ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides

Abstract

The neurotrophin nerve growth factor (NGF) and its precursor proNGF are both bioactive and exert similar or opposite actions depending on the cell target and its milieu. The balance between NGF and proNGF is crucial for cell and tissue homeostasis and it is considered an indicator of pathological conditions. Proteolytical cleavage of proNGF to the mature form results in different fragments, whose function and/or bioactivity is still unclear. The present study was conducted to investigate the distribution of proNGF fragments derived from endogenous cleavage in brain and peripheral tissues of adult rats in the healthy condition and following inflammatory lipopolysaccharide (LPS) challenge. Different anti-proNGF antibodies were tested and the presence of short peptides corresponding to the prodomain sequence (pdNGFpep) was identified. Processing of proNGF was found to be tissue-specific and accumulation of pdNGFpeps was found in inflamed tissues, mainly in testis, intestine and heart, suggesting a possible correlation between organ functions and a response to insults and/or injury. The bioactivity of pdNGFpep was also demonstrated in vitro by using primary hippocampal neurons. Our study supports a biological function for the NGF precursor prodomain and indicates that short peptides from residues 1-60, differing from the 70-110 sequence, induce apoptosis, thereby opening the way for identification of new molecular targets to study pathological conditions.

Keywords: apoptosis; inflammation; nerve growth factor; peptides; proNGF; prodomain.

Fig. 1

(A–C) Dot blot and western blot for commercial antibody selectivity towards proNGF and/or NGF. (A) Detection of NGF, proNGF, C1 and C6 by using the different antibodies against NGF and proNGF: Abcam ab6199, Alomone ANT‐005 and Biosensis M‐1778‐B or S‐080. (B, C) The representative western blots show the ability and selectivity of (B) Abcam anti‐NGF antibody and (C) Biosensis (M‐1778‐B) anti‐proNGF antibody to detect recombinant NGF and/or proNGF.

Fig. 2

Expression of proNGF forms in different organs of adult male rats detected by Biosensis Antibody M‐1778‐B. Representative western blot of proNGF (Biosensis M‐1778‐B) detection in rat cortex, spinal cord and several peripheral tissues. “M” stands for protein marker.

Fig. 3

Expression of pdNGFpeps in different organs of adult male rats detected M‐1778‐B. Representative western blots of proNGF detection in rat testis, intestine, kidney, stomach, liver, spleen and heart. CTRL = healthy rats; LPS = LPS challenged rats. The statistical analysis was conducted using a Student's T‐test. The number of biologically‐independent replicates is 3. Graph bars report the mean + SD. Statistical significance: *P < 0.5; **P < 0.01; ***P < 0.001.

Fig. 4

(A, B) Effects of NGF, its unprocessed precursor proNGF, C1 propeptide and C6 propeptide on hippocampal cells (DIV). (A) Representative immunofluorescence for pJNK in hippocampal cells (DIV) treated for 1 h with NGF, its unprocessed precursor proNGF, C1 propeptide and C6 propeptide alone or with NGF. (B) Quantification of the proapoptotic effects of NGF and proNGF peptides on the activation of cells toward the p75/JNK‐driven proapototic pathway. Number of hippocampal neuronal nuclei positive to pJNK. Values represent the number of nuclei expressing pJNK, as percentage of CTR. Graph bars report the mean + SEM. N = 5 per experimental group. Reported significance is calculated by Student–Newman test. Statistical significance: *P < 0.5; **P < 0.01. Scale bars: 200 μm; inlet: 50 μm.