A decline in the activities of oxidative phosphorylation (OXPHOS) complexes has been consistently reported in amyotrophic lateral sclerosis (ALS) patients and animal models of ALS, although the underlying molecular mechanisms are still elusive. Here, we report that receptor expression enhancing protein 1 (REEP1) acts as an important regulator of complex IV assembly, which is pivotal to preserving motor neurons in SOD1^G93A mice. We found the expression of REEP1 was greatly reduced in transgenic SOD1^G93A mice with ALS. Moreover, forced expression of REEP1 in the spinal cord extended the lifespan, decelerated symptom progression, and improved the motor performance of SOD1^G93A mice. The neuromuscular synaptic loss, gliosis, and even motor neuron loss in SOD1^G93A mice were alleviated by increased REEP1 through augmentation of mitochondrial function. Mechanistically, REEP1 associates with NDUFA4, and plays an important role in preserving the integrity of mitochondrial complex IV. Our findings offer insights into the pathogenic mechanism of REEP1 deficiency in neurodegenerative diseases and suggest a new therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/metabolism* ; Superoxide Dismutase-1/metabolism* ; Superoxide Dismutase/metabolism* ; Mice, Transgenic ; Spinal Cord/pathology* ; Mitochondria/physiology* ; Disease Models, Animal

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord. One important aspect of ALS pathogenesis is superoxide dismutase 1 (SOD1) mutant-mediated mitochondrial toxicity, leading to apoptosis in neurons. This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1 (G-Rg1) and conditioned medium (CM) on a mutational SOD1 cell model, and to explore the underlying mechanisms. We found that the contents of nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells (hUCMSCs) were exposed to neuron differentiation reagents for seven days. CM or G-Rg1 decreased the apoptotic rate of SOD1^G93A-NSC34 cells to a certain extent, but their combination brought about the least apoptosis, compared with CM or G-Rg1 alone. Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups. Proteins associated with mitochondrial apoptotic pathways, such as Bax, caspase 9 (Cas-9), and cytochrome c (Cyt c), were downregulated. Furthermore, CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B (NF-κB) signaling pathway by reducing the phosphorylation of p65 and IκBα. CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid (BetA), an agonist of the NF-κB signaling pathway. In summary, the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^G93A-NSC34 cells through suppressing the NF-κB/Bcl-2 signaling pathway (Fig. 1 is a graphical representation of the abstract).
Humans ; NF-kappa B/metabolism* ; Ginsenosides/pharmacology* ; Amyotrophic Lateral Sclerosis/genetics* ; Culture Media, Conditioned/pharmacology* ; Superoxide Dismutase-1 ; Neurodegenerative Diseases ; Neurons/metabolism* ; Apoptosis

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration.
Amyloidogenic Proteins/metabolism* ; Amyotrophic Lateral Sclerosis/genetics* ; Animals ; COVID-19 ; Cytoplasmic Granules/metabolism* ; Mammals ; SARS-CoV-2 ; Stress Granules

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. At present, no definite ALS biomarkers are available. In this study, exosomes from the plasma of patients with ALS and healthy controls were extracted, and differentially expressed exosomal proteins were compared. Among them, the expression of exosomal coronin-1a (CORO1A) was 5.3-fold higher than that in the controls. CORO1A increased with disease progression at a certain proportion in the plasma of patients with ALS and in the spinal cord of ALS mice. CORO1A was also overexpressed in NSC-34 motor neuron-like cells, and apoptosis, oxidative stress, and autophagic protein expression were evaluated. CORO1A overexpression resulted in increased apoptosis and oxidative stress, overactivated autophagy, and hindered the formation of autolysosomes. Moreover, CORO1A activated Ca²⁺-dependent phosphatase calcineurin, thereby blocking the fusion of autophagosomes and lysosomes. The inhibition of calcineurin activation by cyclosporin A reversed the damaged autolysosomes. In conclusion, the role of CORO1A in ALS pathogenesis was discovered, potentially affecting the disease onset and progression by blocking autophagic flux. Therefore, CORO1A might be a potential biomarker and therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/pathology* ; Calcineurin/metabolism* ; Motor Neurons/pathology* ; Microfilament Proteins/metabolism* ; Cytoskeletal Proteins/metabolism*

Parkinson's disease (PD) is the second most progressive neurodegenerative disorder of the aging population after Alzheimer’s disease (AD). Defects in the lysosomal systems and mitochondria have been suspected to cause the pathogenesis of PD. Nevertheless, the pathogenesis of PD remains obscure. Abnormal cholesterol metabolism is linked to numerous disorders, including atherosclerosis. The brain contains the highest level of cholesterol in the body and abnormal cholesterol metabolism links also many neurodegenerative disorders such as AD, PD, Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS). The blood brain barrier effectively prevents uptake of lipoprotein-bound cholesterol from blood circulation. Accordingly, cholesterol level in the brain is independent from that in peripheral tissues. Because cholesterol metabolism in both peripheral tissue and the brain are quite different, cholesterol metabolism associated with neurodegeneration should be examined separately from that in peripheral tissues. Here, we review and compare cholesterol metabolism in the brain and peripheral tissues. Furthermore, the relationship between alterations in cholesterol metabolism and PD pathogenesis is reviewed.
Aging ; Amyotrophic Lateral Sclerosis ; Atherosclerosis ; Blood Circulation ; Blood-Brain Barrier ; Brain ; Cholesterol ; Metabolism ; Mitochondria ; Neurodegenerative Diseases ; Parkinson Disease

To explore the role of the mutations G38R and D40G of Annexin A11 (ANXA11) in the onset of amyotrophic lateral sclerosis (ALS).
 Methods: The plasmids expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were constructed, respectively. The recombinant plasmids were then transfected into HEK293 cells respectively followed by cycloheximide (CHX) treatment for 0, 2, 4 and 8 h. The protein expressions of ANXA11 wild type, ANXA11 G38R and ANXA11 D40G mutations were determined by Western blot. Gray analysis by Image J was performed to compare the half-life of each protein. The NSC-34 cell lines constantly expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were established. The cells were treated with NP-40 lysis buffer to examine the protein solubility by Western blot.
 Results: Both ANXA11 G38R protein and ANXA11 D40G protein showed a shorter half-life than ANXA11 wild type protein (P<0.05), while there was no difference between ANXA11 G38R protein and ANXA11 D40G protein (P>0.05). There was no visible insoluble substance in the NP-40 lysates for ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein.
 Conclusion: G38R and D40G mutations reduce the stability of ANXA11 protein. G38R and D40G mutations do not alter ANXA11 solubility.
Amyotrophic Lateral Sclerosis ; genetics ; metabolism ; Annexins ; chemistry ; genetics ; metabolism ; HEK293 Cells ; Humans ; Mutation ; Plasmids ; genetics ; Protein Stability ; Solubility ; Transfection

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts of familial ALS patients bearing SOD1 and FUS mutations, respectively. We further generated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing (RNA-seq) analysis of motor neurons derived from SOD1 and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.
Amyotrophic Lateral Sclerosis ; genetics ; metabolism ; therapy ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Therapy ; Genome-Wide Association Study ; Humans ; Induced Pluripotent Stem Cells ; metabolism ; Mutation, Missense ; RNA-Binding Protein FUS ; genetics ; metabolism ; Superoxide Dismutase-1 ; genetics ; metabolism

BACKGROUNDSleep/wake disturbances in patients with amyotrophic lateral sclerosis (ALS) are well-documented, however, no animal or mechanistic studies on these disturbances exist. Orexin is a crucial neurotransmitter in promoting wakefulness in sleep/wake regulation, and may play an important role in sleep disturbances in ALS. In this study, we used SOD1-G93A transgenic mice as an ALS mouse model to investigate the sleep/wake disturbances and their possible mechanisms in ALS.

METHODSElectroencephalogram/electromyogram recordings were performed in SOD1-G93A transgenic mice and their littermate control mice at the ages of 90 and 120 days, and the samples obtained from these groups were subjected to quantitative reverse transcriptase-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay.

RESULTSFor the first time in SOD1-G93A transgenic mice, we observed significantly increased wakefulness, reduced sleep time, and up-regulated orexins (prepro-orexin, orexin A and B) at both 90 and 120 days. Correlation analysis confirmed moderate to high correlations between sleep/wake time (total sleep time, wakefulness time, rapid eye movement [REM] sleep time, non-REM sleep time, and deep sleep time) and increase in orexins (prepro-orexin, orexin A and B).

CONCLUSIONSleep/wake disturbances occur before disease onset in this ALS mouse model. Increased orexins may promote wakefulness and result in these disturbances before and after disease onset, thus making them potential therapeutic targets for amelioration of sleep disturbances in ALS. Further studies are required to elucidate the underlying mechanisms in the future.

Amyotrophic Lateral Sclerosis ; genetics ; metabolism ; Animals ; Female ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Transgenic ; Neuropeptides ; genetics ; metabolism ; Orexins ; Reverse Transcriptase Polymerase Chain Reaction ; Sleep ; physiology ; Superoxide Dismutase ; genetics ; metabolism ; Superoxide Dismutase-1 ; Wakefulness ; physiology

OBJECTIVETo investigate histopathology and proteinopathy in the spinal cord of patients with common neurodegenerative diseases.

METHODSSpinal cord tissues from clinically and neuropathologically confirmed neruodegnerative diseases were enrolled in this study, including 3 cases of multiple system strophy, 4 cases of amyotrophic lateral sclerosis, 5 cases of Alzheimer's disease (AD, included 2 cases of AD combined with Parkinson's disease), 2 cases of progressive supranuclear palsy, 1 case of dementia with lewy body and 1 case of corticobasal degeneration from 1955 to 2013 at Chinese People's Liberation Army General Hospital. Four normal control cases were also included. Routine HE and Gallyas-Braak staining, and immunohistochemical stainings for anti-PHF tau (AT8), anti-α-synuclein, anti-TDP-43 and anti-ubiquitin were performed.

RESULTSExamination of the spinal cord in 3 cases with multiple system strophy revealed severe neuron loss in the intermediolateral nucleus of thoracic segment and Onuf's nucleus of the sacral segment, along with moderate neuron loss in the anterior horn of the cervical segment and mild myelin pallor in the anterior funiculus and anterolateral funiculus in the cervical and thoracic segments. Large amount of argentophilic, ubiquitin and synuclein positive oligodendroglial cytoplasmic inclusions were found widely distributed in the anterior horn and the anterior funiculus and anterolateral funiculus of the full spinal cord. Severe neuron loss and several morphological changes with gliosis in the anterior horn and severe loss of myelin in the anterior funiculus and anterolateral funiculus of the full spinal cord were observed in 4 cases of amyotrophic lateral sclerosis, 2 of which were found with Bunina bodies in neurons of the anterior horn. Three amyotrophic lateral sclerosis cases had ubiquitin-positive neuronal inclusions and TDP-43 positive neuronal and glial inclusions in the anterior horn at cervical and lumbar segments. A few argentophilic, tau positive neurofibrillary tangles (NFTs) and neuropil threads in the anterior horn at cervical and lumbar segments were found in 4 AD cases. Examination of spinal cord in 2 cases with Parkinson's disease combined with AD and 1 case with dementia with lewy body revealed severe neuron loss in the intermediolateral nucleus of thoracic segment, and a few synuclein positive lewy bodies and neuritis were also observed. There was mild neuron loss in the anterior horn at cervical and lumbar segments, along with some argentophilic, tau positive globous NFTs and many argentophilic, tau positive neutrophil threads were observed in 2 progressive supranuclear palsy cases and 1 corticobasal degeneration case.

CONCLUSIONEach common neurodegenerative diseases of the spinal cord including multiple system strophy, amyotrophic lateral sclerosis and Parkinson's disease has its own specific histopathology and proteinopathy characteristics.

Alzheimer Disease ; pathology ; Amyotrophic Lateral Sclerosis ; pathology ; DNA-Binding Proteins ; metabolism ; Humans ; Immunohistochemistry ; Inclusion Bodies ; pathology ; Neurodegenerative Diseases ; pathology ; Neurofibrillary Tangles ; pathology ; Neurons ; pathology ; Parkinson Disease ; pathology ; Spinal Cord ; pathology ; Ubiquitin ; metabolism ; alpha-Synuclein ; metabolism

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into beta-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.
Alzheimer Disease/drug therapy/metabolism ; Amyloid beta-Peptides/metabolism ; Amyotrophic Lateral Sclerosis/drug therapy/metabolism ; Animals ; Autophagy/drug effects ; DNA-Binding Proteins/metabolism ; Humans ; Huntington Disease/drug therapy/genetics/metabolism ; Lysosomes/metabolism ; Molecular Targeted Therapy ; Mutation ; Nerve Tissue Proteins/genetics/metabolism ; Neurodegenerative Diseases/drug therapy/*metabolism ; Parkinson Disease/drug therapy/metabolism ; PrPSc Proteins/metabolism ; Prion Diseases/drug therapy/metabolism ; Proteasome Endopeptidase Complex/metabolism ; Proteolysis ; Proteostasis Deficiencies/metabolism ; Superoxide Dismutase/metabolism ; Ubiquitin/metabolism ; alpha-Synuclein/metabolism ; tau Proteins/metabolism