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1.
Cell ; 187(6): 1387-1401.e13, 2024 Mar 14.
Article in English | MEDLINE | ID: mdl-38412859

ABSTRACT

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.


Subject(s)
Nerve Tissue Proteins , Retinal Degeneration , Animals , Mice , Bacterial Translocation , Eye Proteins/genetics , Leber Congenital Amaurosis/genetics , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Retina/metabolism , Retinal Degeneration/genetics , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Retinitis Pigmentosa/pathology
2.
Cell ; 187(13): 3236-3248.e21, 2024 Jun 20.
Article in English | MEDLINE | ID: mdl-38772369

ABSTRACT

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.


Subject(s)
Gene Regulatory Networks , Single-Cell Analysis , Animals , Female , Humans , Mice , Cerebral Cortex/metabolism , Cerebral Cortex/cytology , CRISPR-Cas Systems/genetics , Dependovirus/genetics , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Genetic Vectors/metabolism , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Neurons/metabolism , Neurons/cytology , Single-Cell Analysis/methods , Transcriptome/genetics , Cell Line , Transcription, Genetic
3.
Cell ; 185(2): 311-327.e24, 2022 01 20.
Article in English | MEDLINE | ID: mdl-35063073

ABSTRACT

The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.


Subject(s)
Vision, Ocular , Visual Cortex/cytology , Visual Cortex/embryology , Animals , Animals, Newborn , Biomarkers/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Glutamic Acid/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , RNA-Seq , Transcriptome/genetics , Vision, Binocular/genetics , gamma-Aminobutyric Acid/metabolism
4.
Cell ; 184(7): 1740-1756.e16, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33705688

ABSTRACT

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2-/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2-/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2-/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them.


Subject(s)
Gastrointestinal Microbiome , Locomotion , Social Behavior , Animals , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Biopterins/analogs & derivatives , Biopterins/metabolism , Disease Models, Animal , Excitatory Postsynaptic Potentials , Fecal Microbiota Transplantation , Feces/microbiology , Limosilactobacillus reuteri/metabolism , Limosilactobacillus reuteri/physiology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/microbiology , Neurodevelopmental Disorders/pathology , Neurodevelopmental Disorders/therapy , Principal Component Analysis , Psychomotor Agitation/pathology , Synaptic Transmission
5.
Cell ; 176(5): 1158-1173.e16, 2019 02 21.
Article in English | MEDLINE | ID: mdl-30712869

ABSTRACT

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.


Subject(s)
Cell Lineage/genetics , Enteroendocrine Cells/metabolism , Gene Expression Profiling/methods , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Cell Lineage/physiology , Enteroendocrine Cells/physiology , Fluorescent Dyes , Homeodomain Proteins/genetics , Intestinal Mucosa/cytology , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Optical Imaging/methods , Organoids , Phenotype , Single-Cell Analysis/methods , Stem Cells , Transcription Factors/genetics , Transcriptome/genetics
6.
Cell ; 179(1): 132-146.e14, 2019 09 19.
Article in English | MEDLINE | ID: mdl-31522887

ABSTRACT

Oligodendrocytes extend elaborate microtubule arbors that contact up to 50 axon segments per cell, then spiral around myelin sheaths, penetrating from outer to inner layers. However, how they establish this complex cytoarchitecture is unclear. Here, we show that oligodendrocytes contain Golgi outposts, an organelle that can function as an acentrosomal microtubule-organizing center (MTOC). We identify a specific marker for Golgi outposts-TPPP (tubulin polymerization promoting protein)-that we use to purify this organelle and characterize its proteome. In in vitro cell-free assays, recombinant TPPP nucleates microtubules. Primary oligodendrocytes from Tppp knockout (KO) mice have aberrant microtubule branching, mixed microtubule polarity, and shorter myelin sheaths when cultured on 3-dimensional (3D) microfibers. Tppp KO mice exhibit hypomyelination with shorter, thinner myelin sheaths and motor coordination deficits. Together, our data demonstrate that microtubule nucleation outside the cell body at Golgi outposts by TPPP is critical for elongation of the myelin sheath.


Subject(s)
Carrier Proteins/metabolism , Golgi Apparatus/metabolism , Microtubules/metabolism , Myelin Sheath/metabolism , Nerve Tissue Proteins/metabolism , Animals , Animals, Newborn , Axons/metabolism , Carrier Proteins/genetics , Cell-Free System/metabolism , Cells, Cultured , Escherichia coli/metabolism , Gene Knockdown Techniques , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Organizing Center/metabolism , Nerve Tissue Proteins/genetics , Oligodendrocyte Precursor Cells/metabolism , Rats , Rats, Sprague-Dawley , Tubulin/metabolism
7.
Cell ; 178(4): 867-886.e24, 2019 08 08.
Article in English | MEDLINE | ID: mdl-31398341

ABSTRACT

Somatosensory over-reactivity is common among patients with autism spectrum disorders (ASDs) and is hypothesized to contribute to core ASD behaviors. However, effective treatments for sensory over-reactivity and ASDs are lacking. We found distinct somatosensory neuron pathophysiological mechanisms underlie tactile abnormalities in different ASD mouse models and contribute to some ASD-related behaviors. Developmental loss of ASD-associated genes Shank3 or Mecp2 in peripheral mechanosensory neurons leads to region-specific brain abnormalities, revealing links between developmental somatosensory over-reactivity and the genesis of aberrant behaviors. Moreover, acute treatment with a peripherally restricted GABAA receptor agonist that acts directly on mechanosensory neurons reduced tactile over-reactivity in six distinct ASD models. Chronic treatment of Mecp2 and Shank3 mutant mice improved body condition, some brain abnormalities, anxiety-like behaviors, and some social impairments but not memory impairments, motor deficits, or overgrooming. Our findings reveal a potential therapeutic strategy targeting peripheral mechanosensory neurons to treat tactile over-reactivity and select ASD-related behaviors.


Subject(s)
Autism Spectrum Disorder/metabolism , GABA Agonists/pharmacology , Isonicotinic Acids/pharmacology , Phenotype , Sensory Receptor Cells/drug effects , Touch/drug effects , Action Potentials/drug effects , Animals , Anxiety/drug therapy , Autism Spectrum Disorder/drug therapy , Autism Spectrum Disorder/genetics , Behavior, Animal/drug effects , Brain/drug effects , Disease Models, Animal , Female , GABA Agonists/therapeutic use , Isonicotinic Acids/therapeutic use , Male , Maze Learning/drug effects , Methyl-CpG-Binding Protein 2/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins , Nerve Tissue Proteins/genetics , Prepulse Inhibition/drug effects , Sensory Receptor Cells/metabolism
8.
Cell ; 172(1-2): 275-288.e18, 2018 01 11.
Article in English | MEDLINE | ID: mdl-29328916

ABSTRACT

The neuronal gene Arc is essential for long-lasting information storage in the mammalian brain, mediates various forms of synaptic plasticity, and has been implicated in neurodevelopmental disorders. However, little is known about Arc's molecular function and evolutionary origins. Here, we show that Arc self-assembles into virus-like capsids that encapsulate RNA. Endogenous Arc protein is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation. Purified Arc capsids are endocytosed and are able to transfer Arc mRNA into the cytoplasm of neurons. These results show that Arc exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis indicates that Arc is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses. These findings suggest that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system.


Subject(s)
Cytoskeletal Proteins/metabolism , Exosomes/metabolism , Gene Products, gag/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism , RNA, Messenger/metabolism , Animals , Cells, Cultured , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Endocytosis , Female , Gene Products, gag/chemistry , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Neurons/physiology
9.
Cell ; 172(4): 706-718.e15, 2018 02 08.
Article in English | MEDLINE | ID: mdl-29398114

ABSTRACT

Dopamine controls essential brain functions through volume transmission. Different from fast synaptic transmission, where neurotransmitter release and receptor activation are tightly coupled by an active zone, dopamine transmission is widespread and may not necessitate these organized release sites. Here, we determine whether striatal dopamine secretion employs specialized machinery for release. Using super resolution microscopy, we identified co-clustering of the active zone scaffolding proteins bassoon, RIM and ELKS in ∼30% of dopamine varicosities. Conditional RIM knockout disrupted this scaffold and, unexpectedly, abolished dopamine release, while ELKS knockout had no effect. Optogenetic experiments revealed that dopamine release was fast and had a high release probability, indicating the presence of protein scaffolds for coupling Ca2+ influx to vesicle fusion. Hence, dopamine secretion is mediated by sparse, mechanistically specialized active zone-like release sites. This architecture supports spatially and temporally precise coding for dopamine and provides molecular machinery for regulation.


Subject(s)
Axons/metabolism , Corpus Striatum/metabolism , Dopamine/metabolism , Synaptic Transmission/physiology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Corpus Striatum/cytology , Dopamine/genetics , Gene Knockdown Techniques , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , rab GTP-Binding Proteins
10.
Cell ; 174(1): 32-43.e15, 2018 06 28.
Article in English | MEDLINE | ID: mdl-29958111

ABSTRACT

The organization of action into sequences underlies complex behaviors that are essential for organismal survival and reproduction. Despite extensive studies of innate sequences in relation to central pattern generators, how learned action sequences are controlled and whether they are organized as a chain or a hierarchy remain largely unknown. By training mice to perform heterogeneous action sequences, we demonstrate that striatal direct and indirect pathways preferentially encode different behavioral levels of sequence structure. State-dependent closed-loop optogenetic stimulation of the striatal direct pathway can selectively insert a single action element into the sequence without disrupting the overall sequence length. Optogenetic manipulation of the striatal indirect pathway completely removes the ongoing subsequence while leaving the following subsequence to be executed with the appropriate timing and length. These results suggest that learned action sequences are not organized in a serial but rather a hierarchical structure that is distinctly controlled by basal ganglia pathways.


Subject(s)
Learning , Neurons/metabolism , Optogenetics , Animals , Behavior, Animal/drug effects , Behavior, Animal/radiation effects , Diphtheria Toxin/pharmacology , Electrodes, Implanted , Evoked Potentials, Visual , Female , Lasers , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscimol/pharmacology , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/drug effects , RGS Proteins/genetics , Receptors, N-Methyl-D-Aspartate/deficiency , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism
11.
Cell ; 174(3): 590-606.e21, 2018 07 26.
Article in English | MEDLINE | ID: mdl-29961574

ABSTRACT

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.


Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurogenesis/genetics , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Animals , Calcium-Binding Proteins , Cerebral Cortex/metabolism , Chick Embryo , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins , Humans , Intercellular Signaling Peptides and Proteins/genetics , Jagged-1 Protein , Jagged-2 Protein , Mammals/embryology , Mice , Mice, Inbred C57BL , Neocortex/physiology , Neural Stem Cells , Neurogenesis/physiology , Neuroglia/physiology , Neurons , PAX6 Transcription Factor/metabolism , Repressor Proteins , Signal Transduction , Snakes/embryology , Roundabout Proteins
12.
Cell ; 172(1-2): 262-274.e11, 2018 01 11.
Article in English | MEDLINE | ID: mdl-29328915

ABSTRACT

Arc/Arg3.1 is required for synaptic plasticity and cognition, and mutations in this gene are linked to autism and schizophrenia. Arc bears a domain resembling retroviral/retrotransposon Gag-like proteins, which multimerize into a capsid that packages viral RNA. The significance of such a domain in a plasticity molecule is uncertain. Here, we report that the Drosophila Arc1 protein forms capsid-like structures that bind darc1 mRNA in neurons and is loaded into extracellular vesicles that are transferred from motorneurons to muscles. This loading and transfer depends on the darc1-mRNA 3' untranslated region, which contains retrotransposon-like sequences. Disrupting transfer blocks synaptic plasticity, suggesting that transfer of dArc1 complexed with its mRNA is required for this function. Notably, cultured cells also release extracellular vesicles containing the Gag region of the Copia retrotransposon complexed with its own mRNA. Taken together, our results point to a trans-synaptic mRNA transport mechanism involving retrovirus-like capsids and extracellular vesicles.


Subject(s)
Cytoskeletal Proteins/metabolism , Gene Products, gag/genetics , Multivesicular Bodies/metabolism , Nerve Tissue Proteins/metabolism , Presynaptic Terminals/metabolism , RNA, Messenger/metabolism , Animals , Biological Transport , Cells, Cultured , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Products, gag/chemistry , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Neuromuscular Junction/metabolism , Neuronal Plasticity , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Presynaptic Terminals/physiology , Protein Binding , Protein Domains , Retroelements/genetics
13.
Cell ; 170(2): 226-247, 2017 Jul 13.
Article in English | MEDLINE | ID: mdl-28708995

ABSTRACT

The nervous system-in particular, the brain and its cognitive abilities-is among humans' most distinctive and impressive attributes. How the nervous system has changed in the human lineage and how it differs from that of closely related primates is not well understood. Here, we consider recent comparative analyses of extant species that are uncovering new evidence for evolutionary changes in the size and the number of neurons in the human nervous system, as well as the cellular and molecular reorganization of its neural circuits. We also discuss the developmental mechanisms and underlying genetic and molecular changes that generate these structural and functional differences. As relevant new information and tools materialize at an unprecedented pace, the field is now ripe for systematic and functionally relevant studies of the development and evolution of human nervous system specializations.


Subject(s)
Biological Evolution , Brain/anatomy & histology , Brain/physiology , Nervous System/anatomy & histology , Nervous System/growth & development , Animals , Brain/cytology , Gene Expression Regulation , Language , Mutation , Nerve Tissue Proteins/genetics , Nervous System/cytology , Nervous System Physiological Phenomena , Primates/genetics , Primates/physiology , Species Specificity
14.
Cell ; 168(5): 775-788.e12, 2017 02 23.
Article in English | MEDLINE | ID: mdl-28235195

ABSTRACT

Stem-cell-based therapies can potentially reverse organ dysfunction and diseases, but the removal of impaired tissue and activation of a program leading to organ regeneration pose major challenges. In mice, a 4-day fasting mimicking diet (FMD) induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing ß cells, resembling that observed during pancreatic development. FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 expression and insulin production. The effects of the FMD are reversed by IGF-1 treatment and recapitulated by PKA and mTOR inhibition. These results indicate that a FMD promotes the reprogramming of pancreatic cells to restore insulin generation in islets from T1D patients and reverse both T1D and T2D phenotypes in mouse models. PAPERCLIP.


Subject(s)
Diabetes Mellitus, Type 1/diet therapy , Diabetes Mellitus, Type 2/diet therapy , Fasting , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Diet , Glucose Tolerance Test , Humans , In Vitro Techniques , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Islets of Langerhans , Mice , Nerve Tissue Proteins/genetics , Pancreas/cytology , Pancreas/metabolism , Signal Transduction , Transcriptome
15.
Cell ; 171(4): 836-848.e13, 2017 Nov 02.
Article in English | MEDLINE | ID: mdl-28988768

ABSTRACT

Adrenergic stimulation promotes lipid mobilization and oxidation in brown and beige adipocytes, where the harnessed energy is dissipated as heat in a process known as adaptive thermogenesis. The signaling cascades and energy-dissipating pathways that facilitate thermogenesis have been extensively described, yet little is known about the counterbalancing negative regulatory mechanisms. Here, we identify a two-pore-domain potassium channel, KCNK3, as a built-in rheostat negatively regulating thermogenesis. Kcnk3 is transcriptionally wired into the thermogenic program by PRDM16, a master regulator of thermogenesis. KCNK3 antagonizes norepinephrine-induced membrane depolarization by promoting potassium efflux in brown adipocytes. This limits calcium influx through voltage-dependent calcium channels and dampens adrenergic signaling, thereby attenuating lipolysis and thermogenic respiration. Adipose-specific Kcnk3 knockout mice display increased energy expenditure and are resistant to hypothermia and obesity. These findings uncover a critical K+-Ca2+-adrenergic signaling axis that acts to dampen thermogenesis, maintain tissue homeostasis, and reveal an electrophysiological regulatory mechanism of adipocyte function.


Subject(s)
Adipose Tissue/metabolism , Nerve Tissue Proteins/metabolism , Obesity/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Receptors, Adrenergic/metabolism , Signal Transduction , Thermogenesis , Adipocytes, Brown/metabolism , Adipose Tissue/pathology , Animals , Cell Separation , Cells, Cultured , Electrophysiological Phenomena , Female , Male , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Obesity/pathology , Potassium Channels, Tandem Pore Domain/genetics
16.
Mol Cell ; 84(20): 3967-3978.e8, 2024 Oct 17.
Article in English | MEDLINE | ID: mdl-39317199

ABSTRACT

While many mRNAs contain more than one translation initiation site (TIS), the functions of most alternative TISs and their corresponding protein isoforms (proteoforms) remain undetermined. Here, we showed that alternative usage of CUG and AUG TISs in neuronal pentraxin receptor (NPR) mRNA produced two proteoforms, of which the ratio was regulated by RNA secondary structure and neuronal activity. Downstream AUG initiation truncated the N-terminal transmembrane domain and produced a secreted NPR proteoform sufficient in promoting synaptic clustering of AMPA-type glutamate receptors. Mutations that altered the ratio of NPR proteoforms reduced AMPA receptors in parvalbumin-positive interneurons and affected learning behaviors in mice. In addition to NPR, upstream AUU-initiated N-terminal extension of C1q-like synaptic organizers anchored these otherwise secreted factors to the membrane. Together, these results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a potentially widespread mechanism in diversifying protein localization and functions.


Subject(s)
Nerve Tissue Proteins , Receptors, AMPA , Synapses , Animals , Mice , Receptors, AMPA/metabolism , Receptors, AMPA/genetics , Synapses/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Humans , Peptide Chain Initiation, Translational , Protein Isoforms/metabolism , Protein Isoforms/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Interneurons/metabolism , HEK293 Cells , Codon, Initiator/genetics , Mice, Inbred C57BL , Male , Neuronal Plasticity/genetics , Mutation , Neurons/metabolism , Parvalbumins/metabolism , Parvalbumins/genetics , C-Reactive Protein , Calcium-Binding Proteins , Neural Cell Adhesion Molecules
17.
Annu Rev Biochem ; 85: 485-514, 2016 Jun 02.
Article in English | MEDLINE | ID: mdl-27145839

ABSTRACT

Radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Of approximately 114,000 of these enzymes, 8 are known to be present in humans: MOCS1, molybdenum cofactor biosynthesis; LIAS, lipoic acid biosynthesis; CDK5RAP1, 2-methylthio-N(6)-isopentenyladenosine biosynthesis; CDKAL1, methylthio-N(6)-threonylcarbamoyladenosine biosynthesis; TYW1, wybutosine biosynthesis; ELP3, 5-methoxycarbonylmethyl uridine; and RSAD1 and viperin, both of unknown function. Aberrations in the genes encoding these proteins result in a variety of diseases. In this review, we summarize the biochemical characterization of these 8 radical S-adenosylmethionine enzymes and, in the context of human health, describe the deleterious effects that result from such genetic mutations.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Heart Defects, Congenital/genetics , Metal Metabolism, Inborn Errors/genetics , Mutation , Neurodegenerative Diseases/genetics , S-Adenosylmethionine/metabolism , Carbon-Carbon Lyases , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/pathology , Gene Expression , Heart Defects, Congenital/enzymology , Heart Defects, Congenital/pathology , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Metal Metabolism, Inborn Errors/enzymology , Metal Metabolism, Inborn Errors/pathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/pathology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Proteins/genetics , Proteins/metabolism , Thioctic Acid/metabolism , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolism
18.
Cell ; 166(1): 245-57, 2016 Jun 30.
Article in English | MEDLINE | ID: mdl-27264607

ABSTRACT

A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.


Subject(s)
Drosophila/physiology , Neurons/metabolism , Visual Pathways , Animals , Calcium/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Kinetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurites/metabolism
19.
Cell ; 165(7): 1734-1748, 2016 Jun 16.
Article in English | MEDLINE | ID: mdl-27238024

ABSTRACT

Odor perception in mammals is mediated by parallel sensory pathways that convey distinct information about the olfactory world. Multiple olfactory subsystems express characteristic seven-transmembrane G-protein-coupled receptors (GPCRs) in a one-receptor-per-neuron pattern that facilitates odor discrimination. Sensory neurons of the "necklace" subsystem are nestled within the recesses of the olfactory epithelium and detect diverse odorants; however, they do not express known GPCR odor receptors. Here, we report that members of the four-pass transmembrane MS4A protein family are chemosensors expressed within necklace sensory neurons. These receptors localize to sensory endings and confer responses to ethologically relevant ligands, including pheromones and fatty acids, in vitro and in vivo. Individual necklace neurons co-express many MS4A proteins and are activated by multiple MS4A ligands; this pooling of information suggests that the necklace is organized more like subsystems for taste than for smell. The MS4As therefore define a distinct mechanism and functional logic for mammalian olfaction.


Subject(s)
Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Smell , Animals , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Odorants , Olfactory Receptor Neurons/metabolism , Phylogeny
20.
Immunity ; 54(8): 1715-1727.e7, 2021 08 10.
Article in English | MEDLINE | ID: mdl-34283971

ABSTRACT

Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.


Subject(s)
Asthma/immunology , Cell Communication/immunology , Connexins/metabolism , Nerve Tissue Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , T-Lymphocytes, Regulatory/immunology , Animals , Cell Line , Cell Proliferation/physiology , Connexins/genetics , Disease Models, Animal , HEK293 Cells , Humans , Jurkat Cells , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Respiratory System/immunology
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