single-cell-calcium-imaging-for-studying-activation-calcium-ion

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...

Beijing University of Chinese Medicine

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder. The purpose of this investigation is to understand the role of mitochondrial haplotypes in the development of LHON associated with ND6 T14484C mutation in Chinese families.

Low penetrance of Leber's hereditary optic neuropathy in ten Han Chinese families carrying the ND6 T11484C mutation.

To investigate the pathophysiology of Leber's hereditary optic neuropathy (LHON), a cohort of 1164 Han Chinese subjects with LHON were screened for ND1 G3460A mutation. A total of 295 subjects from 16 Han Chinese families carrying the G3460A mutation underwent a clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA. The incidence of G3460A mutation was 1.4% in this cohort of Chinese subjects with LHON. Twenty-seven (20 males/7 females) of 109 matrilineal relatives among 10 Chinese pedigrees carrying this mutation exhibited a wide range of severity and age-at-onset in visual impairment. Penetrances of optic neuropathy ranged from 7.1% to 50%, with the average of 24.5%. The age-at-onset of 27 affected matrilineal relatives varied from 10 to 40 years, with the average of 22 years. Molecular analysis identified the homoplasmic G3460A mutation and distinct sets of variants belonging to eight haplogroups. Haplogroup M with G3460A mutation was of higher frequency than those in controls. The penetrances of visual loss in families carrying mitochondrial DNA haplogroups A, B and M were higher than those in other families. Furthermore, haplogroup-specific variants tRNA(Ser(AGY)) A12223G, tRNA(Thr) G15927A and tRNA(Glu) A14693G may enhance the penetrance of visual loss in these families. The G3460A mutation occurred through recurrent origins and founder events in Chinese population. Mitochondrial modifiers may modulate the penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the G3460A mutation. Thus, our findings may provide new insights into the understanding of pathophysiology and valuable information on the management of LHON.

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families.

The m.14484T>C mutation in mitochondrial ND6 gene (MT-ND6) is a primary mutation underlying the development of Leber's hereditary optic neuropathy (LHON) , but by itself not enough to cause visual loss. To explore the role of mitochondrial haplogroups on the expression of LHON for the people carrying the m.14484T>C mutation, we performed systematic and extended mutational screening of MT-ND6 gene in a cohort of 1177 Han Chinese patients with LHON. A total of 67 affected subjects carried the homoplasmic m.14484T>C mutation, accounting for 5.7% of this LHON population. The penetrances of optic neuropathy among 51 pedigrees carrying the m.14484T>C mutation ranged from 5.6% to 100.0%, with the average of 21.5%. The sequence analysis of entire mitochondrial genomes of 51 probands exhibited distinct sets of polymorphisms belonging to 18 Eastern Asian haplogroups. The frequencies of haplogroup A and haplogroup F were sig-nificantly less in the LHON mtDNA samples than those in 106 Chinese controls. On the other hand, the haplogroup M10a accounted for 9.8% of the patient's mtDNA samples but was absent in 106 Chinese controls. Strikingly, the average pene-trance (46.13%) of optic neuropathy for the pedigrees carrying mitochondrial haplogroup M10a was higher than those car-rying other mtDNA haplogroups. These observations indicated that mitochondrial haplogroup M10a may increase the risk of visual loss.

[The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484T >C mutation].

In this report, we investigated the molecular mechanism underlying Leber's hereditary optic neuropathy (LHON)-associated mitochondrial m.3635G>A (p.S110N, ND1) mutation. A mutational screening of ND1 gene in a cohort of 1070 Han Chinese subjects LHON identified the m.3635G>A mutation in nine Chinese families with suggestively maternally transmitted LHON. Thirty-eight (22 males/16 females) of 162 matrilineal relatives in these families exhibited the variable severity and age-at-onset of optic neuropathy. Molecular analysis of their mitochondrial genomes identified the homoplasmic m.3635G>A mutation and distinct sets of polymorphisms belonging to the Asian haplogroups G2a1, R11a, D4, R11a, M7b2, G1a, F1a1, B4, and N9a3, respectively. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from one Chinese family into mtDNA-less (ρ(0)) cells, we showed ~27% decrease in the activity of NADH:ubiquinone oxidoreductase (complex I) in mutant cybrids carrying the m.3635G>A mutation, compared with control cybrids. The respiratory deficiency caused by the m.3635G>A mutation results in decreased efficiency of mitochondrial ATP synthesis. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cybrids. The data provide the direct evidence for the m.3635G>A mutation leading to LHON. Our findings may provide new insights into the understanding of pathophysiology of LHON.

Leber's hereditary optic neuropathy caused by the homoplasmic ND1 m.3635G>A mutation in nine Han Chinese families.

To investigate the prevalence and spectrum of mitochondrial ND4 mutations in subjects with Leber's hereditary optic neuropathy (LHON).

Prevalence of Mitochondrial ND4 Mutations in 1281 Han Chinese Subjects With Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

Leber's hereditary optic neuropathy (LHON) is one of the most common mitochondrial disorders. We report here the clinical, genetic and molecular analysis of mitochondrial DNA (mtDNA) in eight Han Chinese families carrying the known mitochondrial 11778G > A(MT-ND4) mutation. Thirty-seven (26 males/11 females) of 77 matrilineal relatives in these families exhibited the variable severity and age-at-onset of optic neuropathy. The penetrances were from 25% to 75%, with the average of 42%, and the age-at-onset for visual impairment varied from 10 to 25 years, with the average of 17 in these Chinese pedigrees. Molecular analysis of their mtDNA identified distinct sets of variants belonging to the Eastern Asian haplogroupD4j. Except the known m.11778G > A mutation, the m.11696G > A(MT-ND4) mutation caused the substitution of an isoleucine for valineat amino acid position 313, located in a predicted transmembrane region of ND4. And, it is reported that the m.11696G > A mutation was associated with LHON, and appeared to contribute to higher penetrance in these nine Chinese families than other Chinese families carrying only the m.11778G > A mutation. Therefore, the mitochondrial haplogroup D4j specific m.11696G > A mutation may act in synergy with the primary LHON-associated m.11778G > A mutation, thereby increasing the penetrance and expressivity of visual loss in these Chinese families.

Mitochondrial haplogroup D4j specific variant m.11696G > a(MT-ND4) may increase the penetrance and expressivity of the LHON-associated m.11778G > a mutation in Chinese pedigrees.

The purpose of this study was to investigate the mutational incidence and spectrum of mitochondrial ND1 gene in subjects with Leber's hereditary optic neuropathy (LHON).

Mitochondrial ND1 Variants in 1281 Chinese Subjects With Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease. Mitochondrial modifiers are proposed to modify the phenotypic expression of primary LHON-associated mitochondrial DNA (mtDNA) mutations. In this study, we demonstrated that the LHON susceptibility allele (m.14502T > C, p. 58I > V) in the ND6 gene modulated the phenotypic expression of primary LHON-associated m.11778G > A mutation. Twenty-two Han Chinese pedigrees carrying m.14502T > C and m.11778G > A mutations exhibited significantly higher penetrance of optic neuropathy than those carrying only m.11778G > A mutation. We performed functional assays using the cybrid cell models, generated by fusing mtDNA-less ρ cells with enucleated cells from LHON patients carrying both m.11778G > A and m.14502T > C mutations, only m.14502T > C or m.11778G > A mutation and a control belonging to the same mtDNA haplogroup. These cybrids cell lines bearing m.14502T > C mutation exhibited mild effects on mitochondrial functions compared with those carrying only m.11778G > A mutation. However, more severe mitochondrial dysfunctions were observed in cell lines bearing both m.14502T > C and m.11778G > A mutations than those carrying only m.11778G > A or m.14502T > C mutation. In particular, the m.14502T > C mutation altered assemble of complex I, thereby aggravating the respiratory phenotypes associated with m.11778G > A mutation, resulted in a more defective complex I. Furthermore, more reductions in the levels of mitochondrial ATP and increasing production of reactive oxygen species were also observed in mutant cells bearing both m.14502T > C and m.11778G > A mutation than those carrying only 11778G > A mutation. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between primary and secondary mtDNA mutations.

Biochemical evidence for a mitochondrial genetic modifier in the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

Mitochondrial (mt)DNA mutations have been revealed to be associated with Leber's hereditary optic neuropathy (LHON). The present study conducted clinical, genetic and molecular evaluations of two Han Chinese families. A total of 4 (3 men and 1 female) out of 14 matrilineal relatives in the families exhibited visual impairment with variable severity and age of onset. The average age of onset of visual loss was 20.5 years old. Molecular analysis of the complete mitochondrial genome in these pedigrees demonstrated that the three primary mutations associated with LHON were not detected; however, the homoplasmic m.5587T>C mutation was identified, which was localized at the end of the mitochondrially encoded transfer (t)RNA alanine gene and may alter the tertiary structure of this tRNA. Subsequently, this structural alteration may result in tRNA metabolism failure. In addition, distinct sets of mtDNA polymorphisms belonging to haplogroup F1 were detected in both families tested. The findings of the present study suggested that the m.5587T>C mutation may be involved in the pathogenesis of visual impairment. In addition, the mtDNA variant m.15024G>A(p.C93H) in the mitochondrially encoded cytochrome B gene was detected in both families, which exhibited evolutionary conservation, indicating it may serve a potential modifying role in the development of visual impairment associated with m.5587T>C mutation in these families. Furthermore, other modifying factors, including nuclear modifier genes, and environmental and personal factors may also contribute to the development of LHON in subjects carrying this mutation.

Leber's hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees.

In this study, micro-CT was used to observe the microscopic anatomy of the hyoid bone, examine the variation of the trabecular bone inside the hyoid bone, and investigate the internal structure of the hyoid bone.A total of 22 hyoid bones were scanned using micro-CT. The changes in the internal bone trabeculae were assessed with 3D reconstructions, and the fine anatomical structure of the hyoid bone was further analyzed.Micro-CT images showed the microstructure of various parts of the hyoid bone. There were significant differences in total volume, bone volume, bone area, bone density, and volume fraction between the body and greater horns of the hyoid bone (P < .05), but no significant differences in the ratio of bone area/volume and bone surface density were found between the body and greater horns of the hyoid bone (P > .05). In addition, significant differences in the trabecular bone measurements, bone trabecular connectivity, and Euler number were found between the body and greater horns of the hyoid bone (P < .05). Other parameters, including bone trabecular thickness, number of trabecular bones, bone trabecular structure model index, and anisotropy of bone trabeculae, did not differ between the body and greater horns of the hyoid bone (P > .05). There was noticeably ossified healing at the joint between the body and greater horns of the hyoid bone.Micro-CT can adequately display the internal structure of the hyoid bone. The identified bone structure may help clarify the physiological function of the hyoid bone. The present findings provide a theoretical basis for further studies aimed at pathological changes due to hyoid injury in clinical and forensic medicine.

Microstructure of the hyoid bone based on micro-computed tomography findings.

Deubiquitination is the reverse process of ubiquitination, an important protein post-translational modification. Deubiquitination is assisted by deubiquitinating enzymes (DUBs), which catalyze the hydrolysis and removal of ubiquitin chains from targeted proteins and play an important role in regulating protein stability, cell signaling transduction, and programmed cell death. Ubiquitin-specific peptidases 25 and 28 (USP25 and USP28), important members of the USP subfamily of DUBs, are highly homologous, strictly regulated, and closely associated with various diseases, such as cancer and neurodegenerative diseases. Recently, the development of inhibitors targeting USP25 and USP28 for disease treatment has garnered extreme attention. Several non-selective and selective inhibitors have shown potential inhibitory effects. However, the specificity, potency, and action mechanism of these inhibitors remain to be further improved and clarified. Herein, we summarize the structure, regulation, emerging physiological roles, and target inhibition of USP25 and USP28 to provide a basis for the development of highly potent and specific inhibitors for the treatment of diseases, such as colorectal cancer, breast cancer and so on.

Structure and function of the highly homologous deubiquitinases ubiquitin specific peptidase 25 and 28: Insights into their pathophysiological and therapeutic roles.

STIM1 has been identified as a new warm sensor, but the exact molecular mechanism remains unclear. In this study, a variety of mutants of STIM1, Orai1 and Orai3 were generated. The single-cell calcium imaging and confocal analysis were used to evaluate the thermal sensitivity of the resulting STIM mutants and the interaction between STIM1 and Orai mutants in response to temperature. Our results suggested that the CC1-SOAR of STIM1 was a direct activation domain of temperature, leading to subsequent STIM1 activation, and the transmembrane (TM) region and K domain but not EF-SAM were needed for this process. Furthermore, both the TM and SOAR domains exhibited similarities and differences between STIM1-mediated thermal sensation and store-operated calcium entry (SOCE), and the key sites of Orai1 showed similar roles in these two responses. Additionally, the TM23 (comprising TM2, loop2, and TM3) region of Orai1 was identified as the key domain determining the STIM1/Orai1 thermal response pattern, while the temperature reactive mode of STIM1/Orai3 seemed to result from a combined effect of Orai3. These findings provide important support for the specific molecular mechanism of STIM1-induced thermal response, as well as the interaction mechanism of STIM1 with Orai1 and Orai3 after being activated by temperature.

Article	Year
Low penetrance of Leber's hereditary optic neuropathy in ten Han Chinese families carrying the ND6 T11484C mutation. Biochimica et biophysica acta\| PubMed ID: 19733221	2010
Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families. Journal of human genetics\| PubMed ID: 24430572	2014
[The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484T >C mutation]. Yi chuan = Hereditas\| PubMed ID: 24846978	2014
Leber's hereditary optic neuropathy caused by the homoplasmic ND1 m.3635G>A mutation in nine Han Chinese families. Mitochondrion\| PubMed ID: 25194554	2014
Prevalence of Mitochondrial ND4 Mutations in 1281 Han Chinese Subjects With Leber's Hereditary Optic Neuropathy. Investigative ophthalmology & visual science\| PubMed ID: 26218905	2015
The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic ne... Human molecular genetics\| PubMed ID: 26647310	2016
Mitochondrial haplogroup D4j specific variant m.11696G > a(MT-ND4) may increase the penetrance and expressivity of the LHON-associated m.11778G > a mutation in Chinese pedigrees. Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis\| PubMed ID: 27159682	2017
Mitochondrial ND1 Variants in 1281 Chinese Subjects With Leber's Hereditary Optic Neuropathy. Investigative ophthalmology & visual science\| PubMed ID: 27177320	2016
Biochemical evidence for a mitochondrial genetic modifier in the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation. Human molecular genetics\| PubMed ID: 27427386	2016
Leber's hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees. Molecular medicine reports\| PubMed ID: 28990081	2017
Microstructure of the hyoid bone based on micro-computed tomography findings. Medicine\| PubMed ID: 33126297	2020
Structure and function of the highly homologous deubiquitinases ubiquitin specific peptidase 25 and 28: Insights into their pathophysiological and therapeutic roles. Biochemical pharmacology\| PubMed ID: 37245535	2023
Molecular Mechanism Analysis of STIM1 Thermal Sensation. Cells\| PubMed ID: 37998348	2023

Xiaoling Liu

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