Mechanism of life-long maintenance of neuron identity despite molecular fluctuations.

Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. Our simulations identified a possible mechanism that explains life-long maintenance of ASE neuron fate in Caenorhabditis elegans by the terminal selector transcription factor CHE-1. Here, fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensures continued che-1 expression by preferentially binding the che-1 promoter. We provide experimental evidence for this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems

Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands

BACKGROUND: G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro systems. Caenorhabditis elegans is a soil-dwelling, bacteria-feeding nematode that uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds, making this an ideal system for studying in vivo GPCR-ligand interactions. We sought to test this by functionally expressing two medically important mammalian GPCRs, somatostatin receptor 2 (Sstr2) and chemokine receptor 5 (CCR5) in the gustatory neurons of C. elegans. RESULTS: Expression of Sstr2 and CCR5 in gustatory neurons allow C. elegans to specifically detect and respond to somatostatin and MIP-1α respectively in a robust avoidance assay. We demonstrate that mammalian heterologous GPCRs can signal via different endogenous G(α )subunits in C. elegans, depending on which cells it is expressed in. Furthermore, pre-exposure of GPCR transgenic animals to its ligand leads to receptor desensitisation and behavioural adaptation to subsequent ligand exposure, providing further evidence of integration of the mammalian GPCRs into the C. elegans sensory signalling machinery. In structure-function studies using a panel of somatostatin-14 analogues, we identified key residues involved in the interaction of somatostatin-14 with Sstr2. CONCLUSION: Our results illustrate a remarkable evolutionary plasticity in interactions between mammalian GPCRs and C. elegans signalling machinery, spanning 800 million years of evolution. This in vivo system, which imparts novel avoidance behaviour on C. elegans, thus provides a simple means of studying and screening interaction of GPCRs with extracellular agonists, antagonists and intracellular binding partners

DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans

Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT) plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL), resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis

Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein

Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions

Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions

Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair

The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency

EIF2AK3 variants in Dutch patients with Alzheimer's disease

Next-generation sequencing has contributed to our understanding of the genetics of Alzheimer's disease (AD) and has explained a substantial part of the missing heritability of familial AD. We sequenced 19 exomes from 8 Dutch families with a high AD burden and identified EIF2AK3, encoding for protein kinase RNA-like endoplasmic reticulum kinase (PERK), as a candidate gene. Gene-based burden analysis in a Dutch AD exome cohort containing 547 cases and 1070 controls showed a significant association of EIF2AK3 with AD (OR 1.84 [95% CI 1.07–3.17], p-value 0.03), mainly driven by the variant p.R240H. Genotyping of this variant in an additional cohort from the Rotterdam Study showed a trend toward association with AD (p-value 0.1). Immunohistochemical staining with pPERK and peIF2α of 3 EIF2AK3 AD carriers showed an increase in hippocampal neuronal cells expressing these proteins compared with nondemented controls, but no difference was observed in AD noncarriers. This study suggests that rare variants in EIF2AK3 may be associated with disease risk in AD

Poly(GP), neurofilament and grey matter deficits in C9orf72 expansion carriers

Objective: To evaluate poly(GP), a dipeptide repeat protein, and neurofilament light chain (NfL) as biomarkers in presymptomatic C9orf72 repeat expansion carriers and patients with C9orf72-associated frontotemporal dementia. Additionally, to investigate the relationship of poly(GP) with indicators of neurodegeneration as measured by NfL and grey matter volume. Methods: We measured poly(GP) and NfL levels in cerebrospinal fluid (CSF) from 25 presymptomatic C9orf72 expansion carriers, 64 symptomatic expansion carriers with dementia, and 12 noncarriers. We explored associations with grey matter volumes using region of interest and voxel-wise analyses. Results: Poly(GP) was present in C9orf72 expansion carriers and absent in noncarriers (specificity 100%, sensitivity 97%). Presymptomatic carriers had lower poly(GP) levels than symptomatic carriers. NfL levels were higher in symptomatic carriers than in presymptomatic carriers and healthy noncarriers. NfL was highest in patients with concomitant motor neuron disease, and correlated with disease severity and survival. Associations between poly(GP) levels and small grey matter regions emerged but did not survive multiple comparison correction, while higher NfL levels were associated with atrophy in frontotemporoparietal cortices and the thalamus. Interpretation: This study of C9orf72 expansion carriers reveals that: (1) poly(GP) levels discriminate presymptomatic and symptomatic expansion carriers from noncarriers, but are not associated with indicators of neurodegeneration; and (2) NfL levels are associated with grey matter atrophy, disease severity, and shorter survival. Together, poly(GP) and NfL show promise as complementary biomarkers for clinical trials for C9orf72-associated frontotemporal dementia, with poly(GP) as a potential marker for target engagement and NfL as a marker of disease activity and progression

Spatiotemporal analysis for detection of pre-symptomatic shape changes in neurodegenerative diseases: Initial application to the GENFI cohort

Brain atrophy as measured from structural MR images, is one of the primary imaging biomarkers used to track neurodegenerative disease progression. In diseases such as frontotemporal dementia or Alzheimer's disease, atrophy can be observed in key brain structures years before any clinical symptoms are present. Atrophy is most commonly captured as volume change of key structures and the shape changes of these structures are typically not analysed despite being potentially more sensitive than summary volume statistics over the entire structure. In this paper we propose a spatiotemporal analysis pipeline based on Large Diffeomorphic Deformation Metric Mapping (LDDMM) to detect shape changes from volumetric MRI scans. We applied our framework to a cohort of individuals with genetic variants of frontotemporal dementia and healthy controls from the Genetic FTD Initiative (GENFI) study. Our method, take full advantage of the LDDMM framework, and relies on the creation of a population specific average spatiotemporal trajectory of a relevant brain structure of interest, the thalamus in our case. The residuals from each patient data to the average spatiotemporal trajectory are then clustered and studied to assess when presymptomatic mutation carriers differ from healthy control subjects. We found statistical differences in shape in the anterior region of the thalamus at least five years before the mutation carrier subjects develop any clinical symptoms. This region of the thalamus has been shown to be predominantly connected to the frontal lobe, consistent with the pattern of cortical atrophy seen in the disease