LM22B-10

LM22B-10 promotes corneal nerve regeneration through in vitro 3D co-culture model and in vivo corneal injury model

Corneal nerve wounding often causes abnormalities in the cornea and even blindness in severe cases. In this study, we construct a dorsal root ganglion-corneal stromal cell (DRG-CSC, DS) co-culture 3D model to explore the mechanism of corneal nerve regeneration. Firstly, this model consists of DRG collagen grafts sandwiched by orthogonally stacked and orderly arranged CSC-laden plastic compressed collagen. Nerve bundles extend into the entire corneal stroma within 14 days, and they also have orthogonal patterns. This nerve prevents CSCs from apoptosis in the serum withdrawal medium. The conditioned medium (CM) for CSCs in collagen scaffolds contains NT-3, IL-6, and other factors. Among them, NT-3 notably promotes the activation of ERK-CREB in the DRG, leading to the growth of nerve bundles, and IL-6 induces the upregulation of anti-apoptotic genes. Then, LM22B-10, an activator of the NT-3 receptor TrkB/TrkC, can also activate ERK-CREB to enhance nerve growth. After administering LM22B-10 eye drops to regular and diabetic mice with corneal wounding, LM22B-10 significantly improves the healing speed of the corneal epithelium, corneal sensitivity, and corneal nerve density. Overall, the DS co-culture model provides a promising platform and tools for the exploration of corneal physiological and pathological mechanisms, as well as the verification of drug effects in vitro. Meanwhile, we confirm that LM22B-10, as a non-peptide small molecule, has future potential in nerve wound repair. STATEMENT OF SIGNIFICANCE: The cornea accounts for most of the refractive power of the eye. Corneal nerves play an important role in maintaining corneal homeostasis. Once the corneal nerves are damaged, the corneal epithelium and stroma develop lesions. However, the mechanism of the interaction between corneal nerves and corneal cells is still not fully understood. Here, we construct a corneal stroma-nerve co-culture in vitro model and reveal that NT-3 expressed by stromal cells promotes nerve growth by activating the ERK-CREB pathway in nerves. LM22B-10, an activator of NT-3 receptors, can also induce nerve growth in vitro. Moreover, it is used as eye drops to enhance corneal epithelial wound healing, corneal nerve sensitivity and density of nerve plexus in corneal nerve wounding model in vivo.

Dexmedetomidine Inhibits Neuroinflammation by Altering Microglial M1/M2 Polarization Through MAPK/ERK Pathway

Neuroinflammation is critical in the pathogenesis of neurological diseases. Microglial pro-inflammatory (M1) and anti-inflammatory (M2) status determines the outcome of neuroinflammation. Dexmedetomidine exerts anti-inflammatory effects in many neurological conditions. Whether dexmedetomidine functions via modulation of microglia M1/M2 polarization remains to be fully elucidated. In the present study, we investigated the anti-inflammatory effects of dexmedetomidine on the neuroinflammatory cell model and explored the potential mechanism. BV2 cells were stimulated with LPS to establish a neuroinflammatory model. The cell viability was determined with MTT assay. NO levels were assessed using a NO detection kit. The protein levels of IL-10, TNF-α, iNOS, CD206, ERK1/2, and pERK1/2 were quantified using Western blotting. LPS significantly increased pro-inflammatory factors TNF-α and NO, and M1 phenotypic marker iNOS, and decreased anti-inflammatory factor IL-10 and M2 phenotypic marker CD206 in BV2 cells. Furthermore, exposure of BV2 cells to LPS significantly raised pERK1/2 expression. Pretreatment with dexmedetomidine attenuated LPS-elicited changes in p-ERK, iNOS, TNF-α, NO, CD206 and IL-10 levels in BV2 cells. However, co-treatment with dexmedetomidine and LM22B-10, an agonist of ERK, reversed dexmedetomidine-elicited changes in p-ERK, iNOS, TNF-α, NO, CD206 and IL-10 levels in LPS-exposed BV2 cells. We, for the first time, showed that dexmedetomidine increases microglial M2 polarization by inhibiting phosphorylation of ERK1/2, by which it exerts anti-inflammatory effects in BV2 cells.

Targeted activation of ERK1/2 reduces ischemia and reperfusion injury in hyperglycemic myocardium by improving mitochondrial function

Background: Diabetes can increase the risk of coronary heart disease, and also increase the mortality rate of coronary heart disease in diabetic patients. Although reperfusion therapy can preserve the viable myocardium, fatal reperfusion injury can also occur. Studies have shown that diabetes can aggravate myocardial ischemia-reperfusion injury, ERK1/2 can reduce myocardial ischemia-reperfusion injury, but its mechanism in hyperglycemic myocardial ischemia-reperfusion injury is unclear. This study sought to explore the mechanism of extracellular signal-regulated kinase 1/2 (ERK1/2) in hyperglycemic myocardial ischemia reperfusion (I/R) injury.
Methods: H9C2 cardiomyocytes were treated with high-glucose (HG) medium plus I/R stimulation to establish a hyperglycemia I/R model in vitro. The cells were treated with LM22B-10 (an ERK activator) or transfected with the constitutive activation of the mitogen-activated protein kinase 1 (CaMEK) gene. Myocardial cell apoptosis, mitochondria functional-related indicators, the oxidative stress indexes, and the expression levels of ERK1/2 protein were detected.
Results: The HG I/R injury intervention caused an increase in the ratio of apoptotic cardiomyocytes (P<0.05), but the phosphorylation level of the ERK1/2 protein did not increase further. Administering LM22B-10 or transfecting the CaMEK gene significantly activated the phosphorylation levels of ERK1/2 protein and reduced the proportion of cardiomyocyte apoptosis (P<0.05). HG I/R injury increased mitochondrial fission and reduced membrane potential. The intervention reduced the number of punctate mitochondria, increased the average network structure size and median branch length (P<0.01), increased the median network structure size and average branch length (P<0.05), and reduced the colocalization of Drp1 (Dynamin-Related protein1)/TOMM20 (Mitochondrial outer membrane translocation enzyme 20) (P<0.05) and Drp1 with serine 616 phosphorylation (Drp1s616) phosphorylation (P<0.01), thereby reducing mitochondrial fission, increasing membrane potential and mitochondrial function. HG I/R injury increased the level of oxidative stress, while administering LM22B-10 or transfecting the CaMEK gene reduced the level of oxidative stress (P<0.01).
Conclusions: Targeting the activation of ERK1/2 protein phosphorylation reduced mitochondrial fission, increased membrane potential and mitochondrial function, reduced oxidative stress and myocardial cell apoptosis, and alleviated hyperglycemia myocardial I/R injury.

A small molecule TrkB/TrkC neurotrophin receptor co-activator with distinctive effects on neuronal survival and process outgrowth

Neurotrophin (NT) receptors are coupled to numerous signaling networks that play critical roles in neuronal survival and plasticity. Several non-peptide small molecule ligands have recently been reported that bind to and activate specific tropomyosin-receptor kinase (Trk) NT receptors, stimulate their downstream signaling, and cause biologic effects similar to, though not completely overlapping, those of the native NT ligands. Here, in silico screening, coupled with low-throughput neuronal survival screening, identified a compound, LM22B-10, that, unlike prior small molecule Trk ligands, binds to and activates TrkB as well as TrkC. LM22B-10 increased cell survival and strongly accelerated neurite outgrowth, superseding the effects of brain-derived neurotrophic factor (BDNF), NT-3 or the two combined. Additionally, unlike the NTs, LM22B-10 supported substantial early neurite outgrowth in the presence of inhibiting glycoproteins. Examination of the mechanisms of these actions suggested contributions of the activation of both Trks and differential interactions with p75(NTR), as well as a requirement for involvement of the Trk extracellular domain. In aged mice, LM22B-10 activated hippocampal and striatal TrkB and TrkC, and their downstream signaling, and increased hippocampal dendritic spine density. Thus, LM22B-10 may constitute a new tool for the study of TrkB and TrkC signaling and their interactions with p75(NTR), and provides groundwork for the development of ligands that stimulate unique combinations of Trk receptors and activity patterns for application to selected neuronal populations and deficits present in various disease states.

Small molecule modulation of TrkB and TrkC neurotrophin receptors prevents cholinergic neuron atrophy in an Alzheimer's disease mouse model at an advanced pathological stage

Degeneration of basal forebrain cholinergic neurons (BFCNs) in the nucleus basalis of Meynert (NBM) and vertical diagonal band (VDB) along with their connections is a key pathological event leading to memory impairment in Alzheimer's disease (AD). Aberrant neurotrophin signaling via Trks and the p75 neurotrophin receptor (p75^NTR) contributes importantly to BFCN dystrophy. While NGF/TrkA signaling has received the most attention in this regard, TrkB and TrkC signaling also provide trophic support to BFCNs and these receptors may be well located to preserve BFCN connectivity. We previously identified a small molecule TrkB/TrkC ligand, LM22B-10, that promotes cell survival and neurite outgrowth in vitro and activates TrkB/TrkC signaling in the hippocampus of aged mice when given intranasally, but shows poor oral bioavailability. An LM22B-10 derivative, PTX-BD10-2, with improved oral bioavailability has been developed and this study examined its effects on BFCN atrophy in the hAPP^Lond/Swe (APP^L/S) AD mouse model. Oral delivery of PTX-BD10-2 was started after appreciable amyloid and cholinergic pathology was present to parallel the clinical context, as most AD patients start treatment at advanced pathological stages. PTX-BD10-2 restored cholinergic neurite integrity in the NBM and VDB, and reduced NBM neuronal atrophy in symptomatic APP^L/S mice. Dystrophy of cholinergic neurites in BF target regions, including the cortex, hippocampus, and amygdala, was also reduced with treatment. Finally, PTX-BD10-2 reduced NBM tau pathology and improved the survival of cholinergic neurons derived from human induced pluripotent stem cells (iPSCs) after amyloid-β exposure. These data provide evidence that targeting TrkB and TrkC signaling with PTX-BD10-2 may be an effective disease-modifying strategy for combating cholinergic dysfunction in AD. The potential for clinical translation is further supported by the compound's reduction of AD-related degenerative processes that have progressed beyond early stages and its neuroprotective effects in human iPSC-derived cholinergic neurons.