SummaryHere we elucidate the effect of Alzheimer disease (AD)-predisposing genetic backgrounds, APOE4, PSEN1ΔE9, and APPswe, on functionality of human microglia-like cells (iMGLs). We present a physiologically relevant high-yield protocol for producing iMGLs from induced pluripotent stem cells. Differentiation is directed with small molecules through primitive erythromyeloid progenitors to re-create microglial ontogeny from yolk sac. The iMGLs express microglial signature genes and respond to ADP with intracellular Ca2+ release distinguishing them from macrophages. Using 16 iPSC lines from healthy donors, AD patients and isogenic controls, we reveal that the APOE4 genotype has a profound impact on several aspects of microglial functionality, whereas PSEN1ΔE9 and APPswe mutations trigger minor alterations. The APOE4 genotype impairs phagocytosis, migration, and metabolic activity of iMGLs but exacerbates their cytokine secretion. This indicates that APOE4 iMGLs are fundamentally unable to mount normal microglial functionality in AD.

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Background/Objective: Stroke is a leading global cause of adult disability. As the population ages as well as suffers co-morbidities, it is expected that the stroke burden will increase further. There are no established safe and effective restorative treatments to facilitate a good functional outcome in stroke patients. Cell-based therapies, which have a wide therapeutic window, might benefit a large percentage of patients, especially if combined with different restorative strategies. In this study, we tested whether the therapeutic effect of human adipose tissue-derived mesenchymal stem cells (ADMSCs) could be further enhanced by rehabilitation in an experimental model of stroke. Methods: Focal cerebral ischemia was induced in adult male Sprague Dawley rats by permanently occluding the distal middle cerebral artery (MCAO). After the intravenous infusion of vehicle ( n = 46) or ADMSCs (2 × 10 6 ) either at 2 ( n = 37) or 7 ( n = 7) days after the operation, half of the animals were housed in an enriched environment mimicking rehabilitation. Subsequently, their behavioral recovery was assessed by a neurological score, and performance in the cylinder and sticky label tests during a 42-day behavioral follow-up. At the end of the follow-up, rats were perfused for histology to assess the extent of angiogenesis (RECA-1), gliosis (GFAP), and glial scar formation. Results: No adverse effects were observed during the follow-up. Combined ADMSC therapy and rehabilitation improved forelimb use in the cylinder test in comparison to MCAO controls on post-operative days 21 and 42 ( P < 0.01). In the sticky label test, ADMSCs and rehabilitation alone or together, significantly decreased the removal time as compared to MCAO controls on post-operative days 21 and 42. An early initiation of combined therapy seemed to be more effective. Infarct size, measured by MRI on post-operative days 1 and 43, did not differ between the experimental groups. Stereological counting revealed an ischemia-induced increase both in the density of blood vessels and the numbers of glial cells in the perilesional cortex, but there were no differences among MCAO groups. Glial scar volume was also similar in MCAO groups. Conclusion: Early delivery of ADMSCs and combined rehabilitation enhanced behavioral recovery in an experimental stroke model. The mechanisms underlying these treatment effects remain unknown.

MicroRNAs (miRNAs) are small non-coding RNAs that have well-characterized roles in cytoplasmic gene regulation, where they act by binding to mRNA transcripts and inhibiting their translation (i.e. post-transcriptional gene silencing, PTGS). However, miRNAs have also been implicated in transcriptional gene regulation and alternative splicing, events that are restricted to the cell nucleus. Here we performed nuclear-cytoplasmic fractionation in a mouse endothelial cell line and characterized the localization of miRNAs in response to hypoxia using small RNA sequencing. A highly diverse population of abundant miRNA species was detected in the nucleus, of which the majority (56%) was found to be preferentially localized in one compartment or the other. Induction of hypoxia resulted in changes in miRNA levels in both nuclear and cytoplasmic compartments, with the majority of changes being restricted to one location and not the other. Notably, the classical hypoxamiR (miR-210-3p) was highly up-regulated in the nuclear compartment after hypoxic stimulus. These findings reveal a previously unappreciated level of molecular complexity in the physiological response occurring in ischemic tissue. Furthermore, widespread differential miRNA expression in the nucleus strongly suggests that these small RNAs are likely to perform extensive nuclear regulatory functions in the general case.

Developing new therapies for stroke is urgently needed, as this disease is the leading cause of death and disability worldwide, and the existing treatment is only available for a small subset of patients. The interruption of blood flow to the brain during ischemic stroke launches multiple immune responses, characterized by infiltration of peripheral immune cells, the activation of brain microglial cells, and the accumulation of immune mediators. Copper is an essential trace element that is required for many critical processes in the brain. Copper homeostasis is disturbed in chronic neurodegenerative diseases and altered in stroke patients, and targeted copper delivery has been shown to be protective against chronic neurodegeneration. This study was undertaken to assess whether the copper bis(thiosemicarbazone) complex, Cu(atsm), is beneficial in acute brain injury, in preclinical mouse models of ischemic stroke. We demonstrate that the copper complex Cu(atsm) protects neurons from excitotoxicity and N2a cells from OGD in vitro, and is protective in permanent and transient ischemia models in mice as measured by functional outcome and lesion size. Copper delivery in the ischemic brains modulates the inflammatory response, specifically affecting the myeloid cells. It reduces CD45 and Iba1 immunoreactivity, and alters the morphology of Iba1 positive cells in the ischemic brain. Cu(atsm) also protects endogenous microglia against ischemic insult and reduces the proportion of invading monocytes. These results demonstrate that the copper complex Cu(atsm) is an inflammation-modulating compound with high therapeutic potential in stroke and is a strong candidate for the development of therapies for acute brain injury.

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.