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1.
J Neurol ; 270(3): 1615-1623, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36462055

ABSTRACT

OBJECTIVES: Friedreich ataxia is (FRDA) an autosomal recessive neurodegenerative disorder associated with intrinsic oxidative damage, suggesting that decreasing lipid peroxidation (LPO) might ameliorate disease progression. The present study tested the ability of RT001, a deuterated form of linoleic acid (D2-LA), to alter disease severity in patients with FRDA in a double-blind placebo-controlled trial. METHODS: Sixty-five subjects were recruited across six sites and received either placebo or active drug for an 11-month study. Subjects were evaluated at 0, 4, 9, and 11 months, with the primary outcome measure being maximum oxygen consumption (MVO2) during cardiopulmonary exercise testing (CPET). A key secondary outcome measure was a composite statistical test using results from the timed 1-min walk (T1MW), peak workload, and MVO2. RESULTS: Forty-five subjects completed the protocol. RT001 was well tolerated, with no serious adverse events related to drug. Plasma and red blood cell (RBC) membrane levels of D2-LA and its primary metabolite deuterated arachidonic acid (D2-AA) achieved steady-state concentrations by 4 months. No significant changes in MVO2 were observed for RT001 compared to placebo. Similarly, no differences between the groups were found in secondary or exploratory outcome measures. Post hoc evaluations also suggested minimal effects of RT001 at the dosages used in this study. INTERPRETATIONS: The results of this study provide no evidence for a significant benefit of RT001 at the dosages tested in this Friedreich ataxia patient population.


Subject(s)
Friedreich Ataxia , Linoleic Acid , Humans , Friedreich Ataxia/drug therapy , Linoleic Acid/therapeutic use , Linoleic Acids/therapeutic use , Walking , Double-Blind Method
2.
Commun Biol ; 4(1): 1269, 2021 11 05.
Article in English | MEDLINE | ID: mdl-34741098

ABSTRACT

There is currently a dearth of accessible whole genome sequencing (WGS) data for individuals residing in the Americas with Sub-Saharan African ancestry. We generated whole genome sequencing data at intermediate (15×) coverage for 2,294 individuals with large amounts of Sub-Saharan African ancestry, predominantly Atlantic African admixed with varying amounts of European and American ancestry. We performed extensive comparisons of variant callers, phasing algorithms, and variant filtration on these data to construct a high quality imputation panel containing data from 2,269 unrelated individuals. With the exception of the TOPMed imputation server (which notably cannot be downloaded), our panel substantially outperformed other available panels when imputing African American individuals. The raw sequencing data, variant calls and imputation panel for this cohort are all freely available via dbGaP and should prove an invaluable resource for further study of admixed African genetics.


Subject(s)
Genome, Human , Genotype , Adult , Black or African American , Aged , Aged, 80 and over , Humans , Middle Aged , United States , Whole Genome Sequencing , Young Adult
4.
PLoS Genet ; 14(7): e1007394, 2018 07.
Article in English | MEDLINE | ID: mdl-30001343

ABSTRACT

Preterm birth is a leading cause of morbidity and mortality in infants. Genetic and environmental factors play a role in the susceptibility to preterm birth, but despite many investigations, the genetic basis for preterm birth remain largely unknown. Our objective was to identify rare, possibly damaging, nucleotide variants in mothers from families with recurrent spontaneous preterm births (SPTB). DNA samples from 17 Finnish mothers who delivered at least one infant preterm were subjected to whole exome sequencing. All mothers were of northern Finnish origin and were from seven multiplex families. Additional replication samples of European origin consisted of 93 Danish sister pairs (and two sister triads), all with a history of a preterm delivery. Rare exonic variants (frequency <1%) were analyzed to identify genes and pathways likely to affect SPTB susceptibility. We identified rare, possibly damaging, variants in genes that were common to multiple affected individuals. The glucocorticoid receptor signaling pathway was the most significant (p<1.7e-8) with genes containing these variants in a subgroup of ten Finnish mothers, each having had 2-4 SPTBs. This pathway was replicated among the Danish sister pairs. A gene in this pathway, heat shock protein family A (Hsp70) member 1 like (HSPA1L), contains two likely damaging missense alleles that were found in four different Finnish families. One of the variants (rs34620296) had a higher frequency in cases compared to controls (0.0025 vs. 0.0010, p = 0.002) in a large preterm birth genome-wide association study (GWAS) consisting of mothers of general European ancestry. Sister pairs in replication samples also shared rare, likely damaging HSPA1L variants. Furthermore, in silico analysis predicted an additional phosphorylation site generated by rs34620296 that could potentially affect chaperone activity or HSPA1L protein stability. Finally, in vitro functional experiment showed a link between HSPA1L activity and decidualization. In conclusion, rare, likely damaging, variants in HSPA1L were observed in multiple families with recurrent SPTB.


Subject(s)
Genetic Predisposition to Disease , HSP70 Heat-Shock Proteins/genetics , Premature Birth/genetics , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Case-Control Studies , Cell Line , Exome/genetics , Female , Fibroblasts , Finland , Genome-Wide Association Study , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Humans , Infant, Newborn , Male , Models, Molecular , Phosphorylation/genetics , Polymorphism, Single Nucleotide , Pregnancy , Receptors, Glucocorticoid/metabolism , Recurrence , Risk Factors , Signal Transduction/genetics , Exome Sequencing
5.
Methods Mol Biol ; 1755: 197-221, 2018.
Article in English | MEDLINE | ID: mdl-29671272

ABSTRACT

We are now seeing the benefit of investments made over the last decade in high-throughput screening (HTS) that is resulting in large structure activity datasets entering public and open databases such as ChEMBL and PubChem. The growth of academic HTS screening centers and the increasing move to academia for early stage drug discovery suggests a great need for the informatics tools and methods to mine such data and learn from it. Collaborative Drug Discovery, Inc. (CDD) has developed a number of tools for storing, mining, securely and selectively sharing, as well as learning from such HTS data. We present a new web based data mining and visualization module directly within the CDD Vault platform for high-throughput drug discovery data that makes use of a novel technology stack following modern reactive design principles. We also describe CDD Models within the CDD Vault platform that enables researchers to share models, share predictions from models, and create models from distributed, heterogeneous data. Our system is built on top of the Collaborative Drug Discovery Vault Activity and Registration data repository ecosystem which allows users to manipulate and visualize thousands of molecules in real time. This can be performed in any browser on any platform. In this chapter we present examples of its use with public datasets in CDD Vault. Such approaches can complement other cheminformatics tools, whether open source or commercial, in providing approaches for data mining and modeling of HTS data.


Subject(s)
Computational Biology/methods , Data Mining/methods , Databases, Pharmaceutical , Datasets as Topic , Drug Discovery/methods , Software
6.
N Engl J Med ; 377(12): 1156-1167, 2017 09 21.
Article in English | MEDLINE | ID: mdl-28877031

ABSTRACT

BACKGROUND: Despite evidence that genetic factors contribute to the duration of gestation and the risk of preterm birth, robust associations with genetic variants have not been identified. We used large data sets that included the gestational duration to determine possible genetic associations. METHODS: We performed a genomewide association study in a discovery set of samples obtained from 43,568 women of European ancestry using gestational duration as a continuous trait and term or preterm (<37 weeks) birth as a dichotomous outcome. We used samples from three Nordic data sets (involving a total of 8643 women) to test for replication of genomic loci that had significant genomewide association (P<5.0×10-8) or an association with suggestive significance (P<1.0×10-6) in the discovery set. RESULTS: In the discovery and replication data sets, four loci (EBF1, EEFSEC, AGTR2, and WNT4) were significantly associated with gestational duration. Functional analysis showed that an implicated variant in WNT4 alters the binding of the estrogen receptor. The association between variants in ADCY5 and RAP2C and gestational duration had suggestive significance in the discovery set and significant evidence of association in the replication sets; these variants also showed genomewide significance in a joint analysis. Common variants in EBF1, EEFSEC, and AGTR2 showed association with preterm birth with genomewide significance. An analysis of mother-infant dyads suggested that these variants act at the level of the maternal genome. CONCLUSIONS: In this genomewide association study, we found that variants at the EBF1, EEFSEC, AGTR2, WNT4, ADCY5, and RAP2C loci were associated with gestational duration and variants at the EBF1, EEFSEC, and AGTR2 loci with preterm birth. Previously established roles of these genes in uterine development, maternal nutrition, and vascular control support their mechanistic involvement. (Funded by the March of Dimes and others.).


Subject(s)
Genetic Predisposition to Disease , Genetic Variation , Gestational Age , Peptide Elongation Factors/genetics , Premature Birth/genetics , Receptor, Angiotensin, Type 2/genetics , Trans-Activators/genetics , Adenylyl Cyclases/genetics , Datasets as Topic , Female , Genome-Wide Association Study , Humans , Phenotype , Polymorphism, Single Nucleotide , Pregnancy , Regression Analysis , Wnt4 Protein/genetics , ras Proteins/genetics
7.
Cell Rep ; 18(6): 1484-1498, 2017 02 07.
Article in English | MEDLINE | ID: mdl-28178525

ABSTRACT

The mechanism underlying selective motor neuron (MN) death remains an essential question in the MN disease field. The MN disease spinal muscular atrophy (SMA) is attributable to reduced levels of the ubiquitous protein SMN. Here, we report that SMN levels are widely variable in MNs within a single genetic background and that this heterogeneity is seen not only in SMA MNs but also in MNs derived from controls and amyotrophic lateral sclerosis (ALS) patients. Furthermore, cells with low SMN are more susceptible to cell death. These findings raise the important clinical implication that some SMN-elevating therapeutics might be effective in MN diseases besides SMA. Supporting this, we found that increasing SMN across all MN populations using an Nedd8-activating enzyme inhibitor promotes survival in both SMA and ALS-derived MNs. Altogether, our work demonstrates that examination of human neurons at the single-cell level can reveal alternative strategies to be explored in the treatment of degenerative diseases.


Subject(s)
Neuromuscular Diseases/metabolism , SMN Complex Proteins/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Animals , Disease Models, Animal , Humans , Mice , Motor Neurons/metabolism , Muscular Atrophy, Spinal/metabolism , Single-Cell Analysis/methods , Spinal Cord/metabolism
8.
Brain Res ; 1656: 40-48, 2017 02 01.
Article in English | MEDLINE | ID: mdl-27060768

ABSTRACT

Utilizing patient derived cells has enormous promise for discovering new drugs for diseases of the nervous system, a goal that has been historically quite challenging. In this review, we will outline the potential of human stem cell derived neuron models for assessing therapeutics and high-throughput screening and compare to more traditional drug discovery strategies. We summarize recent successes of the approach and discuss special considerations for developing human stem cell based assays. New technologies, such as genome editing, offer improvements to help overcome the challenges that remain. Finally, human neurons derived from patient cells have advantages for translational research beyond drug screening as they can also be used to identify individual efficacy and safety prior to clinical testing and for dissecting disease mechanisms. This article is part of a Special Issue entitled SI: Exploiting human neurons.


Subject(s)
Drug Discovery , Nervous System Diseases/drug therapy , Nervous System Diseases/physiopathology , Neurons/physiology , Animals , Humans
9.
Pharm Res ; 33(1): 194-205, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26311555

ABSTRACT

PURPOSE: We propose a framework with simple proxies to dissect the relative energy contributions responsible for standard drug discovery binding activity. METHODS: We explore a rule of thumb using hydrogen-bond donors, hydrogen-bond acceptors and rotatable bonds as relative proxies for the thermodynamic terms. We apply this methodology to several datasets (e.g., multiple small molecules profiled against kinases, Mycobacterium tuberculosis (Mtb) high throughput screening (HTS) and structure based drug design (SBDD) derived compounds, and FDA approved drugs). RESULTS: We found that Mtb active compounds developed through SBDD methods had statistically significantly larger PEnthalpy values than HTS derived compounds, suggesting these compounds had relatively more hydrogen bond donor and hydrogen bond acceptors compared to rotatable bonds. In recent FDA approved medicines we found that compounds identified via target-based approaches had a more balanced enthalpic relationship between these descriptors compared to compounds identified via phenotypic screens CONCLUSIONS: As it is common to experimentally optimize directly for total binding energy, these computational methods provide alternative calculations and approaches useful for compound optimization alongside other common metrics in available software and databases.


Subject(s)
Drug Discovery/methods , Thermodynamics , Computational Biology , Databases, Factual , Entropy , High-Throughput Screening Assays , Hydrogen Bonding , Mycobacterium tuberculosis/drug effects , Phosphotransferases/chemistry , Receptors, Drug/chemistry , Small Molecule Libraries , Structure-Activity Relationship
10.
F1000Res ; 4: 53, 2015.
Article in English | MEDLINE | ID: mdl-25901280

ABSTRACT

This brief review of current research progress on Charcot-Marie-Tooth (CMT) disease is a summary of discussions initiated at the Hereditary Neuropathy Foundation (HNF) scientific advisory board meeting on November 7, 2014. It covers recent published and unpublished in vitro and in vivo research. We discuss recent promising preclinical work for CMT1A, the development of new biomarkers, the characterization of different animal models, and the analysis of the frequency of gene mutations in patients with CMT. We also describe how progress in related fields may benefit CMT therapeutic development, including the potential of gene therapy and stem cell research. We also discuss the potential to assess and improve the quality of life of CMT patients. This summary of CMT research identifies some of the gaps which may have an impact on upcoming clinical trials. We provide some priorities for CMT research and areas which HNF can support. The goal of this review is to inform the scientific community about ongoing research and to avoid unnecessary overlap, while also highlighting areas ripe for further investigation. The general collaborative approach we have taken may be useful for other rare neurological diseases.

11.
F1000Res ; 4: 38, 2015.
Article in English | MEDLINE | ID: mdl-25713700

ABSTRACT

The recent outbreak of the Ebola virus in West Africa has highlighted the clear shortage of broad-spectrum antiviral drugs for emerging viruses. There are numerous FDA approved drugs and other small molecules described in the literature that could be further evaluated for their potential as antiviral compounds. These molecules are in addition to the few new antivirals that have been tested in Ebola patients but were not originally developed against the Ebola virus, and may play an important role as we await an effective vaccine. The balance between using FDA approved drugs versus novel antivirals with minimal safety and no efficacy data in humans should be considered. We have evaluated 55 molecules from the perspective of an experienced medicinal chemist as well as using simple molecular properties and have highlighted 16 compounds that have desirable qualities as well as those that may be less desirable. In addition we propose that a collaborative database for sharing such published and novel information on small molecules is needed for the research community studying the Ebola virus.

12.
Drug Discov Today ; 20(2): 247-54, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25449658

ABSTRACT

Stem cell research is at an important juncture: despite significant potential for human health and several countries with key initiatives to expedite commercialization, there are gaps in capturing and exploiting the results of past and current research. Here, we propose a concerted plan that could be taken to foster a more collaborative approach and ensure that all research efforts can be leveraged across the community. The creation of a definitive centralized database repository, or at least harmonized data repositories, for stem cell groups in academia and industry, enabling secure selective sharing of data when needed, could provide the core structure that is sought globally and protect intellectual property. The development of minimum information about stem cell experiments (MIASCE) could be key to this development.


Subject(s)
Stem Cell Research , Animals , Cooperative Behavior , Databases, Factual/standards , Humans , Reference Standards
13.
J Med Chem ; 58(5): 2068-76, 2015 Mar 12.
Article in English | MEDLINE | ID: mdl-25415348

ABSTRACT

The availability of structures and linked bioactivity data in databases is powerfully enabling for drug discovery and chemical biology. However, we now review some confounding issues with the divergent expansions of public and commercial sources of chemical structures. These are associated with not only expanding patent extraction but also increasingly large vendor collections amassed via different selection criteria between SciFinder from Chemical Abstracts Service (CAS) and major public sources such as PubChem, ChemSpider, UniChem, and others. These increasingly massive collections may include both real and virtual compounds, as well as so-called prophetic compounds from patents. We address a range of issues raised by the challenges faced resolving the NIH probe compounds. In addition we highlight the confounding of prior-art searching by virtual compounds that could impact the composition of matter patentability of a new medicinal chemistry lead. Finally, we propose some potential solutions.


Subject(s)
Chemistry, Pharmaceutical , Computational Biology/methods , Databases, Factual , Drug Discovery/methods , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Humans , Information Storage and Retrieval , Patents as Topic , Structure-Activity Relationship
14.
J Chem Inf Model ; 54(10): 2996-3004, 2014 Oct 27.
Article in English | MEDLINE | ID: mdl-25244007

ABSTRACT

In a decade with over half a billion dollars of investment, more than 300 chemical probes have been identified to have biological activity through NIH funded screening efforts. We have collected the evaluations of an experienced medicinal chemist on the likely chemistry quality of these probes based on a number of criteria including literature related to the probe and potential chemical reactivity. Over 20% of these probes were found to be undesirable. Analysis of the molecular properties of these compounds scored as desirable suggested higher pKa, molecular weight, heavy atom count, and rotatable bond number. We were particularly interested whether the human evaluation aspect of medicinal chemistry due diligence could be computationally predicted. We used a process of sequential Bayesian model building and iterative testing as we included additional probes. Following external validation of these methods and comparing different machine learning methods, we identified Bayesian models with accuracy comparable to other measures of drug-likeness and filtering rules created to date.


Subject(s)
Artificial Intelligence , Models, Statistical , Molecular Probes/chemistry , Bayes Theorem , Computer Simulation , Humans , Molecular Probes/economics , Molecular Weight , Quality Control , Sensitivity and Specificity
15.
PeerJ ; 2: e524, 2014.
Article in English | MEDLINE | ID: mdl-25165633

ABSTRACT

Bioinformatics and computer aided drug design rely on the curation of a large number of protocols for biological assays that measure the ability of potential drugs to achieve a therapeutic effect. These assay protocols are generally published by scientists in the form of plain text, which needs to be more precisely annotated in order to be useful to software methods. We have developed a pragmatic approach to describing assays according to the semantic definitions of the BioAssay Ontology (BAO) project, using a hybrid of machine learning based on natural language processing, and a simplified user interface designed to help scientists curate their data with minimum effort. We have carried out this work based on the premise that pure machine learning is insufficiently accurate, and that expecting scientists to find the time to annotate their protocols manually is unrealistic. By combining these approaches, we have created an effective prototype for which annotation of bioassay text within the domain of the training set can be accomplished very quickly. Well-trained annotations require single-click user approval, while annotations from outside the training set domain can be identified using the search feature of a well-designed user interface, and subsequently used to improve the underlying models. By drastically reducing the time required for scientists to annotate their assays, we can realistically advocate for semantic annotation to become a standard part of the publication process. Once even a small proportion of the public body of bioassay data is marked up, bioinformatics researchers can begin to construct sophisticated and useful searching and analysis algorithms that will provide a diverse and powerful set of tools for drug discovery researchers.

16.
J Comput Aided Mol Des ; 28(10): 997-1008, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24943138

ABSTRACT

Over the past decade we have seen a growth in the provision of chemistry data and cheminformatics tools as either free websites or software as a service commercial offerings. These have transformed how we find molecule-related data and use such tools in our research. There have also been efforts to improve collaboration between researchers either openly or through secure transactions using commercial tools. A major challenge in the future will be how such databases and software approaches handle larger amounts of data as it accumulates from high throughput screening and enables the user to draw insights, enable predictions and move projects forward. We now discuss how information from some drug discovery datasets can be made more accessible and how privacy of data should not overwhelm the desire to share it at an appropriate time with collaborators. We also discuss additional software tools that could be made available and provide our thoughts on the future of predictive drug discovery in this age of big data. We use some examples from our own research on neglected diseases, collaborations, mobile apps and algorithm development to illustrate these ideas.


Subject(s)
Databases, Factual , Drug Discovery/methods , Software , Statistics as Topic/methods , Cooperative Behavior , Humans , Quantitative Structure-Activity Relationship , Rare Diseases
17.
Science ; 344(6184): 630-4, 2014 May 09.
Article in English | MEDLINE | ID: mdl-24797482

ABSTRACT

In the adult central nervous system, the vasculature of the neurogenic niche regulates neural stem cell behavior by providing circulating and secreted factors. Age-related decline of neurogenesis and cognitive function is associated with reduced blood flow and decreased numbers of neural stem cells. Therefore, restoring the functionality of the niche should counteract some of the negative effects of aging. We show that factors found in young blood induce vascular remodeling, culminating in increased neurogenesis and improved olfactory discrimination in aging mice. Further, we show that GDF11 alone can improve the cerebral vasculature and enhance neurogenesis. The identification of factors that slow the age-dependent deterioration of the neurogenic niche in mice may constitute the basis for new methods of treating age-related neurodegenerative and neurovascular diseases.


Subject(s)
Aging/drug effects , Bone Morphogenetic Proteins/administration & dosage , Brain/drug effects , Cerebrovascular Circulation/drug effects , Growth Differentiation Factors/administration & dosage , Neural Stem Cells/drug effects , Neurogenesis/drug effects , Rejuvenation , Animals , Bone Morphogenetic Proteins/blood , Bone Morphogenetic Proteins/physiology , Brain/blood supply , Cognition/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Growth Differentiation Factors/blood , Growth Differentiation Factors/physiology , Male , Mice , Mice, Inbred C57BL , Neural Stem Cells/cytology , Olfactory Bulb/cytology , Olfactory Bulb/drug effects , Parabiosis , Recombinant Proteins/administration & dosage
18.
F1000Res ; 3: 261, 2014.
Article in English | MEDLINE | ID: mdl-25685324

ABSTRACT

Rare disease research has reached a tipping point, with the confluence of scientific and technologic developments that if appropriately harnessed, could lead to key breakthroughs and treatments for this set of devastating disorders. Industry-wide trends have revealed that the traditional drug discovery research and development (R&D) model is no longer viable, and drug companies are evolving their approach. Rather than only pursue blockbuster therapeutics for heterogeneous, common diseases, drug companies have increasingly begun to shift their focus to rare diseases. In academia, advances in genetics analyses and disease mechanisms have allowed scientific understanding to mature, but the lack of funding and translational capability severely limits the rare disease research that leads to clinical trials. Simultaneously, there is a movement towards increased research collaboration, more data sharing, and heightened engagement and active involvement by patients, advocates, and foundations. The growth in networks and social networking tools presents an opportunity to help reach other patients but also find researchers and build collaborations. The growth of collaborative software that can enable researchers to share their data could also enable rare disease patients and foundations to manage their portfolio of funded projects for developing new therapeutics and suggest drug repurposing opportunities. Still there are many thousands of diseases without treatments and with only fragmented research efforts. We will describe some recent progress in several rare diseases used as examples and propose how collaborations could be facilitated. We propose that the development of a center of excellence that integrates and shares informatics resources for rare diseases sponsored by all of the stakeholders would help foster these initiatives.

19.
J Neurosci ; 33(46): 18015-21, 2013 Nov 13.
Article in English | MEDLINE | ID: mdl-24227713

ABSTRACT

Tuberous sclerosis complex (TSC) is a leading genetic cause of autism. The TSC proteins Tsc1 and Tsc2 control the mTORC1 signaling pathway in diverse cells, but how the mTORC1 pathway is specifically regulated in neurons remains to be elucidated. Here, using an interaction proteomics approach in neural cells including neurons, we uncover the brain-enriched protein huntingtin-associated protein 1 (Hap1) as a novel functional partner of Tsc1. Knockdown of Hap1 promotes specification of supernumerary axons in primary hippocampal neurons and profoundly impairs the positioning of pyramidal neurons in the mouse hippocampus in vivo. The Hap1 knockdown-induced phenotypes in primary neurons and in vivo recapitulate the phenotypes induced by Tsc1 knockdown. We also find that Hap1 knockdown in hippocampal neurons induces the downregulation of Tsc1 and stimulates the activity of mTORC1, as reflected by phosphorylation of the ribosomal protein S6. Inhibition of mTORC1 activity suppresses the Hap1 knockdown-induced polarity phenotype in hippocampal neurons. Collectively, these findings define a novel link between Hap1 and Tsc1 that regulates neuronal mTORC1 signaling and neuronal morphogenesis, with implications for our understanding of developmental disorders of cognition.


Subject(s)
Brain/growth & development , Morphogenesis/physiology , Multiprotein Complexes/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cells, Cultured , Female , HEK293 Cells , Humans , Male , Mechanistic Target of Rapamycin Complex 1 , Mice , Protein Binding/physiology , Rats , Signal Transduction/physiology , Tuberous Sclerosis Complex 1 Protein
20.
PLoS Biol ; 9(5): e1001060, 2011 May.
Article in English | MEDLINE | ID: mdl-21572988

ABSTRACT

The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7(Fbxw8) localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7(Fbxw8) by independent approaches including Fbxw8 knockdown reveals that Cul7(Fbxw8) is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7(Fbxw8) also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7(Fbxw8) in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.


Subject(s)
Cullin Proteins/metabolism , Cytoskeletal Proteins/metabolism , Dendrites/enzymology , F-Box Proteins/metabolism , Golgi Apparatus/enzymology , Animals , Cells, Cultured , Dendrites/physiology , Electroporation , Golgi Apparatus/physiology , Golgi Matrix Proteins , Humans , Membrane Proteins/metabolism , Morphogenesis , Proteomics , Rats , Signal Transduction , Transfection , Ubiquitination
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