Subcellular TSC22D4 Localization in Cerebellum Granule Neurons of the Mouse Depends on Development and Differentiation

We previously demonstrated that TSC22D4, a protein encoded by the TGF-β1-activated gene Tsc22d4 (Thg-1pit) and highly expressed in postnatal and adult mouse cerebellum with multiple post-translationally modified protein forms, moves to nucleus when in vitro differentiated cerebellum granule neurons (CGNs) are committed to apoptosis by hyperpolarizing KCl concentrations in the culture medium. We have now studied TSC22D4 cytoplasmic/nuclear localization in CGNs and Purkinje cells: (1) during CGN differentiation/maturation in vivo, (2) during CGN differentiation in vitro, and (3) by in vitro culturing ex vivo cerebellum slices under conditions favoring/inhibiting CGN/Purkinje cell differentiation. We show that TSC22D4 displays both nuclear and cytoplasmic localizations in undifferentiated, early postnatal cerebellum CGNs, irrespectively of CGN proliferation/migration from external to internal granule cell layer, and that it specifically accumulates in the somatodendritic and synaptic compartments when CGNs mature, as indicated by TSC22D4 abundance at the level of adult cerebellum glomeruli and apparent lack in CGN nuclei. These features were also observed in cerebellum slices cultured in vitro under conditions favoring/inhibiting CGN/Purkinje cell differentiation. In vitro TSC22D4 silencing with siRNAs blocked CGN differentiation and inhibited neurite elongation in N1E-115 neuroblastoma cells, pinpointing the relevance of this protein to CGN differentiation.     

Depolarization-dependent survival of cultured mouse cerebellar granule neurons is strain-restrained

Cerebellar granule neurons (CGNs) isolated from the rat are often used as a model system for the analysis of activity-dependent survival of neurons. These cells do not survive in culture without addition of a depolarizing agent (KCl or glutamate) to the medium. However, it has been reported that mouse CGN behave differently. Here we found that the requirement for depolarization for the survival of the mouse CGN was strain-dependent, which may be important for future analyses using transgenic animals. CGNs from the Balb/C mouse could survive without KCl addition, whereas CGNs from the C57Bl/6 mouse could not (similar to the rat CGN). The survival-promoting activity of the Balb/C mouse CGNs was transferable by coculturing. However, the medium of the Balb/C mouse CGN culture was ineffective, suggesting that the neurotrophic substance that is supposed to be released is labile. Although we are yet to specify the substance, it might be independent of neurotrophins. It is necessary to select the strain of mouse in the production of transgenic animals for the analyses of activity-dependent neuronal survival.     

Eph/ephrin gradients in the retinotectal system of Rana pipiens: Developmental and adult expression patterns

Eph/ephrin‐receptor/ligand A and B families play a variety of roles during CNS development, including patterning the retinotectal projection. However, the alignment of their expression gradients with developing retinotectal maps and gradients of cellular development is not well understood in species whose midbrain tecta undergo a protracted anterior to posterior development. By using anatomical tracing methods and ³H‐thymidine neuronography, we have mapped the retinotectal projection and the spatiotemporal progression of tectal cellular development onto Eph/ephrin expression patterns in the tectum of larval Rana pipiens, as studied by means of in situ affinity analysis with fusion proteins. EphA expression is maximal in anterior tectum (and temporal retina); ephrin‐A expression is maximal at the posterior pole (and nasal retina). EphB expression is graded in the early larva, where it is maximal in the posterior tectum just anterior to the posterior pole (and in the ventral retina). Tectal EphB expression becomes uniform at later stages and remains so in the adult, although its retinal expression remains maximal ventrally. In the early larva, EphA, EphB, and ephrin‐A protein gradients are parallel to each other and align with the temporonasal axis of the retinal projection. The early EphB expression maximum overlaps the boundary between the mantle layer of newly postmitotic cells and the posterior, epithelial region of cell proliferation, suggesting that the expression maximum is associated with the initial migrations of the postmitotic cells. Ephrin‐B expression was detected in the olfactory bulb and dorsal retina at all ages, but not in the tectum. J. Comp. Neurol. 514:30–48, 2009. © 2009 Wiley‐Liss, Inc.     

Differential effects of AMPA receptor activation on survival and neurite integrity during neuronal development

While neuronal cultures are an established model for analyzing excitotoxic brain injury in the adult, in vitro systems have not been extensively employed to study how developing neurons respond to levels of excitatory compounds that are lethal to mature neurons. Recently, we reported that the in vivo differentiation programs of cerebellar granule cells (CGNs) are recapitulated in purified CGN cultures [Manzini M.C., Ward M.S., Zhang Q., Lieberman M.D., Mason C.A. (2006) The stop-signal revised: immature cerebellar granule neurons in the external germinal layer arrest pontine mossy fiber growth. J. Neurosci. 26:6040-6051]. Here, we have used this model system to compare the response of immature and mature neurons to excitotoxic compounds. We found that immature CGNs are less sensitive to AMPA receptor (AMPA-R) activation than mature cells and that levels of AMPA-R expression on the plasma membrane are critical in regulating the balance between death and survival during maturation of these neurons. However, the majority of immature cells that survive excitotoxic treatment bear a degenerating neurite, suggesting that AMPA-R activation can still cause damage in the absence of cell death.     

Localized deposition of M-cadherin in the glomeruli of the granular layer during the postnatal development of mouse cerebellum

M-cadherin is a Ca²⁺-dependent cell adhesion molecule of the cadherin family, initially localized at the areas of contact between myotubes during myogenesis, but also detected in the peripheral nerve and at the adult neuromuscular junction. In this study, searching for the expression of M-cadherin in the adult mouse brain, we observed a restricted expression of M-cadherin in one of the three layers of the cerebellar cortex: the granular layer. M-cadherin was accumulated in structures rich in synapses and other intercellular junctions where mossy fibers connect granule cell dendrites, the glomeruli. This molecule was not expressed in the cerebellum during the first steps of postnatal cerebellar neurogenesis: granule cell proliferation and migration and Purkinje cell alignment. M-cadherin expression was first detected at postnatal day (P)11, after the establishment of the synaptic connections between mossy fibers and granule cell dendrites. It then accumulated in glomeruli during their phase of maturation which is characterized by the formation of puncta adherentia between granule cell dendrites. M-cadherin was undetectable in the cerebella of the weaver and staggerer mutants, lacking granule cells, and therefore mature glomeruli and puncta adherentia. Furthermore, other components classically associated with intercellular junctions, i.e., α-caterin, β-catenin and actin filaments, closely paralleled M-cadherin appearance and colocalized with M-cadherin in the mature glomeruli. M-cadherin, which appears as a molecular marker of glomerulus maturation, might be implicated in the formation, and be the ligand, of adherens junctions encountered in this structure. J. Comp. Neurol. 378:180–195, 1997. © 1997 Wiley-Liss, Inc.     

Action of thyroxine on the survival and neurite maintenance of cerebellar granule neurons in culture

Developmental hypothyroidism causes severe impairments in the cerebellum. To understand the role of thyroid hormones (THs) in cerebellar development, we examined the effect of three different THs, thyroxine (T4), 3,5,3′‐triidothyronine (T3), and 3,3′,5′‐triiodothyronine (reverse T3; rT3), on the survival and morphology of cerebellar granule neurons (CGNs) in culture and found novel actions specific to T4. Rat CGNs obtained at postnatal day 6 were first cultured for 2 days in serum‐containing medium with 25 mM K⁺ (K25), then switched to serum‐free medium with physiological 5 mM K⁺ (K5) or with K25 and cultured for an additional 2 or 4 days. CGNs underwent apoptosis in K5 but survived in K25. Addition of T4 at concentrations of 100–200 nM but not T3 or rT3 rescued CGNs from cell death in K5 in a dose‐dependent manner. Furthermore, 200 nM T4 was also effective in maintaining the neurites of CGNs in K5. In K5, T4 suppressed tau phosphorylation at two developmentally regulated sites as well as phosphorylation of c‐jun N‐terminal kinase (JNK) necessary for its activation and localization to axons. These results suggest that, during cerebellar development, T4 exerts its activity in cell survival and neurite maintenance in a manner distinct from the other two thyroid hormones through regulating the activity and localization of JNK. © 2014 Wiley Periodicals, Inc.     

E‐Cadherin enhances neuregulin signaling and promotes Schwann cell myelination

In myelinating Schwann cells, E‐cadherin is a component of the adherens junctions that stabilize the architecture of the noncompact myelin region. In other cell types, E‐cadherin has been considered as a signaling receptor that modulates intracellular signal transduction and cellular responses. To determine whether E‐cadherin plays a regulatory role during Schwann cell myelination, we investigated the effects of E‐cadherin deletion and over‐expression in Schwann cells. In vivo, Schwann cell‐specific E‐cadherin ablation results in an early myelination delay. In Schwann cell‐dorsal root ganglia neuron co‐cultures, E‐cadherin deletion attenuates myelin formation and shortens the myelin segment length. When over‐expressed in Schwann cells, E‐cadherin improves myelination on Nrg1 type III^+/? neurons and induces myelination on normally non‐myelinated axons of sympathetic neurons. The pro‐myelinating effect of E‐cadherin is associated with an enhanced Nrg1‐erbB receptor signaling, including activation of the downstream Akt and Rac. Accordingly, in the absence of E‐cadherin, Nrg1‐signaling is diminished in Schwann cells. Our data also show that E‐cadherin expression in Schwann cell is induced by axonal Nrg1 type III, indicating a reciprocal interaction between E‐cadherin and the Nrg1 signaling. Altogether, our data suggest a regulatory function of E‐cadherin that modulates Nrg1 signaling and promotes Schwann cell myelin formation. GLIA 2015;63:1522–1536     

Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

1-Methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity has previously been attributed to either caspase-dependent apoptosis or caspase-independent cell death. In the current study, we found that MPP(+) induces a unique, non-apoptotic nuclear morphology coupled with a caspase-independent but calpain-dependent mechanism of cell death in primary cultures of rat cerebellar granule neurons (CGNs). Using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in CGNs exposed to MPP(+), we observed that these neurons are essentially devoid of caspase-dependent DNA fragments indicative of apoptosis. Moreover, proteolysis of a well recognized caspase-3 substrate, poly (ADP ribose) polymerase (PARP), was not observed in CGNs exposed to MPP(+). In contrast, calpain-dependent proteolysis of fodrin and pro-caspases-9 and -3 occurred in this model coupled with inhibition of caspase-3/-7 activities. Notably, several key members of the Bcl-2 protein family appear to be prominent calpain targets in MPP(+)-treated CGNs. Bid and Bax were proteolyzed to truncated forms thought to have greater pro-death activity at mitochondria. Moreover, the pro-survival Bcl-2 protein was degraded to a form predicted to be inactive at mitochondria. Cyclin E was also cleaved by calpain to an active low MW fragment capable of facilitating cell cycle re-entry. Finally, MPP(+)-induced neurotoxicity in CGNs was significantly attenuated by a cocktail of calpain and caspase inhibitors in combination with the antioxidant glutathione. Collectively, these results demonstrate that caspases do not play a central role in CGN toxicity induced by exposure to MPP(+), whereas calpain cleavage of key protein targets, coupled with oxidative stress, plays a critical role in MPP(+)-induced neurotoxicity. Our findings underscore the complexity of MPP(+)-induced neurotoxicity and suggest that calpain may play a fundamental role in causing neuronal death downstream of mitochondrial oxidative stress and dysfunction.     

A peptide derived from a trans‐homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N‐terminal immunoglobulin (Ig)‐like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis‐interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans‐interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding. © 2010 Wiley‐Liss, Inc.     

Microglia mediate diesel exhaust particle-induced cerebellar neuronal toxicity through neuroinflammatory mechanisms

In addition to the well-established effects of air pollution on the cardiovascular and respiratory systems, emerging evidence has implicated it in inducing negative effects on the central nervous system. Diesel exhaust particulate matter (DEP), a major component of air pollution, is a complex mixture of numerous toxicants. Limited studies have shown that DEP-induced dopaminergic neuron dysfunction is mediated by microglia, the resident immune cells of the brain. Here we show that mouse microglia similarly mediate primary cerebellar granule neuron (CGN) death in vitro. While DEP (0, 25, 50, 100 μg/2 cm²) had no effect on CGN viability after 24 h of treatment, in the presence of primary cortical microglia neuronal cell death increased by 2–3-fold after co-treatment with DEP, suggesting that microglia are important contributors to DEP-induced CGN neurotoxicity. DEP (50 μg/2 cm²) treatment of primary microglia for 24 h resulted in morphological changes indicative of microglia activation, suggesting that DEP may induce the release of cytotoxic factors. Microglia-conditioned medium after 24 h treatment with DEP, was also toxic to CGNs. DEP caused a significant increase in reactive oxygen species in microglia, however, antioxidants failed to protect neurons from DEP/microglia-induced toxicity. DEP increased mRNA levels of the pro-inflammatory cytokines IL-6 and IL1-β, and the release of IL-6. The antibiotic minocycline (50 μM) and the peroxisome proliferator-activated receptor-γ agonist pioglitazone (50 μM) attenuated DEP-induced CGN death in the co-culture system. Microglia and CGNs from male mice appeared to be somewhat more susceptible to DEP neurotoxicity than cells from female mice possibly because of lower paraoxonase-2 expression. Together, these results suggest that microglia-induced neuroinflammation may play a critical role in modulating the effect of DEP on neuronal viability. .     

Activation of glycogen synthase kinase 3 beta (GSK-3beta) by platelet activating factor mediates migration and cell death in cerebellar granule neurons

Children with vertically acquired HIV-1 can present with a rapidly progressive encephalopathy and neuronal apoptosis in the first 12-18 months of life. Furthermore, abnormal prenatal platelet activating factor (PAF) signalling may result in lissencephaly, a disorder of neuronal migration. PAF, produced from human immunodeficiency virus type 1 (HIV-1) -infected brain-resident macrophages, induces neuronal apoptosis in cultured cerebellar granule neurons (CGNs) in part by activating glycogen synthase kinase 3 beta (GSK-3beta). However, PAF can also inhibit migration of CGNs that are dispersed and allowed to reaggregate. Therefore, we investigated the biological effects following activation of GSK-3beta by PAF, and whether these effects were dependent on the culture conditions of the CGNs. We show here that activation of neuronal GSK-3beta by PAF is receptor-specific, with similar kinetics of activation in both monolayer cultures of CGNs that have ceased to migrate and reaggregate cultures of CGNs that are actively migrating. However, PAF receptor activation in reaggregated CGNs inhibits neuronal migration and induces approximately half the level of neuronal apoptosis compared with PAF-treated CGN cultures that have ceased to migrate. PAF-mediated inhibition of neuronal migration in reaggregated CGNs or induction of apoptosis in CGNs that have ceased to migrate can be reversed by either PAF receptor antagonists, or the GSK-3beta inhibitors lithium or valproic acid, in a dose-dependent manner. Abnormal PAF signalling that results in GSK-3beta overactivation may represent a common mechanism for pathological defects in neuronal migration in the prenatal period and neuronal apoptosis in the postnatal period.     

Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design‐based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5–10‐year‐old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5–10‐year‐old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.     

Survival promotion of rat cerebellar granule neurons by co-culture with pontine explant

Cultured cerebellar granule neuron (CGN) of the rat is the most frequently used model system for analysis of activity-dependent neuronal survival. CGNs do not survive longer than 2 weeks in a standard culture medium unless KCl (or other excitants such as glutamate) is added. It is assumed that KCl represents synaptic activity, but no tests have been made on whether the survival of CGNs really depends on the synaptic input. Here we co-cultured CGNs with an explant of the pons including the basilar pontine nucleus (BPN), which is one of the input sources of CGNs in vivo, to confirm if synaptic input is really a determinant for the survival of these cells. In this co-culture system, the viability of CGNs was significantly increased without the addition of KCl. The survival promotion was confined to the population of CGNs having contact with neurites of BPN and was cancelled by an application of tetrodotoxin or antagonists of glutamate receptors, indicating that the survival depended on synaptic activity. Explants of other glutamatergic tissues including the hippocampus failed to promote the survival, although neurites grew out from these explants as vigorously as from the BPN explants. Calcium and FM1-43 imaging examinations revealed that the CGNs had formed functional synapses with the BPN explant but not with the hippocampal explant. These results, confirming the assumption that synaptic activity determines neuronal survival, provide evidence for presynaptic contribution to the survival.     

Loss of NECL1, a novel tumor suppressor,can be restored in glioma by HDAC inhibitor‐Trichostatin A through Sp1 binding site

Nectin‐like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue‐specific immunoglobulin‐like cell–cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re‐expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at ?271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor‐Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co‐immunoprecipitation (co‐IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation. © 2008 Wiley‐Liss, Inc.     

Cbln1 downregulates the formation and function of inhibitory synapses in mouse cerebellar Purkinje cells

The formation of excitatory and inhibitory synapses must be tightly coordinated to establish functional neuronal circuitry during development. In the cerebellum, the formation of excitatory synapses between parallel fibers and Purkinje cells is strongly induced by Cbln1, which is released from parallel fibers and binds to the postsynaptic δ2 glutamate receptor (GluD2). However, Cbln1's role, if any, in inhibitory synapse formation has been unknown. Here, we show that Cbln1 downregulates the formation and function of inhibitory synapses between Purkinje cells and interneurons. Immunohistochemical analyses with an anti‐vesicular GABA transporter antibody revealed an increased density of interneuron–Purkinje cell synapses in the cbln1‐null cerebellum. Whole‐cell patch‐clamp recordings from Purkinje cells showed that both the amplitude and frequency of miniature inhibitory postsynaptic currents were increased in cbln1‐null cerebellar slices. A 3‐h incubation with recombinant Cbln1 reversed the increased amplitude of inhibitory currents in Purkinje cells in acutely prepared cbln1‐null slices. Furthermore, an 8‐day incubation with recombinant Cbln1 reversed the increased interneuron–Purkinje cell synapse density in cultured cbln1‐null slices. In contrast, recombinant Cbln1 did not affect cerebellar slices from mice lacking both Cbln1 and GluD2. Finally, we found that tyrosine phosphorylation was upregulated in the cbln1‐null cerebellum, and acute inhibition of Src‐family kinases suppressed the increased inhibitory postsynaptic currents in cbln1‐null Purkinje cells. These findings indicate that Cbln1–GluD2 signaling inhibits the number and function of inhibitory synapses, and shifts the excitatory–inhibitory balance towards excitation in Purkinje cells. Cbln1's effect on inhibitory synaptic transmission is probably mediated by a tyrosine kinase pathway.     

Neurotrophin‐3 promotes cerebellar granule cell exit from the EGL

In the cerebellum, the mRNAs for neurotrophin-3 (NT-3) and its high-affinity tyrosine kinase receptor trkC are expressed by both the differentiated granule cells of the internal granule cell layer (IGL) and their precursors in the external germinal layer (EGL). We have investigated the effects of chronic application of exogenous NT-3 in vivo on cerebellar granule cell genesis and differentiation. NT-3 was applied to the posterior surface of the rat cerebellum from P6 onwards using Elvax implants. At P10 the EGL of cerebellar lobules VII and VIII was significantly reduced in thickness in NT-3 implanted rats when compared with controls. Immunocytochemical analysis of the EGL using antibodies to proliferating cell nuclear antigen (PCNA) revealed that the number of postmitotic, premigratory (PCNA-immunonegative) granule cell precursors was preferentially reduced in the NT-3 implanted rats. In situ DNA fragmentation labelling confirmed that this was not accompanied by increased cell death in the EGL. These results suggest that NT-3 promotes the differentiation of postmitotic, premigratory granule cell precursors, accelerating cell exit from the EGL.     

Expression of AMPA-type glutamate receptors in HEK cells and cerebellar granule neurons impairs CXCL2-mediated chemotaxis

We find that cerebellar granule neurons (CGN) obtained from newborn rats (p3) migrate in response to both CXC chemokine ligand-2 (CXCL2) and -12 (CXCL12), while CGN from p7 rats are unresponsive to CXCL2. The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor 1 (GluR1) greatly impairs the chemotaxis induced by CXCL2 in CXCR2-expressing HEK cells. By immunoprecipitation, we show that CXCR2 is associated with AMPA receptors (AMPARs) in p7 CGN, and with GluR1 co-expressed in HEK cells. Taken together, these results suggest that the association between CXCR2 and AMPARs results in the inhibition of CXCL2-dependent chemotaxis, and may represent a molecular mechanism underlying the modulation of nerve cell migration.