Ultrasound delivery of a TrkA agonist confers neuroprotection to Alzheimer-associated pathologies.

Early degeneration of basal forebrain cholinergic neurons contributes substantially to cognitive decline in Alzheimer's disease. Evidence from preclinical models of neuronal injury and aging support a pivotal role for nerve growth factor (NGF) in neuroprotection, resilience, and cognitive function. However, whether NGF can provide therapeutic benefit in the presence of Alzheimer's disease-related pathologies still unresolved. Perturbations in the NGF signalling system in Alzheimer's disease may render neurons unable to benefit from NGF administration. Additionally, challenges related to brain delivery remain for clinical translation of NGF-based therapies in Alzheimer's disease. To be safe and efficient, NGF-related agents should stimulate the NGF receptor, tropomyosin receptor kinase A (TrkA), avoid activation through the p75 neurotrophin receptor (p75NTR), and be delivered non-invasively to targeted brain areas using real-time monitoring. We addressed these limitations using MRI-guided focused ultrasound (MRIgFUS) to increase blood-brain barrier permeability locally and transiently, allowing an intravenously administered TrkA agonist that does not activate p75NTR, termed D3, to enter targeted brain areas. Here, we report the therapeutic potential of selective TrkA activation in a transgenic mouse model that recapitulates numerous Alzheimer's disease-associated pathologies. Repeated MRIgFUS-mediated delivery of D3 (D3/FUS) improved cognitive function in the TgCRND8 model of Alzheimer's disease. Mechanistically, D3/FUS treatment effectively attenuated cholinergic degeneration and promoted functional recovery. D3/FUS treatment also resulted in widespread reduction of brain amyloid pathology and dystrophic neurites surrounding amyloid plaques. Furthermore, D3/FUS markedly enhanced hippocampal neurogenesis in TgCRND8 mice, implicating TrkA agonism as a novel therapeutic target to promote neurogenesis in the context of Alzheimer's disease-related pathology. Thus, this study provides evidence that selective TrkA agonism confers neuroprotection to effectively counteract Alzheimer's disease-related vulnerability. Recent clinical trials demonstrate that non-invasive blood-brain barrier modulation using MRIgFUS is safe, feasible and reversible in Alzheimer's disease patients. TrkA receptor agonists coupled with MRIgFUS delivery constitute a promising disease-modifying strategy to foster brain health and counteract cognitive decline in Alzheimer's disease.

Therapeutic Neuroprotection by an Engineered Neurotrophin that Selectively Activates Tropomyosin Receptor Kinase (Trk) Family Neurotrophin Receptors but Not the p75 Neurotrophin Receptor.

The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.

Small-molecule agonists of the RET receptor tyrosine kinase activate biased trophic signals that are influenced by the presence of GFRa1 co-receptors.

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET-GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

Antagonism of proNGF or its receptor p75NTR reverses remodelling and improves bladder function in a mouse model of diabetic voiding dysfunction.

AIMS/HYPOTHESIS: Although 80% of diabetic patients will suffer from voiding difficulties and urinary symptoms, defined as diabetic voiding dysfunction (DVD), therapeutic targets and treatment options are limited. We hypothesise that the blockade of the pro-nerve growth factor (NGF)/p75 neurotrophin receptor (p75NTR) axis by an anti-proNGF monoclonal antibody or by a small molecule p75NTR antagonist (THX-B) can restore bladder remodelling (represented by bladder weight) in an animal model of DVD. Secondary outcomes of the study include improvements in bladder compliance, contractility and morphology, as well as in voiding behaviour, proNGF/NGF balance and TNF-α expression. METHODS: In a streptozotocin-induced mouse model of diabetes, diabetic mice received either a blocking anti-proNGF monoclonal antibody or a p75NTR antagonist small molecule as weekly systemic injections for 4 weeks. Animals were tested at baseline (at 2 weeks of diabetes induction), and after 2 and 4 weeks of treatment. Outcomes measured were voiding function with voiding spot assays and cystometry. Bladders were assessed by histological, contractility and protein expression assays. RESULTS: Diabetic mice showed features of DVD as early as 2 weeks after diabetes diagnosis (baseline) presented by hypertrophy, reduced contractility and abnormal cystometric parameters. Following treatment initiation, a twofold increase (p < 0.05) in untreated diabetic mouse bladder weight and thickness compared with non-diabetic controls was observed, and this change was reversed by p75NTR antagonism (37% reduction in bladder weight compared with untreated diabetic mice [95% CI 14%, 60%]) after 4 weeks of treatment. However, blocking proNGF did not help to reverse bladder hypertrophy. While diabetic mice had significantly worse cystometric parameters and contractile responses than non-diabetic controls, proNGF antagonism normalised bladder compliance (0.007 [Q1-Q3; 0.006-0.009] vs 0.015 [Q1-Q3; 0.014-0.029] ml/cmH2O in untreated diabetic mice, representing 62% reduction [95% CI 8%, 110%], p < 0.05) and contractility to KCl, carbachol and electrical field stimulation (p < 0.05 compared with the diabetic group) after 2 weeks of treatment. These effects were not observed after 4 weeks of treatment with proNGF antagonist. p75NTR antagonism did not show important improvements in cystometric parameters after 2 weeks of treatment. Slightly improved bladder compliance (0.01 [Q1-Q3; 0.009-0.012] vs 0.013 [Q1-Q3; 0.011-0.016] ml/cmH2O for untreated diabetic mice) was seen in the p75NTR antagonist-treated group after 4 weeks of treatment with significantly stabilised contractile responses to KCl, carbachol and electric field stimulation (p < 0.05 for each) compared with diabetic mice. Bladder dysfunction observed in diabetic mice was associated with a significant increase in bladder proNGF/NGF ratio (3.1 [±1.2] vs 0.26 [±0.04] ng/pg in control group, p < 0.05 at week 2 of treatment) and TNF-α (p < 0.05). The proNGF/NGF ratio was partially reduced (about 60% reduction) with both treatments (1.03 [±0.6] ng/pg for proNGF antibody-treated group and 1.4 [±0.76] ng/pg for p75NTR blocker-treated group after 2 weeks of treatment), concomitant with a significant decrease in the bladder levels of TNF-α (p < 0.05), despite persistent hyperglycaemia. CONCLUSIONS/INTERPRETATION: Our findings indicate that blockade of proNGF and the p75NTR receptor in diabetes can impede the development and progression of DVD. The reported improvements in morphological and functional features in our DVD model validates the proNGF/p75NTR axis as a potential therapeutic target in this pathology. Graphical abstract.

Small-Molecule Ligands that Bind the RET Receptor Activate Neuroprotective Signals Independent of but Modulated by Coreceptor GFRα1.

Glial cell line-derived neurotrophic factor (GDNF) binds the GFRα1 receptor, and the GDNF-GFRα1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFRα1-RET signaling complex, agents that bind and activate RET directly and independently of GFRα1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFRα1 coexpression. However, RET activation by these ligands is constrained by GFRα1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT: A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor α, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.

Inhibition of CD45 Phosphatase Activity Induces Cell Cycle Arrest and Apoptosis of CD45+ Lymphoid Tumors Ex Vivo and In Vivo.

Src-family kinases (SFK) govern cellular proliferation of bone marrow-derived cells. SFKs are regulated by the protein tyrosine phosphatase enzymatic activity of CD45. All lymphoid cells express CD45, but only proliferating cells are dependent on CD45 activity. We postulated that compound 211 (2-[(4-acetylphenyl)amino]-3-chloronaphthoquinone), a selective inhibitor of CD45 phosphatase activity, could preferentially affect actively proliferating cells but spare resting lymphoid cells. Compound 211 inhibited CD45 and induced inappropriate SFK signaling, leading to a G2/M cell cycle arrest and apoptotic cell death. CD45+ cell lines were sensitive to compound 211 cytotoxicity at low micromolar LD50 while control CD45- cell lines and CD45+ resting primary T cells were spared any toxicity. In two syngeneic tumor models in vivo, compound 211 delayed the growth of established primary tumors and reduced tumor metastasis without causing depletion of resting T cells. This work validates targeting CD45 phosphatase enzymatic activity, which may be a druggable target for cancer therapy.

p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy.

UNLABELLED: In many diseases, expression and ligand-dependent activity of the p75(NTR) receptor can promote pericyte and vascular dysfunction, inflammation, glial activation, and neurodegeneration. Diabetic retinopathy (DR) is characterized by all of these pathological events. However, the mechanisms by which p75(NTR) may be implicated at each stage of DR pathology remain poorly understood. Using a streptozotocin mouse model of diabetic retinopathy, we report that p75(NTR) is upregulated very early in glia and in pericytes to mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. In a mouse model of oxygen-induced retinopathy, mimicking proliferative DR, p75(NTR)-dependent inflammation leads to ischemia and pathological angiogenesis through Semaphorin 3A. The acute use of antagonists of p75(NTR) or antagonists of the ligand proNGF suppresses each distinct phase of pathology, ameliorate disease, and prevent disease progression. Thus, our study documents novel disease mechanisms and validates druggable targets for diabetic retinopathy. SIGNIFICANCE STATEMENT: Diabetic retinopathy (DR) affects an estimated 250 million people and has no effective treatment. Stages of progression comprise pericyte/vascular dysfunction, inflammation, glial activation, and neurodegeneration. The pathophysiology of each stage remains unclear. We postulated that the activity of p75NTR may be implicated. We show that p75NTR in glia and in pericytes mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. p75NTR-promoted inflammation leads to ischemia and angiogenesis through Semaphorin 3A. Antagonists of p75NTR or antagonists of proNGF suppress each distinct phase of pathology, ameliorate disease, and prevent disease progression. Our study documents novel mechanisms in a pervasive disease and validates druggable targets for treatment.

Neurotrophin receptor agonists and antagonists as therapeutic agents: An evolving paradigm.

Neurodegenerative disorders are prevalent, complex and devastating conditions, with very limited treatment options currently available. While they manifest in many forms, there are commonalities that link them together. In this review, we will focus on neurotrophins - a family of related factors involved in neuronal development and maintenance. Neurodegenerative diseases often present with a neurotrophin imbalance, in which there may be decreases in trophic signaling through Trk receptors for example, and/or increases in pro-apoptotic activity through p75. Clinical trials with neurotrophins have continuously failed due to their poor pharmacological properties as well as the unavoidable activation of p75. Thus, there is a need for drugs without such setbacks. Small molecule neurotrophin mimetics are favorable options since they can selectively activate Trks or inactivate p75. In this review, we will initially present a brief outline of how these molecules are synthesized and their mechanisms of action; followed by an update in the current state of neurotrophins and small molecules in major neurodegenerative diseases. Although there has been significant progress in the development of potential therapeutics, more studies are needed to establish clear mechanisms of action and target specificity in order to transition from animal models to the assessment of safety and use in humans.

Angiotensin II type I and prostaglandin F2α receptors cooperatively modulate signaling in vascular smooth muscle cells.

The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gαq signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure.

Allosteric noncompetitive small molecule selective inhibitors of CD45 tyrosine phosphatase suppress T-cell receptor signals and inflammation in vivo.

CD45 is a receptor-like member of the protein tyrosine phosphatase (PTP) family. We screened in silico for small molecules binding at a predicted allosteric pocket unique to the CD45 intracellular domain, and validated inhibitors by in vitro phosphatase assays. Compound 211 exhibited a CD45 IC50 value of 200 nM and had >100-fold selectivity over six related PTPs. The relevance of the allosteric pocket was verified through site-directed mutagenesis. Compound 211 has a noncompetitive mechanism of action, and it is extremely effective at preventing dephosphorylation of substrate Lck phosphotyrosine (pY)-505 versus preventing dephosphorylation of Lck pY-393. In cultured primary T cells, compound 211 prevents T-cell receptor-mediated activation of Lck, Zap-70, and mitogen-activated protein kinase, and interleukin-2 production. In a delayed-type hypersensitivity reaction in vivo, compound 211 abolished inflammation. This work demonstrates a novel approach to develop effective allosteric inhibitors that can be expanded to target the corresponding allosteric domains of other receptor PTPs.

Modulation of p75(NTR) prevents diabetes- and proNGF-induced retinal inflammation and blood-retina barrier breakdown in mice and rats.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by early blood-retina barrier (BRB) breakdown and neurodegeneration. Diabetes causes imbalance of nerve growth factor (NGF), leading to accumulation of the NGF precursor (proNGF), as well as the NGF receptor, p75 neurotrophin receptor (p75(NTR)), suggesting a possible pathological role of the proNGF-p75(NTR) axis in the diabetic retina. To date, the role of this axis in diabetes-induced retinal inflammation and BRB breakdown has not been explored. We hypothesised that modulating p75(NTR) would prevent diabetes- and proNGF-induced retinal inflammation and BRB breakdown. METHODS: Diabetes was induced by streptozotocin in wild-type and p75(NTR) knockout (p75KO) mice. After 5 weeks, the expression of inflammatory mediators, ganglion cell loss and BRB breakdown were determined. Cleavage-resistant proNGF was overexpressed in rodent retinas with and without p75(NTR) short hairpin RNA or with pharmacological inhibitors. In vitro, the effects of proNGF were investigated in retinal Müller glial cell line (rMC-1) and primary Müller cells. RESULTS: Deletion of p75(NTR) blunted the diabetes-induced decrease in retinal NGF expression and increases in proNGF, nuclear factor κB (NFκB), p-NFκB and TNF-α. Deletion of p75(NTR) also abrogated diabetes-induced glial fibrillary acidic protein expression, ganglion cell loss and vascular permeability. Inhibited expression or cleavage of p75(NTR) blunted proNGF-induced retinal inflammation and vascular permeability. In vitro, proNGF induced p75(NTR)-dependent production of inflammatory mediators in primary wild-type Müller and rMC-1 cultures, but not in p75KO Müller cells. CONCLUSIONS/INTERPRETATION: The proNGF-p75(NTR) axis contributes to retinal inflammation and vascular dysfunction in the rodent diabetic retina. These findings underscore the importance of p75(NTR) as a novel regulator of inflammation and potential therapeutic target in diabetic retinopathy.

Alpha-2-macroglobulin prevents platelet aggregation induced by brain-derived neurotrophic factor.

The brain-derived neurotrophic factor (BDNF) has been recently shown to have activating effects in isolated platelets. However, BDNF circulates in plasma and a mechanism to preclude constant activation of platelets appears necessary. Hence, we investigated the mechanism regulating BDNF bioavailability in blood. Protein-protein interactions were predicted by molecular docking and validated through immunoprecipitation. Platelet aggregation was assessed using light transmission aggregometry with washed platelets in response to classical agonists or BDNF, in the absence or presence of alpha-2-macroglobulin (α2M), and in platelet-rich plasma. BDNF signaling was assessed with phospho-blots. As little as 25% autologous plasma was sufficient to completely abolish platelet aggregation in response to BDNF. Docking predicted two forms of BDNF binding to native or activated α2M, in parallel and perpendicular arrangements, and the model suggested that the BDNF-α2M complex cannot bind to the high-affinity BDNF receptor, tropomyosin receptor kinase B (TrkB). Experimentally, native and activated α2M formed stable complexes with BDNF preventing BDNF-induced TrkB activation and signal transduction. Both native and activated α2M inhibited BDNF induced-platelet aggregation in a concentration-dependent manner with comparable half-maximal inhibitory concentrations (IC50≈ 125-150 nM). Our study implicates α2M as a physiological regulator of BDNF bioavailability, and as an inhibitor of BDNF-induced platelet activation in blood.

GD2 and GD3 gangliosides as diagnostic biomarkers for all stages and subtypes of epithelial ovarian cancer.

Background: Ovarian cancer (OC) is the deadliest gynecological cancer, often diagnosed at advanced stages. A fast and accurate diagnostic method for early-stage OC is needed. The tumor marker gangliosides, GD2 and GD3, exhibit properties that make them ideal potential diagnostic biomarkers, but they have never before been quantified in OC. We investigated the diagnostic utility of GD2 and GD3 for diagnosis of all subtypes and stages of OC. Methods: This retrospective study evaluated GD2 and GD3 expression in biobanked tissue and serum samples from patients with invasive epithelial OC, healthy donors, non-malignant gynecological conditions, and other cancers. GD2 and GD3 levels were evaluated in tissue samples by immunohistochemistry (n=299) and in two cohorts of serum samples by quantitative ELISA. A discovery cohort (n=379) showed feasibility of GD2 and GD3 quantitative ELISA for diagnosing OC, and a subsequent model cohort (n=200) was used to train and cross-validate a diagnostic model. Results: GD2 and GD3 were expressed in tissues of all OC subtypes and FIGO stages but not in surrounding healthy tissue or other controls. In serum, GD2 and GD3 were elevated in patients with OC. A diagnostic model that included serum levels of GD2+GD3+age was superior to the standard of care (CA125, p<0.001) in diagnosing OC and early-stage (I/II) OC. Conclusion: GD2 and GD3 expression was associated with high rates of selectivity and specificity for OC. A diagnostic model combining GD2 and GD3 quantification in serum had diagnostic power for all subtypes and all stages of OC, including early stage. Further research exploring the utility of GD2 and GD3 for diagnosis of OC is warranted.

Ligand-dependent TrkA activity in brain differentially affects spatial learning and long-term memory.

In the central nervous system, the nerve growth factor (NGF) receptor TrkA is expressed primarily in cholinergic neurons that are implicated in spatial learning and memory, whereas the NGF receptor p75(NTR) is expressed in many neuronal populations and glia. We asked whether selective TrkA activation may have a different impact on learning, short-term memory, and long-term memory. We also asked whether TrkA activation might affect cognition differently in wild-type mice versus mice with cognitive deficits due to transgenic overexpression of mutant amyloid-precursor protein (APP mice). Mice were treated with wild-type NGF (a ligand of TrkA and p75(NTR)) or with selective pharmacological agonists of TrkA that do not bind to p75(NTR). In APP mice, the selective TrkA agonists significantly improved learning and short-term memory. These improvements are associated with a reduction of soluble Aß levels in the cortex and AKT activation in the cortex and hippocampus. However, this improved phenotype did not translate into improved long-term memory. In normal wild-type mice, none of the treatments affected learning or short-term memory, but a TrkA-selective agonist caused persistent deficits in long-term memory. The deficit in wild-type mice was associated temporally, in the hippocampus, with increased AKT activity, increased brain-derived neurotrophic factor precursor, increased neurotrophin receptor homolog-2 (p75-related protein), and long-term depression. Together, these data indicate that selective TrkA activation affects cognition but does so differently in impaired APP mice versus normal wild-type mice. Understanding mechanisms that govern learning and memory is important for better treatment of cognitive disorders.

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.

A monovalent agonist of TrkA tyrosine kinase receptors can be converted into a bivalent antagonist.

BACKGROUND: Receptor tyrosine kinases (RTK) act through dimerization. Previously it was thought that only bivalent ligands could be agonistic, whereas monovalent ligands should be antagonistic. This notion changed after the demonstration that monovalent ligands can be agonistic, including our report of a small molecule monovalent ligand "D3" that is a partial agonist of the NGF receptor TrkA. A bivalent "D3-linker-D3" was expected to increase agonism. METHODS: Dimeric analogs were synthesized and tested in binding, biochemical, and biological assays. RESULTS: One analog, 1-ss, binds TrkA with higher affinity than D3 and induces or stabilizes receptor dimers. However, 1-ss exhibited antagonistic activity, through two mechanisms. One mechanism is that 1-ss blocks NGF binding, unlike D3 which is non-competitive. Inhibition of NGF binding may be due to the linker of 1-ss filling the inter-receptor space that NGF traverses before docking. In a second mechanism, 1-ss acts as a pure antagonist, inhibiting NGF-independent TrkA activity in cells over-expressing receptors. Inhibition is likely due to 1-ss "freezing" the TrkA dimer in the inactive state. CONCLUSIONS: Dimerization of an RTK can result in antagonism, through two independent mechanisms. GENERAL SIGNIFICANCE: we report a small molecule monovalent agonist being converted to a bivalent antagonist.

An NGF mimetic, MIM-D3, stimulates conjunctival cell glycoconjugate secretion and demonstrates therapeutic efficacy in a rat model of dry eye.

The aim of this study was to evaluate the efficacy of MIM-D3, a small molecule nerve growth factor (NGF) peptidomimetic, as a therapeutic agent in rats with scopolamine induced dry eye. NGF plays an important role in ocular surface maintenance and corneal wound healing and was recently shown to have mucin secretagogue activity in conjunctival cells. We investigated whether MIM-D3 increased glycoconjugate secretion in conjunctival cells in vitro and in rat tear fluids in vivo. Primary rat conjunctival cell cultures were treated with increasing concentrations of MIM-D3 and evaluated for glycoconjugate secretion, proliferation and MAPK1/2 activation. Glycoconjugates were quantitated in tear fluids from normal rats treated topically with increasing doses of MIM-D3 (0.4%, 1% and 2.5%). Dry eye was induced in rats by subcutaneous scopolamine treatment, administered by surgically implanted osmotic pumps for 14 or 28 days. Aqueous tear production, tear clearance, fluorescein corneal staining and tear break-up time (tBUT) were evaluated. Glycoconjugates and NGF were quantitated in the tear fluids by enzyme-linked lectin assay (ELLA) and enzyme-linked immunosorbant assay (ELISA), respectively. We found that 50 µM MIM-D3 statistically significantly induced a 1.3-fold increase in glycoconjugate secretion and a 2.3-fold increase in MAPK1/2 activation without increasing proliferation from conjunctival cell cultures. Application of 2.5% MIM-D3 in normal rat eyes statistically significantly increased tear glycoconjugate concentration by 2.3-fold. In the experimental dry eye model, application of 1% MIM-D3 to rat eyes for either 1 or 17 consecutive days, followed by 1 week of no dosing produced a statistically significant decrease in corneal staining (p < 0.001), a slight increase in tBUT, and increases in tear glycoconjugates (p < 0.05) compared to vehicle. Scopolamine treatment also caused a statistically significant increase of endogenous NGF in tears (p < 0.005). We concluded that the increase in glycoconjugate concentration by the 1% MIM-D3 dose may have improved the quality and stability of the tear film, and thereby improved healing on the ocular surface in dry eye. Therefore, MIM-D3 may have therapeutic potential as a topical agent for the treatment of dry eye.

A kinase-deficient TrkC receptor isoform activates Arf6-Rac1 signaling through the scaffold protein tamalin.

Neurotrophins play an essential role in mammalian development. Most of their functions have been attributed to activation of the kinase-active Trk receptors and the p75 neurotrophin receptor. Truncated Trk receptor isoforms lacking the kinase domain are abundantly expressed during development and in the adult; however, their function and signaling capacity is largely unknown. We show that the neurotrophin-3 (NT3) TrkCT1-truncated receptor binds to the scaffold protein tamalin in a ligand-dependent manner. Moreover, NT3 initiation of this complex leads to activation of the Rac1 GTPase through adenosine diphosphate-ribosylation factor 6 (Arf6). At the cellular level, NT3 binding to TrkCT1-tamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling and the formation of cellular protrusions. Thus, our data identify a new signaling pathway elicited by the kinase-deficient TrkCT1 receptor. Moreover, we establish NT3 as an upstream regulator of Arf6.

Effective chimeric antigen receptor T cells against SARS-CoV-2.

Current therapies to treat coronavirus disease 2019 (COVID-19) involve vaccines against the spike protein S1 of SARS-CoV-2. Here, we outline an alternative approach involving chimeric antigen receptors (CARs) in T cells (CAR-Ts). CAR-T recognition of the SARS-CoV-2 receptor-binding domain (RBD) peptide induced ribosomal protein S6 phosphorylation, the increased expression of activation antigen, CD69 and effectors, interferon-γ, granzyme B, perforin, and Fas-ligand on overlapping subsets of CAR-Ts. CAR-Ts further showed potent in vitro killing of target cells loaded with RBD, S1 peptide, or expressing the S1 protein. The efficacy of killing varied with different sized hinge regions, whereas time-lapse microscopy showed CAR-T cluster formation around RBD-expressing targets. Cytolysis of targets was mediated primarily by the GZMB/perforin pathway. Lastly, we showed in vivo killing of S1-expressing cells by our SARS-CoV-2 CAR-Ts in mice. The successful generation of SARS-CoV-2 CAR-Ts represents a living vaccine approach for the treatment of COVID-19.

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.