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Dysplastic gangliocytoma of the cerebellum (DGC) or Lhermitte-Duclos Disease is a rare lesion (World Health Organization [WHO] grade I) characterized by thickened folia and replacement of the internal granular layer by abnormal ganglion cells. More commonly, the compromised patients are young adults presenting ataxia, seizures, obstructive hydrocephalus, and increased intracranial pressure. Dysplastic gangliocytoma of the cerebellum is intimately associated with Cowden syndrome, a hereditary disorder caused by a germline mutation in the PTEN tumor suppressor gene on chromosome 10q23. Large neurons of DCG show vesicular nuclei with prominent nucleoli. Expansion of the internal granular layer determines vacuolization of the molecular layer and white matter, which can be related to the bright stripes identified on T2-weighted magnetic resonance imaging. Herein, the authors report a female patient who developed long- time recurrence of DGC and discuss pathological findings and differential diagnosis of this rare cerebellar lesion.

Autism spectrum disorders (ASD) are pervasive neurodevelopmental conditions detected during childhood when delayed language onset and social deficits are observed. Children diagnosed with ASD frequently display sensorimotor deficits associated with the cerebellum, suggesting a dysfunction of synaptic circuits. Astroglia are part of the tripartite synapses and postmortem studies reported an increased expression of the glial fibrillary acidic protein (GFAP) in the cerebellum of ASD patients. Astroglia respond to neuronal activity with calcium transients that propagate to neighboring cells, resulting in a functional response known as a calcium wave. This form of intercellular signaling is implicated in proliferation, migration, and differentiation of neural precursors. Prenatal exposure to valproate (VPA) is a preclinical model of ASD in which premature migration and excess of apoptosis occur in the internal granular layer (IGL) of the cerebellum during the early postnatal period. In this study we tested calcium wave propagation in the IGL of mice prenatally exposed to VPA. Sensorimotor deficits were observed and IGL depolarization evoked a calcium wave with astrocyte recruitment. The calcium wave propagation, initial cell recruitment, and mean amplitude of the calcium transients increased significantly in VPA-exposed mice compared to the control group. Astrocyte recruitment was significantly increased in the VPA model, but the mean amplitude of the calcium transients was unchanged. Western blot and histological studies revealed an increased expression of GFAP, higher astroglial density and augmented morphological complexity. We conclude that the functional signature of the IGL is remarkably augmented in the preclinical model of autism.

The cerebellum is a complex system with distinct cortical laminar organization. Alterations in cerebellar microstructure are common and associated with many factors such as genetics, cancer and ageing. Diffusion MRI (dMRI) provides a non-invasive tool to map the brain structural organization, and the recently proposed diffusion-time (td)-dependent dMRI further improves its capability to probe the cellular and axonal/dendritic microstructures by measuring water diffusion at multiple spatial scales. The td-dependent diffusion profile in the cerebellum and its utility in detecting cerebellar disorders, however, are not yet elucidated. Here, we first deciphered the spatial correspondence between dMRI contrast and cerebellar layers, based on which the cerebellar layer-specific td-dependent dMRI patterns were characterized in both euploid and Ts65Dn mice, a mouse model of Down syndrome. Using oscillating gradient dMRI, which accesses diffusion at short td’s by modulating the oscillating frequency, we detected subtle changes in the apparent diffusivity coefficient of the cerebellar internal granular layer and Purkinje cell layer of Ts65Dn mice that were not detectable by conventional pulsed gradient dMRI. The detection sensitivity of oscillating gradient dMRI increased with the oscillating frequency at both the neonatal and adult stages. The td-dependence, quantified by ΔADC map, was reduced in Ts65Dn mice, likely associated with the reduced granule cell density and abnormal dendritic arborization of Purkinje cells as revealed from histological evidence. Our study demonstrates superior sensitivity of short-td diffusion using oscillating gradient dMRI to detect cerebellar microstructural changes in Down syndrome, suggesting the potential application of this technique in cerebellar disorders., Cerebellar hypoplasia is common in people with Down syndrome. Wu et al. decipher the spatial correspondence between cerebellar layers and diffusion MRI contrasts. The layer-specific oscillating gradient diffusion MRI at short diffusion-time shows superior sensitivity to detect microstructure alterations in both developing and mature cerebellar layers of a Down syndrome mouse model., Graphical Abstract Graphical Abstract

Background: Pregabalin is an oral medication that is used to treat neuropathic pain, generalized anxiety disorder and epilepsy; as a therapy for partial seizures. Cerebellar development is not complete during gestation only; but continues after birth to maturation of the cerebellum (postnatal development).Aim of the Work: To study the possible effect of Pregabalin on the postnatal development of the cerebellar cortex of albino rat.Materialandmethods:60 pregnant albino rats were allocated into three groups; Group A: Included 20offspring of 20 control mothers, Group B: Included 20 offspring of 20 mothers treated orally with pregabalin at a dose of 150mg/kg, and Group C:included 25 offspring of 20 mothers treated orally with pregabalin at a dose of 600mg/kg. In the three groups rats were sacrificed at postnatal (age 1, 2, 3 weeks), in addition to adult age (2.5 months).The skulls were opened and the cerebella were removed and processed for light and transmission electron microscope. Results: Pregabalin administration during pregnancy and lactation caused its marked effect during early postnatal life and this effect extended till the adult stage; in the form of increase in the external granular layer (mainly in the 2nd and 3rd weeks); less thickness of the molecular layer in all treated groups; shrunken and abnormal shaped Purkinje cells which appeared with ill-defined cell membranes and destructed nuclei; mainly at higher dose. There were also delayed differentiation of the internal granular layer and granule cells appeared with destructed cytoplasmic membrane and organelles which appeared prominent at higher doses. Conclusion: Pre and postnatal administration of pregabalin in both low and high doses caused loss of cellular components, distortions of cerebellar cortical cells in a dose dependent manner.

Down Syndrome is a chromosomal disorder that affects the development of cerebellar cortical lobules. Impaired neurogenesis in the cerebellum varies among different types of neuronal cells and neuronal layers. In this study, we developed an imaging analysis framework that utilizes gadolinium-enhanced ex vivo mouse brain MRI. We extracted the middle Purkinje layer of the mouse cerebellar cortex, enabling the estimation of the volume, thickness, and surface area of the entire cerebellar cortex, the internal granular layer, and the molecular layer in the Tc1 mouse model of Down Syndrome. The morphometric analysis of our method revealed that a larger proportion of the cerebellar thinning in this model of Down Syndrome resided in the inner granule cell layer, while a larger proportion of the surface area shrinkage was in the molecular layer., Graphical abstract Image, graphical abstract

Inactivating mutations in the human SLC16A2 gene encoding the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in the Allan–Herndon–Dudley syndrome accompanied by severe locomotor deficits. The underlying mechanisms of the associated cerebellar maldevelopment were studied using the chicken as a model. Electroporation of an MCT8-RNAi vector into the cerebellar anlage of a 3-day-old embryo allowed knockdown of MCT8 in Purkinje cell precursors. This resulted in the downregulation of the thyroid hormone-responsive gene RORα and the Purkinje cell-specific differentiation marker LHX1/5 at day 6. MCT8 knockdown also results in a smaller and less complex dendritic tree at day 18 suggesting a pivotal role of MCT8 for cell-autonomous Purkinje cell maturation. Early administration of the thyroid hormone analogue 3,5,3′-triiodothyroacetic acid partially rescued early Purkinje cell differentiation. MCT8-deficient Purkinje cells also induced non-autonomous effects as they led to a reduced granule cell precursor proliferation, a thinner external germinal layer and a loss of PAX6 expression. By contrast, at day 18, the external germinal layer thickness was increased, with an increase in presence of Axonin-1-positive post-mitotic granule cells in the initial stage of radial migration. The concomitant accumulation of presumptive migrating granule cells in the molecular layer, suggests that inward radial migration to the internal granular layer is stalled. In conclusion, early MCT8 deficiency in Purkinje cells results in both cell-autonomous and non-autonomous effects on cerebellar development and indicates that MCT8 expression is essential from very early stages of development, providing a novel insight into the ontogenesis of the Allan–Herndon–Dudley syndrome.

The major anatomical divisions of the cerebellum of the European eel, i.e., corpus cerebelli, lobus vestibulolateralis, and valvula, were studied morphologically and morphometrically. There were differences in cerebellar cytoarchitecture and gross morphology in two stages of the eel life cycle, the trophic stage (yellow eel), and the reproductive stage (silver eel), which are characterized by different degrees of swimming activity. The principal differences between silver and yellow eels in the cytoarchitecture of the corpus cerebelli and the lobus vestibulolateralis were in distribution of Purkinje or Purkinje-like cells in the molecular layer, which is wider in silver eels, in part because of a decreased thickness of the granular cell layer. In the silver eel, the scattering of Purkinje cells was more evident in the lobus vestibulolateralis where the molecular layer is also thicker. The data indicate the transition from the yellow eel to the silver eel is characterized by a migration of granule cells from the ganglionic cell layer to the internal granular layer and by a further development of molecular layer components, e.g., parallel fibers, Purkinje-cell dendrites, etc. In contrast, the thickness of the granular layer and of the Purkinje cell layer, limited to the lower part of the valvula, decreased. There is also a slight increase of cerebellar volume in the silver eel. The volume of the lobus vestibulolateralis was constant. Hypertrophy of the valvula and eminentiae granulares is observed and is due to the migration of cells from the granular layer of the corpus cerebelli whose volume slightly decreases. Perhaps the lobus vestibulolateralis also contributes to the increased volume of eminentiae granulares. Our findings suggest that the cerebellum continues to develop during the passage from the trophic to the reproductive stage of the eel. The appearance of new afferents from the lateral line which becomes more visible in the silver eel probably completes cerebellar ontogeny.

The unfolded protein response (UPR) has been reported during normal development of cortical neurons and cerebellar white matter and may also contribute to the pathogenesis of neurological conditions, such as Marinesco-Sjogren syndrome and Borna virus infection, which result in cerebellar defects. The UPR is initiated when the processing capacity of the endoplasmic reticulum (ER) is overwhelmed. Misfolded proteins accumulate and can activate ER stress sensors; PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activated transcription factor 6 (ATF6) and their downstream targets glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and protein disulfide isomerase (PDI). In order to provide a fuller appreciation of the possible importance of ER stress-associated proteins in the context of cerebellar disease, we have profiled the expression of ER stress sensors and their downstream targets in the developing cerebellar cortex in postnatal rat. Activation of PERK and IRE1 stress sensors was observed for the first time in normally developing granule cell precursors. A second proliferative pPERK-positive population was also detected in the internal granular layer (IGL). In general, the density of UPR protein-positive cells was found to decrease significantly when profiles in early and late postnatal ages were compared. These data may be relevant to studies of medulloblastoma and warrant further investigation.

Propofol is a widely used anesthetic in the clinic while several studies have demonstrated that propofol exposure may cause neurotoxicity in the developing brain. However, the effects of early propofol exposure on cerebellar development are not well understood. Propofol (30 or 60 mg/kg) was administered to mice on postnatal day (P)7; Purkinje cell dendritogenesis and Bergmann glial cell development were evaluated on P8, and granule neuron migration was analyzed on P10. The results indicated that exposure to propofol on P7 resulted in a significant reduction in calbindin-labeled Purkinje cells and their dendrite length. Furthermore, propofol induced impairments in Bergmann glia development, which might be involved in the delay of granule neuron migration from the external granular layer (EGL) to the internal granular layer (IGL) during P8 to P10 at the 60 mg/kg dosage, but not at the 30 mg/kg dosage. Several reports have suggested that the Notch signaling pathway plays instructive roles in the morphogenesis of Bergmann glia. Here, it was revealed that propofol treatment decreased Jagged1 and Notch1 protein levels in the cerebellum on P8. Taken together, exposure to propofol during the neonatal period impairs Bergmann glia development and may therefore lead to cerebellum development defects. Our results may aid in the understanding of the neurotoxic effects of propofol when administrated to infants.

Retinoid-related orphan receptor α (RORα) is a critical master transcription factor that governs postnatal cerebellar development. An RORα-deficient cerebellum has a persistent external granular layer (EGL), rudimentary Purkinje cell (PC) dendrites, grossly reduced numbers of immature parallel fiber (PF)-PC synapses, and multiple climbing fibers (CF) innervating PCs in mice after 3 weeks of age when these features have disappeared in wild-type mice. Functionally, metabotropic glutamate receptor (mGluR)-mediated signaling in PCs is completely abrogated. Here we examined whether these defects could be corrected by lentivirally providing the RORα gene to 3-week-old PCs of RORα-deficient homozygous staggerer (sg/sg) mice. RORα expression in sg/sg PCs significantly increased the numbers of PF–PC synapses, spines on PC dendritic branchlets, and internal granule cells, concomitant with regression of the EGL, suggesting enhanced proliferation in the EGL and migration of post-mitotic progeny into the internal granular layer with augmented synaptogenesis between PFs and PC dendrites. However, the primary dendritic stems were only slightly extended, and mGluR signaling and the loss of redundant CF synapses in sg/sg PCs remained unrestored. These results suggest that the mitogenic and migratory potential of external granule cells in response to RORα was preserved in the >3-week-old sg/sg mouse cerebellum. Moreover, sg/sg PCs sprouted spines and formed synapses with PFs. However, lengthening of the primary dendritic stems, establishment of mGluR signaling, and removal of CF synapses in sg/sg PCs were regressed by 3 weeks of age.

Data on olfactory bulb (OB) development in human fetuses in the stages from the 8th week to birth is provided. Immunohistochemical markers of presynaptic terminals (anti-SNAP-25, synapsin-I, and synaptophysin) were used to evaluate the maturation of the OB. Differentiation of the OB layers begins from the periphery, which indirectly confirms that growth of the olfactory nerve fibers induces not only the anatomical differentiation of the OB but also the differentiation of its functional layers. The sites of the developing glomeruli are revealed using the immunohistochemical procedure prior to the stage in which distinct glomeruli can be identified using a common histological procedure. The OB conductive system demonstrates immunoreactivity with the antibodies to presynaptic proteins at all stages starting with the 10th–11th weeks of fetal development. Four stages of OB development are described. All functional layers of the OB are mature at the 22nd week stage. Further differentiation of the neuroblasts in the OB, including lamina formation of the internal granular layer and the glomerular layer development, and the growth of the OB continue after the 20th–22nd week stage until 38th–40th weeks of fetal development. Patterns of the immunoreactivity with antibodies to SNAP-25, synapsin-I, and synaptophysin at the 38th-40th weeks of prenatal development are fully consistent with those of the adult OB. Complete maturity of the human OB is reached at the 38th–40th week of prenatal development.

Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders.

cerebellum, neurons, development, doublecortin, NeuN. Experiments were performed on the offspring of five mongrel white rats using comparative immunochemical assessments of doublecortin (DCX) and neuronal nuclear antigen (NeuN) expression in neurons in the cortex and nucleus interpositus of the cerebellum in animals during early postnatal ontogeny (days 2-15). DCX expression was seen in postmitotic neurons in the external granular layer and migrating neurons in the cerebellar cortex. DCX expression was greater in the neocerebellum than in the paleocerebellum in rat pups aged two and seven days. NeuN expression was seen in migrating granule neurons, reaching a maximum in more mature neurons in the internal granular layer. DCX expression was not seen in Purkinje cells or in neurons in the nucleus interpositus of the cerebellum. Neurons in the nucleus interpositus showed progressive increases in NeuN from day 2 to day 15 after birth. Thus, comparative immunohistochemical studies of the dynamics of the expression of this pair of molecular markers provide an effective means of evaluating the development of cerebellar granule neurons during early postnatal ontogeny.

Aberrant activation of SHH pathway is a major cause of medulloblastoma (MB), the most frequent brain malignancy of the childhood. A few Hedgehog inhibitors, all antagonizing the membrane transducer Smo, have been approved or are under clinical trials for the treatment of human MB. However, the efficacy of these drugs is limited by the occurrence of novel mutations or by activation of downstream or non-canonical Hedgehog components. Thus, the identification of novel druggable downstream pathways represents a critical step to overcome this problem. In the present work we demonstrate that aerobic glycolysis is a valuable HH-dependent downstream target, since its inhibition significantly counteracts the HH-mediated growth of normal and tumor cells. Hedgehog activation induces transcription of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), two key gatekeepers of glycolysis. The process is mediated by the canonical activation of the Gli transcription factors and causes a robust increase of extracellular lactate concentration. We show that inhibition of glycolysis at different levels blocks the Hedgehog-induced proliferation of granule cell progenitors (GCPs), the cells from which medulloblastoma arises. Remarkably, we demonstrate that this glycolytic transcriptional program is also upregulated in SHH-dependent tumors and that pharmacological targeting with the pyruvate kinase inhibitor dichloroacetate (DCA) efficiently represses MB growth in vitro and in vivo. Together, these data illustrate a previously uncharacterized pharmacological strategy to target Hedgehog dependent growth, which can be exploited for the treatment of medulloblastoma patients.

The strep avidin-biotin-peroxidase complex (SABC) immunohistochemical methods were applied to investigate the localization and semi-quantitative distribution of IFN-γ, IL-1α, NGF-β and TNF-α-immunoreactive cells in the cerebellar cortex of Western Anhui white goose at embryonic day 13, 19, 24, 28 (E13, E19, E24, E28) and postnatal day 7, 15 (P7, P15). The possible roles of IFN-γ、IL-1α、NGF-β and TNF-α in the development of cerebellar cortex were discussed. The results indicated that in the external granular layer, there were IFN-γ and TNF-αpositive cells at E13, E19, E24, E28, P7, IL-1α positive cells at E13, E19, E24, E28 and NGF-β positive cells at E13,E19, E24. The expression levels of these four cytokines all reached peaks at E19 of the six tested periods in this study. In the Purkinje cell layer, there were IFN-γ, IL-1α and TNF-α positive cells at E13, E19, E24, E28, P7, P15 and NGF-βpositive cells at E13, E19, E24, E28, P7. In the internal granular layer, there were IFN-γ positive cells at E13, E19, E24,E28, P7, P15, IL-1α and TNF-α positive cells at E13, E19, E24, E28, P7 and NGF-β positive cells at E13, E19, E24, E28.These results showed that E19 might be the “critical stage” in the cerebellar cortex development of Western Anhui white goose. IFN-γ, IL-1α and TNF-α might be synthesized by cerebellar cortex itself, and NGF-β could be transported from regions which project to Purkinje cells. IFN-γ may interfer the transfer of granular cells, and NGF-β may have neurotrophic functions that are beneficial to the growth and development of Purkinje cells.

Rosette-forming glioneuronal tumor (RGNT) is a recently introduced, indolent neoplasm composed of diminutive circular aggregates of neurocytic-like cells on a noninfiltrative astrocytic background, typically located in the cerebellar midline The traded concept of RGNT being derived from site-specific periventricular precursors may be questioned in the face of extracerebellar examples as well as ones occurring in combination with other representatives of the glioneuronal family. We describe a hitherto not documented example of asymptomatic RGNT discovered during autopsy of a 74-year-old male. Located in the tuberal vermis, this lesion of 6 mm diameter consisted of several microscopic nests of what were felt to represent nascent stages of RGNT, all of them centered on the internal granular layer, and ranging from mucoid dehiscences thereof to fully evolved - if small - tumor foci. Molecular genetic analysis revealed a missense mutation in Exon 20 of the PIK3CA gene involving an A→G transition at Nucleotide 3140. On the other hand, neither codeletion of chromosomes 1p/19q nor pathogenic mutations of IDH1/2 were detected. By analogy with in situ paradigms in other organs, we propose that this tumor is likely to have arisen from the internal granular layer, rather than the plate of the 4th ventricle. A suggestive departure from the wholesale argument of "undifferentiated precursors", this finding also indirectly indicates that a subset of non-classical RGNTs - in particular extracerebellar examples, whose origin cannot be mechanistically accounted for by either of the above structures - may possibly reflect an instance of phenotypic convergence, rather than a lineage-restricted entity.

The immunohistochemical expression of the calcium-binding protein calretinin during human cerebellar development has been investigated in this study. Human cerebellum samples, obtained from 7 fetuses and newborns ranging from 11 to 38 weeks of gestation, were 10% formalin-fixed, routinely processed and paraffin-embedded. 3 μm-tick sections were immunostained with an anti-calretinin antibody. Our study evidenced a different immunoreactivity for calretinin in Purkinje cells and in several cerebellar interneurons at different intrauterine developmental stages. Whereas at 11 weeks of gestation calretinin immunoreactivity was not detected in the developing cerebellum, from the 18th to the 24th week, calretinin expression was found in Purkinje cells migrating from the ventricular neuroepithelium and in migrating cerebellar interneurons. From the 30th to the 38th week, calretinin was expressed by most of Purkinje cells and by migrating cerebellar interneurons. Furthermore, granule cells in the internal granular layer were also immunoreactive for calretinin. Our data show that calretinin, other than for developing Purkinje cells, is a useful marker also for migrating cerebellar interneurons and for some neuronal elements related to the granular layer. Moreover, given the critical role of calcium in a great variety of neuronal processes in the central nervous system, our findings suggest that calretinin may play a pivotal role in the regulation of neuronal excitability during intrauterine cerebellar development.

During cerebellar development, anchoring centers form at the base of each fissure and remain fixed in place while the rest of the cerebellum grows outward. Cerebellar foliation has been extensively studied; yet, the mechanisms that control anchoring center initiation and position remain insufficiently understood. Here we show that a tri-layer model can predict surface wrinkling as a potential mechanism to explain anchoring center initiation and position. Motivated by the cerebellar microstructure, we model the developing cerebellum as a tri-layer system with an external molecular layer and an internal granular layer of similar stiffness and a significantly softer intermediate Purkinje cell layer. Including a weak intermediate layer proves key to predicting surface morphogenesis, even at low stiffness contrasts between the top and bottom layers. The proposed tri-layer model provides insight into the hierarchical formation of anchoring centers and establishes an essential missing link between gene expression and evolution of shape.

The current paper analyzes the development of the male and female rat cerebellum exposed to hydroxyurea (HU) (300 or 600 mg/kg) as embryo and collected at postnatal day 90. Our study reveals that the administration of this drug compromises neither the cytoarchitecture of the cerebellar cortex nor deep nuclei (DCN). However, in comparison with the saline group, we observed that several cerebellar parameters were lower in the HU injected groups. These parameters included area of the cerebellum, cerebellar cortex length, molecular layer area, Purkinje cell number, granule cell counts, internal granular layer, white matter and cerebellar nuclei areas, and number of deep cerebellar nuclei neurons. These features were larger in the rats injected with saline, smaller in those exposed to 300 mg/kg of HU and smallest in the group receiving 600 mg/kg of this agent. No sex differences in the effect of the HU were observed. In addition, we infer the neurogenetic timetables and the neurogenetic gradients of PCs and DCN neurons in rats exposed to either saline or HU as embryos. For this purpose, 5-bromo-2′-deoxyuridine was injected into pregnant rats previously administered with saline or HU. This thymidine analog was administered following a progressively delayed cumulative labeling method. The data presented here show that systematic differences exist in the pattern of neurogenesis and in the spatial location of cerebellar neurons between rats injected with saline or HU. No sex differences in the effect of the HU were observed. These findings have implications for the administration of this compound to women in gestation as the effects of HU on the development of the cerebellum might persist throughout their offsprings’ life.

Medulloblastoma (MB), tumor of the cerebellum, remains a leading cause of cancer-related mortality in childhood.We previously showed, in a mouse model of spontaneous MB (Ptch1+/-/Tis21-/-), that a defect of the migration of cerebellar granule neuron precursor cells (GCPs) correlates with an increased frequency of MB. This occurs because GCPs, rather than migrating internally and differentiating, remain longer in the proliferative area at the cerebellar surface, becoming targets of transforming insults. Furthermore, we identified the chemokine Cxcl3 as responsible for the inward migration of GCPs. As it is known that preneoplastic GCPs (pGCPs) can still migrate and differentiate like normal GCPs, thus exiting the neoplastic program, in this study we tested the hypothesis that pGCPs within a MB lesion could be induced by Cxcl3 to migrate and differentiate. We observed that the administration of Cxcl3 for 28 days within the cerebellum of one-month-old Ptch1+/-/Tis21-/- mice, i.e., when MB lesions are already formed, leads to complete disappearance of the lesions. However, a shorter treatment with Cxcl3 (two weeks) was ineffective, suggesting that the suppression of MB lesions is dependent on the duration of Cxcl3 application. We verified that the treatment with Cxcl3 causes a massive migration of pGCPs from the lesion to the internal granular layer (IGL), where they differentiate.Thus, the induction of migration of pGCPs in medulloblastoma lesions may open new ways to treat MB that exploit the plasticity of the pGCPs, forcing their differentiation. It remains to be tested whether this plasticity continues at advanced stages of medulloblastoma. If so, these findings would set a potential use of the chemokine Cxcl3 as therapeutic agent against MB development in human preclinical studies.

Background: Lhermitte-Duclos is a rare hamartomatous lesion of the cerebellar cortex with both neoplastic and hamartomatous features. Also known as dysplastic cerebellar gangliocytoma, they usually occur in the setting of Cowden’s syndrome, an autosomal dominant condition characterized by multiple hamartomas and neoplastic lesions in skin and internal organs. Although enlargement of the internal granular layer of the cerebellum is a consistent finding in our case, the background setting of Cowden’s syndrome was not present in our case. Case description: We present a unique case of LDD in the absence of Cowdens syndrome in a 58-year old gentleman who presented with cerebellar signs, a first to be reported in literature. Here we describe our clinical vignette. C onclusion: Most of the cases of LDD, reported in literature, were associated with Cowdens disease, however our case is the first to present without Cowdens disease. To date no other similar cases have been reported in literature.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Maternal malnutrition affects every aspect of fetal development. The present study asked the question whether a low-protein diet of the mother could result in motor deficits in the offspring. Further, to examine whether cerebellar pathology was correlated with motor deficits, several parameters of the postnatal development of the cerebellum were assayed. This is especially important because the development of the cerebellum is unique in that the time scale of development is protracted compared with that of the cortex or hippocampus. The most important result of the study is that animals born to protein-deficient mothers showed significant delays in motor development as assessed by rotarod and gait analysis. These animals also showed reduced cell proliferation and reduced thickness in the external granular layer. There was a reduction in the number of calbindin-positive Purkinje cells (PC) and granular cells in the internal granular layer. However, glial fibrillary acidic protein-positive population including Bergmann glia remained unaffected. We therefore conclude that the development of the granular cell layer and the PC is specifically prone to the effects of protein malnutrition potentially due to their protracted developmental period from approximately embryonic day 11 to 13 until about the third postnatal week.

Glucocorticoid (GC) treatment in premature infants may have detrimental effects on the immature brain. Here we show that GCs dexamethasone (Dex) and hydrocortisone (HC) reduce proliferation and induce differentiation of chicken embryo cerebellar neurons in vivo and in vitro. Granule neurons incorporating bromodeoxyuridine were reduced in the internal granular layer (IGL) after 24-h exposure to both substances on embryonic day 17, with Dex about 100-fold more potent than HC. The effects were blocked by GR antagonist RU 38486. Both GCs also increased the expression of neuronal differentiation markers microtubule-associated protein 2 (Map2) and neuronal nuclei protein (NeuN), measured by western blotting of whole cerebellar lysates and immunohistochemistry, respectively. Treatment of cerebellar granule neuron cultures with both GCs significantly reduced the percentage of proliferating-cell nuclear antigen (PCNA) positive neurons and increased NeuN positive neurons, with similar dose-response relationship as in vivo. The cytostatic agent cytosine arabinoside showed comparable effects both on proliferation and differentiation. In conclusion, the effects of Dex and HC on chicken cerebellar granule neuron proliferation are GR mediated and reflect their pharmacological potency. In addition, the effects on differentiation may be related to a cell cycle block per se, since cytosine arabinoside mimicked the effect of the GCs.

While the origin of oligodendroglia in the prosencephalon and spinal cord has been extensively studied and accurately described, the origin of this cell type in the cerebellum is largely unknown. To investigate where cerebellar oligodendrocytes generate and which migratory pathways they follow to reach their final destination in the adult, in ovo transplants were performed using the quail/chick chimeric system. The chimeric embryos were developed up to HH43-49 (17-19 days of incubation) to map the location of donor cells and analyze their phenotype by immunohistochemistry. As a result, mesencephalic homotopic and homochronic transplants generated cellular migratory streams moving from the grafted epithelium into the host cerebellum, crossing the isthmus mainly through the velum medullare and invading the central white matter. From here, these mesencephalic cells invaded all the layers of the cerebellar cortex except the granular layer. The majority of the cells were detected in the central and folial white matter, as well as in superficial regions of the internal granular layer, surrounding the Purkinje cells. In the latter case, the donor cells presented a Bergmann glial morphology and were Vimentin positive, while in other areas they were PLP and Olig2-positive, indicating an oligodendroglial fate. The combinatory analysis of the different grafts allowed us to propose the fate map of chick cerebellar oligodendroglia at the neural tube stage. As a result, the majority of the cerebellar oligodendrocytes originate from the parabasal plate of the mesencephalon. © 2011 Wiley-Liss, Inc., National grants: DIGESIC-MEC.BFU2008-00588, Ingenio 2010 MEC-CONSOLIDER.CSD2007-00023, GVA Prometeo 2009.028, ISCIII/CIBERSAM. RD06/0010/0023 and Tercel. RD06/0010/0023. International grants: ELA Foundation.2006-022I2 and EU LSHG-CT-2004-512003 to S.M.

Liver X receptor α (LXRα) and β (LXRβ) are members of the nuclear receptor superfamily of ligand-activated transcription factors, and expressed in the CNS. We have previously demonstrated that LXRβ is essential for migration of later-born neurons during cerebral cortex development, although the underlying mechanism is not clear. The cerebellum is organized in an exquisitely foliated structure with a simple layered cytoarchitecture and considered to be a good model to study morphogenesis of lamination and neuronal migration. Here, we found that T0901317, a potent LXR receptor agonist, administration to neonatal C57/BL6 mice, increased dendritic growth of Purkinje cell, although the appearance of the cerebellar cortex was not affected. We further demonstrated T0901317 treatment promoted the migration of granule neurons from the external granular layer to the internal granular layer during cerebellum development. Bergmann glial fibers serve as scaffolds for granule cells inward migration during cerebellum postnatal development. T0901317 treatment also inhibited premature differentiation of Bergmann glia during cerebellum development, which is related to the decreased levels of TGF-β1 and Smad4 in the cerebellum. Taken together, our findings suggest that endogenous LXR affects differentiation process of Bergmann glia and subsequently leads to promote the migration of granule neurons.

Thyroid hormone is essential for brain development where it acts mainly through the thyroid hormone receptor α1 (TRα1) isoform. However, the potential for the hormone to act in adult neurons has remained undefined due to difficulties in reliably determining the expression pattern of TR proteins in vivo. We therefore created a mouse strain that expresses TRα1 and green fluorescent protein as a chimeric protein from the Thra locus, allowing examination of TRα1 expression during fetal and postnatal development and in the adult. Furthermore, the use of antibodies against other markers enabled identification of TRα1 expression in subtypes of neurons and during specific stages of their maturation. TRα1 expression was first detected in postmitotic cells of the cortical plate in the embryonic telencephalon and preceded the expression of the mature neuronal protein NeuN. In the cerebellum, TRα1 expression was absent in proliferating cells of the external granular layer, but switched on as the cells migrated towards the internal granular layer. In addition, TRα1 was expressed transiently in developing Purkinje cells, but not in mature cells. Glial expression was found in tanycytes in the hypothalamus and in the cerebellum. In the adult brain, TRα1 expression was detected in essentially all neurons. Our data demonstrate that thyroid hormone, unexpectedly, has the capacity to play an important role in virtually all developing and adult neurons. Because the role of TRα1 in most neuronal cell types in vivo is largely unknown, our findings suggest that novel functions for thyroid hormone remain to be identified in the brain.

The following study analysed apoptosis in proliferative cells and migrating neurons of the developing cerebellum. The external granular layer, Purkinje cell layer and internal granular layer in the developing mouse cerebellar cortex were analysed by active caspase-3 immunohistochemistry, Hoechst 33258 staining and Western blot analysis. Immunocytochemistry results indicated that the peak of apoptosis appeared at postnatal days P8, P5 and P9 in the external granular layer, Purkinje cell layer and internal granular layer, respectively. Subsequently, in each region, the rate of apoptosis decreased with increasing age. In contrast, Western blot results demonstrated the highest expression of activated caspase-3 in the cerebellum at P5, followed by a subsequent decline and disappearance of expression by P14. Activated caspase-8 was expressed maximally at P10, and subsequently disappeared by P30. The results of this study suggest that the key period of neuronal apoptosis in the cerebellar cortex is between P0 and P14, indicating that this developmental period could be susceptible to treatment for congenital neurodegenerative diseases.

In the adult human fronto-opercular cortex, the grade of granularity of cortical layer IV represents a major architectonic feature for distinguishing the opercular area BA44 (which is typically described as being magnopyramidal and intermedio-granular) from the triangular area BA45 (which is typically described as being magnopyramidal and granular). We analyzed postnatal cytoarchitectonic development of internal granular layer (layer IV) of the fronto-opercular region in 23 brains of newborns, infants and children (age range: 15 days to 15 years) and in one adult brain. We found that: (1) the grade of granularity of layer IV in the fronto-opercular cortex displays a great interindividual variability throughout development as well as in the adult brain; (2) this variability is much more pronounced in the opercular area BA44 than in the triangular area BA45. Our findings support the classical interpretation provided by Constantin von Economo (von Economo & Koskinas, 1925) according to which BA44/FCBm represents a specialized variant of the wide premotor cortical zone, while BA45/FDG represents a specialized variant of the typical (associative) frontal granular cortex.

In order to reveal mechanisms of brain damages resulted from fluoride toxicity, we treated adult female mice of Swiss Albinos strain by 500 ppm NaF (226 ppm F−) in their drinking water from the 15th day of pregnancy until the day 14 after delivery. All mice were sacrificed on day 14 after parturition. During treatment, levels of thiobarbituric acid reactive substances (TBARS), the marker of lipid peroxidation extend, increased, while the activities of the antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), and the level of glutathione (tGSH) decreased significantly in both cerebellum and cerebrum compared with those of the control group. These results suggested that fluoride enhanced oxidative stress in the brain, thereby disturbing the antioxidant defense of nursing pups. In addition, acetylcholinesterase (AChE) activity in both cerebellum and cerebrum was inhibited after treatment with fluoride. In our previous studies we have found, a decrease in plasma thyroid hormone levels of nursing pups whose mothers were treated by NaF. In cerebellum of mice, migration of neurons from the external granular layer to the internal granular layer occurred postnatally and was dependent upon the presence of thyroid hormone. In hypothyroidism, several neurons failed to complete their migration and died. Key guidance signals to these migrating neurons were provided by laminin, an extracellular matrix protein fixed to the surface of astrocytes. In the present study we examined the expression and distribution of laminin in cerebellum of 14-day-old mice. Immunoreactive laminin was disappeared by postnatal day 14 in cerebellum parenchyma of control pups and was restricted to vasculature despite the continued presence of granular cells in the external granular layer. In contrast, in cerebellum of NaF treated pups, laminin was deposited in organised punctuate clusters in the molecular layer. These data indicated that the disruption of laminin distribution might play a major role in the profound derangement of neuronal migration observed in cerebellum of NaF treated pups.

Microtubules in typical cells form radial arrays with their plus-ends pointing toward the cell periphery. In contrast, microtubules in dendrites of neurons are free from centrosomes and have a unique arrangement in which about half have a polarity with a minus-end distal orientation. Mechanisms for generation and maintenance of the microtubule arrangement in dendrites are not well understood. Here, we examined dendritic localization of a centrosomal protein, ninein, which has microtubule-anchoring and stabilizing functions. Immunohistochemical analysis of developing mouse cerebral and cerebellar cortices showed that ninein is localized at the centrosome in undifferentiated neural precursors. In contrast, ninein was barely detected in migrating neurons, such as those in the intermediate layer of the cerebral cortex and the internal granular layer of the cerebellar cortex. High expression was observed in thick dendrite-bearing neurons such as pyramidal neurons of the cerebral cortex and Purkinje neurons in the cerebellar cortex. Ninein was not detected at the centrosome of these cells, but was diffusely present in cell soma and dendrites. In cultured cortical neurons, ninein formed granular structures in soma and dendrites, being not associated with gamma-tubulin. About 60% of these structures showed resistance to detergent and association with microtubules. Our observations suggest that the minus-ends of microtubules may be anchored and stabilized by centrosomal proteins localized in dendrites.

The present study was designed in an effort to determine whether changes in fast axonal transport in the mature rat visual system can be directly correlated with the onset of neurological dysfunction. Methyl mercury was administered in the drinking water at a concentration of 54 micrograms Hg/ml. Fast axonal transport of proteins in the optic nerve and tract was quantified by scintillation spectrometry of protein-bound radioactivity along the visual pathway after an intraocular injection of 3H-proline. At 8 hours after injection the labeled protein had reached the lateral geniculate body both in controls and treated animals. However, two-way analysis of variance revealed a significant decrease in the volume of transported protein-bound radioactivity along the visual pathway. Thus, while the rate of fast axonal transport does not seem to be correlated with the onset of motor dysfunction, the onset of neurological symptoms may be associated with abnormal transport capacity. Treatment lowered body weight to the same extent in males and females. Hind limb cross-over occurred after 25.6 +/- 0.8 days and was followed quickly by hind limb paralysis (32 +/- 0.6 days). The cerebellum revealed pyknotic nuclei throughout the internal granular layer. Purkinje cells appeared normal. No pathological changes were noted in the kidneys.

(Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22-C24). In order to study the biological role of CERS2, we have inactivated its coding region in transgenic mice using gene-trapped embryonic stem cells that express lacZ reporter DNA under control of the cers2 promoter. The resulting mice lack ceramide synthase activity toward C24:1 in the brain as well as the liver and show only very low activity toward C18:0-C22:0 in liver and reduced activity toward C22:0 residues in the brain. In addition, these mice exhibit strongly reduced levels of ceramide species with very long fatty acid residues (or=C22) in the liver, kidney, and brain. From early adulthood on, myelin stainability is progressively lost, biochemically accompanied by about 50% loss of compacted myelin and 80% loss of myelin basic protein. Starting around 9 months, both the medullary tree and the internal granular layer of the cerebellum show significant signs of degeneration associated with the formation of microcysts. Predominantly in the peripheral nervous system, we observed vesiculation and multifocal detachment of the inner myelin lamellae in about 20% of the axons. Beyond 7 months, the CERS2-deficient mice developed hepatocarcinomas with local destruction of tissue architecture and discrete gaps in renal parenchyma. Our results indicate that CERS2 activity supports different biological functions: maintenance of myelin, stabilization of the cerebellar as well as renal histological architecture, and protection against hepatocarcinomas.

CNS repair by cell transplantation requires new neurons to integrate into complex recipient networks. We assessed how the migratory route of transplanted granule neurons and the developmental stage of the host rat cerebellum influence engraftment. In both embryonic and postnatal hosts, granule cells can enter the cerebellar cortex and achieve correct placement along their natural migratory pathway. Donor neurons can also reach the internal granular layer from the white matter and integrate following an unusual developmental pattern. Although the frequency of correct positioning declines in parallel with cortical development, in mature recipients correct homing is more frequent through the unusual path. Following depletion of granule cell precursors in the host, more granule neurons engraft, but their ability for achieving correct placement is unchanged. Therefore, while the cerebellar environment remains receptive for granule cells even after the end of development, their full integration is partially hindered by the mature cortical architecture.

In the present study, the effects of methimazole (a major anti-thyroid drug) administration to rat dams on the development of cerebellum of their pups were investigated with morphological, morphometrical and functional procedures. A motor performance in the pups was evaluated by a rota-rod test. Brains removed on 6, 9, 12, 15, 25, and 30 postnatal days were analyzed using the sagittal sections of the cerebellum. Results showed that orally administered methimazole to dams produced a congenital hypothyroid model accompanied with an impaired motor coordination assured by the reduced thyroid hormones. The prominent anomaly was found in the internal granular layer in that there were excess bulges or branching and formation of excess sublobules although the normal lobulation pattern was kept. Three dimensional reconstruction imaging revealed the complex morphological pattern of internal granular layer of the cerebellar hemispheres as well as of the vermis, in which bulges and branches were viewed stereoscopically as the smooth ridges rather than irregular or nodal. In addition, the external granular layer in hypothyroidism survived another several days than that in controls. It is suggested that the complex internal granular layer resulted from the overproduced internal granular cells, which originate in the prolonged external granular layer.

Toward the goal of generating a mouse medulloblastoma model with increased tumor incidence, we developed a homozygous version of our ND2:SmoA1 model. Medulloblastomas form in 94% of homozygous Smo/Smo mice by 2 months of age. Tumor formation is, thus, predictable by age, before the symptomatic appearance of larger lesions. This high incidence and early onset of tumors is ideal for preclinical studies because mice can be enrolled before symptom onset and with a greater latency period before late-stage disease. Smo/Smo tumors also display leptomeningeal dissemination of neoplastic cells to the brain and spine, which occurs in many human cases. Despite an extended proliferation of granule neuron precursors (GNP) in the postnatal external granular layer (EGL), the internal granular layer formed normally in Smo/Smo mice and tumor formation occurred only in localized foci on the superficial surface of the molecular layer. Thus, tumor formation is not simply the result of over proliferation of GNPs within the EGL. Moreover, Smo/Smo medulloblastomas were transplantable and serially passaged in vivo, demonstrating the aggressiveness of tumor cells and their transformation beyond a hyperplastic state. The Smo/Smo model is the first mouse medulloblastoma model to show leptomeningeal spread. The adherence to human pathology, high incidence, and early onset of tumors thus make Smo/Smo mice an efficient model for preclinical studies. [Cancer Res 2008;68(6):1768–76]

Although thyroid hormones are crucial for cerebellar development, and several thyroid hormone-dependent genes are known to be correlated with morphological development of the cerebellum, the precise mechanisms of morphological cerebellar changes in hypothyroidism (HT) remain unknown. To investigate these mechanisms in experimental rat HT induced by the anti-thyroid drug methimazole (MMI-HT rat), we carried out gene expression analysis (sonic hedgehog (Shh), reelin, and Bax) using quantitative real-time PCR. Histological examination revealed cerebellar abnormalities, including reductions in the thickness of the molecular layer and delayed disappearance of the external granular layer (EGL), as well as excess bulges or sublobules in the internal granular layer (IGL). At Postnatal Day (P) 6, Shh expression in MMI-HT rat was comparable to that in controls, thus suggesting that Shh expression was sufficient to form the lobes in the initial phase. However, Shh expression decreased in the later phases, as compared with age-matched controls. This demonstrated that stronger and sustained signaling is necessary for partitioning of the cardinal lobes into lobes and sublobes. Although reelin expression was not clearly different from that in controls, Bax expression decreased at P 15. The attrition of Bax at P 15 as well as Shh in the later phase may be related to irregularities in the IGL and the relatively large numbers of internal granular cells. Taken together, these results suggest that Shh expression is related to the morphological cerebellar changes in experimental hypothyroidism and that sustained signaling by Shh may play a key role in normal development, particularly lobulation, in the cerebellum.

Magali Irla, Murielle Saade, Geneviève Chazal, and Catherine Nguyen contributed equally to this work; International audience; We identified the Spatial (Stromal Protein Associated with Thymii and Lymph-node) gene from an adult thymus mouse library of cDNA clones. By RT-PCR, we reported that Spatial was highly expressed in restricted areas of the central nervous system. Here, we characterize the precise cellular localization of Spatial during mouse brain development in the cerebellum, hippocampus and cortex. Five different transcript isoforms have been described for Spatial and among those, only Spatial-epsilon and -beta present a tightly controlled expression. In the cerebellum, Spatial expression is detected in the external precursor granular layer and persists as these cells migrate and differentiate to form the internal granular layer. It is also expressed in differentiating Purkinje cells with a specific somatodendritic distribution. Spatial expression in the hippocampus is spatially and temporally regulated: it is first expressed in the CA3 field, then in CA1 and later in the dentate gyrus. Interestingly, Spatial-beta expression tightly overlaps with the beginning of neuronal differentiation in both structures. Using cultured hippocampal neurons, we show that Spatial also exhibits a somatodendritic distribution and it is concentrated in some synaptic regions. Moreover, the vesicle-like cellular distribution of Spatial protein in dendrites is similar to that described for the kinesin motor protein KIF17. Immunofluorescence analyses show that Spatial colocalizes with KIF17 in dendrites of hippocampal neurons in primary culture. Additionally, coimmunoprecipitation experiments of endogenous proteins from hippocampus confirmed that Spatial and KIF17 physically interact. These findings suggest that Spatial may play a role in neuronal morphogenesis and synaptic plasticity through its interaction with the kinesin motor KIF17 in dendrites.

Migration of immature neurons is essential for forming the cortical layers and nuclei. Impairment of migration results in aberrant neuronal cytoarchitecture, which leads to various neurological disorders. Neurons alter the mode, tempo and rate of migration when they translocate through different cortical layers, but little is known about the mechanisms underlying this process. Here we show that endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) has short-term and cortical-layer-specific effects on granule cell migration in the early postnatal mouse cerebellum. Application of exogenous PACAP significantly slowed the migration of isolated granule cells and shortened the leading process in the microexplant cultures of the postnatal day (P)0-3 cerebella. Interestingly, in the cerebellar slices of P10 mice, application of exogenous PACAP significantly inhibited granule cell migration in the external granular layer (EGL) and molecular layer (ML), but failed to alter the movement in the Purkinje cell layer (PCL) and internal granular layer (IGL). In contrast, application of PACAP antagonist accelerated granule cell migration in the PCL, but did not change the movement in the EGL, ML and IGL. Inhibition of the cAMP signaling and the activity of phospholipase C significantly reduced the effects of exogenous PACAP on granule cell migration. The PACAP action on granule cell migration was transient, and lasted for approximately 2 h. The duration of PACAP action on granule cell migration was determined by the desensitization of its receptors and prolonged by inhibiting the protein kinase C. Endogenous PACAP was present sporadically in the bottom of the ML, intensively in the PCL, and throughout the IGL. Collectively, these results indicated that PACAP acts on granule cell migration as "a brake (stop signal) for cell movement." Furthermore, these results suggest that endogenous PACAP slows granule cell migration when the cells enter the PACAP-rich PCL, and 2 h later the desensitization of PACAP receptors allows the cells to accelerate the rate of migration and to actively move within the PACAP-rich IGL. Therefore, endogenous PACAP may provide a cue that regulates granule cell migration in a cerebellar cortical-layer-specific manner.

Development of the postnatal cerebellum relies on the tight regulation of cell number by morphogens that control the balance between cell proliferation,survival and differentiation. Here, we analyze the role of the serine-protease inhibitor protease nexin 1 (PN-1; SERPINE2) in the proliferation and differentiation of cerebellar granular neuron precursors (CGNPs) via the modulation of their main mitogenic factor, sonic hedgehog (SHH). Our studies show that PN-1 interacts with low-density lipoprotein receptor-related proteins (LRPs) to antagonize SHH-induced CGNP proliferation and that it inhibits the activity of the SHH transcriptional target GLI1. The binding of PN-1 to LRPs interferes with SHH-induced cyclin D1 expression. CGNPs isolated from Pn-1-deficient mice exhibit enhanced basal proliferation rates due to overactivation of the SHH pathway and show higher sensitivity to exogenous SHH. In vivo, the Pn-1 deficiency alters the expression of SHH target genes. In addition, the onset of CGNP differentiation is delayed,which results in an enlarged outer external granular layer. Furthermore, the Pn-1 deficiency leads to an overproduction of CGNPs and to enlargement of the internal granular layer in a subset of cerebellar lobes during late development and adulthood. We propose that PN-1 contributes to shaping the cerebellum by promoting cell cycle exit.

The neuropathologic findings in 31 cases (aged 6 months to 33 years) of Lesch-Nyhan disease (hypoxanthine-guanine phosphoribosyltransferase deficiency) have been previously reported. Herein 2 additional cases, a 10-year-old boy and a 21-year-old man, are described. Both cases had unusual cerebellar abnormalities comprising multifocal internal granular layer atrophy with sparing of the Purkinje layer, one had a slightly small brain, and neither had striatal abnormalities. Careful review of the literature indicates that the most prevalent neuropathologic abnormalities are small cerebrum (13 of 33 cases) and multifocal cerebellar lesions (9 of 33 cases), although these could be underreported. Other authors have disregarded these abnormalities, focusing on the apparently normal basal nuclei, and they have suggested that the clinical neurologic abnormalities are based solely on changes in neurotransmitters. We discuss potential mechanisms of cerebellar damage, suggest that the cerebellar abnormality could in part explain the clinical syndrome, and recommend that cerebellar structure and function should be more carefully studied in Lesch-Nyhan disease.