Discovery of Small-Molecule CD33 Pre-mRNA Splicing Modulators.

CD33/Siglec 3 is a myeloid lineage cell surface receptor that is known to regulate microglia activity. Multiple genome-wide association studies (GWAS) have identified genetic variants in the CD33 gene that convey protection from late-onset Alzheimer's disease. Furthermore, mechanistic studies into GWAS-linked variants suggest that disease protection is attributed to the alternative splicing of exon 2 of the CD33 pre-mRNA. Using a phenomimetic screen, a series of compounds were found to enhance the exclusion of CD33 exon 2, acting as a chemomimetic of the GWAS-linked gene variants. Additional studies confirmed that meyloid lineage cells treated with several of these compounds have a reduced full-length V-domain containing CD33 protein, while targeted RNA-seq concordantly demonstrated that compound 1 increases exon 2 skipping in cellular mRNA pools. These studies demonstrate how pharmacological interventions can be used to manipulate disease-relevant pre-mRNA splicing and provide a starting point for future efforts to identify small molecules that alter neuroimmune function that is rooted in the human biology of neurodegenerative disease.

Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer's disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid ß (Aß). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aß pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aß and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aß load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.

A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system.

Microglia are resident immune cells of the central nervous system (CNS). The exact role of microglia in CNS disorders is not clear due to lack of tools to discriminate between microglia and infiltrating myeloid cells. Here, we present a novel reporter mouse model targeting a microglia-specific marker, TMEM119, for studying microglia in health and disease. By placing a reporter cassette (GSG-3xFlag-P2A-tdTomato) between the coding sequence of exon 2 and 3'UTR of the Tmem119 gene using CRISPR/Cas9 technology, we generated a Tmem119-tdTomato knock-in mouse strain. Gene expression assay showed no difference of endogenous Tmem119 in the CNS of Tmem119tdTomato/+ relative to wild-type mice. The cells expressing tdTomato were recognized by immunofluorescence staining using commercially available anti-TMEM119 antibodies. Additionally, immunofluorescence and flow cytometry techniques revealed that tdTomato+ cells are detected throughout the CNS, but not in peripheral tissues of Tmem119tdTomato/+ mice. Aging does not influence TMEM119 expression as tdTomato+ cells were detectable in the CNS of older mice (300 and 540 days old). Further immunofluorescence characterization shows that tdTomato+ cells colocalize with Iba1+ cells in the brain, but not with neurons, astrocytes or oligodendrocytes. Moreover, flow cytometry analysis of brain tissues of adult mice demonstrates that the majority of microglia CD45loCD11b+ cells (96.3%) are tdTomato-positive; and a minority of infiltrating CD45hiCD11b+ myeloid cells (5.3%) are also tdTomato-positive, which we further characterized and found that tdTomato expression is in part of choroid plexus macrophages but not in meningeal and perivascular macrophages. Functionally, using an acute injury model, we measured time-lapse activation of tdTomato-labeled microglia by transcranial two-photon microscopy in live Tmem119tdTomato/+ mice. Taken together, the Tmem119-tdTomato reporter mouse model is a valuable tool to specifically study the role of microglia in health and disease.

SRSF1 and PTBP1 Are trans-Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer's Disease Risk.

A single nucleotide polymorphism (SNP) in exon 2 of the CD33 gene is associated with reduced susceptibility to late-onset Alzheimer's disease (AD) and causal for elevated mRNA lacking exon 2. In contrast to full-length CD33, transcripts lacking exon 2 result in CD33 protein unable to suppress activation responses in myeloid cells, including microglia. Currently, little is known about the regulation of CD33 exon 2 splicing. Using functional genomics and proteomic approaches, we found that SRSF1 and PTBP1 act as splicing enhancers to increase CD33 exon 2 inclusion in mRNA. Binding of PTBP1 to RNA sequences proximal to the intron 1-exon 2 splice junction is altered by the SNP and represents a potential mechanism behind the SNP-genotype dependent alternative splicing. Our studies also reveal that binding of SRSF1 to the CD33 RNA is not altered by the SNP genotype. Instead, a putative SRSF1 binding sequence at the 3' end of exon 2 directs CD33 exon 2 inclusion into the mRNA, indicating that PTBP1 and SRSF1 promote full-length isoform expression through different mechanisms. Our findings shed light on molecular interactions that regulate CD33 exon 2 splicing, ultimately impacting receptor expression on the cell surface. These data aid in the understanding of CD33's regulation of microglial signaling underpinning the AD genetic associations.

Adaptation of a Genetic Screen Reveals an Inhibitor for Mitochondrial Protein Import Component Tim44.

Diverse protein import pathways into mitochondria use translocons on the outer membrane (TOM) and inner membrane (TIM). We adapted a genetic screen, based on Ura3 mistargeting from mitochondria to the cytosol, to identify small molecules that attenuated protein import. Small molecule mitochondrial import blockers of the Carla Koehler laboratory (MB)-10 inhibited import of substrates that require the TIM23 translocon. Mutational analysis coupled with molecular docking and molecular dynamics modeling revealed that MB-10 binds to a specific pocket in the C-terminal domain of Tim44 of the protein-associated motor (PAM) complex. This region was proposed to anchor Tim44 to the membrane, but biochemical studies with MB-10 show that this region is required for binding to the translocating precursor and binding to mtHsp70 in low ATP conditions. This study also supports a direct role for the PAM complex in the import of substrates that are laterally sorted to the inner membrane, as well as the mitochondrial matrix. Thus, MB-10 is the first small molecule modulator to attenuate PAM complex activity, likely through binding to the C-terminal region of Tim44.

Application of Imaging-Based Assays in Microplate Formats for High-Content Screening.

The use of multiparametric microscopy-based screens with automated analysis has enabled the large-scale study of biological phenomena that are currently not measurable by any other method. Collectively referred to as high-content screening (HCS), or high-content analysis (HCA), these methods rely on an expanding array of imaging hardware and software automation. Coupled with an ever-growing amount of diverse chemical matter and functional genomic tools, HCS has helped open the door to a new frontier of understanding cell biology through phenotype-driven screening. With the ability to interrogate biology on a cell-by-cell basis in highly parallel microplate-based platforms, the utility of HCS continues to grow as advancements are made in acquisition speed, model system complexity, data management, and analysis systems. This chapter uses an example of screening for genetic factors regulating mitochondrial quality control to exemplify the practical considerations in developing and executing high-content campaigns.

Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter.

Parkin, an E3 ubiquitin ligase, is a central mediator of mitochondrial quality control and is linked to familial forms of Parkinson's disease (PD). Removal of dysfunctional mitochondria from the cell by Parkin is thought to be neuroprotective, and pharmacologically increasing Parkin levels may be a novel therapeutic approach. We used genome-editing to integrate a coincidence reporter into the PARK2 gene locus of a neuroblastoma-derived cell line and developed a quantitative high-throughput screening (qHTS) assay capable of accurately detecting subtle compound-mediated increases in endogenous PARK2 expression. Interrogation of a chemogenomic library revealed diverse chemical classes that up-regulate the PARK2 transcript, including epigenetic agents, drugs controlling cholesterol biosynthesis, and JNK inhibitors. Use of the coincidence reporter eliminated wasted time pursuing reporter-biased false positives accounting for ∼2/3 of the actives and, coupled with titration-based screening, greatly improves the efficiency of compound selection. This approach represents a strategy to revitalize reporter-gene assays for drug discovery.

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy.

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson's disease. Recent studies of the Parkinson's disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.

Role of membrane association and Atg14-dependent phosphorylation in beclin-1-mediated autophagy.

During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.

Innovation in academic chemical screening: filling the gaps in chemical biology.

Academic screening centers across the world have endeavored to discover small molecules that can modulate biological systems. To increase the reach of functional-genomic and chemical screening programs, universities, research institutes, and governments have followed their industrial counterparts in adopting high-throughput paradigms. As academic screening efforts have steadily grown in scope and complexity, so have the ideas of what is possible with the union of technology and biology. This review addresses the recent conceptual and technological innovation that has been propelling academic screening into its own unique niche. In particular, high-content and whole-organism screening are changing how academics search for novel bioactive compounds. Importantly, we recognize examples of successful chemical probe development that have punctuated the changing technology landscape.

A small molecule inhibitor of redox-regulated protein translocation into mitochondria.

The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.

Biology-driven library design for probe discovery.

Libraries of diverse small molecules are important to probe and drug discovery. The current trend toward building massive screening collections to support drug development, a special application of chemical biology, can limit their broader potential. Biology-driven construction methods (Wallace et al., 2011) are rapidly emerging to bring chemical libraries back on a viable path.

Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants.

We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNA(Ser)UCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNA(Ser)UCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNA(Ser)UCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.

Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation.

The TIM22 protein import pathway mediates the import of membrane proteins into the mitochondrial inner membrane and consists of two intermembrane space chaperone complexes, the Tim9-Tim10 and Tim8-Tim13 complexes. To facilitate mechanistic studies, we developed a chemical-genetic approach to identify small molecule agonists that caused lethality to a tim10-1 yeast mutant at the permissive temperature. One molecule, MitoBloCK-1, attenuated the import of the carrier proteins including the ADP/ATP and phosphate carriers, but not proteins that used the TIM23 or the Mia40/Erv1 translocation pathways. MitoBloCK-1 impeded binding of the Tim9-Tim10 complex to the substrate during an early stage of translocation, when the substrate was crossing the outer membrane. As a probe to determine the substrate specificity of the small Tim proteins, MitoBloCK-1 impaired the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex mediates the import of a subset of inner membrane proteins. MitoBloCK-1 also inhibited growth of mammalian cells and import of the ADP/ATP carrier, but not TIM23 substrates, confirming that MitoBloCK-1 can be used to understand mammalian mitochondrial import and dysfunction linked to inherited human disease. Our approach of screening chemical libraries for compounds causing synthetic genetic lethality to identify inhibitors of mitochondrial protein translocation in yeast validates the generation of new probes to facilitate mechanistic studies in yeast and mammalian mitochondria.

Reconstitution of the mia40-erv1 oxidative folding pathway for the small tim proteins.

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of -310 mV via Mia40 with an intermediate midpoint potential of -290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of -150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX(9)C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.

Mutations in the RNA polymerase III subunit Rpc11p that decrease RNA 3' cleavage activity increase 3'-terminal oligo(U) length and La-dependent tRNA processing.

Termination by RNA polymerase III (Pol III) produces RNAs whose 3' oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3' exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3' cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNASerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3' cleavage activity and produce pre-tRNASerUGAM transcripts with elongated 3'-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNASerUGAM contains too few 3' Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3'-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.