Motivated by the cellular heterogeneity in complex tissues, particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models, we developed a complete workflow to reproducibly ...characterize cell types in complex tissues. Our approach combines a flow cytometry (FC) antibody panel with our computational pipeline CelltypeR, enabling dataset aligning, unsupervised clustering optimization, cell type annotating, and statistical comparisons. Applied to human iPSC derived midbrain organoids, it successfully identified the major brain cell types. We performed fluorescence-activated cell sorting of CelltypeR-defined astrocytes, radial glia, and neurons, exploring transcriptional states by single-cell RNA sequencing. Among the sorted neurons, we identified subgroups of dopamine neurons: one reminiscent of substantia nigra cells most vulnerable in Parkinson’s disease. Finally, we used our workflow to track cell types across a time course of organoid differentiation. Overall, our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets in complex tissues.
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•CelltypeR is a flow cytometry and computational pipeline for cell type quantification•Identified brain cell types in midbrain organoids and measured changes in proportions•Enriched selected populations using FACS and characterized by single-cell RNA sequencing•Identified substantia nigra–like dopaminergic neurons sensitive in Parkinson’s disease
Neuroscience; Cell biology; Omics
Social democracy demands existence of freedom, equality, justice and solidarity among masses. Doyens like John Locke, Jean Jacques Rousseau and Karl Marx decrypted the social inequalities that ...deprived people of human rights. Dr B. R. Ambedkar, being the most influential figure vis-à-vis social democracy in Indian context, voiced the deprived status of Dalits. Inspired by John Dewey’s idea of social endosmosis, he concluded that education can help to dissolve the rigid boundaries of caste. He also vociferously advocated education for Dalits to erase the status quo of being a society’s underbelly and overcome the quotidian humiliations. Discourses on Dalits since then have converged to an infectious expansive debate on the concomitant subjugated status of Dalits in the Indian social structure. Many Dalits have procured agency through education and have been vociferously voicing the subjugated position of Dalits in the cultural apparatus of caste. Bama is one such educated Dalit woman who has laid bare through her writings the complexities existing in a Dalit’s life. Her autobiography invocates Dalits to empower themselves through education and transgress the rigid boundaries of caste. The article examines her vision of Dalits’ emancipation vis-à-vis Ambedkar’s notion of social democracy.
•New workflows and methods to both generate and maintain humain midbrain organoids.•Microfabricated disks to scale up the generation of human midbrain organoids.•Decrease the amount of labor and ...variability, while maintaining the viability.
By providing a three-dimensional in vitro culture system with key features of the substantia nigra region in the brain, 3D neuronal organoids derived from human induced pluripotent stem cells (iPSCs) provide living neuronal tissue resembling the midbrain region of the brain. However, a major limitation of conventional brain organoid culture is that it is often labor-intensive, requiring highly specialized personnel for moderate throughput. Additionally, the methods published for long-term cultures require time-consuming maintenance to generate brain organoids in large numbers. With the increasing need for human midbrain organoids (hMOs) to better understand and model Parkinson’s disease (PD) in a dish, there is a need to implement new workflows and methods to both generate and maintain hMOs, while minimizing batch to batch variation. In this study, we developed a method with microfabricated disks to scale up the generation of hMOs. This opens up the possibility to generate larger numbers of hMOs, in a manner that minimizes the amount of labor required, while decreasing variability and maintaining the viability of these hMOs over time. Taken together, producing hMOs in this manner opens up the potential for these to be used to further PD studies.
Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early-onset Parkinson's disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by ...triggering mitophagy mediated by the ubiquitin (Ub) ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane forming a high-molecular-weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates Ub, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 N-terminal-C-terminal extension module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.
•Isotope-labeled D3-leucine can be incorporated in 3D midbrain-like organoids.•Metabolic labeling enables measurement of protein turnover by mass spectrometry.•We describe a robust computational ...method to determine protein half-lives.•Turnover rates for more than 750 proteins can be measured with this method.
Patient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson’s disease (PD) - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain essential as organoid models evolve. To solve this problem, we have designed a workflow to reproducibly extract proteins from brain organoids, measure global turnover using mass spectrometry, and statistically investigate turnover differences between genotypes. We also provide robust methodology for data filtering and statistical treatment of turnover data. Using human midbrain organoids (hMO) as a model system, our method accurately characterized the half-lives of 773 midbrain proteins. We compared these half-lives both to Parkin knockout hMOs and to previously reported data from primary cell cultures and in vivo models. Overall, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.
Parkinson's disease is a devastating neurodegenerative disorder characterized by loss of neuromelanin‐containing dopaminergic neurons. Novel 3D culture systems like human midbrain‐like organoids ...(hMOs) enable new research avenues for patient‐specific therapies, but cannot reach their full potential unless rapid optical imaging of entire organoids is enabled. Currently, hMOs have to undergo tissue clearing processes before imaging to overcome light scattering. Since tissue clearing is a lengthy chemical procedure, large ensemble studies and pharmacological longitudinal studies, which require live cultures, are impossible. To address this obstacle, raster scanning optoacoustic mesoscopy (RSOM) is considered for imaging intact hMOs. RSOM is an optical imaging technique that leverages the optoacoustic effect to overcome the need of tissue clearing. Moreover, by using tomographic principles, large specimens can be imaged within minutes. The results confirm that RSOM can image the neuromelanin distribution in complete hMOs at a single‐cell resolution. Whole hMO volumes of standard size can be imaged in 4 min. Comparison with bright‐field microscopy and histology confirms the ground truth of the RSOM images. This work opens several research opportunities regarding neuromelanin in hMOs with potential to boost research in Parkinson disease.
Optical imaging of human midbrain organoids for Parkinson research poses a problem for current technologies since they require lengthy and invasive clearing methods. Raster‐scanning optoacoustic mesoscopy (RSOM) overcomes this challenge by utilizing intrinsic optical absorption contrast and the optoacoustic effect. It is shown how RSOM provides fast quantitative visualization of the neuromelanin content in intact large organoids at single cell resolution.
Abstract
SNCA, the first gene associated with Parkinson’s disease, encodes the α-synuclein protein, the predominant component within pathological inclusions termed Lewy bodies. The presence of Lewy ...bodies is one of the classical hallmarks found in the brain of patients with Parkinson’s disease, and Lewy bodies have also been observed in patients with other synucleinopathies. However, the study of α-synuclein pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use three-dimensional midbrain organoids, differentiated from human-induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These human midbrain organoids recapitulate key features of α-synuclein pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication human midbrain organoids express elevated levels of α-synuclein and exhibit an age-dependent increase in α-synuclein aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-synuclein. These phosphorylated α-synuclein aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, human midbrain organoids from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson’s disease and provide a powerful system to study the pathogenesis of synucleinopathies.
Mohamed et al. investigate synucleinopathy in human midbrain organoids, derived from patients carrying a triplication of the SNCA gene. Human midbrain organoids recapitulate pathology observed in the brains of patients: an elevated level of α-synuclein, an age-dependent increase in aggregation and a selective reduction in dopaminergic neurons.
Graphical Abstract
Graphical Abstract
One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aβ) peptide into extracellular plaques. Existing research indicates that ...extracellular vesicles (EVs) can carry Aβ associated with AD. However, characterization of the EVs-associated Aβ and its conformational variants has yet to be realized. Raman spectroscopy is a label-free and non-destructive method that is able to assess the biochemical composition of EVs. This study reports for the first time the Raman spectroscopic fingerprint of the Aβ present in the molecular cargo of small extracellular vesicles (sEVs). Raman spectra were measured from sEVs isolated from Alzheimer's disease cell culture model, where secretion of Aβ is regulated by tetracycline promoter, and from midbrain organoids. The averaged spectra of each sEV group showed considerable variation as a reflection of the biochemical content of sEVs. Spectral analysis identified more intense Raman peaks at 1650 cm
and 2930 cm
attributable to the Aβ peptide incorporated in sEVs produced by the Alzheimer's cell culture model. Subsequent analysis of the spectra by principal component analysis differentiated the sEVs of the Alzheimer's disease cell culture model from the control groups of sEVs. Moreover, the results indicate that Aβ associated with secreted sEVs has a α-helical secondary structure and the size of a monomer or small oligomer. Furthermore, by analyzing the lipid content of sEVs we identified altered fatty acid chain lengths in sEVs that carry Aβ that may affect the fluidity of the EV membrane. Overall, our findings provide evidence supporting the use of Raman spectroscopy for the identification and characterization of sEVs associated with potential biomarkers of neurological disorders such as toxic proteins.
One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aβ) peptide into extracellular plaques. Existing research indicates that ...extracellular vesicles (EVs) can carry Aβ associated with AD. However, characterization of the EVs-associated Aβ and its conformational variants has yet to be realized. Raman spectroscopy is a label-free and non-destructive method that is able to assess the biochemical composition of EVs. This study reports for the first time the Raman spectroscopic fingerprint of the Aβ present in the molecular cargo of small extracellular vesicles (sEVs). Raman spectra were measured from sEVs isolated from Alzheimer's disease cell culture model, where secretion of Aβ is regulated by tetracycline promoter, and from midbrain organoids. The averaged spectra of each sEV group showed considerable variation as a reflection of the biochemical content of sEVs. Spectral analysis identified more intense Raman peaks at 1650 cm
−1
and 2930 cm
−1
attributable to the Aβ peptide incorporated in sEVs produced by the Alzheimer's cell culture model. Subsequent analysis of the spectra by principal component analysis differentiated the sEVs of the Alzheimer's disease cell culture model from the control groups of sEVs. Moreover, the results indicate that Aβ associated with secreted sEVs has a α-helical secondary structure and the size of a monomer or small oligomer. Furthermore, by analyzing the lipid content of sEVs we identified altered fatty acid chain lengths in sEVs that carry Aβ that may affect the fluidity of the EV membrane. Overall, our findings provide evidence supporting the use of Raman spectroscopy for the identification and characterization of sEVs associated with potential biomarkers of neurological disorders such as toxic proteins.
Extracellular vesicles can carry Aβ associated with Alzheimer's disease. In this article we identify specific Raman spectroscopic features that can be associated with Aβ present in the molecular cargo of small extracellular vesicles.
Silver is ingested in India in the form of varak, Ayurvedic bhasm and water contained in silver utensils, but nobody knows their effects. We have examined the effect of interaction of silver ingested ...in the form of varak (leaf), bhasm (ash) and water (colloidal solution) on some essential elements (Na, K, Ca, Mg, Cu, Fe and Zn) in blood, liver, spleen and kidneys of chicks. The results showed significant rise of silver in all the tissues and significant fall in copper in the blood. The greatest rise was obtained in blood silver by silver sol while it had the lowest amount of silver. Iron significantly increased in tissues but somewhat decreased in blood. Zinc content increased in all the tissues by leaf and ash but decreased by sol. No consistent pattern was seen in case of other elements.
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