Roy V. Sillitoe

Patterned Purkinje cell degeneration in mouse models of Niemann‐Pick type C disease

Niemann Pick disease type C1 (NPC1) is an inherited, autosomal recessive, lipid‐storage disorder with major neurological involvement. Purkinje cell death is a prominent feature of the neuropathology of NPC. We have investigated Purkinje cell death in two murine models of NPC1, BALB/c npcnih and C57BLKS/J spm. In both cases, extensive Purkinje cell death was found in the cerebellum. The pattern of Purkinje cell death is complex. First, zebrin II‐negative Purkinje cells disappear, to leave survivors aligned in stripes that closely resemble the pattern revealed by using zebrin II immunocytochemistry. Subsequently, as the disease progresses, additional Purkinje cells die. At the terminal stages of the disease, the surviving Purkinje cells are concentrated in lobules IX and X of the posterior lobe vermis. Purkinje cell degeneration is accompanied by the ectopic expression of tyrosine hydroxylase and the small heat shock protein HSP25, both associated preferentially with the surviving cells. The pattern of cell death thus reflects the fundamental compartmentation of the cerebellum into zones and stripes. J. Comp. Neurol. 456:279–291, 2003.

Whole-mount Immunohistochemistry: A High-throughput Screen for Patterning Defects in the Mouse Cerebellum

Large-scale mouse mutagenesis experiments now under way require appropriate screening methods. An important class of potential mutants comprises those with defects in the development of normal cerebellar patterning. Cerebellar defects are likely to be identified often because they typically result in ataxia. Immunohistochemistry (IHC) is commonly used to reveal cerebellar organization. In particular, the antigen zebrin II (=aldolase C), expressed by stripes of Purkinje cells, has been valuable in revealing cerebellar pattern abnormalities. The development of whole-mount procedures in Drosophila, chick, and Xenopus embryos allows complex patterns to be studied in situ while preserving the integrity of the structure. By combining procedures originally designed for embryonic and early postnatal tissue analyses, we have developed a whole-mount IHC protocol using anti-zebrin II, which reveals the complex topography of Purkinje cells in the adult mouse cerebellum. Furthermore, the procedure is effective with a number of other antigens and works well on both perfusion-fixed and immersion-fixed tissue. By use of this approach, normal adult murine cerebellar topography and patterning defects caused by mutation can be studied without the need for three-dimensional reconstruction.

Conservation of the architecture of the anterior lobe vermis of the cerebellum across mammalian species.

Publisher Summary Within the mammalia, the morphology of the cerebellum varies dramatically both in form and size; however, despite numerous anatomical differences, the mammalian cerebellum is classified into 10 lobules, and there is a more fundamental compartmentation of the cerebellum into transverse zones and parasagittal stripes. This chapter focuses on the conserved nature of cerebellar compartmentation, with a specific focus on the anterior vermis, which has a very characteristic appearance in rats and mice. The highly conserved antigen zebrin II has proven useful in comparative studies of cerebellar compartmentation. The results presented in the chapter suggest that regardless of cerebellar size, a characteristic set of stripes is present in the anterior lobe vermis of the mammalian cerebellum, and the topography of zebrin II expression is well conserved. The restriction of the zebrin II epitope to Purkinje cells and the molecular weight of the zebrin II/aldolase C antigen are highly conserved from fish to primates, except amphibia. Differences in the molecular architecture of the cerebellum among phylogenetic groups of mammals are minor, but consistent, such as the pattern of zebrin II expression in the anterior vermis of ferrets resembles that of cats. Several investigations have shown evidence that stripes are determined early in development and are not a response to usage, and several hundred distinct cerebellar modules have been identified based upon expression patterns and afferent terminal fields; however, the function of stripes in the adult cerebellum is a matter of speculation.

Redefining the cerebellar cortex as an assembly of non-uniform Purkinje cell microcircuits

The adult mammalian cerebellar cortex is generally assumed to have a uniform cytoarchitecture. Differences in cerebellar function are thought to arise primarily through distinct patterns of input and output connectivity rather than as a result of variations in cortical microcircuitry. However, evidence from anatomical, physiological and genetic studies is increasingly challenging this orthodoxy, and there are now various lines of evidence indicating that the cerebellar cortex is not uniform. Here, we develop the hypothesis that regional differences in properties of cerebellar cortical microcircuits lead to important differences in information processing.

New roles for the cerebellum in health and disease

The cerebellum has a well-established role in maintaining motor coordination and studies of cerebellar learning suggest that it does this by recognizing neural patterns, which it uses to predict optimal movements. Serious damage to the cerebellum impairs this learning and results in a set of motor disturbances called ataxia. However, recent work implicates the cerebellum in cognition and emotion, and it has been argued that cerebellar dysfunction contributes to non-motor conditions such as autism spectrum disorders (ASD). Based on human and animal model studies, two major questions arise. Does the cerebellum contribute to non-motor as well as motor diseases, and if so, how does altering its function contribute to such diverse symptoms? The architecture and connectivity of cerebellar circuits may hold the answers to these questions. An emerging view is that cerebellar defects can trigger motor and non-motor neurological conditions by globally influencing brain function. Furthermore, during development cerebellar circuits may play a role in wiring events necessary for higher cognitive functions such as social behavior and language. We discuss genetic, electrophysiological, and behavioral evidence that implicates Purkinje cell dysfunction as a major culprit in several diseases and offer a hypothesis as to how canonical cerebellar functions might be at fault in non-motor as well as motor diseases.

Engrailed Homeobox Genes Regulate Establishment of the Cerebellar Afferent Circuit Map

The spatial organization of the cerebellar afferent map has remarkable correspondence to two aspects of intrinsic patterning within the cerebellum embodied by a series of lobules and Purkinje cell (PC)-striped gene expression. Using male and female mice, we tested whether the Engrailed (En) homeobox genes are a common genetic substrate regulating all three systems, since they are expressed in spatially restricted domains within the cerebellum and are critical for patterning PC gene expression and foliation. Indeed, we discovered that En1/2 are necessary for the precise targeting of mossy fibers to distinct lobules, as well as their subsequent resolution into discrete parasagittal bands. Moreover, each En gene coordinately regulates afferent targeting and the striped pattern of PC protein expression (e.g., ZebrinII/AldolaseC) independent of regulating foliation. We further found that En1/2, rather than the presence of a full complement of lobules, are critical for generating PC protein stripes and mossy fiber bands, and that PC striped gene expression is determined before afferent banding. Thus, the En transcription factors not only regulate cerebellum circuit topography, but they also link afferent and efferent neurons precisely enough that alterations in PC protein expression can be used as a read out for underlying defects in circuitry. In summary, our data suggest that En1/2 are master regulators of three-dimensional organization of the cerebellum and coordinately regulate morphology, patterned gene expression, and afferent topography.

Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum.

Underlying the seemingly uniform cellular composition of the adult mammalian cerebellum (Cb) are striking parasagittal stripes of gene expression along the medial-lateral (ML) axis that are organized with respect to the lobules that divide the Cb along the anterior–posterior (AP) axis. Although there is a clear correlation between the organization of gene expression stripes and Cb activity patterns, little is known about the genetic pathways that determine the intrinsic stripe molecular code. Here we establish that ML molecular code patterning is highly dependent on two homeobox transcription factors, Engrailed1 (En1) and En2, both of which are also required for patterning the lobules. Gene expression analysis of an allelic series of En1/2 mutant mice that have an intact Purkinje cell layer revealed severe patterning defects using three known components of the ML molecular code and a new marker of Hsp25 negative stripes (Neurofilament heavy chain, Nfh). Importantly, the complementary expression of ZebrinII/PhospholipaseCβ4 and Hsp25/Nfh changes in unison in each mutant. Furthermore, each En gene has unique as well as overlapping functions in patterning the ML molecular code and each En protein has dominant functions in different AP domains (subsets of lobules). Remarkably, in En1/2 mutants with almost normal foliation, ML molecular code patterning is severely disrupted. Thus, independent mechanisms that use En1/2 must pattern foliation and spatial gene expression separately. Our studies reveal that En1/2 are fundamental components of the genetic pathways that pattern the two intersecting coordinate systems of the Cb, morphological divisions and the molecular code.

Consensus Paper: Cerebellar Development.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal–lysosomal dysfunction

Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium–hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium–hydrogen exchanger 6 deficiency would most likely disrupt the endosomal–lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium–hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for β-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium–hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal–lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert could provide a unified explanation for the cellular and clinical phenotypes in humans with SLC9A6 mutations.

Development of the cerebellum: from gene expression patterns to circuit maps.

The internal structure of the cerebellum reflects an intriguing paradox; its cytoarchitecture is relatively simple and repeated throughout, yet the connections between its neurons are wired into a complex array of gene expression domains and functional circuits. The developmental mechanisms that coordinate the establishment of cerebellar structure and circuitry provide a powerful model for understanding how functional brain networks are formed. Two primary germinal zones generate the cells that make up the cerebellum. Each zone expresses a specific set of genes that establish the cell lineages within the cerebellar anlage. Then, cohorts of differentiated projection neurons and interneuron progenitors migrate into the developing cerebellum. Thereafter, a number of remarkable patterning events occur including transformation of the smooth cerebellar surface into an intricately patterned series of folds, formation of three distinct cellular layers, and the demarcation of parasagittal gene expression domains. Together, these structural and molecular organizations are thought to support the proper connectivity between incoming afferent projections and their target cells. After birth, genetic programs and neural activity repattern synaptic connections into topographic neural networks called modules, which are organized around a longitudinal zone plan and are defined by their molecular, anatomic, and functional properties. WIREs Dev Biol 2013, 2:149–164. doi: 10.1002/wdev.65