Karl Schilling

Astrocyte dysfunction in neurological disorders: a molecular perspective

Recent work on glial cell physiology has revealed that glial cells, and astrocytes in particular, are much more actively involved in brain information processing than previously thought. This finding has stimulated the view that the active brain should no longer be regarded solely as a network of neuronal contacts, but instead as a circuit of integrated, interactive neurons and glial cells. Consequently, glial cells could also have as yet unexpected roles in the diseased brain. An improved understanding of astrocyte biology and heterogeneity and the involvement of these cells in pathogenesis offers the potential for developing novel strategies to treat neurological disorders.

Developmental and cell type-specific expression of the neuronal marker NeuN in the murine cerebellum.

NeuN is a 46/48‐kD nuclear protein antigen used widely to identify postmitotic neurons in both research and diagnostics. It is expressed by neurons throughout the nervous system of a variety of species, including birds, rodents, and man (Mullen et al. [1992] Development 116:201–211). When we sought to use NeuN to follow the developmental progression of murine cerebellar interneurons, we observed that expression of this antigen in the cerebellum was restricted to granule neurons and a small population of cells present in the lower molecular layer of the adult cerebellum. In an attempt to identify these cells, we combined immunostaining for NeuN with a panel of cell type‐specific markers to unambiguously identify neurons that express NeuN in the adult and developing cerebellum. In contrast to postmitotic granule neurons, NeuN was not expressed by any other immunocytochemically identified cerebellar interneurons, which comprised basket and stellate cells, Golgi neurons, unipolar brush cells, and Lugaro cells. NeuN‐positive cells in the molecular layer failed to express any cell type‐specific markers tested. They may represent ectopic granule cells; alternatively, they may represent a hitherto unknown population of cerebellar cells. In vitro experiments suggest that NeuN expression is related closely to granule cell axogenesis. This approach also revealed that the level of NeuN expression could be modulated by chronically depolarizing these cells. Thus, whereas NeuN expression per se is a reliable marker of proliferative capacity, levels of NeuN expression may also be indicative of the physiological status of a postmitotic neuron.

Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization

NeuN (neuronal nuclei) is an antigen used widely in research and diagnostics to identify postmitotic neurons. The present study aims at an initial understanding of the molecular nature and functional significance of this as yet ill‐defined antigen. Using isoelectric focusing, both the 46‐ and 48‐kDa isoforms of NeuN can be separated in multiple spots spanning a pH range of 8–10.5, suggesting that they might be phosphorylated. Enzymatic dephosphorylation abolishes NeuN immunoreactivity, confirming that NeuN is indeed a phosphoprotein, and establishing that binding of the defining antibody depends on its state of phosphorylation. Combined biochemical and immunohistochemical analysis show that both the 46‐ and the 48‐kDa NeuN isoforms can be localized to the cell nucleus as well as in the neuronal cytoplasm. Their relative concentration in these compartments is distinct, however, with the 48‐kDa isoform being the predominant isoform in the cytoplasm. Within the nucleus, NeuN is found preferentially in areas of low chromatin density and virtually excluded from areas containing densely packed DNA. The present identification of multiple differentially phosphorylated isoforms of NeuN, together with recent reports on the dependence of NeuN immunoreactivity levels on a variety of physiologic or pathologic signals, suggests a previously unappreciated level of complexity in the regulation of this enigmatic, neuron‐specific antigen.

Impairment of LTD and cerebellar learning by Purkinje cell–specific ablation of cGMP-dependent protein kinase I

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.

Insulin-Like Growth Factor II Is Involved in the Proliferation Control of Medulloblastoma and Its Cerebellar Precursor Cells

Medulloblastomas (MBs), the most frequent malignant brain tumors of childhood, presumably originate from cerebellar neural precursor cells. An essential fetal mitogen involved in the pathogenesis of different embryonal tumors is insulin-like growth factor II (IGF-II). We screened human MB biopsies of the classic (CMB) and desmoplastic (DMB) variants for IGF2 transcripts of the four IGF2 promoters. We found IGF2 transcription from the imprinted promoter P3 to be significantly increased in the desmoplastic variant compared to the classic subgroup. This was not a result of loss of imprinting of IGF2 in desmoplastic tumors. We next examined the interaction of IGF-II and Sonic hedgehog (Shh), which serves as a critical mitogen for cerebellar granule cell precursors (GCPs) in the external granule cell layer from which DMBs are believed to originate. Mutations of genes encoding components of the Shh-Patched signaling pathway occur in approximately 50% of DMBs. To analyze the effects of IGF-II on Hedgehog signaling, we cultured murine GCP and human MB cells in the presence of Shh and Igf-II. In GCPs, a synergistic effect of Shh and Igf-II on proliferation and gli1 and cyclin D1 mRNA expression was found. Igf-II, but not Shh, induced phosphorylation of Akt and its downstream target Gsk-3beta. In six of nine human MB cell lines IGF-II displayed a growth-promoting effect that was mediated mainly through the IGF-I receptor. Together, our data point to an important role of IGF-II for the proliferation control of both cerebellar neural precursors and MB cells.

Postnatal development of the murine cerebellar cortex: formation and early dispersal of basket, stellate and Golgi neurons.

The cerebellar cortex consists of a small set of neuronal cell types interconnected in a highly stereotyped way. While the development of cerebellar cortical projection neurons, i.e. Purkinje cells, and that of granule cells has been elucidated in considerable detail, that of cerebellar cortical inhibitory interneurons is still rather fragmentarily understood. Here, we use mice expressing green fluorescent protein (GFP) from the Pax2 locus to analyse the ontogenesis of these cells. Numbers of Pax2‐positive inhibitory interneuronal precursors increase following a classical sigmoidal growth curve to yield a total of some 905.000 ± 77.000 cells. Maximal cell increase occurs at about postnatal day (P)5.4, and some 75% of all inhibitory interneurons are generated prior to P7. Conjoint analysis of the developmental accruement of Pax2‐GFP‐positive cells and their cell cycle distribution reveals that, at least at P0 and P3, the numerical increase of these cells results primarily from proliferation of a Pax2‐negative precursor population and suggests that Pax2 expression begins at or around the final mitosis. Following their terminal mitosis, inhibitory cerebellar cortical interneurons go through a protracted quiescent phase in which they maintain expression of the cell cycle marker Ki‐67. During this phase, they translocate into the nascent molecular layer, where they stall next to premigratory granule cell precursors without penetrating this population of cells. These observations provide a quantitative description of cerebellar cortical inhibitory interneuron genesis and early differentiation, and define Pax2 as a marker expressed in basket and stellate cells, from around their final mitosis to their incipient histogenetic integration.

Mice deficient in the chemokine receptor CXCR4 exhibit impaired limb innervation and myogenesis.

The chemokine CXCL12/SDF-1 and its receptor CXCR4 regulate the development and the function of the hematopoietic system and control morphogenesis of distinct brain areas. Here, we demonstrate that inactivation of CXCR4 results in a massive loss of spinal cord motoneurons and dorsal root ganglion neurons and, subsequently, in a reduced innervation of the developing mouse fore- and hindlimbs. However, only the death of sensory neurons seems to be a direct consequence of receptor inactivation as suggested by the observations that DRG neurons, but not motoneurons, of wild-type animals express CXCR4 and respond to CXCL12 with an increase in cell survival. In contrast, the increased death of motoneurons in CXCR4-deficient animals seems to result from impaired limb myogenesis and a subsequent loss of muscle-derived neurotrophic support. In summary, our findings unravel a previously unrecognized complex role of CXCL12/CXCR4 in the control of limb neuromuscular development.

Laminar Fate and Phenotype Specification of Cerebellar GABAergic Interneurons

In most CNS regions, the variety of inhibitory interneurons originates from separate pools of progenitors residing in discrete germinal domains, where they become committed to specific phenotypes and positions during their last mitosis. We show here that GABAergic interneurons of the rodent cerebellum are generated through a different mechanism. Progenitors for these interneurons delaminate from the ventricular neuroepithelium of the embryonic cerebellar primordium and continue to proliferate in the prospective white matter during late embryonic and postnatal development. Young postmitotic interneurons do not migrate immediately to their final destination, but remain in the prospective white matter for several days. The different interneuron categories are produced according to a continuous inside-out positional sequence, and cell identity and laminar placement in the cerebellar cortex are temporally related to birth date. However, terminal commitment does not occur while precursors are still proliferating, and postmitotic cells heterochronically transplanted to developing cerebella consistently adopt host-specific phenotypes and positions. However, solid grafts of prospective white matter implanted into the adult cerebellum, when interneuron genesis has ceased, produce interneuron types characteristic of the donor age. Therefore, specification of cerebellar GABAergic interneurons occurs through a hitherto unknown process, in which postmitotic neurons maintain broad developmental potentialities and their phenotypic choices are dictated by instructive cues provided by the microenvironment of the prospective white matter. Whereas in most CNS regions the repertoire of inhibitory interneurons is produced by recruiting precursors from different origins, in the cerebellum it is achieved by creating phenotypic diversity from a single source.

The SDF‐1/CXCR4 pathway and the development of the cerebellar system

Mice deficient for the chemokine receptor CXCR4 show premature translocation of granule cell neuroblasts from their germinal zone into the nascent cerebellum [Y.‐R. Zuo et al. (1998)Nature, 393, 595–599]. Here, we used CXCR4‐null mice to analyse the early development of cerebellar cortical inhibitory interneurons and pontine neurons which, in the adult, are synaptically integrated with granule cells. Cortical inhibitory interneuronal precursors normally invade the cerebellar anlage of CXCR4‐deficient mice, but their dispersal is impeded by dislocated foci of proliferating granule cells, from which they are excluded. This is reminiscent of the strict exclusion of inhibitory interneuronal precursors from the superficial external granule cell layer. As inhibitory interneuronal precursors readily mingle with post‐mitotic granule cells both in wild‐type and CXCR4‐null mice, these findings indicate that the developmentally regulated interactions between granule and inhibitory interneuronal precursors are independent of SDF‐1/CXCR4 signalling. In contrast, the transit of pontine neurons from the rhombic lip through the anterior extramural stream to the basilar pons is disrupted in CXCR4‐deficient animals. Migrating pontine neurons express CXCR4, and in CXCR4‐null animals these cells are found displaced deep into the brainstem. Consequently, nascent pontine nuclei in CXCR4‐deficient animals are hypoplastic. Moreover, they fail to express plexin D1, suggesting that SDF‐1/CXCR4 signalling may also impinge on axon guidance critical to the orderly formation of granule cell mossy fibre afferents.

Physiological purkinje cell death is spatiotemporally organized in the developing mouse cerebellum.

Physiological cell death is crucial for matching defined cellular populations within the central nervous system. Whereas the time course of developmental cell death in the central nervous system is well analyzed, information about its precise spatial patterning is scarce. Yet, the latter one is needed to appraise its contribution to circuit formation and refinement. Here, we document that during normal cerebellar development, dying Purkinje cells were highly localized within the vermal midline and in a lobule specific, parasagittal pattern along the whole mediolateral axis. In addition, single hot spots of cell death localized to the caudal declive and ventral lobule IX within the posterolateral fissure. These hot spots of dying Purkinje cells partly overlapped with gaps within the Purkinje cell layer which supports the classification of different gaps based on histological and molecular criteria, i.e., midline gap, patchy gaps, and raphes. Areas characterized by a high incidence of Purkinje cell death and gaps colocalize with known molecular and functional boundaries within the cerebellar cortex. Physiological cell death can thus be considered to serve as an important regulator of cerebellar histogenesis.