Monte Carlo modeling of the formation of branched tube structures from cellular aggregates [abstract]

Abstract only availableFaculty Mentor: Gabor Forgacs, Physics and AstronomyMonte Carlo method has been used previously for modeling the fusion of cellular aggregates to produce capillary-like structures and hollow tubes from cell aggregates consisting of smooth muscle cells and endothelial cells.  Here we have extended these modeling efforts to branched tubular structures built of cellular aggregates in the presence of two types of biocompatible gels.  A MATLAB script was created to generate a construct of simulated cell aggregates resembling a branched tube.  All of the simulated cells and gel particles were placed on a uniform 3D lattice.  The main tube consisted of layers of spherical cell aggregates arranged in circles and stacked on top of each other in a close-packed arrangement.  All the aggregates were composed of 30% endothelial cells and 70% smooth muscle cells, randomly distributed, with the exception of a set of three cells adjacent to any branch.  These aggregates were filled to 83% of their radius with gel.  Branches were attached at a site of three gel-filled aggregates, and the aggregates comprising the branch had a radius between 165% and 185% of that of the aggregates in the main tube; these aggregates were gel-filled to approximately 80% of their radius, with the remaining cells distributed as described above.  The lumen of the main tube was filled with the same type of gel as in the centers of the aggregates, and the remainder of the environment was filled with a second type of gel.  The structure was then subjected to Monte Carlo simulation for fifty-thousand steps.  The final equilibrium structure (when simulated with the proper parameters) strongly resembles a branched blood vessel: the cell aggregates fused, and the smooth muscle cells migrated to the outside of the main tube and branch, while the endothelial cells migrated to the interior of the tube and branch.  In addition, the lumen of the branch and main tube were contiguous.  The results of these simulations are currently being used to guide the construction of such structures through in vitro experiments

TDP-43 pathology in Drosophila induces glial-cell type specific toxicity that can be ameliorated by knock-down of SF2/SRSF1.

Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism

TDP-43 overexpression leads to age-dependent loss of each glial cell type.

Representative confocal images (Scale bar = 10μm.) of equivalent anterior section of the Drosophila brain on days 2,5, and 10 post eclosion for flies control (top) vs flies that express TDP-43 in PNGts (A), SPGts (C), CG (E) ALG (G), EG (I). Quantification of the number of each glial cell type in an equivalent anterior section was determined by counting nuclei labeled by Unc84-2X-GFP, which was co-expressed (B,D,F,H,J). Mean and SD are shown. Students T-test used to compare genotypes within each timepoint. * p<0.05, ***p<0.001. Gal4 lines and full genotypes used for each cell type are listed in methods.</p

Genotypes used in each figure.

Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.</div

<i>Drosophila</i> Strains <i>used</i>.

Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.</div

Differentially expressed genes and TEs in response to TDP-43 overexpression in each glial cell type.

Significantly (p methods) upregulated (Red) and downregulated (Blue) genes (A,C,E,G,I) and TEs (B,D,F,H,J) are shown for TDP43 expressing (Green bar, left) vs control (Black bar, right) for each cell type. Genes or TEs that are significantly (p < .05) upregulated and downregulated are shown for PNG (A, B), SPG (C,D), CG (E,F), ALG (G,H) or and EG (I,J). Full genotypes and Gal4 lines are listed in methods.</p

Nucleocytoplasmic transport pathway.

Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.</div

Numbers of fly heads used for TAPIN purification and sequencing based on number of glial cells of each type per brain.

Numbers of fly heads used for TAPIN purification and sequencing based on number of glial cells of each type per brain.</p

Lifespan deficit observed with TDP-43 expression in PNG is not recapitulated by PNG ablation.

(A) confocal images (Scale bar = 10μm) of an anterior section of the Drosophila brain on days 2,5, and 10 for flies expressing of PNGts + TDP-43 (top) or PNGts + rpr (bottom). (B) quantification of PNG cells in equivalent anterior sections was performed on days 2,5, and 10 post induction by counting the number of nuclei labeled with GFP, which was co-expressed using the UAS-WM reporter (Methods). Mean and SD shown for each. * pts + rpr) or TDP-43 (PNGts + TDP-43). TDP-43 expression in PNG yields a significantly shorter lifespan than does rpr expression (Log rank, P<1.085e-20). Gal4 lines and full genotypes are listed in methods.</p

Chromatin organization pathway.

Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.</div