ABSTRACT
Accumulation of α-Synuclein (α-Syn) causes Parkinson's disease (PD) as well as other synucleopathies. α-Syn is the major component of Lewy bodies and Lewy neurites, the proteinaceous aggregates that are a hallmark of sporadic PD. In familial forms of PD, mutations or copy number variations in SNCA (the α-Syn gene) result in a net increase of its protein levels. Furthermore, common risk variants tied to PD are associated with small increases of wild-type α-Syn levels. These findings are further bolstered by animal studies which show that overexpression of α-Syn is sufficient to cause PD-like features. Thus, increased α-Syn levels are intrinsically tied to PD pathogenesis and underscore the importance of identifying the factors that regulate its levels. In this study, we establish a pooled RNAi screening approach and validation pipeline to probe the druggable genome for modifiers of α-Syn levels and identify 60 promising targets. Using a cross-species, tiered validation approach, we validate six strong candidates that modulate α-Syn levels and toxicity in cell lines, Drosophila, human neurons, and mouse brain of both sexes. More broadly, this genetic strategy and validation pipeline can be applied for the identification of therapeutic targets for disorders driven by dosage-sensitive proteins.SIGNIFICANCE STATEMENT We present a research strategy for the systematic identification and validation of genes modulating the levels of α-Synuclein, a protein involved in Parkinson's disease. A cell-based screen of the druggable genome (>7,500 genes that are potential therapeutic targets) yielded many modulators of α-Synuclein that were subsequently confirmed and validated in Drosophila, human neurons, and mouse brain. This approach has broad applicability to the multitude of neurological diseases that are caused by mutations in genes whose dosage is critical for brain function.
Subject(s)
Genome/genetics , Neurons/physiology , RNA Interference/physiology , Sequence Analysis, RNA/methods , alpha-Synuclein/genetics , Animals , Animals, Newborn , Drosophila , Female , HEK293 Cells , Humans , Male , Mice , Reproducibility of Results , Species SpecificityABSTRACT
Alzheimer's and Parkinson's disease are late onset neurodegenerative diseases that will require therapy over decades to mitigate the effects of disease-driving proteins such tau and α-synuclein (α-Syn). Previously we found that TRIM28 regulates the levels and toxicity of α-Syn and tau (
Subject(s)
Aging/metabolism , Gene Deletion , Tripartite Motif-Containing Protein 28/metabolism , alpha-Synuclein/metabolism , tau Proteins/metabolism , Amino Acid Sequence , Animals , Behavior, Animal , Biocatalysis , Brain/metabolism , Brain/pathology , Homeostasis , Iron/metabolism , Mice, Inbred C57BL , Mice, Knockout , Protein Stability , Sumoylation , Tripartite Motif-Containing Protein 28/chemistryABSTRACT
Previous studies suggested that MeCP2 competes with linker histone H1, but this hypothesis has never been tested in vivo. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) of Flag-tagged-H1.0 in mouse forebrain excitatory neurons. Unexpectedly, Flag-H1.0 and MeCP2 occupied similar genomic regions and the Flag-H1.0 binding was not changed upon MeCP2 depletion. Furthermore, mild overexpression of H1.0 did not alter MeCP2 binding, suggesting that the functional binding of MeCP2 and H1.0 are largely independent.
Subject(s)
Histones/genetics , Methyl-CpG-Binding Protein 2/genetics , Animals , Cell Nucleus/chemistry , Cell Nucleus/genetics , Chromatin Immunoprecipitation , DNA Methylation , Genome , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Prosencephalon/cytology , Prosencephalon/metabolism , Protein BindingABSTRACT
Gain-of-function mutations in some genes underlie neurodegenerative conditions, whereas loss-of-function mutations in the same genes have distinct phenotypes. This appears to be the case with the protein ataxin 1 (ATXN1), which forms a transcriptional repressor complex with capicua (CIC). Gain of function of the complex leads to neurodegeneration, but ATXN1-CIC is also essential for survival. We set out to understand the functions of the ATXN1-CIC complex in the developing forebrain and found that losing this complex results in hyperactivity, impaired learning and memory, and abnormal maturation and maintenance of upper-layer cortical neurons. We also found that CIC activity in the hypothalamus and medial amygdala modulates social interactions. Informed by these neurobehavioral features in mouse mutants, we identified five individuals with de novo heterozygous truncating mutations in CIC who share similar clinical features, including intellectual disability, attention deficit/hyperactivity disorder (ADHD), and autism spectrum disorder. Our study demonstrates that loss of ATXN1-CIC complexes causes a spectrum of neurobehavioral phenotypes.
Subject(s)
Ataxin-1/genetics , Autism Spectrum Disorder/genetics , Neurodegenerative Diseases/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Animals , Cerebellum/pathology , Female , Humans , Intellectual Disability/genetics , Interpersonal Relations , Male , Mice , Nerve Tissue Proteins/genetics , PhenotypeABSTRACT
Four mutations in the VAMP/synaptobrevin-associated protein B (VAPB) gene have been linked to amyotrophic lateral sclerosis (ALS) type 8. The mechanism by which VAPB mutations cause motor neuron disease is unclear, but studies of the most common P56S variant suggest both loss of function and dominant-negative sequestration of wild-type protein. Diminished levels of VAPB and its proteolytic cleavage fragment have also been reported in sporadic ALS cases, suggesting that VAPB loss of function may be a common mechanism of disease. Here, we tested whether neuronal overexpression of wild-type human VAPB would attenuate disease in a mouse model of familial ALS1. We used neonatal intraventricular viral injections to express VAPB or YFP throughout the brain and spinal cord of superoxide dismutase (SOD1) G93A transgenic mice. Lifelong elevation of neuronal VAPB slowed the decline of neurological impairment, delayed denervation of hindlimb muscles, and prolonged survival of spinal motor neurons. Collectively, these changes produced a slight but significant extension in lifespan, even in this highly aggressive model of disease. Our findings lend support for a protective role of VAPB in neuromuscular health.
Subject(s)
Neuromuscular Diseases/metabolism , Vesicular Transport Proteins/biosynthesis , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Denervation , Disease Models, Animal , Female , Gene Expression Regulation , Humans , Male , Mice , Mice, Transgenic , Motor Neuron Disease/genetics , Motor Neurons/metabolism , Motor Neurons/pathology , Mutation , Neuromuscular Diseases/genetics , Neurons/metabolism , Spinal Cord/metabolism , Superoxide Dismutase-1/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
The rapid pace of neuroscience research demands equally efficient and flexible methods for genetically manipulating and visualizing selected neurons within the rodent brain. The use of viral vectors for gene delivery saves the time and cost of traditional germline transgenesis and offers the versatility of readily available reagents that can be easily customized to meet individual experimental needs. Here, we present a protocol for widespread neuronal transduction based on intraventricular viral injection of the neonatal mouse brain. Injections can be done either free-hand or assisted by a stereotaxic device to produce lifelong expression of virally delivered transgenes.
Subject(s)
Dependovirus/physiology , Neurons/metabolism , Transduction, Genetic , Animals , Animals, Newborn/virology , Dependovirus/genetics , Genetic Vectors/administration & dosage , Injections, Intraventricular , MiceABSTRACT
Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.
Subject(s)
Nerve Tissue Proteins/genetics , Neurodegenerative Diseases/genetics , Nuclear Proteins/genetics , RNA-Binding Proteins/genetics , 3' Untranslated Regions , Animals , Antigens, Ly/genetics , Ataxin-1 , Ataxins , Brain/metabolism , Gene Knock-In Techniques , Haploinsufficiency , Humans , Membrane Proteins/genetics , Mice , Mice, Knockout , MicroRNAs/metabolism , Mutation , Neurodegenerative Diseases/pathology , Nucleic Acid Conformation , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Messenger/chemistryABSTRACT
With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.
Subject(s)
Brain/physiology , Brain/virology , Dependovirus/physiology , Gene Transfer Techniques , Neurons/physiology , Animals , Animals, Newborn , Brain/cytology , Dependovirus/genetics , Female , Genetic Vectors , Injections, Intraventricular , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Neurons/virology , Pregnancy , Transduction, Genetic , TransgenesABSTRACT
Alzheimer's disease (AD) is associated with an elevated risk for seizures that may be fundamentally connected to cognitive dysfunction. Supporting this link, many mouse models for AD exhibit abnormal electroencephalogram (EEG) activity in addition to the expected neuropathology and cognitive deficits. Here, we used a controllable transgenic system to investigate how network changes develop and are maintained in a model characterized by amyloid ß (Aß) overproduction and progressive amyloid pathology. EEG recordings in tet-off mice overexpressing amyloid precursor protein (APP) from birth display frequent sharp wave discharges (SWDs). Unexpectedly, we found that withholding APP overexpression until adulthood substantially delayed the appearance of epileptiform activity. Together, these findings suggest that juvenile APP overexpression altered cortical development to favor synchronized firing. Regardless of the age at which EEG abnormalities appeared, the phenotype was dependent on continued APP overexpression and abated over several weeks once transgene expression was suppressed. Abnormal EEG discharges were independent of plaque load and could be extinguished without altering deposited amyloid. Selective reduction of Aß with a γ-secretase inhibitor has no effect on the frequency of SWDs, indicating that another APP fragment or the full-length protein was likely responsible for maintaining EEG abnormalities. Moreover, transgene suppression normalized the ratio of excitatory to inhibitory innervation in the cortex, whereas secretase inhibition did not. Our results suggest that APP overexpression, and not Aß overproduction, is responsible for EEG abnormalities in our transgenic mice and can be rescued independently of pathology.
Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/physiopathology , Amyloid beta-Protein Precursor/genetics , Cerebral Cortex/physiopathology , Electroencephalography , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Animals , Disease Models, Animal , Entropy , Female , Gene Knock-In Techniques , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Neurological , Neural Inhibition/physiology , Presenilin-1/genetics , Seizures/chemically induced , Seizures/physiopathology , Suppression, Genetic , Transgenes/physiologyABSTRACT
BACKGROUND: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)-α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus. METHODS: Cultured PDL cells were stimulated with TNF-α and analyzed with an MMP antibody array. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP-3, -10, and -13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP-3, -10, and -13 was observed after blocking the TNFR with an antagonist. Results from real-time PCR, ELISA, and western blot were analyzed by paired t test. RESULTS: The antibody array showed that the protein most strongly upregulated by TNF-α stimulation was MMP-3, followed by MMP-13 and MMP-10. The TNF-α receptor blocker specifically inhibited expression of MMP-3 and -13. In addition, TNF-α increased levels of MMP mRNAs in MMP-3, -13, and -10 (in decreasing order). However, ELISAs showed that MMP-13 was the most upregulated protein, followed by MMP-10 and MMP-3. Western blotting indicated that TNF-α increased MMP-3 and -13 levels but had no significant effect on the level of MMP-10, and zymography showed that TNF-α increased the activities of all forms of MMP-3 and -13, but MMP-10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1. CONCLUSIONS: TNF-α (10 ng/mL) upregulates levels of MMP-3, -10, and -13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.
Subject(s)
Matrix Metalloproteinase 10/drug effects , Matrix Metalloproteinase 13/drug effects , Matrix Metalloproteinase 3/drug effects , Periodontal Ligament/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Adult , Blotting, Western , Cell Culture Techniques , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Humans , Male , Matrix Metalloproteinase Inhibitors/pharmacology , Periodontal Ligament/cytology , Real-Time Polymerase Chain Reaction , Receptors, Tumor Necrosis Factor/analysis , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type I/analysis , Up-RegulationABSTRACT
Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24-84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.
Subject(s)
Brain/metabolism , Capsid/metabolism , Dependovirus/classification , Dependovirus/genetics , Serotyping , Transduction, Genetic/methods , Animals , Animals, Newborn , Astrocytes/virology , Brain/cytology , Brain/virology , Green Fluorescent Proteins/genetics , Injections , Mice , Time Factors , Viral TropismABSTRACT
INTRODUCTION: The purpose of this study was to investigate whether in vitro stimulation of pulp cells leads to increased secretion of matrix metalloproteinases (MMPs) and, if so, to identify which MMPs are affected. METHODS: Cells cultured from dental pulp were stimulated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 24 hours, and lysates were analyzed with an antibody array (Bio-Rad Laboratories, Hercules, CA). The mRNA and protein levels of MMP-3, -10, and -13 were measured by real-time polymerase chain reaction (real-time PCR), enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and zymography. In addition, tumor necrosis factor receptors in the pulp cells were assayed by flow cytometry. The ELISA and real-time PCR results were analyzed by paired t tests. RESULTS: The expression of MMP-3, -10, and -13 was up-regulated in the pulp cells after 24 hours of stimulation with TNF-α (10 ng/mL) as seen in the antibody array, real-time PCR, and ELISA results, but MMP-10 was not detected by Western blotting or casein zymography. Flow cytometry analysis showed that the majority of the pulp cells expressed tumor necrosis factor receptor 1. CONCLUSIONS: In regions of inflammation, TNF-α may initiate the degradation of dental connective tissue by activating MMP-3 and MMP-13. These proteins may play an important pathologic role in the inflammation of dental pulp.
Subject(s)
Dental Pulp/enzymology , Matrix Metalloproteinases/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Cell Culture Techniques , Cells, Cultured , Dental Pulp/cytology , Dental Pulp/drug effects , Humans , Matrix Metalloproteinase 10/analysis , Matrix Metalloproteinase 10/drug effects , Matrix Metalloproteinase 13/analysis , Matrix Metalloproteinase 13/drug effects , Matrix Metalloproteinase 3/analysis , Matrix Metalloproteinase 3/drug effects , Matrix Metalloproteinases/analysis , Receptors, Tumor Necrosis Factor, Type I/analysis , Time Factors , Up-RegulationABSTRACT
The neonatal intraventricular injection of adeno-associated virus has been shown to transduce neurons widely throughout the brain, but its full potential for experimental neuroscience has not been adequately explored. We report a detailed analysis of the method's versatility with an emphasis on experimental applications where tools for genetic manipulation are currently lacking. Viral injection into the neonatal mouse brain is fast, easy, and accesses regions of the brain including the cerebellum and brainstem that have been difficult to target with other techniques such as electroporation. We show that viral transduction produces an inherently mosaic expression pattern that can be exploited by varying the titer to transduce isolated neurons or densely-packed populations. We demonstrate that the expression of virally-encoded proteins is active much sooner than previously believed, allowing genetic perturbation during critical periods of neuronal plasticity, but is also long-lasting and stable, allowing chronic studies of aging. We harness these features to visualise and manipulate neurons in the hindbrain that have been recalcitrant to approaches commonly applied in the cortex. We show that viral labeling aids the analysis of postnatal dendritic maturation in cerebellar Purkinje neurons by allowing individual cells to be readily distinguished, and then demonstrate that the same sparse labeling allows live in vivo imaging of mature Purkinje neurons at a resolution sufficient for complete analytical reconstruction. Given the rising availability of viral constructs, packaging services, and genetically modified animals, these techniques should facilitate a wide range of experiments into brain development, function, and degeneration.
Subject(s)
Brain , Dependovirus/genetics , Mosaicism , Transduction, Genetic/methods , Animals , Animals, Newborn , Genetic Vectors/genetics , MiceABSTRACT
The phosphorylation state of large-conductance calcium-activated potassium (BK(Ca)) channels regulates their activity and is dynamically regulated by protein phosphatases and kinases, including protein kinase C (PKC). In this study, we showed that PKC activators up-regulate the activity of the BK(Ca) channel alpha (alpha)-subunit, Slo1, in cell-attached patches of transfected COS7 cells. In an immune complex kinase assay, BK(Ca) channels isolated from rat brain were phosphorylated in the presence of PKC activators, without the addition of exogenous PKC, which suggests that PKC and BK(Ca) channels functionally interact in vivo. Four different PKC isozymes, including PKCdelta, phosphorylated the C-terminus of Slo1 and the addition of purified PKCdelta-activated BK(Ca) channels in excised patches of transfected HEK293 cells. Our results demonstrate that PKC up-regulates BK(Ca) channels and that PKCdelta may functionally interact with BK(Ca) channel complexes in vivo.
Subject(s)
Brain/metabolism , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Protein Kinase C/metabolism , Animals , COS Cells , Calcium/metabolism , Cell Line , Chlorocebus aethiops , Humans , Isoenzymes/metabolism , Phosphorylation , Protein Kinase C-delta/metabolism , RatsABSTRACT
A 13-yr-old female was admitted to our hospital with fever, seizure, and cervical lymphadenopathy. Laboratory data showed pancytopenia, elevation of serum transaminase, lactate dehydrogenase, triglyceride, and ferritin levels. Lymph node biopsy revealed features of Kikuchi's disease and there were signs of histiocytosis and hemophagocytic phenomenon in bone marrow. She recovered after treatment with intravenous immunoglobulin and corticosteroids therapy. Hemophagocytic syndrome can be associated with Kikuchi's disease especially in childhood and seems to have a less aggressive clinical course and better prognosis.