGROUND/UNASSIGNED:Essential tremor (ET) is characterized by often disabling action tremors. No pharmacological agent has been developed specifically for symptomatic treatment. Anecdotal reports describe tremor improvement with cannabis, but no evidence exists to support these claims. We conducted a phase Ib/II double-blind, placebo-controlled, crossover pilot trial in participants with ET to investigate tolerability, safety, and efficacy of Tilray TN-CT120 LM, an oral pharmaceutical-grade formulation containing tetrahydrocannabinol (THC) 5 mg and cannabidiol (CBD) 100 mg. Our objectives were to determine if short-term THC/CBD exposure improved tremor amplitude and was tolerated. METHODS/UNASSIGNED:Participants with ET were randomized (1:1) to receive either TN-CT120 LM or placebo. Dose titration, driven by tolerability, was attempted every 2-3 days to three capsules daily maximum. Participants remained on the highest tolerated dose for two weeks before returning to complete assessments. After completing the first arm, participants titrated off the agent, underwent a three-week washout, and then returned for the same procedures with the alternate compound. The primary endpoint was tremor amplitude change from baseline using digital spiral assessment. Secondary endpoints explored safety and tolerability. RESULTS/UNASSIGNED:Among thirteen participants screened, seven were eligible and enrolled. Five completed all visits; one withdrew following a serious adverse event, and another did not tolerate the lowest dose. Intent-to-treat analyses performed for six participants did not reveal significant effects on primary or secondary endpoints. CONCLUSIONS/UNASSIGNED:This pilot trial did not detect any signals of efficacy of THC/CBD in ET. Although preliminary due to the small sample size, our data do not support anecdotal reports of cannabinoid effectiveness for ET. HIGHLIGHTS/UNASSIGNED:This double-blind, randomized, placebo-controlled efficacy and tolerability pilot trial did not detect any signals of efficacy of oral cannabidiol and tetrahydrocannabinol in reducing essential tremor amplitude using either digital outcome measures or clinical rating scales. The oral cannabinoids were well-tolerated by most (five out of seven) participants.

INTRODUCTION/UNASSIGNED:Patients with oral cancer often experience intense functional pain due to mechanical stimulation at the cancer site. The role of mechanosensitive ion channels in oral cancer pain, such as TRPV4, is not fully understood. OBJECTIVES/UNASSIGNED:Our objective was to investigate the role of Schwann cell TRPV4 in oral cancer pain. METHODS/UNASSIGNED:imaging, and patch-clamp electrophysiology. The effect of TRPV4 activation on Schwann cell responses to mechanical stimulation was evaluated using a piezo stimulator. Conditioned media (CM) from TRPV4-activated Schwann cells were injected into the mouse paw to evaluate the contribution of TRPV4 in Schwann cells to mechanical hypersensitivity. RESULTS/UNASSIGNED:responses and whole-cell membrane currents in human Schwann cells. Mechanoactivated currents in human Schwann cells were inhibited by the TRPV4 antagonist HC-067047. Schwann cell CM induced mechanical hypersensitivity in mice, which was blocked by pre-treatment with HC-067047. CONCLUSION/UNASSIGNED:TRPV4 activation plays a role in mediating mechanically induced pain of oral cancer.

INTRODUCTION/UNASSIGNED:Individuals with Down syndrome (DS) exhibit neurological deficits throughout life including the development of in Alzheimer's disease (AD) pathology and cognitive impairment. At the cellular level, dysregulation in neuronal gene expression is observed in postmortem human brain and mouse models of DS/AD. To date, RNA-sequencing (RNA-seq) analysis of hippocampal neuronal gene expression including the characterization of discrete circuit-based connectivity in DS remains a major knowledge gap. We postulate that spatially characterized hippocampal neurons display unique gene expression patterns due, in part, to dysfunction of the integrity of intrinsic circuitry. METHODS/UNASSIGNED:We combined laser capture microdissection to microisolate individual neuron populations with single population RNA-seq analysis to determine gene expression analysis of CA1 and CA3 pyramidal neurons and dentate gyrus granule cells located in the hippocampus, a region critical for learning, memory, and synaptic activity. RESULTS/UNASSIGNED:The hippocampus exhibits age-dependent neurodegeneration beginning at ~6 months of age in the Ts65Dn mouse model of DS/AD. Each population of excitatory hippocampal neurons exhibited unique gene expression alterations in Ts65Dn mice. Bioinformatic inquiry revealed unique vulnerabilities and differences with mechanistic implications coinciding with onset of degeneration in this model of DS/AD. CONCLUSIONS/UNASSIGNED:These cell-type specific vulnerabilities may underlie degenerative endophenotypes suggesting precision medicine targeting of individual populations of neurons for rational therapeutic development.

BACKGROUND/UNASSIGNED:The informed consent process has traditionally taken place in person. The introduction of electronic consent (eConsent) has made remote consenting processes possible. Use of eConsent has increased since the COVID-19 pandemic. It has streamlined the process of consenting patients and has been shown to benefit the research study team and participants. ECONSENT IN THE KETAMINE ANALGESIA FOR LONG-LASTING PAIN RELIEF AFTER SURGERY KALPAS STUDY/UNASSIGNED:The KALPAS study is a multicenter, double-blind, randomized controlled study investigating the effectiveness of ketamine in reducing chronic post-mastectomy pain in women undergoing mastectomy for oncologic indication. The study uses a two-part consent form consisting of a master consent with information applicable to all sites and site-specific information. All potential participants receive the full two-part consent form for review. When signing the eConsent, however, all potential participants are provided with a concise summary of the informed consent document, an approach not widely used by multicenter studies. eConsent has been noted to be beneficial to research staff when trying to gather informed consent from participants who live far away from the hospital, want to include their family and friends, and for researchers who can approach patients outside of their clinical appointments. CONCLUSION/UNASSIGNED:The ability to consent patients remotely has allowed for a flexible workflow within sites and a more patient-centric process that focuses on including loved ones in the discussion and scheduling time to speak to a principal investigator. Demand for eConsent will likely continue in the post-COVID era, and use of a concise summary can allow for a more efficient consenting process.

The posterior "tail" region of the striatum receives dense innervation from sensory brain regions and is important for behaviors that require sensorimotor integration. The output neurons of the striatum, D1 and D2 striatal projection neurons (SPNs), which make up the direct and indirect pathways, are thought to play distinct functional roles, although it remains unclear if these neurons show cell-type-specific differences in their response to sensory stimuli. Here, we examine the strength of synaptic inputs onto D1 and D2 SPNs following the stimulation of upstream auditory pathways. We report that auditory-evoked depolarizations onto D1 SPN responses are stronger and faster. This is due to differences in feedforward inhibition, with fast-spiking interneurons forming stronger synapses onto D2 SPNs. Our results support a model in which differences in feedforward inhibition enable the preferential recruitment of D1 SPNs by auditory stimuli, positioning the direct pathway to initiate sound-driven actions.