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Alzheimer’s Disease (AD) is a prevalent neurodegenerative disease characterized by tau hyperphosphorylation, Aβ1-42 aggregation and cognitive dysfunction. Therapeutic agents directed at mitigating tau aggregation and clearing Aβ1-42, and delivery of growth factor genes (BDNF, FGF2), have ameliorated cognitive deficits, but these approaches did not prevent or stop AD progression. Here we report that viral-(AAV) delivery of Neurotrophic Factor-α1/Carboxypeptidase E (NF-α1/CPE) gene in hippocampus at an early age prevented later development of cognitive deficits as assessed by Morris water maze and novel object recognition assays, neurodegeneration, and tau hyperphosphorylation in male 3xTg-AD mice. Additionally, amyloid precursor protein (APP) expression was reduced to near non-AD levels, and insoluble Aβ1-42 was reduced significantly. Pro-survival proteins: mitochondrial Bcl2 and Serpina3g were increased; and mitophagy inhibitor Plin4 and pro-inflammatory protein Card14 were decreased in AAV-NF-α1/CPE treated versus untreated AD mice. Thus NF-α1/CPE gene therapy targets many regulatory components to prevent cognitive deficits in 3xTg-AD mice and has implications as a new therapy to prevent AD progression by promoting cell survival, inhibiting APP overexpression and tau hyperphosphorylation.

Neurotrophic Factor-α1/Carboxypeptidase E (NF-α1/CPE) is a pivotal neuroprotective protein implicated in rescuing cognitive decline associated with Alzheimer's disease (AD). However, its direct role in AD pathogenesis remains unexplored. We utilized the Cre/LoxP system to diminish NF-α1/CPE expression, and employed AAV-mediated overexpression of NF-α1/CPE. NF-α1/CPE expression was significantly down-regulated in advanced stages of AD and with age in 5xFAD mice. Reduced NF-α1/CPE levels in the hippocampus of 5xFAD mice increased plaque burden, microglial cell count, disrupted synaptogenesis, and intensified cognitive impairments at 5 and 7 months. However, by 9 months, no further progression of detrimental effects was observed. Overexpression of NF-α1/CPE markedly decreased amyloid plaque accumulation, mitigated spatial memory deficits, and normalized hippocampal synaptogenesis and microglial anomalies across early and late stages of the disease. NF-α1/CPE is a critical regulator of AD pathogenesis, offering promising therapeutic potential for reducing amyloid beta deposition and toxicity in AD.

Carboxypeptidase E is a peptide processing enzyme, involved in cleaving numerous peptide precursors, including neuropeptides and hormones involved in appetite control and glucose metabolism. Exome sequencing of a morbidly obese female from a consanguineous family revealed homozygosity for a truncating mutation of the CPE gene (c.76_98del; p.E26RfsX68). Analysis detected no CPE expression in whole blood-derived RNA from the proband, consistent with nonsense-mediated decay. The morbid obesity, intellectual disability, abnormal glucose homeostasis and hypogonadotrophic hypogonadism seen in this individual recapitulates phenotypes in the previously described fat/fat and Cpe knockout mouse models, evidencing the importance of this peptide/hormone-processing enzyme in regulating body weight, metabolism, and brain and reproductive function in humans.

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125 I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H 2 O 2 -induced oxidative stress. Cell survival assay in β-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was β-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited β-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of β-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the β-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

Deregulation of mTOR complex 1 (mTORC1) signalling increases the risk for metabolic diseases, including type 2 diabetes. Here we show that β-cell-specific loss of mTORC1 causes diabetes and β-cell failure due to defects in proliferation, autophagy, apoptosis and insulin secretion by using mice with conditional ( βraKO) and inducible ( MIP-βraKO f/f ) raptor deletion. Through genetic reconstitution of mTORC1 downstream targets, we identify mTORC1/S6K pathway as the mechanism by which mTORC1 regulates β-cell apoptosis, size and autophagy, whereas mTORC1/4E-BP2-eIF4E pathway regulates β-cell proliferation. Restoration of both pathways partially recovers β-cell mass and hyperglycaemia. This study also demonstrates a central role of mTORC1 in controlling insulin processing by regulating cap-dependent translation of carboxypeptidase E in a 4EBP2/eIF4E-dependent manner. Rapamycin treatment decreases CPE expression and insulin secretion in mice and human islets. We suggest an important role of mTORC1 in β-cells and identify downstream pathways driving β-cell mass, function and insulin processing. Deregulation of mTORC1 pathway has been associated with several human diseases including diabetes, neurodegeneration and cancer. Here Blandino-Rosano et al . show that mTORC1 signalling controls insulin secretion and β-cell maintenance by regulation of β-cell proliferation, apoptosis and autophagy and insulin processing.

Major depressive disorder is often linked to stress. Although short-term stress is without effect in mice, prolonged stress leads to depressive-like behavior, indicating that an allostatic mechanism exists in this difference. Here we demonstrate that mice after short-term (1 h per day for 7 days) chronic restraint stress (CRS), do not display depressive-like behavior. Analysis of the hippocampus of these mice showed increased levels of neurotrophic factor-α1 (NF-α1; also known as carboxypeptidase E, CPE), concomitant with enhanced fibroblast growth factor 2 (FGF2) expression, and an increase in neurogenesis in the dentate gyrus. In contrast, after prolonged (6 h per day for 21 days) CRS, mice show decreased hippocampal NF-α1 and FGF2 levels and depressive-like responses. In NF-α1-knockout mice, hippocampal FGF2 levels and neurogenesis are reduced. These mice exhibit depressive-like behavior that is reversed by FGF2 administration. Indeed, studies in cultured hippocampal neurons reveal that NF-α1 treatment directly upregulates FGF2 expression through extracellular signal-regulated kinase-Sp1 signaling. Thus, during short-term CRS, hippocampal NF-α1 expression is upregulated and has a key role in preventing the onset of depressive-like behavior through enhanced FGF2-mediated neurogenesis. To evaluate the therapeutic potential of this pathway, we examined, rosiglitazone (Rosi), a PPARγ agonist, which has been shown to have antidepressant activity in rodents and humans. Rosi upregulates FGF2 expression in a NF-α1-dependent manner in hippocampal neurons. Mice fed Rosi show increased hippocampal NF-α1 levels and neurogenesis compared with controls, thereby indicating the antidepressant action of this drug. Development of drugs that activate the NF-α1/FGF2/neurogenesis pathway can offer a new approach to depression therapy.

Carboxypeptidase E (CPE) is a multifunctional protein with many nonenzymatic functions in various systems. Previous studies using CPE knock-out mice have shown that CPE has neuroprotective effects against stress and is involved in learning and memory. However, the functions of CPE in neurons are still largely unknown. Here we used a Camk2a-Cre system to conditionally knockout CPE in neurons. The wild-type, CPEflox/−, and CPEflox/flox mice were weaned, ear-tagged, and tail clipped for genotyping at 3 weeks old, and they underwent open field, object recognition, Y-maze, and fear conditioning tests at 8 weeks old. The CPEflox/flox mice had normal body weight and glucose metabolism. The behavioral tests showed that CPEflox/flox mice had impaired learning and memory compared with wild-type and CPEflox/- mice. Surprisingly, the subiculum (Sub) region of CPEflox/flox mice was completely degenerated, unlike the CPE full knockout mice, which exhibit CA3 region neurodegeneration. In addition, doublecortin immunostaining suggested that neurogenesis in the dentate gyrus of the hippocampus was significantly reduced in CPEflox/flox mice. Interestingly, TrkB phosphorylation in the hippocampus was downregulated in CPEflox/flox mice, but brain-derived neurotrophic factor levels were not. In both the hippocampus and dorsal medial prefrontal cortex, we observed reduced MAP2 and GFAP expression in CPEflox/flox mice. Taken together, the results of this study demonstrate that specific neuronal CPE knockout leads to central nervous system dysfunction in mice, including learning and memory deficits, hippocampal Sub degeneration and impaired neurogenesis.

Carboxypeptidase E (CPE) plays an important role in the biosynthesis of neurotransmitters and peptide hormones including insulin. It also promotes cell proliferation, survival, and invasion of tumor cells. The endoplasmic reticulum stress, hypoxia, and nutrient supply are significant factors of malignant tumor growth including glioblastoma. There are data indicating that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) suppressed glioblastoma cell proliferation and increased invasiveness of these cells. The present study aims to investigate the regulation of the gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Human glioblastoma cells U87MG (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine; for glucose and glutamine deprivations, the cells were cultured in DMEM medium without glucose or glutamine for 16 h, respectively. The expression level of the gene was studied by quantitative RT-PCR and normalized to ACTB. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to a strong up-regulation of gene expression in glioblastoma cells. The expression of this gene also increased in glioblastoma cells after silencing ERN1. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the gene was resistant to hypoxia in control U87MG cells, but increased in cells with ERN1 knockdown. The expression of this gene was up-regulated under glutamine deprivation in control glioblastoma cells, but decreased upon ERN1 knockdown. However, glucose deprivation decreased the expression of gene in both types of used cells, but ERN1 inhibition enhanced this effect. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of pro-oncogenic gene through protein kinase activity of ERN1 and that increased gene expression possibly participates in ERN1 knockdown-mediated invasiveness of glioblastoma cells.

Mice lacking Carboxypeptidase E (CPE) exhibit degeneration of hippocampal neurons caused by stress at weaning while over-expression of CPE in hippocampal neurons protect them against hydrogen peroxide-induced cell death. Here we demonstrate that CPE acts as an extracellular trophic factor to protect neurons. Rat hippocampal neurons pretreated with purified CPE protected the cells against hydrogen peroxide-, staurosporine- and glutamate-induced cell death. This protection was observed even when hippocampal neurons were treated with an enzymatically inactive mutant CPE or with CPE in the presence of its inhibitor, GEMSA. Purified CPE added to the culture medium rescued CPE knock-out hippocampal neurons from cell death. Both ERK and AKT were phosphorylated within 15 min after CPE treatment of hippocampal neurons and, using specific inhibitors, both signaling pathways were shown to be required for the neuroprotective effect. The expression of the anti-apoptotic protein, B-cell lymphoma 2 (BCL-2), was up-regulated after hippocampal neurons were treated with CPE. Furthermore, hydrogen peroxide induced down-regulation of BCL-2 protein and subsequent activation of caspase-3 were inhibited by CPE treatment. Thus, this study has identified CPE as a new neurotrophic factor that can protect neurons against degeneration through the activation of ERK and AKT signaling pathways to up-regulate expression of BCL-2.

Prostate cancer (PCa) is a heterogeneous group of tumors with variable clinical courses. In order to improve patient outcomes, it is critical to clinically separate aggressive PCa (AG) from non-aggressive PCa (NAG). Although recent genomic studies have identified a spectrum of molecular abnormalities associated with aggressive PCa, it is still challenging to separate AG from NAG. To better understand the functional consequences of PCa progression and the unique features of the AG subtype, we studied the proteomic signatures of primary AG, NAG and metastatic PCa. 39 PCa and 10 benign prostate controls in a discovery cohort and 57 PCa in a validation cohort were analyzed using a data-independent acquisition (DIA) SWATH–MS platform. Proteins with the highest variances (top 500 proteins) were annotated for the pathway enrichment analysis. Functional analysis of differentially expressed proteins in NAG and AG was performed. Data was further validated using a validation cohort; and was also compared with a TCGA mRNA expression dataset and confirmed by immunohistochemistry (IHC) using PCa tissue microarray (TMA). 4,415 proteins were identified in the tumor and benign control tissues, including 158 up-regulated and 116 down-regulated proteins in AG tumors. A functional analysis of tumor-associated proteins revealed reduced expressions of several proteinases, including dipeptidyl peptidase 4 (DPP4), carboxypeptidase E (CPE) and prostate specific antigen (KLK3) in AG and metastatic PCa. A targeted analysis further identified that the reduced expression of DPP4 was associated with the accumulation of DPP4 substrates and the reduced ratio of DPP4 cleaved peptide to intact substrate peptide. Findings were further validated using an independently-collected tumor cohort, correlated with a TCGA mRNA dataset, and confirmed by immunohistochemical stains of PCa tumor microarray (TMA). Our study is the first large-scale proteomics analysis of PCa tissue using a DIA SWATH-MS platform. It provides not only an interrogative proteomic signature of PCa subtypes, but also indicates the critical roles played by certain proteinases during tumor progression. The spectrum map and protein profile generated in the study can be used to investigate potential biological mechanisms involved in PCa and for the development of a clinical assay to distinguish aggressive from indolent PCa.