Purinosomes and Purine Metabolism in Mammalian Neural Development: A Review.

Neural stem/progenitor cells (NSPCs) in specific brain regions require precisely regulated metabolite production during critical development periods. Purines-vital components of DNA, RNA, and energy carriers like ATP and GTP-are crucial metabolites in brain development. Purine levels are tightly controlled through two pathways: de novo synthesis and salvage synthesis. Enzymes driving de novo pathway are assembled into a large multienzyme complex termed the "purinosome." Here, we review purine metabolism and purinosomes as spatiotemporal regulators of neural development. Notably, around postnatal day 0 (P0) during mouse cortical development, purine synthesis transitions from the de novo pathway to the salvage pathway. Inhibiting the de novo pathway affects mTORC1 pathway and leads to specific forebrain malformations. In this review, we also explore the importance of protein-protein interactions of a newly identified NSPC protein-NACHT and WD repeat domain-containing 1 (Nwd1)-in purinosome formation. Reduced Nwd1 expression disrupts purinosome formation, impacting NSPC proliferation and neuronal migration, resulting in periventricular heterotopia. Nwd1 interacts directly with phosphoribosylaminoimidazole-succinocarboxamide synthetase (PAICS), an enzyme involved in de novo purine synthesis. We anticipate this review will be valuable for researchers investigating neural development, purine metabolism, and protein-protein interactions.

Spatiotemporal Regulation of De Novo and Salvage Purine Synthesis during Brain Development.

The levels of purines, essential molecules to sustain eukaryotic cell homeostasis, are regulated by the coordination of the de novo and salvage synthesis pathways. In the embryonic central nervous system (CNS), the de novo pathway is considered crucial to meet the requirements for the active proliferation of neural stem/progenitor cells (NSPCs). However, how these two pathways are balanced or separately used during CNS development remains poorly understood. In this study, we showed a dynamic shift in pathway utilization, with greater reliance on the de novo pathway during embryonic stages and on the salvage pathway in postnatal-adult mouse brain. The pharmacological effects of various purine synthesis inhibitors in vitro and the expression profile of purine synthesis enzymes indicated that NSPCs in the embryonic cerebrum mainly use the de novo pathway. Simultaneously, NSPCs in the cerebellum require both the de novo and the salvage pathways. In vivo administration of de novo inhibitors resulted in severe hypoplasia of the forebrain cortical region, indicating a gradient of purine demand along the anteroposterior axis of the embryonic brain, with cortical areas of the dorsal forebrain having higher purine requirements than ventral or posterior areas such as the striatum and thalamus. This histologic defect of the neocortex was accompanied by strong downregulation of the mechanistic target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase (S6K)/S6 signaling cascade, a crucial pathway for cell metabolism, growth, and survival. These findings indicate the importance of the spatiotemporal regulation of both purine pathways for mTORC1 signaling and proper brain development.

Inka2 expression in smooth muscle cells and its involvement in cell migration.

The cell motility of smooth muscle cells (SMCs) is essential for vascular and internal organ development and tissue regeneration in response to damage. Cell migration requires dynamic changes in the actin-cytoskeleton via the p-21 activated kinase (Pak)-Cofilin signaling cascade, which is the central axis of the actin filaments. We previously identified that the Inka2 gene was preferentially expressed in the central nervous system (CNS) and revealed that Inka2 directly binds Pak4 to suppress its kinase activity, thereby regulating actin de-polymerization in dendritic spine formation of the forebrain neurons. However, its physiological significance outside the CNS remains unclear. Here we determined the Inka2 expression profile in various organs using in situ hybridization analysis and lacZ staining on Inka2flox/+ mice. Robust Inka2 expression was consistently detected in the SMCs of many peripheral organs, including the arteries, esophagus, stomach, intestine, and bladder. The scratch assay was used on primary cultured SMCs and revealed that Inka2-/- SMC exhibits accelerated cell migration ability without a change in the cell proliferation rate. Inka2-/- SMCs displayed Cofilin activation/phosphorylation, a downstream molecule of Pak4 signal cascade. These results suggest that Inka2 regulates SMC motility through modulating actin reorganization as the endogenous inhibitor of Pak4.

Identification and expression profile of novel STAND gene Nwd2 in the mouse central nervous system.

In the central nervous system (CNS), neurons need synaptic neurotransmitter release and cellular response for various cellular stress or environmental stimuli. To achieve these highly orchestrated cellular processes, neurons should drive the molecular mechanisms that govern and integrate complex signaling pathways. The signal transduction ATPases with numerous domains (STAND) family of proteins has been shown to play essential roles in diverse signal transduction mechanisms, including apoptosis and innate immunity. However, a comprehensive understanding of STAND genes remains lacking. Previously, we identified the NACHT and WD repeat domain-containing protein 1 (NWD1), a member of STAND family, in the regulation of the assembly of a giant multi-enzyme complex that enables efficient de novo purine biosynthesis during brain development. Here we identified the mouse Nwd2 gene, which is a paralog of Nwd1. A molecular phylogenetic analysis suggested that Nwd1 emerged during the early evolution of the animal kingdom, and that Nwd2 diverged in the process of Nwd1 duplication. RT-PCR and in situ hybridization analyses revealed the unique expression profile of Nwd2 in the developing and adult CNS. Unlike Nwd1, Nwd2 expression was primarily confined to neurons in the medial habenular nucleus, an essential modulating center for diverse psychological states, such as fear, anxiety, and drug addiction. In the adult brain, Nwd2 expression, albeit at a lower level, was also observed in some neuronal populations in the piriform cortex, hippocampus, and substantia nigra pars compacta. NWD2 might play a unique role in the signal transduction required for specific neuronal circuits, especially for cholinergic neurons in the habenula.

Inka2, a novel Pak4 inhibitor, regulates actin dynamics in neuronal development.

The actin filament is a fundamental part of the cytoskeleton defining cell morphology and regulating various physiological processes, including filopodia formation and dendritic spinogenesis of neurons. Serine/threonine-protein kinase Pak4, an essential effector, links Rho GTPases to control actin polymerization. Previously, we identified the Inka2 gene, a novel mammalian protein exhibiting sequence similarity to Inka1, which serves as a possible inhibitor for Pak4. Although Inka2 is dominantly expressed in the nervous system and involved in focal-adhesion dynamics, its molecular role remains unclear. Here, we found that Inka2-iBox directly binds to Pak4 catalytic domain to suppress actin polymerization. Inka2 promoted actin depolymerization and inhibited the formation of cellular protrusion caused by Pak4 activation. We further generated the conditional knockout mice of the Inka2 gene. The beta-galactosidase reporter indicated the preferential Inka2 expression in the dorsal forebrain neurons. Cortical pyramidal neurons of Inka2-/- mice exhibited decreased density and aberrant morphology of dendritic spines with marked activation/phosphorylation of downstream molecules of Pak4 signal cascade, including LIMK and Cofilin. These results uncovered the unexpected function of endogenous Pak4 inhibitor in neurons. Unlike Inka1, Inka2 is a critical mediator for actin reorganization required for dendritic spine development.

Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development.

Brain development is a highly orchestrated process requiring spatiotemporally regulated mitochondrial dynamics. Drp1, a key molecule in the mitochondrial fission machinery, undergoes various post-translational modifications including conjugation to the small ubiquitin-like modifier (SUMO). However, the functional significance of SUMOylation/deSUMOylation on Drp1 remains controversial. SUMO-specific protease 5 (Senp5L) catalyzes the deSUMOylation of Drp1. We revealed that a splicing variant of Senp5L, Senp5S, which lacks peptidase activity, prevents deSUMOylation of Drp1 by competing against other Senps. The altered SUMOylation level of Drp1 induced by Senp5L/5S affects mitochondrial morphology probably through controlling Drp1 ubiquitination and tubulation of the endoplasmic reticulum. A dynamic SUMOylation/deSUMOylation balance controls neuronal polarization and migration during the development of the cerebral cortex. These findings suggest a novel role of post-translational modification, in which deSUMOylation enzyme isoforms competitively regulate mitochondrial dynamics via Drp1 SUMOylation levels, in a tightly controlled process of neuronal differentiation and corticogenesis.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

Engagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called "purinosome." However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

The association between detection of Mycobacterium avium subsp. paratuberculosis DNA in feces and histopathological classification.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic infectious disease that causes intractable diarrhea in ruminants. To control the occurrence of JD in cattle, a national surveillance is conducted in Japan. Since 2013, real-time quantitative PCR has been used for definite diagnosis. In this study, we compared the amount of fecal MAP DNA with histopathological classification of ileocecal lesions. Multinomial logistic regression models enabled us to predict the probability of finding the histopathological classification from the amount of fecal MAP DNA. These results suggest that shedding level of MAP DNA could act as an indicator of JD progression.

Control of cell migration by the novel protein phosphatase-2A interacting protein inka2.

Cell migration is essential for many physiological and pathological processes, including embryonic development, wound healing, immune response and cancer metastasis. Inka2 transcripts are observed in migrating cells during embryonic development, suggesting the involvement of inka2 in cell migration. However, its precise role remains unclear. Here, we found that inka2 controlled focal adhesion dynamics and cell migration, likely by regulating protein phosphatase-2A (PP2A) function. A scratch assay revealed that inka2 shRNA-transfected NIH3T3 cells showed rapid wound closure, indicating an inhibitory effect by inka2 on cell migration. Live-cell imaging of NIH3T3 cells expressing EGFP-paxillin using total internal reflection fluorescence microscopy revealed that inka2 knockdown increased the turnover rate of focal adhesions. Given that PP2A, which consists of catalytic (C), regulatory (B) and scaffolding (A) subunits, is known to regulate focal adhesions, we examined the inka2-PP2A interaction. Immunoprecipitation revealed an association between inka2 and the PP2A C subunit. Binding of Inka2 to the C subunit prevented the association between the A and C subunits, suggesting that inka2 can inhibit PP2A function. Furthermore, both inka2 expression and PP2A inhibition decreased focal adhesion kinase-paxillin interaction, resulting in reduced formation of focal adhesions. We assessed the effect of pharmacological PP2A inhibition on the inka2 knockdown-induced increase in cell migration speed and found that treatment with a PP2A inhibitor negated the accelerated migration of inka2 knockdown cells. These results suggest that inka2 knockdown exerts its effects through PP2A-dependent regulation of focal adhesions. Our findings contribute to a better understanding of the molecular mechanisms underlying cell migration.

Minocycline Alleviates Cluster Formation of Activated Microglia and Age-dependent Dopaminergic Cell Death in the Substantia Nigra of Zitter Mutant Rat.

Microglial activation is a component of neurodegenerative pathology. Here, we examine whether activated microglia participate in age-related dopaminergic (DA) cell death in the substantia nigra pars compacta (SNc) of the zitter (zi/zi) rat, a mutant characterized by deletion of the attractin gene. Confocal microscopy with double-immunohistochemical staining revealed activated microglia-formed cell-clusters surrounding DA neurons in the SNc from 2 weeks after birth. An immunoelectron microscopic study showed that the cytoplasm of activated microglia usually contains phagosome-like vacuoles and lamellar inclusions. Expression levels of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were increased in the midbrain of 2-month-old zi/zi rats. Chronic treatment with the anti-inflammatory agent minocycline altered the morphology of the microglia, reduced cluster formation by the microglia, and attenuated DA cell death in the SNc, and reduced the expression of IL-1ß in the midbrain. These results indicate that activated microglia, at least in part and especially at the initial phase, contribute to DA cell death in the SNc of the zi/zi rat.

Expression profile of the STAND protein Nwd1 in the developing and mature mouse central nervous system.

The orchestrated events required during brain development, as well as the maintenance of adult neuronal plasticity, highly depend on the accurate responses of neuronal cells to various cellular stress or environmental stimuli. Recent studies have defined a previously unrecognized, broad class of multidomain proteins, designated as signal transduction ATPases with numerous domains (STAND), which comprises a large number of proteins, including the apoptotic peptidase activating factor 1 (Apaf1) and nucleotide-binding oligomerization domain-like receptors (NLRs), central players in cell death and innate immune responses, respectively. Although the involvement of STANDs in the central nervous system (CNS) has been postulated in terms of neuronal development and function, it remains largely unclear. Here, we identified Nwd1 (NACHT and WD repeat domain-containing protein 1), as a novel STAND protein, expressed in neural stem/progenitor cells (NSPCs). Structurally, Nwd1 was most analogous to the apoptosis regulator Apaf1, also involved in mitosis and axonal outgrowth regulation in the CNS. Using a specific antibody, we show that, during the embryonic and postnatal period, Nwd1 is expressed in nestin-positive NSPCs in vivo and in vitro, while postnatally it is found in terminally differentiated neurons and blood vessels. At the subcellular level, we demonstrate that Nwd1 is preferentially located in the cytosolic compartment of cultured NSPCs, partially overlapping with cytochrome c. These observations imply that Nwd1 might be involved in the neuronal lineage as a new STAND gene, including having a pro-apoptotic or nonapoptotic role, similar to Apaf1.

Evaluation of fecal shedding and antibody response in dairy cattle infected with paratuberculosis using national surveillance data in Japan.

Paratuberculosis or Johne's disease (JD), is a chronic infectious disease causing intractable diarrhea in cattle, which leads to less productivity, such as decreased milk yield, and lower daily weight gain. As a control measure against JD in cattle, national serological surveillance has been conducted in Japan since 1998. To conduct modeling studies that are useful to evaluate the effectiveness of control measures against JD, reliable parameter values, such as length of time from infection to the start of fecal shedding or antibody expression, are especially important. These parameters in the Japanese cattle population are assumed to be different from those in other countries with a higher prevalence of JD or in experimental infection settings; therefore, they must be estimated for the cattle population in Japan. Data from national surveillance conducted in Tokachi District, Hokkaido Prefecture, were used for this study. Using data from JD diagnostic tests for all cattle in Tokachi District between 1998 and 2014, all testing histories for infected animals were estimated as the number of tested cattle and positive cattle at each age of month for both fecal and antibody tests. A deterministic mathematical model for JD development, from infection to fecal shedding and antibody expression in infected cattle, was constructed to obtain the probability of testing positive when applied to both fecal and antibody tests at a given age. Likelihood was obtained from these estimated test results and best values for parameters were obtained using the Markov Chain Monte-Carlo method. Fifty-five percent of infected cattle were projected to have a transient shedding period, which was estimated to start 12 months after infection and last for 4 months. Persistent shedding was projected to occur in all infected cattle, and estimated to begin 7-84 months from infection. Following persistent shedding, antibody expression was estimated to start 7 months later. These values are useful for developing models to evaluate the status of JD infection and the effectiveness of control measures in the Japanese cattle population.

Synaptic localization of the SUMOylation-regulating protease SENP5 in the adult mouse brain.

Covalent conjugation of small ubiquitin-like modifiers (SUMOs) or SUMOylation is a reversible post-translational modification that regulates the stability and function of target proteins. SUMOs are removed from substrate proteins by sentrin/SUMO-specific proteases (SENPs). Numerous studies have implicated SUMOylation in various physiological and pathological processes in neurons. To understand the functional roles of SUMOylation, it is necessary to determine the distribution of enzymes regulating SUMO conjugation and deconjugation; yet, the localization of SENPs has not been described in detail in intact brain tissue. Here, we report the distribution and subcellular localization of SENP3 and 5 in the adult murine brain. Immunohistochemical analyses revealed the ubiquitous distribution of both SENPs across different brain regions. Within individual cells, SENP3 was confined to the nucleus, consistent with the conventional view that SENPs regulate nuclear events. In contrast, SENP5 was detected in the neuropil but not in cell bodies. Moreover, strong SENP5 immunoreactivity was observed in regions with high numbers of synapses such as the cerebellar glomeruli, suggesting that SENP5 localizes to pre- and/or postsynaptic structures. We performed double immunolabeling in cultured neurons and found that SENP5 co-localized with pre- and post-synaptic markers, as well as a mitochondrial marker. Immunoelectron microscopy confirmed this finding and revealed that SENP5 was localized to presynaptic terminals, postsynaptic spines, and mitochondria in axon terminals. These findings advance the current understanding of the functional roles of SUMOylation in neurons, especially in synaptic regulation, and have implications for future therapeutic strategies in neurodegenerative disorders mediated by mitochondrial dysfunction.

Expression profiles of inka2 in the murine nervous system.

Dynamic rearrangement of the actin cytoskeleton impacts many cellular characteristics in both the developing and adult central nervous systems (CNS), including the migration and adhesion of highly motile neural progenitor cells, axon guidance of immature neurons, and reconstruction of synaptic structures in the adult brain. Inka1, a known regulator of actin cytoskeleton reconstruction, is predominantly expressed by the neural crest cell lineage and regulates the migration and differentiation of these cells. In the present study, we identified a novel gene, designated as inka2, which is related to inka1. Inka2/fam212b is an evolutionarily conserved gene found in different vertebrate species and constitutes a novel gene family together with inka1. Northern blot analysis showed that inka2 mRNA was highly enriched in the nervous system. The spatiotemporal propagation cell profiles of those cells that expressed inka2 transcripts were compatible with those of Olig2-positive oligodendrocyte progenitor cells, which originate in the ventral ventricular zone during embryogenesis. Intense expression of inka2 was also noted in the proliferative neuronal progenitors in the developing cerebellum. On the other hand, immature newborn neurons in the embryonic brain showed no expression of inka2, except for the cells residing in the marginal zone of the embryonic telencephalon, which is known to contain transient cells including the non-subplate pioneer neurons and Cajal-Retzius cells. As brain development proceeds during the postnatal stage, inka2 expression emerged in some populations of immature neurons, including the neocortical pyramidal neurons, hippocampal pyramidal neurons, and granule cells migrating in the cerebellar cortex. In the adult brain, the expression of inka2 was interestingly confined in terminally differentiated neurons in the restricted forebrain regions. Taken together, as a novel regulator of actin cytoskeletons in the CNS, inka2 may be involved in multiple actin-driven processes, including cell migration and establishment of neuronal polarity.

Mutations in CKAP2L, the human homolog of the mouse Radmis gene, cause Filippi syndrome.

Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs(∗)6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.

Spatiotemporal distribution of SUMOylation components during mouse brain development.

Posttranslational modification of proteins might play an important role in brain cellular dynamics via the rapid turnover or functional change of critical proteins controlling neuronal differentiation or synaptic transmission. Small ubiquitin-like modifier protein (SUMO) is a family of ubiquitin-like small proteins that are covalently attached to target proteins to modify their function posttranslationally. Many cellular processes, such as transcription and protein trafficking, are regulated by SUMOylation, but its functional significance in the brain remains unclear. Although developmental regulation of SUMOylation levels in rat brain was recently demonstrated, no comparative immunohistochemical analysis of the cellular distribution profiles of SUMOylation components, including SUMO1, SUMO2/3, and Ubc9, has been undertaken so far. The present study used immunohistochemical and immunoblot analysis with the different developmental stages of mice and demonstrated the developmentally regulated distribution of SUMO1, SUMO2/3, and Ubc9 in the brain. During embryonic development, SUMOylation by SUMO1 and SUMO2/3 occurred in the nucleoplasm of nestin-positive neural stem cells. Although the total amount of SUMO-modified proteins decreased during postnatal brain development, intense and persistent accumulation of SUMO2/3 was detected throughout life in neural progenitor populations in neurogenic regions, including the subventricular zone and the hippocampal subgranular zone. In contrast, many neurons in the adult brain accumulated SUMO1 rather than SUMO2/3. Heavy immunoreactivity of SUMO1 was found in large projection neurons in the brainstem, whereas SUMO2/3 was almost absent from these areas. This heterogeneous distribution implies that both proteins play a specific and unique role in the brain.

Radmis, a novel mitotic spindle protein that functions in cell division of neural progenitors.

Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs.

Neuroprotective effects of melatonin on the nigrostriatal dopamine system in the zitter rat.

Melatonin has ubiquitous actions, both as a direct free-radical scavenger and as an indirect anti-oxidant. The present study examined in vivo neuroprotective effects of melatonin on the nigrostriatal dopaminergic system in zitter (zi/zi) rat, which displays abnormal metabolism of superoxide leading to age-related degeneration of the dopaminergic system. For up to 9 months after weaning, zi/zi rats had ad libitum access to drinking water containing melatonin. Chronic treatment with melatonin attenuated the decreases of dopamine and its metabolite in zi/zi rat caudate-putamen (CPU). Immunohistochemistry for tyrosine hydroxylase (TH) was consistent with neurochemical data in the CPU and demonstrated substantial sparing of the reduction of TH-immunoreactive neurons in the substantia nigra pars compacta. Quantitative polymerase chain reaction (qPCR) was performed to analyze mRNA expressions of pro-inflammatory cytokines (IL-1ß and TNF-α) and the anti-oxidant enzymes (catalase (CAT), superoxide dismutase (SOD) 1 and 2, and glutathione peroxidase (GPx1)) in the striatum and midbrain in order to examine the neuroprotective effect of melatonin. IL-1ß and TNF-α mRNA expressions were significantly increased in both areas of 3-month-old zi/zi rats, whereas there was a significant decrease in CAT mRNA expression in the striatum of 6-month-old zi/zi rat as compared to age-matched controls. With the exception of the high TNF-α mRNA expression in 3-month-old zi/zi midbrains, chronic treatment of melatonin attenuated expressional changes of IL-1ß, CAT, SOD1, and GPx1. These results suggest that besides its direct scavenger effects, chronic melatonin treatment provides a neuroprotective effect against dopaminergic degeneration by suppressing pro-inflammatory cytokines and up-regulating anti-oxidant enzyme expression.

Neural RNA-binding protein Musashi1 controls midline crossing of precerebellar neurons through posttranscriptional regulation of Robo3/Rig-1 expression.

Precisely regulated spatiotemporal gene expression is essential for the establishment of neural circuits. In contrast to the increasing evidence for transcriptional regulation of axon guidance cues and receptors, the role of posttranscriptional regulation in axon guidance, especially in vivo, remains poorly characterized. Here, we demonstrate that the expression of Slit receptor Robo3/Rig-1, which plays crucial roles in axonal midline crossing, is regulated by a neural RNA-binding protein Musashi1 (Msi1). Msi1 binds to Robo3 mRNA through RNA recognition motifs and increases the protein level of Robo3 without affecting its mRNA level. In Msi1-deficient precerebellar neurons, Robo3 protein, but not its mRNA, is dramatically reduced. Moreover, similar to defects in Robo3-deficient mice, axonal midline crossing and neuronal migration of precerebellar neurons are severely impaired in Msi1-deficient mice. Together, these findings indicate that Msi1-mediated posttranscriptional regulation of Robo3 controls midline crossing of precerebellar neurons.

Improved learning in microencephalic rats.

ABSTRACT Environmental enrichment (EE) facilitates recovery from behavioral abnormalities and spatial memory disabilities in several neurological disease models. Exposure to EE improves spatial memory acquisition and enhances the survival of newly generated cells in the dentate gyri of adult rodents. However, the effects of EE on spatial learning and neurogenesis in the methylazoxymethanol acetate-induced microencephalic rat have not been investigated. Depletion of serotonin in the rat hippocampus is known to influence spatial memory and adult neurogenesis, suggesting a role for serotonin in these processes. To confirm this hypothesis, male methylazoxymethanol acetate-induced microencephalic rats were exposed to EE or conventional housing after weaning; half of these rats further received intracisternal 5,7-dihydroxytryptamine on postnatal day 3, to induce long-lasting depletion of serotonin. As adults, these microencephalic rats were observed using the Morris water maze test and examined for hippocampal neurogenesis. EE alleviated the impairment of spatial memory acquisition and enhanced neurogenesis in the dentate gyri of adult microencephalic rats. Injection of 5,7-dihydroxytryptamine during the neonatal period caused pronounced reductions in hippocampal serotonin levels in these rats. Long-lasting depletion of serotonin eliminated the EE-induced alleviation of spatial memory acquisition and neurogenesis impairment in microencephalic rats. The present results suggest that EE alleviates spatial memory performance deficits in microencephalic rats and further indicate that serotonin might be involved in the underlying mechanisms through increased hippocampal neurogenesis. These data provide new insights into therapeutic interventions for individuals with human migration disorders associated with learning disabilities.