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.

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.

Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

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.

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.

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.

Aiming for the Sweet Spot: Glyco-Immune Checkpoints and γδ T Cells in Targeted Immunotherapy.

Though a healthy immune system is capable of recognizing and eliminating emergent cancerous cells, an established tumor is adept at escaping immune surveillance. Altered and tumor-specific expression of immunosuppressive cell surface carbohydrates, also termed the "tumor glycocode," is a prominent mechanism by which tumors can escape anti-tumor immunity. Given their persistent and homogeneous expression, tumor-associated glycans are promising targets to be exploited as biomarkers and therapeutic targets. However, the exploitation of these glycans has been a challenge due to their low immunogenicity, immunosuppressive properties, and the inefficient presentation of glycolipids in a conventional major histocompatibility complex (MHC)-restricted manner. Despite this, a subset of T-cells expressing the gamma and delta chains of the T-cell receptor (γδ T cells) exist with a capacity for MHC-unrestricted antigen recognition and potent inherent anti-tumor properties. In this review, we discuss the role of tumor-associated glycans in anti-tumor immunity, with an emphasis on the potential of γδ T cells to target the tumor glycocode. Understanding the many facets of this interaction holds the potential to unlock new ways to use both tumor-associated glycans and γδ T cells in novel therapeutic interventions.

Alternative Splicing of a Receptor Intracellular Domain Yields Different Ectodomain Conformations, Enabling Isoform-Selective Functional Ligands.

Events at a receptor ectodomain affect the intracellular domain conformation, activating signal transduction (out-to-in conformational effects). We investigated the reverse direction (in-to-out) where the intracellular domain may impact on ectodomain conformation. The primary sequences of naturally occurring TrkC receptor isoforms (TrkC-FL and TrkC.T1) only differ at the intracellular domain. However, owing to their differential association with Protein Disulfide Isomerase the isoforms have different disulfide bonding and conformations at the ectodomain. Conformations were exploited to develop artificial ligands, mAbs, and small molecules, with isoform-specific binding and biased activation. Consistent, the physiological ligands NT-3 and PTP-sigma bind both isoforms, but NT-3 activates all signaling pathways, whereas PTP-sigma activates biased signals. Our data support an "in-to-out" model controlling receptor ectodomain conformation, a strategy that enables heterogeneity in receptors, ligands, and bioactivity. These concepts may be extended to the many wild-type or oncogenic receptors with known isoforms.

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.

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.

BDNF, NT-3 and Trk receptor agonist monoclonal antibodies promote neuron survival, neurite extension, and synapse restoration in rat cochlea ex vivo models relevant for hidden hearing loss.

Neurotrophins and their mimetics are potential treatments for hearing disorders because of their trophic effects on spiral ganglion neurons (SGNs) whose connections to hair cells may be compromised in many forms of hearing loss. Studies in noise or ototoxin-exposed animals have shown that local delivery of NT-3 or BDNF has beneficial effects on SGNs and hearing. We evaluated several TrkB or TrkC monoclonal antibody agonists and small molecules, along with BDNF and NT-3, in rat cochlea ex vivo models. The TrkB agonists BDNF and a monoclonal antibody, M3, had the greatest effects on SGN survival, neurite outgrowth and branching. In organotypic cochlear explants, BDNF and M3 enhanced synapse formation between SGNs and inner hair cells and restored these connections after excitotoxin-induced synaptopathy. Loss of these synapses has recently been implicated in hidden hearing loss, a condition characterized by difficulty hearing speech in the presence of background noise. The unique profile of M3 revealed here warrants further investigation, and the broad activity profile of BDNF observed underpins its continued development as a hearing loss therapeutic.

Pharmacological interrogation of TrkA-mediated mechanisms in hippocampal-dependent memory consolidation.

In the brain, the TrkA receptor for Nerve Growth Factor (NGF) is expressed primarily in the cholinergic system. TrkA/NGF support neuronal health and function, and deficiencies in this axis are associated with progressive cholinergic neuron atrophy and death, and with cognitive deficit in disorders such as Down's syndrome and Alzheimer's disease. These observations led to the hypothesis that TrkA agonists may rescue atrophic cholinergic neurons and benefit cognition. Indeed, a small molecule TrkA partial agonist called D3 normalized TrkA signals and improved memory in cognitive impairment models of ageing and an APP mouse model of Alzheimer's disease. Paradoxically, in young healthy mice chronic delivery of D3 caused impaired memory without impairing learning, a form of anterograde amnesia. Here, we use this as a model to study the mechanisms of impaired memory. In young healthy mice acute or chronic treatment with D3 induces hyperactivation of TrkA-mediated signals in hippocampus, and causes a deficit in hippocampal-dependent memory consolidation proximal to drug exposure, without affecting learning or memory retrieval. The impairment after acute drug exposure is reversible. The impairment after long-term drug exposure is irreversible, likely due to a decrease in hippocampal CA1 neuron basal arborization. These findings support the notion of a homeostatic role for TrkA in memory, and demonstrate the differential outcomes of TrkA (hyper)activation in healthy versus disease states.

Vaccination with Tumor-Ganglioside Glycomimetics Activates a Selective Immunity that Affords Cancer Therapy.

Immune targeting of (glyco)protein tumor markers has been useful to develop cancer and virus vaccines. However, the ganglioside family of tumor-associated glycolipids remains intractable to vaccine approaches. Here we show that synthetic antigens mimicking the carbohydrate moiety of GD2 or GD3 gangliosides can be used as vaccines to activate a selective humoral and cellular immunity that is therapeutic against several cancers expressing GD2 or GD3. Adoptive transfer of T cells generated after vaccination elicits tumor-infiltrating lymphocytes of the γδ T cell receptor and CD8+ phenotypes; and affords a high therapeutic index. The glycomimetic vaccine principles can be expanded to target the family of tumor gangliosides and other carbohydrates expressed primarily in pathological states.

The route of administration influences the therapeutic index of an anti-proNGF neutralizing mAb for experimental treatment of Diabetic Retinopathy.

Many neurodegenerative retinal diseases are treated with monoclonal antibodies (mAb) delivered by invasive intravitreal injection (IVT). In Diabetic Retinopathy there is a scarcity of effective agents that can be delivered using non-invasive methods, and there are significant challenges in the validation of novel therapeutic targets. ProNGF represents a potential novel target, and IVT administration of a function-blocking anti-proNGF mAb is therapeutic in a mouse model of DR. We therefore compared invasive IVT to less invasive systemic intravenous (IV) and local subconjunctival (SCJ) administration, for therapy of Diabetic Retinopathy. The IV and SCJ routes are safe, afford sustained pharmacokinetics and tissue penetration of anti-proNGF mAb, and result in long-term therapeutic efficacy that blocks retinal inflammation, edema, and neuronal death. SCJ may be a more convenient and less-invasive approach for ophthalmic use and may enable reduced frequency of intervention for the treatment of retinal pathologies.

UNG-1 and APN-1 are the major enzymes to efficiently repair 5-hydroxymethyluracil DNA lesions in C. elegans.

In Caenorhabditis elegans, two DNA glycosylases, UNG-1 and NTH-1, and two AP endonucleases, APN-1 and EXO-3, have been characterized from the base-excision repair (BER) pathway that repairs oxidatively modified DNA bases. UNG-1 removes uracil, while NTH-1 can remove 5-hydroxymethyluracil (5-hmU), an oxidation product of thymine, as well as other lesions. Both APN-1 and EXO-3 can incise AP sites and remove 3'-blocking lesions at DNA single strand breaks, and only APN-1 possesses 3'- to 5'-exonulease and nucleotide incision repair activities. We used C. elegans mutants to study the role of the BER pathway in processing 5-hmU. We observe that ung-1 mutants exhibited a decrease in brood size and lifespan, and an elevated level of germ cell apoptosis when challenged with 5-hmU. These phenotypes were exacerbated by RNAi downregulation of apn-1 in the ung-1 mutant. The nth-1 or exo-3 mutants displayed wild type phenotypes towards 5-hmU. We show that partially purified UNG-1 can act on 5-hmU lesion in vitro. We propose that UNG-1 removes 5-hmU incorporated into the genome and the resulting AP site is cleaved by APN-1 or EXO-3. In the absence of UNG-1, the 5-hmU is removed by NTH-1 creating a genotoxic 3'-blocking lesion that requires the action of APN-1.

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.

In retinitis pigmentosa TrkC.T1-dependent vectorial Erk activity upregulates glial TNF-α, causing selective neuronal death.

In some diseases the TrkC.T1 isoform is upregulated in glia, associated with glial TNF-α production and neuronal death. What remains unknown are the activating signals in glia, and how paracrine signals may be selective for a targeted neuron while sparing other proximate neurons. We studied these questions in the retina, where Müller glia contacts photoreceptors on one side and retinal ganglion cells on the other. In a mutant Rhodopsin mouse model of retinitis pigmentosa (RP) causing progressive photoreceptor death-but sparing retinal ganglion cells-TrkC.T1 and NT-3 ligand are upregulated in Müller glia. TrkC.T1 activity generates p-Erk, which causes increased TNF-α. These sequential events take place predominantly in Müller fibers contacting stressed photoreceptors, and culminate in selective death. Each event and photoreceptor death can be prevented by reduction of TrkC.T1 expression, by pharmacological antagonism of TrkC or by pharmacological inhibition Erk. Unmasking the sequence of non-cell autologous events and mechanisms causing selective neuronal death may help rationalize therapies.

p75NTR antagonists attenuate photoreceptor cell loss in murine models of retinitis pigmentosa.

ProNGF signaling through p75NTR has been associated with neurodegenerative disorders. Retinitis pigmentosa (RP) comprises a group of inherited retinal dystrophies that causes progressive photoreceptor cell degeneration and death, at a rate dependent on the genetic mutation. There are more than 300 mutations causing RP, and this is a challenge to therapy. Our study was designed to explore a common mechanism for p75NTR in the progression of RP, and assess its potential value as a therapeutic target. The proNGF/p75NTR system is present in the dystrophic retina of the rd10 RP mouse model. Compared with wild-type (WT) retina, the levels of unprocessed proNGF were increased in the rd10 retina at early degenerative stages, before the peak of photoreceptor cell death. Conversely, processed NGF levels were similar in rd10 and WT retinas. ProNGF remained elevated throughout the period of photoreceptor cell loss, correlating with increased expression of α2-macroglobulin, an inhibitor of proNGF processing. The neuroprotective effect of blocking p75NTR was assessed in organotypic retinal cultures from rd10 and RhoP mouse models. Retinal explants treated with p75NTR antagonists showed significantly reduced photoreceptor cell death, as determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and by preservation of the thickness of the outer nuclear layer (ONL), where photoreceptor nuclei are located. This effect was accompanied by decreased retinal-reactive gliosis and reduced TNFα secretion. Use of p75NTR antagonist THX-B (1,3-diisopropyl-1-[2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-purin-7-yl)-acetyl]-urea) in vivo in the rd10 and RhoP mouse models, by a single intravitreal or subconjunctival injection, afforded neuroprotection to photoreceptor cells, with preservation of the ONL. This study demonstrates a role of the p75NTR/proNGF axis in the progression of RP, and validates these proteins as therapeutic targets in two different RP models, suggesting utility irrespective of etiology.