Mapping the peripheral nervous system in the whole mouse via compressed sensing tractography.

Objective.The peripheral nervous system (PNS) connects the central nervous system with the rest of the body to regulate many physiological functions and is therapeutically targeted to treat diseases such as epilepsy, depression, intestinal dysmotility, chronic pain, and more. However, we still lack understanding of PNS innervation in most organs because the large span, diffuse nature, and small terminal nerve bundle fibers have precluded whole-organism, high resolution mapping of the PNS. We sought to produce a comprehensive peripheral nerve atlas for use in future interrogation of neural circuitry and selection of targets for neuromodulation.Approach.We used diffusion tensor magnetic resonance imaging (DT-MRI) with high-speed compressed sensing to generate a tractogram of the whole mouse PNS. The tractography generated from the DT-MRI data is validated using lightsheet microscopy on optically cleared, antibody stained tissue.Main results.Herein we demonstrate the first comprehensive PNS tractography in a whole mouse. Using this technique, we scanned the whole mouse in 28 h and mapped PNS innervation and fiber network in multiple organs including heart, lung, liver, kidneys, stomach, intestines, and bladder at 70µm resolution. This whole-body PNS tractography map has provided unparalleled information; for example, it delineates the innervation along the gastrointestinal tract by multiple sacral levels and by the vagal nerves. The map enabled a quantitative tractogram that revealed relative innervation of the major organs by each vertebral foramen as well as the vagus nerve.Significance.This novel high-resolution nerve atlas provides a potential roadmap for future neuromodulation therapies and other investigations into the neural circuits which drive homeostasis and disease throughout the body.

The frontier of live tissue imaging across space and time.

What you see is what you get-imaging techniques have long been essential for visualization and understanding of tissue development, homeostasis, and regeneration, which are driven by stem cell self-renewal and differentiation. Advances in molecular and tissue modeling techniques in the last decade are providing new imaging modalities to explore tissue heterogeneity and plasticity. Here we describe current state-of-the-art imaging modalities for tissue research at multiple scales, with a focus on explaining key tradeoffs such as spatial resolution, penetration depth, capture time/frequency, and moieties. We explore emerging tissue modeling and molecular tools that improve resolution, specificity, and throughput.

Intravital imaging of mouse embryos.

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.

An intravital window to image the colon in real time.

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.

Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately.

The extreme N-terminus of TDP-43 mediates the cytoplasmic aggregation of TDP-43 and associated toxicity in vivo.

Inclusions of Tar DNA- binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Pathological TDP-43 exhibits the disease-specific biochemical signatures, which include its ubiquitination, phosphorylation and truncation. Recently, we demonstrated that the extreme N-terminus of TDP-43 regulates formation of abnormal cytoplasmic TDP-43 aggregation in cultured cells and primary neurons. However, it remained unclear whether this N-terminal domain mediates TDP-43 aggregation and the associated toxicity in vivo. To investigate this, we expressed a GFP-tagged TDP-43 with a nuclear localization signal mutation (GFP-TDP-43NLSm) and a truncated form without the extreme N-terminus (GFP-TDP-4310-414-NLSm) by adeno-associated viral (AAV) vectors in mouse primary cortical neurons and murine central nervous system. Compared to neurons containing GFP alone, expression of GFP-TDP-43NLSm resulted in the formation of ubiquitin-positive cytoplasmic inclusions and activation of caspase-3, an indicator of cell death. Moreover, mice expressing GFP-TDP-43NLSm proteins show reactive gliosis and develop neurological abnormalities. However, by deletion of TDP-43's extreme N-terminus, these pathological alterations can be abrogated. Together, our study provides further evidence confirming the critical role of the extreme N-terminus of TDP-43 in regulating protein structure as well as mediating toxicity associated with its aggregation. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.

C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins.

Neuronal inclusions of poly(GA), a protein unconventionally translated from G4C2 repeat expansions in C9ORF72, are abundant in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) caused by this mutation. To investigate poly(GA) toxicity, we generated mice that exhibit poly(GA) pathology, neurodegeneration and behavioral abnormalities reminiscent of FTD and ALS. These phenotypes occurred in the absence of TDP-43 pathology and required poly(GA) aggregation. HR23 proteins involved in proteasomal degradation and proteins involved in nucleocytoplasmic transport were sequestered by poly(GA) in these mice. HR23A and HR23B similarly colocalized to poly(GA) inclusions in C9ORF72 expansion carriers. Sequestration was accompanied by an accumulation of ubiquitinated proteins and decreased xeroderma pigmentosum C (XPC) levels in mice, indicative of HR23A and HR23B dysfunction. Restoring HR23B levels attenuated poly(GA) aggregation and rescued poly(GA)-induced toxicity in neuronal cultures. These data demonstrate that sequestration and impairment of nuclear HR23 and nucleocytoplasmic transport proteins is an outcome of, and a contributor to, poly(GA) pathology.

Microscopic matrix remodeling precedes endothelial morphological changes during capillary morphogenesis.

The formation of microvascular networks (MVNs) is influenced by many aspects of the microenvironment, including soluble and insoluble biochemical factors and the biophysical properties of the surrounding matrix. It has also become clear that a dynamic and reciprocal interaction between the matrix and cells influences cell behavior. In particular, local matrix remodeling may play a role in driving cellular behaviors, such as MVN formation. In order to explore the role of matrix remodeling, an in vitro model of MVN formation involving suspending human umbilical vein endothelial cells within collagen hydrogels was used. The resulting cell and matrix morphology were microscopically observed and quantitative metrics of MVN formation and collagen gathering were applied to the resulting images. The macroscopic compaction of collagen gels correlates with the extent of MVN formation in gels of different stiffness values, with compaction preceding elongation leading to MVN formation. Furthermore, the microscopic analysis of collagen between cells at early timepoints demonstrates the alignment and gathering of collagen between individual adjacent cells. The results presented are consistent with the hypothesis that endothelial cells need to gather and align collagen between them as an early step in MVN formation.