Fraternine, a Novel Wasp Peptide, Protects against Motor Impairments in 6-OHDA Model of Parkinsonism

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative condition that affects the Central Nervous System (CNS). Insect venoms show high molecular variability and selectivity in the CNS of mammals and present potential for the development of new drugs for the treatment of PD. In this study, we isolated and identified a component of the venom of the social wasp Parachartergus fraternus and evaluated its neuroprotective activity in the murine model of PD. For this purpose, the venom was filtered and separated through HPLC; fractions were analyzed through mass spectrometry and the active fraction was identified as a novel peptide, called Fraternine. We performed two behavioral tests to evaluate motor discoordination, as well as an apomorphine-induced rotation test. We also conducted an immunohistochemical assay to assess protection in TH+ neurons in the Substantia Nigra (SN) region. Group treated with 10 μg/animal of Fraternine remained longer in the rotarod compared to the lesioned group. In the apomorphine test, Fraternine decreased the number of rotations between treatments. This dose also inhibited dopaminergic neuronal loss, as indicated by immunohistochemical analysis. This study identified a novel peptide able to prevent the death of dopaminergic neurons of the SN and recover motor deficit in a 6-OHDA-induced murine model of PD.

Keywords: Parkinson’s disease; antiparkinsonian activity; motor behavior; neuroprotective effect; wasp venom.

Figure 1

Chromatographic profile of the ultrafiltered venom of Parachartegus fraternus in a semi preparative column C18 Phenomenex. ACN gradient is shown in the dark line. Numbers represent fractions of the venom that were collected for further analysis. Fraction 6 corresponds to the compound denominated Fraternine.

Figure 2

Alignment with peptide from wasp venom. Toxins are presented by their UniProt KB codes or name. Capital letters denote amino acids. (*) represents identical amino acid residue. (:) designates a conservative modification in the residue of amino acid. (.) means semi-conservative modification. The white space represents an absence of identity between amino acid residues. Cys residues shaded in blue are connected by a disulfide bond. Ambiguities of isoleucine and leucine are shaded in yellow. AA means amino acid residues, and %Id is the percentage of sequence identity with Fraternine.

Figure 3

Motor coordination in the Rota-rod of mice SHAM (n = 5) or 6-OHDA lesioned and treated with vehicle (6-OHDA, n = 7), L-DOPA + benserazide (6 mg/Kg and 5 mg/kg, respectively) via i.p. (L-Dopa, n = 6), Fraternine 0.01 µg/animal (n = 7), Fraternine 0.1 µg/animal (n = 5), Fraternine 1.0 µg/animal (n = 7) or Fraternine 10 μg/animal (n = 6). (a) Performance of mice in the rotarod test on 3 consecutive days (days 3, 4 and 5). (b) Performance of mice in the R-TET, which was executed at 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360 min after the treatments on day 5. The symbol * represents significant difference (p < 0.05) with SHAM group and # represents difference (p < 0.05) with 6-OHDA group.

Figure 4

Number of turns exhibited on the apomorphine-induced turning behavior test, which is a dopamine agonist that was injected via s.c. (5 µg/animal) on day 7. Mice were divided into the following groups: SHAM (n = 5) or 6-OHDA lesioned and treated with vehicle (6-OHDA, n= 10), L-DOPA + benserazide (L-Dopa, n = 8), Fraternine 0.01 µg/animal (n = 8), Fraternine 0.1 µg/animal (n = 9), Fraternine 1.0 µg/animal (n = 8) or Fraternine 10 μg/animal (n = 9). The symbol * represents difference (p < 0.05) with SHAM and # represents difference (p < 0.05) with 6-OHDA.

Figure 5

(A) Percentage of neurons reactive for tyrosine-hydroxylase in immunohistochemistry compared to the unlesioned side of the brain of mice that were SHAM (n = 7) or 6-OHDA lesioned and treated with vehicle (6-OHDA, n = 8), L-DOPA + benserazide (L-Dopa, n = 5), Fraternine 0.01 μg/animal (n = 3), Fraternine 0.1 µg/animal (n = 4), Fraternine 1.0 μg/animal (n = 3) or Fraternine 10 μg/animal (n = 3). (B) Photo in the microscope (200× magnification) of a section of Substantia nigra of mice in the groups SHAM, 6-OHDA and Fraternine 10 µg/animal stained for tyrosine hydroxylase (TH⁺-green) and DAPI (blue). The white scale bar in the right corner of the image represents 50 µm. The symbol * represents difference (p < 0.05) with SHAM and # represents difference (p < 0.05) with 6-OHDA.

Figure 6

Graphical representation of the administration regimen and behavioral evaluation of animals treated with Fraternine peptide, L-DOPA/Benserazide or saline after 6-OHDA lesioning or SHAM (without lesion). On day one, animals received an intrastriatal injection of 40.6 ug of 6-OHDA (red line) or saline (dotted line) and a guide cannula was implanted in the left ventricle. Treatments was performed via i.c.v. injection for Fraternine (blue rectangle) and saline (white rectangle) and were administered twice a day via i.p. for L-DOPA/Benserazide (yellow squares). The behavioral tests consisted of rotarod motor discoordination test (Rotarod—green circle), six-hour rotarod temporal evaluation test (R-TET-green ellipse) and apomorphine-induced rotation test (purple triangle).