The use of a SOX10 reporter toward ameliorating oligodendrocyte lineage differentiation from human induced pluripotent stem cells.

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC-derived OLs.

Transcriptional Dysregulation and Impaired Neuronal Activity in FMR1 Knock-Out and Fragile X Patients' iPSC-Derived Models.

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.

FOXG1 dose tunes cell proliferation dynamics in human forebrain progenitor cells.

Heterozygous loss-of-function mutations in Forkhead box G1 (FOXG1), a uniquely brain-expressed gene, cause microcephaly, seizures, and severe intellectual disability, whereas increased FOXG1 expression is frequently observed in glioblastoma. To investigate the role of FOXG1 in forebrain cell proliferation, we modeled FOXG1 syndrome using cells from three clinically diagnosed cases with two sex-matched healthy parents and one unrelated sex-matched control. Cells with heterozygous FOXG1 loss showed significant reduction in cell proliferation, increased ratio of cells in G0/G1 stage of the cell cycle, and increased frequency of primary cilia. Engineered loss of FOXG1 recapitulated this effect, while isogenic repair of a patient mutation reverted output markers to wild type. An engineered inducible FOXG1 cell line derived from a FOXG1 syndrome case demonstrated that FOXG1 dose-dependently affects all cell proliferation outputs measured. These findings provide strong support for the critical importance of FOXG1 levels in controlling human brain cell growth in health and disease.

Rapid Generation of Ventral Spinal Cord-like Astrocytes from Human iPSCs for Modeling Non-Cell Autonomous Mechanisms of Lower Motor Neuron Disease.

Astrocytes play important roles in the function and survival of neuronal cells. Dysfunctions of astrocytes are associated with numerous disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Human-induced pluripotent stem cell (iPSC)-based approaches are becoming increasingly important for the study of the mechanisms underlying the involvement of astrocytes in non-cell autonomous processes of motor neuron degeneration in ALS. These studies must account for the molecular and functional diversity among astrocytes in different regions of the brain and spinal cord. It is essential that the most pathologically relevant astrocyte preparations are used when investigating non-cell autonomous mechanisms of either upper or lower motor neuron degeneration in ALS. Here, we describe the efficient and streamlined generation of human iPSC-derived astrocytes with molecular and biological properties similar to physiological astrocytes in the ventral spinal cord. These induced astrocytes exhibit spontaneous and ATP-induced calcium transients, and lack signs of overt activation. Human iPSC-derived astrocytes with ventral spinal cord features offer advantages over more generic astrocyte preparations for the study of both ventral spinal cord astrocyte biology and the involvement of astrocytes in mechanisms of lower motor neuron degeneration in ALS.

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy.

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.

A new functional role of mitochondria-anchored protein ligase in peroxisome morphology in mammalian cells.

Mitochondria and peroxisomes are metabolically interconnected and functionally active subcellular organelles. These two dynamic organelles, share a number of common biochemical functions such as ß-oxidation of fatty acids and detoxification of peroxides. The biogenesis and morphology of both these organelles in the mammalian cells is controlled by common transcription factors like PGC1α, and by a common fission machinery comprising of fission proteins like DRP1, Mff, and hFis1, respectively. In addition, the outer membrane mitochondria-anchored protein ligase (MAPL), the first mitochondrial SUMO E3 ligase with a RING-finger domain, also regulates mitochondrial morphology inducing mitochondrial fragmentation upon its overexpression. This fragmentation is dependent on both the RING domain of MAPL and the presence of the mitochondrial fission GTPase dynamin-related protein-1 (DRP1). Earlier studies have demonstrated that mitochondrial-derived vesicles are formed independently of the known mitochondrial fission GTPase, DRP1 are enriched for MAPL and are targeted to peroxisomes. The current study shows that MAPL regulates morphology of peroxisomes in a cell-type specific manner. Fascinatingly, the peroxisome elongation caused either due to silencing of DRP1 or by addition of polyunsaturated fatty acid, docosahexaenoic acid was blocked by overexpressing MAPL in mammalian cell lines. Furthermore, the transfection and colocalisation studies of MAPL with peroxisome membrane marker, PMP70, in different cell lines clearly revealed a cell-type specificity of transport of MAPL to peroxisomes. Previous work has placed the Vps35 (retromer component) as vital for delivery of MAPL to peroxisomes, placing the retromer as critical for the formation of MAPL-positive mitochondrial-derived vesicles. The results of polyethylene glycol-based cell-cell fusion assay signified that the enrichment of MAPL in peroxisomes is through vesicles and a retromer dependent phenomenon. Thus, a novel function for MAPL in peroxisomes is established to regulate peroxisome elongation and morphology under growth conditions and thus possibly modulate peroxisome fission.

Pharmacological Inhibition of Brain EGFR Activation By a BBB-penetrating Inhibitor, AZD3759, Attenuates α-synuclein Pathology in a Mouse Model of α-Synuclein Propagation.

Aggregation and deposition of α-synuclein (α-syn) in Lewy bodies within dopamine neurons of substantia nigra (SN) is the pathological hallmark of Parkinson's disease (PD). These toxic α-syn aggregates are believed to propagate from neuron-to-neuron and spread the α-syn pathology throughout the brain beyond dopamine neurons in a prion-like manner. Targeting propagation of such α-syn aggregates is of high interest but requires identifying pathways involving in this process. Evidence from previous Alzheimer's disease reports suggests that EGFR may be involved in the prion-like propagation and seeding of amyloid-ß. We show here that EGFR regulates the uptake of exogenous α-syn-PFFs and the levels of endogenous α-syn in cell cultures and a mouse model of α-syn propagation, respectively. Thus, we tested the therapeutic potentials of AZD3759, a highly selective BBB-penetrating EGFR inhibitor, in a preclinical mouse model of α-syn propagation. AZD3759 decreases activated EGFR levels in the brain and reduces phosphorylated α-synuclein (pSyn) pathology in brain sections, including striatum and SN. As AZD3759 is already in the clinic, this paper's results suggest a possible repositioning of AZD3759 as a disease-modifying approach for PD.

Characterization of human iPSC-derived astrocytes with potential for disease modeling and drug discovery.

Astrocytes play a number of key functions in health and disease. Activated astrocytes are present in most, if not all, neurological diseases. Most current information on the mechanisms underlying reactive astrocyte emergence derives from studies using animal experimental systems, mainly because the ability to study human astrocytes under healthy and pathological conditions has been hampered by the difficulty in obtaining primary human astrocytes. Here we describe robust and reliable derivation of astrocytes from human induced pluripotent stem cells (iPSCs). Phenotypically characterized human iPSC-derived astrocytes exhibit typical traits of physiological astrocytes, including spontaneous and induced calcium transients. Moreover, human iPSC-derived astrocytes respond to stimulation with a pro-inflammatory combination of tumor necrosis factor-alpha, interleukin 1-alpha, and complement component C1q by undergoing changes in gene expression patterns suggesting acquisition of a reactive astrocyte phenotype. Together, these findings provide evidence suggesting that human iPSC-derived astrocytes are a suitable experimental model system to study astrocyte function and reactivation in healthy and pathological conditions of the human nervous system.

Stimulation of L-type calcium channels increases tyrosine hydroxylase and dopamine in ventral midbrain cells induced from somatic cells.

Making high-quality dopamine (DA)-producing cells for basic biological or small molecule screening studies is critical for the development of novel therapeutics for disorders of the ventral midbrain. Currently, many ventral midbrain assays have low signal-to-noise ratio due to low levels of cellular DA and the rate-limiting enzyme of DA synthesis, tyrosine hydroxylase (TH), hampering discovery efforts. Using intensively characterized ventral midbrain cells derived from human skin, which demonstrate calcium pacemaking activity and classical electrophysiological properties, we show that an L-type calcium agonist can significantly increase TH protein levels and DA content and release. Live calcium imaging suggests that it is the immediate influx of calcium occurring simultaneously in all cells that drives this effect. Genome-wide expression profiling suggests that L-type calcium channel stimulation has a significant effect on specific genes related to DA synthesis and affects expression of L-type calcium receptor subunits from the CACNA1 and CACNA2D families. Together, our findings provide an advance in the ability to increase DA and TH levels to improve the accuracy of disease modeling and small molecule screening for disorders of the ventral midbrain, including Parkinson's disease.

Nuclear factor-kappaB regulates multiple steps of gliogenesis in the developing murine cerebral cortex.

Nuclear factor-kappaB (NF-κB) is activated in neural progenitor cells in the developing murine cerebral cortex during the neurogenic phase, when it acts to prevent premature neuronal differentiation. Here we show that NF-κB activation continues in mouse neocortical neural progenitor cells during the neurogenic-to-gliogenic switch. Blockade of endogenous NF-κB activity during neocortical gliogenesis leads to the formation of supernumerary committed gliogenic progenitors and premature glial cell differentiation. Conversely, forced NF-κB activation during the neocortical neurogenic-to-gliogenic transition causes delayed gliogenic commitment and decreased astroglial gene expression. NF-κB activation continues in neocortical gliogenic progenitors following commitment and is important to inhibit the differentiation of oligodendrocyte precursor cells and to maintain persistent expression of glial fibrillary acidic protein in maturing astrocytes. These results reveal a number of previously uncharacterized roles for NF-κB during different phases of neocortical gliogenesis and identify NF-κB as an inhibitor of early oligodendrocyte development in the cerebral cortex.

The Nogo Receptor Ligand LGI1 Regulates Synapse Number and Synaptic Activity in Hippocampal and Cortical Neurons.

Leucine-rich glioma-inactivated protein 1 (LGI1) is a secreted neuronal protein and a Nogo receptor 1 (NgR1) ligand. Mutations in LGI1 in humans causes autosomal dominant lateral temporal lobe epilepsy and homozygous deletion of LGI1 in mice results in severe epileptic seizures that cause early postnatal death. NgR1 plays an important role in the development of CNS synapses and circuitry by limiting plasticity in the adult cortex via the activation of RhoA. These relationships and functions prompted us to examine the effect of LGI1 on synapse formation in vitro and in vivo. We report that application of LGI1 increases synaptic density in neuronal culture and that LGI1 null hippocampus has fewer dendritic mushroom spines than in wild-type (WT) littermates. Further, our electrophysiological investigations demonstrate that LGI1 null hippocampal neurons possess fewer and weaker synapses. RhoA activity is significantly increased in cortical cultures derived from LGI1 null mice and using a reconstituted system; we show directly that LGI1 antagonizes NgR1-tumor necrosis factor receptor orphan Y (TROY) signaling. Our data suggests that LGI1 enhances synapse formation in cortical and hippocampal neurons by reducing NgR1 signaling.

Disruption of GRIN2B Impairs Differentiation in Human Neurons.

Heterozygous loss-of-function mutations in GRIN2B, a subunit of the NMDA receptor, cause intellectual disability and language impairment. We developed clonal models of GRIN2B deletion and loss-of-function mutations in a region coding for the glutamate binding domain in human cells and generated neurons from a patient harboring a missense mutation in the same domain. Transcriptome analysis revealed extensive increases in genes associated with cell proliferation and decreases in genes associated with neuron differentiation, a result supported by extensive protein analyses. Using electrophysiology and calcium imaging, we demonstrate that NMDA receptors are present on neural progenitor cells and that human mutations in GRIN2B can impair calcium influx and membrane depolarization even in a presumed undifferentiated cell state, highlighting an important role for non-synaptic NMDA receptors. It may be this function, in part, which underlies the neurological disease observed in patients with GRIN2B mutations.

NF-κB Signalling in Glioblastoma.

Nuclear factor-κB (NF-κB) is a transcription factor regulating a wide array of genes mediating numerous cellular processes such as proliferation, differentiation, motility and survival, to name a few. Aberrant activation of NF-κB is a frequent event in numerous cancers, including glioblastoma, the most common and lethal form of brain tumours of glial cell origin (collectively termed gliomas). Glioblastoma is characterized by high cellular heterogeneity, resistance to therapy and almost inevitable recurrence after surgery and treatment. NF-κB is aberrantly activated in response to a variety of stimuli in glioblastoma, where its activity has been implicated in processes ranging from maintenance of cancer stem-like cells, stimulation of cancer cell invasion, promotion of mesenchymal identity, and resistance to radiotherapy. This review examines the mechanisms of NF-κB activation in glioblastoma, the involvement of NF-κB in several mechanisms underlying glioblastoma propagation, and discusses some of the important questions of future research into the roles of NF-κB in glioblastoma.

HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology.

Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease.

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control.

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.

A vesicular transport pathway shuttles cargo from mitochondria to lysosomes.

Mitochondrial respiration relies on electron transport, an essential yet dangerous process in that it leads to the generation of reactive oxygen species (ROS). ROS can be neutralized within the mitochondria through enzymatic activity, yet the mechanism for steady-state removal of oxidized mitochondrial protein complexes and lipids is not well understood. We have previously characterized vesicular profiles budding from the mitochondria that carry selected cargo. At least one population of these mitochondria-derived vesicles (MDVs) targets the peroxisomes; however, the fate of the majority of MDVs was unclear. Here, we demonstrate that MDVs carry selected cargo to the lysosomes. Using a combination of confocal and electron microscopy, we observe MDVs in steady state and demonstrate that they are stimulated as an early response to oxidative stress, the extent of which is determined by the respiratory status of the mitochondria. Delivery to the lysosomes does not require mitochondrial depolarization and is independent of ATG5 and LC3, suggesting that vesicle delivery complements mitophagy. Consistent with this, ultrastructural analysis of MDV formation revealed Tom20-positive structures within the vesicles of multivesicular bodies. These data characterize a novel vesicle transport route between the mitochondria and lysosomes, providing insights into the basic mechanisms of mitochondrial quality control.

Reconstitution of mitochondria derived vesicle formation demonstrates selective enrichment of oxidized cargo.

The mechanisms that ensure the removal of damaged mitochondrial proteins and lipids are critical for the health of the cell, and errors in these pathways are implicated in numerous degenerative diseases. We recently uncovered a new pathway for the selective removal of proteins mediated by mitochondrial derived vesicular carriers (MDVs) that transit to the lysosome. However, it was not determined whether these vesicles were selectively enriched for oxidized, or damaged proteins, and the extent to which the complexes of the electron transport chain and the mtDNA-containing nucloids may have been incorporated. In this study, we have developed a cell-free mitochondrial budding reaction in vitro in order to better dissect the pathway. Our data confirm that MDVs are stimulated upon various forms of mitochondrial stress, and the vesicles incorporated quantitative amounts of cargo, whose identity depended upon the nature of the stress. Under the conditions examined, MDVs did not incorporate complexes I and V, nor were any nucleoids present, demonstrating the specificity of cargo incorporation. Stress-induced MDVs are selectively enriched for oxidized proteins, suggesting that conformational changes induced by oxidation may initiate their incorporation into the vesicles. Ultrastructural analyses of MDVs isolated on sucrose flotation gradients revealed the formation of both single and double membranes vesicles of unique densities and uniform diameter. This work provides a framework for a reductionist approach towards a detailed examination of the mechanisms of MDV formation and cargo incorporation, and supports the emerging concept that MDVs are critical contributors to mitochondrial quality control.

Formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 and Su e/g.

Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F(1)F(O)-ATP synthase (F(1)F(O)) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F(1)F(O) supercomplexes. Impairment of F(1)F(O) oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F(1)F(O) oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips.