Mature Cerebellum Research Articles

Subcellular Distribution of Patched and Smoothened in the Cerebellar Neurons

The Sonic hedgehog (Shh) signaling pathway carries out a wide range of biological functions such as patterning of the embryonic neural tube and expansion of cerebellar granule cell precursors. We previously have found that the Shh signaling receptors, Patched1 (Ptch1) and Smoothened (Smo), are expressed in hippocampal neurons of developing and adult rats, suggesting the continued presence of Shh signaling in postmitotic, differentiated neurons. Here, we report that Ptch1 and Smo are present in the processes and growth cones of immature neurons in the developing cerebellum, and that, in the mature cerebellum, Ptch1 and Smo are expressed by several types of neurons including Purkinje cells, granule cells, and interneurons. Within these neurons, Ptch1 and Smo are predominantly localized in the postsynaptic side of the synapses, a distribution pattern similar to that found in hippocampal neurons. Our findings provide morphological evidence that Shh signaling events are not confined to neuronal precursors and are likely to have ongoing roles within the postmitotic neurons of the developing and adult cerebellum.

Prostaglandin E2 is an endogenous modulator of cerebellar development and complex behavior during a sensitive postnatal period

Prostaglandins are lipid-derived molecules that mediate the generation of fever in the central nervous system. In addition to their proinflammatory role, prostaglandins also impact neuronal development and synaptic plasticity, sometimes in a sex-specific manner. The cerebellum has a high expression of prostaglandin receptors during development, but the role that these molecules play during normal cerebellar maturation is unknown. We demonstrate here that disrupting prostaglandin synthesis with cyclo-oxygenase inhibitors during a time-sensitive window in early postnatal life alters cerebellar Purkinje cell development in rats, resulting in initially increased dendritic growth in both sexes. We show that this results in later cerebellar atrophy in males only, resulting in a sex-specific loss of cerebellar volume. Further, although performance in motor tasks is spared, social interaction and the sensory threshold are altered in males developmentally exposed to cyclo-oxygenase inhibitors. This work demonstrates a previously unknown role for prostaglandins in cerebellar development and emphasizes the role that the cerebellum plays outside motor tasks, in cognitive and sensory domains that may help to explain its connection to complex neurodevelopmental disorders such as autism.

Neuronal Profilin Isoforms Are Addressed by Different Signalling Pathways

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

Allelic Variation Within the Putative Autism Spectrum Disorder Risk Gene Homeobox A1 and Cerebellar Maturation in Typically Developing Children and Adolescents

Homeobox A1 (HOXA1) has been proposed as a candidate gene for autism spectrum disorder (ASD) as it regulates embryological patterning of hind-brain structures implicated in autism neurobiology. In line with this notion, a nonsynonymous single nucleotide polymorphism within a highly conserved domain of HOXA1--A218G (rs10951154)--has been linked to both ASD risk, and cross-sectional differences in superior posterior lobar cerebellar anatomy in late adulthood. Despite evidence for early onset and developmentally dynamic cerebellar involvement in ASD, little is known of the relationship between A218G genotype and maturation of the cerebellum over early development. We addressed this issue using 296 longitudinally acquired structural magnetic resonance imaging brain scans from 116 healthy individuals between 5 and 23 years of age. Mixed models were used to compare the relationship between age and semi-automated measures of cerebellar volume in A-homozygotes (AA) and carriers of the G allele (Gcar). Total cerebellar volume increased between ages of 5 and 23 years in both groups. However, this was accelerated in the Gcar relative to the AA group (Genotype-by-age interaction term, P = 0.03), and driven by genotype-dependent differences in the rate of bilateral superior posterior lobar volume change with age (P = 0.002). Resultantly, although superior posterior lobar volume did not differ significantly between genotype groups at age 5 (P = 0.9), by age 23 it was 12% greater in Gcar than AA (P = 0.002). Our results suggest that common genetic variation within this putative ASD risk gene has the capacity to modify the development of cerebellar systems implicated in ASD neurobiology.

Regional Cerebellar Volumes Predict Functional Outcome in Children with Cerebellar Malformations

The cerebellum has recently been recognized for its role in high-order functions, including cognition, language, and behavior. Recent studies have also begun to describe a functional topography of the mature cerebellum that includes organization on a mediolateral axis. However, no study to date has examined the relationship between regional cerebellar volume and developmental disabilities in children with cerebellar malformations. The objective of this study was to estimate the extent to which total and regional cerebellar volumes are associated with developmental disabilities in a cohort of children with cerebellar malformations. Children aged 1 to 6years with a diagnosis of cerebellar malformation underwent standardized outcome measures and quantitative magnetic resonance scanning. The cerebellum was parcellated into seven mediolateral zones (three for each hemisphere plus the vermis) for regional volume analysis. In children with cerebellar malformations, decreased total cerebellar volume was associated with delays in global development, expressive language, cognition, as well as gross and fine motor function. Decreased volume in the right lateral cerebellar hemisphere was related to impaired cognition, expressive language, and gross motor function. Additionally, reduced vermis volume was associated with impaired global development, cognition, expressive language, and gross and fine motor skills, as well as behavior problems and a higher rate of positive autism spectrum screening test. These results begin to define the structural topography of functional outcome in children with cerebellar malformations and should lead to greater accuracy of prognostication as well as timely early developmental interventions.

Responses of cerebellar Purkinje cells to conditioned stimulus change after acquisition of conditioned fear in goldfish

The changing pattern of ChE staining in the cerebellar cortex was examined in rats 0, 3, 7, 10, 15, 21, and 30 days old. For correlative purposes, Golgi-impregnated sections from rats of the same ages were also studied. The following conclusions were drawn about the mature cerebellum. (a) Most of the nerve cells of the cerebellar cortex (Purkinje, granule, basket, and stellate cells) are noncholinergic; the Golgi cells are cholinergic. (b) Of the noncholinergic neurons, the Purkinje cells are noncholinoceptive because the climbing fibers, parallel fibers, and the basket terminals are noncholinergic. (c) A large proportion of the mossy fibers that apparently originate directly or indirectly in the acousticovestibular system are cholinergic; the others are predominantly noncholinergic. (d) The granule cells are cholinoceptive. This is due to Golgi-cell terminals in the glomeruli in all lobules and to the additional cholinergic mossy fiber terminal in the lobules that receive acousticovestibular projection (nodulus, flocculus, part of uvula, tuber, folium, and lingula). (e) Available evidence in the literature about other neurochemical transmitters in the cerebellar cortex was considered. The following conclusions were drawn about the postnatal development of the “wiring pattern” of the cerebellar cortex. (a) In the newborn rat the only input to the Purkinje cell is by way of climbing fibers which contact their soma. (b) A few days after birth the cholinergic mossy fibers reach the zone of Purkinje cells. The transient ChE staining of Purkinje cells and the development of transient perisomatic processes suggest that mossy fibers may establish direct contact with the Purkinje cells during this period. (c) The cessation of Purkinje-cell staining during the second week, the descent of the precursors of granule cells from the external into the internal granular layer, and the increased staining of the glomeruli indicate that the mossy fibers establish contact with the granule cells.

Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 ( Ink4c ), a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.

Morphological development of dendritic spines on rat cerebellar Purkinje cells

The posterior cerebellum is strongly involved in motor coordination and its maturation parallels the development of motor control. Climbing and mossy fibers from the spinal cord and inferior olivary complex, respectively, provide excitatory afferents to cerebellar Purkinje neurons. From post-natal day 19 climbing fibers form synapses with thorn-like spines located on the lower primary and secondary dendrites of Purkinje cells. By contrast, mossy fibers transmit synaptic information to Purkinje cells trans-synaptically through granule cells. This communication occurs via excitatory synapses between the parallel fibers of granule cells and spines on the upper dendritic branchlets of Purkinje neurons that are first evident at post-natal day 21. Dendritic spines influence the transmission of synaptic information through plastic changes in their distribution, density and geometric shape, which may be related to cerebellar maturation. Thus, spine density and shape was studied in the upper dendritic branchlets of rat Purkinje cells, at post-natal days 21, 30 and 90. At 90 days the number of thin, mushroom and thorn-like spines was greater than at 21 and 30 days, while the filopodia, stubby and wide spines diminished. Thin and mushroom spines are associated with increased synaptic strength, suggesting more efficient transmission of synaptic impulses than stubby or wide spines. Hence, the changes found suggest that the development of motor control may be closely linked to the distinct developmental patterns of dendritic spines on Purkinje cells, which has important implications for future studies of cerebellar dysfunctions.

Expression of vascular endothelial growth factor receptor-3 mRNA in the developing rat cerebellum.

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, has recently been suggested to play an important role during neuronal development. To characterize its potential role in CNS ontogenesis, we investigated the spatiotemporal and cellular expression of VEGFR-3 in developing and mature rat cerebellum using in situ hybridization. VEGFR-3 expression appeared as early as E15, and was restricted to the ventricular zone of the cerebellar primordium, the germinative neuroepithelium, but was absent by E20. Instead, the expression area of VEGFR-3 in the cerebellum grew in parallel with cerebellar development. From E20 on, two populations of VEGFR-3-expressing cells can be clearly distinguished in the developing cerebellum: a population of differentiating and postmitotic neurons and the Bergmann glia. VEGFR-3 expression in neurons occurred during the period of neuronal differentiation, and increased with maturation. In particular, the expression of VEGFR-3 mRNA revealed different temporal patterns in different neuronal populations. Neurons generated early, Purkinje cells, and deep nuclear neurons expressed VEGFR-3 mRNA during late embryonic stages, whereas VEGFR-3 transcription in local interneurons appeared by P14 with weaker expression. In addition, Bergmann glia expressed VEGFR-3 throughout cerebellar maturation into adulthood. However, receptor expression was absent in the progenitors in the external granular layer and during further migration. The results of this study suggest that VEGFR-3 has even broader functions than previously thought, regulating both developmental processes and adult neuronal function in the cerebellum.

Purkinje Cell NMDA Receptors Assume a Key Role in Synaptic Gain Control in the Mature Cerebellum

A classic view in cerebellar physiology holds that Purkinje cells do not express functional NMDA receptors and that, therefore, postsynaptic NMDA receptors are not involved in the induction of long-term depression (LTD) at parallel fiber (PF) to Purkinje cell synapses. Recently, it has been demonstrated that functional NMDA receptors are postsynaptically expressed at climbing fiber (CF) to Purkinje cell synapses in mice, reaching full expression levels at ∼2 months after birth. Here, we show that in the mature mouse cerebellum LTD (induced by paired PF and CF activation), but not long-term potentiation (LTP; PF stimulation alone) at PF to Purkinje cell synapses is blocked by bath application of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV). A blockade of LTD, but not LTP, was also observed when the noncompetitive NMDA channel blocker MK-801 was added to the patch-pipette saline, suggesting that postsynaptically expressed NMDA receptors are required for LTD induction. Using confocal calcium imaging, we show that CF-evoked calcium transients in dendritic spines are reduced in the presence of D-APV. This observation confirms that NMDA receptor signaling occurs at CF synapses and suggests that NMDA receptor-mediated calcium transients at the CF input site might contribute to LTD induction. Finally, we performed dendritic patch-clamp recordings from rat Purkinje cells. Dendritically recorded CF responses were reduced when D-APV was bath applied. Together, these data suggest that the late developmental expression of postsynaptic NMDA receptors at CF synapses onto Purkinje cells is associated with a switch toward an NMDA receptor-dependent LTD induction mechanism.

Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia

The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we describe a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5-trisphosphate (IP(3) ) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber-Purkinje cell synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP(3) signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum.

Astroglial cells in the external granular layer are precursors of cerebellar granule neurons in neonates

It is well established that cerebellar granule cell precursors (GCPs) initially derive from progenitors in the rhombic lip of the embryonic cerebellar primordium. GCPs proliferate and migrate tangentially across the cerebellum to form the external granule cell layer (EGL) in late embryogenesis and early postnatal development. It is unclear whether GCPs are specified exclusively in the embryonic rhombic lip or whether their precursor persists in the neonate. Using transgenic mice expressing DsRed under the human glial fibrillary acidic protein ( hGFAP) promoter, we found 2 populations of DsRed + cells in the EGL in the first postnatal week defined by bright and faint DsRed-fluorescent signal. Bright DsRed + cells have a protein expression profile and electrophysiological characteristics typical of astrocytes, but faint DsRed + cells in the EGL and internal granule cell layer (IGL) express markers and physiological properties of immature neurons. To determine if these astroglial cells gave rise to GCPs, we genetically tagged them with EGFP or βgal reporter genes at postnatal day (P)3–P5 using a hGFAP promoter driven inducible Cre recombinase. We found that GFAP promoter + cells in the EGL are proliferative and express glial and neural stem cell markers. In addition, immature granule cells (GCs) en route to the IGL at P12 as well as GCs in the mature cerebellum, 30 days after recombination, express the reporter protein, suggesting that GFAP promoter + cells in the EGL generate a subset of granule cells. The identification of glial cells which function as neuronal progenitor cells profoundly impacts our understanding of cellular plasticity in the developing cerebellum.

Cerebellar Lingula Thickness as a Novel Risk Factor for Alcohol and Drug Abuse

The identification of developmental risk factors for alco-holism and drug abuse is a matter of intense research.Human twin and adoption studies have shown that alcoholintake is a highly heritable trait [1, 2]. Also, large-samplestudies of alcoholism have documented moderately stronggenetic contributions to variation in alcohol dependence,typically accounting for 40% to 60% of variation in risk [3].The genetic background interacts with environmentalevents to produce risk for substance abuse [4]. A classicexample is the individual with a low level of response toalcohol. Indeed, subjects who require greater amounts ofalcohol to exhibit balance deficits or impaired psychomotorperformance are at risk for developing alcohol use disorders[5].Genetic susceptibility interacts with experience to deter-mine outcome [6]. Exposure to childhood adversity is nowconsidered as a major experiential risk factor. There is adose-dependent relationship between the number of adversechildhood experiences, such as recurrent physical abuse orrecurrent emotional abuse, and risk for alcoholism or drugabuse [7]. The understanding of this relationship is highlyrelevant.Cerebellum is a key target of alcohol toxicity in thecentral nervous system [8]. Gait disturbances and ataxia inlower limbs are common both in acute intoxication and inchronic alcoholism. As a result of the closed loops betweencerebellar circuitry and cerebral cortex, cerebellar damagemight contribute to the visuospatial difficulties and im-paired verbal learning observed in alcoholism [9].Individual differences in cerebellar morphology seem toplay a role in the development of alcohol dependence andsubstance abuse [10]. Cerebellar vermis is particularlyvulnerable to alcohol abuse [11]. The anterior vermis istypically affected both in case of fetal exposure and inadults [12, 13]. Atrophy of this region of the cerebellum isone of the first consequences visible on brain magneticresonance imaging. Other areas of the cerebellum such asthe flocculonodular lobe and the central white matter alsoshow volume loss [14]. A 42% atrophy of vermal whitematter has been found in ataxic alcoholics and appears tosignificantly contribute to ataxia [14]. However, cerebellardegeneration may be discovered in alcoholics without signsof ataxia [15]. Purkinje cell loss and impaired dendriticnetworks in the molecular layer are common [16, 17].Successive episodes of ethanol withdrawal also causes aloss of Purkinje cells [18]. Granule cells are less vulnerablethan Purkinje neurons [19].The mechanisms of ethanol-induced cerebellar degen-eration include thiamine deficiency and defects in energyproduction, deficits in growth factors, and apoptosis [20].Thiamine is a cofactor of key enzymes of the energymetabolism. Not only chronic alcoholics present a deficitin thiamine resulting from inadequate intake, but alsoethanol inhibits the conversion into its active metaboliteby blocking the pyrophosphokinase [21]. Thiamine defi-ciency contributes to the pathogenesis of Wernickeencephalopathy. Alcohol interacts with neurotrophins,whose roles in the developing and mature cerebellum arecritical [22, 23]. Excitotoxicity is another detrimental actorof the cascade of events implicated in ethanol-inducedcerebellar degeneration. Acute doses potentiate GABA-Areceptors, hence, the antianxiety effect. Intranuclearadministration of ethanol decreases the levels of GABAand raises the concentrations of nitric oxide [20]. Chronic

Novel Approaches to Studying the Genetic Basis of Cerebellar Development

The list of genes that when mutated cause disruptions in cerebellar development is rapidly increasing. The study of both spontaneous and engineered mouse mutants has been essential to this progress, as it has revealed much of our current understanding of the developmental processes required to construct the mature cerebellum. Improvements in brain imaging, such as magnetic resonance imaging (MRI) and the emergence of better classification schemes for human cerebellar malformations, have recently led to the identification of a number of genes which cause human cerebellar disorders. In this review we argue that synergistic approaches combining classical molecular techniques, genomics, and mouse models of human malformations will be essential to fuel additional discoveries of cerebellar developmental genes and mechanisms.

Young age and low temperature, but not female gender delay ATP loss and glutamate release, and protect Purkinje cells during simulated ischemia in cerebellar slices

Excessive activation of glutamate receptors contributes to Purkinje cell (PC) damage during brain ischemia, but the mechanisms of glutamate release are contentious. Age, gender and temperature all strongly influence ischemic brain damage, but the mechanisms underlying their influence are not fully understood. We determined how age, gender and temperature influence ATP loss, glutamate release, glutamate receptor activation and PC damage during cerebellar ischemia. We used voltage-clamped PCs to monitor glutamate release during simulated ischemia in slices of cerebellum of different ages and genders, and at different temperatures. While gender did not affect ischemic glutamate release, both young age and low temperature dramatically delayed the onset of glutamate release without affecting its magnitude. Glutamate receptor and transporter density were similar around young and old PCs, but the rate of ATP decline during ischemia was dramatically slowed in young animals and by lowered temperature. Bypassing the ischemia-induced loss of ATP, and disrupting ionic gradients directly by pharmacologically inhibiting the Na(+)/K(+)-ATPase, reduced the difference in timing of glutamate release in newborn and mature cerebellum. Ischemic damage in newborn and mature cerebellum paralleled ATP loss and glutamate release, but blocking glutamate receptors did not prevent ischemic damage. Thus, protection against brain ischemia provided by young age or lowered temperature is due to slower consumption and hence delayed loss of ATP, with a corresponding delay in glutamate release and other undetermined damage mechanisms. The protection afforded by female gender must occur downstream of ATP decline, glutamate release, and activation of glutamate receptors on PCs.

Dynamic distribution and stem cell characteristics of Sox1-expressing cells in the cerebellar cortex

Bergmann glia cells are a discrete radial glia population surrounding Purkinje cells in the cerebellar cortex. Although Bergmann glia are essential for the development and correct arborization of Purkinje cells, little is known about the regulation of this cell population after the developmental phase. In an effort to characterize this population at the molecular level, we have analyzed marker expression and established that adult Bergmann glia express Sox1, Sox2 and Sox9, a feature otherwise associated with neural stem cells (NSCs). In the present study, we have further analyzed the developmental pattern of Sox1-expressing cells in the developing cerebellum. We report that before becoming restricted to the Purkinje cell layer, Sox1-positive cells are present throughout the immature tissue, and that these cells show characteristics of Bergmann glia progenitors. Our study shows that these progenitors express Sox1, Sox2 and Sox9, a signature maintained throughout cerebellar maturation into adulthood. When isolated in culture, the Sox1-expressing cerebellar population exhibited neurosphere-forming ability, NSC-marker characteristics, and demonstrated multipotency at the clonal level. Our results show that the Bergmann glia population expresses Sox1 during cerebellar development, and that these cells can be isolated and show stem cell characteristics in vitro, suggesting that they could hold a broader potential than previously thought.

GluRdelta2 expression in the mature cerebellum of hotfoot mice promotes parallel fiber synaptogenesis and axonal competition.

Glutamate receptor delta 2 (GluRδ2) is selectively expressed in the cerebellum, exclusively in the spines of the Purkinje cells (PCs) that are in contact with parallel fibers (PFs). Although its structure is similar to ionotropic glutamate receptors, it has no channel function and its ligand is unknown. The GluRδ2-null mice, such as knockout and hotfoot have profoundly altered cerebellar circuitry, which causes ataxia and impaired motor learning. Notably, GluRδ2 in PC-PF synapses regulates their maturation and strengthening and induces long term depression (LTD). In addition, GluRδ2 participates in the highly territorial competition between the two excitatory inputs to the PC; the climbing fiber (CF), which innervates the proximal dendritic compartment, and the PF, which is connected to spiny distal branchlets. Recently, studies have suggested that GluRδ2 acts as an adhesion molecule in PF synaptogenesis. Here, we provide in vivo and in vitro evidence that supports this hypothesis. Through lentiviral rescue in hotfoot mice, we noted a recovery of PC-PF contacts in the distal dendritic domain. In the proximal domain, we observed the formation of new spines that were innervated by PFs and a reduction in contact with the CF; ie, the pattern of innervation in the PC shifted to favor the PF input. Moreover, ectopic expression of GluRδ2 in HEK293 cells that were cocultured with granule cells or in cerebellar Golgi cells in the mature brain induced the formation of new PF contacts. Collectively, our observations show that GluRδ2 is an adhesion molecule that induces the formation of PF contacts independently of its cellular localization and promotes heterosynaptic competition in the PC proximal dendritic domain.

PTPRR Protein Tyrosine Phosphatase Isoforms and Locomotion of Vesicles and Mice

Protein tyrosine phosphatases (PTPs) are central players in many different cellular processes and their aberrant activity is associated with multiple human pathologies. In this review, we present current knowledge on the PTPRR subfamily of classical PTPs that is expressed in neuronal cells and comprises receptor-type (PTPBR7, PTP-SL) as well as cytosolic (PTPPBSγ-37, PTPPBSγ-42) isoforms. The two receptor-type isoforms PTPBR7 and PTP-SL both localize in late endosomes and the Golgi area. PTPBR7, however, is additionally localized at the cell surface and on early endosomes. During cerebellar maturation, PTPBR7 expression in developing Purkinje cells ceases and is replaced by PTP-SL expression in the mature Purkinje cells. All PTPRR isoforms contain a kinase interacting motif that makes them mitogen-activated protein kinase phosphatases. The distinct subcellular localization of the different PTPRR isoforms may reflect differential roles in growth-factor-induced MAPK-mediated retrograde signaling cascades. Studies in PTPRR-deficient mice established that PTPRR isoforms are physiological regulators of MAPK phosphorylation levels. Surprisingly, PTPRR-deficient mice display defects in motor coordination and balancing skills, while cerebellar morphological abnormalities, which are often encountered in ataxic mouse models, are absent. This is reminiscent of the phenotype observed in a handful of mouse mutants that have alterations in cerebellar calcium ion homeostasis. Elucidation of the molecular mechanisms by which PTPRR deficiency imposes impairment of cerebellar neurons and motor coordination may provide candidate molecules for hereditary cerebellar ataxias that still await identification of the corresponding disease genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s12311-008-0088-y) contains supplementary material, which is available to authorized users.

Insulin-like Growth Factor 2 Is Required for Progression to Advanced Medulloblastoma in patched1 Heterozygous Mice

Medulloblastoma (MB) can arise in the cerebellum due to genetic activation of the Sonic Hedgehog (Shh) signaling pathway. During normal cerebellum development, Shh spurs the proliferation of granule neuron precursors (GNP), the precursor cells of MB. Mutations in the Shh receptor gene patched1 (ptc1+/-) lead to increased MB incidence in humans and mice. MB tumorigenesis in mice heterozygous for ptc1+/- shows distinct steps of progression. Most ptc1+/- mice form clusters of preneoplastic cells on the surface of the mature cerebellum that actively transcribe Shh target genes. In approximately 15% of mice, these preneoplastic cells will become fast-growing, lethal tumors. It was previously shown that the loss of function of insulin-like growth factor 2 (igf2) suppresses MB formation in ptc1+/- mice. We found that igf2 is not expressed in preneoplastic lesions but is induced as these lesions progress to more advanced MB tumors. Igf2 is not required for formation of preneoplastic lesions but is necessary for progression to advanced tumors. Exogenous Igf2 protein promoted proliferation of MB precursor cells (GNP) and a MB cell line, PZp53(MED). Blocking igf2 signaling inhibited growth of PZp53(MED) cells, implicating igf2 as a potential clinical target.

Species differences in the expression of Ahi1, a protein implicated in the neurodevelopmental disorder Joubert syndrome, with preferential accumulation to stigmoid bodies

Joubert syndrome (JBTS) is an autosomal recessive disorder characterized by cerebellum and brainstem malformations. Individuals with JBTS have abnormal breathing and eye movements, ataxia, hypotonia, and cognitive difficulty, and they display mirror movements. Mutations in the Abelson-helper integration site-1 gene (AHI1) cause JBTS in humans, suggesting that AHI1 is required for hindbrain development; however AHI1 may also be required for neuronal function. Support for this idea comes from studies demonstrating that the AHI1 locus is associated with schizophrenia. To gain further insight into the function of AHI1 in both the developing and mature central nervous system, we determined the spatial and temporal expression patterns of the gene products of AHI1 orthologs throughout development, in human, mouse, and zebrafish. Murine Ahi1 was distributed throughout the cytoplasm, dendrites, and axons of neurons, but was absent in glial cells. Ahi1 expression in the mouse brain was observed as early as embryonic day 10.5 and persisted into adulthood, with peak expression during the first postnatal week. Murine Ahi1 was observed in neurons of the hindbrain, midbrain, and ventral forebrain. Generally, the AHI1/Ahi1/ahi1 orthologs had a conserved distribution pattern in human, mouse, and zebrafish, but mouse Ahi1 was not present in the developing and mature cerebellum. Ahi1 was also observed consistently in the stigmoid body, a poorly characterized cytoplasmic organelle found in neurons. Overall, these results suggest roles for AHI1 in neurodevelopmental processes that underlie most of the neuroanatomical defects in JBTS, and perhaps in neuronal functions that contribute to schizophrenia.