Neuronal lineage tracing from progenitors in human cortical organoids reveals novel mechanisms of human neuronal production, diversity, and disease.

The contribution of progenitor subtypes to generate the billions of neurons during human cortical neurogenesis is not well understood. We developed the Cortical ORganoid Lineage Tracing (COR-LT) system for human cortical organoids. Differential fluorescent reporter activation in distinct progenitor cells leads to permanent reporter expression, enabling the progenitor cell lineage of neurons to be determined. Surprisingly, nearly all neurons produced in cortical organoids were generated indirectly from intermediate progenitor cells. Additionally, neurons of different progenitor lineages were transcriptionally distinct. Isogenic lines made from an autistic individual with and without a likely pathogenic variant in the CTNNB1 gene demonstrated that the variant substantially altered the proportion of neurons derived from specific progenitor cell lineages, as well as the lineage-specific transcriptional profiles of these neurons, suggesting a pathogenic mechanism for this mutation. These results suggest individual progenitor subtypes play unique roles in generating the diverse neurons of the human cerebral cortex.

Generation of advanced cerebellar organoids for neurogenesis and neuronal network development.

Neurons within the cerebellum form temporal-spatial connections through the cerebellum, and the entire brain. Organoid models provide an opportunity to model the early differentiation of the developing human cerebellum, which is difficult to study in vivo, and affords the opportunity to study neurodegenerative and neurodevelopmental diseases of the cerebellum. Previous cerebellar organoid models focused on early neuron generation and single cell activity. Here, we modify previous protocols to generate more mature cerebellar organoids that allow for the establishment of several classes of mature neurons during cerebellar differentiation and development, including the establishment of neural networks during whole-organoid maturation. This will provide a means to study the generation of several more mature cerebellar cell types, including Purkinje cells, granule cells and interneurons expression as well as neuronal communication for biomedical, clinical and pharmaceutical applications.

Autism-specific PTEN p.Ile135Leu variant and an autism genetic background combine to dysregulate cortical neurogenesis.

Alterations in cortical neurogenesis are implicated in neurodevelopmental disorders including autism spectrum disorders (ASDs). The contribution of genetic backgrounds, in addition to ASD risk genes, on cortical neurogenesis remains understudied. Here, using isogenic induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) and cortical organoid models, we report that a heterozygous PTEN c.403A>C (p.Ile135Leu) variant found in an ASD-affected individual with macrocephaly dysregulates cortical neurogenesis in an ASD-genetic-background-dependent fashion. Transcriptome analysis at both bulk and single-cell level revealed that the PTEN c.403A>C variant and ASD genetic background affected genes involved in neurogenesis, neural development, and synapse signaling. We also found that this PTEN p.Ile135Leu variant led to overproduction of NPC subtypes as well as neuronal subtypes including both deep and upper layer neurons in its ASD background, but not when introduced into a control genetic background. These findings provide experimental evidence that both the PTEN p.Ile135Leu variant and ASD genetic background contribute to cellular features consistent with ASD associated with macrocephaly.

The effect of zonal harmonics on dynamical structures in the circular restricted three-body problem near the secondary body.

The circular restricted three-body model is widely used for astrodynamical studies in systems where two major bodies are present. However, this model relies on many simplifications, such as point-mass gravity and planar, circular orbits of the bodies, and limiting its accuracy. In an effort to achieve higher-fidelity results while maintaining the autonomous simplicity of the classic model, we employ zonal harmonic perturbations since they are symmetric about the z-axis, thus bearing no time-dependent terms. In this study, we focus on how these perturbations affect the dynamic environment near the secondary body in real systems. Concise, easily implementable equations for gravitational potential, particle motion, and modified Jacobi constant in the perturbed model are presented. These perturbations cause a change in the normalized mean motion, and two different formulations are addressed for assigning this new value. The shifting of collinear equilibrium points in many real systems due to J 2 of each body is reported, and we study how families of common periodic orbits-Lyapunov, vertical, and southern halo-shift and distort when J 2 , J 4 , and J 6 of the primary and J 2 of the secondary body are accounted for in the Jupiter-Europa and Saturn-Enceladus systems. It is found that these families of periodic orbits change shape, position, and energy, which can lead to dramatically different dynamical behavior in some cases. The primary focus is on moons of the outer planets, many of which have very small odd zonal harmonic terms, or no measured value at all, so while the developed equations are meant for any and all zonal harmonic terms, only even terms are considered in the simulations. Early utilization of this refined CR3BP model in mission design will result in a more smooth transition to full ephemeris model.

Spatio-temporal dynamics of neocortical presynaptic terminal development using multi-photon imaging of the corpus callosum in vivo.

Within the developing central nervous system, the dynamics of synapse formation and elimination are insufficiently understood. It is ideal to study these processes in vivo, where neurons form synapses within appropriate behavioral and anatomical contexts. In vivo analysis is particularly important for long-range connections, since their development cannot be adequately studied in vitro. The corpus callosum (CC) represents a clinically-relevant long-range connection since several neurodevelopmental diseases involve CC defects. Here, we present a novel strategy for in vivo longitudinal and rapid time-lapse imaging of CC presynaptic terminal development. In postnatal mice, the time-course of CC presynaptic terminal formation and elimination was highly variable between axons or groups of axons. Young presynaptic terminals were remarkably dynamic - moving, dividing to generate more boutons, and merging to consolidate small terminals into large boutons. As synaptic networks matured, presynaptic mobility decreased. These rapid dynamics may be important for establishing initial synaptic contacts with postsynaptic partners, refining connectivity patterns or modifying synapse strength during development. Ultimately, this in vivo imaging approach will facilitate investigation of synapse development in other long-range connections and neurodevelopmental disease models.

Concise Review: Induced Pluripotent Stem Cell Models for Neuropsychiatric Diseases.

The major neuropsychiatric conditions of schizophrenia, affective disorders, and infantile autism are characterized by chronic symptoms of episodic, stable, or progressive nature that result in significant morbidity. Symptomatic treatments are the mainstay but do not resolve the underlying disease processes, which are themselves poorly understood. The prototype psychotropic drugs are of variable efficacy, with therapeutic mechanisms of action that are still uncertain. Thus, neuropsychiatric disorders are ripe for new technologies and approaches with the potential to revolutionize mechanistic understanding and drive the development of novel targeted treatments. The advent of methods to produce patient-derived stem cell models and three-dimensional organoids with the capacity to differentiate into neurons and the various neuronal cellular lineages mark such an advance. We discuss numerous techniques involved, their applications, and areas that require further optimization. Stem Cells Translational Medicine 2017;6:2062-2070.

Building a Terminal: Mechanisms of Presynaptic Development in the CNS.

To create a presynaptic terminal, molecular signaling events must be orchestrated across a number of subcellular compartments. In the soma, presynaptic proteins need to be synthesized, packaged together, and attached to microtubule motors for shipment through the axon. Within the axon, transport of presynaptic packages is regulated to ensure that developing synapses receive an adequate supply of components. At individual axonal sites, extracellular interactions must be translated into intracellular signals that can incorporate mobile transport vesicles into the nascent presynaptic terminal. Even once the initial recruitment process is complete, the components and subsequent functionality of presynaptic terminals need to constantly be remodeled. Perhaps most remarkably, all of these processes need to be coordinated in space and time. In this review, we discuss how these dynamic cellular processes occur in neurons of the central nervous system in order to generate presynaptic terminals in the brain.

Presynaptic NMDA receptors - dynamics and distribution in developing axons in vitro and in vivo.

During cortical development, N-methyl-D-aspartate (NMDA) receptors (NMDARs) facilitate presynaptic terminal formation, enhance neurotransmitter release and are required in presynaptic neurons for spike-timing-dependent long-term depression (tLTD). However, the extent to which NMDARs are found within cortical presynaptic terminals has remained controversial, and the sub-synaptic localization and dynamics of axonal NMDARs are unknown. Here, using live confocal imaging and biochemical purification of presynaptic membranes, we provide strong evidence that NMDARs localize to presynaptic terminals in vitro and in vivo in a developmentally regulated manner. The NR1 and NR2B subunits (also known as GRIN1 and GRIN2B, respectively) were found within the active zone membrane, where they could respond to synaptic glutamate release. Surprisingly, NR1 also appeared in glutamatergic and GABAergic synaptic vesicles. During synaptogenesis, NR1 was mobile throughout axons - including growth cones and filopodia, structures that are involved in synaptogenesis. Upon synaptogenic contact, NMDA receptors were quickly recruited to terminals by neuroligin-1 signaling. Unlike dendrites, the trafficking and distribution of axonal NR1 were insensitive to activity changes, including NMDA exposure, local glutamate uncaging or action potential blockade. These results support the idea that presynaptic NMDARs play an early role in presynaptic development.

Dynamic mechanisms of neuroligin-dependent presynaptic terminal assembly in living cortical neurons.

BACKGROUND: Synapse formation occurs when synaptogenic signals trigger coordinated development of pre and postsynaptic structures. One of the best-characterized synaptogenic signals is trans-synaptic adhesion. However, it remains unclear how synaptic proteins are recruited to sites of adhesion. In particular, it is unknown whether synaptogenic signals attract synaptic vesicle (SV) and active zone (AZ) proteins to nascent synapses or instead predominantly function to create sites that are capable of forming synapses. It is also unclear how labile synaptic proteins are at developing synapses after their initial recruitment. To address these issues, we used long-term, live confocal imaging of presynaptic terminal formation in cultured cortical neurons after contact with the synaptogenic postsynaptic adhesion proteins neuroligin-1 or SynCAM-1. RESULTS: Surprisingly, we find that trans-synaptic adhesion does not attract SV or AZ proteins nor alter their transport. In addition, although neurexin (the presynaptic partner of neuroligin) typically accumulates over the entire region of contact between axons and neuroligin-1-expressing cells, SV proteins selectively assemble at spots of enhanced neurexin clustering. The arrival and maintenance of SV proteins at these sites is highly variable over the course of minutes to hours, and this variability correlates with neurexin levels at individual synapses. CONCLUSIONS: Together, our data support a model of synaptogenesis where presynaptic proteins are trapped at specific axonal sites, where they are stabilized by trans-synaptic adhesion signaling.

Coordinated trafficking of synaptic vesicle and active zone proteins prior to synapse formation.

BACKGROUND: The proteins required for synaptic transmission are rapidly assembled at nascent synapses, but the mechanisms through which these proteins are delivered to developing presynaptic terminals are not understood. Prior to synapse formation, active zone proteins and synaptic vesicle proteins are transported along axons in distinct organelles referred to as piccolo-bassoon transport vesicles (PTVs) and synaptic vesicle protein transport vesicles (STVs), respectively. Although both PTVs and STVs are recruited to the same site in the axon, often within minutes of axo-dendritic contact, it is not known whether or how PTV and STV trafficking is coordinated before synapse formation. RESULTS: Here, using time-lapse confocal imaging of the dynamics of PTVs and STVs in the same axon, we show that vesicle trafficking is coordinated through at least two mechanisms. First, a significant proportion of STVs and PTVs are transported together before forming a stable terminal. Second, individual PTVs and STVs share pause sites within the axon. Importantly, for both STVs and PTVs, encountering the other type of vesicle increases their propensity to pause. To determine if PTV-STV interactions are important for pausing, PTV density was reduced in axons by expression of a dominant negative construct corresponding to the syntaxin binding domain of syntabulin, which links PTVs with their KIF5B motor. This reduction in PTVs had a minimal effect on STV pausing and movement, suggesting that an interaction between STVs and PTVs is not responsible for enhancing STV pausing. CONCLUSIONS: Our results indicate that trafficking of STVs and PTVs is coordinated even prior to synapse development. This novel coordination of transport and pausing might provide mechanisms through which all of the components of a presynaptic terminal can be rapidly accumulated at sites of synapse formation.

How it's made: the synapse.

How are synapses made? This question is one of the most important issues in neurobiology today and has been the subject of intense study in recent years. This review focuses on the mechanisms of presynaptic terminal formation in the mammalian central nervous system. Building a synapse requires stabilization of contacts between axons and dendrites and formation of synaptic subcellular structures. Here, we discuss what determines where and when synapses form, how components of the nascent presynaptic terminal accumulate at the site of synapse formation, and whether assembly occurs via an ordered process dependent on a master organizer. Understanding synapse formation in the central nervous system is relevant for understanding and treating brain diseases as diverse as autism, epilepsy, anxiety disorders, brain injury, and Alzheimer's disease.

Small vessel ischemic disease of the brain and brain metastases in lung cancer patients.

BACKGROUND: Brain metastases occur commonly in patients with lung cancer. Small vessel ischemic disease is frequently found when imaging the brain to detect metastases. We aimed to determine if the presence of small vessel ischemic disease (SVID) of the brain is protective against the development of brain metastases in lung cancer patients. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective cohort of 523 patients with biopsy confirmed lung cancer who had received magnetic resonance imaging of the brain as part of their standard initial staging evaluation was reviewed. Information collected included demographics, comorbidities, details of the lung cancer, and the presence of SVID of the brain. A portion of the cohort had the degree of SVID graded. The primary outcome measure was the portion of study subjects with and without SVID of the brain who had evidence of brain metastases at the time of initial staging of their lung cancer.109 patients (20.8%) had evidence of brain metastases at presentation and 345 (66.0%) had evidence of SVID. 13.9% of those with SVID and 34.3% of those without SVID presented with brain metastases (p<0.0001). In a model including age, diabetes mellitus, hypertension, hyperlipidemia, and tobacco use, SVID of the brain was found to be the only protective factor against the development of brain metastases, with an OR of 0.31 (0.20, 0.48; p<0.001). The grade of SVID was higher in those without brain metastases. CONCLUSIONS/SIGNIFICANCE: These findings suggest that vascular changes in the brain are protective against the development of brain metastases in lung cancer patients.