Bioorthogonal labeling reveals different expression of glycans in mouse hippocampal neuron cultures during their development

The following are available online. Scheme S1: Synthesis of peracetylated azidomannose (Ac4ManNAz); Scheme S2: Alternative synthesis of GlcNAz using chloroacetic anhydride and NaOH as a base; Scheme S3: Synthesis of peracetylated azidofucose (Ac4FucAz); Figure S1: 1H-NMR spectra of ManNAz (D2O, 300 MHz); Figure S2: 1H-NMR spectra of Ac4ManNAz (CDCl3 , 300 MHz), mixture of anomers; Figure S3: HPLC chromatogram of purified Ac4ManNAz showing the two anomers; Figure S4: 1H-NMR spectra of GlcNAz (D2O, 300MHz); Figure S5: 1H-NMR spectra of Ac4GlcNAz (CDCl3 , 300 MHz), mixture of anomers; Figure S6: HPLC chromatogram of purified Ac4GlcNAz showing the two anomers; Figure S7: 1H-NMR spectra of 6-azido-1,2,3,4-tetra-O-acetyl-6-deoxy-α,β-L-galactopyranose Ac4FucAz (CDCl3 , 300 MHz): mixture of anomers; Table S1: Primer sequences used in qRT-PCR.The expression of different glycans at the cell surface dictates cell interactions with their environment and other cells, being crucial for the cell fate. The development of the central nervous system is associated with tremendous changes in the cell glycome that is tightly regulated. Herein, we have employed biorthogonal Cu-free click chemistry to image temporal distribution of different glycans in live mouse hippocampal neurons during their maturation in vitro. We show development-dependent glycan patterns with increased fucose and decreased mannose expression at the end of the maturation process. We also demonstrate that this approach is biocompatible and does not affect glycan transport although it relies on an administration of modified glycans. The applicability of this strategy to tissue sections unlocks new opportunities to study the glycan dynamics under more complex physiological conditions.This research was funded by the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement n.º 668983—FoReCaST, by European Union’s Horizon 2020 Research and Innovation programme, under the Grant Agreement number 739572–The Discoveries CTR, and the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

The choroid plexus transcriptome reveals changes in type I and II interferon responses in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a marked decline in cognition and memory function. Increasing evidence highlights the essential role of neuroinflammatory and immune-related molecules, including those produced at the brain barriers, on brain immune surveillance, cellular dysfunction and amyloid beta (Aß) pathology in AD. Therefore, understanding the response at the brain barriers may unravel novel pathways of relevance for the pathophysiology of AD. Herein, we focused on the study of the choroid plexus (CP), which constitutes the blood-cerebrospinal fluid barrier, in aging and in AD. Specifically, we used the PDGFB-APPSwInd (J20) transgenic mouse model of AD, which presents early memory decline and progressive Aß accumulation, and littermate age-matched wild-type (WT) mice, to characterize the CP transcriptome at 3, 5-6 and 11-12months of age. The most striking observation was that the CP of J20 mice displayed an overall overexpression of type I interferon (IFN) response genes at all ages. Moreover, J20 mice presented a high expression of type II IFN genes in the CP at 3months, which became lower than WT at 5-6 and 11-12months. Importantly, along with a marked memory impairment and increased glial activation, J20 mice also presented a similar overexpression of type I IFN genes in the dorsal hippocampus at 3months. Altogether, these findings provide new insights on a possible interplay between type I and II IFN responses in AD and point to IFNs as targets for modulation in cognitive decline.The authors would like to acknowledge Pedro Moreira, Madalena Esteves and Dr. Patricio Costa (all from the ICVS/3B's - PT Government Associate Laboratory) for comments and scientific input regarding the statistical analysis of this study. Sandro Da Mesquita and Ana C. Ferreira are recipients of PhD fellowships by the Fundacao para a Ciencia e Tecnologia (FCT, Portugal)/FEDER and Programa Operacional Potencial Humano (POPH/FSE), references SFRH/BD/69706/2010 and SFRH/BD/51989/2012, respectively. Fernanda Marques is an assistant researcher and recipient of a FCT Investigator Grant with the reference IF/00231/2013. This work was supported by FCT and COMPETE through the project EXPL/NEU-OSD/2196/2013 and the Bial Foundation through the Grant 217/12

From the periphery to the brain: Lipocalin-2, a friend or foe?

Lipocalin-2 (LCN2) is an acute-phase protein that, by binding to iron-loaded siderophores, acts as a potent bacteriostatic agent in the iron-depletion strategy of the immune system to control pathogens. The recent identification of a mammalian siderophore also suggests a physiological role for LCN2 in iron homeostasis, specifically in iron delivery to cells via a transferrin-independent mechanism. LCN2 participates, as well, in a variety of cellular processes, including cell proliferation, cell differentiation and apoptosis, and has been mostly found up-regulated in various tissues and under inflammatory states, being its expression regulated by several inducers. In the central nervous system less is known about the processes involving LCN2, namely by which cells it is produced/secreted, and its impact on cell proliferation and death, or in neuronal plasticity and behaviour. Importantly, LCN2 recently emerged as a potential clinical biomarker in multiple sclerosis and in ageing-related cognitive decline. Still, there are conflicting views on the role of LCN2 in pathophysiological processes, with some studies pointing to its neurodeleterious effects, while others indicate neuroprotection. Herein, these various perspectives are reviewed and a comprehensive and cohesive view of the general function of LCN2, particularly in the brain, is provided.Ana Catarina Ferreira and Sandro Da Mesquita are recipients of PhD fellowships by the Fundação para a Ciência e Tecnologia (FCT, Portugal)/FEDER. Fernanda Marques is an assistant researcher IF/ 00231/2013 of the Fundação para a Ciência e Tecnologia (FCT, Portugal). This work was supported by Fundação para a Ciência e Tecnologia (FCT) and COMPETE through the project: EXPL/NEUOSD/2196/2013 (to Marques F). The authors thank Nadine Santos for the helpful comments on the manuscript

Envelhecimento e doença de Alzheimer: Em busca de novas pistas moleculares

Tese de Doutoramento em Ciências da SaúdeIncreased life expectancy contributed to a marked increase in aging-associated maladies. Among those, we can find dementias, which are often accompanied by neurodegeneration and behavioral alterations, and may culminate in a loss of independence and autonomy. Alzheimer's disease (AD) represents the most prevalent form of age-associated dementia and is characterized by severe memory loss and cognitive deficits. This neurodegenerative disease exists as either a less common, early-onset, autosomal dominant familial form and a more prevalent, late-onset, sporadic form. Yet, both types of AD are characterized by common brain pathological hallmarks, namely the presence of extracellular senile plaques, which are highly enriched in different forms of aggregated amyloid beta (Aβ) peptides, and of intracellular neurofibrillary tangles, that result from the aggregation of hyperphosphorylated Tau protein. The discoveries made in the past two decades shed some light on the mechanisms that trigger neuroinflammation, cellular dysfunction and brain pathology, as well as their role as mediators of behavioral alterations, in AD. However, certain aspects of the disease are still poorly understood and are, sometimes, controversial. This thesis emerged with the objective of using different models to explore the role of novel and promising cellular and molecular targets in the context of aging and AD. Initially, we focused on the role of the protein lipocalin 2 (LCN2), which is closely involved in the innate immune response and in iron metabolism, two different mechanisms that can equally modulate the amyloidogenic pathway and Aβ toxicity in AD. Herein, we show that LCN2 is overproduced both in vitro and in vivo by specific brain cell types, namely choroid plexus (CP) epithelial cells and astrocytes, in response to Aβ1-42 peptides. Importantly, we show that the overexpression of iron metabolism and innate-like proinflammatory genes, as well as the marked astrogliosis, observed in response to the toxic levels of Aβ1-42, are ablated in the absence of LCN2. These results point to a role of LCN2 in the modulation of Aβ-induced toxicity and prompt for further investigation regarding the impact of LCN2 on brain pathology and behavior impairment in AD. Next, taking into account the important function of the brain barriers in the maintenance of central nervous system (CNS) homeostasis in health and in disease, we analyzed the response at the level of the blood-cerebrospinal fluid (CSF) barrier, formed by the CP cells, in AD. For that, we took advantage of an AD transgenic mouse model, the PDGFB-APPSwInd (J20) mice, which overexpress the human gene for amyloid precursor protein (APP) with two mutations that favor its amyloidogenic processing and an increased formation of Aβ peptides. Specifically, we analyzed the response in the CP of J20 mice and of age matched wild-type (WT) littermate controls at 3, 5-6 and 11-12 months. By microarray analysis, we found that aging influences different gene pathways in the CP of 11-12 months-old mice, such as the ones involved in the regulation of the cellular circadian cycle, transport of nutrients and lipid metabolism. These alterations support the idea that there is a clear dysfunction of the CP with aging, which impacts on the production of CSF and on the integrity of the blood-CSF barrier. Strikingly, we found an early overexpression of genes implicated in the type I interferon (IFN) response and a decreased expression of the gene encoding for IFN-γ, the major type II IFN cytokine, at later ages, in the CP of J20 mice when compared to age-matched WT mice. Trying to figure out the extent of these changes, we also investigated the response in the brain parenchyma, namely in the dorsal hippocampus (dHPC), and in the periphery, particularly in the liver. Interestingly, the manifestation of deficits in spatial reference-memory by J20 mice, at 3 months, and by WT mice, at 11-12 months, was accompanied by an increased expression of type I IFN genes in the dHPC. Noticeably, we observed a significant correlation between the levels of IFN-α in the CSF and the performance in a memory-dependent task. Regarding the responses in the brain and in the periphery, we found a similar pattern of type II IFN gene expression in the CP and in the liver of J20 mice. However, with aging, there was an overexpression of type I IFN genes and a decrease in the number of perivascular Aβ-loaded macrophages in the liver of J20 mice. These observations suggest that changes in the levels of different types of IFNs may influence the deposition and/or clearance of Aβ, as well as memory and cognition. Altogether, the work presented here highlights some important functions and effects of different types of inflammatory molecules, such as LCN2 and IFNs, in the context of neurodegeneration and of behavioral alterations in AD, opening new doors for future research in the field.O aumento da esperança média de vida contribuiu para um aumento acentuado das doenças associadas ao envelhecimento. Entre essas, podemos encontrar as demências, que são muitas vezes acompanhadas por neurodegeneração e alterações comportamentais, e poderão culminar numa perda de independência e autonomia. A Doença de Alzheimer (DA) representa a forma mais prevalente de demência associada ao envelhecimento, e é caracterizada por perda de memória e défices cognitivos acentuados. Esta doença neurodegenerativa pode manifestar-se numa forma autossómica dominante menos comum, de início precoce, ou numa forma esporádica mais comum, de início tardio. No entanto, ambas as formas de DA são caracterizadas por marcas patológicas cerebrais comuns, nomeadamente a presença de placas senis extracelulares, ricas em péptidos beta-amiloide (Aβ) em diferentes estados de agregação, e emaranhados neurofibrilares intracelulares, resultantes da agregação da proteína Tau hiperfosforilada. As descobertas realizadas nas últimas duas décadas contribuiram para uma melhor compreensão dos mecanismos que desencadeiam neuroinflamação, disfunção celular e patologia cerebral, assim como o seu papel como mediadores de alterações comportamentais, na DA. No entanto, alguns pormenores acerca da doença não são ainda completamente claros, sendo até, por vezes, controversos. Esta tese surgiu com o objetivo de utilizar modelos diferentes para explorar o papel de alvos celulares e moleculares novos e promissores no contexto do envelhecimento e da DA. Inicialmente, focámo-nos no papel da proteína lipocalina 2 (LCN2), que está intimamente envolvida na resposta imune inata e no metabolismo do ferro, dois mecanismos diferentes que estão envolvidos na modulação da via amiloidogénica e da toxicidade da Aβ na DA. Neste trabalho, mostramos que a LCN2 é produzida em maior quantidade, tanto in vitro como in vivo, por certos tipos de células cerebrais, nomeadamente por células epiteliais do plexo coroide (CP) e astrócitos, em resposta a péptidos Aβ1-42. É importante salientar que os níveis de expressão de genes do metabolismo do ferro e da resposta pró-inflamatória inata, assim como a astrogliose acentuada, observados em resposta a níveis tóxicos de Aβ1-42, estão reduzidos na ausência de LCN2. Estes resultados apontam para um papel da LCN2 na modulação da toxicidade induzida por péptidos Aβ e para a necessidade de aprofundar a investigação acerca do impacto da LCN2 na patologia cerebral e défices comportamentais na DA. De seguida, tendo em conta a importante função das barreiras do cérebro na manutenção da homeostasia do sistema nervoso central (SNC) na saúde e na doença, analisámos a resposta ao nível da barreira sangue-líquido cefalorraquidiano (LCR), formado pela células do CP, na DA. Nesse sentido, utilizámos um modelo da DA, os ratinhos transgénicos PDGFB-APPSwInd (J20), que sobreexpressam o gene humano que codifica a proteína precursora da amiloide (APP) com duas mutações que favorecem o seu processamento pela via amiloidogénica e um aumento da formação de péptidos Aβ. Especificamente, analisámos a resposta no CP de ratinhos J20 e do tipo selvagem (WT), da mesma idade e ninhada, aos 3, 5-6 e 11-12 meses. Através da análise por microarray, descobrimos que o envelhecimento influencia genes de vias diferentes no CP de ratinhos com 11- 12 meses de idade, nomeadamente as que estão envolvidas na regulação do ciclo circadiano celular, transporte de nutrientes e metabolismo de lípidos. Estas alterações apoiam a ideia de que há uma clara disfunção do CP com o envelhecimento, e que esta tem um impacto sobre a produção de LCR e sobre a integridade da barreira sangue-LCR. Surpreendentemente, descobrimos uma sobre-expressão precoce de genes envolvidos na resposta de interferão (IFN) do tipo I e uma diminuição da expressão do gene que codifica IFN-γ, a principal citocina de IFN tipo II, em idades posteriores, no CP de ratinhos J20 em comparação com ratinhos WT da mesma idade. Numa tentativa de descobrir a extensão destas alterações, também investigámos a resposta no parênquima cerebral, nomeadamente no hipocampo dorsal (dHPC), e na periferia, particularmente no fígado. Curiosamente, a manifestação de défices de memória por ratinhos J20, aos 3 meses, e por ratinhos WT, aos 11-12 meses, foi acompanhada por um aumento da expressão de genes de IFN tipo I no dHPC. Notavelmente, observámos uma correlação significativa entre os níveis de IFN-α no LCR e o desempenho numa tarefa dependente da memória. Relativamente às respostas no cérebro e na periferia, encontrámos um padrão semelhante de expressão de genes de IFN tipo II no CP e no fígado de ratinhos J20. No entanto, com o envelhecimento, houve uma sobre-expressão de genes de IFN tipo I e uma diminuição no número de macrófagos perivasculares positivos para Aβ no fígado de ratinhos J20. Estas observações sugerem que alterações nos níveis de diferentes tipos de IFNs podem influenciar a deposição e/ou a excreção de Aβ, bem como a memória e cognição. No seu todo, o trabalho aqui apresentado destaca importantes funções e efeitos de diferentes moléculas inflamatórias, como a LCN2 e os IFNs, no contexto da neurodegeneração e das alterações comportamentais na DA, abrindo novas portas para futura investigação na área

Insights on the pathophysiology of Alzheimer's disease: the crosstalk between amyloid pathology, neuroinflammation and the peripheral immune system

Alzheimer's disease (AD) is the most common form of dementia, whose prevalence is growing along with the increased life expectancy. Although the accumulation and deposition of amyloid beta (Aß) peptides in the brain is viewed as one of the pathological hallmarks of AD and underlies, at least in part, brain cell dysfunction and behavior alterations, the etiology of this neurodegenerative disease is still poorly understood. Noticeably, increased amyloid load is accompanied by marked inflammatory alterations, both at the level of the brain parenchyma and at the barriers of the brain. However, it is debatable whether the neuroinflammation observed in aging and in AD, together with alterations in the peripheral immune system, are responsible for increased amyloidogenesis, decreased clearance of Aß out of the brain and/or the marked deficits in memory and cognition manifested by AD patients. Herein, we scrutinize some important traits of the pathophysiology of aging and AD, focusing on the interplay between the amyloidogenic pathway, neuroinflammation and the peripheral immune system.Fundação Calouste Gulbenkian (Programa Gulbenkian Inovar em Saúde), with the reference UMINHO/BI/155/2015. (FCT, Portugal)/FEDER and Programa Operacional Potencial Humano (POPH/FSE); This work was supported by FCT and COMPETE through the project EXPL/NEU-OSD/2196/2013 and the Bial Foundation through the Grant 217/1

Plasmacytoid and conventional dendritic cells are early producers of IL-12 in Neospora caninum-infected mice

Neospora caninum is a coccidian parasite causative of clinical infections in a wide range of animal hosts. The maturation and activation of splenic conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) were studied here in BALB/c mice challenged intraperitoneal with N. caninum tachyzoites. The number of cDCs was found to decrease in the spleen of the infected mice 12 h and 2 days after the parasitic challenge, whereas at day 5 after infection it was significantly above that of mock-infected controls. In contrast, the number of splenic pDCs did not change significantly on infection. In the infected mice, both cell subtypes displayed an activated phenotype with upregulation of costimulatory and MHC class II molecules. This stimulatory effect was more marked at the earliest assessed time point after infection, 12 h, when a clear increase in the frequency of cDCs (CD8alpha(+) and CD8alpha(-)) and pDCs producing interleukin-12 (IL-12) was also observed. N. caninum tachyzoites could be observed by confocal microscopy associated with sorted DCs. Overall, these results present the first evidence that both cDCs and pDCs mediate in vivo the innate immune response to N. caninum infection through the production of IL-12, a key cytokine for host resistance to neosporosis.We are indebted to Dr Andreia Lino, Dr Elsa Seixas and Dr Flávia Lima forfruitful discussions. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) and FEDER grant no POCTI/CVT/38791/MGI/2001 andSUDOE-FEDER IMMUNONET SOE1/P1/E014. Luzia Teixeira was financedby FCT fellowship SFRH/BD/12983/200