Targeting the Multiple Complex Processes of Hypoxia-Ischemia to Achieve Neuroprotection.

Hypoxic-ischemic encephalopathy (HIE) is a major cause of newborn brain damage stemming from a lack of oxygenated blood flow in the neonatal period. Twenty-five to fifty percent of asphyxiated infants who develop HIE die in the neonatal period, and about sixty percent of survivors develop long-term neurological disabilities. From the first minutes to months after the injury, a cascade of events occurs, leading to blood-brain barrier (BBB) opening, neuronal death and inflammation. To date, the only approach proposed in some cases is therapeutic hypothermia (TH). Unfortunately, TH is only partially protective and is not applicable to all neonates. This review synthesizes current knowledge on the basic molecular mechanisms of brain damage in hypoxia-ischemia (HI) and on the different therapeutic strategies in HI that have been used and explores a major limitation of unsuccessful therapeutic approaches.

Deciphering neuronal deficit and protein profile changes in human brain organoids from patients with creatine transporter deficiency.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3ß, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3ß, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.

Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease.

Human brain organoids (mini-brains) consist of self-organized three-dimensional (3D) neural tissue which can be derived from reprogrammed adult cells and maintained for months in culture. These 3D structures manifest substantial potential for the modeling of neurodegenerative diseases and pave the way for personalized medicine. However, as these 3D brain models can express the whole human genetic complexity, it is critical to have access to isogenic mini-brains that only differ in specific and controlled genetic variables. Genetic engineering based on retroviral vectors is incompatible with the long-term modeling needed here and implies a risk of random integration while methods using CRISPR-Cas9 are still too complex to adapt to stem cells. We demonstrate in this study that our strategy which relies on an episomal plasmid vector derived from the Epstein-Barr virus (EBV) offers a simple and robust approach, avoiding the remaining caveats of mini-brain models. For this proof-of-concept, we used a normal tau protein with a fluorescent tag and a mutant genetic form (P301S) leading to Fronto-Temporal Dementia. Isogenic cell lines were obtained which were stable for more than 30 passages expressing either form. We show that the presence of the plasmid in the cells does not interfere with the mini-brain differentiation protocol and obtain the development of a pathologically relevant phenotype in cerebral organoids, with pathological hyperphosphorylation of the tau protein. Such a simple and versatile genetic strategy opens up the full potential of human organoids to contribute to disease modeling, personalized medicine and testing of therapeutics.

3-O-sulfated heparan sulfate interactors target synaptic adhesion molecules from neonatal mouse brain and inhibit neural activity and synaptogenesis in vitro.

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.

Glycosaminoglycans from Alzheimer's disease hippocampus have altered capacities to bind and regulate growth factors activities and to bind tau.

Glycosaminoglycans (GAGs), including heparan sulfates and chondroitin sulfates, are major components of the extracellular matrix. Upon interacting with heparin binding growth factors (HBGF), GAGs participate to the maintaintenance of tissue homeostasis and contribute to self-healing. Although several processes regulated by HBGF are altered in Alzheimer's disease, it is unknown whether the brain GAG capacities to bind and regulate the function of HBGF or of other heparin binding proteins, as tau, are modified in this disease. Here, we show that total sulfated GAGs from hippocampus of Alzheimer's disease have altered capacities to bind and potentiate the activities of growth factors including FGF-2, VEGF, and BDNF while their capacity to bind to tau is remarkable increased. Alterations of GAG structures and capacities to interact with and regulate the activity of heparin binding proteins might contribute to impaired tissue homeostasis in the Alzheimer's disease brain.

The role of heparan sulfates in protein aggregation and their potential impact on neurodegeneration.

Neurodegenerative disorders, such as Alzheimer's, Parkinson's, and prion diseases, are directly linked to the formation and accumulation of protein aggregates in the brain. These aggregates, principally made of proteins or peptides that clamp together after acquisition of ß-folded structures, also contain heparan sulfates. Several lines of evidence suggest that heparan sulfates centrally participate in the protein aggregation process. In vitro, they trigger misfolding, oligomerization, and fibrillation of amyloidogenic proteins, such as Aß, tau, α-synuclein, prion protein, etc. They participate in the stabilization of protein aggregates, protect them from proteolysis, and act as cell-surface receptors for the cellular uptake of proteopathic seeds during their spreading. This review focuses attention on the importance of heparan sulfates in protein aggregation in brain disorders including Alzheimer's, Parkinson's, and prion diseases. The presence of these sulfated polysaccharides in protein inclusions in vivo and their capacity to trigger protein aggregation in vitro strongly suggest that they might play critical roles in the neurodegenerative process. Further advances in glyco-neurobiology will improve our understanding of the molecular and cellular mechanisms leading to protein aggregation and neurodegeneration.

Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis.

Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.

Gallium-68 Complexes Conjugated to Pittsburgh Compound B: Radiolabeling and Biological Evaluation.

PURPOSE: The aim of this work is to develop an efficient and fully automated radiosynthesis of three derivatives of the Pittsburgh compound B labeled with gallium-68 for the detection of amyloid plaques. PROCEDURES: The radiolabeling of the precursors and purification of the radiolabeled agents by high pressure liquid chromatography has been studied prior to their in vitro and in vivo evaluations. RESULTS: The complete process led, in 50 min, to pure Ga-68 products in a 12-38 % yield and with appreciable specific radioactivity (SRA, 85-168 GBq/µmol) which enabled us to demonstrate a considerable in vivo stability of the products. Unfortunately, this result was associated with a poor blood-brain barrier (BBB) permeability and a limited uptake of our compounds by amyloid deposits was observed by in vitro autoradiography. CONCLUSION: Although we have not yet identified a compound able to significantly mark cerebral amyloidosis, this present investigation will likely contribute to the development of more successful Ga-68 radiotracers.

Heparán sulfatos, amiloidosis y neurodegeneración / Heparan sulphates, amyloidosis and neurodegeneration

Introducción. Numerosos trastornos neurodegenerativos se han asociado directamente a la acumulación de fibras amiloides. Estas fibras están formadas por proteínas o péptidos con conformaciones alteradas y que se agregan in vivo en asociación con polisacáridos de tipo heparán sulfatos. Objetivos. Examinar los conceptos más recientes sobre la biología de los heparán sulfatos y su papel en la agregación del péptido Abeta, de la proteína tau, de la alfa-sinucleína y de los priones, y analizar sus implicaciones en trastornos neurodegenerativos como las enfermedades de Alzheimer y de Parkinson y las enfermedades priónicas. Desarrollo. In vitro, los heparán sulfatos han desempeñado un papel importante en el proceso de oligomerización y fibrilación de proteínas o péptidos amiloidógenos, en la estabilización de estos cuerpos y su resistencia a la proteólisis, participando así en la formación de una gran variedad de fibras amiloides. Los heparán sulfatos se han relacionado también con el proceso de internalización de fibras proamiloides durante el proceso de propagación intercelular (spreading) considerado como central en la evolución de las proteinopatías, cuyo mejor ejemplo es la enfermedad de Alzheimer. Conclusión. Este trabajo sugiere que las estructuras finas de los heparán sulfatos, sus localizaciones celulares y tisulares, así como sus concentraciones locales, pueden regular los procesos de amiloidosis. Avances en la comprensión de esta área de la gliconeurobiología permitirán mejorar la comprensión de los mecanismos celulares y moleculares del proceso neurodegenerativo (AU)

Introduction. A number of neurodegenerative disorders have been linked directly to the accumulation of amyloid fibres. These fibres are made up of proteins or peptides with altered structures and which join together in vivo in association with heparan sulphate-type polysaccharides. AIMS. To examine the most recent concepts in the biology of heparan sulphates and their role in the aggregation of the peptide Abeta, of tau protein, of alpha-synuclein and of prions. The study also seeks to analyse their implications in neurodegenerative disorders such as Alzheimers and Parkinson’s disease and prion diseases. Development. In vitro, heparan sulphates have played an important role in the process of oligomerisation and fibrillation of amyloidogenic proteins or peptides, in the stabilisation of these bodies and their resistance to proteolysis, thereby participating in the formation of a wide range of amyloid fibres. Heparan sulphates have also been related to the internalisation of pro-amyloid fibres during the process of intercellular propagation (spreading), which is considered to be crucial in the development of proteinopathies, the best example of which is Alzheimers disease. Conclusion This study suggests that the fine structures of heparan sulphates, their localisation in cells and tissues, together with their local concentration, may regulate the amyloidosis processes. The advances made in the understanding of this area of glyconeurobiology will make it possible to improve the understanding of the cell and molecular mechanisms underlying the neurodegenerative process (AU)