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Axonal transport of organelles visualized by light microscopy: cinemicrographic and computer analysis.
Rapid movements of intra-axonal organelles in acutely isolated single myelinated fibers from bullfrog sciatic nerve were visualized by dark-field microscopy. The movements were recorded by cinemicrography, and analyzed by computer-based methods. The movements are saltatory and bidirectional, but each particle moves mainly in a single direction. For more than 90% of the particles, the predominant movement direction is retrograde, i.e. toward the cell body. Quantitative measurements on a variety of parameters of the organelle movements are presented. Different particles in the same axon show a broad range of mean speeds. The average mean speed of movement in the retrograde direction at 28 degrees C was 1.08 micrometer/sec (S.D. - 0.41), equivalent to an axonal transport rate of 93 mm/day. Disperse distributions were also found for other parameters such as the instantaneous velocities of individual particles. Quantal velocities, periodic movement patterns, and specific 'channels' were not detected. When the data from a population of particles is treated statistically, the average mean speed, the distribution of velocities, and other statistical parameters are found to be similar in different axons studied at the same temperature. Direct microscopical observation of axonal organelle movement is a technique which provides information about axonal transport which is different from and complementary to that obtained from enzyme accumulation of radioactive tracer methods.
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Selective inhibition of retrograde axonal transport by erythro-9-[3-(2-hydroxynonyl)]adenine.
Retrograde transport of microscopically visible organelles in isolated lobster axons was selectively inhibited by erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), with little or no effect on anterograde transport. The retrograde velocity was reduced both in living axons and in permeabilized axons in which movement was reactivated with exogenous ATP. This is the first report of an agent that selectively inhibits axonal transport in one direction.
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Chromatin accessibility analysis reveals regulatory dynamics of developing human retina and hiPSC-derived retinal organoids.
Retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs) provide potential opportunities for studying human retinal development and disorders; however, to what extent ROs recapitulate the epigenetic features of human retinal development is unknown. In this study, we systematically profiled chromatin accessibility and transcriptional dynamics over long-term human retinal and RO development. Our results showed that ROs recapitulated the human retinogenesis to a great extent, but divergent chromatin features were also discovered. We further reconstructed the transcriptional regulatory network governing human and RO retinogenesis in vivo. Notably, NFIB and THRA were identified as regulators in human retinal development. The chromatin modifications between developing human and mouse retina were also cross-analyzed. Notably, we revealed an enriched bivalent modification of H3K4me3 and H3K27me3 in human but not in murine retinogenesis, suggesting a more dedicated epigenetic regulation on human genome.
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Production of human spinal-cord organoids recapitulating neural-tube morphogenesis.
Human spinal-cord-like tissues induced from human pluripotent stem cells are typically insufficiently mature and do not mimic the morphological features of neurulation. Here, we report a three-dimensional culture system and protocol for the production of human spinal-cord-like organoids (hSCOs) recapitulating the neurulation-like tube-forming morphogenesis of the early spinal cord. The hSCOs exhibited neurulation-like tube-forming morphogenesis, cellular differentiation into the major types of spinal-cord neurons as well as glial cells, and mature synaptic functional activities, among other features of the development of the spinal cord. We used the hSCOs to screen for antiepileptic drugs that can cause neural-tube defects. hSCOs may also facilitate the study of the development of the human spinal cord and the modelling of diseases associated with neural-tube defects.
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Spatiotemporal cellular map of the developing human reproductive tract.
The human reproductive tract is essential for species perpetuation and overall health. Its development involves complex processes of sex specification, tissue patterning and morphogenesis, the disruption of which can cause lifelong issues, including infertility1-5. Here we present an extensive single-cell and spatial multi-omic atlas of the human reproductive tract during prenatal development to provide insights beyond those that are possible with smaller-scale, organ-focused studies. We describe potential regulators of sexual dimorphism in reproductive organs and pinpoint previously unknown genes involved in Müllerian duct emergence and regression and urethral canalization of the penis. By combining histological features with gene expression and chromatin accessibility data, we define transcription factors and signalling events potentially involved in the regionalization of the Müllerian and Wolffian ducts. We also refine how the HOX code is established in distinct reproductive organs and reveal that the expression of thoracic HOX genes is increased in the rostral mesenchyme of the fallopian tube and epididymis. Our findings further indicate that epithelial regionalization of the fallopian tube and epididymis, which probably contribute to sperm maturation and capacitation, is established during development. By contrast, later events are necessary for regionalization of the uterocervical canal epithelium. Finally, on the basis of single-cell data and fetal-derived organoids, we show that the fetal uterine epithelium is vulnerable to oestrogen-mimicking endocrine disruptors. By mapping sex-specific reproductive tract regionalization and differentiation at the cellular level, our study provides valuable insights into causes and potential treatments of developmental reproductive disorders.
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Bipolar disorder-iPSC derived neural progenitor cells exhibit dysregulation of store-operated Ca2+ entry and accelerated differentiation.
While most of the efforts to uncover mechanisms contributing to bipolar disorder (BD) focused on phenotypes at the mature neuron stage, little research has considered events that may occur during earlier timepoints of neurodevelopment. Further, although aberrant calcium (Ca2+) signaling has been implicated in the etiology of this condition, the possible contribution of store-operated Ca2+ entry (SOCE) is not well understood. Here, we report Ca2+ and developmental dysregulations related to SOCE in BD patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells (BD-NPCs) and cortical-like glutamatergic neurons. First, using a Ca2+ re-addition assay we found that BD-NPCs and neurons had attenuated SOCE. Intrigued by this finding, we then performed RNA-sequencing and uncovered a unique transcriptome profile in BD-NPCs suggesting accelerated neurodifferentiation. Consistent with these results, we measured a slower rate of proliferation, increased neurite outgrowth, and decreased size in neurosphere formations with BD-NPCs. Also, we observed decreased subventricular areas in developing BD cerebral organoids. Finally, BD NPCs demonstrated high expression of the let-7 family while BD neurons had increased miR-34a, both being microRNAs previously implicated in neurodevelopmental deviations and BD etiology. In summary, we present evidence supporting an accelerated transition towards the neuronal stage in BD-NPCs that may be indicative of early pathophysiological features of the disorder.
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Neuronal branching in stem cell models of mitochondrial and neurological diseases.
Neuronal branching, the extension and arborization of neurites, is critical for establishing and maintaining functional neural circuits. Emerging evidence suggests that mitochondria play an important role in regulating this process. In this review, we explore how the use of human induced pluripotent stem cell (iPSC)-derived neuronal models in two dimensions (2D) and three dimensions (3D) could help uncover possible mechanisms linking mitochondrial function and dysfunction to neuronal branching capacity. We highlight examples of iPSC-based models of mitochondrial and neurological diseases where aberrant neurite growth has been observed and discuss the potential therapeutic implications. Additionally, we review current methodologies for assessing neurite outgrowth in 2D and 3D neuronal models, addressing their strengths and limitations. Insights gained from these models emphasize the significance of mitochondrial health in neuronal branching and demonstrate the potential of iPSC-derived neurons and brain organoids for studying disrupted neuronal morphology. Harnessing these human stem cell models to devise phenotypic drug discovery platforms can eventually pave the way for innovative therapeutic interventions, particularly in the context of disorders with poorly understood genetic mechanisms and limited therapeutic options.
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Effects of neurotoxic industrial solvents on cultured neuroblastoma cells: methyl n-butyl ketone, n-hexane and derivatives.
The neurotoxic effects of the commercial organic solvents n-hexane and methyl n-butyl ketone (MBK), recently discovered to cause profound peripheral neuropathy in man, were studied in neuronal-like cells in tissue culture. These agents are known to induce marked proliferation of 10nm neurofilaments in peripheral and central axons of both humans and rats. In a murine neuroblastoma cell line, previously reported to show filamentous hyperplasia when exposed to aluminum ions, both MBK and n-hexane induced a highly reproducible series of cytotoxic effects at the light and electron microscopic levels and caused dose-dependent inhibition of cellular proliferation. In contrast, two closely related but clinically non-toxic solvents, methyl isobutyl ketone and methyl ethyl ketone, caused little or no cytopathological or growth inhibiting effects. MBK and its major water soluble derivative, 2,5-hexane dione (HD), produced identical cytotoxic changes in vitro, supporting the postulate that HD is the toxically active agent in victims exposed to MBK. Although MBKlthought MBK and n-hexand adversely affected the extension of maintenance of neuritic processes, electron microscopy and immunofluorescent reaction failed to reveal any proliferation of 10 nm cytoplasmic filaments in the intoxicated cells. Also, these agents had no deleterious effect on in vitro brain microtubule polymerization. In contrast, aluminum ions produced a doserelated inhibition of neurotubule assembly, similar to that seen with the filament-inducing agents colchicine and vinblastine. The results suggest that the fibrous cytoskeleton may not be the primary or essential target of MBK n-hexane and related human neurotoxins.
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[A specific organelle in the vascular endothelial cells of cerebellar neoplasms (author's transl)].
A "rod-shaped tubulated body" (tubular body) was first described by Weibel and Palade in the vascular endothelial cells of various organs in both man and animals. This is now considered to be an organelle specific to the endothelial cell, but its function is still unknown. Both in experimental and human pathology this organelle has been observed more often in either seemingly young or abnormal endothelial cells of the blood vessels in tissue regeneration, inflammation, brain tumors among others. This report deals with ultrastructural study of two surgical cases of cerebellar neoplasm, in which the vascular endothelium was examined for a tubular body. The first case was a 12-year-old boy with cerebellar hemangioblastoma, and the second a 36-year-old female who had a history of renal cell carcinoma removed approximately 5 years previously. Histological diagnosis of the cerebellar tumor in the latter case was indetermined, because a part of the tumor consisted of clear cells suggestive of clear cell carcinoma and another part of well developed endothelial cells and vascular channels apparently indicative of hemangioblastoma. The findings of the ultrastructural study were rather compatible with that of renal cell carcinoma metastatic to the cerebellum although inconclusive. The tubular body observed in the endothelial cells of those tumor vessels consisted of a membrane-limited round, oval or elongated shaped intracytoplasmic body which contained tubules of 170 to 200 A outer diameter with approximately 50 to 60 A thickness. The tubules were arranged mostly in a parallel fashion along their long axis. In the first type of tubular body they were embedded in a relatively pale matrix, and in the second their arrangement appeared to be more compact. The third tubular body, so far undescribed in human endothelial cells except in our previous communication, showed an irregularly and markedly enlarged matrix, surrounded by a limiting membrane which was occasionally observed connected with either a coated vesicle or cytoplasmic membrane. Abundant tubules were intermingled without showing a particular arrangement. Morphological and functional significance of the third type tubular body is unknown, but it might represent a pathological change of a tubular body in cerebellar neoplasms. These findings might give us some clues in understanding a tumor angiogenesis.
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Thyroid hormone promotes fetal neurogenesis.
Maternal low thyroxine (T4) serum levels during the first trimester of pregnancy correlate with cerebral cortex volume and mental development of the progeny, but why neural cells during early fetal brain development are vulnerable to maternal T4 levels remains unknown. In this study, using iPSCs obtained from a boy with a loss-of-function mutation in MCT8 - a transporter previously identified as critical for thyroid hormone uptake and action in neural cells - we demonstrate that thyroid hormone induces transcriptional changes that promote the progression of human neural precursor cells along the dorsal projection trajectory. Consistent with these findings, single-cell, spatial, and bulk transcriptomics from MCT8-deficient cerebral organoids and cultures of human neural precursor cells underscored the necessity for optimal thyroid hormone levels for these cells to differentiate into neurons. The controlled intracellular activation of T4 signaling occurs through the transient expression of the enzyme type 2 deiodinase, which converts T4 into its active form, T3, alongside the coordinated expression of thyroid hormone nuclear receptors. The intracellular activation of T4 in neural precursor cells results in transcriptional changes important for their division mode and cell cycle progression. Thus, T4 is essential for fetal neurogenesis, highlighting the importance of adequate treatment for mothers with hypothyroidism.