Early Life Exposure to Deltamethrin Impairs Synaptic Function by Altering the Brain-Derived Extracellular Vesicle Proteome

Abstract

Pyrethroid pesticides have been associated with neurodevelopmental disorders including attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). While behavioral effects of pyrethroid exposure have been previously reported, the underlying mechanisms remain unclear. Here, we hypothesized that exposure to deltamethrin (DM), a widely used pyrethroid pesticide known for its neurotoxicity during early developmental stages, induces brain dysfunction through alterations in brain-derived extracellular vesicle (BDEV) signaling. Using a well-established rodent model of early life DM exposure within the recommended no observable effect level, we isolated BDEVs from postnatal 30-day-old vehicle-exposed (control) and DM-exposed mice using a differential sucrose density gradient. Following ZetaView nanoparticle tracking and electron microscopy characterization, quantitative mass spectrometry-based proteomics revealed 89 differentially expressed proteins (DEPs) in BDEVs from DM exposed animals compared to control BDEVs. Bioinformatic analysis identified convergence of DEPs on pathways associated with mitochondrial function and synaptic plasticity. PKH67-green conjugated BDEVs derived from either control or DM-exposed mice were bilaterally injected intracerebroventricularly into naive adult mice, and the brain distribution of labeled BDEVs was verified prior to extracellular field recording experiments. Strikingly, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses, a functional correlate of learning and memory, was intact in control BDEVs but absent in naive mice receiving BDEVs from DM exposed mice. Notably, exogenously delivering LRRTM1, one of the DEPs found in DM BDEVs, disrupts synaptic transmission in CA1 neurons consistent with impaired LTP. Thus, differentially regulated signaling in BDEVs represents a novel mechanism of DM neurotoxicity.

Keywords: Hebbian plasticity; exosome; hippocampus; neurodevelopment; pyrethroid.

Graphical abstract

Fig. 1

Characterization of DM BDEVs.A, representative TEM and ZetaView analysis of Control and DM BDEV size and concentration. B, ZetaView nanoparticle tracking analysis shows no change in BDEV concentration between control or DM treated animals as determined by an unpaired t test following a Welch’s correction (n = 3 control and 3 DM litters, 2 samples per litter). C, The mean BDEV size is not altered between control and DM treated animals using an unpaired t test. D, relative size distribution is not altered by DM as determined by a two-way ANOVA, indicating that there is no change in different types of extracellular vesicles. E, Western blot analysis on BDEVs isolated from control and DM animals shows the expression of the positive markers Alix, Hsp-70, B-Actin, and CD81 both in control and DM BDEVs (n = 3 control and 3 DM litters).

Fig. 2

DM exposure alters BDEV proteome.A, Heatmap showing the DEPs in the control (n = 3) versus DM treated (n = 4) litters. B, Volcano plot showing significantly (p < 0.05) changed proteins. Proteins of interest have been labelled.

Fig. 3

Bioinformatic analysis of altered DM BDEV proteins. A, percentage of genes per group mapped to biological processes. B, histogram of altered genes per term in ClueGo analysis. C, networks show the interaction of proteins and their biological process for the DEPs (Control n = 3 and DM treated n = 4) using ClueGo. The asterisks represent the group term p-value representing each category. ∗∗p < 0.01.

Fig. 4

DM BDEVs alter LTP but not PPF in naive mice.A, bilateral ICV injection of control or DM BDEVs into male naive mice. B, scan of hippocampus after ICV injection of BDEVs (green) and DAPI (white) with overlay of hippocampus anatomy (scale bar indicates 250 μm). Lower zoomed in images on the CA1 region show DAPI and BDEVs and then a merge of both channels (scale bar indicates 20 μm). C, fEPSP slope plotted as a function of stimulation intensity to assess basic synaptic responses in slices. D, PPF ratio comparison in CA1 of injected mice indicates that DM BDEVs do not alter presynaptic function as determined by an unpaired t test (control n = 10 and DM n = 12) E, representative traces from male control mice before (black/left) and after (gray) and male DM BDEV before (black/right) and after (blue) LTP induction by tetanic stimulation of Schaffer collaterals (100 Hz, 1s). Field excitatory postsynaptic potential (fEPSP) from the striatum radiatum (CA1) of the hippocampus were recorded for 30 min prior to tetanic stimulation from males injected with control BDEVs (gray) and DM BDEVs (blue). A total of 70 min was recorded and slope change was calculated for each slice. Then, analysis of fEPSP slope change indicates DM BDEV injection results in a loss of LTP in males as indicated by the lack of change in slope of fEPSPs following tetanic stimulation as determined by an unpaired t test with a Welch’s correction (control n = 10 and DM n = 12) ∗∗∗∗indicates p < 0.001 by unpaired t test.

Fig. 5

Effects of LRRTM1 on EPSCs in CA1 pyramidal neurons.A, AiryScan image following injection of BDEVs (Green), DAPI (blue) and NeuN (red). Arrows indicate BDEVs imaged within close proximity to NeuN-labeled neurons. Scale bar indicates 10 μm. B, % of BDEVS near (Class I: gray) and at a distance from (Class II: blue) NeuN-labeled neurons compared to the total number of BDEVs. C, representative traces of sEPSCs recorded from CA1 pyramidal neurons from hippocampal slices treated with either vehicle (0.01% BSA, gray) or 100 ng/ml LRRTM1 (blue). LRRTM1 exposure results in a decrease in frequency of sEPSCs (D and E). This effect was accompanied by altered cumulative distribution of sEPSC amplitudes (F). Although this was effect was not observed in direct comparison of mean EPSC amplitudes (G). In (E and G), data points represent the total number of EPSCs observed during 1 min of recording (E) and the average amplitude of observed EPSCs (G) from each recording (control n = 6 and LRRTM1 n = 5). Data in (C and E) are mean ± SEM; ∗∗∗∗ indicates p < 0.01 as determined by an unpaired t test. Data in D, F are cumulative distributions of means; ∗∗∗∗ indicates p < 0.0001 as measured by a Kolmogorov-Smirnov test.