Cerebellum Lecture: the Cerebellar Nuclei-Core of the Cerebellum.

The cerebellum is a key player in many brain functions and a major topic of neuroscience research. However, the cerebellar nuclei (CN), the main output structures of the cerebellum, are often overlooked. This neglect is because research on the cerebellum typically focuses on the cortex and tends to treat the CN as relatively simple output nuclei conveying an inverted signal from the cerebellar cortex to the rest of the brain. In this review, by adopting a nucleocentric perspective we aim to rectify this impression. First, we describe CN anatomy and modularity and comprehensively integrate CN architecture with its highly organized but complex afferent and efferent connectivity. This is followed by a novel classification of the specific neuronal classes the CN comprise and speculate on the implications of CN structure and physiology for our understanding of adult cerebellar function. Based on this thorough review of the adult literature we provide a comprehensive overview of CN embryonic development and, by comparing cerebellar structures in various chordate clades, propose an interpretation of CN evolution. Despite their critical importance in cerebellar function, from a clinical perspective intriguingly few, if any, neurological disorders appear to primarily affect the CN. To highlight this curious anomaly, and encourage future nucleocentric interpretations, we build on our review to provide a brief overview of the various syndromes in which the CN are currently implicated. Finally, we summarize the specific perspectives that a nucleocentric view of the cerebellum brings, move major outstanding issues in CN biology to the limelight, and provide a roadmap to the key questions that need to be answered in order to create a comprehensive integrated model of CN structure, function, development, and evolution.

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

Ablation of Projection Glutamatergic Neurons in the Lateral Cerebellar Nuclei Alters Motor Coordination in Vglut2-Cre+ Mice.

Cerebellar nuclei (CN) constitute the sole cerebellar output to the rest of the central nervous system and play a central role in cerebellar circuits. Accumulating evidence from both human genetics and animal studies point to a crucial role for CN connectivity in neurological diseases, including several types of ataxia. However, because of the compact and restricted topography and close functional connection between the CN and the cerebellar cortex, identifying cerebellar deficits exclusively linked to CN is challenging. In this study, we have experimentally ablated large projection glutamatergic neurons of the lateral CN and evaluated the impact of this selective manipulation on motor coordination in mice. To this end, through stereotaxic surgery, we injected the lateral CN of Vglut2-Cre+ mice with an adeno-associated virus (AAV) encoding a Cre-dependent diphtheria toxin receptor (DTR), followed by an intraperitoneal injection of diphtheria toxin (DT) to ablate the glutamatergic neurons of the lateral nucleus. Double immunostaining of cerebellar sections with anti-SMI32 and -GFP antibodies revealed GFP expression and provided evidence of SMI32+ neuron degeneration at the site of AAV injection in the lateral nucleus of Vglut2-Cre+ mice. No changes were observed in Vglut2-Cre negative mice. Analysis of motor coordination by rotarod test indicated that the latency to fall was significantly different before and after AAV/DT injection in the Vglut2-Cre+ group. Elapsed time and number of steps in the beam walking test were significantly higher in AAV/DT injected Vglut2-Cre+ AAV/DT mice compared to controls. We demonstrate for the first time that partial degeneration of glutamatergic neurons in the lateral CN is sufficient to induce an ataxic phenotype.

The First 50 Years of Postnatal Neurogenesis in the Cerebellum: a Long Journey Across Phenomena, Mechanisms, and Human Disease.

The discovery by Altman and coworkers of adult-born microneurons in the olfactory bulb and dentate gyrus has triggered a long stream of studies and many attempts to harness adult neurogenesis, promote regeneration after injury, and contrast cognitive decline in the elderly. Likewise, the discovery of postnatal neurogenesis in the cerebellum has provided the framework for many subsequent molecular studies, including investigations of developmental processes and the assessment of GC progenitor (GCP) clonal expansion in the context of human disease. Here, I will briefly discuss some of the discoveries made in the field of cerebellar development over the years building upon the findings of Altman and his colleagues, touching upon signaling pathways that regulate granule cell neurogenesis and their involvement in developmental and neoplastic disorders of the cerebellum.

Upregulation of Neural Cell Adhesion Molecule 1 and Excessive Migration of Purkinje Cells in Cerebellar Cortex.

Purkinje cells (PCs) are large GABAergic projection neurons of the cerebellar cortex, endowed with elaborate dendrites that receive a multitude of excitatory inputs. Being the only efferent neuron of the cerebellar cortex, PCs project to cerebellar nuclei and control behaviors ranging from movement to cognition and social interaction. Neural cell adhesion molecule 1 (NCAM1) is widely expressed in the embryonic and postnatal development of the brain and plays essential roles in neuronal migration, axon pathfinding and synapse assembly. However, despite its high expression levels in cerebellum, little is known to date regarding the role(s) of NCAM1 in PCs development. Among other aspects, elucidating how the expression of NCAM1 in PCs could impact their postnatal migration would be a significant achievement. We analyzed the Acp2 mutant mouse (nax: naked and ataxia), which displays excessive PC migration into the molecular layer, and investigated how the excessive migration of PCs along Bergmann glia could correlate to NCAM1 expression pattern in early postnatal days. Our Western blot and RT-qPCR analysis of the whole cerebellum show that the protein and mRNA of NCAM1 in wild type are not different during PC dispersal from the cluster stage to monolayer formation. However, RT-qPCR analysis from FACS-based isolated PCs shows that Ncam1 is significantly upregulated when PCs fail to align and instead overmigrate into the molecular layer. Our results suggest two alternative interpretations: (1) NCAM1 promotes excessive PC migration along Bergmann glia, or (2) NCAM1 upregulation is an attempt to prevent PCs from invading the molecular layer. If the latter scenario proves true, NCAM1 may play a key role in PC monolayer formation.

ZFP423 regulates early patterning and multiciliogenesis in the hindbrain choroid plexus.

The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423, which encodes a Kruppel-type zinc-finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia/Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate, from which the IV ventricle ChP arises, and, later, in mesenchymal cells, which give rise to the stroma and leptomeninges. Mouse Zfp423 mutants display a marked reduction of the hindbrain ChP (hChP), which: (1) fails to express established markers of its secretory function and genes implicated in its development and maintenance (Lmx1a and Otx2); (2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); and (3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis (Gmnc). Our results propose that Zfp423 is a master gene and one of the earliest known determinants of hChP development.

Origins, Development, and Compartmentation of the Granule Cells of the Cerebellum.

Granule cells (GCs) are the most numerous cell type in the cerebellum and indeed, in the brain: at least 99% of all cerebellar neurons are granule cells. In this review article, we first consider the formation of the upper rhombic lip, from which all granule cell precursors arise, and the way by which the upper rhombic lip generates the external granular layer, a secondary germinal epithelium that serves to amplify the upper rhombic lip precursors. Next, we review the mechanisms by which postmitotic granule cells are generated in the external granular layer and migrate radially to settle in the granular layer. In addition, we review the evidence that far from being a homogeneous population, granule cells come in multiple phenotypes with distinct topographical distributions and consider ways in which the heterogeneity of granule cells might arise during development.

Taste bud formation depends on taste nerves.

It has been known for more than a century that, in adult vertebrates, the maintenance of taste buds depends on their afferent nerves. However, the initial formation of taste buds is proposed to be nerve-independent in amphibians, and evidence to the contrary in mammals has been endlessly debated, mostly due to indirect and incomplete means to impede innervation during the protracted perinatal period of taste bud differentiation. Here, by genetically ablating, in mice, all somatic (i.e. touch) or visceral (i.e. taste) neurons for the oral cavity, we show that the latter but not the former are absolutely required for the proper formation of their target organs, the taste buds.

Dynamic Expression and New Functions of Early B Cell Factor 2 in Cerebellar Development.

The collier/Olf1/EBF family genes encode helix-loop-helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls, fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.

An ancient role for collier/Olf/Ebf (COE)-type transcription factors in axial motor neuron development.

BACKGROUND: Mammalian motor circuits display remarkable cellular diversity with hundreds of motor neuron (MN) subtypes innervating hundreds of different muscles. Extensive research on limb muscle-innervating MNs has begun to elucidate the genetic programs that control animal locomotion. In striking contrast, the molecular mechanisms underlying the development of axial muscle-innervating MNs, which control breathing and spinal alignment, are poorly studied. METHODS: Our previous studies indicated that the function of the Collier/Olf/Ebf (COE) family of transcription factors (TFs) in axial MN development may be conserved from nematodes to simple chordates. Here, we examine the expression pattern of all four mouse COE family members (mEbf1-mEbf4) in spinal MNs and employ genetic approaches in both nematodes and mice to investigate their function in axial MN development. RESULTS: We report that mEbf1 and mEbf2 are expressed in distinct MN clusters (termed "columns") that innervate different axial muscles. Mouse Ebf1 is expressed in MNs of the hypaxial motor column (HMC), which is necessary for breathing, while mEbf2 is expressed in MNs of the medial motor column (MMC) that control spinal alignment. Our characterization of Ebf2 knock-out mice uncovered a requirement for Ebf2 in the differentiation program of a subset of MMC MNs and revealed for the first time molecular diversity within MMC neurons. Intriguingly, transgenic expression of mEbf1 or mEbf2 can rescue axial MN differentiation and locomotory defects in nematodes (Caenorhabditis elegans) lacking unc-3, the sole C. elegans ortholog of the COE family, suggesting functional conservation among mEbf1, mEbf2 and nematode UNC-3. CONCLUSIONS: These findings support the hypothesis that genetic programs controlling axial MN development are deeply conserved across species, and further advance our understanding of such programs by revealing an essential role for Ebf2 in mouse axial MNs. Because human mutations in COE orthologs lead to neurodevelopmental disorders characterized by motor developmental delay, our findings may advance our understanding of these human conditions.

Zfp423/ZNF423 regulates cell cycle progression, the mode of cell division and the DNA-damage response in Purkinje neuron progenitors.

The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.

An intersectional gene regulatory strategy defines subclass diversity of C. elegans motor neurons.

A core principle of nervous system organization is the diversification of neuron classes into subclasses that share large sets of features but differ in select traits. We describe here a molecular mechanism necessary for motor neurons to acquire subclass-specific traits in the nematode Caenorhabditis elegans. Cholinergic motor neuron classes of the ventral nerve cord can be subdivided into subclasses along the anterior-posterior (A-P) axis based on synaptic connectivity patterns and molecular features. The conserved COE-type terminal selector UNC-3 not only controls the expression of traits shared by all members of a neuron class, but is also required for subclass-specific traits expressed along the A-P axis. UNC-3, which is not regionally restricted, requires region-specific cofactors in the form of Hox proteins to co-activate subclass-specific effector genes in post-mitotic motor neurons. This intersectional gene regulatory principle for neuronal subclass diversification may be conserved from nematodes to mice.

The Transcription Factors EBF1 and EBF2 Are Positive Regulators of Myelination in Schwann Cells.

Myelin formation by Schwann cells is tightly controlled by multiple pathways and regulatory molecules. The Ebf2 gene, belonging to the Ebf family of transcription factors regulating cell development and differentiation, is expressed in Schwann cells, and Ebf2 knockout mice show peripheral nerve defects. We also found that Ebf1 is expressed in Schwann cells. To investigate Ebf function in myelination, we silenced Ebf genes in myelinating dorsal root ganglia cultures. Combined downregulation of Ebf genes leads to a severe impairment of myelin formation that is completely rescued by their specific overexpression, suggesting that the expression level of Ebf genes strongly influences axon myelination. In addition, by profiling Ebf target genes, we found several transcripts belonging to pathways actively involved in peripheral myelination, including Gliomedin, a gene with a role in the formation of the nodes of Ranvier and recently implicated in the pathogenesis of the nodo-paranodopathies. Our results suggest that Ebf genes act as positive regulators of myelination and directly regulate the promoter of Gliomedin.

Layer 4 pyramidal neurons exhibit robust dendritic spine plasticity in vivo after input deprivation.

Pyramidal neurons in layers 2/3 and 5 of primary somatosensory cortex (S1) exhibit somewhat modest synaptic plasticity after whisker input deprivation. Whether neurons involved at earlier steps of sensory processing show more or less plasticity has not yet been examined. Here, we used longitudinal in vivo two-photon microscopy to investigate dendritic spine dynamics in apical tufts of GFP-expressing layer 4 (L4) pyramidal neurons of the vibrissal (barrel) S1 after unilateral whisker trimming. First, we characterize the molecular, anatomical, and electrophysiological properties of identified L4 neurons in Ebf2-Cre transgenic mice. Next, we show that input deprivation results in a substantial (∼50%) increase in the rate of dendritic spine loss, acutely (4-8 d) after whisker trimming. This robust synaptic plasticity in L4 suggests that primary thalamic recipient pyramidal neurons in S1 may be particularly sensitive to changes in sensory experience. Ebf2-Cre mice thus provide a useful tool for future assessment of initial steps of sensory processing in S1.

The gene encoding the mouse contactin-1 axonal glycoprotein is regulated by the collier/Olf1/EBF family early B-Cell factor 2 transcription factor.

The Contactin-1 axonal glycoprotein (formerly F3/Contactin) plays a relevant role in cerebellar ontogenesis, as shown in Contactin-1 KO-mice and in transgenic mice misexpressing the corresponding cDNA from a heterologous promoter. Likewise, null mutant mice for the Collier/Olf1/Early B-cell family transcription factor EBF2, in which Purkinje neuron development is primarily affected, exhibit abnormalities in cerebellar corticogenesis. Here, to evaluate the contribution to the Ebf2 null phenotype of changes in the profile of Contactin-1, we study its expression in Ebf2 null mice. In addition, we explore the activation profile of the Cntn1 gene promoter upon transferring the Ebf2 mutation to transgenic mice expressing an enhanced green fluorescent protein reporter under control of Cntn1 gene regulatory sequences. In Ebf2 null mice, Contactin-1 protein expression and Cntn1 gene promoter activity are both downregulated during embryonic and early postnatal cerebellar development, both in the rostral and caudal folia, while in the latter an upregulation is observed at postnatal day 8. In vitro, vectors driving EBF1,2,3 transcription factors from a cytomegalovirus (CMV) promoter transactivate a Cntn1-Choline acetyltransferse (CAT) promoter-reporter construct in cotransfection assays and, accordingly, by chromatin immunoprecipitation, we show that the Cntn1 gene 5' flanking region is bound by the EBF2 transcription factor, consistent with the evidence that this region bears the cognate deoxyribonucleic acid (DNA) consensus sequences. These data indicate that Contactin-1 expression is dependent upon EBF factors, suggesting that the Cntn1 gene belongs to the expanding regulatory cascade driven by these transcriptional regulators so that changes in its activation may contribute to the phenotype of Ebf2 null mutant mice.

A conditional transgenic reporter of presynaptic terminals reveals novel features of the mouse corticospinal tract.

In many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), synaptic alterations precede the demise of the neuronal cell, making synapses a useful vantage point from which to monitor the onset and progression of clinical signs and pathological changes. While murine models of ALS display many features in common with the clinical picture observed in patients, corticospinal tract (CST) involvement is usually less severe in mice than the picture observed in humans. In this paper we describe the characterization of a new conditional transgenic line obtained by targeted integration of a GFP-VAMP2 fusion gene into the Rosa26 locus, and devised to permit the detection of genetically defined presynaptic terminals in wild type mice and murine models of neural disorders. This reporter molecule is selectively enriched in presynaptic boutons, significantly reducing the background signal produced by fibers of passage. The specific features of this reporter line allow us to strongly support the view that murine CST terminals give rise to very few direct contacts with spinal motor neurons. Moreover, the evidence described here reveals the existence of previously uncharacterized, putative direct connections between CST presynaptic boutons and Renshaw neurons in the spinal cord. These results constitute a proof of concept for the potential application of this indicator line to morphological analyses of wild type and diseased synapses.

Rac1 and rac3 GTPases control synergistically the development of cortical and hippocampal GABAergic interneurons.

The intracellular mechanisms driving postmitotic development of cortical γ-aminobutyric acid (GABA)ergic interneurons are poorly understood. We have addressed the function of Rac GTPases in cortical and hippocampal interneuron development. Developing neurons express both Rac1 and Rac3. Previous work has shown that Rac1 ablation does not affect the development of migrating cortical interneurons. Analysis of mice with double deletion of Rac1 and Rac3 shows that these GTPases are required during postmitotic interneuron development. The number of parvalbumin-positive cells was affected in the hippocampus and cortex of double knockout mice. Rac depletion also influences the maturation of interneurons that reach their destination, with reduction of inhibitory synapses in both hippocampal CA1 and cortical pyramidal cells. The decreased number of cortical migrating interneurons and their altered morphology indicate a role of Rac1 and Rac3 in regulating the motility of cortical interneurons, thus interfering with their final localization. While electrophysiological passive and active properties of pyramidal neurons including membrane capacity, resting potential, and spike amplitude and duration were normal, these cells showed reduced spontaneous inhibitory currents and increased excitability. Our results show that Rac1 and Rac3 contribute synergistically to postmitotic development of specific populations of GABAergic cells, suggesting that these proteins regulate their migration and differentiation.

Modeling human brain development with cerebral organoids.

The recent discovery of a new three-dimensional culture system for the derivation of cerebral organoids from human induced pluripotent stem cells provides developmental neurobiologists with the first example of a three-dimensional framework for the study of human brain development. This innovative approach permits the in vitro assembly of a human embryonic brain rudiment that recapitulates the developing human cerebrum. Organoids contain progenitor populations that develop to yield mature cortical neuron subtypes, potentially allowing investigators to study complex brain diseases that lack appropriate animal models.

Neural stem cells engrafted in the adult brain fuse with endogenous neurons.

Neural stem cells (NSCs) have become promising tools for basic research and regenerative medicine. Intracerebral transplantation studies have suggested that these cells may be able to adopt neuronal phenotypes typical of their engraftment site and to establish appropriate connections in the recipient circuitries. Here, we examined the in vivo neurogenic competence of well-characterized NSC lines subjected to in vitro priming and subsequent implantation into the adult intact mouse brain. Upon implantation into the hippocampus and, less frequently, in the striatum and in the cerebral cortex, numerous green fluorescent protein (GFP)-tagged cells acquired differentiated features indistinguishable from resident neurons. Upon closer examination, however, we found that this outcome resulted from fusion of donor cells with local neuronal elements generating long-term persistent GFP(+) neuronal hybrids. This fusogenic behavior of NSCs was unexpected and also observed in coculture with E18 hippocampal immature neural cells, but not with microglia or astrocytes. Similar findings were consistently obtained with different NSC lines, mouse recipients, and donor cell-labeling methods. The frequent and cell type-specific fusion of donor NSCs with host neurons highlights a previously underestimated biological property of the nervous tissue that might prove profitable for basic and therapeutically oriented studies.