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1.
Mol Neurodegener ; 19(1): 42, 2024 May 27.
Article in English | MEDLINE | ID: mdl-38802940

ABSTRACT

Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-ß (Aß) deposition. Mice expressing CD33M have increased Aß levels, more diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to 5XFAD control mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.


Subject(s)
Alzheimer Disease , Disease Models, Animal , Mice, Transgenic , Microglia , Protein Isoforms , Sialic Acid Binding Ig-like Lectin 3 , Animals , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Microglia/metabolism , Sialic Acid Binding Ig-like Lectin 3/metabolism , Humans , Mice , Protein Isoforms/metabolism , Amyloid beta-Peptides/metabolism , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology
2.
FEBS Lett ; 598(4): 415-436, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38320753

ABSTRACT

Matrin-3 (MATR3) is an RNA-binding protein implicated in neurodegenerative and neurodevelopmental diseases. However, little is known regarding the role of MATR3 in cryptic splicing within the context of functional genes and how disease-associated variants impact this function. We show that loss of MATR3 leads to cryptic exon inclusion in many transcripts. We reveal that ALS-linked S85C pathogenic variant reduces MATR3 solubility but does not impair RNA binding. In parallel, we report a novel neurodevelopmental disease-associated M548T variant, located in the RRM2 domain, which reduces protein solubility and impairs RNA binding and cryptic splicing repression functions of MATR3. Altogether, our research identifies cryptic events within functional genes and demonstrates how disease-associated variants impact MATR3 cryptic splicing repression function.


Subject(s)
Amyotrophic Lateral Sclerosis , Humans , Amyotrophic Lateral Sclerosis/genetics , Exons/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA , Nuclear Matrix-Associated Proteins/genetics
3.
eNeuro ; 10(10)2023 10.
Article in English | MEDLINE | ID: mdl-37775311

ABSTRACT

Cajal-Retzius (CR) cells are transient neurons with long-lasting effects on the architecture and circuitry of the neocortex and hippocampus. Contrary to the prevailing assumption that CR cells completely disappear in rodents shortly after birth, a substantial portion of these cells persist in the hippocampus throughout adulthood. The role of these surviving CR cells in the adult hippocampus is largely unknown, partly because of the paucity of suitable tools to dissect their functions in the adult versus the embryonic brain. Here, we show that genetic crosses of the ΔNp73-Cre mouse line, widely used to target CR cells, to reporter mice induce reporter expression not only in CR cells, but also progressively in postnatal dentate gyrus granule neurons. Such a lack of specificity may confound studies of CR cell function in the adult hippocampus. To overcome this, we devise a method that not only leverages the temporary CR cell-targeting specificity of the ΔNp73-Cre mice before the first postnatal week, but also capitalizes on the simplicity and effectiveness of freehand neonatal intracerebroventricular injection of adeno-associated virus. We achieve robust Cre-mediated recombination that remains largely restricted to hippocampal CR cells from early postnatal age to adulthood. We further demonstrate the utility of this method to manipulate neuronal activity of CR cells in the adult hippocampus. This versatile and scalable strategy will facilitate experiments of CR cell-specific gene knockdown and/or overexpression, lineage tracing, and neural activity modulation in the postnatal and adult brain.


Subject(s)
Hippocampus , Neocortex , Mice , Animals , Hippocampus/metabolism , Neurons/physiology , Cell Movement
4.
Hum Mutat ; 43(7): 889-899, 2022 07.
Article in English | MEDLINE | ID: mdl-35165976

ABSTRACT

Heterozygous pathogenic variants in CIC, which encodes a transcriptional repressor, have been identified in individuals with neurodevelopmental phenotypes. To date, 11 CIC variants have been associated with the CIC-related neurodevelopmental syndrome. Here, we describe three novel and one previously reported CIC variants in four individuals with neurodevelopmental delay. Notably, we report for the first time a de novo frameshift variant specific to the long isoform of CIC (CIC-L, NM_001304815.1:c.1100dup, p.Pro368AlafsTer16) in an individual with speech delay, intellectual disability, and autism spectrum disorder. Our investigation into the function of CIC-L reveals that partial loss of CIC-L leads to transcriptional derepression of CIC target genes. We also describe a missense variant (NM_015125.3:c.683G>A, p.Arg228Gln) in an individual with a history of speech delay and relapsed pre-B acute lymphoblastic leukemia. Functional studies of this variant suggest a partial loss of CIC transcriptional repressor activity. Our study expands the list of CIC pathogenic variants and contributes to the accumulating evidence that CIC haploinsufficiency or partial loss of function is a pathogenic mechanism causing neurodevelopmental phenotypes.


Subject(s)
Autism Spectrum Disorder , Intellectual Disability , Language Development Disorders , Neurodevelopmental Disorders , Autism Spectrum Disorder/genetics , Heterozygote , Humans , Intellectual Disability/genetics , Intellectual Disability/pathology , Language Development Disorders/genetics , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/pathology , Phenotype
5.
Cell Rep ; 38(7): 110386, 2022 02 15.
Article in English | MEDLINE | ID: mdl-35172136

ABSTRACT

B-1 cell development mainly occurs via fetal and neonatal hematopoiesis and is suppressed in adult bone marrow hematopoiesis. However, little is known about the factors inhibiting B-1 cell development at the adult stage. We report that capicua (CIC) suppresses postnatal B-1a cell development and survival. CIC levels are high in B-1a cells and gradually increase in transitional B-1a (TrB-1a) cells with age. B-cell-specific Cic-null mice exhibit expansion of the B-1a cell population and a gradual increase in TrB-1a cell frequency with age but attenuated B-2 cell development. CIC deficiency enhances B cell receptor (BCR) signaling in transitional B cells and B-1a cell viability. Mechanistically, CIC-deficiency-mediated Per2 derepression upregulates Bhlhe41 levels by inhibiting CRY-mediated transcriptional repression for Bhlhe41, consequently promoting B-1a cell formation in Cic-null mice. Taken together, CIC is a key transcription factor that limits the B-1a cell population at the adult stage and balances B-1 versus B-2 cell formation.


Subject(s)
B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Period Circadian Proteins/metabolism , Repressor Proteins/metabolism , Signal Transduction , Animals , Animals, Newborn , Apoptosis , Base Sequence , Bone Marrow/embryology , Cell Differentiation , Cell Survival , Child , Child, Preschool , Fetus/embryology , HEK293 Cells , Humans , Liver/embryology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , NIH 3T3 Cells , Receptors, Antigen, B-Cell/metabolism
6.
Sci Rep ; 11(1): 11725, 2021 06 03.
Article in English | MEDLINE | ID: mdl-34083623

ABSTRACT

New neurons continuously arise from neural progenitor cells in the dentate gyrus of the adult hippocampus to support ongoing learning and memory formation. To generate functional adult-born neurons, neural progenitor cells proliferate to expand the precursor cell pool and differentiate into neurons. Newly generated cells then undergo postmitotic maturation to migrate to their final destination and develop elaborate dendritic branching, which allows them to receive input signals. Little is known about factors that regulate neuronal differentiation, migration, and dendrite maturation during adult hippocampal neurogenesis. Here, we show that the transcriptional repressor protein capicua (CIC) exhibits dynamic expression in the adult dentate gyrus. Conditional deletion of Cic from the mouse dentate gyrus compromises the adult neural progenitor cell pool without altering their proliferative potential. We further demonstrate that the loss of Cic impedes neuronal lineage development and disrupts dendritic arborization and migration of adult-born neurons. Our study uncovers a previously unrecognized role of CIC in neurogenesis of the adult dentate gyrus.


Subject(s)
Hippocampus/cytology , Neurogenesis/genetics , Pyramidal Cells/cytology , Pyramidal Cells/metabolism , Repressor Proteins/genetics , Animals , Cell Differentiation , Dentate Gyrus/cytology , Mice , Mice, Knockout , Neural Stem Cells/cytology , Neural Stem Cells/metabolism
7.
Nat Commun ; 11(1): 5304, 2020 10 20.
Article in English | MEDLINE | ID: mdl-33082323

ABSTRACT

A missense mutation, S85C, in the MATR3 gene is a genetic cause for amyotrophic lateral sclerosis (ALS). It is unclear how the S85C mutation affects MATR3 function and contributes to disease. Here, we develop a mouse model that harbors the S85C mutation in the endogenous Matr3 locus using the CRISPR/Cas9 system. MATR3 S85C knock-in mice recapitulate behavioral and neuropathological features of early-stage ALS including motor impairment, muscle atrophy, neuromuscular junction defects, Purkinje cell degeneration and neuroinflammation in the cerebellum and spinal cord. Our neuropathology data reveals a loss of MATR3 S85C protein in the cell bodies of Purkinje cells and motor neurons, suggesting that a decrease in functional MATR3 levels or loss of MATR3 function contributes to neuronal defects. Our findings demonstrate that the MATR3 S85C mouse model mimics aspects of early-stage ALS and would be a promising tool for future basic and preclinical research.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Motor Neurons/metabolism , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Amyotrophic Lateral Sclerosis/genetics , Animals , Disease Models, Animal , Female , Gene Knock-In Techniques , Humans , Loss of Function Mutation , Male , Mice , Mutation, Missense , Purkinje Cells/metabolism
8.
Proc Natl Acad Sci U S A ; 117(38): 23742-23750, 2020 09 22.
Article in English | MEDLINE | ID: mdl-32878998

ABSTRACT

Ataxin-1 (ATXN1) is a ubiquitous polyglutamine protein expressed primarily in the nucleus where it binds chromatin and functions as a transcriptional repressor. Mutant forms of ataxin-1 containing expanded glutamine stretches cause the movement disorder spinocerebellar ataxia type 1 (SCA1) through a toxic gain-of-function mechanism in the cerebellum. Conversely, ATXN1 loss-of-function is implicated in cancer development and Alzheimer's disease (AD) pathogenesis. ATXN1 was recently nominated as a susceptibility locus for multiple sclerosis (MS). Here, we show that Atxn1-null mice develop a more severe experimental autoimmune encephalomyelitis (EAE) course compared to wildtype mice. The aggravated phenotype is mediated by increased T helper type 1 (Th1) cell polarization, which in turn results from the dysregulation of B cell activity. Ataxin-1 ablation in B cells leads to aberrant expression of key costimulatory molecules involved in proinflammatory T cell differentiation, including cluster of differentiation (CD)44 and CD80. In addition, comprehensive phosphoflow cytometry and transcriptional profiling link the exaggerated proliferation of ataxin-1 deficient B cells to the activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) pathways. Lastly, selective deletion of the physiological binding partner capicua (CIC) demonstrates the importance of ATXN1 native interactions for correct B cell functioning. Altogether, we report a immunomodulatory role for ataxin-1 and provide a functional description of the ATXN1 locus genetic association with MS risk.


Subject(s)
Ataxin-1/metabolism , B-Lymphocytes/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Animals , Antigen Presentation , Cell Proliferation , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Mice , Mice, Knockout , Multiple Sclerosis , Signal Transduction
9.
Chem Pharm Bull (Tokyo) ; 67(7): 713-716, 2019 Jul 01.
Article in English | MEDLINE | ID: mdl-31006725

ABSTRACT

A reverse phase (RP)-HPLC method for separation and determination of Schisandrin A and Schisandrin B was presented, using a C18 Bondclone column, with methanol-water (v/v = 68 : 32) as mobile phase at a flow-rate of 1.00 mL·min-1, and UV detection at 220 nm. The tested parameters included mobile phase composition and UV detection wavelength. Good linearities were observed within concentration ranges of Schisandrin A 0.008-4.8 mg·L-1 (r = 0.9996), and Schisandrin B 0.005-3.1 mg·L-1 (r = 0.9994), respectively. The limit of detection (LOD) (S/N = 3) were 0.005 mg·L-1 Schisandrin A and 0.002 mg·L-1 Schisandrin B, respectively. The method was applied to determine the 2 compounds in a traditional Chinese medicine preparation for treatment of hepatic diseases, Huganpian tablet. To eliminate matrix effect, Oasis hydrophilic lipophilic balance (HLB) solid-phase extraction (SPE) was used to purify the ultra-sonicately extracted solution of the drug sample. Combined with the HLB SPE purification procedure, the HPLC method gave satisfactory results for quantitation of Schisandrin A and Schisandrin B in 3 types of Huganpian tablet samples, with spiking recoveries ca. 98% (relative standard deviation (R.S.D.) ≤ 3.5%) (n = 5).


Subject(s)
Chromatography, High Pressure Liquid , Cyclooctanes/analysis , Lignans/analysis , Polycyclic Compounds/analysis , Tablets/chemistry , Chromatography, Reverse-Phase , Cyclooctanes/isolation & purification , Lignans/isolation & purification , Limit of Detection , Medicine, Chinese Traditional , Polycyclic Compounds/isolation & purification , Solid Phase Extraction
10.
Neurobiol Learn Mem ; 165: 106902, 2019 11.
Article in English | MEDLINE | ID: mdl-30030131

ABSTRACT

Animal models have been the mainstay of biological and medical research. Although there are drawbacks to any research tool, we argue that mice have been under-utilized as a tool for predicting human diseases. Here we review four examples from our research group where studying the consequences of altered gene dosage in a mouse led to the discovery of previously unrecognized human syndromes: MECP2 duplication syndrome, SHANK3 duplication syndrome, CIC haploinsufficiency syndrome, and PUM1-related disorders. We also describe the clinical phenotypes of two individuals with CIC haploinsufficiency syndrome who have not been reported previously. To help bring biological insights gained from model systems a step closer to disease gene discovery, we discuss tools and resources that will facilitate this process. Moving back and forth between the lab and the clinic, between studies of mouse models and human patients, will continue to drive disease gene discovery and lead to better understanding of gene functions and disease mechanisms, laying the groundwork for future therapeutic interventions.


Subject(s)
Disease Models, Animal , Nervous System Diseases/genetics , Animals , Genetic Association Studies/methods , Haploinsufficiency/genetics , Humans , Mental Retardation, X-Linked/genetics , Mice , Nerve Tissue Proteins/genetics , RNA-Binding Proteins/genetics , Repressor Proteins/genetics
11.
J Exp Bot ; 69(21): 5205-5219, 2018 10 12.
Article in English | MEDLINE | ID: mdl-30113690

ABSTRACT

Legumes fix atmospheric nitrogen through a symbiotic relationship with bacteroids in root nodules. Following fixation in pea (Pisum sativum L.) nodules, nitrogen is reduced to amino acids that are exported via the nodule xylem to the shoot, and in the phloem to roots in support of growth. However, the mechanisms involved in amino acid movement towards the nodule vasculature, and their importance for nodule function and plant nutrition, were unknown. We found that in pea nodules the apoplasmic pathway is an essential route for amino acid partitioning from infected cells to the vascular bundles, and that amino acid permease PsAAP6 is a key player in nitrogen retrieval from the apoplasm into inner cortex cells for nodule export. Using an miRNA interference (miR) approach, it was demonstrated that PsAAP6 function in nodules, and probably in roots, and affects both shoot and root nitrogen supply, which were strongly decreased in PsAAP6-miR plants. Further, reduced transporter function resulted in increased nodule levels of ammonium, asparagine, and other amino acids. Surprisingly, nitrogen fixation and nodule metabolism were up-regulated in PsAAP6-miR plants, indicating that under shoot nitrogen deficiency, or when plant nitrogen demand is high, systemic signaling leads to an increase in nodule activity, independent of the nodule nitrogen status.


Subject(s)
Amino Acid Transport Systems/genetics , Nitrogen Fixation , Nitrogen/metabolism , Pisum sativum/physiology , Plant Proteins/genetics , Root Nodules, Plant/metabolism , Amino Acid Transport Systems/metabolism , Amino Acids/metabolism , Gene Expression Profiling , Nutrients/metabolism , Plant Proteins/metabolism , Plant Vascular Bundle/metabolism
12.
Hum Mol Genet ; 27(16): 2863-2873, 2018 08 15.
Article in English | MEDLINE | ID: mdl-29860311

ABSTRACT

Spinocerebellar ataxia type 1 (SCA1) is caused by the expansion of a trinucleotide repeat that encodes a polyglutamine tract in ataxin-1 (ATXN1). The expanded polyglutamine in ATXN1 increases the protein's stability and results in its accumulation and toxicity. Previous studies have demonstrated that decreasing ATXN1 levels ameliorates SCA1 phenotypes and pathology in mouse models. We rationalized that reducing ATXN1 levels through pharmacological inhibition of its modulators could provide a therapeutic avenue for SCA1. Here, through a forward genetic screen in Drosophila we identified, p21-activated kinase 3 (Pak3) as a modulator of ATXN1 levels. Loss-of-function of fly Pak3 or Pak1, whose mammalian homologs belong to Group I of PAK proteins, reduces ATXN1 levels, and accordingly, improves disease pathology in a Drosophila model of SCA1. Knockdown of PAK1 potently reduces ATXN1 levels in mammalian cells independent of the well-characterized S776 phosphorylation site (known to stabilize ATXN1) thus revealing a novel molecular pathway that regulates ATXN1 levels. Furthermore, pharmacological inhibition of PAKs decreases ATXN1 levels in a mouse model of SCA1. To explore the potential of using PAK inhibitors in combination therapy, we combined the pharmacological inhibition of PAK with MSK1, a previously identified modulator of ATXN1, and examined their effects on ATXN1 levels. We found that inhibition of both pathways results in an additive decrease in ATXN1 levels. Together, this study identifies PAK signaling as a distinct molecular pathway that regulates ATXN1 levels and presents a promising opportunity to pursue for developing potential therapeutics for SCA1.


Subject(s)
Ataxin-1/genetics , Spinocerebellar Ataxias/genetics , p21-Activated Kinases/genetics , Animals , Ataxin-1/antagonists & inhibitors , Cerebellum/metabolism , Cerebellum/pathology , Disease Models, Animal , Drosophila melanogaster/genetics , Enzyme Inhibitors/administration & dosage , Gene Knockdown Techniques , Humans , Mice , Peptides/genetics , Phosphorylation , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Signal Transduction/genetics , Spinocerebellar Ataxias/physiopathology , p21-Activated Kinases/antagonists & inhibitors
13.
Neuron ; 97(6): 1235-1243.e5, 2018 03 21.
Article in English | MEDLINE | ID: mdl-29526553

ABSTRACT

Polyglutamine (polyQ) diseases are caused by expansion of translated CAG repeats in distinct genes leading to altered protein function. In spinocerebellar ataxia type 1 (SCA1), a gain of function of polyQ-expanded ataxin-1 (ATXN1) contributes to cerebellar pathology. The extent to which cerebellar toxicity depends on its cognate partner capicua (CIC), versus other interactors, remains unclear. It is also not established whether loss of the ATXN1-CIC complex in the cerebellum contributes to disease pathogenesis. In this study, we exclusively disrupt the ATXN1-CIC interaction in vivo and show that it is at the crux of cerebellar toxicity in SCA1. Importantly, loss of CIC in the cerebellum does not cause ataxia or Purkinje cell degeneration. Expression profiling of these gain- and loss-of-function models, coupled with data from iPSC-derived neurons from SCA1 patients, supports a mechanism in which gain of function of the ATXN1-CIC complex is the major driver of toxicity.


Subject(s)
Ataxin-1/deficiency , Cerebellum/metabolism , Gain of Function Mutation/physiology , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/metabolism , Animals , Ataxin-1/genetics , Cells, Cultured , Cerebellum/pathology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Spinocerebellar Ataxias/pathology
14.
Proc Natl Acad Sci U S A ; 115(7): E1511-E1519, 2018 02 13.
Article in English | MEDLINE | ID: mdl-29382756

ABSTRACT

Capicua (CIC) regulates a transcriptional network downstream of the RAS/MAPK signaling cascade. In Drosophila, CIC is important for many developmental processes, including embryonic patterning and specification of wing veins. In humans, CIC has been implicated in neurological diseases, including spinocerebellar ataxia type 1 (SCA1) and a neurodevelopmental syndrome. Additionally, we and others have reported mutations in CIC in several cancers. However, whether CIC is a tumor suppressor remains to be formally tested. In this study, we found that deletion of Cic in adult mice causes T cell acute lymphoblastic leukemia/lymphoma (T-ALL). Using hematopoietic-specific deletion and bone marrow transplantation studies, we show that loss of Cic from hematopoietic cells is sufficient to drive T-ALL. Cic-null tumors show up-regulation of the KRAS pathway as well as activation of the NOTCH1 and MYC transcriptional programs. In sum, we demonstrate that loss of CIC causes T-ALL, establishing it as a tumor suppressor for lymphoid malignancies. Moreover, we show that mouse models lacking CIC in the hematopoietic system are robust models for studying the role of RAS signaling as well as NOTCH1 and MYC transcriptional programs in T-ALL.


Subject(s)
Cell Differentiation , Disease Susceptibility , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/etiology , Repressor Proteins/physiology , T-Lymphocytes/pathology , Animals , Cells, Cultured , Mice , Mice, Knockout , Mutation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Signal Transduction , T-Lymphocytes/metabolism , ras Proteins/genetics , ras Proteins/metabolism
15.
Nat Genet ; 49(4): 527-536, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28288114

ABSTRACT

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 , Phenotype
16.
Hum Mol Genet ; 25(23): 5083-5093, 2016 12 01.
Article in English | MEDLINE | ID: mdl-28007900

ABSTRACT

Splicing regulation is an important step of post-transcriptional gene regulation. It is a highly dynamic process orchestrated by RNA-binding proteins (RBPs). RBP dysfunction and global splicing dysregulation have been implicated in many human diseases, but the in vivo functions of most RBPs and the splicing outcome upon their loss remain largely unexplored. Here we report that constitutive deletion of Rbm17, which encodes an RBP with a putative role in splicing, causes early embryonic lethality in mice and that its loss in Purkinje neurons leads to rapid degeneration. Transcriptome profiling of Rbm17-deficient and control neurons and subsequent splicing analyses using CrypSplice, a new computational method that we developed, revealed that more than half of RBM17-dependent splicing changes are cryptic. Importantly, RBM17 represses cryptic splicing of genes that likely contribute to motor coordination and cell survival. This finding prompted us to re-analyze published datasets from a recent report on TDP-43, an RBP implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as it was demonstrated that TDP-43 represses cryptic exon splicing to promote cell survival. We uncovered a large number of TDP-43-dependent splicing defects that were not previously discovered, revealing that TDP-43 extensively regulates cryptic splicing. Moreover, we found a significant overlap in genes that undergo both RBM17- and TDP-43-dependent cryptic splicing repression, many of which are associated with survival. We propose that repression of cryptic splicing by RBPs is critical for neuronal health and survival. CrypSplice is available at www.liuzlab.org/CrypSplice.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , DNA-Binding Proteins/genetics , Frontotemporal Dementia/genetics , Nerve Degeneration/genetics , Nerve Tissue Proteins/genetics , RNA Splicing Factors/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Computational Biology/methods , Disease Models, Animal , Exons/genetics , Frontotemporal Dementia/physiopathology , Gene Expression Regulation, Developmental , Humans , Mice , Nerve Degeneration/pathology , Nerve Tissue Proteins/biosynthesis , Purkinje Cells/metabolism , Purkinje Cells/pathology , RNA Splicing/genetics , RNA Splicing Factors/biosynthesis , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics
17.
Nature ; 498(7454): 325-331, 2013 Jun 20.
Article in English | MEDLINE | ID: mdl-23719381

ABSTRACT

Many neurodegenerative disorders, such as Alzheimer's, Parkinson's and polyglutamine diseases, share a common pathogenic mechanism: the abnormal accumulation of disease-causing proteins, due to either the mutant protein's resistance to degradation or overexpression of the wild-type protein. We have developed a strategy to identify therapeutic entry points for such neurodegenerative disorders by screening for genetic networks that influence the levels of disease-driving proteins. We applied this approach, which integrates parallel cell-based and Drosophila genetic screens, to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of a polyglutamine tract in ataxin 1 (ATXN1). Our approach revealed that downregulation of several components of the RAS-MAPK-MSK1 pathway decreases ATXN1 levels and suppresses neurodegeneration in Drosophila and mice. Importantly, pharmacological inhibitors of components of this pathway also decrease ATXN1 levels, suggesting that these components represent new therapeutic targets in mitigating SCA1. Collectively, these data reveal new therapeutic entry points for SCA1 and provide a proof-of-principle for tackling other classes of intractable neurodegenerative diseases.


Subject(s)
Drosophila melanogaster/metabolism , Mitogen-Activated Protein Kinases/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Spinocerebellar Ataxias/metabolism , Spinocerebellar Ataxias/pathology , ras Proteins/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Ataxin-1 , Ataxins , Cell Line, Tumor , Disease Models, Animal , Down-Regulation/drug effects , Drosophila melanogaster/genetics , Female , Humans , MAP Kinase Signaling System/drug effects , Male , Mice , Molecular Sequence Data , Molecular Targeted Therapy , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphorylation , Protein Stability/drug effects , Ribosomal Protein S6 Kinases, 90-kDa/deficiency , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Transgenes
18.
Plant Cell ; 22(11): 3603-20, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21075769

ABSTRACT

Seed development and nitrogen (N) storage depend on delivery of amino acids to seed sinks. For efficient translocation to seeds, amino acids are loaded into the phloem in source leaves and along the long distance transport pathway through xylem-phloem transfer. We demonstrate that Arabidopsis thaliana AMINO ACID PERMEASE2 (AAP2) localizes to the phloem throughout the plant. AAP2 T-DNA insertion lines showed changes in source-sink translocation of amino acids and a decrease in the amount of seed total N and storage proteins, supporting AAP2 function in phloem loading and amino acid distribution to the embryo. Interestingly, in aap2 seeds, total carbon (C) levels were unchanged, while fatty acid levels were elevated. Moreover, branch and silique numbers per plant and seed yield were strongly increased. This suggests changes in N and C delivery to sinks and subsequent modulations of sink development and seed metabolism. This is supported by tracer experiments, expression studies of genes of N/C transport and metabolism in source and sink, and by phenotypic and metabolite analyses of aap2 plants. Thus, AAP2 is key for xylem to phloem transfer and sink N and C supply; moreover, modifications of N allocation can positively affect C assimilation and source-sink transport and benefit sink development and oil yield.


Subject(s)
Amino Acids/metabolism , Arabidopsis/chemistry , Arabidopsis/metabolism , Phloem/metabolism , Plant Oils , Seeds , Xylem/metabolism , Amino Acid Transport Systems, Acidic/genetics , Amino Acid Transport Systems, Acidic/metabolism , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Biological Transport , Carbon/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Nitrogen/metabolism , Onions/cytology , Onions/genetics , Onions/metabolism , Plant Leaves/chemistry , Plant Leaves/cytology , Plant Leaves/metabolism , Plant Oils/chemistry , Plant Oils/metabolism , Plants, Genetically Modified/chemistry , Plants, Genetically Modified/cytology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Seeds/chemistry , Seeds/metabolism
19.
Plant Physiol ; 154(4): 1886-96, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20923886

ABSTRACT

Seeds of grain legumes are important energy and food sources for humans and animals. However, the yield and quality of legume seeds are limited by the amount of sulfur (S) partitioned to the seeds. The amino acid S-methylmethionine (SMM), a methionine derivative, has been proposed to be an important long-distance transport form of reduced S, and we analyzed whether SMM phloem loading and source-sink translocation are important for the metabolism and growth of pea (Pisum sativum) plants. Transgenic plants were produced in which the expression of a yeast SMM transporter, S-Methylmethionine Permease1 (MMP1, YLL061W), was targeted to the phloem and seeds. Phloem exudate analysis showed that concentrations of SMM are elevated in MMP1 plants, suggesting increased phloem loading. Furthermore, expression studies of genes involved in S transport and metabolism in source organs, as well as xylem sap analyses, support that S uptake and assimilation are positively affected in MMP1 roots. Concomitantly, nitrogen (N) assimilation in root and leaf and xylem amino acid profiles were changed, resulting in increased phloem loading of amino acids. When investigating the effects of increased S and N phloem transport on seed metabolism, we found that protein levels were improved in MMP1 seeds. In addition, changes in SMM phloem loading affected plant growth and seed number, leading to an overall increase in seed S, N, and protein content in MMP1 plants. Together, these results suggest that phloem loading and source-sink partitioning of SMM are important for plant S and N metabolism and transport as well as seed set.


Subject(s)
Nitrogen/metabolism , Phloem/metabolism , Pisum sativum/metabolism , Seeds/growth & development , Sulfur/metabolism , Vitamin U/metabolism , Pisum sativum/embryology , Seeds/metabolism
20.
Funct Plant Biol ; 34(11): 1019-1028, 2007 Dec.
Article in English | MEDLINE | ID: mdl-32689430

ABSTRACT

Expression of the amino acid permeases PsAAP1 and PsAAP2 was analysed in developing pea (Pisum sativum L.) plants. Both transporters were expressed in seed coats and cotyledon epidermal transfer cells and storage parenchyma cells. AAP expression is developmentally regulated and coincides with the onset of storage protein synthesis. Nitrogen was shown to induce AAP expression and AAP transcript levels were upregulated during the photoperiod. Analysis of Arabidopsis thaliana AAP1 promoter activity in pea, using promoter-ß-glucuronidase (promotor-GUS) studies, revealed targeting of GUS to seed coats and cotyledon epidermal transfer cells. Expression was found in the nutritious endosperm during the early stages of seed development, whereas GUS staining in embryos was detected from the heart stage onward. In addition, AAP1 expression was observed in the phloem throughout the plant. This finding equally applied to PsAAP1 expression as shown by in situ mRNA hybridisation, which also demonstrated that PsAAP1 expression was localised to companion cells. Overall, PsAAP1 expression patterns and cellular localisation point to a function of the transporter in phloem loading of amino acids for translocation to sinks and in seed loading for development and storage protein accumulation.

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