Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.

Missense mutations in the gene encoding the microtubule-associated protein TAU (current and approved symbol is MAPT) cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human TAU in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas-mediated gene editing to model frontotemporal dementia caused by the TAU P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for Tau P251L display age-dependent neurodegeneration, display metabolic defects, and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies, we performed single-cell RNA sequencing on approximately 130,000 cells from brains of Tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified. Gene expression was also altered in glial cells, suggestive of non-cell-autonomous regulation. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell type-specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy that faithfully recapitulates the genetic context and phenotypic features of the human disease, and use the results of comprehensive single-cell sequencing analysis to outline pathways of neurotoxicity and highlight the potential role of non-cell-autonomous changes in glia.

Cell type- and tissue-specific functions of ecto-5'-nucleotidase (CD73).

Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene NT5E cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.

An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.

Vimentin is a cytoskeletal intermediate filament protein that is expressed in mesenchymal cells and cancer cells during the epithelial-mesenchymal transition. The goal of this study was to identify vimentin-targeting small molecules by using the Tocriscreen library of 1120 biochemically active compounds. We monitored vimentin filament reorganization and bundling in adrenal carcinoma SW13 vimentin-positive (SW13-vim+) cells via indirect immunofluorescence. The screen identified 18 pharmacologically diverse hits that included 2 statins-simvastatin and mevastatin. Simvastatin induced vimentin reorganization within 15-30 min and significant perinuclear bundling within 60 min (IC50 = 6.7 nM). Early filament reorganization coincided with increased vimentin solubility. Mevastatin produced similar effects at >1 µM, whereas the structurally related pravastatin and lovastatin did not affect vimentin. In vitro vimentin filament assembly assays revealed a direct targeting mechanism, as determined biochemically and by electron microscopy. In SW13-vim+ cells, simvastatin, but not pravastatin, reduced total cell numbers (IC50 = 48.1 nM) and promoted apoptosis after 24 h. In contrast, SW13-vim- cell viability was unaffected by simvastatin, unless vimentin was ectopically expressed. Simvastatin similarly targeted vimentin filaments and induced cell death in MDA-MB-231 (vim+), but lacked effect in MCF7 (vim-) breast cancer cells. In conclusion, this study identified vimentin as a direct molecular target that mediates simvastatin-induced cell death in 2 different cancer cell lines.-Trogden, K. P., Battaglia, R. A., Kabiraj, P., Madden, V. J., Herrmann, H., Snider, N. T. An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.

Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.

Missense mutations in the gene encoding the microtubule-associated protein tau cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human tau in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease, but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas mediated gene editing to model frontotemporal dementia caused by the tau P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for tau P251L display age-dependent neurodegeneration, metabolic defects and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies we performed single-cell RNA sequencing on approximately 130,000 cells from brains of tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified and non-cell autonomously in glial cells. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell-type specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy, which faithfully recapitulates the genetic context and phenotypic features of the human disease and use the results of comprehensive single cell sequencing analysis to outline pathways of neurotoxicity and highlight the role of non-cell autonomous changes in glia.

Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging.

Aß peptides derived from the amyloid precursor protein (APP) have been strongly implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP and the importance of that role in neurodegenerative disease is less clear. We recover the Drosophila ortholog of APP, Appl, in an unbiased forward genetic screen for neurodegeneration mutants. We perform comprehensive single cell transcriptional and proteomic studies of Appl mutant flies to investigate Appl function in the aging brain. We find an unexpected role for Appl in control of multiple cellular pathways, including translation, mitochondrial function, nucleic acid and lipid metabolism, cellular signaling and proteostasis. We mechanistically define a role for Appl in regulating autophagy through TGFß signaling and document the broader relevance of our findings using mouse genetic, human iPSC and in vivo tauopathy models. Our results demonstrate a conserved role for APP in controlling age-dependent proteostasis with plausible relevance to Alzheimer's disease.

Intermediate filament dysregulation in astrocytes in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN).

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, which regulates intermediate filament (IF) turnover. Previous neuropathological studies and examination of postmortem brain tissue in the current study revealed involvement of astrocytes in GAN. To develop a clinically-relevant model, we reprogrammed skin fibroblasts from seven GAN patients to pluripotent stem cells (iPSCs), which were used to generate neural progenitor cells (NPCs), astrocytes, and brain organoids. Multiple isogenic control clones were derived via CRISPR/Cas9 gene editing of one patient line carrying the G332R gigaxonin mutation. All GAN iPSCs were deficient for gigaxonin and displayed patient-specific increased vimentin expression. GAN NPCs had lower nestin expression and fewer nestin-positive cells compared to isogenic controls, but nestin morphology was unaffected. GAN brain organoids were marked by the presence of neurofilament and GFAP aggregates. GAN iPSC-astrocytes displayed striking dense perinuclear vimentin and GFAP accumulations and abnormal nuclear morphology. In over-expression systems, GFAP oligomerization and perinuclear aggregation were augmented in the presence of vimentin. GAN patient cells with large perinuclear vimentin aggregates accumulated significantly more nuclear KLHL16 mRNA compared to cells without vimentin aggregates. As an early effector of KLHL16 mutations, vimentin may be a potential target in GAN.

Intermediate filament dysregulation and astrocytopathy in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN).

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, a regulator of intermediate filament (IF) protein turnover. Previous neuropathological studies and our own examination of postmortem GAN brain tissue in the current study revealed astrocyte involvement in GAN. To study the underlying mechanisms, we reprogrammed skin fibroblasts from seven GAN patients carrying different KLHL16 mutations to iPSCs. Isogenic controls with restored IF phenotypes were derived via CRISPR/Cas9 editing of one patient carrying a homozygous missense mutation (G332R). Neural progenitor cells (NPCs), astrocytes, and brain organoids were generated through directed differentiation. All GAN iPSC lines were deficient for gigaxonin, which was restored in the isogenic control. GAN iPSCs displayed patient-specific increased vimentin expression, while GAN NPCs had decreased nestin expression compared to isogenic control. The most striking phenotypes were observed in GAN iPSC-astrocytes and brain organoids, which exhibited dense perinuclear IF accumulations and abnormal nuclear morphology. GAN patient cells with large perinuclear vimentin aggregates accumulated nuclear KLHL16 mRNA. In over-expression studies, GFAP oligomerization and perinuclear aggregation were potentiated in the presence of vimentin. As an early effector of KLHL16 mutations, vimentin may serve as a potential therapeutic target in GAN.

Borealin directs recruitment of the CPC to oocyte chromosomes and movement to the microtubules.

The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, we developed a strategy to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. We found that an interaction between Borealin and the chromatin is crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. We propose that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation.

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.

Vimentin on the move: new developments in cell migration.

The vimentin gene ( VIM) encodes one of the 71 human intermediate filament (IF) proteins, which are the building blocks of highly ordered, dynamic, and cell type-specific fiber networks. Vimentin is a multi-functional 466 amino acid protein with a high degree of evolutionary conservation among vertebrates. Vim -/- mice, though viable, exhibit systemic defects related to development and wound repair, which may have implications for understanding human disease pathogenesis. Vimentin IFs are required for the plasticity of mesenchymal cells under normal physiological conditions and for the migration of cancer cells that have undergone epithelial-mesenchymal transition. Although it was observed years ago that vimentin promotes cell migration, the molecular mechanisms were not completely understood. Recent advances in microscopic techniques, combined with computational image analysis, have helped illuminate vimentin dynamics and function in migrating cells on a precise scale. This review includes a brief historical account of early studies that unveiled vimentin as a unique component of the cell cytoskeleton followed by an overview of the physiological vimentin functions documented in studies on Vim -/- mice. The primary focus of the discussion is on novel mechanisms related to how vimentin coordinates cell migration. The current hypothesis is that vimentin promotes cell migration by integrating mechanical input from the environment and modulating the dynamics of microtubules and the actomyosin network. These new findings undoubtedly will open up multiple avenues to study the broader function of vimentin and other IF proteins in cell biology and will lead to critical insights into the relevance of different vimentin levels for the invasive behaviors of metastatic cancer cells.

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton - a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes.

Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule-chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation.

The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes.

The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore-microtubule attachment in oocytes.