Analysis of neuropeptide expression and localization in adult drosophila melanogaster central nervous system by affinity cell-capture mass spectrometry

Abstract

A combined approach using mass spectrometry, a novel neuron affinity capture technique, and Drosophila melanogaster genetic manipulation has been developed to characterize the expression and localization of neuropeptides in the adult D. melanogaster brain. In extract from the whole adult brain, 42 neuropeptides from 18 peptide families were sequenced. Neuropeptide profiling also was performed on targeted populations of cells which were enriched with immunoaffinity purification using a genetically expressed marker.

Figure 1

Examples of mass spectra obtained by LC-ESI-QTOF-CID MS and off-line HPLC/MALDI-FTICR MS analysis from extracts of pooled D. melanogaster brain tissue. (A) ESI-Q-TOF MS/MS analysis of the isolated protonated molecular ion with m/z 1247.70 generated almost complete y- and b-ion series and resulted in identification of this molecule as Dromyosuppressin. The fragment ions are labeled according to the nomenclature of Biemann. The sequence is consistent with the predicted product encoded by gene CG6440. (B) MALDI-FT mass spectrum of one LC fraction obtained by HPLC separation of the extract. The quasimolecular ions [M + H]⁺ of several neuropeptides or intermediate processing products were present in this LC fraction including the AKH processing intermediate (m/z 1161.56), an Allatostatin A peptide (m/z 1276.68), a member of the SIFamide family (m/z 1395.76), an Allatostatin B peptide (m/z 1603.84), and a member of the Nplp1 family (m/z 1423.84).

Figure 2

Outline of method used to enrich for labeled populations of neurons in the adult D. melanogaster brain. Adult brains genetically labeled with the surface marker mCD8-GFP using the GAL4-UAS expression system are dissociated into single cell preparations and incubated with paramagnetic beads conjugated to a monoclonal anti-mCD8 antibody. The mixture is placed in a magnetic column allowing antibody-captured cells to be retained in the column. When the magnet is removed, the cells are eluted. Following an acidified methanol extraction, the contents of the immunoaffinity-purified cells are analyzed by MALDI-TOF mass spectrometry.

Figure 3

The efficiency of the immunoaffinity enrichment method was assessed by FACS analysis. The histograms demonstrate the counts of DRAQ5-positive cells expressing GFP in cell suspension after immunoaffinity enrichment. With the use of the dimm (c929)-GAL4 driver, 55% of the cells were GFP-positive (A). The line indicates the gate intensity threshold for GFP-positive fluorescence. With the use of the Ddc-GAL4 driver, 35% of the cells were GFP-positive (B). Live cells were distinguished from necrotic cells using an imaging flow cytometer which provided simultaneous analysis of morphological features (brightfield illumination), GFP fluorescence (green), and DRAQ5 staining (red). Representative images of cells isolated with the dimm (c929)-GAL4 driver are shown (C).

Figure 4

Frontal confocal microscope images of D. melanogaster adult brain tissue genetically labeled with the cell-surface antigen mCD8-GFP (green) and the corresponding mass spectral profile of extract from cell populations enriched using GAL4-UAS-mediated immunoaffinity purification. Counterstaining with the anti-NC82 antibody was used to mark the neuropil regions (blue). The dimm (c929)- (A) or Ddc-GAL4 (C) driver lines targeted, respectively, primarily peptidergic or dopaminergic and serotonergic cells. Dorsal and ventral aspects of the fly brain are indicated. MALDI-TOF MS analysis of extract from neuronal subpopulations enriched using the dimm (c929)-GAL4 driver gave the broadest peptide profile (B), whereas isolation with the Ddc-GAL4 driver (D) resulted in simpler profile. Signals of identified peptide ion species have been labeled with the predicted amino acid sequence. PR, protocerebrum; ME, medulla; SOG, subesophageal ganglion. Scale bar: 50 μm.

Figure 5

Frontal images of D. melanogaster adult brain tissue labeled using immunocytochemistry and the GAL4-UAS expression system. Immmunocytochemical staining was performed to confirm the colocalization of short Neuropeptide F (NPF) expression within dimm (c929)-GAL4 circuitry and with the biogenic amine serotonin (5HT). Colocalization of anti-short NPF antibody staining (red) and dimm (c929)-GAL4-driven GFP expression (green) was observed in paired cells in the protocerebrum and in the subesophageal ganglion (yellow, A). A higher magnification view is shown of the framed region in A (B–D). Neuropil regions were labeled with the anti-NC82 antibody (blue). Colocalization of anti-5HT staining (red) with NPF-GAL4-driven GFP expression was observed in the central protocerebrum (yellow, E). Higher magnification images of the framed area showed colocalization in cell bodies and processes in the protocerebral bridge (F) and central complex neuropils (G). These observations were consistent with the results of the immunoaffinity cell-enrichment/MS approach which indicated that short NPFs are expressed within dimm (c929)-GAL4 and Ddc-GAL4-labeled cells. CC, central complex; ME, medulla: PR, protocerebrum; PR BR, protocerebral bridge; SOG, subesophageal ganglion; VTI, ventral tritocerebrum.