Lihong Guan

Neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are characterized by the progressive loss of neuronal function and structure, leading to severe morbidity and mortality. Current therapeutic approaches are ineffective at stopping or reversing disease progression. Stem cell therapy has emerged as a promising candidate in research and treatment. Mesenchymal stem cells (MSCs) are considered ideal candidates for regenerative medicine because of their high proliferation rate and multi-differentiation potential. MSCs can differentiate into neurons and glial cells, modulate immune responses, and reduce inflammation, and their exosomes can promote neural repair and regulate neuronal function; thus, MSCs offer unique advantages for treating neurodegenerative diseases. However, challenges remain in optimizing cell delivery methods, ensuring the long-term survival and integration of transplanted cells, and fully understanding their therapeutic effects. This article primarily outlines the functions of MSCs in neurodegenerative diseases, with the intention that further research will fully harness their potential and translate these findings into clinical applications, offering new hope for patients suffering from neurodegenerative diseases.

Niemann–Pick disease (NPD) is a rare autosomal recessive neurodegenerative disease characterized by hepatosplenomegaly, neuropathy, and a significantly shortened lifespan. Lipid metabolism disorder is the main pathological feature of NPD. Currently, the exact pathogenesis of NPD remains unclear, and drug therapy is largely palliative, focusing on symptom management, but it has side effects. Mesenchymal stem cells (MSCs) possess several advantageous properties, including their differentiation potential, wide availability, low immunogenicity, and the ability to secrete regulatory factors, which have led to their extensive application in basic research targeting neurodegenerative diseases. Studies have demonstrated that transplantation of MSCs from different sources into animal models of NPD can delay the loss of Purkinje cells in the cerebellum, reduce lipid deposition, improve motor coordination, slow the rate of weight loss, and extend lifespan. This review explores the therapeutic potential of MSCs in the treatment of NPD, highlighting their emerging role in addressing this challenging condition.

The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein (Cas) system is a gene editing technology guided by RNA endonuclease. The CRISPR-Cas12a (also known as CRISPR-Cpf1) system is extensively utilized in genome editing research due to its accuracy and high efficiency. In this paper, we primarily focus on the application of CRISPR-Cpf1 technology in the construction of disease models and gene therapy. Firstly, the structure and mechanism of the CRISPR-Cas system are introduced. Secondly, the similarities and differences between CRISPR-Cpf1 and CRISPR-Cas9 technologies are compared. Thirdly, the main focus is on the application of the CRISPR-Cpf1 system in cell and animal genome editing. Finally, the challenges faced by CRISPR-Cpf1 technology and corresponding strategies are analyzed. Although CRISPR-Cpf1 technology has certain off-target effects, it can effectively and accurately edit cell and animal genomes, and has significant advantages in the preclinical research.

Niemann-Pick disease type C1 (NPC1) is a hereditary neurodegenerative disorder caused by a mutation in the NPC1 gene. This gene encodes a transmembrane protein found in lysosomes. This disease characterized by hepatosplenomegaly, neurological impairments and premature death. Recent preclinical studies have shown promising results in using mesenchymal stem cells (MSCs) to alleviate the symptoms of NPC1. One type of MSCs, known as human menstrual blood-derived endometrial stem cells (MenSCs), has attracted attention due to its accessibility, abundant supply, and strong proliferation and regeneration capabilities. However, it remains uncertain whether the conditioned medium of MenSCs (MenSCs-CM) can effectively relieve the symptoms of NPC1. To investigate this further, we employed the CRISPR-Cas9 technique to successfully create a Npc1 gene knockout N2a cell line (Npc1^KO N2a). Sanger sequencing confirmed the occurrence of Npc1 gene mutation in these cells, while western blotting revealed a lack of NPC1 protein expression. Filipin staining provided visual evidence of unesterified cholesterol accumulation in Npc1^KO N2a cells. Moreover, Npc1^KO N2a cells exhibited significantly decreased viability, increased inflammation, and heightened cell apoptosis. Notably, our study demonstrated that the viability of Npc1^KO N2a cells was most significantly improved after being cultured by 36 h-collected MenSCs-CM for 0.5 days. Additionally, MenSCs-CM exhibited the ability to effectively reduce inflammation, counteract cell apoptosis, and ameliorate unesterified cholesterol accumulation in Npc1^KO N2a cells. This groundbreaking finding establishes, for the first time, the protective effect of MenSCs-CM on N2a cells with Npc1 gene deletion. These findings suggest that the potential of MenSCs-CM as a beneficial therapeutic approach for NPC1 and other neurodegenerative diseases.

NPC1 gene encodes a transmembrane glycoprotein on the late endosome/lysosomal membrane. Its mutation leads to a rare and aggravated autosomal recessive neurovisceral condition, termed Niemann-Pick disease type C1 (NPC1), which is characterized by progressive neurodegeneration, visceral symptoms, and premature death. To investigate the influence of NPC1 gene deletion on cell morphology, adhesion, proliferation, and apoptosis, CRISPR-Cas9 technology was used to knockout the NPC1 gene in HEK 293 T cells. Sanger sequencing, western blotting, and immunofluorescence were used to confirm successful NPC1 ablation. Filipin staining results indicated that deletion of NPC1 gene led to accumulation of unesterified cholesterol in HEK 293 T cells. Phalloidin staining results revealed cell aggregation, synapse shortening, nuclear enlargement, and cytoskeleton filamentous actin thinning in HEK 293 T cells with NPC1 gene mutation. Furthermore, NPC1 gene mutated HEK 293 T cell showed enhanced cell adhesion, inhibited cell proliferation, and increased cell apoptosis. In addition, NPC1 gene mutations significantly increased the protein expression levels of E-cadherin and γ-catenin and significantly decreased the protein expression levels of Wnt 3a, c-Myc, and cyclin D1. These results suggest that NPC1 may regulate cell adhesion by affecting the cadherin–catenin complex through E-cadherin, and that the classical Wnt signaling pathway may be inhibited by restricting β-catenin from entering the nucleus to inhibit cell proliferation.

Niemann–Pick disease type C1 (NPC1) is a neurodegenerative disorder characterized by lysosomal storage of free cholesterol. 2-Hydroxypropyl-β-cyclodextrin (HPβCD) is a cyclic oligosaccharide derivative that is being developed to treat NPC1. Recently, metformin was reported to be beneficial in various neurodegenerative diseases, such as Alzheimer’s and Huntington’s diseases. In this study, we examined the effects of combined treatment with HPβCD and metformin on Npc1^−/− mice. Unfortunately, body weight and survival rates showed that cotreatment with metformin did not extend survival time and increase the body weight of HPβCD-treated Npc1^−/− mice. However, cotreatment with metformin reduced inflammatory response and inhibited the proinflammatory cytokine release in the brain, liver and spleen of HPβCD-treated Npc1^−/− mice. Furthermore, metformin did not reduce the free cholesterol levels in Npc1^−/− brain tissue or fibroblasts. In conclusion, our results demonstrate that metformin does not show beneficial effects on body weight or survival time but reduced the inflammatory response in a mouse model of NPC1 when combined with HPβCD.

Menstrual blood-derived endometrial stem cells (MenSCs) emerge as an ideal source for cell-based treatment in regenerative medicine and immunotherapy. However, the major obstacle is the low survival rate in tissues and the limited expansion number. Autophagy is an intracellular metabolic self-degradative process which plays important roles in normal cellular division and survival, and the present study aimed to explore the related mechanisms between autophagy and survival of MenSCs in vitro and in vivo. The MenSCs were obtained from menstrual blood procured from healthy female donors. In vitro, MenSCs were exposed to rapamycin and Earle’s balanced salts solution (EBSS). We evaluated the MenSCs immunophenotypic cell cycle distribution by propidium iodide (PI) staining and cell apoptosis by Annexin V/PI staining as well as their proliferative potential by the MTT assay. We also assessed the expression of genes associated with the cell cycle and Gsk3β signaling pathway by western blot analysis. We depressed Atg5 and Gsk3β expression by short hairpin RNA (shRNA) and undertook the experiments. Moreover, the labeled MenSCs were observed and counted with DiI after transplantation into the mice via the tail vein by microscopy in vivo. In vitro, rapamycin and starvation induced autophagy of MenSCs. Hyperactive autophagy significantly induced G0/G1 arrest and slightly promoted apoptosis of MenSCs. Meanwhile, autophagy could stimulate p-GSK3β expression in MenSCs. Further, knockdown GSK3β can accelerate the proliferation of MenSCs by shRNA and CHIR99021. Moreover, the shGSK3β MenSCs showed strong proliferative activity in vitro and in vivo. Our results indicate that autophagy induced G0/G1 arrest and apoptosis of MenSCs via GSK3β/β-catenin pathway. Inhibiting autophagy or reduced GSK3β levels may improve survival rate in vivo, thus playing roles in MenSCs therapy.

Hypoxia-inducible factor (HIF) is a master regulator that mediates major changes in gene expression under hypoxic conditions. Though HIF family has been identified in many organisms, little is known about this family in schizothoracine fish. Duplicated hif-α (hif-1αA, hif-1αB, hif-2αA, and hif-2αB) genes were identified in schizothoracine fish. All the deduced HIF-α proteins contain the main domains (bHLH-PAS, ODDD, and TAD), also found in humans. Evidence suggests a Cyprinidae-specific deletion, specifically, a conserved proline hydroxylation motif LxxLAP, in the NODD domain of schizothoracine fish HIF-1αA. In addition, a schizothoracine-specific mutation was observed in the CODD domain of the specialized and highly specialized schizothoracine fish HIF-1αB, which is the proline hydroxylation motif mutated into PxxLAP. Standard and stochastic branch-site codon model analysis indicated that only HIF-1αB has undergone positive selection, which may have led to changes in function. To confirm this hypothesis, HIF-αs tagged with Myc were transfected into HEK 293 T cells. Each HIF-1αB was found to significantly upregulate luciferase activity under normoxic and hypoxic conditions, which indicated that the HIF-1αB protein was more stable than other HIF-αs. All deduced HIF-α proteins of schizothoracine fish contain important domains, like their mammalian counterparts, and each HIF-α is shorter than that of human. Our experiments reveal that teleost-specific duplicated HIF-α genes played different roles under hypoxic conditions, and HIF-1αB may be the most important regulator in the adaptation of schizothoracine fish to the environment of the Tibetan Plateau.

Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA^Ala molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA^Ala-derived region at the 5′ end, a tRNA-unrelated region, and AT-rich region at the 3′ end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA^Ala gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA^Ala. The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.