Loss of Inpp5d has disease-relevant and sex-specific effects on glial transcriptomes.

INTRODUCTION: Inpp5d is genetically associated with Alzheimer's disease risk. Loss of Inpp5d alters amyloid pathology in models of amyloidosis. Inpp5d is expressed predominantly in microglia but its function in brain is poorly understood. METHODS: We performed single-cell RNA sequencing to study the effect of Inpp5d loss on wild-type mouse brain transcriptomes. RESULTS: Loss of Inpp5d has sex-specific effects on the brain transcriptome. Affected genes are enriched for multiple neurodegeneration terms. Network analyses reveal a gene co-expression module centered around Inpp5d in female mice. Inpp5d loss alters Pleotrophin (PTN), Prosaposin (PSAP), and Vascular Endothelial Growth Factor A (VEGFA) signaling probability between cell types. DISCUSSION: Our data suggest that the normal function of Inpp5d is entangled with mechanisms involved in neurodegeneration. We report the effect of Inpp5d loss without pathology and show that this has dramatic effects on gene expression. Our study provides a critical reference for researchers of neurodegeneration, allowing separation of disease-specific changes mediated by Inpp5d in disease from baseline effects of Inpp5d loss. HIGHLIGHTS: Loss of Inpp5d has different effects in male and female mice. Genes dysregulated by Inpp5d loss relate to neurodegeneration. Total loss of Inpp5d in female mice collapses a conserved gene co-expression module. Loss of microglial Inpp5d affects the transcriptome of other cell types.

TONSL Is an Immortalizing Oncogene and a Therapeutic Target in Breast Cancer.

Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we used healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku-like, DNA repair protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in approximately 20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor-positive adenocarcinomas in mice. Analysis of a breast tumor microarray with approximately 600 tumors revealed poor overall and progression-free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors, including NF-κB and limited access to the tumor-suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi anemia pathways. Consistent with these results, TONSL-overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL-FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy. SIGNIFICANCE: The chr.8q24.3 amplicon-resident gene TONSL is upregulated during the initial steps of tumorigenesis to support neoplastic transformation by increasing DNA repair and represents a potential therapeutic target for treating breast cancer.

INPP5D deficiency attenuates amyloid pathology in a mouse model of Alzheimer's disease.

INTRODUCTION: Inositol polyphosphate-5-phosphatase (INPP5D) is a microglia-enriched lipid phosphatase in the central nervous system. A non-coding variant (rs35349669) in INPP5D increases the risk for Alzheimer's disease (AD), and elevated INPP5D expression is associated with increased plaque deposition. INPP5D negatively regulates signaling via several microglial cell surface receptors, including triggering receptor expressed on myeloid cells 2 (TREM2); however, the impact of INPP5D inhibition on AD pathology remains unclear. METHODS: We used the 5xFAD mouse model of amyloidosis to assess how Inpp5d haplodeficiency regulates amyloid pathogenesis. RESULTS: Inpp5d haplodeficiency perturbs the microglial intracellular signaling pathways regulating the immune response, including phagocytosis and clearing of amyloid beta (Aß). It is important to note that Inpp5d haploinsufficiency leads to the preservation of cognitive function. Spatial transcriptomic analysis revealed that pathways altered by Inpp5d haploinsufficiency are related to synaptic regulation and immune cell activation. CONCLUSION: These data demonstrate that Inpp5d haplodeficiency enhances microglial functions by increasing plaque clearance and preserves cognitive abilities in 5xFAD mice. Inhibition of INPP5D is a potential therapeutic strategy for AD.

Ultrasound-controllable engineered bacteria for cancer immunotherapy.

Rapid advances in synthetic biology are driving the development of genetically engineered microbes as therapeutic agents for a multitude of human diseases, including cancer. The immunosuppressive microenvironment of solid tumors, in particular, creates a favorable niche for systemically administered bacteria to engraft and release therapeutic payloads. However, such payloads can be harmful if released outside the tumor in healthy tissues where the bacteria also engraft in smaller numbers. To address this limitation, we engineer therapeutic bacteria to be controlled by focused ultrasound, a form of energy that can be applied noninvasively to specific anatomical sites such as solid tumors. This control is provided by a temperature-actuated genetic state switch that produces lasting therapeutic output in response to briefly applied focused ultrasound hyperthermia. Using a combination of rational design and high-throughput screening we optimize the switching circuits of engineered cells and connect their activity to the release of immune checkpoint inhibitors. In a clinically relevant cancer model, ultrasound-activated therapeutic microbes successfully turn on in situ and induce a marked suppression of tumor growth. This technology provides a critical tool for the spatiotemporal targeting of potent bacterial therapeutics in a variety of biological and clinical scenarios.

Acoustically triggered mechanotherapy using genetically encoded gas vesicles.

Recent advances in molecular engineering and synthetic biology provide biomolecular and cell-based therapies with a high degree of molecular specificity, but limited spatiotemporal control. Here we show that biomolecules and cells can be engineered to deliver potent mechanical effects at specific locations inside the body through ultrasound-induced inertial cavitation. This capability is enabled by gas vesicles, a unique class of genetically encodable air-filled protein nanostructures. We show that low-frequency ultrasound can convert these biomolecules into micrometre-scale cavitating bubbles, unleashing strong local mechanical effects. This enables engineered gas vesicles to serve as remotely actuated cell-killing and tissue-disrupting agents, and allows genetically engineered cells to lyse, release molecular payloads and produce local mechanical damage on command. We demonstrate the capabilities of biomolecular inertial cavitation in vitro, in cellulo and in vivo, including in a mouse model of tumour-homing probiotic therapy.

Ultraparamagnetic Cells Formed through Intracellular Oxidation and Chelation of Paramagnetic Iron.

Making cells magnetic is a long-standing goal of chemical biology, aiming to enable the separation of cells from complex biological samples and their visualization in vivo using magnetic resonance imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have been largely unsuccessful due to the stringent required chemical conditions. Here, we introduce an alternative approach to making cells magnetic, focused on biochemically maximizing cellular paramagnetism. We show that a novel genetic construct combining the functions of ferroxidation and iron chelation enables engineered bacterial cells to accumulate iron in "ultraparamagnetic" macromolecular complexes, allowing these cells to be trapped with magnetic fields and imaged with MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, nuclear magnetic resonance, biochemical assays, and computational modeling to elucidate the unique mechanisms and capabilities of this paramagnetic concept.

Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures.

Non-invasive biological imaging requires materials capable of interacting with deeply penetrant forms of energy such as magnetic fields and sound waves. Here, we show that gas vesicles (GVs), a unique class of gas-filled protein nanostructures with differential magnetic susceptibility relative to water, can produce robust contrast in magnetic resonance imaging (MRI) at sub-nanomolar concentrations, and that this contrast can be inactivated with ultrasound in situ to enable background-free imaging. We demonstrate this capability in vitro, in cells expressing these nanostructures as genetically encoded reporters, and in three model in vivo scenarios. Genetic variants of GVs, differing in their magnetic or mechanical phenotypes, allow multiplexed imaging using parametric MRI and differential acoustic sensitivity. Additionally, clustering-induced changes in MRI contrast enable the design of dynamic molecular sensors. By coupling the complementary physics of MRI and ultrasound, this nanomaterial gives rise to a distinct modality for molecular imaging with unique advantages and capabilities.

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts.

The mammalian microbiome has many important roles in health and disease, and genetic engineering is enabling the development of microbial therapeutics and diagnostics. A key determinant of the activity of both natural and engineered microorganisms in vivo is their location within the host organism. However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance owing to light scattering or require radioactive tracers. Here we introduce acoustic reporter genes, which are genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available inexpensive technique with deep tissue penetration and high spatial resolution. These constructs are based on gas vesicles, a unique class of gas-filled protein nanostructures that are expressed primarily in water-dwelling photosynthetic organisms as a means to regulate buoyancy. Heterologous expression of engineered gene clusters encoding gas vesicles allows Escherichia coli and Salmonella typhimurium to be imaged noninvasively at volumetric densities below 0.01% with a resolution of less than 100 µm. We demonstrate the imaging of engineered cells in vivo in proof-of-concept models of gastrointestinal and tumour localization, and develop acoustically distinct reporters that enable multiplexed imaging of cellular populations. This technology equips microbial cells with a means to be visualized deep inside mammalian hosts, facilitating the study of the mammalian microbiome and the development of diagnostic and therapeutic cellular agents.

Acoustically targeted chemogenetics for the non-invasive control of neural circuits.

Neurological and psychiatric disorders are often characterized by dysfunctional neural circuits in specific regions of the brain. Existing treatment strategies, including the use of drugs and implantable brain stimulators, aim to modulate the activity of these circuits. However, they are not cell-type-specific, lack spatial targeting or require invasive procedures. Here, we report a cell-type-specific and non-invasive approach based on acoustically targeted chemogenetics that enables the modulation of neural circuits with spatiotemporal specificity. The approach uses ultrasound waves to transiently open the blood-brain barrier and transduce neurons at specific locations in the brain with virally encoded engineered G-protein-coupled receptors. The engineered neurons subsequently respond to systemically administered designer compounds to activate or inhibit their activity. In a mouse model of memory formation, the approach can modify and subsequently activate or inhibit excitatory neurons within the hippocampus, with selective control over individual brain regions. This technology overcomes some of the key limitations associated with conventional brain therapies.

Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI.

Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that are detectable at subnanomolar concentrations and whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and MRI. Here we provide a protocol for isolating GVs from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and MRI. GVs can be isolated from natural cyanobacterial and haloarchaeal host organisms or from Escherichia coli expressing a heterologous GV gene cluster and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, whereas dynamic light scattering (DLS)and transmission electron microscopy (TEM) are used to determine nanoparticle size and morphology, respectively. GVs can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between GV-bound and dissolved xenon-a technique currently implemented in vitro. Taking 3-8 d to prepare, these genetically encodable nanostructures enable multimodal, noninvasive biological imaging with high sensitivity and potential for molecular targeting.

Molecular Engineering of Acoustic Protein Nanostructures.

Ultrasound is among the most widely used biomedical imaging modalities, but has limited ability to image specific molecular targets due to the lack of suitable nanoscale contrast agents. Gas vesicles-genetically encoded protein nanostructures isolated from buoyant photosynthetic microbes-have recently been identified as nanoscale reporters for ultrasound. Their unique physical properties give gas vesicles significant advantages over conventional microbubble contrast agents, including nanoscale dimensions and inherent physical stability. Furthermore, as a genetically encoded material, gas vesicles present the possibility that the nanoscale mechanical, acoustic, and targeting properties of an imaging agent can be engineered at the level of its constituent proteins. Here, we demonstrate that genetic engineering of gas vesicles results in nanostructures with new mechanical, acoustic, surface, and functional properties to enable harmonic, multiplexed, and multimodal ultrasound imaging as well as cell-specific molecular targeting. These results establish a biomolecular platform for the engineering of acoustic nanomaterials.