Decreased peripheral brain-derived neurotrophic factor levels in Alzheimer's disease: a meta-analysis study (N=7277).

Studies suggest that dysfunction of brain-derived neurotrophic factor (BDNF) is a possible contributor to the pathology and symptoms of Alzheimer's disease (AD). Several studies report reduced peripheral blood levels of BDNF in AD, but findings are inconsistent. This study sought to quantitatively summarize the clinical BDNF data in patients with AD and mild cognitive impairment (MCI, a prodromal stage of AD) with a meta-analytical technique. A systematic search of Pubmed, PsycINFO and the Cochrane Library identified 29 articles for inclusion in the meta-analysis. Random-effects meta-analysis showed that patients with AD had significantly decreased baseline peripheral blood levels of BDNF compared with healthy control (HC) subjects (24 studies, Hedges' g=-0.339, 95% confidence interval (CI)=-0.572 to -0.106, P=0.004). MCI subjects showed a trend for decreased BDNF levels compared with HC subjects (14 studies, Hedges' g=-0.201, 95% CI=-0.413 to 0.010, P=0.062). No differences were found between AD and MCI subjects in BDNF levels (11 studies, Hedges' g=0.058, 95% CI=-0.120 to 0.236, P=0.522). Interestingly, the effective sizes and statistical significance improved after excluding studies with reported medication in patients (between AD and HC: 18 studies, Hedges' g=-0.492, P<0.001; between MCI and HC: 11 studies, Hedges' g=-0.339, P=0.003). These results strengthen the clinical evidence that AD or MCI is accompanied by reduced peripheral blood BDNF levels, supporting an association between the decreasing levels of BDNF and the progression of AD.

A human carboxypeptidase E/NF-α1 gene mutation in an Alzheimer's disease patient leads to dementia and depression in mice.

Patients with Alzheimer's disease (AD), a common dementia among the aging population, often also suffer from depression. This comorbidity is poorly understood. Although most forms of AD are not genetically inherited, we have identified a new human mutation in the carboxypeptidase E (CPE)/neurotrophic factor-α1 (NF-α1) gene from an AD patient that caused memory deficit and depressive-like behavior in transgenic mice. This mutation consists of three adenosine inserts, introducing nine amino acids, including two glutamines into the mutant protein, herein called CPE-QQ. Expression of CPE-QQ in Neuro2a cells demonstrated that it was not secreted, but accumulated in the endoplasmic reticulum and was subsequently degraded by proteasomes. Expression of CPE-QQ in rat hippocampal neurons resulted in cell death, through increased ER stress and decreased expression of pro-survival protein, BCL-2. Transgenic mice expressing CPE-QQ did not show any difference in the processing enzyme activity of CPE compared with wild-type mice. However, the transgenic mice exhibited poor memory, depressive-like behavior, severely decreased dendrites in the hippocampal CA3 region and medial prefrontal cortex indicative of neurodegeneration, hyperphosphorylation of tau at Ser396, and diminished neurogenesis in the dentate gyrus at 50 weeks old. All these pathologies are associated with AD and the latter with depression and were observed in 50-week-old mice. Interestingly, the younger CPE-QQ mice (11 weeks old) did not show deficits in dendrite outgrowth and neurogenesis. This study has uncovered a human CPE/NF-α1 gene mutation that could lead to comorbidity of dementia and depression, emphasizing the importance of this gene in cognitive function.

Neurotrophic factor-α1 prevents stress-induced depression through enhancement of neurogenesis and is activated by rosiglitazone.

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.

Toward gene therapy for growth hormone deficiency via salivary gland expression of growth hormone.

OBJECTIVES: Salivary glands are useful targets for gene therapeutics. After gene transfer into salivary glands, regulated secretory pathway proteins, such as human growth hormone, are secreted into saliva, whereas constitutive secretory pathway proteins, such as erythropoietin, are secreted into the bloodstream. Secretion of human growth hormone (hGH) into the saliva is not therapeutically useful. In this study, we attempted to redirect the secretion of transgenic hGH from the saliva to the serum by site-directed mutagenesis. MATERIALS AND METHODS: We tested hGH mutants first in vitro with AtT20 cells, a model endocrine cell line that exhibits polarized secretion of regulated secretory pathway proteins. Selected mutants were further studied in vivo using adenoviral-mediated gene transfer to rat submandibular glands. RESULTS: We identified two mutants with differences in secretion behavior compared to wild-type hGH. One mutant, ΔN1-6 , was detected in the serum of transduced rats, demonstrating that expression of this mutant in the salivary gland resulted in its secretion through the constitutive secretory pathway. CONCLUSION: This study demonstrates that mutagenesis of therapeutic proteins normally destined for the regulated secretory pathway may result in their secretion via the constitutive secretory pathway into the circulation for potential therapeutic benefit.

Carboxypeptidase E protects hippocampal neurons during stress in male mice by up-regulating prosurvival BCL2 protein expression.

Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocampus during chronic restraint stress (CRS), and the molecular mechanisms involved. Quantitative RT-PCR/in situ hybridization and Western blots were used to assay for mRNA and protein. After mild CRS (1 h/d for 7 d), CPE protein and mRNA were significantly elevated in the hippocampal CA3 region, compared to naïve littermates. In addition, luciferase reporter assays identified a functional glucocorticoid regulatory element within the cpe promoter that mediated the up-regulation of CPE expression in primary hippocampal neurons following dexamethasone treatment, suggesting that circulating plasma glucocorticoids could evoke a similar effect on CPE in the hippocampus in vivo. Overexpression of CPE in hippocampal neurons, or CRS in mice, resulted in elevated prosurvival BCL2 protein/mRNA and p-AKT levels in the hippocampus; however, CPE(-/-) mice showed a decrease. Thus, during mild CRS, CPE expression is up-regulated, possibly contributed by glucocorticoids, to mediate neuroprotection of the hippocampus by enhancing BCL2 expression through AKT signaling, and thereby maintaining allostasis.

Serpinins: role in granule biogenesis, inhibition of cell death and cardiac function.

Serpinins are a family of peptides derived from proteolytic cleavage of the penultimate and the last pair of basic residues at the C-terminus of Chromogranin A. Three forms of naturally occurring serpinin have been found in AtT-20 pituitary cells and rat heart. They are serpinin, pyrogutaminated (pGlu) -serpinin and a C-terminally extended form, serpinin-RRG. In addition pGlu-serpinin has been found in brain, primarily in neurites and nerve terminals and shown to have protective effects against oxidative stress on neurons and pituitary cells. Serpinin has also been demonstrated to regulate granule biogenesis in endocrine cells by up-regulating the protease inhibitor, protease nexin-1 transcription via a cAMP-PKA-sp1 pathway. This leads to inhibition of granule protein degradation in the Golgi complex which in turn promotes granule formation. More recently, pGlu-serpinin has been demonstrated to enhance both myocardial contractility (inotropy) and relaxation (lusitropy). In the Langendorff perfused rat heart, pGlu-serpinin showed a concentration-dependent positive inotropic effect exerted through a cAMP-PKA dependent pathway. In conclusion, the serpinin peptides have profound effects at many levels that affect the endocrine and nervous systems and cardiac function.

Oligomerization of pro-opiomelanocortin is independent of pH, calcium and the sorting signal for the regulated secretory pathway.

Studies indicate that pro-opiomelanocortin (POMC) is sorted to the regulated secretory pathway by binding to a sorting receptor identified as membrane-bound carboxypeptidase E (CPE) [Cool et al. (1997) Cell 88, 73-83]. The efficiency of this sorting mechanism could be enhanced if POMC molecules were to self-associate to form oligomers, prior or subsequent to binding to CPE. Using cross-linking and gel filtration techniques, we demonstrated that POMC forms oligomers at both neutral and acidic pHs and calcium was not necessary. delta N-POMC, which lacks the N-terminal sorting signal for the regulated secretory pathway, also formed similar oligomers, indicating that the sorting and oligomerization domains are different.

Identification and characterization of Saccharomyces cerevisiae yapsin 3, a new member of the yapsin family of aspartic proteases encoded by the YPS3 gene.

A new aspartic protease from Saccharomyces cerevisiae, with a high degree of similarity with yapsin 1 and yapsin 2 and a specificity for basic residue cleavage sites of prohormones, has been cloned. This enzyme was named yapsin 3. Expression of a C-terminally truncated non-membrane anchored yapsin 3 in yeast yielded a heterogeneous protein between 135-200 kDa which, upon treatment with endoglycosidase H, migrated as a 60 kDa form. Amino-acid analysis of the N-terminus of expressed yapsin 3 revealed two different N-terminal residues, serine-48 and phenylalanine-54, which followed a dibasic and a monobasic residue respectively. Cleavage of several prohormones by non-anchored yapsin 3 revealed a specificity distinct from that of yapsin 1.

Cleavage efficiency of the novel aspartic protease yapsin 1 (Yap3p) enhanced for substrates with arginine residues flanking the P1 site: correlation with electronegative active-site pockets predicted by molecular modeling.

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.

Activation and processing of non-anchored yapsin 1 (Yap3p).

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel sub-class of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding approximately 1 microg of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala68. The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated alpha and beta, that were associated to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at approximately 4.0. When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme. The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.

Immunological identification and localization of yeast aspartic protease 3-like prohormone-processing enzymes in mammalian brain and pituitary.

The novel aspartic proteases, yeast aspartic protease 3 and the mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D. A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blots of bovine intermediate lobe where PCE activity has been found. YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules. Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus. In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA). Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus. These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.

Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P2, P1 and P2' positions.

The yeast aspartic protease Yap3 is localised to the secretory pathway and correctly cleaves pro-alpha-mating factor at its dibasic sites. We determined the specificity of Yap3 for mono-, di-, and multi-basic cleavage sites in the context of 15 residue synthetic proalbumin peptides. Yap3 cleaved after dibasic ArgArg and LysArg sites but not after monobasic Arg sites even when there was an additional arginine at -6 and/or -4. Yap3 did not cleave a tetra-arginine site and tri-basic sites (RRR and RRK) were poor substrates. Cleavage always occurred C-terminal to the last arginine in the di- or tri-basic sequence. The optimal cleavage site sequence was RR DR and this substrate was cleaved 8-9-fold faster than the normal RR DA sequence. In contrast to Kex2, Yap3 did not remove the propeptide from normal proalbumin or a range of natural or recombinant proalbumin variants. However at pH 4.0 Yap3 slowly cleaved proalbumin and albumin between domains 2 and 3.

Specificity and kinetic studies on the cleavage of various prohormone mono- and paired-basic residue sites by yeast aspartic protease 3.

The specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) m-1 s-1 and 1.6 x 10(4) m-1 s-1, respectively.

Processing of prothyrotropin-releasing hormone (Pro-TRH) by bovine intermediate lobe secretory vesicle membrane PC1 and PC2 enzymes.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretion of yeast aspartic protease 3 is regulated by its carboxy-terminal tail: characterization of secreted YAP3p.

Yeast aspartic protease 3 (YAP3p), a basic-residue specific proprotein processing enzyme, was shown to be a membrane-associated protease. The membrane association of YAP3p was demonstrated to be through a glycophosphatidylinositol anchor situated in the carboxy terminus of the enzyme. Carboxy-terminal truncation of YAP3p by 37 amino acids resulted in secretion of YAP3p into the growth medium. Western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two secreted forms of YAP3p with apparent molecular masses of approximately 180 and approximately 90 kDa. YAP3p has an isoelectric point of approximately 4.5 as determined by isoelectric focusing gel electrophoresis. Treatment of YAP3p with endoglycosidase H reduced the size of both forms of the protein to approximately 65 kDa, consistent with the presence of 10 potential N-linked glycosylation sites in the deduced amino acid sequence of this protein. Removal of the N-linked sugars did not affect the enzymatic activity of YAP3p. Analysis of the effect of temperature on the stability and the rate of enzymatic activity of YAP3p showed that the enzyme retained 100% of its activity when incubated for 1 h at 37 degrees C, while incubation at 50 degrees C for 1 h resulted in approximately 80% loss of activity. The dependence of activity on temperature demonstrated a calculated Q10 of 1.95.

Purified yeast aspartic protease 3 cleaves anglerfish pro-somatostatin I and II at di- and monobasic sites to generate somatostatin-14 and -28.

Anglerfish somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are derived from pro-somatostatin I (aPSS-I) and pro-somatostatin II (PSS-II), respectively. Purified yeast aspartic protease 3 (YAP3), was shown to cleave aPSS-I at the Arg81-Lys82 to yield SS-14 and Lys-1SS-14. In contrast, YAP3 cleaved aPSS-II only at the monobasic residue, Arg73 to yield aSS-28. Since the paired basic and monobasic sites are present in both precursors, the results indicate that the structure and conformation of these substrates dictate where cleavage occurs. Furthermore, the data show that YAP3 has specificity for both monobasic and paired basic residues.