Ganglioside GD3 regulates neural stem cell quiescence and controls postnatal neurogenesis.

The postnatal neural stem cell (NSC) pool hosts quiescent and activated radial glia-like NSCs contributing to neurogenesis throughout adulthood. However, the underlying regulatory mechanism during the transition from quiescent NSCs to activated NSCs in the postnatal NSC niche is not fully understood. Lipid metabolism and lipid composition play important roles in regulating NSC fate determination. Biological lipid membranes define the individual cellular shape and help maintain cellular organization and are highly heterogeneous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycosphingolipids. An often overlooked but key aspect is that the functional activities of proteins and genes are highly dependent on their molecular environments. We previously reported that ganglioside GD3 is the predominant species in NSCs and that the reduced postnatal NSC pools are observed in global GD3-synthase knockout (GD3S-KO) mouse brains. The specific roles of GD3 in determining the stage and cell-lineage determination of NSCs remain unclear, since global GD3S-KO mice cannot distinguish if GD3 regulates postnatal neurogenesis or developmental impacts. Here, we show that inducible GD3 deletion in postnatal radial glia-like NSCs promotes NSC activation, resulting in the loss of the long-term maintenance of the adult NSC pools. The reduced neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of GD3S-conditional-knockout mice led to the impaired olfactory and memory functions. Thus, our results provide convincing evidence that postnatal GD3 maintains the quiescent state of radial glia-like NSCs in the adult NSC niche.

Identification of genotype-biochemical phenotype correlations associated with fructose 1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. Due to the rarity of FBPase deficiency, the mechanism by which the mutations cause enzyme activity loss still remains unclear. Here we identify compound heterozygous missense mutations of FBP1, c.491G>A (p.G164D) and c.581T>C (p.F194S), in an adult patient with hypoglycemic lactic acidosis. The G164D and F194S FBP1 mutants exhibit decreased FBP1 protein expression and a loss of FBPase enzyme activity. The biochemical phenotypes of all previously reported FBP1 missense mutations in addition to G164D and F194S are classified into three functional categories. Type 1 mutations are located at pivotal residues in enzyme activity motifs and have no effects on protein expression. Type 2 mutations structurally cluster around the substrate binding pocket and are associated with decreased protein expression due to protein misfolding. Type 3 mutations are likely nonpathogenic. These findings demonstrate a key role of protein misfolding in mediating the pathogenesis of FBPase deficiency, particularly for Type 2 mutations. This study provides important insights that certain patients with Type 2 mutations may respond to chaperone molecules.

Ganglioside GD3 regulates neural stem cell quiescence and controls postnatal neurogenesis.

The postnatal neural stem cell (NSC) pool hosts quiescent and activated radial glia-like NSCs contributing to neurogenesis throughout adulthood. However, the underlying regulatory mechanism during the transition from quiescent NSCs to activated NSCs in the postnatal NSC niche is not fully understood. Lipid metabolism and lipid composition play important roles in regulating NSC fate determination. Biological lipid membranes define the individual cellular shape and help maintain cellular organization and are highly heterogenous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycosphingolipids. An often overlooked but key aspect is that the functional activities of proteins and genes are highly dependent upon their molecular environments. We previously reported that ganglioside GD3 is the predominant species in NSCs and that the reduced postnatal NSC pools are observed in global GD3-synthase knockout (GD3S-KO) mouse brains. The specific roles of GD3 in determining the stage and cell-lineage determination of NSCs remain unclear, since global GD3S-KO mice cannot distinguish if GD3 regulates postnatal neurogenesis or developmental impacts. Here we show that inducible GD3 deletion in postnatal radial glia-like NSCs promotes the NSC activation, resulting in the loss of the long-term maintenance of the adult NSC pools. The reduced neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of GD3S-conditional-knockout mice led to impaired olfactory and memory functions. Thus, our results provide convincing evidence that postnatal GD3 maintains the quiescent state of radial glia-like NSCs in the adult NSC niche.

Restoration of Adult Neurogenesis by Intranasal Administration of Gangliosides GD3 and GM1 in The Olfactory Bulb of A53T Alpha-Synuclein-Expressing Parkinson's-Disease Model Mice.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting the body and mind of millions of people in the world. As PD progresses, bradykinesia, rigidity, and tremor worsen. These motor symptoms are associated with the neurodegeneration of dopaminergic neurons in the substantia nigra. PD is also associated with non-motor symptoms, including loss of smell (hyposmia), sleep disturbances, depression, anxiety, and cognitive impairment. This broad spectrum of non-motor symptoms is in part due to olfactory and hippocampal dysfunctions. These non-motor functions are suggested to be linked with adult neurogenesis. We have reported that ganglioside GD3 is required to maintain the neural stem cell (NSC) pool in the subventricular zone (SVZ) of the lateral ventricles and the subgranular layer of the dentate gyrus (DG) in the hippocampus. In this study, we used nasal infusion of GD3 to restore impaired neurogenesis in A53T alpha-synuclein-expressing mice (A53T mice). Intriguingly, intranasal GD3 administration rescued the number of bromodeoxyuridine + (BrdU +)/Sox2 + NSCs in the SVZ. Furthermore, the administration of gangliosides GD3 and GM1 increases doublecortin (DCX)-expressing immature neurons in the olfactory bulb, and nasal ganglioside administration recovered the neuronal populations in the periglomerular layer of A53T mice. Given the relevance of decreased ganglioside on olfactory impairment, we discovered that GD3 has an essential role in olfactory functions. Our results demonstrated that intranasal GD3 infusion restored the self-renewal ability of the NSCs, and intranasal GM1 infusion promoted neurogenesis in the adult brain. Using a combination of GD3 and GM1 has the potential to slow down disease progression and rescue dysfunctional neurons in neurodegenerative brains.

Functional Impairment of the Nervous System with Glycolipid Deficiencies.

Patients with nervous system disorders suffer from impaired cognitive, sensory and motor functions that greatly inconvenience their daily life and usually burdens their family and society. It is difficult to achieve functional recovery for the damaged central nervous system (CNS) because of its limited ability to regenerate. Glycosphingolipids (GSLs) are abundant in the CNS and are known to play essential roles in cell-cell recognition, adhesion, signal transduction, and cellular migration, that are crucial in all phases of neurogenesis. Despite intense investigation of CNS regeneration, the roles of GSLs in neural regeneration remain unclear. Here we focus on the respective potentials of glycolipids to promote regeneration and repair of the CNS. Mice lacking glucosylceramide, lactosylceramide or gangliosides show lethal phenotypes. More importantly, patients with ganglioside deficiencies exhibit severe clinical phenotypes. Further, neurodegenerative diseases and mental health disorders are associated with altered GSL expression. Accumulating studies demonstrate that GSLs not only delimit physical regions but also play central roles in the maintenance of the biological functions of neurons and glia. We anticipate that the ability of GSLs to modulate behavior of a variety of molecules will enable them to ameliorate biochemical and neurobiological defects in patients. The use of GSLs to treat such defects in the human CNS will be a paradigm-shift in approach since GSL-replacement therapy has not yet been achieved in this manner clinically.

Intranasal infusion of GD3 and GM1 gangliosides downregulates alpha-synuclein and controls tyrosine hydroxylase gene in a PD model mouse.

Parkinson's disease (PD) is characterized by Lewy bodies (composed predominantly of alpha-synuclein [aSyn]) and loss of pigmented midbrain dopaminergic neurons comprising the nigrostriatal pathway. Most PD patients show significant deficiency of gangliosides, including GM1, in the brain, and GM1 ganglioside appears to keep dopaminergic neurons functioning properly. Thus, supplementation of GM1 could potentially provide some rescuing effects. In this study, we demonstrate that intranasal infusion of GD3 and GM1 gangliosides reduces intracellular aSyn levels. GM1 also significantly enhances expression of tyrosine hydroxylase (TH) in the substantia nigra pars compacta of the A53T aSyn overexpressing mouse, following restored nuclear expression of nuclear receptor related 1 (Nurr1, also known as NR4A2), an essential transcription factor for differentiation, maturation, and maintenance of midbrain dopaminergic neurons. GM1 induces epigenetic activation of the TH gene, including augmentation of acetylated histones and recruitment of Nurr1 to the TH promoter region. Our data indicate that intranasal administration of gangliosides could reduce neurotoxic proteins and restore functional neurons via modulating chromatin status by nuclear gangliosides.

Life-Long Neural Stem Cells Are Fate-Specified at an Early Developmental Stage.

The origin and life-long fate of quiescent neural stem cells (NSCs) in the adult mammalian brain remain largely unknown. A few neural precursor cells in the embryonic brain elongate their cell cycle time and subsequently become quiescent postnatally, suggesting the possibility that life-long NSCs are selected at an early embryonic stage. Here, we utilized a GFP-expressing lentivirus to investigate the fate of progeny from individual lentivirus-infected NSCs by identifying the lentiviral integration site. Our data suggest that NSCs become specified to two or more lineages prior to embryonic day 13.5 in mice: one NSC lineage produces cells only for the cortex and another provides neurons to the olfactory bulb. The majority of neurosphere-forming NSCs in the adult brain are relatively dormant and generate very few cells, if any, in the olfactory bulb or cortex, and this NSC population could serve as a reservoir that is occasionally reactivated later in life.

Early Maternal and Social Deprivation Expands Neural Stem Cell Population Size and Reduces Hippocampus/Amygdala-Dependent Fear Memory.

Early life stress can exert detrimental or beneficial effects on neural development and postnatal behavior depending on the timing, duration, strength, and ability to control the stressors. In this study, we utilized a maternal and social deprivation (MSD) model to investigate the effects of early life stress on neural stem cells (NSCs) and neurogenesis in the adult brain. We found that MSD during the stress-hyporesponsive period (SHRP) (early-MSD), when corticosterone secretion is suppressed, increased the size of the NSC population, whereas the same stress beyond the SHRP abrogated these effects. Early-MSD enhanced neurogenesis not only in the dentate gyrus of the hippocampus, one of the classic neurogenic regions, but also in the amygdala. In addition, mice exposed to early-MSD exhibited a reduction in amygdala/hippocampus-dependent fear memory. These results suggest that animals exposed to early life stress during the SHRP have reinforced stress resilience to cope with perceived stressors to maintain a normal homeostatic state.

Minocycline Directly Enhances the Self-Renewal of Adult Neural Precursor Cells.

Minocycline not only has antibacterial action but also produces a variety of pharmacological effects. It has drawn considerable attention as a therapeutic agent for symptoms caused by inflammation in many neurological disorders, leading to several clinical trials. Although some of these effects are mediated through its function of suppressing microglial activation, it is not clear whether minocycline acts on other cell types in the adult brain. In this study, we utilized a colony-forming neurosphere assay, in which neural stem cells (NSCs) clonally proliferate to form floating colonies, called neurospheres. We found that minocycline (at therapeutically relevant concentrations in cerebrospinal fluid) enhances the self-renewal capability of NSCs derived from the subependymal zone of adult mouse brain and facilitates their differentiation into oligodendrocytes. Importantly, these effects were independent of a suppression of microglial activation and were specifically observed with minocycline (among tetracycline derivatives). In addition, the size of the NSC population in the adult brain was increased when minocycline was infused into the lateral ventricle by an osmotic minipump in vivo. While precise molecular mechanisms of how minocycline alters the behavior of adult NSCs remain unknown, our data provide a basis for the clinical use of minocycline to treat neurodegenerative and demyelinating diseases.

Bre1a, a histone H2B ubiquitin ligase, regulates the cell cycle and differentiation of neural precursor cells.

Cell cycle regulation is crucial for the maintenance of stem cell populations in adult mammalian tissues. During development, the cell cycle length in neural stem cells increases, which could be associated with their capabilities for self-renewal. However, the molecular mechanisms that regulate differentiation and cell cycle progression in embryonic neural stem cells remain largely unknown. Here, we investigated the function of Bre1a, a histone H2B ubiquitylation factor, which is expressed in most but not all of neural precursor cells (NPCs) in the developing mouse brain. We found that the knockdown of Bre1a in NPCs lengthened their cell cycle through the upregulation of p57(kip2) and the downregulation of Cdk2. In addition, the knockdown of Bre1a increased the expression of Hes5, an effector gene of Notch signaling, through the action of Fezf1 and Fezf2 genes and suppressed the differentiation of NPCs. Our data suggest that Bre1a could be a bifunctional gene that regulates both the differentiation status and cell cycle length of NPCs. We propose a novel model that the Bre1a-negative cells in the ventricular zone of early embryonic brains remain undifferentiated and are selected as self-renewing neural stem cells, which increase their cell cycle time during development.

Dab1-mediated colocalization of multi-adaptor protein CIN85 with Reelin receptors, ApoER2 and VLDLR, in neurons.

Reelin-Dab1 signaling is indispensable for proper positioning of neurons in mammalian brain. Reelin is a glycoprotein secreted from Cajal-Reztuis cells in marginal zone of cerebral cortex, and its receptors are Apolipoprotein E receptor 2 (ApoER2) or very low density lipoprotein receptor (VLDLR) expressed on migrating neurons. When Reelin binds to ApoER2 or VLDLR, an adaptor protein Dab1 bound to the receptors undergoes Tyr phosphorylation that is essential for Reelin signaling. We reported previously that Cdk5-p35 phosphorylates Dab1 at Ser400 and Ser491 and the phosphorylation regulates its binding to CIN85, which is an SH3-containing multiadaptor protein involved in endocytic downregulation of receptor-tyrosine kinases. However, the interaction of CIN85 with Dab1 has not been addressed in neurons. We examined here a possibility that CIN85 has a role in Reelin signaling. We found nonpho-sphorylated Dab1-mediated colocalization of CIN85 with ApoER2. The colocalization of CIN85 with ApoER2 was increased in neurons stimulated with Reelin repeats 3-6, an active Reelin fragment. The stimulation recruited CIN85 to domains in plasma membrane where it colocalized with ApoER2 and Dab1 and then to EEA1-labeled early endosomes in the cytoplasm. In addition, Tyr phosphorylation of Dab1 strengthened the binding to CIN85. These results suggest that CIN85 participates in Reelin signaling through the binding to Dab1.

Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.

Cyclin-dependent kinase 5 (Cdk5) is activated by binding to its activators, p35 and p39. The level of Cdk5 activity is determined by the amount of p35 and p39, which is regulated not only by transcription but also via proteasomal degradation. Alternatively, calpain-induced cleavage of p35 to p25 can induce aberrant Cdk5 activation. As the regulation of p35 and p39 proteolysis is not well understood, we have studied here the mechanisms governing their degradation and cleavage. We find that p35 and p39 undergo proteasomal degradation in neurons, with p39 showing a slower degradation rate than p35. Degradation of the activators is dependent on their respective N-terminal p10 region, as indicated by experiments in which cognate p10 regions were swapped between p35 and p39. The effect of the p10 region on degradation and cleavage could be assigned to its membrane binding properties, mediated predominantly by myristoylation. Together, these results indicate that both proteasomal degradation and calpain cleavage of p35 and p39 are stimulated by membrane association, which is in turn mediated via myristoylation of their p10 regions. However, p35 and p39 show differences in degradation and cleavage rates, which may in fact underlie the distinct physiological and pathological functions of these two Cdk5 activators.

Regulation of the interaction of Disabled-1 with CIN85 by phosphorylation with Cyclin-dependent kinase 5.

Disabled-1 (Dab1) is an adaptor protein mediating Reelin signaling in neuronal migration during brain development. Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase also involved in neuronal migration. The interaction between Dab1 and Cdk5 is in need of investigation. Dab1 was phosphorylated at Ser400 and Ser491 by Cdk5 in vivo. We search for proteins that interact with Dab1 in a phosphorylation-dependent manner at these sites, and identified CIN85, an SH3-containing adaptor protein involved in endocytosis, and CPalpha/CPbeta, which are subunits of barbed end F-actin-capping proteins (CP), as proteins bound to unphosphorylated Dab1 by mass spectrometric analysis. It was shown that the PTPAPR sequence of Dab1, conforming to the PxxxPR atypical SH3-binding motif, was the binding site for SH3 domains of CIN85. The results that phosphorylation at Ser491 close to the PTPAPR sequence inhibited association with CIN85 may provide a mechanism regulating the interaction between the PxxxPR motif proteins and SH3 domains of CIN85 family proteins. Together with previous results that CIN85 regulates actin assembly, present results raise the possibility that Cdk5 modulates actin dynamics through regulation of CIN85-Dab1 interaction by the Dab1 phosphorylation.