Accelerated neurodegeneration of basal forebrain cholinergic neurons in HIV-1 gp120 transgenic mice: Critical role of the p75 neurotrophin receptor.

Human Immunodeficiency Virus-1 (HIV) infection of the brain induces HIV-associated neurocognitive disorders (HAND). The set of molecular events employed by HIV to drive cognitive impairments in people living with HIV are diverse and remain not completely understood. We have shown that the HIV envelope protein gp120 promotes loss of synapses and decreases performance on cognitive tasks through the p75 neurotrophin receptor (p75NTR). This receptor is abundant on cholinergic neurons of the basal forebrain and contributes to cognitive impairment in various neurological disorders. In this study, we examined cholinergic neurons of gp120 transgenic (gp120tg) mice for signs of degeneration. We observed that the number of choline acetyltransferase-expressing cells is decreased in old (12-14-month-old) gp120tg mice when compared to age matched wild type. In the same animals, we observed an increase in the levels of pro-nerve growth factor, a ligand of p75NTR, as well as a disruption of consolidation of extinction of conditioned fear, a behavior regulated by cholinergic neurons of the basal forebrain. Both biochemical and behavioral outcomes of gp120tg mice were rescued by the deletion of the p75NTR gene, strongly supporting the role that this receptor plays in the neurotoxic effects of gp120. These data indicate that future p75NTR-directed pharmacotherapies could provide an adjunct therapy against synaptic simplification caused by HIV.

ProNGF Expression and Targeting in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most lethal adult brain cancer. Temozolomide (TMZ), the standard chemotherapeutic drug used in GBM, has limited benefit and alternate therapies are needed to improve GBM treatment. Nerve growth factor (NGF) and its precursor proNGF are increasingly recognized as stimulators of human tumor progression. The expression and stimulatory effect of NGF on GBM cell growth has previously been reported, but the status of proNGF in GBM is unreported. In this study, we have investigated proNGF expression and biological activity in GBM. A clinical cohort of GBM (n = 72) and low-grade glioma (n = 20) was analyzed by immunohistochemistry for proNGF and digital quantification. ProNGF expression was significantly increased in GBM compared to low grade gliomas and proNGF was also detected in patient plasma samples. ProNGF was also detected in most GBM cell lines by Western blotting. Although anti-proNGF blocking antibodies inhibited cell growth in GBM cells with methylated MGMT gene promoter, targeting proNGF could not potentiate the efficacy of TMZ. In subcutaneous xenograft of human GBM cells, anti-proNGF antibodies slightly reduced tumor volume but had no impact on TMZ efficacy. In conclusion, this data reveals that proNGF is overexpressed in GBM and can stimulate cancer cell growth. The potential of proNGF as a clinical biomarker and therapeutic target warrants further investigations.

Expression of NGF/proNGF and Their Receptors TrkA, p75NTR and Sortilin in Melanoma.

There is increasing evidence that nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA), the common neurotrophin receptor (NGFR/p75NTR) and the membrane receptor sortilin, participate in cancer growth. In melanoma, there have been some reports suggesting that NGF, TrkA and p75NTR are dysregulated, but the expression of the NGF precursor (proNGF) and its membrane receptor sortilin is unknown. In this study, we investigated the expression of NGF, proNGF, TrkA, p75NTR and sortilin by immunohistochemistry in a series of human tissue samples (n = 100), including non-cancerous nevi (n = 20), primary melanomas (n = 40), lymph node metastases (n = 20) and distant metastases (n = 20). Immunostaining was digitally quantified and revealed NGF and proNGF were expressed in all nevi and primary melanomas, and that the level of expression decreased from primary tumors to melanoma metastases (p = 0.0179 and p < 0.0001, respectively). Interestingly, TrkA protein expression was high in nevi and thin primary tumors but was strongly downregulated in thick primary tumors (p < 0.0001) and metastases (p < 0.0001). While p75NTR and sortilin were both expressed in most nevi and melanomas, there was no significant difference in expression between them. Together, these results pointed to a downregulation of NGF/ProNGF and TrkA in melanoma, and thus did not provide evidence to support the use of anti-proNGF/NGF or anti-TrkA therapies in advanced and metastatic forms of melanoma.

A new role for matrix metalloproteinase-3 in the NGF metabolic pathway: Proteolysis of mature NGF and sex-specific differences in the continuum of Alzheimer's pathology.

Matrix metalloproteinase-3 (MMP-3) has been associated with risk of Alzheimer's disease (AD). In this study we introduce a novel role for MMP-3 in degrading nerve growth factor (NGF) in vivo and examine its mRNA and protein expression across the continuum of AD pathology. We provide evidence that MMP-3 participates in the degradation of mature NGF in vitro and in vivo and that it is secreted from the rat cerebral cortex in an activity-dependent manner. We show that cortical MMP-3 is upregulated in the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis. A similar upregulation was found in AD and MCI brains as well as in cognitively normal individuals with elevated amyloid deposition. We also observed that frontal cortex MMP-3 protein levels are higher in males. MMP-3 protein correlated with more AD neuropathology, markers of NGF metabolism, and lower cognitive scores in males but not in females. These results suggest that MMP-3 upregulation in AD might contribute to NGF dysmetabolism, and therefore to cholinergic atrophy and cognitive deficits, in a sex-specific manner. MMP-3 should be further investigated as a biomarker candidate or as a therapeutic target in AD.

Oral Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Improves Erectile Function in a Mouse Model of Cavernous Nerve Injury.

BACKGROUND: Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI), but also causes cavernous hypoxia and cavernous structural changes, which lead to a poor response to phosphodiesterase 5 inhibitors. AIM: To investigate the therapeutic effect of oral administration of LM11A-31, a small molecule p75 neurotrophin receptor (p75NTR) ligand and proNGF antagonist, in a mouse model of bilateral CNI, which mimics nerve injury-induced erectile dysfunction after radical prostatectomy. METHODS: 8-week-old male C57BL/6 mice were divided into sham operation and CNI groups. Each group was divided into 2 subgroups: phosphate-buffered saline and LM11A-31 (50 mg/kg/day) being administered once daily starting 3 days before CNI via oral gavage. 2 weeks after CNI, we measured erectile function by electrical stimulation of the bilateral cavernous nerve. The penis was harvested for histologic examination and Western blot analysis. The major pelvic ganglia was harvested and cultured for assays of ex vivo neurite outgrowth. OUTCOMES: Intracavernous pressure, neurovascular regeneration in the penis, in vivo or ex vivo functional evaluation, and cell survival signaling were measured. RESULTS: Erectile function was decreased in the CNI group (44% of the sham operation group), while administration of LM11A-31 led to a significant improvement of erectile function (70% of the sham operation group) in association with increased neurovascular content, including cavernous endothelial cells, pericytes, and neuronal processes. Immunohistochemical and Western blot analyses showed significantly increased p75NTR expression in the dorsal nerve of CNI mice, which was attenuated by LM11A-31 treatment. Protein expression of active PI3K, AKT, and endothelial nitric oxide synthase was increased, and cell death and c-Jun N-terminal kinase signaling was significantly attenuated after LM11A-31 treatment. Furthermore, LM11A-31 promoted neurite sprouting in cultured major pelvic ganglia after lipopolysaccharide exposure. CLINICAL IMPLICATIONS: LM11A-31 may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: Unlike biological therapeutics, such as proteins, gene therapies, or stem cells, the clinical application of LM11A-31 would likely be relatively less complex and low cost. Our study has some limitations. Future studies will assess the optimal dosing and duration of the compound. Given its plasma half-life of approximately 1 hour, it is possible that dosing more than once per day will provide added efficacy. CONCLUSION: Specific inhibition of the proNGF-p75NTR degenerative signaling via oral administration of LM11A-31 represents a novel therapeutic strategy for erectile dysfunction induced by nerve injury. Yin GN, Ock J, Limanjaya A, et al. Oral Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Improves Erectile Function in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2021;18:17-28.

Imbalance of nerve growth factor metabolism in aging women with overactive bladder syndrome.

PURPOSE: Given the disputable link between nerve growth factor (NGF) and overactive bladder syndrome (OAB) and the lack of studies on its precursor (proNGF) in OAB, the aim of the study was to identify changes in the urinary levels of NGF and its proteolytic enzymes in aging women with OAB. METHODS: We examined the urinary proNGF/NGF ratio and its processing enzymes in aging women (50-80 years), comparing 20 controls and 20 subjects with OAB. RESULTS: In contrast to previous reports correlating NGF to OAB symptoms, we found that proNGF/NGF ratio in the OAB group was twice as high compared to controls (p = 0.009) with a lower NGF levels in women with OAB without statistical significance [1.36 (Q1, Q3: 0.668, 2.39) vs. 1.7 (Q1, Q3: 1.27, 3.045) pg/mg creatinine in control group, p = 0.05]. Enzymatic activity of MMP-7, the main enzyme for extracellular proNGF maturation, was significantly increased in the OAB group and correlated positively with scores of OAB symptoms questionnaires. However, this was counteracted by several-folds increase in the MMP-9 enzyme responsible for NGF proteolysis. While these findings highlight the importance of changes in the proteolytic enzymes to maintain proNGF/NGF balance in OAB, analysis of covariates showed that these changes were attributed to age, insulin resistance and renal function. CONCLUSION: NGF proteolysis imbalance can be clinically meaningful in OAB related to aging, rendering it as a potential therapeutic target. However, other age-related factors such as insulin resistance and renal function may contribute to the relationship between NGF and aging-related OAB phenotype.

Nerve growth factor (NGF) pathway biomarkers in Down syndrome prior to and after the onset of clinical Alzheimer's disease: A paired CSF and plasma study.

BACKGROUND: The discovery that nerve growth factor (NGF) metabolism is altered in Down syndrome (DS) and Alzheimer's disease (AD) brains offered a framework for the identification of novel biomarkers signalling NGF deregulation in AD pathology. METHODS: We examined levels of NGF pathway proteins (proNGF, neuroserpin, tissue plasminogen activator [tPA], and metalloproteases [MMP]) in matched cerebrospinal fluid (CSF)/plasma samples from AD-symptomatic (DSAD) and AD-asymptomatic (aDS) individuals with DS, as well as controls (HC). RESULTS: ProNGF and MMP-3 were elevated while tPA was decreased in plasma from individuals with DS. CSF from individuals with DS showed elevated proNGF, neuroserpin, MMP-3, and MMP-9. ProNGF and MMP-9 in CSF differentiated DSAD from aDS (area under the curve = 0.86, 0.87). NGF pathway markers associated with CSF amyloid beta and tau and differed by sex. DISCUSSION: Brain NGF metabolism changes can be monitored in plasma and CSF, supporting relevance in AD pathology. These markers could assist staging, subtyping, or precision medicine for AD in DS.

proNGF Involvement in the Adult Neurogenesis Dysfunction in Alzheimer's Disease.

In recent decades, neurogenesis in the adult brain has been well demonstrated in a number of animal species, including humans. Interestingly, work with rodents has shown that adult neurogenesis in the dentate gyrus (DG) of the hippocampus is vital for some cognitive aspects, as increasing neurogenesis improves memory, while its disruption triggers the opposite effect. Adult neurogenesis declines with age and has been suggested to play a role in impaired progressive learning and memory loss seen in Alzheimer's disease (AD). Therefore, therapeutic strategies designed to boost adult hippocampal neurogenesis may be beneficial for the treatment of AD. The precursor forms of neurotrophins, such as pro-NGF, display remarkable increase during AD in the hippocampus and entorhinal cortex. In contrast to mature NGF, pro-NGF exerts adverse functions in survival, proliferation, and differentiation. Hence, we hypothesized that pro-NGF and its p75 neurotrophin receptor (p75NTR) contribute to disrupting adult hippocampal neurogenesis during AD. To test this hypothesis, in this study, we took advantage of the availability of mouse models of AD (APP/PS1), which display memory impairment, and AD human samples to address the role of pro-NGF/p75NTR signaling in different aspects of adult neurogenesis. First, we observed that DG doublecortin (DCX) + progenitors express p75NTR both, in healthy humans and control animals, although the percentage of DCX+ cells are significantly reduced in AD. Interestingly, the expression of p75NTR in these progenitors is significantly decreased in AD conditions compared to controls. In order to assess the contribution of the pro-NGF/p75NTR pathway to the memory deficits of APP/PS1 mice, we injected pro-NGF neutralizing antibodies (anti-proNGF) into the DG of control and APP/PS1 mice and animals are subjected to a Morris water maze test. Intriguingly, we observed that anti-pro-NGF significantly restored memory performance of APP/PS1 animals and significantly increase the percentage of DCX+ progenitors in the DG region of these animals. In summary, our results suggest that pro-NGF is involved in disrupting spatial memory in AD, at least in part by blocking adult neurogenesis. Moreover, we propose that adult neurogenesis alteration should be taken into consideration for better understanding of AD pathology. Additionally, we provide a new molecular entry point (pro-NGF/p75NTR signaling) as a promising therapeutic target in AD.

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.

ProNGF Is a Cell-Type-Specific Mitogen for Adult Hippocampal and for Induced Neural Stem Cells.

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.

Expression and Prognostic Significance of Neurotrophins and Their Receptors in Canine Mammary Tumors.

Accumulating data highlight the role of neurotrophins and their receptors in human breast cancer. This family includes nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), both synthetized as proneurotrophins (proNGF and proBDNF). (pro)NGF and (pro)BDNF initiate their biological effects by binding to both their specific receptors TrkA and TrkB, respectively, and the common receptor p75NTR. Currently, no data are available about their expression and potential role in canine mammary tumors. The aim of this study was to investigate expression of proNGF and BDNF as well as their receptors TrkA, TrkB, and p75NTR in canine mammary carcinomas, and to correlate them with clinicopathological parameters (grade, histological type, lymph node status, recurrence, and distant metastasis) and survival. Immunohistochemistry was performed on serial sections of 96 canine mammary carcinomas with antibodies against proNGF, BDNF, TrkA, TrkB, and p75NTR. Of the 96 carcinomas, proNGF expression was detected in 71 (74%), BDNF in 79 (82%), TrkA in 94 (98%), TrkB in 35 (37%), and p75NTR in 44 (46%). No association was observed between proNGF, BDNF, or TrkA expression and either clinicopathological parameters or survival. TrkB and p75NTR expression were associated with favorable clinicopathological parameters as well as better overall survival.

Modulation of the p75 neurotrophin receptor using LM11A-31 prevents diabetes-induced retinal vascular permeability in mice via inhibition of inflammation and the RhoA kinase pathway.

AIMS/HYPOTHESIS: Breakdown of the inner blood-retinal barrier (BRB) is an early event in the pathogenesis of diabetic macular oedema, that eventually leads to vision loss. We have previously shown that diabetes causes an imbalance of nerve growth factor (NGF) isoforms resulting in accumulation of its precursor proNGF and upregulation of the p75 neurotrophin receptor (p75NTR), with consequent increases in the activation of Ras homologue gene family, member A (RhoA). We also showed that genetic deletion of p75NTR in diabetes preserved the BRB and prevented inflammatory mediators in retinas. This study aims to examine the therapeutic potential of LM11A-31, a small-molecule p75NTR modulator and proNGF antagonist, in preventing diabetes-induced BRB breakdown. The study also examined the role of p75NTR/RhoA downstream signalling in mediating cell permeability. METHODS: Male C57BL/6 J mice were rendered diabetic using streptozotocin injection. After 2 weeks of diabetes, mice received oral gavage of LM11A-31 (50 mg kg-1 day-1) or saline (NaCl 154 mmol/l) for an additional 4 weeks. BRB breakdown was assessed by extravasation of BSA-AlexaFluor-488. Direct effects of proNGF were examined in human retinal endothelial (HRE) cells in the presence or absence of LM11A-31 or the Rho kinase inhibitor Y-27632. RESULTS: Diabetes triggered BRB breakdown and caused significant increases in circulatory and retinal TNF-α and IL-1ß levels. These effects coincided with significant decreases in retinal NGF and increases in vascular endothelial growth factor and proNGF expression, as well as activation of RhoA. Interventional modulation of p75NTR activity through treatment of mouse models of diabetes with LM11A-31 significantly mitigated proNGF accumulation and preserved BRB integrity. In HRE cells, treatment with mutant proNGF (10 ng/ml) triggered increased cell permeability with marked reduction of expression of tight junction proteins, zona occludens-1 (ZO-1) and claudin-5, compared with control, independent of inflammatory mediators or cell death. Modulating p75NTR significantly inhibited proNGF-mediated RhoA activation, occludin phosphorylation (at serine 490) and cell permeability. ProNGF induced redistribution of ZO-1 in the cell wall and formation of F-actin stress fibres; these effects were mitigated by LM11A-31. CONCLUSIONS/INTERPRETATION: Targeting p75NTR signalling using LM11A-31, an orally bioavailable receptor modulator, may offer an effective, safe and non-invasive therapeutic strategy for treating macular oedema, a major cause of blindness in diabetes.

Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse.

INTRODUCTION: Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in diabetic patients, which causes poor response to oral phosphodiesterase-5 inhibitors. Nerve growth factor precursor (proNGF) and its p75 neurotrophin receptor (p75NTR) have been known to be involved in microvascular complications and neurodegeneration. AIM: To examine the role of proNGF and its receptor p75NTR signaling pathway in diabetic ED, and to determine the effectiveness of proNGF neutralizing antibody (proNGF-Ab) in restoring erectile function in streptozotocin (STZ)-induced diabetic mice. METHODS: Diabetes mellitus was induced by intraperitoneal injection of STZ (50 mg/kg) into 8-week-old C57BL/6 male mice for 5 consecutive days. At 8 weeks after the induction of diabetes mellitus, the animals were distributed into 3 groups: controls and STZ-induced diabetic mice receiving 2 intracavernous injections of either saline (days -3 and 0; 20 µL) or proNGF-Ab (days -3 and 0; 20 µg in 20 µL of saline). We also examined the effect of proNGF-Ab or p75NTR small interfering RNA in primary cultured mouse cavernous endothelial cells, pericytes, and major pelvic ganglion. MAIN OUTCOME MEASURES: Erectile function was measured by electrical stimulation of the cavernous nerve at 2 weeks after treatment, and the penis was then harvested for histologic and biochemical studies. RESULTS: The cavernous expression of proNGF and p75NTR was upregulated under diabetic conditions. Intracavernous injection of proNGF-Ab successfully restored erectile function in diabetic mice, which reach 93-96% of control values. ProNGF-Ab significantly restored cavernous endothelial cell, pericyte, and neuronal cell content, and increased endothelial cell-to-cell junction proteins in the diabetic mice. Under the high-glucose condition, proNGF-Ab or p75NTR small interfering RNA promoted tube formation in mouse cavernous endothelial cells and pericytes, decreased apoptosis of endothelial cells and pericytes, and enhanced neurite sprouting from major pelvic ganglion. CLINICAL IMPLICATIONS: The ProNGF/p75NTR pathway will be a new therapeutic target for diabetic ED. STRENGTH & LIMITATIONS: This is the first study demonstrating the efficacy of the inhibition of proNGF/p75NTR pathway in diabetic ED. Further studies are needed to test whether a different dosing of proNGF-Ab would induce more durable erectile function recovery. CONCLUSION: Our findings suggest that inhibition of the proNGF/p75NTR signaling pathway is a promising therapeutic strategy for diabetic ED. Nguyen NM, Song K-M, Choi M-J, et al. Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse. J Sex Med 2019;16:351-364.

The precursor for nerve growth factor (proNGF) is not a serum or biopsy-rinse biomarker for thyroid cancer diagnosis.

BACKGROUND: Nerves and neurotrophic growth factors are emerging promoters of cancer growth. The precursor for Nerve Growth Factor (proNGF) is overexpressed in thyroid cancer, but its potential role as a clinical biomarker has not been reported. Here we have examined the value of proNGF as a serum and biopsy-rinse biomarker for thyroid cancer diagnosis. METHODS: Patients presenting for thyroid surgery or biopsy were enrolled in separate cohorts examining serum (n = 204, including 46 cases of thyroid cancer) and biopsy-rinse specimens (n = 188, including 26 cases of thyroid cancer). ProNGF levels in clinical samples were analysed by ELISA. Univariate and multivariate statistical analyses were used to compare proNGF levels with malignancy status and clinicopathological parameters. RESULTS: ProNGF was not detected in the majority of serum samples (176/204, 86%) and the detection of proNGF was not associated with thyroid cancer diagnosis. In the few cases where proNGF was detected in the serum, thyroidectomy did not affect proNGF concentration, demonstrating that the thyroid was not the source of serum proNGF. Intriguingly, an association between hyperthyroidism and serum proNGF was observed (OR 3.3, 95% CI 1.6-8.7 p = 0.02). In biopsy-rinse, proNGF was detected in 73/188 (39%) cases, with no association between proNGF and thyroid cancer. However, a significant positive association between follicular lesions and biopsy-rinse proNGF was found (OR 3.3, 95% CI 1.2-8.7, p = 0.02). CONCLUSIONS: ProNGF levels in serum and biopsy-rinse are not increased in thyroid cancer and therefore proNGF is not a clinical biomarker for this condition.

The Precursor for Nerve Growth Factor (proNGF) in Thyroid Cancer Lymph Node Metastases: Correlation with Primary Tumour and Pathological Variables.

Metastases in thyroid cancer are associated with aggressive disease and increased patient morbidity, but the factors driving metastatic progression are unclear. The precursor for nerve growth factor (proNGF) is increased in primary thyroid cancers, but its expression or significance in metastases is not known. In this study, we analysed the expression of proNGF in a retrospective cohort of thyroid cancer lymph node metastases (n = 56), linked with corresponding primary tumours, by automated immunohistochemistry and digital quantification. Potential associations of proNGF immunostaining with clinical and pathological parameters were investigated. ProNGF staining intensity (defined by the median h-score) was significantly higher in lymph node metastases (h-score 94, interquartile range (IQR) 50-147) than in corresponding primary tumours (57, IQR 42-84) (p = 0.002). There was a correlation between proNGF expression in primary tumours and corresponding metastases, where there was a 0.68 (95% CI 0 to 1.2) increase in metastatic tumour h-score for each unit increase in the primary tumour h-score. However, larger tumours (both primary and metastatic) had lower proNGF expression. In a multivariate model, proNGF expression in nodal metastases was negatively correlated with lateral neck disease and being male. In conclusion, ProNGF is expressed in locoregional metastases of thyroid cancer and is higher in lymph node metastases than in primary tumours, but is not associated with high-risk clinical features.

Decreased serum proNGF concentration in patients with Parkinson's disease.

Parkinson's disease (PD), a progressive and age-related neurodegenerative condition, is a common neurodegenerative disorder. However, no validated biomarkers for PD have been identified to date. Accumulating evidence supports the role of proNGF-p75NTR-sortilin signaling in the neurodegeneration and pathogenesis of PD. The aim of our study was to investigate alterations in serum proNGF concentrations in PD patients and related anxiety. Seventy-seven consecutive PD patients and 39 healthy controls were enrolled, and clinical data were collected. Modified Hoehn-Yahr Staging Scale, Unified Parkinson's Disease Rating Scale (UPDRS), and Hamilton Anxiety (HAMA) Scale scores were assessed upon admission. Serum proNGF concentration was compared between that of PD patients and healthy controls. Pearson correlation coefficients were determined to explore the relationship between proNGF concentration and UPDRS, Hoehn-Yahr, and HAMA scores. Received operating characteristic (ROC) curves and proNGF optimal cutoff point were used to distinguish PD and related anxiety. The median concentration of proNGF was significantly lower (p = 0.000) in PD patients (94.91 ng/L, range 85.92-118.06 ng/L) compared with that of healthy controls (106.67 ng/L, range 102.39-122.06 ng/L). The optimal proNGF cutoff point for distinguishing PD patients was 102.29 ng/L, and the sensitivity and specificity values were 87.0 and 100%, respectively. proNGF concentration positively correlated with UPDRS (r = 0.281, p = 0.013), Hoehn-Yahr (r = 0.260, p = 0.023), and HAMA (r = 0.276, p = 0.015) scores. Our results indicate that serum proNGF concentration may represent a biomarker for PD and its role in the pathogenesis of PD thus warrants further investigation.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF.

Differential deregulation of NGF and BDNF neurotrophins in a transgenic rat model of Alzheimer's disease.

Evidence from human neuropathological studies indicates that the levels of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are compromised in Alzheimer's disease. However, the causes and temporal (pathology-dependent) evolution of these alterations are not completely understood. To elucidate these issues, we investigated the McGill-R-Thy1-APP transgenic rat, which exhibits progressive intracellular and extracellular amyloid-beta (Aß) pathology and ensuing cognitive deficits. Neurochemical analyses revealed a differential dysregulation of NGF and BDNF transcripts and protein expression. While BDNF mRNA levels were significantly reduced at very early stages of amyloid pathology, before plaques appeared, there were no changes in NGF mRNA expression even at advanced stages. Paradoxically, the protein levels of the NGF precursor were increased. These changes in neurotrophin expression are identical to those seen during the progression of Alzheimer's disease. At advanced pathological stages, deficits in the protease cascade controlling the maturation and degradation of NGF were evident in McGill transgenic rats, in line with the paradoxical upregulation of proNGF, as seen in Alzheimer's disease, in the absence of changes in NGF mRNA. The compromise in NGF metabolism and BDNF levels was accompanied by downregulation of cortical cholinergic synapses; strengthening the evidence that neurotrophin dysregulation affects cholinergic synapses and synaptic plasticity. Our findings suggest a differential temporal deregulation of NGF and BDNF neurotrophins, whereby deficits in BDNF mRNA appear at early stages of intraneuronal Aß pathology, before alterations in NGF metabolism and cholinergic synapse loss manifest.

ProNGF, but Not NGF, Switches from Neurotrophic to Apoptotic Activity in Response to Reductions in TrkA Receptor Levels.

Nerve growth factor (NGF) promotes the survival and differentiation of neurons. NGF is initially synthesized as a precursor, proNGF, which is the predominant form in the central nervous system. NGF and proNGF bind to TrkA/p75NTR to mediate cell survival and to sortilin/p75NTR to promote apoptosis. The ratio of TrkA to p75NTR affects whether proNGF and mature NGF signal cell survival or apoptosis. The purpose of this study was to determine whether the loss of TrkA influences p75NTR or sortilin expression levels, and to establish whether proNGF and mature NGF have a similar ability to switch between cell survival and cell death. We systematically altered TrkA receptor levels by priming cells with NGF, using small interfering RNA, and using the mutagenized PC12nnr5 cell line. We found that both NGF and proNGF can support cell survival in cells expressing TrkA, even in the presence of p75NTR and sortilin. However, when TrkA is reduced, proNGF signals cell death, while NGF exhibits no activity. In the absence of TrkA, proNGF-induced cell death occurs, even when p75NTR and sortilin levels are reduced. These results show that proNGF can switch between neurotrophic and apoptotic activity in response to changes in TrkA receptor levels, whereas mature NGF cannot. These results also support the model that proNGF is neurotrophic under normal circumstances, but that a loss in TrkA in the presence of p75NTR and sortilin, as occurs in neurodegenerative disease or injury, shifts proNGF, but not NGF, signalling from cell survival to cell death.

Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation.

The p75 neurotrophin receptor (p75(NTR)) is a multifunctional receptor that participates in many critical processes in the nervous system, ranging from apoptosis to synaptic plasticity and morphological events. It is a member of the tumor necrosis factor receptor (TNFR) superfamily, whose members undergo trimeric oligomerization. Interestingly, p75(NTR) interacts with dimeric ligands (i.e., proneurotrophins or mature neurotrophins), but several of the intracellular adaptors that mediate p75(NTR) signaling are trimeric (i.e., TNFR-associated factor 6 or TRAF6). Consequently, the active receptor signaling unit remains uncertain. To identify the functional receptor complex, we evaluated its oligomerization in vitro and in mice brain tissues using a combination of biochemical techniques. We found that the most abundant homotypic arrangement for p75(NTR) is a trimer and that monomers and trimers coexist at the cell surface. Interestingly, trimers are not required for ligand-independent or ligand-dependent p75(NTR) activation in a growth cone retraction functional assay. However, monomers are capable of inducing acute morphological effects in neurons. We propose that p75(NTR) activation is regulated by its oligomerization status and its levels of expression. These results indicate that the oligomeric state of p75(NTR) confers differential responses and offers an explanation for the diverse and contradictory actions of this receptor in the nervous system. SIGNIFICANCE STATEMENT: The p75 neurotrophin receptor (p75(NTR)) regulates a wide range of cellular functions, including apoptosis, neuronal processes remodeling, and synaptic plasticity. The goal of our work was to inquire whether oligomers of the receptor are required for function. Here we report that p75(NTR) predominantly assembles as a trimer, similar to other tumor necrosis factor receptors. Interestingly, monomers and trimers coexist at the cell surface, but trimers are not required for p75(NTR) activation in a functional assay. However, monomers are capable of inducing acute morphological effects in neurons. Identification of the oligomerization state of p75(NTR) begins to provide insights to the mechanisms of signal initiation of this noncatalytic receptor, as well as to develop therapeutic interventions to diminish its activity.