Identification of Fluorescent Small Molecule Compounds for Synaptic Labeling by Image-Based, High-Content Screening.

Few tools are available for noninvasive imaging of synapses in the living mammalian brain. Current paradigms require the use of genetically modified mice or viral delivery of genetic material to the brain. To develop an alternative chemical approach, utilizing the recognition of synaptic components by organic small molecules, we designed an imaging-based, high-content screen in cultured cortical neurons to identify molecules based on their colocalization with fluorescently tagged synaptic proteins. We used this approach to screen a library of ∼7000 novel fluorescent dyes, and identified a series of compounds in the xanthone family that exhibited consistent synaptic labeling. Follow-up studies with one of these compounds, CX-G3, demonstrated its ability to label acidic organelles and in particular synaptic vesicles at glutamatergic synapses in cultured neurons and murine brain tissue, indicating the potential of this screening approach to identify promising lead compounds for use as synaptic markers, sensors, and targeting devices.

Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway.

UNLABELLED: Synaptic vesicle (SV) pools must maintain a functional repertoire of proteins to efficiently release neurotransmitter. The accumulation of old or damaged proteins on SV membranes is linked to synaptic dysfunction and neurodegeneration. However, despite the importance of SV protein turnover for neuronal health, the molecular mechanisms underlying this process are largely unknown. Here, we have used dissociated rat hippocampal neurons to investigate the pathway for SV protein degradation. We find that neuronal activity drives the degradation of a subset of SV proteins and that the endosomal sorting complex required for transport (ESCRT) machinery and SV-associated GTPase Rab35 are key elements of this use-dependent degradative pathway. Specifically, neuronal activity induces Rab35 activation and binding to the ESCRT-0 protein Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to SV pools, thereby initiating SV protein degradation via the ESCRT pathway. Our findings show that the Rab35/ESCRT pathway facilitates the activity-dependent removal of specific proteins from SV pools, thereby maintaining presynaptic protein homeostasis. SIGNIFICANCE STATEMENT: Synaptic transmission is mediated by the release of chemical neurotransmitters from synaptic vesicles (SVs). This tightly regulated process requires a functional pool of SVs, necessitating cellular mechanisms for removing old or damaged proteins that could impair SV cycling. Here, we show that a subset of SV proteins is degraded in an activity-dependent manner and that key steps in this degradative pathway are the activation of the small GTPase Rab35 and the subsequent recruitment of the endosomal sorting complex required for transport (ESCRT) machinery to SV pools. Further, we demonstrate that ESCRT-0 component Hrs is an effector of Rab35, thus providing novel mechanistic insight into the coupling of neuronal activity with SV protein degradation and the maintenance of functional SV pools.

The Oct1 homolog Nubbin is a repressor of NF-κB-dependent immune gene expression that increases the tolerance to gut microbiota.

BACKGROUND: Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood. RESULTS: We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism. CONCLUSIONS: This study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.

Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells.

In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here, we report that nuclear factor erythroid-derived 2-related factor 1 (Nrf1), a transcription factor of the cap "n" collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1(-/-) MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101, and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.

A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation.

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97(Ufd1/Npl4) complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.

Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts.

Regulatory transcription factors (rTFs), which bind specific DNA sequences in the regulatory regions of genes and subsequently activate or repress transcription, play a central role in programming genomic expression. The number of rTFs in a species might therefore reflect its functional complexity. For simple organisms like yeast, a relatively small number of rTFs might be expected that is fairly constant between yeast species. We show that the budding yeast, Saccharomyces cerevisiae, contains 201 rTfs, which is one of the largest rTF numbers found in yeast species for which genome sequences are available. This is a much higher number than the 129 rTFs found in the fission yeast, Schizosaccharomyces pombe, which is currently the yeast with the lowest number of rTFs. Comparative analysis of several different budding yeast species shows that most of the 'extra' rTFs found in S. cerevisiae were probably acquired as a result of a whole genome duplication (WGD) event that occurred in an ancestor of a subset of budding yeast species. However, we also show that budding yeast species that have not been affected by the WGD contain a greater number of rTFs than S. pombe (mean = 145). Thus, two or more mechanisms have led to the 60% increase in rTFs in S. cerevisiae compared to S. pombe. This difference may correlate with a more extensive functional divergence in budding yeasts compared to fission yeasts. The relatively small number of rTFs in S. pombe make this organism an attractive model for global studies of mechanisms that programme gene expression.