Melanie Hayden Gephart

New tools for studying microglia in the mouse and human CNS

Significance Microglia are the tissue resident macrophages of the brain and spinal cord, implicated in important developmental, homeostatic, and disease processes, although our understanding of their roles is complicated by an inability to distinguish microglia from related cell types. Although they share many features with other macrophages, microglia have distinct developmental origins and functions. Here we validate a stable and robustly expressed microglial marker for both mouse and human, transmembrane protein 119 (Tmem119). We use custom-made antibodies against Tmem119 to perform deep RNA sequencing of developing microglia, and demonstrate that microglia mature by the second postnatal week in mice. The antibodies, cell isolation methods, and RNAseq profiles presented here will greatly facilitate our understanding of microglial function in health and disease. The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

A survey of human brain transcriptome diversity at the single cell level

Significance The brain comprises an immense number of cells and cellular connections. We describe the first, to our knowledge, single cell whole transcriptome analysis of human adult cortical samples. We have established an experimental and analytical framework with which the complexity of the human brain can be dissected on the single cell level. Using this approach, we were able to identify all major cell types of the brain and characterize subtypes of neuronal cells. We observed changes in neurons from early developmental to late differentiated stages in the adult. We found a subset of adult neurons which express major histocompatibility complex class I genes and thus are not immune privileged. The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma.

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvβ3, αvβ5, and α5β1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5β1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F–Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F–Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5–Fc as targeted molecular probes for brain tumor imaging.

Neuropilins are positive regulators of Hedgehog signal transduction

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.

Venous thromboembolism after thoracic/thoracolumbar spinal fusion.

OBJECTIVE Venous thromboembolism (VTE), which includes deep venous thrombosis and pulmonary embolism, is a serious and potentially fatal surgical complication. The goal of our study was to examine preoperative characteristics, incidence, and outcomes of patients with VTE after elective thoracic/thoracolumbar level spine fusion. METHODS We identified 430,081 patients from the Nationwide Inpatient Sample database who underwent spinal fusion between 2002 and 2008. Patients undergoing thoracic/thoracolumbar level fusion (n = 8617) were found to have the greatest concurrent rate of VTE. We then performed multivariate analyses on this cohort to identify predictors of and outcomes after VTE in patients undergoing thoracic/thoracolumbar level fusion. RESULTS The overall VTE rate in spinal fusion surgery was 0.40% (cervical = 0.22%, thoracic/thoracolumbar = 1.90%, lumbar/lumbosacral = 0.49%, re-fusions = 0.64%, and fusions not otherwise specified = 0.84%). On multivariate logistic regression analysis of patients undergoing spinal fusion at the thoracic/thoracolumbar level, increasing age, Medicare insurance coverage (vs. private insurance), urban teaching hospital (vs. urban nonteaching hospital), combined anterior/posterior surgical approach (vs. posterior-only approach), and the presence of congestive heart failure or weight loss (Elixhauser comorbidity groups) were each independently associated with an increased odds ratio of VTE complication. VTE after thoracic/thoracolumbar surgery was significantly associated with longer hospital stays (16.6 vs. 6.74 days), increased total hospital costs (

Cochlea Radiation Dose Correlates with Hearing Loss After Stereotactic Radiosurgery of Vestibular Schwannoma

260,208 vs.

Retrospective, Propensity Score-Matched Cohort Study Examining Timing of Fracture Fixation for Traumatic Thoracolumbar Fractures

115,474), and increased mortality (4.33% vs. 0.33%). CONCLUSIONS Multivariate logistic regression analysis reveals age, insurance status, hospital type, combined anterior/posterior surgical approach, and the presence of congestive heart failure or weight loss to be independently associated with an increased odds ratio of VTE complication. This complication is associated with increased hospital costs, length of stay, and overall mortality.

Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma

OBJECTIVE For multisession radiosurgery, no published data relate the volume and dose of cochlear irradiation to quantified risk of hearing loss. We conducted a retrospective, dosimetric study to evaluate the relationship between hearing loss after stereotactic radiosurgery (SRS) and the dose-volume of irradiated cochlea. METHODS Cochlear dose data were retrospectively collected on consecutive patients who underwent SRS (18 Gy in 3 sessions) for vestibular schwannoma between 1999 and 2005 at Stanford University Hospital. Inclusion criteria included Gardner-Robertson (GR) grade I or II hearing prior to radiosurgical treatment, complete audiograms, and magnetic resonance imaging (MRI) follow-up. A cochlea dose-volume histogram was generated for each of the 94 patients who qualified for this study. RESULTS GR grade I-II hearing posttreatment was maintained in 74% of patients (70/94). Median time to last follow-up audiogram was 2.4 years (range 0.4-8.9) and to last MRI was 3.6 years (range 0.5-9.4). Each higher level of cochlear irradiation was associated with increased risk of hearing loss. Larger cochlear volume was associated with lower risk of hearing loss. Controlling for differences in cochlear volume among subjects, each additional mm(3) of cochlea receiving 10 to 16 Gy (single session equivalent doses of 6.6-10.1 Gy3) significantly increased the odds of hearing loss by approximately 5%. CONCLUSIONS Larger cochlear volume is associated with lower risk of hearing loss following trisession SRS for vestibular schwannoma. Controlling for this phenomenon, higher radiation dose and larger irradiated cochlear volume are significantly associated with higher risk of hearing loss. This study confirms and quantifies the risk of hearing loss following trisession SRS for vestibular schwannoma.

A Population-Based Study of Inpatient Outcomes After Operative Management of Nontraumatic Intracerebral Hemorrhage in the United States

The timing of surgery in patients with traumatic thoracic/thoracolumbar fractures, with or without spinal cord injury, remains controversial. The objective of this study was to determine the importance of the timing of surgery for complications and resource utilization following fixation of traumatic thoracic/thoracolumbar fractures. In this retrospective cohort study, the 2003-2008 California Inpatient Databases were searched for patients receiving traumatic thoracic/thoracolumbar fracture fixation. Patients were classified as having early (<72 h) or late (>72 h) surgery. Propensity score modeling produced a matched cohort balanced on age, comorbidity, trauma severity, and other factors. Complications, mortality, length of stay, and hospital charges were assessed. Multivariate logistic regression was used to determine the impact of delayed surgery on in-hospital complications after balancing and controlling for other important factors. Early surgery (<72 h) for traumatic thoracic/thoracolumbar fractures was associated with a significantly lower overall complication rate (including cardiac, thromboembolic, and respiratory complications), and decreased hospital stay. In-hospital charges were significantly lower (

Cushing's disease: predicting long-term remission after surgical treatment

38,120 difference) in the early surgery group. Multivariate analysis identified time to surgery as the strongest predictor of in-hospital complications, although age, medical comorbidities, and injury severity score were also independently associated with increased complications. We reinforce the beneficial impact of early spinal surgery (prior to 72 h) in traumatic thoracic/thoracolumbar fractures to reduce in-hospital complications, hospital stay, and resource utilization. These results provide further support to the emerging literature and professional consensus regarding the importance of early thoracic/thoracolumbar spine stabilization of traumatic fractures to improve patient outcomes and limit hospitalization costs.