Honokiol alleviated neurodegeneration by reducing oxidative stress and improving mitochondrial function in mutant SOD1 cellular and mouse models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting both upper and lower motor neurons (MNs) with large unmet medical needs. Multiple pathological mechanisms are considered to contribute to the progression of ALS, including neuronal oxidative stress and mitochondrial dysfunction. Honokiol (HNK) has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke, Alzheimer's disease and Parkinson's disease. Here we found that honokiol also exhibited protective effects in ALS disease models both in vitro and in vivo. Honokiol improved the viability of NSC-34 motor neuron-like cells that expressed the mutant G93A SOD1 proteins (SOD1-G93A cells for short). Mechanistical studies revealed that honokiol alleviated cellular oxidative stress by enhancing glutathione (GSH) synthesis and activating the nuclear factor erythroid 2-related factor 2 (NRF2)-antioxidant response element (ARE) pathway. Also, honokiol improved both mitochondrial function and morphology via fine-tuning mitochondrial dynamics in SOD1-G93A cells. Importantly, honokiol extended the lifespan of the SOD1-G93A transgenic mice and improved the motor function. The improvement of antioxidant capacity and mitochondrial function was further confirmed in the spinal cord and gastrocnemius muscle in mice. Overall, honokiol showed promising preclinical potential as a multiple target drug for ALS treatment.

Modificações em proteínas induzidas por compostos eletrofílicos. possível papel em esclerose lateral amiotrófica / Modifications in proteins induced by electrophilic compounds. Possible role in amyotrophic lateral

Danos em biomoléculas podem ocorrer a partir de uma interação direta entre as biomoléculas e espécies reativas de oxigênio e nitrogênio como também, pela reação de produtos secundários dessas espécies como eletrófilos gerados na peroxidação lipídica. Alguns desses produtos secundários possuem estabilidade química maior que as espécies reativas das quais foram derivadas e podem se ligar covalentemente as biomoléculas comprometendo o funcionamento normal das mesmas. Portanto, modificações em proteínas por aldeídos gerados na lipoperoxidação têm sido investigadas por suas implicações com desordens patológicas relacionadas à agregação proteica, e modificações em diversas vias de sinalização amplificando os efeitos deletérios em sistemas biológicos. Os objetivos desse trabalho foi contribuir na elucidação dos mecanismos moleculares associados ao desenvolvimento da esclerose lateral amiotrófica (ELA) através da identificação, caracterização e quantificação de modificações póstraducionais em proteínas pelos aldeídos 4-hidroxi-2-hexenal (HHE) e trans-4-hidroxi-2-nonenal (HNE) in vitro (citocromo c) e in vivo (modelo ELA) a partir de técnicas de Western blot, imunoprecipitação e espectrometria de massa com abordagem proteômica de "shotgun" em ratosSOD1G93A modelo de esclerose lateral amiotrófica (ELA). Estudos com citocromo c mostraram a ligação dos aldeídos ao citocromo c e mecanismos de reação foram propostos. Foram encontrados seis peptídeos modificados por HHE e um para o HNE, e o peptídeo TGPNLHGLFGR se mostrou modificado pelos dois aldeídos paralelamente. Foi demonstrado que a histidina 33 é um "hot spot" frente as adições pelos aldeídos. Nas análises por western blot das proteínas ligadas a aldeídos foi possível observar uma tendência de aumento na concentração de proteínas ligadas ao HNE nos animais ELA, mais acentuada nas amostras de 70 dias comparadas ao controle. Com relação aos resultados obtidos com HHE tanto os animais pré-sintomáticos quanto os sintomáticos não apresentaram diferenças de HHE-proteína, tantonos controles quanto nos animais ELA. Nas amostras dos animais sintomáticos não detectamos diferença significativa na concentração de aldeído-proteína entre os grupos. Já as análises proteômicas revelaram 24 proteínas diferencialmente expressas, com destaque para proteínas com os maiores valores de significância (p-value), como a ubiquitina no grupo dos pré- sintomáticos e a neurogranina, no grupo dos animais sintomáticos e várias proteínas de metabolismo energético, de neurofilamentos, proteínas de processos redox e proteínas ligadas o metabolismo de cálcio (fundamentais na fisiopatologia em ELA). Algumas proteínas importantes foram encontradas com exclusividades nos grupos pré-sintomáticos e sintomáticos pelo diagrama de Venn. Com relação a proteínas modificadas pelos aldeídos, foram encontradas algumas relevantes como a proteína 2 de interação com a polimerase delta que foi modificada por HNE via adição de Michael encontrada nos animais ELA pré-sintomáticos e sintomáticos, a catalase que foi encontrada modificada por HNE via base de Schiff apenas nos ELA pré- sintomáticos, e a tiol redutase induzível por interferon gama no grupo dos animais ELA sintomáticos. Com relação a proteínas modificadas por HHE, foram encontradas a Janus quinase e proteína 3 de interação com microtúbulo, modificadas tanto por adição de Michael quanto via base de Schiff nos animais ELA sintomáticos. É interessante ressaltar que algumas modificações encontradas em proteínas não caracterizadas podem indicar proteínas novas ainda não descritas como modificadas por esses aldeídos. Os resultados mostram que algumas das proteínas modificadas por HNE e HHE encontradas neste trabalho, estão relacionadas ao estresse redox, vias metabólicas energéticas, proteínas envolvidas na resposta a danos oxidativos, e processos inflamatórios. Tais modificações ocorrem não só no modelo de neurodegeneração, mas foram previamente descritas em outros processos patológicos, como doença cardiovascular, lesão hepática por uso crônico de álcool

Damage to biomolecules can occur from a direct interaction between biomolecules and reactive of oxygen and nitrogen species as well as from the reaction of secondary products of these species as electrophiles generated in lipid peroxidation. Some of these by-products have greater chemical stability than the derived reactive species and can bind to biomolecules compromising their normal function. Therefore, protein modifications by aldehydes generated during lipoperoxidation have been investigated for their implications with pathological disorders related to protein aggregation and modifications in signaling pathways amplifying the deleterious effects in biological systems. The aim of this work was to contribute to the elucidation of the molecular mechanisms associated with the development of amyotrophic lateral sclerosis (ALS) through the identification, characterization and quantification of posttranslational modifications in proteins by 4-hydroxy-2-hexenal (HHE) and trans-4-hydroxy-2- nonenal (HNE) in vitro, cytochrome c, and in vivo, rat model (SOD1G93A) of amyotrophic lateral sclerosis (ALS), throught Western blot techniques, and mass spectrometry with shotgun proteomics approach. The results showed the binding of aldehydes to cytochrome c. Six peptides were modified by HHE and one by HNE. The peptide TGPNLHGLFGR was modified by the two aldehydes. Histidine 33 has been shown to be a hot spot against aldehydes additions. By western blot analysis of the aldehyde-bound proteins, it was possible to observe a tendency of increase in the concentration of HNE-bound proteins in the ALS animals, more pronounced in the samples of 70 days compared to control samples. Regarding the results obtained with HHE, both pre-symptomatic and symptomatic animals did not show HHE-protein differences, both in controls and in ALS animals. We did not detect a significant difference in the aldehyde-protein concentration between the groups in the samples of the symptomatic animals. Proteomic analysis revealed 24 differentially expressed proteins, with emphasis on proteins with thehighest values of significance (p-value), such as the ubiquitin in the pre-symptomatic group and neurogranin in the group of the symptomatic animals and several proteins of the energetic metabolism pathways, neurofilaments, proteins of redox processes and proteins linked to calcium metabolism (fundamental in the pathophysiology of ALS). Some important proteins were found exclusivity in the pre-symptomatic and symptomatic groups by the Venn diagram. With regard to aldehyde-modified proteins, some relevant ones such as Delta-2 polymerase interaction protein, that was modified by HNE via the addition of Michael found in presymptomatic and symptomatic ELA animals, catalase that was found to be modified by HNE via Schiff's base only in pre-symptomatic ALS, and gamma interferon-inducible thiol reductase in the group of symptomatic ALS animals. Janus kinase and microtubule interaction protein 3, were found to be modified by Michael addition and Schiff base pathway respectively in symptomatic ALS animals. It is interesting to note that some modifications found in uncharacterized proteins may indicate new proteins not yet described as modified by these aldehydes. The results show that some of the proteins modified by HNE and HHE found in this work are related to redox stress, energetic metabolic pathways, proteins involved in the response to oxidative damage, and inflammatory processes. Such modifications occur not only in the neurodegeneration model, but were previously described in other pathological processes, such as cardiovascular disease, liver injury due to chronic alcohol use

Region-specific changes in the immunoreactivity of Atg9A in the central nervous system of SOD1(G93A) transgenic mice / 대한해부학회지

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.

Region-specific changes in the immunoreactivity of Atg9A in the central nervous system of SOD1(G93A) transgenic mice / 대한해부학회지

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.

Effectiveness and pathologic changes of transplantation of human mesenchymal stem cells in SOD1-G93A mouse model of familial amyotrophic lateral sclerosis / 中国病理生理杂志

AIM:To study intravenous transplantation of human mesenchymal stem cells (hMSCs) on the life span and pathological change of SOD1-G93A amyotrophic lateral sclerosis (ALS) mice. METHODS:hMSCs were cultured and expanded from heparinized bone marrow cells from healthy donors and the purity and features were identified with FCM. hMSCs (3×10~6) resuspended in 0.3 mL DMEM or 0.3 mL DMEM only were injected into the tail vein of genotyped SOD1-G93A ALS mice. The mice were evaluated for signs of motor deficit with 4-point scoring system according to Weydt and the onset and life span were assessed. The pathological change was observed with Nissl staining and number of motor neuron was counted. RESULTS:The onset symptoms in untreated SOD1-G93A ALS mice appeared at (156.6±3.6) d of age and the average life span was (188.3±3.5) d. hMSCs transplantation delayed the onset of ALS type symptoms about 14 d and prolonged the life span about 18 d compared to the untreated SOD1-G93A littermates. The loss of motor neurons in untreated mice was much faster and severer than that in hMSCs transplanted mice. At 16 th week and 20 th week,motor neurons of untreated mice were significantly fewer than those of transplanted mice. β-globin gene in brain was detected in transplanted ALS mice. CONCLUSION:hMSCs migrate to central nervous system after intravenous transplantation,prolong the life span and delay the onset and motor neuron loss in SOD1-G93A ALS mice.

Immunohistochemical Study on the Distribution of Glycogen Synthase Kinase (GSK) 3beta in the Central Nervous System of SOD1G93A Transgenic Mice / 체질인류학회지

In the present study, we investigated influences of glycogen synthase kinase (GSK) 3beta on the development and/or progression of amyotrophic lateral sclerosis (ALS). We used transgenic mice expressing a human Cu/Zn superoxide dismutase mutant (SOD1G93A) as an in vivo model of ALS and examined expressional changes of GSK3beta immunohistochemically in the spinal cord, brain stem and cerebellum. With these experiments we demonstrate that the neurons in these regions of symptomatic SOD1G93A transgenic mice showed increased GSK3beta immunoreactivities compared with wild-type SOD1 transgenic mice. In contrast to symptomatic SOD1G93A transgenic mice, few GSK3beta immunoreactivity changes were detected in 8w- and 13w-old presymptomatic SOD1G93A transgenic mice. These data suggest the possibility that GSK3 functions as a modulating factor of apoptosis-related alterations in ALS and that GSK3beta exert differential functions in the development and/or progression of ALS. But the exact functional significances of these changes require further elucidation.