Manho Kim

Role of GSK-3β activity in motor neuronal cell death induced by G93A or A4V mutant hSOD1 gene

Point mutations such as G93A and A4V in the human Cu/Zn‐superoxide dismutase gene (hSOD1) cause familial amyotrophic lateral sclerosis (fALS). In spite of several theories to explain the pathogenic mechanisms, the mechanism remains largely unclear. Increased activity of glycogen synthase kinase‐3 (GSK‐3) has recently been emphasized as an important pathogenic mechanism of neurodegenerative diseases, including Alzheimers disease and ALS. To investigate the effects of G93A or A4V mutations on the phosphatidylinositol‐3‐kinase (PI3‐K)/Akt and GSK‐3 pathway as well as the caspase‐3 pathway, VSC4.1 motoneuron cells were transfected with G93A‐ or A4V‐mutant types of hSOD1 (G93A and A4V cells, respectively) and, 24u2003h after neuronal differentiation, their viability and intracellular signals, including PI3‐K/Akt, GSK‐3, heat shock transcription factor‐1 (HSTF‐1), cytochromeu2003c, caspase‐3 and poly(ADP‐ribose) polymerase (PARP), were compared with those of wild type (wild cells). Furthermore, to elucidate the role of the GSK‐3β‐mediated cell death mechanism, alterations of viability and intracellular signals in those mutant motoneurons were investigated after treating the cells with GSK‐3β inhibitor. Compared with wild cells, viability was greatly reduced in the G93A and A4V cells. However, the treatment of G93A and A4V cells with GSK‐3β inhibitor increased their viability by activating HSTF‐1 and by reducing cytochromeu2003c release, caspase‐3 activation and PARP cleavage. However, the treatment did not affect the expression of PI3‐K/Akt and GSK‐3β. These results suggest that the G93A or A4V mutations inhibit PI3‐K/Akt and activate GSK‐3β and caspase‐3, thus becoming vulnerable to oxidative stress, and that the GSK‐3β‐mediated cell death mechanism is important in G93A and A4V cell death.

Altered Expression of the Long Noncoding RNA NEAT1 in Huntington’s Disease

Huntington’s disease (HD) is a devastating neurodegenerative disease caused by cytosine-adenine-guanine trinucleotide repeat expansion in the huntingtin gene. Growing evidence supports the regulatory functions of long noncoding RNAs (lncRNAs) in the disease process, but little is known about the association between lncRNAs and neuronal death in HD. Here, we evaluated the altered expression profiles of lncRNA in HD by using microarrays. Among dysregulated lncRNAs, we focused on the upregulation of nuclear paraspeckle assembly transcript 1 (NEAT1). Quantitative PCR analysis validated increased NEAT1 levels in the R6/2 mouse brain as well as the human HD postmortem brain. To determine the biological effects of NEAT1 on neuronal survival, neuro2A cells were transfected with the NEAT1 short isoform vector and were subjected to H2O2-induced injury. Subsequently, NEAT1-transfected cells showed increased viability under oxidative stress. Our observations support the notion that NEAT1 upregulation in HD contributes to the neuroprotective mechanism against neuronal injury rather than the pathological process underlying neurodegeneration in HD.

Adipose-derived stem cell exosomes alleviate pathology of amyotrophic lateral sclerosis in vitro

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder that involves the death of motor neurons in the cortex, brain stem, and spinal cord. Adipose-derived stem cells (ADSCs) are considered as a perspective remedy for therapy of neurodegenerative diseases including ALS. Stem cells secrete various factors which can modulate a hostile environment, called paracrine effect. Exosomes are small extracellular vesicles containing cell derived factors and mediate paracrine effect of cells. Thus, exosomes from ADSCs (ADSC-exo) can be a potential candidate of therapeutic effects of stem cells. To investigate the effect of ADSC-exo on the cellular phenotypes of ALS, we used neuronal stem cells (NSCs), which can be differentiated into neuronal cells, isolated from wild type or G93A ALS mice model. ADSC-exo was treated to neuronal cells from G93A ALS mice model. Immunocytochemistry and dot-blot assay result showed that ADSC-exo alleviated aggregation of superoxide dismutase 1 (SOD1). Reduction of cytosolic SOD1 level by ADSC-exo was also confirmed by western blot. Mitochondria display various abnormalities in ALS and the decrease of phospho-CREB and PGC-1α were observed in the G93A cells. ADSC-exo treatment showed normalization of phospho-CREB/CREB ratio and PGC-1α expression level. Our results suggest that ADSC-exo modulates cellular phenotypes of ALS including SOD-1 aggregation and mitochondrial dysfunction, and can be a therapeutic candidate for ALS.

Altered Functional Connectivity in Idiopathic Rapid Eye Movement Sleep Behavior Disorder: A Resting-State EEG Study

Study ObjectivesnIdiopathic rapid eye movement sleep behavior disorder (iRBD) is considered as a prodromal stage of synucleinopathy. Although loss of functional connectivity is implicated in neurodegenerative diseases, network characteristics of electroencephalography (EEG) in iRBD are unknown. Therefore, we evaluated resting-state EEG functional connectivity to identify the brain network changes in patients with iRBD.nnnMethodsnWe prospectively enrolled 20 patients with polysomnography-confirmed iRBD and 16 controls. Four patients with mild cognitive impairment were excluded from the analysis after cognitive function tests. EEG was recorded during relaxed wakefulness. We computed the weighted phase lag index as a measure of functional connectivity from EEG recordings.nnnResultsnAll patients with iRBD (mean age 64.3 years; men, 68.8%) had no overt manifestations of neurodegenerative diseases such as Parkinsonism or dementia. The mean duration from symptom onset was 4.8 years. Overall connectivity strength did not differ between the two groups in all frequency bands. However, comparisons of each functional connection with the nonparametric permutation test demonstrated iRBD had decreased delta-band functional connectivity in the frontal regions. There were no significantly increased functional connections in all frequencies. The altered connections had a significant correlation with RBD questionnaire scores. Notably, delta-band weighted phase lag index between left frontal and central regions was correlated with verbal fluency performance (r = 0.486, p = .007).nnnConclusionsnResting-state brain network of iRBD was characterized by a loss of delta-band functional connectivity. Therefore, functional networks in iRBD are altered at the early phase of disease.

The protective effect of growth hormone on Cu/Zn superoxide dismutase-mutant motor neurons

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by selective degeneration of motor neurons. The gene encoding Cu/Zn superoxide dismutase (SOD1) is responsible for 20% of familial ALS cases. Growth hormone (GH) concentrations are low in the cerebrospinal fluid of patients with ALS; however, its association with motoneuronal death is not known. We tested the neuroprotective effects of GH on human SOD-1-expressing cultured motor neurons and SOD1G93A transgenic mice.ResultsIn cultured motor neurons, cytotoxicity was induced by A23187, GNSO, or homocysteine, and the effects of GH were determined by MTT, bax, PARP cleavage pattern, Hoechst nuclear staining, MAPK, and PI3K assay. In SOD-1 transgenic mice, rotarod motor performance was evaluated. Survival analysis of motoneuronal loss was done using cresyl violet, GFAP, and Bcl-2 staining. GH prevents motorneuronal death caused by GSNO and homocysteine, but not that by A23187. It activates MAPK and PI3K. GH-treated mice showed prolonged survival with improved motor performance and weight loss. GH decreased cresyl violet positive motoneuronal loss with strong Bcl-2 and less GFAP immunoreactivity.ConclusionsOur results demonstrate that GH has a protective effect on mutant SOD-1-expressing motor neurons.

Novel compound heterozygous mutations of the SPG11 gene in Korean families with hereditary spastic paraplegia with thin corpus callosum

Hereditary spastic paraplegia with thin corpus callosum (HSP-TCC) is one of the most common complicated forms of autosomal recessive hereditary spastic paraplegia (HSP). Mutation in SPG11 gene, which is mapped to chromosome 15q21, was recently found to be a major cause of this variant form of HSP. The aim of this study is to investigate SPG11 mutations and clinical manifestations in two Korean families with HSP-TCC. Direct sequencing of the 40 coding exons and boundaries of exon–intron in SPG11 gene, and descriptions of clinical findings in two nonconsanguineous families with HSP-TCC are presented. Three novel and one known compound heterozygous mutations were found in two affected families, which were not found in controls, including one deletion in exon (c.5410_5411delTG), two insertions (c.1834_1835InsT and c.2163_2164InsT), and one missense mutation (c.3291+1G>T). Both of our patients had impairments in frontal lobe functions. We present the first SPG11 mutations in Korean families, three of which are novel. SPG11 mutation should be suspected in Korean patients having HSP with TCC and executive dysfunction.

Influence of storage condition on exosome recovery

Most of mammalian cells release extracellular vesicles including exosomes which mediate intercellular communication by delivering a variety of molecules. Despite of their importance in normal physiology and disease progression, the standard criteria of storage condition is indefinite and controversial. Therefore, we investigated exosome’s recovery yield and stability by various storage conditions. To investigate the effect of short-term storage temperature on exosome stability, exosomes were incubated at temperatures ranging from -70 to 90°C for 30 min. Immunoblot results showed that all exosome-associated proteins incubated at 90°C were mostly degraded for a short period of time. To examine the effect of long-term storage, isolated exosomes were incubated for 10 days at from -70°C to room temperature (RT), and exosomal protein, RNA and exosome markers were examined. Protein and RNA amounts were most reduced at RT compared with -70 and 4°C. Incubation at 4°C and RT resulted in major loss of CD63, and decreasing level of HSP70 was shown at only RT. In addition, flow cytometry result showed that exosome population became more dispersed after RT incubation for 10 days compared with -70°C incubated or freshly isolated exosomes. In summary, our results indicate that different storage temperature and period influences recovery yield and morphology of exosome, and storage at below -70°C is the favorable condition for preservation of fresh exosomes for clinical application and basic researches.

Neuroprotective Effect of Human Adipose Stem Cell-Derived Extract in Amyotrophic Lateral Sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disease. The precise pathogenic mechanisms of the disease remain uncertain, and as of yet, there is no effective cure. Human adipose stem cells (hASC) can be easily obtained during operative procedures. hASC have a clinically feasible potential to treat neurodegenerative disorders, since cytosolic extract of hASC contain a number of essential neurotrophic factors. In this study, we investigated effects of hASC extract on the SOD1 G93A mouse model of ALS and in vitro test. Administration of hASC extract improved motor function and prolonged the time until symptom onset, rotarod failure, and death in ALS mice. In the hASC extracts group, choline acetyltransferase immunostaining in the ventral horn of the lumbar spinal cord showed a large number of motor neurons, suggesting normal morphology. The neuroprotective effect of hASC extract in ALS mice was also suggested by western blot analysis of spinal cord extract from ALS mice and in vitro test. hASC extract treatment significantly increased expression of p-Akt, p-CREB, and PGC-1α in SOD1 G93A mouse model and in vitro test. Our results indicated that hASC extract reduced apoptotic cell death and recovered mutant SOD1-induced mitochondrial dysfunction. Moreover, hASC extract reduced mitochondrial membrane potential. In conclusion, we have demonstrated, for the first time, that hASC extract exert a potential therapeutic action in the SOD1 G93A mouse model of ALS and in vitro test. These findings suggest that hASC hold promise as a novel therapeutic strategy for treating ALS.

Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells

Stem-cell-based therapies are regarded as promising treatments for neurological disorders, and adipose-derived stem cells (ASCs) are a feasible source of clinical application of stem cell. Recent studies have shown that stem cells have a therapeutic potential for use in the treatment of various illnesses through paracrine action. To examine the effects of cell components of ASCs on neural stem cells (NSCs), we treated cell-free extracts of ASCs (CFE-ASCs) containing various components with brain-derived NSCs. To elucidate the effects of CFE-ASCs in NSC proliferation, we treated mouse subventricular zone-derived cultured NSCs with various doses of CFE-ASCs. As a result, CFE-ASCs were found to induce the proliferation of NSCs under conditions of growth factor deprivation in a dose-dependent manner (p<0.01). CFE-ASCs increase the expression of neuron and astrocyte differentiation markers including Tuj-1 (p<0.05) and glial fibrillary acidic protein (p<0.01) without altering the cell’s fate in differentiating NSCs. In addition, treatment with CFE-ASCs induces an increase in neurite numbers (p<0.01) and lengths of NSCs (p<0.05). Furthermore, CFE-ASCs rescue the hydrogen peroxide-induced reduction of NSCs’ viability (p<0.05) and neurite branching (p<0.01). Findings from our study indicate that CFE-ASCs support the survival, proliferation and differentiation of NSCs accompanied with neurite outgrowth, suggesting that CFE-ASCs can modulate neurogenesis in the central nervous system.

Psychiatric symptoms delay the diagnosis of anti-LGI1 encephalitis

The aim of this study was to analyze the detailed characteristics of the psychiatric symptoms in patients with anti-LGI1 encephalitis. Of 16 patients, ten showed psychiatric symptoms as the initial manifestations. All 10 patients experienced mood-related symptoms. The time to immune therapy was longer in those with initial psychiatric symptoms compared to those without them. Initial manifestation of psychiatric symptoms in patients with anti-LGI1 encephalitis may be a poor prognostic factor, at least in the short term, in that it misleads both the patients and the clinicians to neglect the typically accompanied symptoms of the disease such as faciobrachial dystonic seizure, delaying the timing of immune therapy.