Communication defects with astroglia contribute to early impairments in the motor cortex plasticity of SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, involving the selective degeneration of cortical upper synapses in the primary motor cortex (M1). Excitotoxicity in ALS occurs due to an imbalance between excitation and inhibition, closely linked to the loss/gain of astrocytic function. Using the ALS SOD1G93A mice, we investigated the astrocytic contribution for the electrophysiological alterations observed in the M1 of SOD1G93A mice, throughout disease progression. Results showed that astrocytes are involved in synaptic dysfunction observed in presymptomatic SOD1G93A mice, since astrocytic glutamate transport currents are diminished and pharmacological inhibition of astrocytes only impaired long-term potentiation and basal transmission in wild-type mice. Proteomic analysis revealed major differences in neuronal transmission, metabolism, and immune system in upper synapses, confirming early communication deficits between neurons and astroglia. These results provide valuable insights into the early impact of upper synapses in ALS and the lack of supportive functions of cortical astrocytes, highlighting the possibility of manipulating astrocytes to improve synaptic function.

Dietary NMN supplementation enhances motor and NMJ function in ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that causes the degeneration of motor neurons in the motor cortex and spinal cord. Patients with ALS experience muscle weakness and atrophy in the limbs which eventually leads to paralysis and death. NAD+ is critical for energy metabolism, such as glycolysis and oxidative phosphorylation, but is also involved in non-metabolic cellular reactions. In the current study, we determined whether the supplementation of nicotinamide mononucleotide (NMN), an NAD+ precursor, in the diet had beneficial impacts on disease progression using a SOD1G93A mouse model of ALS. We found that the ALS mice fed with an NMN-supplemented diet (ALS+NMN mice) had modestly extended lifespan and exhibited delayed motor dysfunction. Using electrophysiology, we studied the effect of NMN on synaptic transmission at neuromuscular junctions (NMJs) in symptomatic of ALS mice (18 weeks old). ALS+NMN mice had larger end-plate potential (EPP) amplitudes and maintained better responses than ALS mice, and also had restored EPP facilitation. While quantal content was not affected by NMN, miniature EPP (mEPP) amplitude and frequency were elevated in ALS+NMN mice. NMN supplementation in diet also improved NMJ morphology, innervation, mitochondrial structure, and reduced reactive astrogliosis in the ventral horn of the lumbar spinal cord. Overall, our results indicate that dietary consumption of NMN can slow motor impairment, enhance NMJ function and improve healthspan of ALS mice.

2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.

Induced pluripotent stem cells-based disease modeling, drug screening, clinical trials, and reverse translational research for amyotrophic lateral sclerosis.

It has been more than 10 years since the hopes for disease modeling and drug discovery using induced pluripotent stem cell (iPSC) technology boomed. Recently, clinical trials have been conducted with drugs identified using this technology, and some promising results have been reported. For amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, several groups have identified candidate drugs, ezogabine (retigabine), bosutinib, and ropinirole, using iPSCs-based drug discovery, and clinical trials using these drugs have been conducted, yielding interesting results. In our previous study, an iPSCs-based drug repurposing approach was utilized to show the potential of ropinirole hydrochloride (ROPI) in reducing ALS-specific pathological phenotypes. Recently, a phase 1/2a trial was conducted to investigate the effects of ropinirole on ALS further. This double-blind, randomized, placebo-controlled study confirmed the safety and tolerability of and provided evidence of its ability to delay disease progression and prolong the time to respiratory failure in ALS patients. Furthermore, in the reverse translational research, in vitro characterization of patient-derived iPSCs-motor neurons (MNs) mimicked the therapeutic effects of ROPI in vivo, suggesting the potential application of this technology to the precision medicine of ALS. Interestingly, RNA-seq data showed that ROPI treatment suppressed the sterol regulatory element-binding protein 2-dependent cholesterol biosynthesis pathway. Therefore, this pathway may be involved in the therapeutic effect of ROPI on ALS. The possibility that this pathway may be involved in the therapeutic effect of ALS was demonstrated. Finally, new future strategies for ALS using iPSCs technology will be discussed in this paper.

Effect of electroacupuncture intervention on the spinal cord PPIA/NF-κB signaling pathway in mice with amyotrophic lateral sclerosis. / ç”µé’ˆå¯¹è‚ŒèŽç¼©ä¾§ç´¢ç¡¬åŒ–ç—‡å°é¼ è„Šé«“PPIA/NF-κB信号通路的影响.

OBJECTIVES: To observe the effects of electroacupuncture (EA) on motor function, expression of extracellular cyclophile A(PPIA) and PPIA/nuclear factor-κB (NF-κB) signaling pathway in spinal cord of amyotrophic la-teral sclerosis (ALS) mice, so as to explore the mechanism of EA intervention in regulating extracellular PPIA on neuroinflammation in ALS mice. METHODS: Thirty ALS-SOD1G93A mice with hSOD1-G93A gene were randomly divided into model, EA and Riluzole groups , with 10 mice in each group, and other 10 ALS-SOD1G93A negative mice were used as the blank group. EA was applied to bilateral "Yanglingquan"(GB34) and "Zusanli"(ST36) for 20 min once daily, 5 days a week for 2 weeks. In the Riluzole group, riluzole solution (30 mg·kg-1·d-1) was administrated intragastrically, and the treatment time was the same as that in the EA group.Rotating rod experiment and open field experiment were used to evaluate the changes in motor function of mice .The morphology of motor neurons in the anterior horn of spinal cord was observed by HE staining.The relative protein expression levels of PPIA, TDP-43 and NF-κB in the spinal cord were detected by Western blot.The positive expression level of TDP-43 in the spinal cord was detected by immunohistochemistry. The positive expression level of PPIA in spinal cord was marked by immunofluorescence. Serum PPIA content was determined by ELISA. RESULTS: Compared with the blank group, the time of rod dropping and the total distance of open field movement in the model group were shortened (P<0.01), the number of motor neurons in the anterior horn of the spinal cord was reduced, the cell morphology was incomplete, the cell body was atrophied, the protein expression and positive expression of TDP-43 were increased (P<0.01), the protein expressions of PPIA and NF-κB in the spinal cord were increased(P<0.01), the serum content of PPIA and immunofluorescence expression of PPIA in spinal cord were increased (P<0.01). Compared with the model group, the time of rod dropping and the total distance of open field movement of mice in the EA group and the Riluzole group were prolonged (P<0.05, P<0.01), and the injury of motor neuron in the anterior horn of the spinal cord was decreased, the protein expression and positive expression of TDP-43 in the spinal cord were decreased (P<0.05, P<0.01)；the relative expression levels of PPIA and NF-κB proteins were decreased (P<0.05, P<0.01), and the content of PPIA in serum and the immunofluorescence expression of PPIA in the spinal cord were decreased (P<0.05, P<0.01) in the EA group；the relative protein expression of NF-κB and fluorescence expression of PPIA in spinal cord of mice in the Riluzole group were decreased (P<0.05). CONCLUSIONS: EA intervention can improve motor function in ALS mice, and its mechanism may be related to the inhibition of PPIA/NF-κB signaling pathway by EA to alleviating neuroinflammatory response.

Therapeutic Effects of Combination of Nebivolol and Donepezil: Targeting Multifactorial Mechanisms in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive loss of motor neurons in the spinal cord. Although the disease's pathophysiological mechanism remains poorly understood, multifactorial mechanisms affecting motor neuron loss converge to worsen the disease. Although two FDA-approved drugs, riluzole and edaravone, targeting excitotoxicity and oxidative stress, respectively, are available, their efficacies are limited to extending survival by only a few months. Here, we developed combinatorial drugs targeting multifactorial mechanisms underlying key components in ALS disease progression. Using data analysis based on the genetic information of patients with ALS-derived cells and pharmacogenomic data of the drugs, a combination of nebivolol and donepezil (nebivolol-donepezil) was identified for ALS therapy. Here, nebivolol-donepezil markedly reduced the levels of cytokines in the microglial cell line, inhibited nuclear factor-κB (NF-κB) nucleus translocation in the HeLa cell and substantially protected against excitotoxicity-induced neuronal loss by regulating the PI3K-Akt pathway. Nebivolol-donepezil significantly promoted the differentiation of neural progenitor cells (NPC) into motor neurons. Furthermore, we verified the low dose efficacy of nebivolol-donepezil on multiple indices corresponding to the quality of life of patients with ALS in vivo using SOD1G93A mice. Nebivolol-donepezil delayed motor function deterioration and halted motor neuronal loss in the spinal cord. Drug administration effectively suppressed muscle atrophy by mitigating the proportion of smaller myofibers and substantially reducing phospho-neurofilament heavy chain (pNF-H) levels in the serum, a promising ALS biomarker. High-dose nebivolol-donepezil significantly prolonged survival and delayed disease onset compared with vehicle-treated mice. These results indicate that the combination of nebivolol-donepezil efficiently prevents ALS disease progression, benefiting the patients' quality of life and life expectancy.

Persistent NRG1 Type III Overexpression in Spinal Motor Neurons Has No Therapeutic Effect on ALS-Related Pathology in SOD1G93A Mice.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons (MNs). Neuregulin-1 (NRG1) is a pleiotropic growth factor that has been shown to be potentially valuable for ALS when supplemented by means of viral-mediated gene therapy. However, these results are inconsistent with other reports. An alternative approach for investigating the therapeutic impact of NRG1 on ALS is the use of transgenic mouse lines with genetically defined NRG1 overexpression. Here, we took advantage of a mouse line with NRG1 type III overexpression in spinal cord α motor neurons (MN) to determine the impact of steadily enhanced NRG1 signalling on mutant superoxide dismutase 1 (SOD1)-induced disease. The phenotype of SOD1G93A-NRG1 double transgenic mice was analysed in detail, including neuropathology and extensive behavioural testing. At least 3 animals per condition and sex were histopathologically assessed, and a minimum of 10 mice per condition and sex were clinically evaluated. The accumulation of misfolded SOD1 (mfSOD1), MN degeneration, and a glia-mediated neuroinflammatory response are pathological hallmarks of ALS progression in SOD1G93A mice. None of these aspects was significantly improved when examined in double transgenic NRG1-SOD1G93A mice. In addition, behavioural testing revealed that NRG1 type III overexpression did not affect the survival of SOD1G93A mice but accelerated disease onset and worsened the motor phenotype.

Elevated α5 integrin expression on myeloid cells in motor areas in amyotrophic lateral sclerosis is a therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a fatal disease affecting upper and lower motor neurons. Microglia directly interact with motor neurons and participate in the progression of ALS. Single-cell mass cytometry (CyTOF) analysis revealed prominent expression of α5 integrin in microglia and macrophages in a superoxide dismutase-1 G93A mouse model of ALS (SOD1G93A). In postmortem tissues from ALS patients with various clinical ALS phenotypes and disease duration, α5 integrin is prominent in motor pathways of the central and peripheral nervous system and in perivascular zones associated with the blood-brain barrier. In SOD1G93A mice, administration of a monoclonal antibody against α5 integrin increased survival compared to an isotype control and improved motor function on behavioral testing. Together, these findings in mice and in humans suggest that α5 integrin is a potential therapeutic target in ALS.

Integrated Omic Analysis Delineates Pathways Modulating Toxic TDP-43 Protein Aggregates in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multi-systemic, incurable, amyloid disease affecting the motor neurons, resulting in the death of patients. The disease is either sporadic or familial with SOD1, C9orf72, FUS, and TDP-43 constituting the majority of familial ALS. Multi-omics studies on patients and model systems like mice and yeast have helped in understanding the association of various signaling and metabolic pathways with the disease. The yeast model system has played a pivotal role in elucidating the gene amyloid interactions. We carried out an integrated transcriptomic and metabolomic analysis of the TDP-43 expressing yeast model to elucidate deregulated pathways associated with the disease. The analysis shows the deregulation of the TCA cycle, single carbon metabolism, glutathione metabolism, and fatty acid metabolism. Transcriptomic analysis of GEO datasets of TDP-43 expressing motor neurons from mice models of ALS and ALS patients shows considerable overlap with experimental results. Furthermore, a yeast model was used to validate the obtained results using metabolite addition and gene knock-out experiments. Taken together, our result shows a potential role for the TCA cycle, cellular redox pathway, NAD metabolism, and fatty acid metabolism in disease. Supplementation of reduced glutathione, nicotinate, and the keto diet might help to manage the disease.

A Glutamate Scavenging Protocol Combined with Deanna Protocol in SOD1-G93A Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive disease of neuronal degeneration in the motor cortex, brainstem, and spinal cord, resulting in impaired motor function and premature demise as a result of insufficient respiratory drive. ALS is associated with dysfunctions in neurons, neuroglia, muscle cells, energy metabolism, and glutamate balance. Currently, there is not a widely accepted, effective treatment for this condition. Prior work from our lab has demonstrated the efficacy of supplemental nutrition with the Deanna Protocol (DP). In the present study, we tested the effects of three different treatments in a mouse model of ALS. These treatments were the DP alone, a glutamate scavenging protocol (GSP) alone, and a combination of the two treatments. Outcome measures included body weight, food intake, behavioral assessments, neurological score, and lifespan. Compared to the control group, DP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan despite a greater loss of weight. GSP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan. DP+GSP had a significantly slower decline in neurological score with a trend toward increased lifespan, despite a greater loss of weight. While each of the treatment groups fared better than the control group, the combination of the DP+GSP was not better than either of the individual treatments. We conclude that the beneficial effects of the DP and the GSP in this ALS mouse model are distinct, and appear to offer no additional benefit when combined.

In Silico Exploration of Metabolically Active Peptides as Potential Therapeutic Agents against Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is regarded as a fatal neurodegenerative disease that is featured by progressive damage of the upper and lower motor neurons. To date, over 45 genes have been found to be connected with ALS pathology. The aim of this work was to computationally identify unique sets of protein hydrolysate peptides that could serve as therapeutic agents against ALS. Computational methods which include target prediction, protein-protein interaction, and peptide-protein molecular docking were used. The results showed that the network of critical ALS-associated genes consists of ATG16L2, SCFD1, VAC15, VEGFA, KEAP1, KIF5A, FIG4, TUBA4A, SIGMAR1, SETX, ANXA11, HNRNPL, NEK1, C9orf72, VCP, RPSA, ATP5B, and SOD1 together with predicted kinases such as AKT1, CDK4, DNAPK, MAPK14, and ERK2 in addition to transcription factors such as MYC, RELA, ZMIZ1, EGR1, TRIM28, and FOXA2. The identified molecular targets of the peptides that support multi-metabolic components in ALS pathogenesis include cyclooxygenase-2, angiotensin I-converting enzyme, dipeptidyl peptidase IV, X-linked inhibitor of apoptosis protein 3, and endothelin receptor ET-A. Overall, the results showed that AGL, APL, AVK, IIW, PVI, and VAY peptides are promising candidates for further study. Future work would be needed to validate the therapeutic properties of these hydrolysate peptides by in vitro and in vivo approaches.

Proteomics analysis indicates the involvement of immunity and inflammation in the onset stage of SOD1-G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease, and the pathogenic mechanism that underlies ALS is still unclear. We analyzed the differentially expressed proteins (DEPs) in the spinal cord between SOD1-G93A transgenic mice at the onset stage and non-transgenic (NTG) littermates based on 4D label-free quantitative proteomics (4D-LFQ) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In our study, 189 DEPs were screened, of which 166 were up-regulated and 23 down-regulated. Clusters of Orthologous Groups (COG)/ EuKaryotic Orthologous Groups (KOG) classification, subcellular localization annotation, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, clustering analysis and protein-protein interaction (PPI) network analyses were performed. Parallel reaction monitoring (PRM) analysis validated 48 proteins from immunity and inflammation-related pathways of KEGG. We described the function and distribution of DEPs, most of which were involved in the following pathways: complement and coagulation cascades, antigen processing and presentation, NF-kappa B signaling pathway, Retinoic acid-inducible gene I (RIG) -I-like receptor signaling pathway, the extracellular matrix-receptor (ECM-receptor) interaction, focal adhesion, phagosome and lysosome. PPI network analysis identified Fn1, Fga, Serpina1e and Serpina3n as potential biomarkers. Our discoveries broaden the view and expand our understanding of immunity and inflammation in ALS. SIGNIFICANCE: This study gives a comprehensive description of DEPs in the spinal cord proteomics of SOD1-G93A mice at the onset period. Compared with a previous study focusing on progressive stage, we showed that immunity and inflammation play an important role at the onset stage of ALS. Several pathways validated by PRM bring new insight to the pathological mechanisms of ALS. The participation of RIG-I-like signaling pathway in ALS and potential biomarkers Fga, Fn1, Serpina1e and Serpina3n are supplements to existing knowledge.

High Dose Pharmaceutical Grade Biotin (MD1003) Accelerates Differentiation of Murine and Grafted Human Oligodendrocyte Progenitor Cells In Vivo.

Accumulating evidences suggest a strong correlation between metabolic changes and neurodegeneration in CNS demyelinating diseases such as multiple sclerosis (MS). Biotin, an essential cofactor for five carboxylases, is expressed by oligodendrocytes and involved in fatty acid synthesis and energy production. The metabolic effect of biotin or high-dose-biotin (MD1003) has been reported on rodent oligodendrocytes in vitro, and in neurodegenerative or demyelinating animal models. However, clinical studies, showed mild or no beneficial effect of MD1003 in amyotrophic lateral sclerosis (ALS) or MS. Here, we took advantage of a mouse model of myelin deficiency to study the effects of MD1003 on the behavior of murine and grafted human oligodendrocytes in vivo. We show that MD1003 increases the number and the differentiation potential of endogenous murine oligodendroglia over time. Moreover, the levels of MD1003 are increased in the plasma and brain of pups born to treated mothers, indicating that MD1003 can pass through the mother's milk. The histological analysis of the grafted animals shows that MD1003 increased proliferation and accelerates differentiation of human oligodendroglia, but without enhancing their myelination potential. These findings provide important insights into the role of MD1003 on murine and human oligodendrocyte maturation/myelination that may explain the mitigated outcome of ALS/MS clinical trials.

Molecular Analysis of the Melanogenesis Inhibitory Effect of Saponins-Rich Fraction of Argania spinosa Leaves Extract.

Plant saponins are abundant and diverse natural products with a great potential for use in drug-discovery research. Here, we evaluated extracts of saponins-rich fractions of argan leaves and argan oil extraction byproducts (shell, pulp, press cake) for their effect on melanogenesis. Results show that from among the samples tested, only the saponins-rich fraction from leaves (ALS) inhibited melanin production in B16 murine melanoma (B16) cells. The mechanism of the melanogenesis inhibition was elucidated by determining the protein and mRNA expression of melanogenesis-associated enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT), and microphthalmia-associated transcription factor (MITF), and performing DNA microarray analysis. Results showed that 10 µg/mL ALS significantly inhibited melanogenesis in B16 cells and human epidermal melanocytes by 59% and 48%, respectively, without cytotoxicity. The effect of ALS on melanogenesis can be attributed to the decrease in TYR, TRP1, and MITF expression at the protein and mRNA levels. MITF inhibition naturally led to the downregulation of the expression of Tyr and Trp1 genes. Results of the DNA microarray analysis revealed the effect on melanogenesis-associated cAMP and Wnt signaling pathways' genes. The results of this study suggest that ALS may be used in cosmeceuticals preparations for hyperpigmentation treatment.

Treatment with Herbal Formula Extract in the hSOD1G93A Mouse Model Attenuates Muscle and Spinal Cord Dysfunction via Anti-Inflammation.

Amyotrophic lateral sclerosis (ALS), a multicomplex neurodegenerative disease, has multiple underlying pathological factors and can induce other neuromuscular diseases, leading to muscle atrophy and respiratory failure. Currently, there is no effective drug for treating patients with ALS. Herbal medicine, used to treat various diseases, has multitarget effects and does not usually induce side effects. Each bioactive component in such herbal combinations can exert a mechanism of action to increase therapeutic efficacy. Herein, we investigated the efficacy of an herbal formula, comprising Achyranthes bidentata Blume, Eucommia ulmoides Oliver, and Paeonia lactiflora Pallas, in suppressing the pathological mechanism of ALS in male hSOD1G93A mice. Herbal formula extract (HFE) (1 mg/g) were orally administered once daily for six weeks, starting at eight weeks of age, in hSOD1G93A transgenic mice. To evaluate the effects of HFE, we performed footprint behavioral tests, western blotting, and immunohistochemistry to detect protein expression and quantitative PCR to detect mRNA levels in the muscles and spinal cord of hSOD1G93A mice. HFE-treated hSOD1G93A mice showed increased anti-inflammation, antioxidation, and regulation of autophagy in the muscles and spinal cord. Thus, HEF can be therapeutic candidates for inhibiting disease progression in patients with ALS. This study has some limitations. Although this experiment was performed only in male hSOD1G93A mice, studies that investigate the efficacy of HEF in various ALS models including female mice, such as mice modeling TAR DNA-binding protein 43 (TDP43) and ORF 72 on chromosome 9 (C9orf72) ALS, are required before it can be established that HEF are therapeutic candidates for patients with ALS.

In silico design of a TLR4-mediating multiepitope chimeric vaccine against amyotrophic lateral sclerosis via advanced immunoinformatics.

Amyotrophic lateral sclerosis (ALS) is the most prevalent motor neuron disorder worldwide. In ALS, progressing disease can result from misfolding and aggregation of superoxide dismutase-1 (SOD1) or TAR DNA-binding protein 43 kDa (TDP43). An efficient immunotherapy for ALS should spare intact SOD1 while eliminating its dysfunctional variant. We utilized advanced immunoinformatics to suggest a potential vaccine candidate against ALS by proposing a model of dynamic TLR4 mediation and induction of a specific Th2-biased shift against mutant SOD1, TDP43, and TRAF6, a protein that specifically interacts with dysfunctional SOD1. SOD1, TDP43, and TRAF6 were retrieved in FASTA. Immune Epitopes Database and CTLpred suggested T/B-cell epitopes from disease-specific regions of selected antigens. A TLR4-mediating adjuvant, RS01, was used. Sequences were assembled via suitable linkers. Tertiary structure of the protein was calculated. Refined protein structure and physicochemical features of the 3D structure were verified in silico. Differential immune induction was assessed via C-ImmSim. GROningen MAchine for Chemical Simulation was used to assess evolution of the docked vaccine-TLR4 complex in blood. Our protein showed high structural quality and was nonallergenic and immune inducing. Also, the vaccine-TLR4 complex stability was verified by RMSD, RMSF, gyration, and visual analyses of the molecular dynamic trajectory. Contact residues in the vaccine-TLR4 complex showed favorable binding energies. Immune stimulation analyses of the proposed candidate demonstrated a sustained memory cell response and a strong adaptive immune reaction. We proposed a potential vaccine candidate against ALS and verified its physicochemical and immune inducing features. Future studies should assess this vaccine in animal studies.

Unique molecular features and cellular responses differentiate two populations of motor cortical layer 5b neurons in a preclinical model of ALS.

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding the unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type-specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Stem and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell-type-specific processes in neurodegenerative disease.

Infantile SOD1 deficiency syndrome caused by a homozygous SOD1 variant with absence of enzyme activity.

Pathogenic variants in SOD1, encoding superoxide dismutase 1, are responsible for about 20% of all familial amyotrophic lateral sclerosis cases, through a gain-of-function mechanism. Recently, two reports showed that a specific homozygous SOD1 loss-of-function variant is associated with an infantile progressive motor-neurological syndrome. Exome sequencing followed by molecular studies, including cDNA analysis, SOD1 protein levels and enzymatic activity, and plasma neurofilament light chain levels, were undertaken in an infant with severe global developmental delay, axial hypotonia and limb spasticity. We identified a homozygous 3-bp in-frame deletion in SOD1. cDNA analysis predicted the loss of a single valine residue from a tandem pair (p.Val119/Val120) in the wild-type protein, yet expression levels and splicing were preserved. Analysis of SOD1 activity and protein levels in erythrocyte lysates showed essentially no enzymatic activity and undetectable SOD1 protein in the child, whereas the parents had ∼50% protein expression and activity relative to controls. Neurofilament light chain levels in plasma were elevated, implying ongoing axonal injury and neurodegeneration. Thus, we provide confirmatory evidence of a second biallelic variant in an infant with a severe neurological syndrome and suggest that the in-frame deletion causes instability and subsequent degeneration of SOD1. We highlight the importance of the valine residues at positions V119-120, and suggest possible implications for future therapeutics research.

Therapeutic effect of a novel curcumin derivative GT863 on a mouse model of amyotrophic lateral sclerosis.

The present study investigated the therapeutic effects of the curcumin derivative 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl]ethenyl]-1H-pyrazole (GT863) in amyotrophic lateral sclerosis (ALS). The inhibitory effect of GT863 on superoxide dismutase 1 (SOD1) aggregation was evaluated in cell-free assays. GT863 interfered with the conformational changes of the SOD1 protein and later, oligomeric aggregation. Furthermore, its antioxidant, anti-inflammatory, and neuroprotective effects were evaluated in cell-free and cultured cell assays. GT863 inhibited H2O2- and glutamate-induced cytotoxicity and activated an antioxidant responsive element pathway. Additionally, in vivo effects of GT863 in the ALS mice model were evaluated by its oral administration to H46R mutant SOD1 transgenic mice. Rotarod test showed that GT863 administration significantly slowed the progression of motor dysfunction in the mice. In addition, GT863 substantially reduced highly-aggregated SOD1, further preserving large neurons in the spinal cord of GT863-treated mice. Collectively, these results indicated that GT863 could be a viable therapeutic agent with multiple vital actions for the treatment of ALS.

Combining structural and metabolic markers in a quantitative MRI study of motor neuron diseases.

OBJECTIVE: To assess the performance of a combination of three quantitative MRI markers (iron deposition, basal neuronal metabolism, and regional atrophy) for differential diagnosis between amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). METHODS: In total, 33 ALS, 12 PLS, and 28 healthy control (HC) subjects underwent a 3T MRI study including single- and multi-echo sequences for gray matter (GM) volumetry and quantitative susceptibility mapping (QSM) and a pseudo-continuous arterial spin labeling (ASL) sequence for cerebral blood flow (CBF) measurement. Mean values of QSM, CBF, and GM volumes were extracted in the motor cortex, basal ganglia, thalamus, amygdala, and hippocampus. A generalized linear model was applied to the three measures to binary discriminate between groups. The diagnostic performances were evaluated via receiver operating characteristic analyses. RESULTS: A significant discrimination was obtained: between ALS and HCs in the left and right motor cortex, where QSM increases were respectively associated with disability scores and disease duration; between PLS and ALS in the left motor cortex, where PLS patients resulted significantly more atrophic; between ALS and HC in the right motor cortex, where GM volumes were associated with upper motor neuron scores. Significant discrimination between ALS and HC was achieved in subcortical structures only combining all three parameters. INTERPRETATION: While increased QSM values in the motor cortex of ALS patients is a consolidated finding, combining QSM, CBF, and GM volumetry shows higher diagnostic potential for differentiating ALS patients from HC subjects and, in the motor cortex, between ALS and PLS.