Gamma secretase modulators and BACE inhibitors reduce Aß production without altering gene expression in Alzheimer's disease iPSC-derived neurons and mice.

In drug discovery, as well as in the study of disease biology, it is fundamental to develop models that recapitulate aspects of a disorder, in order to understand the pathology and test therapeutic approaches. Patient-derived induced pluripotent stem cells (iPSCs) offer the potential of obtaining tissue-specific cells with a given human genotype. Here we derived neural cultures from Alzheimer's disease patient iPSCs and characterized their response to three classes of compounds that reduce the production of Aß42, a major driving force of this pathology. We characterized their effect on the cells, looking at Tau proteostasis and gene expression changes by RNAseq. ß-secretase inhibitor and γ-secretase modulators left the transcriptional balance of the cells virtually unaffected, while γ-secretase inhibitors caused drastic gene expression changes due to Notch inhibition. We observed similar effects in vivo, treating mice with the same compound classes. Our results show that ß-secretase inhibitors and γ-secretase modulators are attractive candidates for modulating Aß production in Alzheimer's disease. Moreover, we demonstrate that the response to compounds obtained with iPSC-derived neurons is similar to the one observable in vivo.

Embryonic stem cell-derived neural stem cells fuse with microglia and mature neurons.

Transplantation of neural stem cells (NSCs) is a novel strategy to restore function in the diseased brain, acting through multiple mechanisms, for example, neuronal replacement, neuroprotection, and modulation of inflammation. Whether transplanted NSCs can operate by fusing with microglial cells or mature neurons is largely unknown. Here, we have studied the interaction of a mouse embryonic stem cell-derived neural stem (NS) cell line with rat and mouse microglia and neurons in vitro and in vivo. We show that NS cells spontaneously fuse with cocultured cortical neurons, and that this process requires the presence of microglia. Our in vitro data indicate that the NS cells can first fuse with microglia and then with neurons. The fused NS/microglial cells express markers and retain genetic and functional characteristics of both parental cell types, being able to respond to microglia-specific stimuli (LPS and IL-4/IL-13) and to differentiate to neurons and astrocytes. The NS cells fuse with microglia, at least partly, through interaction between phosphatidylserine exposed on the surface of NS cells and CD36 receptor on microglia. Transplantation of NS cells into rodent cortex results in fusion with mature pyramidal neurons, which often carry two nuclei, a process probably mediated by microglia. The fusogenic role of microglia could be even more important after NSC transplantation into brains affected by neurodegenerative diseases associated with microglia activation. It remains to be elucidated how the occurrence of the fused cells will influence the functional outcome after NSC transplantation in the diseased brain.

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aß Levels Mediated by Brain Shuttle Transport.

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidß (Aß) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aß levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aß. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Precursor States of Brain Tumor Initiating Cell Lines Are Predictive of Survival in Xenografts and Associated with Glioblastoma Subtypes.

In glioblastoma multiforme (GBM), brain-tumor-initiating cells (BTICs) with cancer stem cell characteristics have been identified and proposed as primordial cells responsible for disease initiation, recurrence, and therapeutic resistance. However, the extent to which individual, patient-derived BTIC lines reflect the heterogeneity of GBM remains poorly understood. Here we applied a stem cell biology approach and compared self-renewal, marker expression, label retention, and asymmetric cell division in 20 BTIC lines. Through cluster analysis, we identified two subgroups of BTIC lines with distinct precursor states, stem- or progenitor-like, predictive of survival after xenograft. Moreover, stem and progenitor transcriptomic signatures were identified, which showed a strong association with the proneural and mesenchymal subtypes, respectively, in the TCGA cohort. This study proposes a different framework for the study and use of BTIC lines and provides precursor biology insights into GBM.

Human fetal striatum-derived neural stem (NS) cells differentiate to mature neurons in vitro and in vivo.

Clonogenic neural stem (NS) cell lines grown in adherent cultures have previously been established from embryonic stem cells and fetal and adult CNS in rodents and from human fetal brain and spinal cord. Here we describe the isolation of a new cell line from human fetal striatum (hNS cells). These cells showed properties of NS cells in vitro such as monolayer growth, high proliferation rate and expression of radial glia markers. The hNS cells expressed an early neuronal marker while being in the proliferative state. Under appropriate conditions, the hNS cells were efficiently differentiated to neurons, and after 4 weeks about 50% of the cells were ßIII tubulin positive. They also expressed the mature neuronal marker NeuN and markers of neuronal subtypes, GABA, calbindin, and DARPP32. After intrastriatal implantation into newborn rats, the hNS cells survived and many of them migrated outside the transplant core into the surrounding tissue. A high percentage of cells in the grafts expressed the neuroblast marker DCX, indicating their neurogenic potential, and some of the cells differentiated to NeuN+ mature neurons. The human fetal striatum-derived NS cell line described here should be a useful tool for studies on cell replacement strategies in models of the striatal neuronal loss occurring in Huntington's disease and stroke.

Adult periventricular neural stem cells: outstanding progress and outstanding issues.

Twenty years have past since the existence of neural stem cells (NSCs) within the walls of the adult lateral ventricles was discovered. During this period of time, great strides have been made in every facet of our understanding of this adult periventricular NSC population. In this review, some of the fields' major advancements regarding the nature and function of adult periventricular NSCs are examined. We bring attention to issues related to NSC identity, potential, and the role of Notch signaling in regulating quiescence and activation that warrant further investigation. Progress in the understanding of human adult NSCs will aid in the development of tools required to advance therapies not only for brain repair after injury or disease but may also lead to novel therapeutics for brain tumors.

Cell number and timing of transplantation determine survival of human neural stem cell grafts in stroke-damaged rat brain.

Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survive in stroke-damaged striatum, migrate from the implantation site, and differentiate into mature neurons. Here, we investigated how various steps of neurogenesis are affected by intrastriatal transplantation of human NSCs at different time points after stroke and with different numbers of cells in each implant. Rats were subjected to middle cerebral artery occlusion and then received intrastriatal transplants of NSCs. Transplantation shortly after stroke (48 hours) resulted in better cell survival than did transplantation 6 weeks after stroke, but the delayed transplantation did not influence the magnitude of migration, neuronal differentiation, and cell proliferation in the grafts. Transplanting greater numbers of grafted NSCs did not result in a greater number of surviving cells or increased neuronal differentiation. A substantial number of activated microglia was observed at 48 hours after the insult in the injured striatum, but reached maximum levels 1 to 6 weeks after stroke. Our findings show that the best survival of grafted human NSCs in stroke-damaged brain requires optimum numbers of cells to be transplanted in the early poststroke phase, before the inflammatory response is established. These findings, therefore, have direct clinical implications.