Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. ...Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-β (Aβ). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aβ. Furthermore, in mhCOs, we observed reduced expression of Aβ-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer's disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia in response to Aβ using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.
The diencephalon, an integral component of the forebrain, governs a spectrum of crucial functions, ranging from sensory processing to emotional regulation. Yet, unraveling its unique development, ...intricate connectivity, and its role in neurodevelopmental disorders has long been hampered by the scarcity of human brain tissue and ethical constraints. Recent advancements in stem cell technology, particularly the emergence of brain organoids, have heralded a new era in neuroscience research. Although most brain organoid methodologies have hitherto concentrated on directing stem cells toward telencephalic fates, novel techniques now permit the generation of region-specific brain organoids that faithfully replicate precise diencephalic identities. These models mirror the complexity of the human diencephalon, providing unprecedented opportunities for investigating diencephalic development, functionality, connectivity, and pathophysiology
. This review summarizes the development, function, and connectivity of diencephalic structures and touches upon developmental brain disorders linked to diencephalic abnormalities. Furthermore, it presents current diencephalic organoid models and their applications in unraveling the intricacies of diencephalic development, function, and pathology in humans. Lastly, it highlights thalamocortical assembloid models, adept at capturing human-specific aspects of thalamocortical connections, along with their relevance in neurodevelopmental disorders.
Brain wiring is remarkably precise, yet most neurons readily form synapses with incorrect partners when given the opportunity. Dynamic axon-dendritic positioning can restrict synaptogenic encounters, ...but the spatiotemporal interaction kinetics and their regulation remain essentially unknown inside developing brains. Here we show that the kinetics of axonal filopodia restrict synapse formation and partner choice for neurons that are not otherwise prevented from making incorrect synapses. Using 4D imaging in developing Drosophila brains, we show that filopodial kinetics are regulated by autophagy, a prevalent degradation mechanism whose role in brain development remains poorly understood. With surprising specificity, autophagosomes form in synaptogenic filopodia, followed by filopodial collapse. Altered autophagic degradation of synaptic building material quantitatively regulates synapse formation as shown by computational modeling and genetic experiments. Increased filopodial stability enables incorrect synaptic partnerships. Hence, filopodial autophagy restricts inappropriate partner choice through a process of kinetic exclusion that critically contributes to wiring specificity.
Defects in membrane trafficking are hallmarks of neurodegeneration. Rab GTPases are key regulators of membrane trafficking. Alterations of Rab GTPases, or the membrane compartments they regulate, are ...associated with virtually all neuronal activities in health and disease. The observation that many Rab GTPases are associated with neurodegeneration has proven a challenge in the quest for cause and effect. Neurodegeneration can be a direct consequence of a defect in membrane trafficking. Alternatively, changes in membrane trafficking may be secondary consequences or cellular responses. The secondary consequences and cellular responses, in turn, may protect, represent inconsequential correlates or function as drivers of pathology. Here, we attempt to disentangle the different roles of membrane trafficking in neurodegeneration by focusing on selected associations with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and selected neuropathies. We provide an overview of current knowledge on Rab GTPase functions in neurons and review the associations of Rab GTPases with neurodegeneration with respect to the following classifications: primary cause, secondary cause driving pathology or secondary correlate. This analysis is devised to aid the interpretation of frequently observed membrane trafficking defects in neurodegeneration and facilitate the identification of true causes of pathology.
Kiral et al. review the known functions of Rab GTPases in neurons and disentangle the causal and correlative relationships between Rab-associated membrane trafficking defects and neurodegeneration.
Dyslexia, also known as reading disability, is defined as difficulty processing written language in individuals with normal intellectual capacity and educational opportunity. The prevalence of ...dyslexia is between 5 and 17%, and the heritability ranges from 44 to 75%. Genetic linkage analysis and association studies have identified several genes and regulatory elements linked to dyslexia and reading ability. However, their functions and molecular mechanisms are not well understood. Prominent among these is
, encoded in the DYX2 locus of human chromosome 6p22. The association of
with reading performance has been replicated in independent studies and different languages. Rodent models suggest that
is involved in neuronal migration, but its role throughout the cortical development is largely unknown. In order to define the function of
in human cortical development, we applied the neural developmental model of a human embryonic stem cell. We knocked down
expression in hESCs and performed the cortical neuroectodermal differentiation. We found that neuroepithelial cell differentiation is one of the first stages of hESC differentiation that are affected by
knocked down could affect radial migration and thus differentiation into diverse neural populations at the cortical layers.
In this issue of Cell Stem Cell, Jin et al. report that human Down syndrome microglia exhibit enhanced synaptic engulfment and accelerated tau-induced cellular senescence in human-mouse chimeric ...brains. They show that inhibiting interferon signaling rescues both developmental and tau-associated phenotypes, rendering it a potential therapeutic target for Down syndrome.
In this issue of Cell Stem Cell, Jin et al. report that human Down syndrome microglia exhibit enhanced synaptic engulfment and accelerated tau-induced cellular senescence in human-mouse chimeric brains. They show that inhibiting interferon signaling rescues both developmental and tau-associated phenotypes, rendering it a potential therapeutic target for Down syndrome.
In the central nervous system (CNS), microglia carry out multiple tasks related to brain development, maintenance of brain homeostasis, and function of the CNS. Recent advanced in vitro model systems ...allow us to perform more detailed and specific analyses of microglial functions in the CNS. The development of human pluripotent stem cells (hPSCs)-based 2D and 3D cell culture methods, particularly advancements in brain organoid models, offers a better platform to dissect microglial function in various contexts. Despite the improvement of these methods, there are still definite restrictions. Understanding their drawbacks and benefits ensures their proper use. In this primer, we review current developments regarding in vitro microglial production and characterization and their use to address fundamental questions about microglial function in healthy and diseased states, and we discuss potential future improvements with a particular emphasis on brain organoid models.
In this Neuron primer, Cakir, Kiral, and Park outline recent in vitro microglial generation and characterization achievements, applications of in vitro approaches to questions of microglial functions in health and disease, and prospective future enhancements focusing on brain organoid models.
Region specific brain organoids are brain organoids derived by patterning protocols using extrinsic signals as opposed to cerebral organoids obtained by self-patterning. The main focus of this review ...is to discuss various region-specific brain organoids developed so far and their application in modeling neurodevelopmental disease. We first discuss the principles of neural axis formation by series of growth factors, such as SHH, WNT, BMP signalings, that are critical to generate various region-specific brain organoids. Then we discuss various neurodevelopmental disorders modeled so far with these region-specific brain organoids, and findings made on mechanism and treatment options for neurodevelopmental disorders (NDD).
Neurons are highly polarized cells that require continuous turnover of membrane proteins at axon terminals to develop, function, and survive. Yet, it is still unclear whether membrane protein ...degradation requires transport back to the cell body or whether degradation also occurs locally at the axon terminal, where live observation of sorting and degradation has remained a challenge. Here, we report direct observation of two cargo-specific membrane protein degradation mechanisms at axon terminals based on a live-imaging approach in intact Drosophila brains. We show that different acidification-sensing cargo probes are sorted into distinct classes of degradative “hub” compartments for synaptic vesicle proteins and plasma membrane proteins at axon terminals. Sorting and degradation of the two cargoes in the separate hubs are molecularly distinct. Local sorting of synaptic vesicle proteins for degradation at the axon terminal is, surprisingly, Rab7 independent, whereas sorting of plasma membrane proteins is Rab7 dependent. The cathepsin-like protease CP1 is specific to synaptic vesicle hubs, and its delivery requires the vesicle SNARE neuronal synaptobrevin. Cargo separation only occurs at the axon terminal, whereas degradative compartments at the cell body are mixed. These data show that at least two local, molecularly distinct pathways sort membrane cargo for degradation specifically at the axon terminal, whereas degradation can occur both at the terminal and en route to the cell body.
•Membrane protein degradation can occur at axon terminals•Two parallel mechanisms degrade synaptic vesicle and plasma membrane proteins•The two mechanisms are molecularly distinct and employ different cathepsins•Local “hub” compartments bud off retrograde vesicles at any maturation stage
Jin et al. develop a live-imaging method based on acidification-sensing probes in intact Drosophila brains to reveal two molecularly distinct endolysosomal degradation pathways at axon terminals. Independent of the canonical Rab7-dependent pathway, synaptic vesicle protein degradation requires n-Syb, V100, and Cathepsin L-like protease Cp1.
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