The impact of rattlesnake venom on mice cerebellum proteomics points to synaptic inhibition and tissue damage

Highlights

• Mice injected with C. d. terrificus venom show several proteins abundancies changing in the cerebellum.

• Several biochemical processes changed over time with the injection of venom, such as metabolic processes and tissue damage.

• Each time point analyzed showed an increase or decrease of components such as oxidative stress, DNA repair, and synapses.

• A network analysis showed disturbances of diverse proteins that might be directly related to Crotalus envenomation.

Abstract

Snake envenomation is responsible for more than 130,000 deaths worldwide. In Brazil, the Crotalus rattlesnake is responsible for the second largest number of accidental snake bites in the country. Although there are many descriptions of the clinical and biochemical effects of Crotalus envenoming, there are few works describing the molecular events in the central nervous system of an organism due to envenomation. In this study, we analyzed the proteomic effect of Crotalus durissus terrificus snake venom on mice cerebellums. To monitor the envenomation over time, changes in the protein abundance were evaluated at 1 h, 6 h, 12 h and 24 h after venom injection by mass spectrometry. The analysis of the variation of over 4600 identified proteins over time showed a reduction in components of inhibitory synapse signaling, oxidative stress, and maintenance of neuronal cells, which paralleled increasing tissue damage and apoptosis factors. These analyses revealed the potential protein targets of the C. d. terrificus venom on the murine cerebellum, showing new aspects of the snake envenomation effect. These data may contribute to new therapeutic approaches (i.e., approaches directed at protein targets affected by the envenomation) on the treatment of envenomation by the neurotoxic C. d. terrificus snake venom.

Significance

Snakebites are a neglected global health problem that affects mostly rural and tropical areas of developing countries. It is estimated that over 5.4 million people are bitten by snakes each year, from which 2.7 million people are bitten by venomous snakes, resulting in disabilities such as amputations and in some cases leading to death.

The C. d. terrificus snake is the most lethal snake in Brazil. Studying the molecular changes upon envenomation in a specific tissue may lead to a better understanding of the envenomation process by C. d. terrificus snakebites.

Introduction

According to the World Health Organization, snakebites are a neglected health problem that affects mostly tropical and subtropical countries. Every year, ~2.7 million venomous snakes accidents occur worldwide, resulting in 130,000 deaths, 490,000 amputations, and other severe health problems [1]. In Brazil, according to the Brazilian Ministry of Health, 22,000 accidents occur with venomous snakes every year [2]. These accidents are mainly caused by four genera of venomous snakes found in the Brazilian territory: Bothrops, responsible for 90.5% of the accidents; Micrurus, responsible for 0.4% of the accidents; Lachesis, responsible for 1.4% of the accidents; and Crotalus, known as rattlesnakes, responsible for 7.7% of the accidents. Although Crotalus accidents are the second largest cause of snakebite accidents in the country, their lethality is the highest, reaching 1.87% [3].

Crotalus durissus snake venom is mainly composed of a mixture of peptides and other minor chemical compounds. Among these compounds are presynaptic neurotoxins that act on nerve terminals, inhibiting acetylcholine and causing motor paralysis [4,5]. Within the first six hours of accidental venom inoculation, patients typically report several eye-related problems, such as palpebral, uni- or bilateral ptosis, ophthalmoplegia, blurred vision, and diplopia, which is a consequence of the paralysis of the extrinsic and intrinsic musculature of the eyeball after impairment of the third pair of cranial nerves [6]. In addition to its neurotoxic activity, myotoxic and abnormal coagulant activities of the venom have been observed. The myotoxic activity can be observed in the most serious cases, where various painful systemic lesions occur against the muscular fibers, leading to a release of enzymes and myoglobin to the blood, which are excreted by the urine and turn it a reddish-brown color. The abnormal coagulant activity comes from a fraction containing a thrombin-like enzyme, which causes disturbances in blood coagulation in approximately 40% of patients [7]. In addition, C. durissus venom has also been described to be neurotoxic [8], nephrotoxic [9], and cardiotoxic [10,11].

A composition analysis of C. durissus venom revealed four major components. Crotoxin is the most abundant toxin in the venom (65% of dry weight) and has neurotoxic activity and a secondary hemolytic activity [12]. Crotamine is related to myotoxic effects [13]. Gyroxin causes strong involuntary contractions of the muscles and a severe loss of balance, making mice spin on their own axis after subjecting the animals to toxin injection [14]; additionally, gyroxin has also been described to transpose the blood-brain barrier [15]. Convulxin has been described to present the same effects of gyroxin in tests performed in mice, in addition to an abrupt and transient fall of arterial blood pressure and late hypotension in dogs [16].

These descriptions show the severity of the venom action in crotalic accidents and the need to rapidly neutralize these toxins in an injured individual. Currently, the antivenom antiophidic sera, first described by Albert Calmette in 1859 [17], is still used to treat snakebites. These sera are developed by injecting small amounts of snake venom in horses and then purifying the antibodies against the venom. However, many countries that present high incidences of snakebite accidents, especially in Asia and Africa, have few companies that produce the antiophidic sera, making obtaining the sera an aggravating problem for the treatment of this type of medical emergency [1].

Several studies have described the clinical features of C. durissus snakebites and many others have studied the venom composition of these snakes at the genomic, transcriptomic, and proteomic levels. Previous studies also evaluated the biological and therapeutic effects of specific molecules isolated from Crotalus snake venom. However, even though C. d. terrificus is responsible for the second largest number of envenomation accidents and the highest lethality in Brazil, no previous studies evaluated the effect of its venom on the central nervous system.

To obtain a comprehensive proteomic characterization of the effects of the C. d. terrificus venom in mice cerebellums, which is responsible for equilibrium, coordination, balance, motor learning, motor memory, and motor consolidation, we used high-resolution mass spectrometry-based proteomics to analyze mice cerebellums injected with the venom at different time points over the period when patients seek the hospital after a snakebite [18]. The results indicated effects not previously described in the literature, such as the perturbation of proteins involved in the blood brain barrier, synaptic transmission, and tissue damage. These data add a greater understanding of the mechanism of action for venom in the context of the ophidian accidents, thus yielding new approaches to the study of ophidian accidents.