Faculty Bibliography

To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the blood-CSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse blood-CSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.

The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.

This study describes a combined transcriptome and proteome analysis of Monodelphis domestica response to spinal cord injury at two different postnatal ages. Previously we showed that complete transection at postnatal day 7 (P7) is followed by profuse axon growth across the lesion with near-normal locomotion and swimming when adult. In contrast, at P28 there is no axon growth across the lesion, the animals exhibit weight-bearing locomotion, but cannot use hind limbs when swimming. Here we examined changes in gene and protein expression in the segment of spinal cord rostral to the lesion at 24 h after transection at P7 and at P28. Following injury at P7 only forty genes changed (all increased expression); most were immune/inflammatory genes. Following injury at P28 many more genes changed their expression and the magnitude of change for some genes was strikingly greater. Again many were associated with the immune/inflammation response. In functional groups known to be inhibitory to regeneration in adult cords the expression changes were generally muted, in some cases opposite to that required to account for neurite inhibition. For example myelin basic protein expression was reduced following injury at P28 both at the gene and protein levels. Only four genes from families with extracellular matrix functions thought to influence neurite outgrowth in adult injured cords showed substantial changes in expression following injury at P28: Olfactomedin 4 (Olfm4, 480 fold compared to controls), matrix metallopeptidase (Mmp1, 104 fold), papilin (Papln, 152 fold) and integrin alpha4 (Itga4, 57 fold). These data provide a resource for investigation of a priori hypotheses in future studies of mechanisms of spinal cord regeneration in immature animals compared to lack of regeneration at more mature stages.

Effective visualization is central to the exploration and comprehension of brain imaging data. While MRI data are acquired in three-dimensional space, the methods for visualizing such data have rarely taken advantage of three-dimensional stereoscopic technologies. We present here results of stereoscopic visualization of clinical data, as well as an atlas of whole-brain functional connectivity. In comparison with traditional 3D rendering techniques, we demonstrate the utility of stereoscopic visualizations to provide an intuitive description of the exact location and the relative sizes of various brain landmarks, structures and lesions. In the case of resting state fMRI, stereoscopic 3D visualization facilitated comprehension of the anatomical position of complex large-scale functional connectivity patterns. Overall, stereoscopic visualization improves the intuitive visual comprehension of image contents, and brings increased dimensionality to visualization of traditional MRI data, as well as patterns of functional connectivity.

The identification of phenotypic associations in high-dimensional brain connectivity data represents the next frontier in the neuroimaging connectomics era. Exploration of brain-phenotype relationships remains limited by statistical approaches that are computationally intensive, depend on a priori hypotheses, or require stringent correction for multiple comparisons. Here, we propose a computationally efficient, data-driven technique for connectome-wide association studies (CWAS) that provides a comprehensive voxel-wise survey of brain-behavior relationships across the connectome; the approach identifies voxels whose whole-brain connectivity patterns vary significantly with a phenotypic variable. Using resting state fMRI data, we demonstrate the utility of our analytic framework by identifying significant connectivity-phenotype relationships for full-scale IQ and assessing their overlap with existent neuroimaging findings, as synthesized by openly available automated meta-analysis (www.neurosynth.org). The results appeared to be robust to the removal of nuisance covariates (i.e., mean connectivity, global signal, and motion) and varying brain resolution (i.e., voxelwise results are highly similar to results using 800 parcellations). We show that CWAS findings can be used to guide subsequent seed-based correlation analyses. Finally, we demonstrate the applicability of the approach by examining CWAS for three additional datasets, each encompassing a distinct phenotypic variable: neurotypical development, Attention-Deficit/Hyperactivity Disorder diagnostic status, and L-DOPA pharmacological manipulation. For each phenotype, our approach to CWAS identified distinct connectome-wide association profiles, not previously attainable in a single study utilizing traditional univariate approaches. As a computationally efficient, extensible, and scalable method, our CWAS framework can accelerate the discovery of brain-behavior relationships in the connectome.

Purpose: Secondary cell death via gap junctions plays a critical role in the cell loss associated with neurodegenerative diseases (Belousov and Fontes 2013). In the retina we reported that secondary cell death accounts for most cell loss that occurs under excitotoxic and ischemic conditions (ARVO, 2011). Secondary cell death in CNS may be connexin specific and connexins may be up- or down-regulated under different pathological conditions (Rouach et al., 2002). Here we examined whether secondary cell death of retinal ganglion cells (RGCs) under excitotoxic and ischemic conditions is connexin-specific and whether the expression of Cx36 and Cx45 in inner retina is differentially effected. Methods: Excitotoxicity was induced in vitro by incubation of mouse retinas in NMDA. Transient retinal ischemia was induced in vivo by elevation of IOP. Levels of cell death were assayed histologically and antibodies against Cx36 and Cx45 were used to assess their levels in the IPL. Results: Consistent with our earlier work, we found that excitotoxic and ischemic conditions produced a significant loss of RGCs. Ablation of Cx36 in the Cx36 knockout (KO) mouse retina resulted in an ~70% decrease in RGC loss under excitotoxic conditions, whereas RGC loss in the Cx45 KO retina was not statistically different than that seen in WT mice. In contrast, RGC loss with ischemia was significantly reduced in Cx45 KO retinas, whereas the loss in Cx36 KO retinas was similar to that in the WT. In WT retinas the expression of Cx36 and Cx45 in the IPL followed a punctuate pattern typical for gap junctions. Under excitotoxic conditions the expression of Cx45 was down-regulated, whereas there were no detectable changes in Cx36 expression. In contrast, induction of ischemic conditions produced a dramatic change in Cx36 expression, which appeared as dense clusters around nuclei rather than as puncta. We found no change in the control punctate labeling pattern of Cx45 expression in ischemic retinas. Conclusions: Secondary cell death of RGCs is connexin specific where Cx36 gap junctions play a role under excitotoxic conditions and Cx45 gap junctions play a role during ischemia. These results are consistent with changes in connexin expression seen under these two conditions. These results suggest that targeting of specific connexins can be a novel therapeutic strategy for reducing RGC loss under different pathological conditions

Purpose: In retinal pathologies, microglia have been implicated in regulating trophic influences to retinal neurons and gliosis in macroglia. However, whether microglia exert such regulatory functions in a constitutive manner in the healthy adult retina is unclear. We explore this question by examining the effects of sustained microglial depletion in the adult mouse retina. Methods: Transgenic mice on a C57Bl6 background in which the Cx3cr1 gene was replaced by a sequence encoding a mutant Cre protein with a tamoxifen (TAM)- dependent estrogen ligand-binding domain (Cx3cr1^CreER ) were crossed with mice carrying lox-P-flanked ('floxed') diphtheria toxin (DTA) to create Cx3cr1^CreER/+ DTA/+ (TG) mice. Depletion of retinal microglia in TG mice was induced by TAM gavage and was sustained over 30 days by repeated administration. Histological changes in the retina were monitored in retinal sections and fiat-mounted preparations. Results: Following 30 days of sustained microglial depletion (<1% of microglia remaining), histological analyses revealed that overall retinal organization relative to untreated controls was maintained without detectable changes in retinal lamination or thicknesses in retinal cell layers. Apoptosis, as detected by TUNEL staining, was not induced in any retinal layer. The structure and laminar organization of retinal vasculature, which are normally in close proximity to microglia, remained unaffected. The morphologies of retinal astrocytes and Muller cells, as revealed by immunohistochemical labeling for GFAP and glutamine synthetase respectively, were similar to controls; signs of Muller cell gliosis, as evidenced by process hypertrophy or GFAP upregulation, were also absent. Conclusions: Microglial absence in the mouse retina for intervals up to 1 month did not induce detectable neuronal cell death, vascular change, or alterations in macroglial morphology and activation. Retinal microglia do not appear necessary for retinal cell survival or for the maintenance of astrocyte and Muller cell morphology or physiology. Studies to discover any constitutive microglial contribution to retinal cell function and retinal synaptic structure are currently ongoing