Small GSK-3 Inhibitor Shows Efficacy in a Motor Neuron Disease Murine Model Modulating Autophagy.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron degenerative disease that has no effective treatment up to date. Drug discovery tasks have been hampered due to the lack of knowledge in its molecular etiology together with the limited animal models for research. Recently, a motor neuron disease animal model has been developed using ß-N-methylamino-L-alanine (L-BMAA), a neurotoxic amino acid related to the appearing of ALS. In the present work, the neuroprotective role of VP2.51, a small heterocyclic GSK-3 inhibitor, is analysed in this novel murine model together with the analysis of autophagy. VP2.51 daily administration for two weeks, starting the first day after L-BMAA treatment, leads to total recovery of neurological symptoms and prevents the activation of autophagic processes in rats. These results show that the L-BMAA murine model can be used to test the efficacy of new drugs. In addition, the results confirm the therapeutic potential of GSK-3 inhibitors, and specially VP2.51, for the disease-modifying future treatment of motor neuron disorders like ALS.

Morphometric and neurochemical alterations found in l-BMAA treated rats.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive muscle paralysis that reflects the motoneurons' degeneration. Several studies support the relationship between ß-N-methylamino-l-alanine (l-BMAA), a neurotoxic amino acid produced by cyanobacteria and diatoms, and the sporadic occurrence of ALS and other neurodegenerative diseases. Therefore, the study of its neurotoxicity mechanisms has assumed great relevance in recent years. Recently, our research team has proposed a sporadic ALS animal model by l-BMAA administration in rats, which displays many pathophysiological features of human ALS. In this paper, we deepen the characterization of this model corroborating the occurrence of alterations present in ALS patients such as decreased muscle volume, thinning of the motor cortex, enlarged brain's lateral ventricles, and alteration of both bulbar nuclei and neurotransmitters' levels. Therefore, we conclude that l-BMAA treated rats could be a good model which mimics degenerative features that ALS causes in humans.

ß-N-methylamino-l-alanine causes neurological and pathological phenotypes mimicking Amyotrophic Lateral Sclerosis (ALS): the first step towards an experimental model for sporadic ALS.

ß-N-methylamino-l-alanine (L-BMAA) is a neurotoxic amino acid that has been related to various neurodegenerative diseases. The aim of this work was to analyze the biotoxicity produced by L-BMAA in vivo in rats, trying to elucidate its physiopathological mechanisms and to search for analogies between the found effects and pathologies like Amyotrophic Lateral Sclerosis (ALS). Our data demonstrated that the neurotoxic effects in vivo were dosage-dependent. For evaluating the state of the animals, a neurological evaluation scale was developed as well as a set of functional tests. Ultrastructural cell analysis of spinal motoneurons has revealed alterations both in endoplasmic reticulum and mitochondria. Since GSK3ß could play a role in some neuropathological processes, we analyzed the alterations occurring in GSK3ß levels in L-BMAA treated rats, we have observed an increase in the active form of GSK3ß levels in lumbar spinal cord and motor cerebral cortex. On the other hand, (TAR)-DNA-binding protein 43 (TDP-43) increased in L-BMAA treated animals. Our results indicated that N-acetylaspartate (NAA) declined in animals treated with L-BMAA, and the ratio of N-acetylaspartate/choline (NAA/Cho), N-acetylaspartate/creatine (NAA/Cr) and N-acetylaspartate/choline+creatine (NAA/Cho+Cr) tended to decrease in lumbar spinal cord and motor cortex. This project offers some encouraging results that could help establishing the progress in the development of an animal model of sporadic ALS and L-BMAA could be a useful tool for this purpose.

ß-N-methylamino-L-alanine induces changes in both GSK3 and TDP-43 in human neuroblastoma.

ß-N-methylamino-L-alanine (L-BMAA) is a neurotoxic amino acid produced by most cyanobacteria, which are extensively distributed in different environments all over the world. L-BMAA has been linked to a variety of neurodegenerative diseases. This work aims to analyze the toxicological action of L-BMAA related to alterations observed in different neurodegenerative illness as Alzheimer disease and amyotrophic lateral sclerosis. Our results demonstrate that neuroblastoma cells treated with L-BMAA show an increase in glycogen synthase kinase 3 ß (GSk3ß) and induce accumulation of TAR DNA-binding protein 43 (TDP-43) truncated forms (C-terminal fragments), phosphorylated  and high molecular weight forms of TDP-43, that appears frequently in some neurodegenerative diseases.

Anisakis antigens detected in fish muscle infested with Anisakis simplex L3.

Anisakis simplex is a fish parasite that is a public health risk to those consuming raw or poorly cooked marine fish and cephalopods because of the possibility of becoming infested with live larvae. In humans, penetration of the larvae into the gastrointestinal track can cause acute and chronic symptoms and allergic anisakiasis. Excretion and secretion products released by the larvae are thought to play a role in migration through the tissues and induce an immunoglobulin E-mediated immune response. The aim of this preliminary study was to detect parasite antigens and allergens in fish tissues surrounding the migrating larvae. Hake and anchovy fillets were artificially parasitized with Anisakis larvae and stored in chilled conditions for 5 days. Larvae were evaluated for fluorescence, fish muscle tissue was examined with transmission electron microscopy, and immunohistochemical reactions of two rabbit polyclonal antisera against a parasite crude extract and the allergen Ani s 4 were recorded. Larvae immediately migrated into the fish muscle, and no emission of bluish fluorescence was observed. Fish muscle areas in contact with the parasite showed disruptions in the structure and inclusion of granules within sarcomeres. Both parasite antigens and the Ani s 4 allergen were located in areas close to the larvae and where sarcomere structure was preserved. These findings indicate that parasite antigens and allergens are dispersed into the muscle and might cause allergic symptoms such as dyspnea, vomiting, diarrhea, urticaria, angioedema, or anaphylaxis in some individuals sensitive to A. simplex.

Allergenic properties and cuticle microstructure of Anisakis simplex L3 after freezing and pepsin digestion.

This article examines the viability of and the alterations to the larval cuticle and the pattern of the antigens released when live or frozen Anisakis simplex larvae were treated with acid and pepsin. The results showed that freezing did not greatly alter the larva body. If ruptures were observed, the antigen release to the incubation media was not enhanced, and most of the antigenic content was retained inside the bodies of the larvae. The immunoblotting assay demonstrated that most of the antigens released, including the allergen Ani s 4, were resistant to pepsin. Freezing killed the larvae, but their survival was not compromised by acid treatment or pepsin digestion when kept chilled. All these findings support recommendations about freezing fish for consumption raw or undercooked to prevent human infection by A. simplex larvae. However, our data show that the antigenicity of the larvae is preserved after freezing and may explain why some sensitized patients develop symptoms after ingestion of infested frozen fish.