In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

The cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is one of the most prominent examples within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis as well as interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2 and cytoplasmic dynein-1. We also describe pH-dependent conformational changes of BicD2 using cryoEM, template-free structure predictions, and biophysical tools. Our results will help defining the biochemical parameters for the invitro reconstitution of higher order BicD2 protein complexes.

Engrafted glial progenitor cells yield long-term integration and sensory improvement in aged mice

Aging causes astrocyte morphological degeneration and functional deficiency, which impairs neuronal functions. Until now, whether age-induced neuronal deficiency could be alleviated by engraftment of glial progenitor cell (GPC) derived astrocytes remained unknown. In the current study, GPCs were generated from embryonic cortical neural stem cells in vitro and transplanted into the brains of aged mice. Their integration and intervention effects in the aged brain were examined 12 months after transplantation. Results indicated that these in-vitro-generated GPC-derived astrocytes possessed normal functional properties. After transplantation they could migrate, differentiate, achieve long-term integration, and maintain much younger morphology in the aged brain. Additionally, these GPC-derived astrocytes established endfeet expressing aquaporin-4 (AQP4) and ameliorate AQP4 polarization in the aged neocortex. More importantly, age-dependent sensory response degeneration was reversed by GPC transplantation. This work demonstrates that rejuvenation of the astrocyte niche is a promising treatment to prevent age-induced degradation of neuronal and behavioral functions.

Therapeutic Effects of hiPSC-Derived Glial and Neuronal Progenitor Cells-Conditioned Medium in Experimental Ischemic Stroke in Rats

Transplantation of various types of stem cells as a possible therapy for stroke has been tested for years, and the results are promising. Recent investigations have shown that the administration of the conditioned media obtained after stem cell cultivation can also be effective in the therapy of the central nervous system pathology (hypothesis of their paracrine action). The aim of this study was to evaluate the therapeutic effects of the conditioned medium of hiPSC-derived glial and neuronal progenitor cells in the rat middle cerebral artery occlusion model of the ischemic stroke. Secretory activity of the cultured neuronal and glial progenitor cells was evaluated by proteomic and immunosorbent-based approaches. Therapeutic effects were assessed by overall survival, neurologic deficit and infarct volume dynamics, as well as by the end-point values of the apoptosis- and inflammation-related gene expression levels, the extent of microglia/macrophage infiltration and the numbers of formed blood vessels in the affected area of the brain. As a result, 31% of the protein species discovered in glial progenitor cells-conditioned medium and 45% in neuronal progenitor cells-conditioned medium were cell type specific. The glial progenitor cell-conditioned media showed a higher content of neurotrophins (BDNF, GDNF, CNTF and NGF). We showed that intra-arterial administration of glial progenitor cells-conditioned medium promoted a faster decrease in neurological deficit compared to the control group, reduced microglia/macrophage infiltration, reduced expression of pro-apoptotic gene Bax and pro-inflammatory cytokine gene Tnf, increased expression of anti-inflammatory cytokine genes (Il4, Il10, Il13) and promoted the formation of blood vessels within the damaged area. None of these effects were exerted by the neuronal progenitor cell-conditioned media. The results indicate pronounced cytoprotective, anti-inflammatory and angiogenic properties of soluble factors secreted by glial progenitor cells.

Secretome-Mediated Neuroprotective Effects of the hiPSC-derived Glial and Neuronal Progenitor Cells in the Hypoxia-Induced Neuronal Damage (Comparative Study)

Background: Stem cell secretomes hold great promise for regenerative medicine. This study is focused on the secretome-mediated neuroprotective effects of the human induced pluripotent stem cell-derived neuronal and glial progenitor cells. Therapeutic properties of the secretomes were assessed under conditions of the hypoxia-induced neuronal damage in vitro and in vivo. Methods: Secretory activity of the cultured neuronal and glial progenitor cells was analyzed by proteomic and immunosorbent-based approaches. Conditioned media collected from the cultures was tested for neuroprotective properties in vitro and in vivo.In vitro experiments involved exposure of SH-SY5Y cells to the conditioned media during the recovery from the cobalt chloride-induced hypoxia. Neuroprotective effects were assessed by cell survival and neurite outgrowth. Cell survival indicators included MTT and LDH tests, vital staining with propidium iodide and Hoechst 33342, and polymerase chain reaction assay for the expression of apoptosis-related genes. Neurite outgrowth was assessed by alterations in SH-SY5Y cell morphology and MAP2/GAP43 gene expression dynamics. In vivo experiments involved intra-arterial administration of the conditioned media to laboratory rats during the recovery from experimental ischemic stroke. Neuroprotective effects were assessed by overall survival, neurologic deficit and infarct volume dynamics, as well as by the end-point values of the apoptosis- and inflammation-related gene expression levels, the extent of microglia/macrophage infiltration, and the numbers of newly formed blood vessels in the affected area of the brain. Results: Secretomes of glial and neuronal progenitor cells partially overlapped, with specific proteins (found in secretome of one of the studied cultures and absent from the other) constituting, respectively, 31% and 45%. The glial progenitor cell-conditioned media showed higher content of neurotrophins (BDNF, GDNF, CNTF and NGF).Moreover, the glial progenitor cell-conditioned media was superior to the neuronal progenitor cell-conditioned media in facilitating neurite outgrowth and increasing SHSY-5Y cell survival after the cobalt dichloride-induced hypoxia. In addition, intra-arterial infusion of the glial progenitor cell-conditioned media to the animals after experimental ischemic stroke significantly enhanced functional recovery and promoted tissue repair at the site of brain damage, as indicated by reduced microglia/macrophage infiltration, decreased expression of pro-apoptotic gene Bax and pro-inflammatory cytokine gene Tnf, increased expression of anti-inflammatory cytokine genes (Il4, Il10, Il13), and increased numbers of newly formed blood vessels within the damaged area. None of these effects were exerted by the neuronal progenitor cell-conditioned media. Conclusions: The results indicate pronounced cytoprotective, anti-inflammatory and angionenic properties of soluble factors secreted by glial progenitor cells.

Direct Conversion of Human Stem Cell-Derived Glial Progenitor Cells into GABAergic Interneurons

Glial progenitor cells are widely distributed in brain parenchyma and represent a suitable target for future therapeutic interventions that generate new neurons via in situ reprogramming. Previous studies have shown successful reprogramming of mouse glia into neurons whereas the conversion of human glial cells remains challenging due to the limited accessibility of human brain tissue. Here, we have used a recently developed stem cell-based model of human glia progenitor cells (hGPCs) for direct neural reprogramming by overexpressing a set of transcription factors involved in GABAergic interneuron fate specification. GABAergic interneurons play a key role in balancing excitatory and inhibitory neural circuitry in the brain and loss or dysfunction of these have been implicated in several neurological disorders such as epilepsy, schizophrenia, and autism. Our results demonstrate that hGPCs successfully convert into functional induced neurons with postsynaptic activity within a month. The induced neurons have properties of GABAergic neurons, express subtype-specific interneuron markers (e.g. parvalbumin) and exhibit a complex neuronal morphology with extensive dendritic trees. The possibility of inducing GABAergic interneurons from a renewable in vitro hGPC system could provide a foundation for the development of therapies for interneuron pathologies.

Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly

Primary microcephaly and megalencephaly are severe brain malformations defined by reduced and increased brain size, respectively. Whether these two pathologies arise from related alterations at the molecular level is unclear. Microcephaly has been largely associated with centrosomal defects, leading to cell death. Here, we investigated the consequences of WDR81 loss of function, which cause severe microcephaly in patients. We show that WDR81 regulates endosomal trafficking of EGFR, and that loss of function leads to reduced MAP kinase pathway activation. Mouse radial glial progenitor cells knocked-out for WDR81 display reduced proliferation rates, leading to reduced brain size. These proliferation defects are rescued in vivo by the expression of megalencephaly-causing mutated Cyclin D2. Our results identify the endosomal machinery as an important regulator of RG cell proliferation rates and brain growth. They demonstrate that microcephaly and megalencephaly can be due to opposite effects on the proliferation rate of radial glial progenitors.