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. 2018 Jul 3;4(7):e00673.
doi: 10.1016/j.heliyon.2018.e00673. eCollection 2018 Jul.

Human Brain Gene Expression Profiles of the Cathepsin V and Cathepsin L Cysteine Proteases, With the PC1/3 and PC2 Serine Proteases, Involved in Neuropeptide Production

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Free PMC article

Human Brain Gene Expression Profiles of the Cathepsin V and Cathepsin L Cysteine Proteases, With the PC1/3 and PC2 Serine Proteases, Involved in Neuropeptide Production

Sonia Podvin et al. Heliyon. .
Free PMC article

Abstract

Proteases are required to generate active peptide neurotransmitters, known as neuropeptides, from pro-neuropeptides. Model animal systems have recently illustrated roles for the cathepsin V (CTSV) and cathepsin L (CTSL) cysteine proteases, combined with the serine proteases PC1/3 (PCSK1) and PC2 (PCSK2), and exopeptidases in the production of neuropeptides. There is notable interest in the human-specific cathepsin V gene which is not present in rodent and other animal models used in prior studies of neuropeptide production. A gap in the field is knowledge of the human brain gene expression patterns of these neuropeptide-producing protease systems. Therefore, the goal of this study was to characterize the expression profiles of these pro-neuropeptide processing proteases in human brain. Quantitative gene expression microarray data for 169 human brain regions was obtained from the Allen Institute Human Brain Atlas resource, analyzed as log2 of gene expression intensity normalized to the mean of human genes (21,245 genes) expressed in human brain. These proteases had log2 values of 2-12, indicating expression levels above the average of all genes in the human brain, with varying expression levels among the 169 brain regions. CTSV and CTSL displayed moderate to high expression values of 1.9-8.6 and 7.1-10.6, respectively. Interestingly, CTSV and CTSL showed high expression in white matter composed of myelinated axons, consistent with the knowledge that neuropeptide production occurs in axons within transported neuropeptide secretory vesicles to nerve terminals. PCSK1 had a broad range of moderate to very high expression with log2 of 2-12. PCSK2 had somewhat lower expression levels than PCSK1. The exopeptidase genes RNPEP, CTSH, and CPE each showed fairly even levels of expression throughout the brain, with CPE displaying high expression. The prevalence of these processing proteases throughout human brain regions, including areas rich in neuropeptides such as hypothalamus, is consistent with their roles for neuropeptide production. Further, proenkephalin and NPY precursors, substrates of CTSV and CTSL shown in prior model animal studies, were co-expressed with CTSV and CTSL. These data demonstrate that the human brain expresses the neuropeptide-producing cysteine and serine proteases, with exopeptidases, throughout a multitude of brain regions.

Keywords: Neuroscience.

Figures

Fig. 1
Fig. 1
Cysteine and serine protease pathways for neuropeptide production. Cysteine and serine subtilisin-like protease pathways are involved in neuropeptide production from pro-neuropeptide precursors (9–11). The cathepsin V (CTSV) and cathepsin L (CTSL) cysteine proteases cleave pro-neuropeptides at paired basic residues. Resultant peptide intermediates require removal of N-terminal basic residues by aminopeptidase B (RNPEP) or cathepsin H (CTSH), and removal of C-terminal basic residues by carboxypeptidase E (CPE). The cathepsin V cysteine protease functions in human cells for neuropeptide production, but not in rodent or other mammalian species previously used for studies of neuropeptide-producing proteases (28–33) since these species do not have the cathepsin V gene (23–26). The serine protease pathway consists of the subtilisin-like pro-protein convertases (PC) PC1/3 and PC2 (PCSK1 and PCSK2, respectively) that cleave at paired basic residues. Resultant peptide intermediates require removal of C-terminal basic residues by CPE.
Fig. 2
Fig. 2
Human brain gene expression analyses by microarrays and data normalization. Gene expression data for 169 human brain regions was achieved by microarrays conducted by the Allen Human Brain Atlas resource as described in the methods. Neurotypical brains (from six donors) were dissected, RNA was isolated, and cRNA-Cy3 was synthesized for microaray chip hybridzation. The chip hybridization fluorescence raw data was obtained by optical feature extraction. This data was normalized between chips by internal controls, subjected to batch normalization with pooled tissue controls. Data were normalized as log2 expression intensity values where the 75th percentile of expression was approximately log = 5, and the 95th percentile of expression was approximately log2 = 7, calculated as described by Rizzo et al., (2014) (40). Log2 expression values presented in graphs of this study are log2 expression values that are averages ± MAD (median absolute deviation) for six donor brains (n = 6).
Fig. 3
Fig. 3
169 Human brain regions. 169 human brain regions are listed numbered #1–169, named according to main region (color coded) and subregions. These color-coded brain regions are used in the graphs to display gene expression values for the neuropeptide-producing protease genes (Figs. 4, 5, and 6) and the pro-neuropeptide genes of PENK and NPY (Fig. 7).
Fig. 4
Fig. 4
Gene expression of cathepsin V and cathepsin L cysteine proteases in human brain. Quantitative gene expression values for (a) cathepsin V (CTSV), and (b) cathepsin L (CTSL) are illustrated for 169 adult human brain regions. Microarray probes used for gene expression analyses are listed in Fig. S1. Log2 quantitative gene expression values were calculated for 169 human brain regions, by normalization to the average expression of all genes human genes analyzed by the Allen Human Brain Atlas resource utilized for this study, and expressed as the average log2 ± MAD (median absolute deviation) (n = 6 human donor brains). Brain regions are indicated by the colored bars (at top) indicating the main regions of: FL: Frontal lobe, Insula; CgG: Cingulate gyri; HiF: Hippocampal formation; OL: Occipital lobe; PL: Parietal lobe; TL: Temporal lobe; Amg: Amygdala; BsFb: Basal forebrain; Str: Striatum; Clstr: Claustrum; Epithal: Epithalamus; Hypothalamus; Thal: Thalamus; Subthal: Subthalamus; MES: Mesencephalon; CbCtx: Cerebellar cortex; CbN: Cerebellar nuclei; Pons; MY: Myelencephalon; WM: White matter structures; CP: Choroid plexus of the lateral ventricles. Color keys for these regions, with subregions numbered 1–169 are shown in Fig. 3.
Fig. 5
Fig. 5
Gene expression of the pro-protein convertase PC1/3 and PC2 serine proteases in human brain. Quantitative log2 gene expression values for (a) pro-protein convertase PC1/3 (PCSK1) and (b) pro-protein convertase PC2 (PCSK2) are illustrated for 169 adult human brain regions. Values shown are the average of median log2 expression ± median absolute deviation (MAD) (from six donor brains). The 169 brain regions and subregions numbered 1–169 are shown in Fig. 3, with color keys.
Fig. 6
Fig. 6
Gene expression of aminopeptidase B (RNPEP), cathepsin H (CTSH), and carboxypeptidase E (CPE) exopeptidases in human brain. Quantitative log2 gene expression values for (a) aminopeptidase B (RNPEP), (b) cathepsin H (CTSH), and (c) carboxypeptidase E (CPE) are illustrated for 169 human brain regions. Values shown are the average of median log2 expression ± median absolute deviation (MAD) (from six donor brains). The 169 brain regions and subregions numbered 1–169 are shown in Fig. 3, with color keys.
Fig. 7
Fig. 7
Expression of proenkephalin and pro-NPY, substrates of CTSV and CTSL. (a) Illustration of the proenkephalin and pro-NPY pro-neuropeptide substrates of cathepsin V (i) and cathepsin L (ii) are shown. (b) Quantitative log2 gene expression values for the proenkephalin (PENK) (i) and the NPY (NPY) (ii) genes are illustrated for 169 brain regions (brain regions listed in Fig. 3). Values shown are the average of median log2 expression ± median absolute deviation (MAD) (from six donor brains).

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