The missense mutation C667F in murine ß-dystroglycan causes embryonic lethality, myopathy and blood-brain barrier destabilization.

Dystroglycan (DG) is an extracellular matrix receptor consisting of an α- and a ß-DG subunit encoded by the DAG1 gene. The homozygous mutation (c.2006G>T, p.Cys669Phe) in ß-DG causes muscle-eye-brain disease with multicystic leukodystrophy in humans. In a mouse model of this primary dystroglycanopathy, approximately two-thirds of homozygous embryos fail to develop to term. Mutant mice that are born undergo a normal postnatal development but show a late-onset myopathy with partially penetrant histopathological changes and an impaired performance on an activity wheel. Their brains and eyes are structurally normal, but the localization of mutant ß-DG is altered in the glial perivascular end-feet, resulting in a perturbed protein composition of the blood-brain and blood-retina barrier. In addition, α- and ß-DG protein levels are significantly reduced in muscle and brain of mutant mice. Owing to the partially penetrant developmental phenotype of the C669F ß-DG mice, they represent a novel and highly valuable mouse model with which to study the molecular effects of ß-DG functional alterations both during embryogenesis and in mature muscle, brain and eye, and to gain insight into the pathogenesis of primary dystroglycanopathies.

Paroxysmal dystonia results from the loss of RIM4 in Purkinje cells.

Full-length RIM1 and 2 are key components of the presynaptic active zone that ubiquitously control excitatory and inhibitory neurotransmitter release. Here, we report that the function of the small RIM isoform RIM4, consisting of a single C2 domain, is strikingly different from that of the long isoforms. RIM4 is dispensable for neurotransmitter release but plays a postsynaptic, cell-type specific role in cerebellar Purkinje cells that is essential for normal motor function. In the absence of RIM4, Purkinje cell intrinsic firing is reduced and caffeine-sensitive, and dendritic integration of climbing fibre input is disturbed. Mice lacking RIM4, but not mice lacking RIM1/2, selectively in Purkinje cells exhibit a severe, hours-long paroxysmal dystonia. These episodes can also be induced by caffeine, ethanol or stress and closely resemble the deficits seen with mutations of the PNKD (paroxysmal non-kinesigenic dystonia) gene. Our data reveal essential postsynaptic functions of RIM proteins and show non-overlapping specialized functions of a small isoform despite high homology to a single domain in the full-length proteins.

Cell type specificity for circuit output in the midbrain dopaminergic system.

Midbrain dopaminergic neurons are a relatively small group of neurons in the mammalian brain controlling a wide range of behaviors. In recent years, increasingly sophisticated tracing, imaging, transcriptomic, and machine learning approaches have provided substantial insights into the anatomical, molecular, and functional heterogeneity of dopaminergic neurons. Despite this wealth of new knowledge, it remains unclear whether and how the diverse features defining dopaminergic subclasses converge to delineate functional ensembles within the dopaminergic system. Here, we review recent studies investigating various aspects of dopaminergic heterogeneity and discuss how development, behavior, and disease influence subtype characteristics. We then outline what further approaches could be pursued to gain a more inclusive picture of dopaminergic diversity, which could be crucial to understanding the functional architecture of this system.

Targeting aberrant dendritic integration to treat cognitive comorbidities of epilepsy.

Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding and deficits in spatial memory. Two-photon glutamate uncaging experiments revealed a marked increase in the fraction of hippocampal first-order CA1 pyramidal cell dendrites capable of generating dendritic spikes in the kainate model of chronic epilepsy. Moreover, in epileptic mice dendritic spikes were generated with lower input synchrony, and with a lower threshold. The Nav1.3/1.1 selective Na+ channel blocker ICA-121431 reversed dendritic hyperexcitability in epileptic mice, while the Nav1.2/1.6 preferring anticonvulsant S-Lic did not. We used in vivo two-photon imaging to determine if aberrant dendritic excitability is associated with altered place-related firing of CA1 neurons. We show that ICA-121431 improves degraded hippocampal spatial representations in epileptic mice. Finally, behavioural experiments show that reversing aberrant dendritic excitability with ICA-121431 reverses hippocampal memory deficits. Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition.

Lineage Analysis of Cxcr4-Expressing Cells in the Developing Midbrain Suggests That Progressive Competence Restriction in Dopaminergic Progenitor Cells Contributes to the Establishment of Dopaminergic Neuronal Diversity.

Midbrain dopaminergic (mDA) neurons are generated from a ventral midbrain progenitor zone over a time span of several days [embryonic day 10.0 (E10.0) to E14.5 in mouse]. Within this neurogenic period, a progressively changing fate potential of mDA progenitors could contribute to the generation of diverse mDA neuronal subpopulations. To test this idea, we combined inducible genetic fate mapping and intersectional labeling approaches to trace the lineage of cells expressing the chemokine receptor CXCR4. The Cxcr4 transcript is expressed in mDA progenitors and precursors, but not in differentiated mDA neurons. Cxcr4-expressing mDA progenitors/precursors labeled at E11.5 develop into a broad range of mDA neurons, whereas labeling of the Cxcr4 lineage at later time points (E12.5-E15.5) results in an increasingly restricted contribution to mDA neurons proceeding from lateral to medial in the substantia nigra and from dorsal to ventral in the ventral tegmental area. In parallel, the innervation of dopaminergic projection targets by mDA neurons derived from Cxcr4-expressing cells is becoming more restricted: the late-generated mDA neurons innervate only the medial-rostral regions in the dorsal striatum and only the medial shell in the nucleus accumbens. Our results suggest that mDA progenitor cells become increasingly restricted in their cell fate potential over time.

The Development of the Mesoprefrontal Dopaminergic System in Health and Disease.

Midbrain dopaminergic neurons located in the substantia nigra and the ventral tegmental area are the main source of dopamine in the brain. They send out projections to a variety of forebrain structures, including dorsal striatum, nucleus accumbens, and prefrontal cortex (PFC), establishing the nigrostriatal, mesolimbic, and mesoprefrontal pathways, respectively. The dopaminergic input to the PFC is essential for the performance of higher cognitive functions such as working memory, attention, planning, and decision making. The gradual maturation of these cognitive skills during postnatal development correlates with the maturation of PFC local circuits, which undergo a lengthy functional remodeling process during the neonatal and adolescence stage. During this period, the mesoprefrontal dopaminergic innervation also matures: the fibers are rather sparse at prenatal stages and slowly increase in density during postnatal development to finally reach a stable pattern in early adulthood. Despite the prominent role of dopamine in the regulation of PFC function, relatively little is known about how the dopaminergic innervation is established in the PFC, whether and how it influences the maturation of local circuits and how exactly it facilitates cognitive functions in the PFC. In this review, we provide an overview of the development of the mesoprefrontal dopaminergic system in rodents and primates and discuss the role of altered dopaminergic signaling in neuropsychiatric and neurodevelopmental disorders.

The transcription factor BCL11A defines distinct subsets of midbrain dopaminergic neurons.

Midbrain dopaminergic (mDA) neurons are diverse in their projection targets, effect on behavior, and susceptibility to neurodegeneration. Little is known about the molecular mechanisms establishing this diversity during development. We show that the transcription factor BCL11A is expressed in a subset of mDA neurons in the developing and adult murine brain and in a subpopulation of pluripotent-stem-cell-derived human mDA neurons. By combining intersectional labeling and viral-mediated tracing, we demonstrate that Bcl11a-expressing mDA neurons form a highly specific subcircuit within the murine dopaminergic system. In the substantia nigra, the Bcl11a-expressing mDA subset is particularly vulnerable to neurodegeneration upon α-synuclein overexpression or oxidative stress. Inactivation of Bcl11a in murine mDA neurons increases this susceptibility further, alters the distribution of mDA neurons, and results in deficits in skilled motor behavior. In summary, BCL11A defines mDA subpopulations with highly distinctive characteristics and is required for establishing and maintaining their normal physiology.

The BBSome: a nexus controlling energy metabolism in the brain.

Bardet-Biedl syndrome (BBS) is a syndromic ciliopathy that has obesity as a cardinal feature. BBS is caused by mutations in BBS genes. BBS proteins control primary cilia function, and BBS mutations therefore lead to dysfunctional primary cilia. Obesity in patients with BBS is mainly caused by hyperphagia due to dysregulated neuronal function in the brain, in particular in the hypothalamus. However, the mechanism by which mutations in BBS genes result in dysfunction in hypothalamic neurons is not well understood. In this issue of the JCI, Wang et al. used BBS and non-BBS patient-derived induced pluripotent stem cells to generate neurons and hypothalamic neurons. Using this human model system, the authors demonstrated that mutations in BBS genes affected primary cilia function, neuronal morphology, and signaling pathways regulating the function of hypothalamic neurons, which control energy homeostasis. This study provides important insights into the mechanisms of BBS-induced obesity.

The Impact of Mitochondrial Dysfunction on Dopaminergic Neurons in the Olfactory Bulb and Odor Detection.

Understanding non-motor symptoms of Parkinson's disease is important in order to unravel the underlying molecular mechanisms of the disease. Olfactory dysfunction is an early stage, non-motor symptom which occurs in 95% of Parkinson's disease patients. Mitochondrial dysfunction is a key feature in Parkinson's disease and importantly contributes to the selective loss of dopaminergic neurons the substantia nigra pars compacta. The olfactory bulb, the first olfactory processing station, also contains dopaminergic neurons, which modulate odor information and thereby enable odor detection as well as odor discrimination. MitoPark mice are a genetic model for Parkinson's disease with severe mitochondrial dysfunction, reproducing the differential vulnerability of dopaminergic neurons in the midbrain. These animals were used to investigate the impact of mitochondrial dysfunction on olfactory-related behavior and olfactory bulb dopaminergic neuron survival. Odor detection was severely impaired in MitoPark mice. Interestingly, only the small anaxonic dopaminergic subpopulation, which is continuously replenished by neurogenesis, was moderately reduced in number, much less compared with dopaminergic neurons in the midbrain. As a potential compensatory response, an enhanced mobilization of progenitor cells was found in the subventricular zone. These results reveal a high robustness of dopaminergic neurons located in the olfactory bulb towards mitochondrial impairment, in striking contrast to their midbrain counterparts.

Calcium Channel Subunit α2δ4 Is Regulated by Early Growth Response 1 and Facilitates Epileptogenesis.

Transient brain insults, including status epilepticus (SE), can trigger a period of epileptogenesis during which functional and structural reorganization of neuronal networks occurs resulting in the onset of focal epileptic seizures. In recent years, mechanisms that regulate the dynamic transcription of individual genes during epileptogenesis and thereby contribute to the development of a hyperexcitable neuronal network have been elucidated. Our own results have shown early growth response 1 (Egr1) to transiently increase expression of the T-type voltage-dependent Ca2+ channel (VDCC) subunit CaV3.2, a key proepileptogenic protein. However, epileptogenesis involves complex and dynamic transcriptomic alterations; and so far, our understanding of the transcriptional control mechanism of gene regulatory networks that act in the same processes is limited. Here, we have analyzed whether Egr1 acts as a key transcriptional regulator for genes contributing to the development of hyperexcitability during epileptogenesis. We found Egr1 to drive the expression of the VDCC subunit α2δ4, which was augmented early and persistently after pilocarpine-induced SE. Furthermore, we show that increasing levels of α2δ4 in the CA1 region of the hippocampus elevate seizure susceptibility of mice by slightly decreasing local network activity. Interestingly, we also detected increased expression levels of Egr1 and α2δ4 in human hippocampal biopsies obtained from epilepsy surgery. In conclusion, Egr1 controls the abundance of the VDCC subunits CaV3.2 and α2δ4, which act synergistically in epileptogenesis, and thereby contributes to a seizure-induced "transcriptional Ca2+ channelopathy."SIGNIFICANCE STATEMENT The onset of focal recurrent seizures often occurs after an epileptogenic process induced by transient insults to the brain. Recently, transcriptional control mechanisms for individual genes involved in converting neurons hyperexcitable have been identified, including early growth response 1 (Egr1), which activates transcription of the T-type Ca2+ channel subunit CaV3.2. Here, we find Egr1 to regulate also the expression of the voltage-dependent Ca2+ channel subunit α2δ4, which was augmented after pilocarpine- and kainic acid-induced status epilepticus. In addition, we observed that α2δ4 affected spontaneous network activity and the susceptibility for seizure induction. Furthermore, we detected corresponding dynamics in human biopsies from epilepsy patients. In conclusion, Egr1 orchestrates a seizure-induced "transcriptional Ca2+ channelopathy" consisting of CaV3.2 and α2δ4, which act synergistically in epileptogenesis.

Correct setup of the substantia nigra requires Reelin-mediated fast, laterally-directed migration of dopaminergic neurons.

Midbrain dopaminergic (mDA) neurons migrate to form the laterally-located substantia nigra pars compacta (SN) and medially-located ventral tegmental area (VTA), but little is known about the underlying cellular and molecular processes. Here we visualize the dynamic cell morphologies of tangentially migrating SN-mDA neurons in 3D and identify two distinct migration modes. Slow migration is the default mode in SN-mDA neurons, while fast, laterally-directed migration occurs infrequently and is strongly associated with bipolar cell morphology. Tangential migration of SN-mDA neurons is altered in absence of Reelin signaling, but it is unclear whether Reelin acts directly on migrating SN-mDA neurons and how it affects their cell morphology and migratory behavior. By specifically inactivating Reelin signaling in mDA neurons we demonstrate its direct role in SN-mDA tangential migration. Reelin promotes laterally-biased movements in mDA neurons during their slow migration mode, stabilizes leading process morphology and increases the probability of fast, laterally-directed migration.

Induced pluripotent stem cell-based modeling of mutant LRRK2-associated Parkinson's disease.

Recent advances in cell reprogramming have enabled assessment of disease-related cellular traits in patient-derived somatic cells, thus providing a versatile platform for disease modeling and drug development. Given the limited access to vital human brain cells, this technology is especially relevant for neurodegenerative disorders such as Parkinson's disease (PD) as a tool to decipher underlying pathomechanisms. Importantly, recent progress in genome-editing technologies has provided an ability to analyze isogenic induced pluripotent stem cell (iPSC) pairs that differ only in a single genetic change, thus allowing a thorough assessment of the molecular and cellular phenotypes that result from monogenetic risk factors. In this review, we summarize the current state of iPSC-based modeling of PD with a focus on leucine-rich repeat kinase 2 (LRRK2), one of the most prominent monogenetic risk factors for PD linked to both familial and idiopathic forms. The LRRK2 protein is a primarily cytosolic multi-domain protein contributing to regulation of several pathways including autophagy, mitochondrial function, vesicle transport, nuclear architecture and cell morphology. We summarize iPSC-based studies that contributed to improving our understanding of the function of LRRK2 and its variants in the context of PD etiopathology. These data, along with results obtained in our own studies, underscore the multifaceted role of LRRK2 in regulating cellular homeostasis on several levels, including proteostasis, mitochondrial dynamics and regulation of the cytoskeleton. Finally, we expound advantages and limitations of reprogramming technologies for disease modeling and drug development and provide an outlook on future challenges and expectations offered by this exciting technology.

Crosstalk of Intercellular Signaling Pathways in the Generation of Midbrain Dopaminergic Neurons In Vivo and from Stem Cells.

Dopamine-synthesizing neurons located in the mammalian ventral midbrain are at the center stage of biomedical research due to their involvement in severe human neuropsychiatric and neurodegenerative disorders, most prominently Parkinson's Disease (PD). The induction of midbrain dopaminergic (mDA) neurons depends on two important signaling centers of the mammalian embryo: the ventral midline or floor plate (FP) of the neural tube, and the isthmic organizer (IsO) at the mid-/hindbrain boundary (MHB). Cells located within and close to the FP secrete sonic hedgehog (SHH), and members of the wingless-type MMTV integration site family (WNT1/5A), as well as bone morphogenetic protein (BMP) family. The IsO cells secrete WNT1 and the fibroblast growth factor 8 (FGF8). Accordingly, the FGF8, SHH, WNT, and BMP signaling pathways play crucial roles during the development of the mDA neurons in the mammalian embryo. Moreover, these morphogens are essential for the generation of stem cell-derived mDA neurons, which are critical for the modeling, drug screening, and cell replacement therapy of PD. This review summarizes our current knowledge about the functions and crosstalk of these signaling pathways in mammalian mDA neuron development in vivo and their applications in stem cell-based paradigms for the efficient derivation of these neurons in vitro.

The zinc-finger transcription factor GLI3 is a regulator of precerebellar neuronal migration.

Hindbrain precerebellar neurons arise from progenitor pools at the dorsal edge of the embryonic hindbrain: the caudal rhombic lip. These neurons follow distinct migratory routes to establish nuclei that provide climbing or mossy fiber inputs to the cerebellum. Gli3, a zinc-finger transcription factor in the Sonic hedgehog signaling pathway, is an important regulator of dorsal brain development. We demonstrate that in Gli3-null mutant mice, disrupted neuronal migratory streams lead to a disorganization of precerebellar nuclei. Precerebellar progenitors are properly established in Gli3-null embryos and, using conditional gene inactivation, we provide evidence that Gli3 does not play a cell-autonomous role in migrating precerebellar neurons. Thus, GLI3 likely regulates the development of other hindbrain structures, such as non-precerebellar nuclei or cranial ganglia and their respective projections, which may in turn influence precerebellar migration. Although the organization of non-precerebellar hindbrain nuclei appears to be largely unaffected in absence of Gli3, trigeminal ganglia and their central descending tracts are disrupted. We show that rostrally migrating precerebellar neurons are normally in close contact with these tracts, but are detached in Gli3-null embryos.

BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells.

The embryonic formation of midbrain dopaminergic (mDA) neurons in vivo provides critical guidelines for the in vitro differentiation of mDA neurons from stem cells, which are currently being developed for Parkinson's disease cell replacement therapy. Bone morphogenetic protein (BMP)/SMAD inhibition is routinely used during early steps of stem cell differentiation protocols, including for the generation of mDA neurons. However, the function of the BMP/SMAD pathway for in vivo specification of mammalian mDA neurons is virtually unknown. Here, we report that BMP5/7-deficient mice (Bmp5-/-; Bmp7-/-) lack mDA neurons due to reduced neurogenesis in the mDA progenitor domain. As molecular mechanisms accounting for these alterations in Bmp5-/-; Bmp7-/- mutants, we have identified expression changes of the BMP/SMAD target genes MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog). Conditionally inactivating SMAD1 in neural stem cells of mice in vivo (Smad1Nes) hampered the differentiation of progenitor cells into mDA neurons by preventing cell cycle exit, especially of TH+SOX6+ (tyrosine hydroxylase, SRY-box 6) and TH+GIRK2+ (potassium voltage-gated channel subfamily-J member-6) substantia nigra neurons. BMP5/7 robustly increased the in vitro differentiation of human induced pluripotent stem cells and induced neural stem cells to mDA neurons by up to threefold. In conclusion, we have identified BMP/SMAD signaling as a novel critical pathway orchestrating essential steps of mammalian mDA neurogenesis in vivo that balances progenitor proliferation and differentiation. Moreover, we demonstrate the potential of BMPs to improve the generation of stem-cell-derived mDA neurons in vitro, highlighting the importance of sequential BMP/SMAD inhibition and activation in this process.SIGNIFICANCE STATEMENT We identify bone morphogenetic protein (BMP)/SMAD signaling as a novel essential pathway regulating the development of mammalian midbrain dopaminergic (mDA) neurons in vivo and provide insights into the molecular mechanisms of this process. BMP5/7 regulate MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog) expression to direct mDA neurogenesis. Moreover, the BMP signaling component SMAD1 controls the differentiation of mDA progenitors, particularly to substantia nigra neurons, by directing their cell cycle exit. Importantly, BMP5/7 increase robustly the differentiation of human induced pluripotent and induced neural stem cells to mDA neurons. BMP/SMAD are routinely inhibited in initial stages of stem cell differentiation protocols currently being developed for Parkinson's disease cell replacement therapies. Therefore, our findings on opposing roles of the BMP/SMAD pathway during in vitro mDA neurogenesis might improve these procedures significantly.

In vitro segregation and isolation of human pluripotent stem cell-derived neural crest cells.

The neural crest (NC) is a transient embryonic cell population with remarkable characteristics. After delaminating from the neural tube, NC cells (NCCs) migrate extensively, populate nearly every tissue of the body and differentiate into highly diverse cell types such as peripheral neurons and glia, but also mesenchymal cells including chondrocytes, osteocytes, and adipocytes. While the NC has been extensively studied in several animal models, little is known about human NC development. A number of methods have been established to derive NCCs in vitro from human pluripotent stem cells (hPSC). Typically, these protocols comprise several cell culture steps to enrich for NCCs in the neural derivatives of the differentiating hPSCs. Here we report on a remarkable and hitherto unnoticed in vitro segregation phenomenon that enables direct extraction of virtually pure NCCs during the earliest stages of hPSC differentiation. Upon aggregation to embryoid bodies (EB) and replating, differentiating hPSCs give rise to a population of NCCs, which spontaneously segregate from the EB outgrowth to form conspicuous, macroscopically visible atoll-shaped clusters in the periphery of the EB outgrowth. Isolation of these NC clusters yields p75NTR(+)/SOXE(+) NCCs, which differentiate to peripheral neurons and glia as well as mesenchymal derivatives. Our data indicate that differentiating hPSC cultures recapitulate, in a simplified manner, the physical segregation of central nervous system (CNS) tissue and NCCs. This phenomenon may be exploited for NCC purification and for studying segregation and differentiation processes observed during early human NC development in vitro.

Evaluation of the effect of a floxed Neo cassette within the dystroglycan (Dag1) gene.

OBJECTIVE: Dystroglycan (DG) is an adhesion complex formed by two subunits, α-DG and ß-DG. In skeletal muscle, DG is part of the dystrophin-glycoprotein complex that is crucial for sarcolemma stability and it is involved in a plethora of muscular dystrophy phenotypes. Due to the important role played by DG in skeletal muscle stability as well as in a wide variety of other tissues including brain and the peripheral nervous system, it is essential to investigate its genetic assembly and transcriptional regulation. RESULTS: Herein, we analyze the effect of the insertion of a floxed neomycin (Neo) cassette within the 3' portion of the universally conserved IG1-intron of the DG gene (Dag1). We analyzed the transcription level of Dag1 and the expression of the DG protein in skeletal muscle of targeted mice compared to wild-type and we did not find any alterations that might be attributed to the gene targeting. However, we found an increase of the cross-sectional areas of tibialis anterior that might have some physiological significance that needs to be assessed in the future. Moreover, in targeted mice the skeletal muscle morphology and its regeneration capacity after injury did not show any evident alterations. We confirmed that the targeting of Dag1 with a floxed Neo-cassette did not produce any gross undesired effects.

Reelin Signaling in the Migration of Ventral Brain Stem and Spinal Cord Neurons.

The extracellular matrix protein Reelin is an important orchestrator of neuronal migration during the development of the central nervous system. While its role and mechanism of action have been extensively studied and reviewed in the formation of dorsal laminar brain structures like the cerebral cortex, hippocampus, and cerebellum, its functions during the neuronal migration events that result in the nuclear organization of the ventral central nervous system are less well understood. In an attempt to delineate an underlying pattern of Reelin action in the formation of neuronal cell clusters, this review highlights the role of Reelin signaling in the migration of neuronal populations that originate in the ventral brain stem and the spinal cord.

Definition of a critical spatiotemporal window within which primary cilia control midbrain dopaminergic neurogenesis.

Midbrain dopaminergic (mDA) neurons are generated in the ventral midbrain floor plate depending on Sonic Hedgehog (SHH) signaling for induction. Primary cilia transduce canonical SHH signals. Loss of intraflagellar transport protein IFT88, essential for ciliary function, disrupts SHH signaling in the ventral midbrain and results in the reduction in mDA progenitors and neurons. We investigate whether conditional inactivation of the kinesin motor protein KIF3A recapitulates phenotypes observed in conditional Ift88 mutants. Conditional Kif3a inactivation reduced the mDA progenitor domain size, but did not result in mDA neuron reduction, most likely because of a delayed loss of cilia and delayed inactivation of SHH signaling. We thereby define a precise spatiotemporal window within which primary cilia-dependent SHH signaling determines mDA fate.