Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly

Primary microcephaly and megalencephaly are severe brain malformations defined by reduced and increased brain size, respectively. Whether these two pathologies arise from related alterations at the molecular level is unclear. Microcephaly has been largely associated with centrosomal defects, leading to cell death. Here, we investigate the consequences of WDR81 loss of function, which causes severe microcephaly in patients. We show that WDR81 regulates endosomal trafficking of EGFR and that loss of function leads to reduced MAP kinase pathway activation. Mouse radial glial progenitor cells knocked-out for WDR81 exhibit reduced proliferation rate, subsequently leading to reduced brain size. These proliferation defects are rescued in vivo by expressing a megalencephaly-causing mutant form of Cyclin D2. Our results identify the endosomal machinery as an important regulator of proliferation rates and brain growth, demonstrating that microcephaly and megalencephaly can be caused by opposite effects on the proliferation rate of radial glial progenitors.

Hexosamine pathway activation improves memory but does not extend lifespan in mice.

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension in various species. Thus, there is considerable interest in the potential health benefits of dietary supplementation with glucosamine or other HBP metabolites in people. The HBP is a sensor for energy availability and its activation has been implicated in tumor progression and diabetes in higher organisms. As the activation of the HBP has been linked to longevity in lower animals, it is imperative to explore the long-term effects of chronic HBP activation in mammals, which has not been examined so far. To address this issue, we activated the HBP in mice both genetically and through metabolite supplementation, and evaluated metabolism, memory, and survival. GlcNAc supplementation in the drinking water had no adverse effect on weight gain in males but increased weight in young female mice. Glucose or insulin tolerance were not affected up to 20 months of age. Of note, we observed improved memory in the Morris water maze in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc supplementation. To assess the effects of genetic HBP activation we overexpressed the key enzyme GFAT1 as well as a constitutively activated mutant form in all mouse tissues. We detected elevated UDP-GlcNAc levels in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.

Lymphocyte Cytosolic Protein 1 (L-plastin) I232F Mutation Impairs Granulocytic Proliferation and Causes Neutropenia

Neutrophils migrate into inflamed tissue, engage in phagocytosis, and clear pathogens or apoptotic cells. These processes require well-coordinated events involving the actin cytoskeleton. We describe a child with severe neutropenia and episodes of soft tissue infections and pneumonia. Bone marrow examination showed granulocytic hypoplasia with dysplasia. Whole exome sequencing revealed a de novo heterozygous missense mutation in LCP1, which encodes the F-actin binding protein Lymphocyte Cytosolic Protein 1. To determine its pathophysiologic significance, we stably transduced cells with a doxycycline-inducible wild type LCP1 and LCP1 I232F lentiviral constructs. We observed dysplastic granulocytic 32D cells expressing LCP1 I232F cells. These cells showed decreased proliferation without a block in differentiation. Additionally, expression of LCP1 I232F resulted in a cell cycle arrest at G2/M phase, but it did not lead to increased levels of genes involved in apoptosis or the unfolded protein response. Both 32D and HeLa cells expressing mutant LCP1 showed impaired cell motility and invasiveness. Flow cytometry showed increased F-actin. However, mutant LCP1-expressing 32D cells demonstrated normal oxidative burst upon stimulation. Confocal imaging and subcellular fractionation revealed diffuse intracellular localization of LCP1, but only the mutant form was found in the nucleus. We conclude that LCP1 is a new gene involved in granulopoiesis, and the missense variant LCP1 I232F leads to neutropenia and granulocytic dysplasia with aberrant actin dynamics. Our work supports a model of neutropenia due to aberrant actin regulation.

Frameshifting at collided ribosomes is modulated by elongation factor eEF3 and by Integrated Stress Response regulators Gcn1 and Gcn20

Ribosome stalls can result in ribosome collisions that elicit quality control responses, one function of which is to prevent ribosome frameshifting, an activity that entails interaction of the conserved yeast protein Mbf1 with uS3 on colliding ribosomes. However, the full spectrum of factors that mediate frameshifting during ribosome collisions is unknown. To delineate such factors in the yeast Saccharomyces cerevisiae, we used genetic selections for mutants that affect frameshifting from a known ribosome stall site, CGA codon repeats. We show that the general translation elongation factor eEF3 and the Integrated Stress Response (ISR) pathway components Gcn1 and Gcn20 modulate frameshifting in opposing manners. We found a mutant form of eEF3 that specifically suppressed frameshifting, but not translation inhibition by CGA codons. Thus, we infer that frameshifting at collided ribosomes requires eEF3, which facilitates tRNA-mRNA translocation and E-site tRNA release in yeast and other single cell organisms. By contrast, we found that removal of either Gcn1 or Gcn20, which bind collided ribosomes with Mbf1, increased frameshifting. Thus, we conclude that frameshifting is suppressed by Gcn1 and Gcn20, although these effects are not mediated primarily through activation of the ISR. Furthermore, we examined the relationship between eEF3-mediated frameshifting and other quality control mechanisms, finding that Mbf1 requires either Hel2 or Gcn1 to suppress frameshifting with wild type eEF3. Thus, these results provide evidence of a direct link between translation elongation and frameshifting at collided ribosomes, as well as evidence that frameshifting is constrained by quality control mechanisms that act on collided ribosomes.

PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells

Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-β-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-β increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-β. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-β induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-β treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.

Tau Modulates mRNA Transcription, Alternative Polyadenylation Profiles of hnRNPs, Chromatin Remodeling and Spliceosome Complexes

Tau protein is a known contributor in several neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). It is well-established that tau forms pathological aggregates and fibrils in these diseases. Tau has been observed within the nuclei of neurons, but there is a gap in understanding regarding the mechanism by which tau modulates transcription. We are interested in the P301L mutation of tau, which has been associated with FTD and increased tau aggregation. Our study utilized tau-inducible HEK (iHEK) cells to reveal that WT and P301L tau distinctively alter the transcription and alternative polyadenylation (APA) profiles of numerous nuclear precursors mRNAs, which then translate to form proteins involved in chromatin remodeling and splicing. We isolated total mRNA before and after over-expressing tau and then performed Poly(A)-ClickSeq (PAC-Seq) to characterize mRNA expression and APA profiles. We characterized changes in Gene Ontology (GO) pathways using EnrichR and Gene Set Enrichment Analysis (GSEA). We observed that P301L tau up-regulates genes associated with reactive oxygen species responsiveness as well as genes involved in dendrite, microtubule, and nuclear body/speckle formation. The number of genes regulated by WT tau is greater than the mutant form, which indicates that the P301L mutation causes loss-of-function at the transcriptional level. WT tau up-regulates genes contributing to cytoskeleton-dependent intracellular transport, microglial activation, microtubule and nuclear chromatin organization, formation of nuclear bodies and speckles. Interestingly, both WT and P301L tau commonly down-regulate genes responsible for ubiquitin-proteosome system. In addition, WT tau significantly down-regulates several genes implicated in chromatin remodeling and nucleosome organization. Although there are limitations inherent to the model systems used, this study will improve understanding regarding the nuclear impact of tau at the transcriptional and post-transcriptional level. This study also illustrates the potential impact of P301L tau on the human brain genome during early phases of pathogenesis.

Improved SARS-CoV-2 Spike Glycoproteins for Pseudotyping Lentiviral Vectors

The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the native CD4 tropic envelope protein of the virus and thereby either limit or expand the target cell population. We generated a modified S glycoprotein of SARS-Cov-2 to pseudotype lentiviral vectors which efficiently transduced ACE2-expressing cells with high specificity and contain minimal off-target transduction of ACE2 negative cells. By utilizing optimized codons, modifying the S cytoplasmic tail domain, and including a mutant form of the spike protein, we generated an expression plasmid encoding an optimized protein that produces S-pseudotyped lentiviral vectors at an infectious titer (TU/mL) 1000-fold higher than the unmodified S protein and 4 to 10-fold more specific than the widely used delta-19 S-pseudotyped lentiviral vectors. S-pseudotyped replication-defective lentiviral vectors eliminate the need for biosafety-level-3 laboratories required when developing therapeutics against SARS-CoV-2 with live infectious virus. Furthermore, S-pseudotyped vectors with high activity and specificity may be used as tools to understand the development of immunity against SARS-CoV-2, to develop assays of neutralizing antibodies and other agents that block viral binding, and to allow in vivo imaging studies of ACE2-expressing cells.

Impaired oligodendrocyte maturation is an early feature in SCA3 disease pathogenesis

Spinocerebellar ataxia type 3 (SCA3), the most common dominantly inherited ataxia, is a polyglutamine neurodegenerative disease for which there is no disease-modifying therapy. The polyglutamine-encoding CAG repeat expansion in the ATXN3 gene results in expression of a mutant form of the ATXN3 protein, a deubiquitinase that causes selective neurodegeneration despite being widely expressed. The mechanisms driving neurodegeneration in SCA3 are unclear. Research to date, however, has focused almost exclusively on neurons. Here, using equal male and female age-matched transgenic mice expressing full-length human mutant ATXN3, we identified early and robust transcriptional changes in selectively vulnerable brain regions that implicate oligodendrocytes in disease pathogenesis. We mapped transcriptional changes across early, mid, and late stages of disease in two selectively vulnerable brain regions, the cerebellum and brainstem. The most significant disease-associated module through weighted gene co-expression network analysis revealed dysfunction in SCA3 oligodendrocyte maturation. These results reflect a toxic gain of function mechanism, as ATXN3 knockout mice do not exhibit any impairments in oligodendrocyte maturation. Genetic crosses to reporter mice revealed a marked reduction in mature oligodendrocytes in SCA3-disease vulnerable brain regions and ultrastructural microscopy confirmed abnormalities in axonal myelination. Further study of isolated oligodendrocyte precursor cells from SCA3 mice established that this impairment in oligodendrocyte maturation is a cell autonomous process. We conclude that SCA3 is not simply a disease of neurons and the search for therapeutic strategies and disease biomarkers will need to account for non-neuronal involvement in SCA3 pathogenesis.

Prothrombotic Phenotype of a Protein S Mutant Lacking Anticoagulant Activity

Protein S is a vitamin K-dependent protein that plays an important role in balancing pro- and anti-thrombotic responses to vascular injury. On one hand, it circulates bound to activated protein C, functioning as an anticoagulant complex that downregulates the activities of coagulation factors V and VIII. On the other hand, protein S is a ligand for platelet TYRO3 and MERTK, which, along with the third paralog AXL, constitute the TAM family of receptor tyrosine kinases that functions to potentiate the action of platelet-activating agonists, ultimately resulting in activation of the α IIbβ3 integrin. The relative importance of these two activities in vivo, however, is not known. To better understand the importance of the TYRO3/MERTK-stimulating ability of protein S and thus gain additional insight into its role in platelet activation, we used CRISPR/Cas9 technology to generate mice with a D136A mutant form of protein S that lacks the ability to bind protein C and function as an anticoagulant, while retaining its ability to bind platelet-activating TAM receptors - hereafter termed Pros1 D136A mice. Since homozygosity for this variant was embryonically lethal, all assays were carried out in wild-type animals and their heterozygous littermates comprising a wild-type Pros1 allele and an allele encoding Pros1 D136A. Though there was no significant difference between Pros1 D136A mice and their wild-type littermates in either a collagen/epinephrine-induced pulmonary embolism model or in tail vein bleeding times, platelets from Pros1 D136A mice accumulated at the injury site to a significantly greater degree following in vivo laser injury to the cremaster muscle microvasculature. Taken together with the embryonic phenotype of Pros1 D136A homozygous mice, these data support the notion that protein S can function to augment platelet responsiveness. Further studies on this and other ligands for the TAM family of receptor tyrosine kinases should provide additional insights into their roles in physiological platelet activation. Disclosures Branchford: Bio Products Laboratory: Honoraria; Novo Nordisk: Honoraria.