In vitro modeling of the human dopaminergic system using spatially arranged ventral midbrain-striatum-cortex assembloids.

Ventral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson's disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson's disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain-striatum-cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.

More than a small adult brain: Lessons from chemotherapy-induced cognitive impairment for modelling paediatric brain disorders.

Childhood is recognised as a period of immense physical and emotional development, and this, in part, is driven by underlying neurophysiological transformations. These neurodevelopmental processes are unique to the paediatric brain and are facilitated by augmented rates of neuroplasticity and expanded neural stem cell populations within neurogenic niches. However, given the immaturity of the developing central nervous system, innate protective mechanisms such as neuroimmune and antioxidant responses are functionally naïve which results in periods of heightened sensitivity to neurotoxic insult. This is highly relevant in the context of paediatric cancer, and in particular, the neurocognitive symptoms associated with treatment, such as surgery, radio- and chemotherapy. The vulnerability of the developing brain may increase susceptibility to damage and persistent symptomology, aligning with reports of more severe neurocognitive dysfunction in children compared to adults. It is therefore surprising, given this intensified neurocognitive burden, that most of the pre-clinical, mechanistic research focuses exclusively on adult populations and extrapolates findings to paediatric cohorts. Given this dearth of age-specific research, throughout this review we will draw comparisons with neurodevelopmental disorders which share comparable pathways to cancer treatment related side-effects. Furthermore, we will examine the unique nuances of the paediatric brain along with the somatic systems which influence neurological function. In doing so, we will highlight the importance of developing in vitro and in vivo paediatric disease models to produce age-specific discovery and clinically translatable research.

Patch-seq: Past, Present, and Future.

Single-cell transcriptomic approaches are revolutionizing neuroscience. Integrating this wealth of data with morphology and physiology, for the comprehensive study of neuronal biology, requires multiplexing gene expression data with complementary techniques. To meet this need, multiple groups in parallel have developed "Patch-seq," a modification of whole-cell patch-clamp protocols that enables mRNA sequencing of cell contents after electrophysiological recordings from individual neurons and morphologic reconstruction of the same cells. In this review, we first outline the critical technical developments that enabled robust Patch-seq experimental efforts and analytical solutions to interpret the rich multimodal data generated. We then review recent applications of Patch-seq that address novel and long-standing questions in neuroscience. These include the following: (1) targeted study of specific neuronal populations based on their anatomic location, functional properties, lineage, or a combination of these factors; (2) the compilation and integration of multimodal cell type atlases; and (3) the investigation of the molecular basis of morphologic and functional diversity. Finally, we highlight potential opportunities for further technical development and lines of research that may benefit from implementing the Patch-seq technique. As a multimodal approach at the intersection of molecular neurobiology and physiology, Patch-seq is uniquely positioned to directly link gene expression to brain function.

A human neurodevelopmental model for Williams syndrome.

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes, with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioural pathologies in humans, remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome, we narrowed this cellular phenotype to a single gene candidate, frizzled 9 (FZD9). At the neuronal stage, layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.

The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis.

Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely because of activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival.

Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience.

The scarcity of live human brain cells for experimental access has for a long time limited our ability to study complex human neurological disorders and elucidate basic neuroscientific mechanisms. A decade ago, the development of methods to reprogramme somatic human cells into induced pluripotent stem cells enabled the in vitro generation of a wide range of neural cells from virtually any human individual. The growth of methods to generate more robust and defined neural cell types through reprogramming and direct conversion into induced neurons has led to the establishment of various human reprogramming-based neural disease models.

Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells.

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-ß precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-ß(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3ß (aGSK-3ß). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with ß-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3ß levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-ß, in GSK-3ß activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.

Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro.

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.

Cord blood-derived neuronal cells by ectopic expression of Sox2 and c-Myc.

The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133(+) cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies.

Neuronal and tumourigenic boundaries of glioblastoma plasticity.

Glioblastoma (GBM) remains the most lethal primary brain cancer largely due to recurrence of treatment-resistant disease. Current therapies are ultimately ineffective as GBM tumour cells adapt their identity to escape treatment. Recent advances in single-cell epigenetics and transcriptomics highlight heterogeneous cell populations in GBM tumours originating from unique cancerous genetic aberrations. However, they also suggest that tumour cells conserve molecular properties of parent neuronal cells, with their permissive epigenetic profiles enabling them to morph along a finite number of reprogramming routes to evade treatment. Here, we review the known tumourigenic, neurodevelopmental and brain-injury boundaries of GBM plasticity, and propose that effective treatment of GBM requires the addition of therapeutics that restrain GBM plasticity.

Human cerebrospinal fluid affects chemoradiotherapy sensitivities in tumor cells from patients with glioblastoma.

Cancers in the central nervous system resist therapies effective in other cancers, possibly due to the unique biochemistry of the human brain microenvironment composed of cerebrospinal fluid (CSF). However, the impact of CSF on cancer cells and therapeutic efficacy is unknown. Here, we examined the effect of human CSF on glioblastoma (GBM) tumors from 25 patients. We found that CSF induces tumor cell plasticity and resistance to standard GBM treatments (temozolomide and irradiation). We identified nuclear protein 1 (NUPR1), a transcription factor hampering ferroptosis, as a mediator of therapeutic resistance in CSF. NUPR1 inhibition with a repurposed antipsychotic, trifluoperazine, enhanced the killing of GBM cells resistant to chemoradiation in CSF. The same chemo-effective doses of trifluoperazine were safe for human neurons and astrocytes derived from pluripotent stem cells. These findings reveal that chemoradiation efficacy decreases in human CSF and suggest that combining trifluoperazine with standard care may improve the survival of patients with GBM.

Recapitulation of Perturbed Striatal Gene Expression Dynamics of Donors' Brains With Ventral Forebrain Organoids Derived From the Same Individuals With Schizophrenia.

OBJECTIVE: Schizophrenia is a brain disorder that originates during neurodevelopment and has complex genetic and environmental etiologies. Despite decades of clinical evidence of altered striatal function in affected patients, studies examining its cellular and molecular mechanisms in humans are limited. To explore neurodevelopmental alterations in the striatum associated with schizophrenia, the authors established a method for the differentiation of induced pluripotent stem cells (iPSCs) into ventral forebrain organoids (VFOs). METHODS: VFOs were generated from postmortem dural fibroblast-derived iPSCs of four individuals with schizophrenia and four neurotypical control individuals for whom postmortem caudate genotypes and transcriptomic data were profiled in the BrainSeq neurogenomics consortium. Individuals were selected such that the two groups had nonoverlapping schizophrenia polygenic risk scores (PRSs). RESULTS: Single-cell RNA sequencing analyses of VFOs revealed differences in developmental trajectory between schizophrenia and control individuals in which inhibitory neuronal cells from the patients exhibited accelerated maturation. Furthermore, upregulated genes in inhibitory neurons in schizophrenia VFOs showed a significant overlap with upregulated genes in postmortem caudate tissue of individuals with schizophrenia compared with control individuals, including the donors of the iPSC cohort. CONCLUSIONS: The findings suggest that striatal neurons derived from high-PRS individuals with schizophrenia carry abnormalities that originated during early brain development and that the VFO model can recapitulate disease-relevant cell type-specific neurodevelopmental phenotypes in a dish.

Druggable transcriptomic pathways revealed in Parkinson's patient-derived midbrain neurons.

Complex genetic predispositions accelerate the chronic degeneration of midbrain substantia nigra neurons in Parkinson's disease (PD). Deciphering the human molecular makeup of PD pathophysiology can guide the discovery of therapeutics to slow the disease progression. However, insights from human postmortem brain studies only portray the latter stages of PD, and there is a lack of data surrounding molecular events preceding the neuronal loss in patients. We address this gap by identifying the gene dysregulation of live midbrain neurons reprogrammed in vitro from the skin cells of 42 individuals, including sporadic and familial PD patients and matched healthy controls. To minimize bias resulting from neuronal reprogramming and RNA-seq methods, we developed an analysis pipeline integrating PD transcriptomes from different RNA-seq datasets (unsorted and sorted bulk vs. single-cell and Patch-seq) and reprogramming strategies (induced pluripotency vs. direct conversion). This PD cohort's transcriptome is enriched for human genes associated with known clinical phenotypes of PD, regulation of locomotion, bradykinesia and rigidity. Dysregulated gene expression emerges strongest in pathways underlying synaptic transmission, metabolism, intracellular trafficking, neural morphogenesis and cellular stress/immune responses. We confirmed a synaptic impairment with patch-clamping and identified pesticides and endoplasmic reticulum stressors as the most significant gene-chemical interactions in PD. Subsequently, we associated the PD transcriptomic profile with candidate pharmaceuticals in a large database and a registry of current clinical trials. This study highlights human transcriptomic pathways that can be targeted therapeutically before the irreversible neuronal loss. Furthermore, it demonstrates the preclinical relevance of unbiased large transcriptomic assays of reprogrammed patient neurons.

Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings.

Advances in cellular reprogramming have radically increased the use of patient-derived cells for neurological research in vitro. However, adherence of human neurons on tissue cultureware is unreliable over the extended periods required for electrophysiological maturation. Adherence issues are particularly prominent for transferable glass coverslips, hindering imaging and electrophysiological assays. Here, we assessed thin-film plasma polymer treatments, polymeric factors, and extracellular matrix coatings for extending the adherence of human neuronal cultures on glass. We find that positive-charged, amine-based plasma polymers improve the adherence of a range of human brain cells. Diaminopropane (DAP) treatment with laminin-based coating optimally supports long-term maturation of fundamental ion channel properties and synaptic activity of human neurons. As proof of concept, we demonstrated that DAP-treated glass is ideal for live imaging, patch-clamping, and optogenetics. A DAP-treated glass surface reduces the technical variability of human neuronal models and enhances electrophysiological maturation, allowing more reliable discoveries of treatments for neurological and psychiatric disorders.

Reduced synaptic activity and dysregulated extracellular matrix pathways in midbrain neurons from Parkinson's disease patients.

Several mutations that cause Parkinson's disease (PD) have been identified over the past decade. These account for 15-25% of PD cases; the rest of the cases are considered sporadic. Currently, it is accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from the induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, show changes that are central and convergent to PD and suggest a strong involvement of the tetra-partite synapse in PD pathophysiology.

How, when, and where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.

Adult-born neurons continuously incorporate into the olfactory bulb where they rapidly establish contacts with a variety of synaptic inputs. Little is known, however, about the functional properties of their output. Characterization of synaptic outputs from new neurons is essential to assess the functional impact of adult neurogenesis on mature circuits. Here, we used optogenetics to control neurotransmitter release from new neurons. We found that light-induced synaptic GABA release from adult-born neurons leads to profound modifications of postsynaptic target firing patterns. We revealed that functional output synapses form just after new cells acquire the faculty to spike, but most synapses were made a month later. Despite discrepancies in the timing of new synapse recruitment, the properties of postsynaptic signals remain constant. Remarkably, we found that all major cell types of the olfactory bulb circuit, including output neurons and several distinct subtypes of local interneurons, were contacted by adult-born neurons. Thus, this study provides new insights into how new neurons integrate into the adult neural network and may influence the sense of smell.

Retromer regulates the lysosomal clearance of MAPT/tau.

The macroautophagy/autophagy-lysosome axis enables the clearance and degradation of cytoplasmic components including protein aggregates, damaged organelles and invading pathogens. Protein aggregation and lysosomal system dysfunction in the brain are common features of several late-onset neurological disorders including Alzheimer disease. Spatial overlap between depletion of the endosomal-sorting complex retromer and MAPT/tau aggregation in the brain have been previously reported. However, whether retromer dysfunction plays a direct role in mediating MAPT aggregation remains unclear. Here, we demonstrate that the autophagy-lysosome axis is the primary mode for the clearance of aggregated species of MAPT using both chemical and genetic approaches in cell models of amyloid MAPT aggregation. We show that depletion of the central retromer component VPS35 causes a block in the resolution of autophagy. We establish that this defect underlies marked accumulation of cytoplasmic MAPT aggregates upon VPS35 depletion, and that VPS35 overexpression has the opposite effect. This work illustrates how retromer complex integrity regulates the autophagy-lysosome axis to suppress MAPT aggregation and spread.

Early synapse formation in developing interneurons of the adult olfactory bulb.

New olfactory bulb granule cells (GCs) are GABAergic interneurons continuously arising from neuronal progenitors and integrating into preexisting bulbar circuits. They receive both GABAergic and glutamatergic synaptic inputs from olfactory bulb intrinsic neurons and centrifugal afferents. Here, we investigated the spatiotemporal dynamic of newborn GC synaptogenesis in adult mouse olfactory bulb. First, we established that GABAergic synapses onto mature GC dendrites contain the GABA(A) receptor alpha2 subunit along with the postsynaptic scaffolding protein gephyrin. Next, we characterized morphologically and electrophysiologically the development of GABAergic and glutamatergic inputs onto newborn GCs labeled with eGFP (enhanced green fluorescent protein) using lentiviral vectors. Already when reaching the GC layer (GCL), at 3 d post-vector injection (dpi), newborn GCs exhibited tiny voltage-dependent sodium currents and received functional GABAergic and glutamatergic synapses, recognized immunohistochemically by apposition of specific presynaptic and postsynaptic markers. Thereafter, GABAergic and glutamatergic synaptic contacts increased differentially in the GCL, and at 7 dpi, PSD-95 clusters outnumbered gephyrin clusters. Thus, the weight of GABAergic input was predominant at early stages of GC maturation, but not later. Newborn GC dendrites first reached the external plexiform layer at 4 dpi, where they received functional GABAergic contacts at 5 dpi. Reciprocal synapses initially were formed on GC dendritic shafts, where they might contribute to spine formation. Their presence was confirmed ultrastructurally at 7 dpi. Together, our findings unravel rapid synaptic integration of newborn GCs in adult mouse olfactory bulb, with GABAergic and glutamatergic influences being established proximally before formation of output synapses by apical GC dendrites onto mitral/tufted cells.

Genetic predispositions of Parkinson's disease revealed in patient-derived brain cells.

Parkinson's disease (PD) is the second most prevalent neurological disorder and has been the focus of intense investigations to understand its etiology and progression, but it still lacks a cure. Modeling diseases of the central nervous system in vitro with human induced pluripotent stem cells (hiPSC) is still in its infancy but has the potential to expedite the discovery and validation of new treatments. Here, we discuss the interplay between genetic predispositions and midbrain neuronal impairments in people living with PD. We first summarize the prevalence of causal Parkinson's genes and risk factors reported in 74 epidemiological and genomic studies. We then present a meta-analysis of 385 hiPSC-derived neuronal lines from 67 recent independent original research articles, which point towards specific impairments in neurons from Parkinson's patients, within the context of genetic predispositions. Despite the heterogeneous nature of the disease, current iPSC models reveal converging molecular pathways underlying neurodegeneration in a range of familial and sporadic forms of Parkinson's disease. Altogether, consolidating our understanding of robust cellular phenotypes across genetic cohorts of Parkinson's patients may guide future personalized drug screens in preclinical research.

BrainPhys neuronal medium optimized for imaging and optogenetics in vitro.

The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology are advancing. In parallel, cellular reprogramming and organoid engineering are expanding the use of human neuronal models in vitro. This creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identify multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (i.e., suboptimal fluorescence signals, phototoxicity, and unphysiological neuronal activity). To overcome these issues, we develop a neuromedium called BrainPhys™ Imaging (BPI) in which we optimize the concentrations of fluorescent and phototoxic compounds. BPI is based on the formulation of the original BrainPhys medium. We benchmark available neuronal media and show that BPI enhances fluorescence signals, reduces phototoxicity and optimally supports the electrical and synaptic activity of neurons in culture. We also show the superior capacity of BPI for optogenetics and calcium imaging of human neurons. Altogether, our study shows that BPI improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting optimal neuronal viability and function.