Preclinical Study on Biodistribution of Mesenchymal Stem Cells after Local Transplantation into the Brain.

Therapeutic efficacy of mesenchymal stem cells (MSCs) is determined by biodistribution and engraftment in vivo. Compared to intravenous infusion, biodistribution of locally transplanted MSCs are partially understood. Here, we performed a pharmacokinetics (PK) study of MSCs after local transplantation. We grafted human MSCs into the brains of immune-compromised nude mice. Then we extracted genomic DNA from brains, lungs, and livers after transplantation over a month. Using quantitative polymerase chain reaction with human Alu-specific primers, we analyzed biodistribution of the transplanted cells. To evaluate the role of residual immune response in the brain, MSCs expressing a cytosine deaminase (MSCs/CD) were used to ablate resident immune cells at the injection site. The majority of the Alu signals mostly remained at the injection site and decreased over a week, finally becoming undetectable after one month. Negligible signals were transiently detected in the lung and liver during the first week. Suppression of Iba1-positive microglia in the vicinity of the injection site using MSCs/CD prolonged the presence of the Alu signals. After local transplantation in xenograft animal models, human MSCs remain predominantly near the injection site for limited time without disseminating to other organs. Transplantation of human MSCs can locally elicit an immune response in immune compromised animals, and suppressing resident immune cells can prolong the presence of transplanted cells. Our study provides valuable insights into the in vivo fate of locally transplanted stem cells and a local delivery is effective to achieve desired dosages for neurological diseases.

Cell Type-specific Knockout with Gli1-mediated Cre Recombination in the Developing Cerebellum.

The inducible Cre-loxP system provides a useful tool for inducing the selective deletion of genes that are essential for proper development and enables the study of gene functions in properly developed animals. Here, we show that inducible Cre-loxP driven by the Gli1-promoter can induce cell-type-specific deletion of target genes in cerebellar cortical neurons. We used reporter mice containing the YFP (yellow fluorescence protein) gene at the Gt(ROSA)26Sor locus with a loxP-flanked transcriptional stop sequence, in which successful Cre-mediated excision of the stop sequence is indicated by YFP expression in Cre-expressing cells. Administration of tamoxifen during early postnatal days (P4~7) induces Cre-dependent excision of stop sequences and allows YFP expression in proliferating neuronal progenitor cells in the external granule layer and Bergmann glia in the Purkinje cell layer. A substantial number of YFP-positive progenitor cells in the external granule layer migrated to the internal granule cell layer and became granule cell neurons. By comparison, injection of tamoxifen during late postnatal days (P19~22) induces YFP expression only in Bergmann glia, and most granule cell neurons were devoid of YFP expression. The results indicate that the Gli1 promoter is temporarily active in progenitor cells in the external granule layer during the early postnatal period but constitutively active in Bergmann glia. We propose that the Gli1-mediated CreER system can be applied for the conditional deletion of genes of interest from cerebellar granule cell neurons and/or Bergmann glia.

ERK Regulates NeuroD1-mediated Neurite Outgrowth via Proteasomal Degradation.

Neurogenic differentiation 1 (NeuroD1) is a class B basic helix-loop-helix (bHLH) transcription factor and regulates differentiation and survival of neuronal and endocrine cells by means of several protein kinases, including extracellular signal-regulated kinase (ERK). However, the effect of phosphorylation on the functions of NeuroD1 by ERK has sparked controversy based on context-dependent differences across diverse species and cell types. Here, we evidenced that ERK-dependent phosphorylation controlled the stability of NeuroD1 and consequently, regulated proneural activity in neuronal cells. A null mutation at the ERK-dependent phosphorylation site, S274A, increased the half-life of NeuroD1 by blocking its ubiquitin-dependent proteasomal degradation. The S274A mutation did not interfere with either the nuclear translocation of NeuroD1 or its heterodimerization with E47, its ubiquitous partner and class A bHLH transcription factor. However, the S274A mutant increased transactivation of the E-box-mediated gene and neurite outgrowth in F11 neuroblastoma cells, compared to the wild-type NeuroD1. Transcriptome and Gene Ontology enrichment analyses indicated that genes involved in axonogenesis and dendrite development were downregulated in NeuroD1 knockout (KO) mice. Overexpression of the S274A mutant salvaged neurite outgrowth in NeuroD1-deficient mice, whereas neurite outgrowth was minimal with S274D, a phosphomimicking mutant. Our data indicated that a longer protein half-life enhanced the overall activity of NeuroD1 in stimulating downstream genes and neuronal differentiation. We propose that blocking ubiquitin-dependent proteasomal degradation may serve as a strategy to promote neuronal activity by stimulating the expression of neuron-specific genes in differentiating neurons.

Deletion of the α subunit of the heterotrimeric Go protein impairs cerebellar cortical development in mice.

Go is a member of the pertussis toxin-sensitive Gi/o family. Despite its abundance in the central nervous system, the precise role of Go remains largely unknown compared to other G proteins. In the present study, we explored the functions of Go in the developing cerebellar cortex by deleting its gene, Gnao. We performed a histological analysis with cerebellar sections of adult mice by cresyl violet- and immunostaining. Global deletion of Gnao induced cerebellar hypoplasia, reduced arborization of Purkinje cell dendrites, and atrophied Purkinje cell dendritic spines and the terminal boutons of climbing fibers from the inferior olivary nucleus. These results indicate that Go-mediated signaling pathway regulates maturation of presynaptic parallel fibers from granule cells and climbing fibers during the cerebellar cortical development.

Spatiotemporal Protein Atlas of Cell Death-Related Molecules in the Rat MCAO Stroke Model.

Ischemic stroke and cerebral infarction triggered by the blockage of blood supply can cause damage to the brain via a complex series of pathological changes. Recently, diverse therapies have emerged as promising candidates for the treatment of stroke. These treatments exert therapeutic effects by acting on diverse target molecules and cells in different time windows from the acute to chronic phases. Here, using immunohistochemistry, we show pathophysiological changes in the brain microenvironment at the hyperacute (within 6 h), acute (1~3 days), subacute (7 days), and chronic (1 month) phases following ischemic injury. Ischemic injury in rats was induced by occluding the middle cerebral artery and was validated by magnetic resonance imaging. The progression of damage to the brain was evaluated by immunohistochemistry for NeuN+ neurons, GFAP+ astrocytes, and Iba1+ microglia, and by the emergence of the cell death-related molecules such as AIF, FAF1, and activated caspase-3. Our data regarding the spatial and temporal information on pathophysiological changes may warrant the investigation of the timing of administration of therapeutic treatments in preclinical studies with an animal model of stroke.

Development of the main olfactory system and main olfactory epithelium-dependent male mating behavior are altered in Go-deficient mice.

In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). The heterotrimeric GTP-binding protein Go is widely expressed in the MOE and VNO of mice. Early studies indicated that Go expression in VNO sensory neurons is critical for directing social and sexual behaviors in female mice [Oboti L, et al. (2014) BMC Biol 12:31]. However, the physiological functions of Go in the MOE have remained poorly defined. Here, we examined the role of Go in the MOE using mice lacking the α subunit of Go Development of the olfactory bulb (OB) was perturbed in mutant mice as a result of reduced neurogenesis and increased cell death. The balance between cell types of OB interneurons was altered in mutant mice, with an increase in the number of tyrosine hydroxylase-positive interneurons at the expense of calbindin-positive interneurons. Sexual behavior toward female mice and preference for female urine odors by olfactory sensory neurons in the MOE were abolished in mutant male mice. Our data suggest that Go signaling is essential for the structural and functional integrity of the MOE and for specification of OB interneurons, which in turn are required for the transmission of pheromone signals and the initiation of mating behavior with the opposite sex.

AKT-independent Reelin signaling requires interactions of heterotrimeric Go and Src.

Reelin, a large secreted extracellular matrix glycoprotein, plays a key role in neuronal migration during cortical development and promotes neuronal maturation. The signaling pathway regulating neuronal maturation in the postnatal period are relatively less well understood. In this study, we demonstrated that a heterotrimeric G protein, Go, is a novel target of Reelin-induced signaling to promote neurite outgrowth. In primary hippocampal neurons of Reelin-deficient reeler mice, neurite outgrowth was significantly reduced and rescued upon addition of Reelin. Pertussis toxin (PTX) treatment or transfection with Gαo-siRNA suppressed Reelin-mediated neurite outgrowth in wild-type neurons. Additionally, Reelin treatment led to increased phosphorylation of AKT, GSK3ß, and JNK, which were all effectively blocked by the PI3K inhibitor, LY294002. By comparison, PTX specifically blocked JNK activation, but not AKT and GSK3ß. Immunoprecipitation assays disclosed that Reelin increases the active forms of both Src and Gαo and promotes their direct association. Notably, Dab1, a cytoplasmic adaptor molecule that mediates Reelin signaling, did not interact with Gαo. Neurite outgrowth by Reelin was induced via activating Src kinase, which directly stimulated Gαo, activity, leading to JNK activation. Based on the collective findings, we suggest that Reelin-dependent signaling mechanisms may be split into Src-AKT-dependent and Src-Go-dependent pathways. Our results additionally provide evidence that Reelin receptors cross-communicate with heterologous G protein-coupled receptors (GPCR) independently of the cognate ligands of GPCR.

Erratum: Analysis of Dosage Mutation in PARK2 among Korean Patients with Early-Onset or Familial Parkinson's Disease.

[This corrects the article on p. 244 in vol. 10, PMID: 25045378.].

Analysis of Dosage Mutation in PARK2 among Korean Patients with Early-Onset or Familial Parkinson's Disease.

BACKGROUND AND PURPOSE: There is some controversy regarding heterozygous mutations of the gene encoding parkin (PARK2) as risk factors for Parkinson's disease (PD), and all previous studies have been performed in non-Asian populations. Dosage mutation of PARK2, rather than a point mutation or small insertion/deletion mutation, was reported to be a risk factor for familial PD; dosage mutation of PARK2 is common in Asian populations. METHODS: We performed a gene-dosage analysis of PARK2 using real-time polymerase chain reaction for 189 patients with early-onset PD or familial PD, and 191 control individuals. In the case of PD patients with heterozygous gene-dosage mutation, we performed a sequencing analysis to exclude compound heterozygous mutations. The association between heterozygous mutation of PARK2 and PD was tested. RESULTS: We identified 22 PD patients with PARK2 mutations (11.6%). Five patients (2.6%) had compound heterozygous mutations, and 13 patients (6.9%) had a heterozygous mutation. The phase could not be determined in one patient. Three small sequence variations were found in 30 mutated alleles (10.0%). Gene-dosage mutation accounted for 90% of all of the mutations found. The frequency of a heterozygous PARK2 gene-dosage mutation was higher in PD patients than in the controls. CONCLUSIONS: Heterozygous gene-dosage mutation of PARK2 is a genetic risk factor for patients with early-onset or familial PD in Koreans.

Simultaneous deletion of floxed genes mediated by CaMKIIα-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goα.

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIα-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIα-Cre(+/Cre) mice generated by crossing CaMKIIα-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIα-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIα-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Δ)) without inheriting the Cre transgene. The Gnao(Δ/Δ) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIα-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIα-Cre mice as breeding pairs.

Association of mutations in the glucocerebrosidase gene with Parkinson disease in a Korean population.

Recent studies have shown an association between Parkinson disease (PD) and mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA), which is deficient in patients with Gaucher disease. In Asian populations, 2 mutational analysis studies have been performed in all exons of GBA; one study in a Japanese population showed the highest odds ratio among all ethnic groups, whereas the other study in ethnic Chinese observed a trend of a higher frequency of GBA mutation in PD patients without statistical significance. To investigate whether there is an association between PD and mutations of GBA in a Korean population, we analyzed mutations of GBA and compared mutation frequencies between Korean PD patients and a control population. We analyzed mutations in GBA by sequencing exons of GBA in 277 Korean PD patients and 291 control subjects. All exons of GBA were sequenced in all PD cases and 100 control subjects. Exon 2 and exons 5-11, where mutations of GBA were found in our PD patients, were analyzed in an additional 191 control subjects. Five different pathogenic heterozygous GBA mutations, including N188S, P201H, R257Q, S271G, and L444P, were identified in 9 PD cases (3.2%), whereas there were no GBA mutations found in control subjects (p<0.01, OR 20.6, 95% CI 1.2-356.4). The mean age-at-onset of heterozygous GBA variants carriers was younger than that of non-carriers (48.6±11.9 versus 57.9±13.5, p<0.05, Mann-Whitney test). Our results suggest that heterozygous mutations of GBA represent a risk factor for PD in Koreans.

Analysis of mutations and the association between polymorphisms in the cerebral dopamine neurotrophic factor (CDNF) gene and Parkinson disease.

Neurotrophic factors support the survival of dopaminergic neurons. The cerebral dopamine neurotrophic factor (CDNF) is a novel neurotrophic factor with strong trophic activity on dopaminergic neurons comparable to that of glial cell line-derived neurotrophic factor (GDNF). To investigate whether rare or common variants in CDNF are associated with Parkinson disease (PD), we performed mutation analysis of CDNF and a genetic association study between CDNF polymorphisms and PD. We screened 110 early-onset Parkinson disease (EOPD) patients for CDNF mutations. Allelic and genotype frequencies of 3 CDNF single nucleotide polymorphisms (SNPs) (rs1901650, rs7094179, and rs11259365) were compared in 215 PD patients and age- and sex-matched controls. We failed to identify any mutations in CDNF among the EOPD patient sample population. We observed a trend towards increased risk for PD in patients carrying the C allele of SNP rs7094179 (odds ratio (OR)=1.27, 95% confidence interval (CI) 0.96-1.67). Patients carrying the C allele were susceptible to PD in both dominant (CC+CA vs. AA; OR=7.20, 95% CI 0.88-59.1) and recessive (CA+AA vs. CC; OR=0.64, 95% CI 0.41-0.99) models. Genotype and allele frequencies of SNPs rs1901650 and rs11259365 did not differ between PD patients and controls. Our study suggests that the C allele of an intronic CDNF SNP (rs7094179) might be an allele for susceptibility to PD. Further studies with larger sample size are required to confirm our results.

Analysis of PARK genes in a Korean cohort of early-onset Parkinson disease.

Mutations in five PARK genes (SNCA, PARKIN, DJ-1, PINK1, and LRRK2) are well-established genetic causes of Parkinson disease (PD). Recently, G2385R substitution in LRRK2 has been determined as a susceptibility allele in Asian PD. The objective of this study is to determine the frequency of mutations in these PARK genes in a Korean early-onset Parkinson disease (EOPD) cohort. The authors sequenced 35 exons in SNCA, PARKIN, DJ-1, PINK1, and LRRK2 in 72 unrelated EOPD (age-at-onset

Compartmentalization of protein kinase A signaling by the heterotrimeric G protein Go.

G(o), a member of the G(o/i) family, is the most abundant heterotrimeric G protein in brain. Most functions of G(o) are mediated by the G(betagamma) dimer; effector(s) for its alpha-subunit have not been clearly defined. Here we report that G(oalpha) interacts directly with cAMP-dependent protein kinase (PKA) through its GTPase domain. This interaction did not inhibit the kinase function of PKA but interfered with nuclear translocation of PKA while sparing its cytosolic function. This regulatory mechanism by which G(o) bifurcates PKA signaling may provide insights into how G(o) regulates complex processes such as neuritogenesis, synaptic plasticity, and cell transformation.

Differential regulation of tyrosine hydroxylase expression by sonic hedgehog.

Sonic hedgehog functions to induce floor plate in early stages, and spinal motor neurons and midbrain dopaminergic neurons in later stages of development. Here, we investigated the effects of sonic hedgehog on tyrosine hydroxylase expression in three cell lines that correspond to different stages of neural development. Sonic hedgehog increased the tyrosine hydroxylase gene expression in pluripotent P19 cells but repressed it in tyrosine hydroxylase-producing PC12 cells. Promoter analysis in mouse neural stem cells indicated that the N-terminal of sonic hedgehog repressed both the basal and cAMP-dependent protein kinase A-mediated tyrosine hydroxylase activity. These results suggest that the N-terminal of sonic hedgehog increases tyrosine hydroxylase gene expression in cells to acquire dopaminergic phenotypes, but decreases expression in late born neurons by antagonizing the protein kinase A cAMP-responsive element binding protein pathway.

cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK.

Mesenchymal stem cells are able to trans-differentiate into nonmesodermal lineage cells. Here, we identified downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels. We found that forskolin induced neuron-like morphology and expression of neuron-specific enolase and neurofilament-200 in mesenchymal stem cells. Forskolin sequentially activated protein kinase A and B-regulation of alpha-fetoprotein (Raf), which led to phosphorylation of extracellular signal-regulated kinase. Importantly, blockade of extracellular signal-regulated kinase phosphorylation with a mitogen-activated protein kinase (MAPK) kinase inhibitor abrogated the forskolin-induced morphological changes and induction of neuronal proteins. These results indicate that extracellular signal-regulated kinase/MAPK mediates both cAMP-induced early cytoskeletal rearrangement and the later induction of neuronal markers in mesenchymal stem cells.

Changes in EEG of children during brain respiration-training.

Brain Respiration (BR)-training is a unique form of breathing exercise that develops potential ability by facilitating brain function. It is recognized as an effective method of improving the scholastic aptitude and emotional stability of children. The present study was designed to investigate the characteristics of the EEG during this training. Spectral analysis was used to examine the relative power in the EEG of 12 children while they practiced BR-training, and these were compared to those of 12 matched controls. BR-trainees showed a lower theta rhythm than the controls before the training session began and lower beta2 power before, during and after the session. In contrast, the BR subjects showed greater relative alpha1 power than the controls in the left frontal region during BR-training, which persisted throughout the BR-training schedule. There is evidence that decreased theta and beta waves may be correlated with emotional maturation, whilst increased alpha waves are associated with educational achievement. These findings enhance our understanding of the neurophysiological basis of the effects of BR-training upon emotion and maturation.