Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

Role of the actin-binding protein profilin1 in radial migration and glial cell adhesion of granule neurons in the cerebellum

Profilins are small G-actin-binding proteins essential for cytoskeletal dynamics. Of the four mammalian profilin isoforms, profilin1 shows a broad expression pattern, profilin2 is abundant in the brain, and profilin3 and profilin4 are restricted to the testis. In vitro studies on cancer and epithelial cell lines suggested a role for profilins in cell migration and cell-cell adhesion. Genetic studies in mice revealed the importance of profilin1 in neuronal migration, while profilin2 has apparently acquired a specific function in synaptic physiology. We recently reported a mouse mutant line lacking profilin1 in the brain; animals display morphological defects that are typical for impaired neuronal migration. We found that during cerebellar development, profilin1 is specifically required for radial migration and glial cell adhesion of granule neurons. Profilin1 mutants showed cerebellar hypoplasia and aberrant organization of cerebellar cortex layers, with ectopically arranged granule neurons. In this commentary, we briefly introduce the profilin family and summarize the current knowledge on profilin activity in cell migration and adhesion. Employing cerebellar granule cells as a model, we shed some light on the mechanisms by which profilin1 may control radial migration and glial cell adhesion. Finally, a potential implication of profilin1 in human developmental neuropathies is discussed.     

Role of the actin-binding protein profilin1 in radial migration and glial cell adhesion of granule neurons in the cerebellum

Profilins are small G-actin-binding proteins essential for cytoskeletal dynamics. Of the four mammalian profilin isoforms, profilin1 shows a broad expression pattern, profilin2 is abundant in the brain, and profilin3 and profilin4 are restricted to the testis. In vitro studies on cancer and epithelial cell lines suggested a role for profilins in cell migration and cell-cell adhesion. Genetic studies in mice revealed the importance of profilin1 in neuronal migration, while profilin2 has apparently acquired a specific function in synaptic physiology. We recently reported a mouse mutant line lacking profilin1 in the brain; animals display morphological defects that are typical for impaired neuronal migration. We found that during cerebellar development, profilin1 is specifically required for radial migration and glial cell adhesion of granule neurons. Profilin1 mutants showed cerebellar hypoplasia and aberrant organization of cerebellar cortex layers, with ectopically arranged granule neurons. In this commentary, we briefly introduce the profilin family and summarize the current knowledge on profilin activity in cell migration and adhesion. Employing cerebellar granule cells as a model, we shed some light on the mechanisms by which profilin1 may control radial migration and glial cell adhesion. Finally, a potential implication of profilin1 in human developmental neuropathies is discussed.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

The Effects of Tag-1 on the Maturation of Mouse Cerebellar Granule Neurons

The cell adhesion molecule Tag-1 is highly expressed in immature cerebellar granule neurons (CGNs) during axonogenesis and is down-regulated prior to onset of radial migration. However, its precise role(s) during development of mammalian CGNs has been unclear. Here we studied the effects of anti-Tag-1 function blocking antibodies on the development of mouse CGNs in primary cell culture and in situ. Interfering antibodies inhibited axon formation by mouse CGNs in both cell cultures and in cerebellar slices. Effects on axon extension in cell cultures were observed under conditions of homotypic cell–cell contact, consistent with inhibition of cell adhesion activity. Further, when used as a substratum Tag-1 protein strongly stimulated neurite outgrowth by CGNs. Antagonism of Tag-1 also enhanced CGN migration in modified Boyden chamber assays. Radial migration was inhibited by Tag-1 antibodies in cerebellar slices, possibly reflecting a block in early CGN maturation in situ. These findings are consistent with a regulatory role for Tag-1 in axon emergence as well as migratory behavior by developing mouse CGNs.     

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.     

The Immp2l mutation causes age‐dependent degeneration of cerebellar granule neurons prevented by antioxidant treatment

Reactive oxygen species are implicated in age‐associated neurodegeneration, although direct in vivo evidence is lacking. We recently showed that mice with a mutation in the Inner Mitochondrial Membrane Peptidase 2‐like (Immp2l) gene had elevated levels of mitochondrial superoxide, impaired fertility and age‐associated phenotypes, including kyphosis and ataxia. Here we show that ataxia and cerebellar hypoplasia occur in old mutant mice (> 16 months). Cerebellar granule neurons (CGNs) are significantly underrepresented; Purkinje cells and cells in the molecular layer are not affected. Treating mutant mice with the mitochondria‐targeted antioxidant SkQ1 from 6 weeks to 21 months protected cerebellar granule neurons. Apoptotic granule neurons were observed in mutant mice but not in age‐matched normal control mice or SkQ1‐treated mice. Old mutant mice showed increased serum protein carbonyl content, cerebellar 4‐hydroxynonenal (HNE), and nitrotyrosine modification compared to old normal control mice. SOD2 expression was increased in Purkinje cells but decreased in granule neurons of old mutant mice. Mitochondrial marker protein VDAC1 also was decreased in CGNs of old mutant mice, suggesting decreased mitochondrial number. SkQ1 treatment decreased HNE and nitrotyrosine modification, and restored SOD2 and VDAC1 expression in CGNs of old mutant mice. Neuronal expression of nitric oxide synthase was increased in cerebella of young mutant mice but decreased in old mutant mice. Our work provides evidence for a causal role of oxidative stress in neurodegeneration of Immp2l mutant mice. The Immp2l mutant mouse model could be valuable in elucidating the role of oxidative stress in age‐associated neurodegeneration.     

Rap1 is involved in the signal transduction of myelin-associated glycoprotein

Myelin-associated glycoprotein (MAG) is a well-characterized axon growth inhibitor in the adult vertebrate nervous system. Several signals that play roles in inhibiting axon growth have been identified. Here, we report that soluble MAG induces activation of Rap1 in postnatal cerebellar granule neurons (CGNs) and dorsal root ganglion (DRG) neurons. The p75 receptor associates with activated Rap1 and is internalized in response to MAG. After MAG is applied to the distal axons of the sciatic nerves, the activated Rap1, internalized p75 receptor, and MAG are retrogradely trafficked via axons to the cell bodies of the DRG neurons. Rap1 activity is required for survival of the DRG neurons as well as CGNs when treated with MAG. The transport of the signaling complex containing the p75 receptor and Rap1 may play a role in the effect of MAG.     

Tachykinin neuropeptides in cerebellar granule neurons: an immunocytochemical study

We previously demonstrated that exogenously administered neurokinin A and neurokinin B, but not substance P, increased the sensitivity of cultured cerebellar granule neurons (CGNs) to glutamate. In the present study, the presence of tachykinin neuropeptides in CGNs was tested by confocal-based immunofluorescence. We found that neurokinin A and neurokinin B are present in CGNs but absent in astrocytes while substance P is abundant in astrocytes but absent in CGNs. It is postulated that the different localization of tachykinin neuropeptides in CGNs and astroglial cells has a physiological role in the modulation of excitatory transmission.     

LINGO-1-Fc蛋白对低钾诱导小脑颗粒神经元凋亡的保护作用

髓鞘抑制因子Nogo-A、MAG和OMgp通过共同的受体信号复合物NgR/p75NTR(或者TROY)发挥对中枢神经纤维再生的抑制作用.新近克隆的跨膜蛋白LINGO-1是该信号途径的另一个重要组成成分和调节分子.LINGO-1特异表达于中枢神经系统,神经元上的LINGO-1被证明参与调节中枢神经再生的抑制信号,而少突胶质细胞表达的LINGO-1分子参与负调节少突胶质细胞的髓鞘化过程.为探讨LINGO-1分子在神经元凋亡过程中的作用,利用包含LINGO-1分子胞外段LRR和IgC2结构域的Fc融合蛋白作为功能性拮抗剂,研究LINGO-1对低钾诱导的小脑颗粒神经元凋亡的保护作用.利用成熟的Hoechst标记凋亡细胞的方法,观察到经LINGO-1-Fc蛋白预处理2h能够显著阻止小脑颗粒神经元的凋亡.仅包括LRR结构域的GST-LINGO-1与LINGO-1-Fc蛋白,虽同样具有与颗粒神经元的结合活性,但是GST-LINGO-1不能有效地阻止低钾诱导的细胞凋亡.这些结果提示,LINGO-1-Fc蛋白能够阻止低钾诱导的小脑颗粒神经元凋亡,并且这种作用可能是IgC2结构域依赖的.     

Mitochondrial calcium uniporter protein MCU is involved in oxidative stress-induced cell death

Mitochondrial calcium uniporter (MCU) is a conserved Ca²⁺ transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stressinduced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca²⁺ uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca²⁺ uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.     

Effects of ethanol and NAP on cerebellar expression of the neural cell adhesion molecule L1

The neural cell adhesion molecule L1 is critical for brain development and plays a role in learning and memory in the adult. Ethanol inhibits L1-mediated cell adhesion and neurite outgrowth in cerebellar granule neurons (CGNs), and these actions might underlie the cerebellar dysmorphology of fetal alcohol spectrum disorders. The peptide NAP potently blocks ethanol inhibition of L1 adhesion and prevents ethanol teratogenesis. We used quantitative RT-PCR and Western blotting of extracts of cerebellar slices, CGNs, and astrocytes from postnatal day 7 (PD7) rats to investigate whether ethanol and NAP act in part by regulating the expression of L1. Treatment of cerebellar slices with 20 mM ethanol, 10(-12) M NAP, or both for 4 hours, 24 hours, and 10 days did not significantly affect L1 mRNA and protein levels. Similar treatment for 4 or 24 hours did not regulate L1 expression in primary cultures of CGNs and astrocytes, the predominant cerebellar cell types. Because ethanol also damages the adult cerebellum, we studied the effects of chronic ethanol exposure in adult rats. One year of binge drinking did not alter L1 gene and protein expression in extracts from whole cerebellum. Thus, ethanol does not alter L1 expression in the developing or adult cerebellum; more likely, ethanol disrupts L1 function by modifying its conformation and signaling. Likewise, NAP antagonizes the actions of ethanol without altering L1 expression.     

Involvement of a Caspase-3-Like Cysteine Protease in 1-Methyl-4-Phenylpyridinium-Mediated Apoptosis of Cultured Cerebellar Granule Neurons

Abstract: Exposure of various neuronal cells or cell lines to high concentrations of 1-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), results in cell death. Recently, it has been reported that low concentrations of MPP⁺ induce apoptosis in susceptible neurons. We have further characterized MPP⁺-mediated toxicity of cultured cerebellar granule neurons (CGNs) and found that exposure of CGNs to relatively low concentrations of MPP⁺ results in apoptosis, whereas higher concentrations result in necrosis. Cotreatment of CGNs with MPP⁺ and the tetrapeptide inhibitor of caspase-3-like proteases, acetyl-DEVD-CHO, markedly attenuates apoptotic but not necrotic death of these neurons. The more specific inhibitor of caspase-1-like proteases, acetyl-YVAD-CHO, however, was ineffective against MPP⁺ neurotoxicity. Moreover, cytoplasmic extracts prepared from MPP⁺-treated CGNs contain markedly increased protease activity that cleaves the caspase-3 substrate acetyl-DEVD- p -nitroaniline. Finally, the cytoplasmic concentration of the apoptogenic protein cytochrome c was increased in a time-dependent fashion in MPP⁺-treated CGNs before the onset of apoptosis. Our data confirm that the neurotoxicity of MPP⁺ is due to both necrosis and apoptosis and suggest that the latter is mediated by activation of a caspase-3-like protease.     

Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit

How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.     

Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

In mouse cerebellar granule neurons (CGNs) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase and phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs.