The dynamic brain N-glycome.

The attachment of carbohydrates to other macromolecules, such as proteins or lipids, is an important regulatory mechanism termed glycosylation. One subtype of protein glycosylation is asparagine-linked glycosylation (N-glycosylation) which plays a key role in the development and normal functioning of the vertebrate brain. To better understand the role of N-glycans in neurobiology, it's imperative we analyse not only the functional roles of individual structures, but also the collective impact of large-scale changes in the brain N-glycome. The systematic study of the brain N-glycome is still in its infancy and data are relatively scarce. Nevertheless, the prevailing view has been that the neuroglycome is inherently restricted with limited capacity for variation. The development of improved methods for N-glycomics analysis of brain tissue has facilitated comprehensive characterisation of the complete brain N-glycome under various experimental conditions on a larger scale. Consequently, accumulating data suggest that it's more dynamic than previously recognised and that, within a general framework, it has a given capacity to change in response to both intrinsic and extrinsic stimuli. Here, we provide an overview of the many factors that can alter the brain N-glycome, including neurodevelopment, ageing, diet, stress, neuroinflammation, injury, and disease. Given this emerging evidence, we propose that the neuroglycome has a hitherto underappreciated plasticity and we discuss the therapeutic implications of this regarding the possible reversal of pathological changes via interventions. We also briefly review the merits and limitations of N-glycomics as an analytical method before reflecting on some of the outstanding questions in the field.

Post-natal developmental changes in the composition of the rat neocortical N-glycome.

Asparagine-linked glycosylation (N-glycosylation) plays a key role in many neurodevelopmental processes, including neural cell adhesion, neurite outgrowth and axon targeting. However, little is known about the dynamics of N-glycosylation during brain development and, in particular, how the N-glycome of the developing neocortex differs from that of the adult. The aim of this study, therefore, was to perform a thorough characterization of N-glycosylation in both the adult and neonatal rat neocortex in order to gain insights into the types of changes occurring in the N-glycome during neurodevelopment. To this end, we used hydrophilic interaction ultraperformance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry to compare the adult neocortical N-glycome with that of 24- and 48-h neonates. We report that the abundance of complex N-glycans is significantly lower in adults compared with neonates. Furthermore, the proportion of charged complex N-glycans is also greatly reduced. This decrease in the abundance of complex N-glycans is offset by a corresponding increase in the proportion of truncated and, to a lesser extent, hybrid N-glycans. Lastly, we report that although the proportion of oligomannose N-glycans remains constant at around 24%, the distribution of high-mannose subtypes shifts from predominantly large subtypes in neonates to smaller subtypes in the adult. In summary, our findings indicate that N-glycan synthesis in the rat neocortex is fundamentally different in neonates compared with adults with a general shift occurring from large, sialylated N-glycans towards smaller, neutral structures as neonates develop into adults, coupled with a parallel shift towards smaller oligomannose structures.

Replication of 15 loci involved in human plasma protein N-glycosylation in 4802 samples from four cohorts.

Human protein glycosylation is a complex process, and its in vivo regulation is poorly understood. Changes in glycosylation patterns are associated with many human diseases and conditions. Understanding the biological determinants of protein glycome provides a basis for future diagnostic and therapeutic applications. Genome-wide association studies (GWAS) allow to study biology via a hypothesis-free search of loci and genetic variants associated with a trait of interest. Sixteen loci were identified by three previous GWAS of human plasma proteome N-glycosylation. However, the possibility that some of these loci are false positives needs to be eliminated by replication studies, which have been limited so far. Here, we use the largest set of samples so far (4802 individuals) to replicate the previously identified loci. For all but one locus, the expected replication power exceeded 95%. Of the 16 loci reported previously, 15 were replicated in our study. For the remaining locus (near the KREMEN1 gene), the replication power was low, and hence, replication results were inconclusive. The very high replication rate highlights the general robustness of the GWAS findings as well as the high standards adopted by the community that studies genetic regulation of protein glycosylation. The 15 replicated loci present a good target for further functional studies. Among these, eight loci contain genes encoding glycosyltransferases: MGAT5, B3GAT1, FUT8, FUT6, ST6GAL1, B4GALT1, ST3GAL4 and MGAT3. The remaining seven loci offer starting points for further functional follow-up investigation into molecules and mechanisms that regulate human protein N-glycosylation in vivo.

Increased yield of enzymatic synthesis by chromatographic selection of different N-glycoforms of yeast invertase.

Invertases are glycosidases applied for synthesis of alkyl glycosides that are important and effective surfactants. Stability of invertases in the environment with increased content of organic solvent is crucial for increase of productivity of glycosidases. Their stability is significantly influenced by N-glycosylation. However, yeast N-glycosylation pathways may synthesize plethora of N-glycan structures. A total natural crude mixture of invertase glycoforms (EINV) extracted from Saccharomyces cerevisiae was subfractionated by anion-exchange chromatography on industrial monolithic supports to obtain different glycoforms (EINV1-EINV3). Separated glycoforms exhibited different stabilities in water-alcohol solutions that are in direct correlation with the amount of phosphate bound to N-glycans. Observed differences in stability of different invertase glycoforms were used to improve productivity of methyl ß-d-fructofuranoside (MF) synthesis. The efficiency and yield of MF synthesis were improved more than 50% when the most stabile glycoform bearing the lowest amount of phosphorylated N-glycans is selected and utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrate different impact of N-glycans on the surface charge and enzyme stability in regard to particular reaction environment, and provide a platform for improvement of yield of industrial enzymatic synthesis by chromatographic selection of glycoforms on monolithic supports.

Searching for glycomic biomarkers for predicting resilience and vulnerability in a rat model of posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is triggered by traumatic events in 10-20% of exposed subjects. N-linked glycosylation, by modifying protein functions, may provide an important environmental link predicting vulnerability. Our goals were (1) to find alterations in plasma N-glycome predicting stress-vulnerability; (2) to investigate how trauma affects N-glycome in the plasma (PGP) and in three PTSD-related brain regions (prefrontal cortex, hippocampus and amygdala; BGP), hence, uncover specific targets for PTSD treatment. We examined male (1) controls, (2) traumatized vulnerable and (3) traumatized resilient rats both before and several weeks after electric footshock. Vulnerable and resilient groups were separated by z-score analysis of behavior. Higher freezing behavior and decreased social interest were detected in vulnerable groups compared to control and resilient rats. Innate anxiety did not predict vulnerability, but pretrauma levels of PGP10(FA1G1Ga1), PGP11(FA2G2), and PGP15(FA3G2) correlated positively with it, the last one being the most sensitive. Traumatic stress induced a shift from large, elaborate N-glycans toward simpler neutral structures in the plasma of all traumatized animals and specifically in the prefrontal cortex of vulnerable rats. In plasma trauma increased PGP17(A2G2S) level in vulnerable animals. In all three brain regions, BGP11(F(6)A2B) was more abundant in vulnerable rats, while most behavioral correlations occurred in the prefrontal cortex. In conclusion, we found N-glycans (especially PGP15(FA3G2)) in plasma as possible biomarkers of vulnerability to trauma that warrants further investigation. Posttrauma PGP17(A2G2S1) increase showed overlap with human results highlighting the utility and relevance of this animal model. Prefrontal cortex is a key site of trauma-induced glycosylation changes that could modulate the behavioral outcome.

The Role of the Neuroprotective Factor Npas4 in Cerebral Ischemia.

Stroke is one of the leading causes of death and adult disability in the world. Although many molecules have been documented to have a neuroprotective effect, the majority of these molecules failed to improve the neurological outcomes for patients with brain ischemia. It has been proposed that neuroprotection alone may, in fact, not be adequate for improving the prognosis of ischemic stroke. Neuroprotectants that can regulate other processes which occur in the brain during ischemia could potentially be targets for the development of effective therapeutic interventions in stroke. Neuronal Per-Arnt-Sim domain protein 4 (Npas4) is an activity-dependent transcription factor whose expression is induced in various brain insults, including cerebral ischemia. It has been shown that Npas4 plays an important role in protecting neurons against many types of neurodegenerative insult. Recently, it was demonstrated that Npas4 indeed has a neuroprotective role in ischemic stroke and that Npas4 might be involved in modulating the cell death pathway and inflammatory response. In this review, we summarize the current knowledge of the roles that Npas4 may play in neuroinflammation and ischemia. Understanding how ischemic lesion size in stroke may be reduced through modulation of Npas4-dependent apoptotic and inflammatory pathways could lead to the development of new stroke therapies.

Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia.

The neuronal Per-Arnt-Sim domain protein 4 (Npas4) is an important transcriptional regulator of synaptic plasticity and cognition. The present study characterises the in vivo neuroanatomical expression pattern of the Npas4 protein in a rat model of focal cerebral ischemia. Animals were subjected to unilateral middle cerebral artery occlusion for 2 h, after which the spatiotemporal and neuronal profiles of Npas4 protein expression were analysed by immunohistochemistry at different time points post-reperfusion. Focal cerebral ischemia induced an early, transient and robust upregulation of Npas4 in a brain region-dependent manner involving predominantly principal neurons. Interestingly, we observed a unique differential induction of Npas4 protein expression in corticolimbic regions of the rat brain that are critically linked to cognition and emotion. These findings suggest that stroke-induced Npas4 upregulation may be involved in a transcriptional regulatory program within the corticolimbic circuitry following an ischemic insult.

Human-specific features and developmental dynamics of the brain N-glycome.

Comparative "omics" studies have revealed unique aspects of human neurobiology, yet an evolutionary perspective of the brain N-glycome is lacking. Here, we performed multi-regional characterization of rat, macaque, chimpanzee, and human brain N-glycomes using chromatography and mass spectrometry, then integrated these data with complementary glycotranscriptomic data. We found that in primates the brain N-glycome has evolved more rapidly than the underlying transcriptomic framework, providing a mechanism for generating additional diversity. We show that brain N-glycome evolution in hominids has been characterized by an increase in complexity and α(2-6)-linked N-acetylneuraminic acid along with human-specific cell-type expression of key glycogenes. Finally, by comparing the prenatal and adult human brain N-glycome, we identify region-specific neurodevelopmental pathways that lead to distinct spatial N-glycosylation profiles in the mature brain. One-Sentence Summary: Evolution of the human brain N-glycome has been marked by an increase in complexity and a shift in sialic acid linkage.

Human-specific features and developmental dynamics of the brain N-glycome.

Comparative "omics" studies have revealed unique aspects of human neurobiology, yet an evolutionary perspective of the brain N-glycome is lacking. We performed multiregional characterization of rat, macaque, chimpanzee, and human brain N-glycomes using chromatography and mass spectrometry and then integrated these data with complementary glycotranscriptomic data. We found that, in primates, the brain N-glycome has diverged more rapidly than the underlying transcriptomic framework, providing a means for rapidly generating additional interspecies diversity. Our data suggest that brain N-glycome evolution in hominids has been characterized by an overall increase in complexity coupled with a shift toward increased usage of α(2-6)-linked N-acetylneuraminic acid. Moreover, interspecies differences in the cell type expression pattern of key glycogenes were identified, including some human-specific differences, which may underpin this evolutionary divergence. Last, by comparing the prenatal and adult human brain N-glycomes, we uncovered region-specific neurodevelopmental pathways that lead to distinct spatial N-glycosylation profiles in the mature brain.

HILIC-UPLC Analysis of Brain Tissue N-Glycans.

Extraction of N-glycans from intact tissue presents a unique set of challenges which makes it a relatively laborious and time-consuming process in comparison to other sample types, such as plasma. Here we present an approach designed for the extraction, purification, and labeling of free N-glycans from brain tissue. Using this method, up to 16 samples can be processed at once which translates to an output of 48 samples per week when rounds of extraction are staggered. Moreover, although intended for brain tissue, the method could easily be adapted to other tissue types as well. The protocol involves several stages. First, the tissue is homogenized and total proteins are isolated using chloroform-methanol extraction. The proteins are then deglycosylated using the Peptide N-Glycosidase F (PNGase F) enzyme in a reaction lasting two days. The released N-glycans are subsequently cleaned up from the reaction mixture using a centrifugal filter device and dried overnight. Next, the N-glycans are resuspended, labeled with 2-aminobenzamide (2-AB) and once again cleaned up using a filter plate. The purified N-glycans are released from the filter using ultrapure water and are then ready for analysis by for hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC).

miR-744 and miR-224 Downregulate Npas4 and Affect Lineage Differentiation Potential and Neurite Development During Neural Differentiation of Mouse Embryonic Stem Cells.

Neuronal PAS domain protein 4 (Npas4) is a brain-specific transcription factor whose expression is enriched in neurogenic regions of the brain. In addition, it was demonstrated that Npas4 expression is dynamic and highly regulated during neural differentiation of embryonic stem cells (ESCs). While these findings implicate a role for Npas4 in neurogenesis, the underlying mechanisms of regulation remain unknown. Given that growing evidence suggests that microRNAs (miRNAs) play important roles in both embryonic and adult neurogenesis, we reasoned that miRNAs are good candidates for regulating Npas4 expression during neural differentiation of ESCs. In this study, we utilized the small RNA sequencing method to profile miRNA expression during neural differentiation of mouse ESCs. Two differentially expressed miRNAs were identified to be able to significantly reduce reporter gene activity by targeting the Npas4 3'UTR, namely miR-744 and miR-224. More importantly, ectopic expression of these miRNAs during neural differentiation resulted in downregulation of endogenous Npas4 expression. Subsequent functional analysis revealed that overexpression of either miR-744 or miR-224 delayed early neural differentiation, reduced GABAergic neuron production and inhibited neurite outgrowth. Collectively, our findings indicate that Npas4 not only functions at the early stages of neural differentiation but may also, in part, contribute to neuronal subtype specification and neurite development.

Reduction of the neuroprotective transcription factor Npas4 results in increased neuronal necrosis, inflammation and brain lesion size following ischaemia.

Stroke is the second leading cause of death and the most frequent cause of adult disability. Neuronal Per-Arnt-Sim domain protein 4 (Npas4) is an activity-dependent transcription factor whose expression is induced in various brain insults, including cerebral ischaemia. Although previous studies have demonstrated that Npas4 plays a critical role in protecting neurons against neurodegenerative insults, the neuroprotective effect of Npas4 in response to ischaemic brain injury remains unknown. In this study, we used a loss-of-function approach to examine the neuroprotective potential of Npas4 in the context of ischaemic damage. Using oxygen and glucose deprivation, we demonstrated that the knockdown of Npas4 in mouse cortical neurons resulted in increased susceptibility to cell death. The protective effect of Npas4 was further investigated in vivo using a photochemically-induced stroke model in mice. We found a significantly larger lesion size and increased neurodegeneration in Npas4 knockout mice as compared to wild-type mice. Moreover, we also showed that ablation of Npas4 caused an increase in activated astrocytes and microglia, pro-inflammatory cytokines interleukin-6 and tumour necrosis factor alpha levels and a switch from apoptotic to necrotic cell death. Taken together, these data suggest that Npas4 plays a neuroprotective role in ischaemic stroke by limiting progressive neurodegeneration and neuroinflammation.

Effects of Npas4 deficiency on anxiety, depression-like, cognition and sociability behaviour.

The transcription factor neuronal PAS domain-containing protein 4 (Npas4), which regulates the formation of inhibitory synapses on excitatory neurons, has been suggested as a candidate gene for neurological and psychiatric conditions such as bipolar depression, autism spectrum and cognitive disorders. A mouse model of Npas4 deficiency has been developed to investigate any role in these disorders. Behavioural characterisation of Npas4(-/-), Npas4(+/-) and Npas4(+/+) mice has been conducted using the open field, elevated zero maze (EZM), Y-maze, sociability test and forced swim test (FST) to investigate a range of behaviours. Npas4(-/-) mice spent more time in the open arm of the EZM than other genotypes, suggesting decreased anxiety-like behaviour. Npas4(+/-) mice, however, were more immobile in the FST than other genotypes, suggesting increased depression-like behaviour, and also showed impaired spatial recognition memory in the Y-maze. There were no differences between genotype in social behaviour. These results suggest that differential levels of Npas4 expression in the brain may regulate anxiety, depression and cognition related disorders.

Activity-dependent expression of neuronal PAS domain-containing protein 4 (npas4a) in the developing zebrafish brain.

In rodents, the Npas4 gene has recently been identified as being an important regulator of synaptic plasticity and memory. Homologs of Npas4 have been found in invertebrate species though their functions appear to be too divergent for them to be studied as a proxy for the mammalian proteins. The aim of this study, therefore, was to ascertain the suitability of the zebrafish as a model organism for investigating the function of Npas4 genes. We show here that the expression and regulation of the zebrafish Npas4 homolog, npas4a, is remarkably similar to that of the rodent Npas4 genes. As in mammals, expression of the zebrafish npas4a gene is restricted to the brain where it is up-regulated in response to neuronal activity. Furthermore, we also show that knockdown of npas4a during embryonic development results in a number of forebrain-specific defects including increased apoptosis and misexpression of the forebrain marker genes dlx1a and shha. Our work demonstrates that the zebrafish is a suitable model organism for investigating the role of the npas4a gene and one that is likely to provide valuable insights into the function of the mammalian homologs. Furthermore, our findings highlight a potential role for npas4a in forebrain development.

A reduction in Npas4 expression results in delayed neural differentiation of mouse embryonic stem cells.

INTRODUCTION: Npas4 is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. It is known that in the adult brain Npas4 plays an important role in several key aspects of neurobiology including inhibitory synapse formation, neuroprotection and memory, yet very little is known about the role of Npas4 during neurodevelopment. The aim of this study was to examine the expression and function of Npas4 during nervous system development by using a combination of in vivo experiments in the developing mouse embryo and neural differentiation of embryonic stem cells (ESCs) as an in vitro model of the early stages of embryogenesis. METHODS: Two different neural differentiation paradigms were used to investigate Npas4 expression during neurodevelopment in vitro; adherent monolayer differentiation of mouse ESCs in N2B27 medium and Noggin-induced differentiation of human ESCs. This work was complemented by direct analysis of Npas4 expression in the mouse embryo. The function of Npas4 in the context of neurodevelopment was investigated using loss-of-function experiments in vitro. We created several mouse ESC lines in which Npas4 expression was reduced during neural differentiation through RNA interference and we then analyzed the ability of these Npas4 knockdown mouse ESCs lines to undergo neural differentiation. RESULTS: We found that while Npas4 is not expressed in undifferentiated ESCs, it becomes transiently up-regulated during neural differentiation of both mouse and human ESCs at a stage of differentiation that is characterized by proliferation of neural progenitor cells. This was corroborated by analysis of Npas4 expression in the mouse embryo where the Npas4 transcript was detected specifically in the developing forebrain beginning at embryonic day 9.5. Finally, knockdown of Npas4 expression in mouse ESCs undergoing neural differentiation affected their ability to differentiate appropriately, resulting in delayed neural differentiation. CONCLUSIONS: Here we provide the first evidence that Npas4 is expressed during embryonic development and that it may have a developmental role that is unrelated to its function in the adult brain.

Generating and measuring photochemical changes inside the brain using optical fibers: exploring stroke.

We report here on the development of a method for inducing a stroke in a specific location within a mouse brain through the use of an optical fiber. By capturing the emitted fluorescence signal generated using the same fiber it is possible to monitor photochemical changes within the brain in real-time, and directly measure the concentration of the stroke-inducing dye, Rose Bengal, at the infarct site. This technique reduces the requirement for post-operative histology to determine if a stroke has successfully been induced within the animal, and therefore opens up the opportunity to explore the recovery of the brain after the stroke event.