Gene expression and functional deficits underlie TREM2-knockout microglia responses in human models of Alzheimer's disease.

The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.

Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo.

iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Aß-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Aß-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in vivo function of patient-derived and genetically modified microglia.

Development and validation of a simplified method to generate human microglia from pluripotent stem cells.

BACKGROUND: Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS: iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFß-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFß signaling (IDE1) can be used to replace recombinant TGFß1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered. CONCLUSION: We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.

4-Hydroxytamoxifen probes for light-dependent spatiotemporal control of Cre-ER mediated reporter gene expression.

The tamoxifen inducible Cre-ER/loxP system provides tissue specific temporal control of gene recombination events, and can be used to induce expression of reporter genes (e.g. GFP, LacZ) for lineage tracing studies. Cre enzyme fused with estrogen receptor (Cre-ER) is released upon tamoxifen binding, resulting in permanent activation of reporter genes within cells and their progeny. Tamoxifen and its active metabolite, hydroxytamoxifen (4OHT) diffuses rapidly in vivo, making it difficult to restrict labeling to specific locations. In this study, we developed a photocaged 4OHT molecule by covalently attaching 4OHT to an ortho-nitrobenzyl (ONB1) group, rendering 4OHT inactive. Exposure to UV radiation cleaves the bond between ONB1 and 4OHT, freeing the 4OHT to bind Cre-ER to result in downstream genetic recombination and reporter activation. We show that caged ONB1-4OHT crosses the cell membrane and uncages after short UV exposure, resulting in Cre-driven genetic recombination that can be localized to specific regions or tissues. ONB1-4OHT can provide spatial control of reporter activation and be adapted with any existing Cre-ER/loxP based system.

Expression of Oct4 in human embryonic stem cells is dependent on nanotopographical configuration.

The fate of adult stem cells can be influenced by physical cues, including nanotopography. However, the response of human embryonic stem cells (hESCs) to dimensionally well-defined nanotopography is unknown. Using imprint lithography, we prepared well-defined nanotopography of hexagonal (HEX) and honeycomb (HNY) configurations with various spacings between the nanostructures. In serum-free hESC culture medium, basic fibroblast growth factor (bFGF) is required to maintain expression of Oct4, a pluripotent gene. Unexpectedly, hESCs cultured on nanotopography could maintain Oct4 expression without bFGF supplementation. With bFGF supplementation, the HEX nanotopography maintained Oct4 expression whereas the HNY configuration caused down-regulation of Oct4 expression. Thus, we observed that the lattice configurations of the nanotopography cause hESCs to respond to bFGF in different ways. This differential response to a biochemical cue by nanotopography was unforeseen, but its discovery could lead to novel differentiation pathways. Consistent with studies of other cells, we observed that nanotopography affects focal adhesion formation in hESCs. We posit that this can in turn affect cell-matrix tension, focal adhesion kinase signaling and integrin-growth factor receptor crosstalk, which eventually modulates Oct4 expression in hESCs.

ß-Amyloid impairs axonal BDNF retrograde trafficking.

The neurotrophin, brain-derived neurotrophic factor (BDNF), is essential for synaptic function, plasticity and neuronal survival. At the axon terminal, when BDNF binds to its receptor, tropomyosin-related kinase B (TrkB), the signal is propagated along the axon to the cell body, via retrograde transport, regulating gene expression and neuronal function. Alzheimer disease (AD) is characterized by early impairments in synaptic function that may result in part from neurotrophin signaling deficits. Growing evidence suggests that soluble ß-amyloid (Aß) assemblies cause synaptic dysfunction by disrupting both neurotransmitter and neurotrophin signaling. Utilizing a novel microfluidic culture chamber, we demonstrate a BDNF retrograde signaling deficit in AD transgenic mouse neurons (Tg2576) that can be reversed by γ-secretase inhibitors. Using BDNF-GFP, we show that BDNF-mediated TrkB retrograde trafficking is impaired in Tg2576 axons. Furthermore, Aß oligomers alone impair BDNF retrograde transport. Thus, Aß reduces BDNF signaling by impairing axonal transport and this may underlie the synaptic dysfunction observed in AD.

Axonal mRNA in uninjured and regenerating cortical mammalian axons.

Using a novel microfluidic chamber that allows the isolation of axons without contamination by nonaxonal material, we have for the first time purified mRNA from naive, matured CNS axons, and identified the presence of >300 mRNA transcripts. We demonstrate that the transcripts are axonal in nature, and that many of the transcripts present in uninjured CNS axons overlap with those previously identified in PNS injury-conditioned DRG axons. The axonal transcripts detected in matured cortical axons are enriched for protein translational machinery, transport, cytoskeletal components, and mitochondrial maintenance. We next investigated how the axonal mRNA pool changes after axotomy, revealing that numerous gene transcripts related to intracellular transport, mitochondria and the cytoskeleton show decreased localization 2 d after injury. In contrast, gene transcripts related to axonal targeting and synaptic function show increased localization in regenerating cortical axons, suggesting that there is an increased capacity for axonal outgrowth and targeting, and increased support for synapse formation and presynaptic function in regenerating CNS axons after injury. Our data demonstrate that CNS axons contain many mRNA species of diverse functions, and suggest that, like invertebrate and PNS axons, CNS axons synthesize proteins locally, maintaining a degree of autonomy from the cell body.

Differential regulation of inhibitors of apoptosis proteins in Alzheimer's disease brains.

Neuronal degeneration linked to apoptosis can be inhibited by a family of proteins known as inhibitors of apoptosis proteins (IAPs). We examined three members of the IAP family that are implicated in the regulation of neuronal death. We assessed NAIP, XIAP, and cIAP-2 protein levels in the entorhinal cortex of non-demented, cognitively impaired and Alzheimer's disease cases. Levels of paired helical filament-1 (PHF-1), a marker of neurofibrillary tangles, were assessed to determine their relationship to IAP levels. NAIP was decreased in AD cases compared to mildly impaired and unimpaired cases, and this decrease was associated with increased PHF-1 levels. Low NAIP levels were associated with higher Braak and Braak tangle stage and cognitive dysfunction. XIAP levels were higher in AD cases and cIAP-2 levels did not vary with clinical status. Our data suggest that decreased NAIP may place neurons at risk for the development of tangles and apoptosis.

Patterned cell culture inside microfluidic devices.

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.

Prevention of apoptotic but not necrotic cell death following neuronal injury by neurotrophins signaling through the tyrosine kinase receptor.

OBJECT: Neurotrophins prevent the death of neurons during embryonal development and have potential as therapeutic agents. During development, neuronal death occurs only by apoptosis and not by necrosis. Following injury, however, neurons can die by both processes. Data from prior studies have not clearly indicated whether neurotrophins can decrease apoptosis compared with necrosis. The goal of this study was to determine the effect of neurotrophin treatment on each of these processes following injury and to characterize the receptor(s) required. METHODS: The authors used an in vitro model of injury with the aid of primary cortical neurons obtained from rat embryos. After 9 days in culture and the elimination of glia, homogeneous and mature neurons were available for experimentation. Noxious stimuli were applied, including radiation, hypoxia, and ischemia. Subsequent cell death by apoptosis or necrosis was noted based on morphological and enzymatic assessments (such as lactate dehydrogenase [LDH] release) and assays for DNA fragmentation. The effect of treatment with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 was determined. Finally, Western blot analyses were performed to note the neurotrophin receptor status in the neurons (tyrosine kinase receptors [Trks] and p75). The authors studied different stimuli-induced cell death by using different processes. With the application of radiation, cells died primarily by apoptosis, as evidenced by cell shrinkage, the presence of apoptotic bodies, and specific DNA fragmentation. This was a delayed process (> 6 hours) that could be reduced by gene transcription or protein synthesis inhibitors. With ischemia, cells died immediately by necrosis, showing cell enlargement and rupture. Ischemic cell death was not affected by the inhibition of macromolecular synthesis. Hypoxia produced a mixture of the two cell death processes. Both BDNF and neurotrophin-3 demonstrated protection against apoptotic cell death only. Statistically significant decreases of both LDH release and apoptosis-specific DNA fragmentation were noted following radiation and hypoxia, but not for ischemia. Nerve growth factor, unlike the other neurotrophins, did not affect apoptosis because a functional receptor, Trk A, was not expressed by the cortical neurons. There was expression of both Trk B and Trk C, which bind BDNF and neurotrophin-3. CONCLUSIONS: These findings have significant clinical implications. Neurotrophins may only be effective in disorders in which apoptosis, and not necrosis, is the major process. Furthermore, the Trk signaling cascade must be activated for this response to occur. Because the expression of these receptors diminishes in adulthood, neurotrophin application may be most appropriate in the pediatric population.

Microfluidic Multicompartment Device for Neuroscience Research.

This paper describes and characterizes a novel microfabricated neuronal culture device. This device combines microfabrication, microfluidic, and surface micropatterning techniques to create a multicompartment neuronal culturing device that can be used in a number of neuroscience research applications. The device is fabricated in poly(dimethylsiloxane), PDMS, using soft lithography techniques. The PDMS device is placed on a tissue culture dish (polystyrene) or glass substrate, forming two compartments with volumes of less than 2 µL each. These two compartments are separated by a physical barrier in which a number of micron-size grooves are embedded to allow growth of neurites across the compartments while maintaining fluidic isolation. Cells are plated into the somal (cell body) compartment, and after 3-4 days, neurites extend into the neuritic compartment via the grooves. Viability of the neurons in the devices is between 50 and 70% after 7 days in culture; this is slightly lower than but comparable to values for a control grown on tissue culture dishes. Healthy neuron morphology is evident in both the devices and controls. We demonstrate the ability to use hydrostatic pressure to isolate insults to one compartment and, thus, expose localized areas of neurons to insults applied in soluble form. Due to the high resistance of the microgrooves for fluid transport, insults are contained in the neuritic compartment without appreciable leakage into the somal compartment for over 15 h. Finally, we demonstrate the use of polylysine patterning in combination with the microfabricated device to facilitate identification and visualization of neurons. The ability to direct sites of neuronal attachment and orientation of neurite outgrowth by micropatterning techniques, combined with fluidically isolated compartments within the culture area, offers significant advantages over standard open culture methods and other conventional methods for manipulating distinct neuronal microenvironments.