Deciphering the Neurotoxic Effects of Karenia selliformis

Abstract

Karenia selliformis is a globally recognized dinoflagellate associated with harmful algal blooms and massive fish kills along southern Chilean coasts. Its toxicity varies with environmental factors and genetic diversity. While K. selliformis is traditionally linked to neurotoxins like gymnodimines (GYMs), analysis of the strain CREAN-KS02 from Chile's Aysén Region (43° S) revealed no presence of toxins associated with this genus, such as gymnodimines, brevetoxins, or brevenal. Given the high toxicity and impact on marine life, our study aimed to functionally characterize the neurotoxic metabolites in the exudate of K. selliformis cultures. Cytotoxicity was evaluated using a Neuro-2a cell-based assay (CBA), determining an IC₅₀ of 2.41 ± 0.02 μg mL^-1. The incubation of Neuro-2a cells with the bioactive lipophilic extract obtained from the exudate of K. selliformis and the ouabain/veratridine couple showed activation of voltage-gated ion channels. Electrophysiological recordings on cultured mouse hippocampal neurons showed that the extract increased cell excitability in a dose-dependent manner, modulating action potential firing and exhibiting an opposed effect to tetrodotoxin. These findings indicate the presence of excitatory neurotoxic compounds affecting mammalian cells. This study provides the first mechanistic evidence of K. selliformis toxicity and highlights potential risks associated with its proliferation in marine environments.

Keywords: dinoflagellate; harmful algal blooms; neurotoxic compounds.

Figure 1

Cytotoxic curve of ExKs of Karenia selliformis (CREAN_KS02) on mouse neuroblastoma cells (Neuro-2a). The cells were incubated with extract of ExKs (0–5 µg mL⁻¹) during 24 h (black dots, R² = 0.985) or extract of culture medium (blue triangles). The calculated IC₅₀ was 2.41 ± 0.02 µg mL⁻¹. The graph shows three experiments performed in different weeks, with three replicates each one (n = 3, N = 3).

Figure 2

ExKs increases cytotoxicity in Neuro-2a cells in presence of ouabain (150 µM) and veratridine (15 µM). Neuro-2a cell assay for voltage-dependent sodium-channel-activating toxins exposed to ExKs, in the presence (black dot) or absence (blue triangles) of 100 nM saxitoxin. The graph shows a representative experiment of two experiments performed in different weeks, with three replicates each. (n = 2, N = 3).

Figure 3

Karenia selliformis (CREAN_KS02) exudate extract (ExKs) induced a potentiation in the synaptic neurotransmission. (A) Illustrative scheme of perfusion for electrophysiological recordings. Representative traces of postsynaptic currents illustrate the effect of ExKs (1 µg mL⁻¹) and TTX (50 nM) applications. The graph bars show the changes in the amplitude (pA) of the recordings (left panel). The values are mean ± SEM obtained from n = 8. (B) ExKs concentration–response curve, n = 7. The curve describes the potentiation of the neuronal synaptic currents elicited by ExKs (right panel) (n = 3, N = 12, *** p < 0.001).

Figure 4

Modulation of excitatory neurotransmission by Karenia selliformis (CREAN_KS02) exudate extract (ExKs). (A) Current traces showing the comparison of APs firing in control and ExKs (1 μg mL⁻¹) conditions, obtained in primary hippocampal neurons using electrophysiology patch clamp technique in current-clamp mode, and quantification of the relationship between the number of AP spikes and the injected current intensity, and the quantification of total spikes (bottom panel). The values represent mean ± SEM, obtained from n = 7 hippocampal neurons. Unpaired Student’s t-test was used for statistical analyses (n = 7–8 per group). (B) Miniature excitatory postsynaptic currents (mEPSC) were isolated using bicuculline (5 μM), and TTX (0.3 μM), and glutamatergic synaptic activity before and during the application of ExKs (1 μg mL⁻¹). The bar plot summarizes the effects of ExKs on the amplitude of the frequency of mEPSC (Hz) in hippocampal neurons in vitro (bottom panel, n = 8). TTX (50 nM) was used as positive control (n = 3, N = 8,** p < 0.01, *** p < 0.001).

Figure 5

Karenia selliformis (CREAN_KS02) exudate extract (ExKs) induced Ca²⁺ influxes. (A) Representative traces of ExKs-induced (1 μg mL⁻¹) changes in intracellular Ca²⁺ in hippocampal neurons after depolarizing stimulus of High K+ solution pulse (90 mM). (B) Quantification of areas under the curve (AUC) for each condition, n = 5–6. *** p < 0.001 compared to the control group.

Figure 6

Karenia selliformis (CREAN_KS02) exudate extract (ExKs) induced SV2 depletion. (A) Representative confocal microscopy images show SV2 (green) and DAPI (cyan) staining. Top: control condition; bottom: acute treatment with ExKs (1 μg mL⁻¹). (B) Quantification of the number of SV2 puncta in primary processes per 20 μm from panel A. Number of primary processes quantified: Control (21 processes from 15 neurons); ExKs (24 processes from 17 neurons). *** p < 0.001 compared to the control group.