ACS Chemical Neuroscience最新文献

Visualizing signaling systems in the brain with high spatial resolution is critical to understanding brain function and to develop therapeutics. Especially, enzymes are often regulated on the post-translational level, resulting in a disconnect between protein levels and activity. Conventional antibody-based methods have limitations, including potential cross-reactivity and the inability of antibodies to discriminate between active and inactive enzyme states. Monoacylglycerol lipase (MAGL), an enzyme degrading the neuroprotective endocannabinoid 2-arachidonoylglycerol, is the target of inhibitors currently in clinical trials for the treatment of several neurological disorders. To support translational and (pre)clinical studies and fully realize the therapeutic opportunities of MAGL inhibitors, it is essential to map the spatial distribution of MAGL activity throughout the brain in both health and disease. Here, we introduce selective fluorescent activity-based probes for MAGL enabling direct visualization of its enzymatic activity in lysates, cultured cells, and tissue sections. We show that oxidative stress, which inactivates MAGL through the oxidation of regulatory cysteines, reduces probe labeling, thereby validating the probes activity-dependence. Extending this approach, we developed an activity-based histology protocol to visualize MAGL activity in fresh-frozen mouse and human brain tissues. This approach revealed robust MAGL activity in astrocytes and presynaptic terminals within the mouse hippocampus and further allows detection of MAGL activity in the human cerebral cortex. Collectively, these findings establish selective activity-based probes as powerful tools mapping MAGL activity with high spatial resolution across mammalian brain tissue.

We recently disclosed VU0467319, a muscarinic acetylcholine receptor subtype 1 (M₁) Positive Allosteric Modulator (PAM) clinical candidate that had successfully completed a Phase I Single Ascending Dose (SAD) clinical trial, but the identification of an inactive metabolite constituting a major portion of the total plasma AUC detracted from the molecules’ pharmacokinetic profile and contributed to clinical development discontinuation. Attempts to block metabolism with the incorporation of deuterium atoms proved successful in vitro and in vivo at low exposures; however, in high-dose nonclinical toxicology studies, the degree of oxidative metabolism and metabolite accumulation was comparable to that of the proteo-congener. Here, we describe a second-generation back-up effort based on the VU0467319 scaffold to discover VU6052254. Strategic placement of a tertiary hydroxyl moiety afforded VU6052254, a potent M₁ PAM (EC₅₀ = 59 nM, 79% ACh max), with high CNS exposure (rat K_p = 1.07; K_p,uu = 1.27; P-gp ER = 1.97, P_app = 23 × 10^–6 cm/s), reduced metabolism across species, excellent pharmacodynamic responses (MED in rat NOR = 1 mg/kg PO; MED in rat CFC = 0.3 mg/kg PO), excellent multispecies PK (Cl_ps < 10 mL/min/kg, %F > 65), and favorable human PK and dose projections. Based on these beneficial attributes, VU6052254 was nominated for further nonclinical development. However, possible CYP₄₅₀ induction liability as well as uncertain projected margins for human efficacy at those systemic concentrations where dose/exposure-related clinical and anatomic pathology kidney findings were observed in a 14-day exploratory toxicity study in male rats, precluded further development.

Microglia-mediated neuroinflammation constitutes a pivotal secondary injury mechanism after traumatic brain injury (TBI). Recent studies have unveiled the role of Nr1d1 in neuroinflammation and glial activation in the Central Nervous System (CNS) injury, found the activation of Nr1d1 appears to prevent inflammation and apoptosis cell death. However, the role of Nr1d1 in the regulation of M1/M2 polarization and neuroinflammatory responses in TBI remains unclear. The purpose of this study is to investigate the effects of Nr1d1 on neuroinflammatory responses in the acute phase of TBI. SR9009 (100 mg/kg) was administered by intraperitoneal injection to activate Nr1d1. Neurological impairments were assessed using the modified neurological severity score (mNSS). Molecular levels were evaluated through Western Blotting and quantitative real-time polymerase chain reaction. Measurement of the water content of brain tissue was used to assess cerebral edema, and the damaged area of brain tissue was evaluated by Hematoxylin-Eosin (H&E) staining. The functional behavioral assessment was used to evaluate the cognitive impairments and emotional change. Our study, for the first time, demonstrates that the circadian rhythm of Nr1d1 is disrupted during the acute phase of TBI. We also found Nr1d1 prevented nerve dysfunction and contributed to the recovery of neurological impairment, promoted the transformation of microglia phenotype, and reduced the damage to neurons, synaptic structures, and the neuroinflammation. These findings unveiled that Nr1d1 may represent a promising therapeutic target for the successful treatment of TBI and for improving neurological deficits during the acute phase of TBI.

Transmembrane channel-like protein 1 (TMC1) forms the pore of the mechanotransduction channel in cochlear and vestibular hair cells, converting mechanical stimuli from sound and head movements into electrochemical signals. Recent evidence supports a dimeric structure for TMC1, with each monomer harboring an independent ion-conducting pore. The p.(M654V) variant, in which methionine 654 is substituted with valine, is associated with non-syndromic autosomal recessive deafness. In the present work, we used molecular dynamics (MD) simulations to compare the structural and biophysical properties of the wild-type and M654V-TMC1 variants, providing atomistic-level insights into subtle alterations in the mechanotransduction system. Our analysis reveals specific alterations in pore size, lipid composition of the pore walls, and the electrostatic environment. The results suggest that the two monomers function independently and underscore the critical role of lipids in shaping the pore architecture. Potential molecular mechanisms of M654V-associated pathogenicity include disrupted local interactions between transmembrane α-helices and residue 654, leading to reduced pore flexibility, a shifted choke point, and fewer lipid molecules incorporated into the pore walls. These findings provide mechanistic insights into TMC1 function and its impairment in deafness-associated variants.

In order to understand the retinal network, it is essential to identify functional connectivity among retinal neurons. For this purpose, imaging neuronal activity through fluorescent indicator proteins has been a promising approach, offering simultaneous measurements of neuronal activities from different regions of the circuit. In this study, we used genetically encoded indicators─Bongwoori-R3 for voltage or GCaMP6f for calcium─to visualize membrane voltage or calcium dynamics, respectively, as spatial maps within individual retinal ganglion cells from retinal tissues of photoreceptor-degenerated rd1 mice. Retinal voltage imaging was able to show current-evoked somatic spiking as well as subthreshold voltage changes, while calcium imaging showed changes in calcium concentrations evoked by current pulses in retinal ganglion cells. These results indicate that the combination of fluorescent protein sensors and high-speed imaging methods permits the imaging of electrical activity with cellular precision and millisecond resolution. Hence, we expect our method will provide a potent experimental platform for the study of retinal signaling pathways, as well as the development of retinal stimulation strategies in visual prosthesis.

The electrical membrane voltage (V_m) characterizes the functional state of biological cells, thus requiring precise, noninvasive V_m-sensing techniques. While voltage-dependent fluorescence intensity changes from genetically encoded voltage indicators (GEVIs) indicate V_m changes, variability in sensor expression confounds the determination of absolute V_m. Fluorescence lifetime imaging microscopy (FLIM) promises a solution to this problem, as fluorescence lifetime is expected to be unaffected by sensor expression and excitation intensity. By examining ASAP1, ASAP3, JEDI-1P, rEstus, and rEstus-NI (G138N:T141I) with one-photon-excited FLIM measurements, we demonstrate that all sensors display a voltage-dependent lifetime. Based on the highest lifetime change in the V_m range of −120 to 60 mV, rEstus-NI (798 ps) and ASAP3 (726 ps) are preferred for FLIM recordings. At a physiologically relevant V_m of −30 mV, the voltage sensitivity of rEstus-NI (6.6 ps/mV) is 3.6 and 1.4 times greater than that of ASAP1 and rEstus, respectively. As a proof of concept, we successfully used rEstus-NI to estimate absolute resting V_m in HEK293T, A375 melanoma, and MCF7 breast cancer cells and quantified spontaneous V_m fluctuations in A375 cells.

Central nervous system (CNS) disorders such as Alzheimer’s diseases, Parkinson’s diseases, stroke, and glioma remain among the most challenging to treat, largely due to the restrictive nature of the blood–brain barrier (BBB). In recent years, intranasal administration has emerged as a noninvasive route for CNS drug delivery. Due to its anatomical advantage over the traditional route, the nose-to-brain route can easily bypass the BBB and deliver drugs directly to the brain. Parallel advances in the interface of synthetic biology and materials engineering have led to the development of engineered living materials (ELMs) dynamic structures that embed mammalian cells, bacteria, or viruses within self-renewing or engineered matrices. These bioengineered systems have been developed as next-generation therapeutic platforms for various biomedical applications, utilizing intrinsic or engineered capabilities such as disease-targeted migration, localized therapeutic production, adaptive delivery, immune activation, and metabolic regulation. Therefore, developing a bioengineered commensal based delivery system that uses the intranasal route to effectively deliver drug across the BBB could represent a transformative strategy for treating CNS disorder and advancing neurotherapeutic research.

Mitragynine is a psychoactive alkaloid in Mitragyna speciosa with unique polypharmacology at G protein-coupled receptors. In addition to its well-known partial agonist activity at opioid receptors, mitragynine is an antagonist at human α_2A-adrenoceptors (α_2ARs), as measured in an in vitro GTPγS G protein assay. Mitragynine’s in vitro α_2AR antagonist pharmacology contrasts with rat behavioral pharmacology studies that suggest mitragynine behaves as an in vivo agonist at rat α₂Rs. This study investigates this apparent discrepancy using recombinant α-adrenoceptors and a range of orthogonal signal transducers. We evaluated whether mitragynine activates any of seven Gα_i/o proteins coupled to α_2A, α_2B, and α_2CRs, as well as Gα_q and Gα₁₁ coupled to α_1AR. Additionally, we examined rat and human α_2AR-mediated cAMP inhibition, α_2AR-mediated β-arrestin2 recruitment, and tested α₂R or α₁R-mediated ERK phosphorylation in wild-type, β-arrestin 1/2 knockout, and Gα_q/11 knockout cells. Finally, we report binding and enzyme-inhibition profiling results for mitragynine and its major metabolites, 7-Hydroxymitragynine and 9-Hydroxycorynantheidine, at 99 targets. The results did not support the hypothesis that mitragynine (or its primary metabolites) activates α₂Rs, but, aligned with our previous GTPγS results, demonstrate that mitragynine is a low-potency, competitive α_2AR antagonist at Gα_i1, cAMP, and β-arrestin2 transducers. However, we show that mitragynine is a low-potency (EC₅₀ ∼3 μM), partial agonist at α_1AR-Gα₁₁ and stimulates ERK phosphorylation via Gα_q/11-coupled α₁Rs, supporting in vivo studies that suggest mitragynine is an α₁R agonist. Nevertheless, the agonist effects of mitragynine at α_1AR-Gα₁₁ were modest compared to clonidine, a partial agonist control that also activated all α₂R transducers. Mitragynine’s dual α_2AR antagonist/α₁R partial agonist pharmacology might contribute to mitragynine’s psychostimulant-like properties.

This study provides evidence of lymphatic vessels in the mouse brain, specifically in the cortex, thalamus, and hippocampus. Using confocal microscopy, Western blotting, and real-time PCR, lymphatic vessels were identified by the expression of Lyve1, Prox1, and VEGFC, alongside endothelial markers CD31 and CD34. Paraffin-embedded brain slices from wild-type Balb/C mice were stained with antibodies against Lyve1, CD31, and CD34, revealing lymphatic vessels through 2D and 3D imaging. A coiled 3D structure of lymphatic vessels was observed in the hippocampus, indicating a complex drainage network. Western blot and real-time PCR analyses confirmed the presence of lymphatic and endothelial markers at the protein and mRNA levels. These findings demonstrate a lymphatic system extending from the meninges into deeper brain regions, offering insights into immune and waste clearance pathways in the central nervous system and potential therapeutic targets for neurological disorders.

Spinal cord injury (SCI) leads to complex pathological cascades, including endothelial cell dysfunction and vascular degeneration. In this study, we employed label-free quantitative proteomics to profile spinal cord tissue following injury, and identify altered molecular pathways. Proteomic analysis identified lactate dehydrogenase A (LDHA) as significantly upregulated at day post-injury 7 (DPI-7) and a potential regulator of vascular endothelial growth factor (VEGF)-VEGFR2 signaling. Pharmacological inhibition of LDHA using FX-11 led to increased oxidative stress in endothelial cells, reduced cell proliferation, impaired angiogenesis, and aggravated neuronal damage at the lesion epicenter. These findings suggest that LDHA functions as a metabolic regulator supporting endothelial cell survival under injury conditions. Notably, systemic lactate treatment counteracted the detrimental effects of LDHA inhibition and promoted functional recovery post-SCI. Overall, this study identifies LDHA as a critical regulator of VEGF–VEGFR2 signaling post-SCI and proposes lactate treatment as a potential therapeutic strategy to enhance vascular repair.