ABCA7 deficiency causes neuronal dysregulation by altering mitochondrial lipid metabolism.

ABCA7 loss-of-function variants are associated with increased risk of Alzheimer's disease (AD). Using ABCA7 knockout human iPSC models generated with CRISPR/Cas9, we investigated the impacts of ABCA7 deficiency on neuronal metabolism and function. Lipidomics revealed that mitochondria-related phospholipids, such as phosphatidylglycerol and cardiolipin were reduced in the ABCA7-deficient iPSC-derived cortical organoids. Consistently, ABCA7 deficiency-induced alterations of mitochondrial morphology accompanied by reduced ATP synthase activity and exacerbated oxidative damage in the organoids. Furthermore, ABCA7-deficient iPSC-derived neurons showed compromised mitochondrial respiration and excess ROS generation, as well as enlarged mitochondrial morphology compared to the isogenic controls. ABCA7 deficiency also decreased spontaneous synaptic firing and network formation in iPSC-derived neurons, in which the effects were rescued by supplementation with phosphatidylglycerol or NAD+ precursor, nicotinamide mononucleotide. Importantly, effects of ABCA7 deficiency on mitochondria morphology and synapses were recapitulated in synaptosomes isolated from the brain of neuron-specific Abca7 knockout mice. Together, our results provide evidence that ABCA7 loss-of-function contributes to AD risk by modulating mitochondria lipid metabolism.

Loss of Tmem106b exacerbates FTLD pathologies and causes motor deficits in progranulin-deficient mice.

Progranulin (PGRN) and transmembrane protein 106B (TMEM106B) are important lysosomal proteins implicated in frontotemporal lobar degeneration (FTLD) and other neurodegenerative disorders. Loss-of-function mutations in progranulin (GRN) are a common cause of FTLD, while TMEM106B variants have been shown to act as disease modifiers in FTLD. Overexpression of TMEM106B leads to lysosomal dysfunction, while loss of Tmem106b ameliorates lysosomal and FTLD-related pathologies in young Grn-/- mice, suggesting that lowering TMEM106B might be an attractive strategy for therapeutic treatment of FTLD-GRN. Here, we generate and characterize older Tmem106b-/- Grn-/- double knockout mice, which unexpectedly show severe motor deficits and spinal cord motor neuron and myelin loss, leading to paralysis and premature death at 11-12 months. Compared to Grn-/- , Tmem106b-/- Grn-/- mice have exacerbated FTLD-related pathologies, including microgliosis, astrogliosis, ubiquitin, and phospho-Tdp43 inclusions, as well as worsening of lysosomal and autophagic deficits. Our findings confirm a functional interaction between Tmem106b and Pgrn and underscore the need to rethink whether modulating TMEM106B levels is a viable therapeutic strategy.

Loss of TMEM106B leads to myelination deficits: implications for frontotemporal dementia treatment strategies.

Genetic variants that define two distinct haplotypes at the TMEM106B locus have been implicated in multiple neurodegenerative diseases and in healthy brain ageing. In frontotemporal dementia (FTD), the high expressing TMEM106B risk haplotype was shown to increase susceptibility for FTD with TDP-43 inclusions (FTD-TDP) and to modify disease penetrance in progranulin mutation carriers (FTD-GRN). To elucidate the biological function of TMEM106B and determine whether lowering TMEM106B may be a viable therapeutic strategy, we performed brain transcriptomic analyses in 8-month-old animals from our recently developed Tmem106b-/- mouse model. We included 10 Tmem106b+/+ (wild-type), 10 Tmem106b+/- and 10 Tmem106-/- mice. The most differentially expressed genes (153 downregulated and 60 upregulated) were identified between Tmem106b-/- and wild-type animals, with an enrichment for genes implicated in myelination-related cellular processes including axon ensheathment and oligodendrocyte differentiation. Co-expression analysis also revealed that the most downregulated group of correlated genes was enriched for myelination-related processes. We further detected a significant loss of OLIG2-positive cells in the corpus callosum of Tmem106b-/- mice, which was present already in young animals (21 days) and persisted until old age (23 months), without worsening. Quantitative polymerase chain reaction revealed a reduction of differentiated but not undifferentiated oligodendrocytes cellular markers. While no obvious changes in myelin were observed at the ultrastructure levels in unchallenged animals, treatment with cuprizone revealed that Tmem106b-/- mice are more susceptible to cuprizone-induced demyelination and have a reduced capacity to remyelinate, a finding which we were able to replicate in a newly generated Tmem106b CRISPR/cas9 knock-out mouse model. Finally, using a TMEM106B HeLa knock-out cell line and primary cultured oligodendrocytes, we determined that loss of TMEM106B leads to abnormalities in the distribution of lysosomes and PLP1. Together these findings reveal an important function for TMEM106B in myelination with possible consequences for therapeutic strategies aimed at lowering TMEM106B levels.

Parkinson's disease: experimental models and reality.

Parkinson's disease (PD) is a chronic, progressive movement disorder of adults and the second most common neurodegenerative disease after Alzheimer's disease. Neuropathologic diagnosis of PD requires moderate-to-marked neuronal loss in the ventrolateral substantia nigra pars compacta and α-synuclein (αS) Lewy body pathology. Nigrostriatal dopaminergic neurodegeneration correlates with the Parkinsonian motor features, but involvement of other peripheral and central nervous system regions leads to a wide range of non-motor features. Nigrostriatal dopaminergic neurodegeneration is shared with other parkinsonian disorders, including some genetic forms of parkinsonism, but many of these disorders do not have Lewy bodies. An ideal animal model for PD, therefore, should exhibit age-dependent and progressive dopaminergic neurodegeneration, motor dysfunction, and abnormal αS pathology. Rodent models of PD using genetic or toxin based strategies have been widely used in the past several decades to investigate the pathogenesis and therapeutics of PD, but few recapitulate all the major clinical and pathologic features of PD. It is likely that new strategies or better understanding of fundamental disease processes may facilitate development of better animal models. In this review, we highlight progress in generating rodent models of PD based on impairments of four major cellular functions: mitochondrial oxidative phosphorylation, autophagy-lysosomal metabolism, ubiquitin-proteasome protein degradation, and endoplasmic reticulum stress/unfolded protein response. We attempt to evaluate how impairment of these major cellular systems contribute to PD and how they can be exploited in rodent models. In addition, we review recent cell biological studies suggesting a link between αS aggregation and impairment of nuclear membrane integrity, as observed during cellular models of apoptosis. We also briefly discuss the role of incompetent phagocytic clearance and how this may be a factor to consider in developing new rodent models of PD.

Histones facilitate α-synuclein aggregation during neuronal apoptosis.

Ample in vitro and in vivo experimental evidence supports the hypothesis that intercellular transmission of α-synuclein (αS) is a mechanism underlying the spread of αS pathology in Parkinson's disease and related disorders. What remains unexplained is where and how initial transmissible αS aggregates form. In a previous study, we demonstrated that αS aggregates rapidly form in neurons with impaired nuclear membrane integrity due to the interaction between nuclear proaggregant factor(s) and αS and that such aggregates may serve as a source for αS seeding. In the present study, we identify histones as a potential nuclear proaggregant factor for αS aggregation in both apoptotic neurons and brains with αS pathology. We further demonstrate that histone-induced aggregates contain a range of αS oligomers, including protofibrils and mature fibrils, and that these αS aggregates can seed additional aggregation. Importantly, we demonstrate transmissibility in mouse brains from stereotaxic injection. This study provides new clues to the mechanism underlying initial pathological aggregation of αS in PD and related disorders, and could lead to novel diagnostic and therapeutic approaches.

Proaggregant nuclear factor(s) trigger rapid formation of α-synuclein aggregates in apoptotic neurons.

Cell-to-cell transmission of α-synuclein (αS) aggregates has been proposed to be responsible for progressive αS pathology in Parkinson disease (PD) and related disorders, including dementia with Lewy bodies. In support of this concept, a growing body of in vitro and in vivo experimental evidence shows that exogenously introduced αS aggregates can spread into surrounding cells and trigger PD-like pathology. It remains to be determined what factor(s) lead to initiation of αS aggregation that is capable of seeding subsequent propagation. In this study we demonstrate that filamentous αS aggregates form in neurons in response to apoptosis induced by staurosporine or other toxins-6-hydroxy-dopamine and 1-methyl-4-phenylpyridinium (MPP+). Interaction between αS and proaggregant nuclear factor(s) is associated with disruption of nuclear envelope integrity. Knocking down a key nuclear envelop constituent protein, lamin B1, enhances αS aggregation. Moreover, in vitro and in vivo experimental models demonstrate that aggregates released upon cell breakdown can be taken up by surrounding cells. Accordingly, we suggest that at least some αS aggregation might be related to neuronal apoptosis or loss of nuclear membrane integrity, exposing cytosolic α-synuclein to proaggregant nuclear factors. These findings provide new clues to the pathogenesis of PD and related disorders that can lead to novel treatments of these disorders. Specifically, finding ways to limit the effects of apoptosis on αS aggregation, deposition, local uptake and subsequent propagation might significantly impact progression of disease.

Genetic modification of H2AX renders mesenchymal stromal cell-derived dopamine neurons more resistant to DNA damage and subsequent apoptosis.

BACKGROUND AIMS: Aberrant production of reactive oxygen species (ROS) and its impact on the integrity of genomic DNA have been considered one of the major risk factors for the loss of dopaminergic neurons in Parkinson's disease (PD). Stem cell transplantation as a strategy to replenish new functional neurons has great potential for PD treatment. However, limited survival of stem cells post-transplantation has always been an obstacle ascribed to the existence of neurotoxic environment in PD patients. METHODS: To improve the survival of transplanted stem cells for PD treatment, we explored a new strategy based on the function of the H2AX gene (H2A histone family, member X) in determination of DNA repair and cell apoptosis. We introduced a mutant form Y142F of H2AX into dopamine (DA) neuron-like cells differentiated from bone marrow-derived mesenchymal stromal cells (BMSCs). RESULTS: Expression of H2AX(Y142F) renders DA neuron-like cells more resistant to DNA damage and subsequent cell death induced by ultraviolet irradiation and 1-methyl-4-phenylpyridinium (MPP+) treatment. DISCUSSION: This is a meaningful attempt to improve the sustainability of BMSC-derived dopamine neurons under a brain neurotoxic environment. Further studies are needed to evaluate the implications of our findings in stem cell therapy for PD and related diseases.

Assessment of Skin Biopsy as a Diagnostic Biomarker in CSF1R-Related Disorder.

OBJECTIVES: To validate a recently published study in which skin biopsy was reported as a valuable alternative to brain biopsy in diagnosing CSF1R-related disorder (CSF1R-RD). METHODS: Blinded evaluation of skin samples was performed by independent reviewers using light and electron microscopy collected from a group of CSF1R variant carriers (n = 10) with various genotypes (mono and biallelic), different stages of the disease (asymptomatic and symptomatic), and exposed to different therapies (glucocorticoids, hematopoietic stem cell transplantation, and TREM2 agonist), and from a group of healthy controls (n = 5). RESULTS: Biopsies from patients with CSF1R-RD at various disease stages were indistinguishable from controls determined using light microscopy and electron microscopy. DISCUSSION: We found no distinctive axonal pathology in skin biopsies collected from CSF1R variant carriers at all stages of the disease. Our results are consistent with clinical and neurophysiologic features of the CSF1R-RD, in that peripheral nervous system involvement has not been reported. Studies aiming to discover new biomarkers are important, but the results must be validated with larger numbers of patients and healthy controls. Based on blinded light and electron microscopic studies of skin biopsies, there is no evidence that CSF1R-RD is associated with distinctive changes in cutaneous peripheral nerves. This suggests that skin biopsy is not useful in diagnosis of CSF1R-RD. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that skin biopsy does not distinguish those with CSF1R-RD, or carriers, from normal controls.

Comprehensive characterization of human brain-derived extracellular vesicles using multiple isolation methods: Implications for diagnostic and therapeutic applications.

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain-derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size-exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG-UC). Our results showed that SG-UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass-tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG-UC showing the best enrichment of common EV proteins with less contamination of non-EV proteins. In addition, SG-UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell-substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single-particle analysis using the super-resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.

Apoptotic Neuron-Derived Histone Amyloid Fibrils Induce α-Synuclein Aggregation.

Cell-to-cell transfer of α-synuclein (αS) is increasingly thought to play an important role in propagation of αS pathology, but mechanisms responsible for formation of initial αS seeds and factors facilitating their propagation remain unclear. We previously demonstrated that αS aggregates are formed rapidly in apoptotic neurons and that interaction between cytoplasmic αS and proaggregant nuclear factors generates seed-competent αS. We also provided initial evidence that histones have proaggregant properties. Since histones are released from cells undergoing apoptosis or cell stress, we hypothesized that internalization of histones into αS expressing cells could lead to intracellular αS aggregation. Here using mCherry-tagged histone, we show that nuclear extracts from apoptotic cells can induce intracellular αS inclusions after uptake into susceptible cells, while extracts from non-apoptotic cells did not. We also demonstrate that nuclear extracts from apoptotic cells contained histone-immunoreactive amyloid fibrils. Moreover, recombinant histone-derived amyloid fibrils are able to induce αS aggregation in cellular and animal models. Induction of αS aggregation by histone amyloid fibrils is associated with endocytosis-mediated rupture of lysosomes, and this effect can be enhanced in cells with chemically induced lysosomal membrane defects. These studies provide initial descriptions of the contribution of histone amyloid fibrils to αS aggregation.

Nanoparticles With Affinity for α-Synuclein Sequester α-Synuclein to Form Toxic Aggregates in Neurons With Endolysosomal Impairment.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. It is characterized pathologically by the aggregation of α-synuclein (αS) in the form of Lewy bodies and Lewy neurites. A major challenge in PD therapy is poor efficiency of drug delivery to the brain due to the blood-brain barrier (BBB). For this reason, nanomaterials, with significant advantages in drug delivery, have gained attention. On the other hand, recent studies have shown that nanoparticles can promote αS aggregation in salt solution. Therefore, we tested if nanoparticles could have the same effect in cell models. We found that nanoparticle can induce cells to form αS inclusions as shown in immunocytochemistry, and detergent-resistant αS aggregates as shown in biochemical analysis; and nanoparticles of smaller size can induce more αS inclusions. Moreover, the induction of αS inclusions is in part dependent on endolysosomal impairment and the affinity of αS to nanoparticles. More importantly, we found that the abnormally high level of endogenous lysosomotropic biomolecules (e.g., sphingosine), due to impairing the integrity of endolysosomes could be a determinant factor for the susceptibility of cells to nanoparticle-induced αS aggregation; and deletion of GBA1 gene to increase the level of intracellular sphingosine can render cultured cells more susceptible to the formation of αS inclusions in response to nanoparticle treatment. Ultrastructural examination of nanoparticle-treated cells revealed that the induced inclusions contained αS-immunopositive membranous structures, which were also observed in inclusions seeded by αS fibrils. These results suggest caution in the use of nanoparticles in PD therapy. Moreover, this study further supports the role of endolysosomal impairment in PD pathogenesis and suggests a possible mechanism underlying the formation of membrane-associated αS pathology.

C9orf72 poly(GR) aggregation induces TDP-43 proteinopathy.

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G4C2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G4C2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR)200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G4C2 repeat-expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G4C2-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.

Using leucine zipper to facilitate alpha-synuclein assembly.

The accumulation of filamentous alpha-synuclein (alpha-S) is associated with Parkinson's disease. It remains controversial as to the mode (antiparallel or parallel) of alpha-S self-assembly and whether an exact alignment of the central hydrophobic region is essential. In the present study, we performed in vitro assembly using alpha-S with or without the attachment of artificial leucine zippers (Zips) capable of forming either parallel or antiparallel coiled coils and included a spacer in one derivative. Results showed that Zips accelerate filament assembly in both the parallel and antiparallel fashions, that a precise alignment of the central hydrophobic region is not essential, and that the antiparallel pairs displayed the highest thioflavin T signals. More importantly, cells expressing Zip-fused alpha-S, but not alpha-S alone, formed alpha-S immunopositive and thioflavin S-positive inclusions in 7 days. The results suggest that alpha-S can assemble in both parallel and antiparallel modes but have a higher tendency to assemble in the latter mode and that cells overexpressing Zip-fused alpha-S may be used to screen alpha-S assembly inhibitors due to enhanced ability to form inclusions.

Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxy-terminally truncated species.

Alpha-synuclein is likely to play a key role in the development of Parkinson's disease as well as other synucleinopathies. In animal models, overexpression of full-length or carboxy-terminally truncated alpha-synuclein has been shown to produce pathology. Although the proteosome and lysosome have been proposed to play a role in the degradation of alpha-synuclein, the enzyme(s) involved in alpha-synuclein clearance and generation of its carboxy-terminally truncated species have not been identified. In this study, the role of cathepsin D and calpain I in these processes was analyzed. In vitro experiments, using either recombinant or endogenous alpha-synuclein as substrates and purified cathepsin D or lysosomes, demonstrated that cathepsin D degraded alpha-synuclein very efficiently, and that limited proteolysis resulted in the generation of carboxy-terminally truncated species. Purified calpain I also cleaved alpha-synuclein, but carboxy-terminally truncated species were not the main cleavage products, and calpain I activity present in cellular lysates was not able to degrade the protein. Knockdown of cathepsin D in cells overexpressing wild-type alpha-synuclein increased total alpha-synuclein levels by 28% and lysosomal alpha-synuclein by 2-fold. In in vitro experiments, pepstatin A completely blocked the degradation of alpha-synuclein in purified lysosomes. Furthermore, lysosomes isolated from cathepsin D knockdown cells showed a marked reduction in alpha-synuclein degrading activity, indicating that cathepsin D is the main lysosomal enzyme involved in alpha-synuclein degradation. Our findings suggest that upregulation of cathepsin D could be an additional therapeutic strategy to lessen alpha-synuclein burden in synucleinopathies.

Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates.

In neurodegenerative diseases, seeding is a process initiated by the internalization of exogenous protein aggregates. Multiple pathways for internalization of aggregates have been proposed, including direct membrane penetration and endocytosis. To decipher the seeding mechanisms of alpha-synuclein (αS) aggregates in human cells, we visualized αS aggregation, endo-lysosome distribution, and endo-lysosome rupture in real-time. Our data suggest that exogenous αS can seed endogenous cytoplasmic αS by either directly penetrating the plasma membrane or via endocytosis-mediated endo-lysosome rupture, leading to formation of endo-lysosome-free or endo-lysosome-associated αS aggregates, respectively. Further, we demonstrate that αS aggregates isolated from postmortem human brains with diffuse Lewy body disease (DLBD) preferentially show endocytosis-mediated seeding associated with endo-lysosome rupture and have significantly reduced seeding activity compared to recombinant αS aggregates. Colocalization of αS pathology with galectin-3 (a marker of endo-lysosomal membrane rupture) in the basal forebrain of DLBD, but not in age-matched controls, suggests endo-lysosome rupture is involved in the formation of αS pathology in humans. Interestingly, cells with endo-lysosomal membrane permeabilization (LMP) are more vulnerable to the seeding effects of αS aggregates. This study suggests that endo-lysosomal impairment in neurons might play an important role in PD progression.

[Effects of different electrofusion parameters on activation and early development of mouse embryos reconstructed by cumulus cell nuclear transfer].

OBJECTIVE: To study the effects of different parameters for electrofusion on the activation and early development of mouse embryos reconstructed by cumulus cell nuclear transfer, and explore optimal parameters for electrofusion. METHODS: A C57BL/6j mouse cumulus cell nucleus 10-12 mm in diameter was inserted into the perivitelline space of an enucleated oocyte. The fusion of donor-recipient pairs was induced with different parameters for electrofusion (with variation in electric field intensity, pulse duration and pulse times). Successful formation of the reconstructed embryos from donor-recipient pairs and the reconstructed embryos developing into the early embryonic stages (2-cell, 4-8-cell and morula stages) were observed and counted. RESULTS: The electric field intensity and pulse duration allowed variation during electrofusion within the range of 1 000-2 000 kV/cm and 40-160 ms, respectively. The donor-recipient pairs fused at very low rate when the parameters were below the allowed ranges, and disintegration or even death might occur when the parameters were above the ranges. Within these allowed ranges, variation of the electrofusion parameters did not produce significant impact on the ratio of the reconstructed embryos in 2-cell, 4-8-cell or morula stages (P<0.05). In addition, we suggested that pulse times be limited to 1-2. CONCLUSION: Optimal parameters for electrofusion are crucial for the fusion of donor-recipient pairs and the activation of the reconstructed embryo.

[Analysis of protein expression maps of two metastatic colorectal carcinoma cell lines generated by two-dimensional gel electrophoresis].

OBJECTIVE: To analyze the differentially expressed proteins in metastatic colorectal carcinoma and provide clues for the molecular mechanisms of colorectal cancer metastasis. METHODS: A pair of colorectal carcinoma cell lines SW620 and SW480 with different metastatic potentials from the same parent cell lines were studied. The protein expression maps of the two cell lines were obtained and optimized using two-dimensional gel electrophoresis (2-DE), and the differentially expressed protein spots associated with metastasis were analyzed by Melanie III software. RESULTS: The 2-DE expression maps of the two cell lines were well matched. The resolution of 2-DE was increased after using the nonlinear pH3-10 or overlapping narrow immobilized pH gradient strips, and 11 differentially expressed protein spots were identified after analysis using the software. CONCLUSION: Nonlinear pH3-10 and overlapping narrow immobilized pH gradient strips have higher separation efficiency for protein in 2-DE. The differentially expressed proteins separated will be useful for identifying metastasis-associated proteins of colorectal carcinoma and for establishing 2-DE database of the metastatic colorectal carcinoma cell lines.

[A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nuclei].

OBJECTIVE: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.

Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction.

The pathologic hallmark of Parkinson's disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation and/or aggregation. Using 2 neuronal cell models, we found that extracellular adenosine triphosphate (ATP) induced a significant increase in intracellular αsyn levels between 24 and 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells' response to extracellular ATP. Although Ca(2+) influx via P2X1 receptor is necessary for αsyn accumulation, Ca(2+) influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD.

Infection of immortalized human epithelial cell line Hacat with high-concentration Epstein-Barr virus.

OBJECTIVE: To study the mechanism by which Epstein-Barr (EB) virus infects human epithelial cells. METHODS: Large-scale culture of marmoset lymphocytes B958 was carried out to extract and condense EB virus therein. Titrated by lymphocytes from fetal umbilical blood, the EB virus of high concentration was used to infect immortalized human epithelial cell line Hacat, followed by genomic DNA extraction from the Hacat cells and amplification of the special DNA sequence Bam HIw fragments of EBV genomic DNA by PCR. Southern blotting and in situ hybridization were employed to confirm the result of PCR. RESULTS: The results of PCR, Southern blotting and in situ hybridization all indicated that high-concentration EBV could infect Hacat cell line, but the rate of infection was rather low. CONCLUSION: There is an unknown pathway of infection for EBV entry into the epithelial cells other than the mediation by CR2 or pIgR.