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1.
Nat Cell Biol ; 26(9): 1482-1495, 2024 Sep.
Article in English | MEDLINE | ID: mdl-39117796

ABSTRACT

As lifelong interphase cells, neurons face an array of unique challenges. A key challenge is regulating nuclear pore complex (NPC) biogenesis and localization, the mechanisms of which are largely unknown. Here we identify neuronal maturation as a period of strongly upregulated NPC biogenesis. We demonstrate that the AAA+ protein torsinA, whose dysfunction causes the neurodevelopmental movement disorder DYT-TOR1A dystonia and co-ordinates NPC spatial organization without impacting total NPC density. We generated an endogenous Nup107-HaloTag mouse line to directly visualize NPC organization in developing neurons and find that torsinA is essential for proper NPC localization. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized nascent NPCs, and the formation of complete NPCs is delayed. Our work demonstrates that NPC spatial organization and number are independently determined and identifies NPC biogenesis as a process vulnerable to neurodevelopmental disease insults.


Subject(s)
Molecular Chaperones , Neurons , Nuclear Pore Complex Proteins , Nuclear Pore , Animals , Nuclear Pore/metabolism , Nuclear Pore/genetics , Neurons/metabolism , Molecular Chaperones/metabolism , Molecular Chaperones/genetics , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore Complex Proteins/genetics , Mice , Neurogenesis , Humans , Mice, Knockout , Mice, Inbred C57BL
2.
Nature ; 2024 Aug 28.
Article in English | MEDLINE | ID: mdl-39198643

ABSTRACT

Life-threatening thrombotic events and neurological symptoms are prevalent in COVID-19 and are persistent in patients with long COVID experiencing post-acute sequelae of SARS-CoV-2 infection1-4. Despite the clinical evidence1,5-7, the underlying mechanisms of coagulopathy in COVID-19 and its consequences in inflammation and neuropathology remain poorly understood and treatment options are insufficient. Fibrinogen, the central structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, correlates with disease severity and is a predictive biomarker for post-COVID-19 cognitive deficits1,5,8-10. Here we show that fibrin binds to the SARS-CoV-2 spike protein, forming proinflammatory blood clots that drive systemic thromboinflammation and neuropathology in COVID-19. Fibrin, acting through its inflammatory domain, is required for oxidative stress and macrophage activation in the lungs, whereas it suppresses natural killer cells, after SARS-CoV-2 infection. Fibrin promotes neuroinflammation and neuronal loss after infection, as well as innate immune activation in the brain and lungs independently of active infection. A monoclonal antibody targeting the inflammatory fibrin domain provides protection from microglial activation and neuronal injury, as well as from thromboinflammation in the lung after infection. Thus, fibrin drives inflammation and neuropathology in SARS-CoV-2 infection, and fibrin-targeting immunotherapy may represent a therapeutic intervention for patients with acute COVID-19 and long COVID.

3.
bioRxiv ; 2024 Aug 10.
Article in English | MEDLINE | ID: mdl-39149258

ABSTRACT

The first steps in vision take place in photoreceptor cells, which are highly compartmentalized neurons exhibiting significant structural variation across species. The light-sensitive ciliary compartment, called the outer segment, is located atop of the cell soma, called the inner segment. In this study, we present an ultrastructural analysis of human photoreceptors, which reveals that, in contrast to this classic arrangement, the inner segment of human rods extends alongside the outer segment to form a structure hereby termed the "accessory inner segment". While reminiscent of the actin-based microvilli known as "calyceal processes" observed in other species, the accessory inner segment is a unique structure: (1) it contains an extensive microtubule-based cytoskeleton, (2) it extends far alongside the outer segment, (3) its diameter is comparable to that of the outer segment, (4) it contains numerous mitochondria, and (5) it forms electron-dense structures that likely mediate adhesion to the outer segment. Given that the spacing of extrafoveal human photoreceptors is more sparse than in non-primate species, with vast amounts of interphotoreceptor matrix present between cells, the closely apposed accessory inner segment likely provides structural support to the outer segment. This discovery expands our understanding of the human retina and directs future studies of human photoreceptor function in health and disease.

4.
Curr Biol ; 34(17): 4056-4061.e2, 2024 Sep 09.
Article in English | MEDLINE | ID: mdl-39127047

ABSTRACT

In animals, overt circadian rhythms of physiology and behavior are centrally regulated by a circadian clock located in specific brain regions. In the fruit fly Drosophila and in mammals, these clocks rely on single-cell oscillators, but critical for their function as central circadian pacemakers are network properties that change dynamically throughout the circadian cycle as well as in response to environmental stimuli.1,2,3 In the fly, this plasticity involves circadian rhythms of expansion and retraction of clock neuron fibers.4,5,6,7,8,9,10,11,12,13,14 Whether these drastic structural changes are a universal property of central neuronal pacemakers is unknown. To address this question, we studied neurons of the mouse suprachiasmatic nucleus (SCN) that express vasoactive intestinal polypeptide (VIP), which are critical for the SCN to function as a central circadian pacemaker. By targeting the expression of the fluorescent protein tdTomato to these neurons and using tissue clearing techniques to visualize all SCN VIPergic neurons and their fibers, we show that, similar to clock neurons in the fly, VIPergic fibers undergo a daily rhythm of expansion and retraction, with maximal branching during the day. This rhythm is circadian, as it persists under constant environmental conditions and is present in both males and females. We propose that circadian structural remodeling of clock neurons represents a key feature of central circadian pacemakers that is likely critical to regulate network properties, the response to environmental stimuli, and the regulation of circadian outputs.


Subject(s)
Circadian Rhythm , Suprachiasmatic Nucleus , Vasoactive Intestinal Peptide , Animals , Vasoactive Intestinal Peptide/metabolism , Mice , Suprachiasmatic Nucleus/physiology , Suprachiasmatic Nucleus/metabolism , Circadian Rhythm/physiology , Male , Female , Neurons/physiology , Neurons/metabolism , Mice, Inbred C57BL
5.
J Mol Biol ; 436(10): 168559, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38580077

ABSTRACT

Upstream open reading frames (uORFs) are cis-acting elements that can dynamically regulate the translation of downstream ORFs by suppressing downstream translation under basal conditions and, in some cases, increasing downstream translation under stress conditions. Computational and empirical methods have identified uORFs in the 5'-UTRs of approximately half of all mouse and human transcripts, making uORFs one of the largest regulatory elements known. Because the prevailing dogma was that eukaryotic mRNAs produce a single functional protein, the peptides and small proteins, or microproteins, encoded by uORFs were rarely studied. We hypothesized that a uORF in the SLC35A4 mRNA is producing a functional microprotein (SLC35A4-MP) because of its conserved amino acid sequence. Through a series of biochemical and cellular experiments, we find that the 103-amino acid SLC35A4-MP is a single-pass transmembrane inner mitochondrial membrane (IMM) microprotein. The IMM contains the protein machinery crucial for cellular respiration and ATP generation, and loss of function studies with SLC35A4-MP significantly diminish maximal cellular respiration, indicating a vital role for this microprotein in cellular metabolism. The findings add SLC35A4-MP to the growing list of functional microproteins and, more generally, indicate that uORFs that encode conserved microproteins are an untapped reservoir of functional microproteins.


Subject(s)
Mitochondrial Membranes , Mitochondrial Proteins , Nucleotide Transport Proteins , Open Reading Frames , Humans , 5' Untranslated Regions/genetics , Amino Acid Sequence , Mitochondria/metabolism , Mitochondria/genetics , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Open Reading Frames/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Nucleotide Transport Proteins/genetics , Nucleotide Transport Proteins/metabolism , HEK293 Cells
6.
bioRxiv ; 2024 Mar 27.
Article in English | MEDLINE | ID: mdl-38586011

ABSTRACT

Microglia-driven neuroinflammation plays an important role in the development of Alzheimer's disease (AD). Microglia activation is accompanied by the formation and chronic maintenance of TLR4 inflammarafts, defined as enlarged and cholesterol-rich lipid rafts serving as an assembly platform for TLR4 dimers and complexes of other inflammatory receptors. The secreted apoA-I binding protein (APOA1BP or AIBP) binds TLR4 and selectively targets cholesterol depletion machinery to TLR4 inflammaraft expressing inflammatory, but not homeostatic microglia. Here we demonstrated that amyloid-beta (Aß) induced formation of TLR4 inflammarafts in microglia in vitro and in the brain of APP/PS1 mice. Mitochondria in Apoa1bp-/- APP/PS1 microglia were hyperbranched and cupped, which was accompanied by increased ROS and the dilated ER. The size and number of Aß plaques and neuronal cell death were significantly increased, and the animal survival was decreased in Apoa1bp-/- APP/PS1 compared to APP/PS1 female mice. These results suggest that AIBP exerts control of TLR4 inflammarafts and mitochondrial dynamics in microglia and plays a protective role in AD associated oxidative stress and neurodegeneration.

7.
Mol Psychiatry ; 29(7): 1951-1967, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38355784

ABSTRACT

Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.


Subject(s)
Neurons , Rabies virus , Animals , Mice , Neurons/virology , Neurons/metabolism , Brain/virology , Connectome/methods , Brain Mapping/methods , Alzheimer Disease/virology , Alzheimer Disease/pathology , Disease Models, Animal , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Genetic Vectors , Mice, Inbred C57BL , Microscopy, Fluorescence/methods , Rabies/virology , Humans , Nerve Net/virology , Nerve Net/metabolism
8.
bioRxiv ; 2024 Jan 21.
Article in English | MEDLINE | ID: mdl-38293120

ABSTRACT

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

9.
J Comp Neurol ; 532(2): e25552, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37916792

ABSTRACT

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination.


Subject(s)
Astrocytes , Myelin Sheath , Animals , Mice , Axons/ultrastructure , Oligodendroglia/physiology , Brain Stem/physiology
10.
Sci Rep ; 13(1): 21462, 2023 12 05.
Article in English | MEDLINE | ID: mdl-38052818

ABSTRACT

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.


Subject(s)
Nucleic Acids , RNA/metabolism , Microscopy, Electron , DNA , Cell Nucleolus/metabolism
11.
bioRxiv ; 2023 Nov 30.
Article in English | MEDLINE | ID: mdl-38076839

ABSTRACT

Neuronal extracellular matrix (ECM) and a specific form of ECM called the perineuronal net (PNN) are important structures for central nervous system (CNS) integrity and synaptic plasticity. PNNs are distinctive, dense extracellular structures that surround parvalbumin (PV)-positive inhibitory interneurons with openings at mature synapses. Enzyme-mediated PNN disruption can erase established memories and re-open critical periods in animals, suggesting that PNNs are important for memory stabilization and conservation. Here, we characterized the structure and distribution of several ECM/PNN molecules around neurons in culture, brain slice, and whole mouse brain. While specific lectins are well-established as PNN markers and label a distinct, fenestrated structure around PV neurons, we show that other CNS neurons possess similar extracellular structures assembled around hyaluronic acid, suggesting a PNN-like structure of different composition that is more widespread. We additionally report that genetically encoded labeling of hyaluronan and proteoglycan link protein 1 (HAPLN1) reveals a PNN-like structure around many neurons in vitro and in vivo. Our findings add to our understanding of neuronal extracellular structures and describe a new mouse model for monitoring live ECM dynamics.

12.
EMBO J ; 42(24): e114054, 2023 Dec 11.
Article in English | MEDLINE | ID: mdl-37933600

ABSTRACT

Cristae are high-curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous lipid-based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low-oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.


Subject(s)
Cardiolipins , Lipidomics , Cardiolipins/metabolism , Mitochondrial Membranes/metabolism , Phospholipids/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphate/metabolism
13.
bioRxiv ; 2023 Sep 25.
Article in English | MEDLINE | ID: mdl-37808832

ABSTRACT

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.

14.
bioRxiv ; 2023 Oct 04.
Article in English | MEDLINE | ID: mdl-37662194

ABSTRACT

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

15.
PLoS Comput Biol ; 19(9): e1011464, 2023 09.
Article in English | MEDLINE | ID: mdl-37729344

ABSTRACT

Astrocytes with their specialised morphology are essential for brain homeostasis as metabolic mediators between blood vessels and neurons. In neurodegenerative diseases such as Alzheimer's disease (AD), astrocytes adopt reactive profiles with molecular and morphological changes that could lead to the impairment of their metabolic support and impact disease progression. However, the underlying mechanisms of how the metabolic function of human astrocytes is impaired by their morphological changes in AD are still elusive. To address this challenge, we developed and applied a metabolic multiscale modelling approach integrating the dynamics of metabolic energy pathways and physiological astrocyte morphologies acquired in human AD and age-matched control brain samples. The results demonstrate that the complex cell shape and intracellular organisation of energetic pathways determine the metabolic profile and support capacity of astrocytes in health and AD conditions. Thus, our mechanistic approach indicates the importance of spatial orchestration in metabolism and allows for the identification of protective mechanisms against disease-associated metabolic impairments.


Subject(s)
Alzheimer Disease , Humans , Alzheimer Disease/metabolism , Astrocytes/metabolism , Brain/metabolism , Energy Metabolism
16.
J Physiol ; 2023 Sep 05.
Article in English | MEDLINE | ID: mdl-37668020

ABSTRACT

Deleterious Ca2+ accumulation is central to hypoxic cell death in the brain of most mammals. Conversely, hypoxia-mediated increases in cytosolic Ca2+ are retarded in hypoxia-tolerant naked mole-rat brain. We hypothesized that naked mole-rat brain mitochondria have an enhanced capacity to buffer exogenous Ca2+ and examined Ca2+ handling in naked mole-rat cortical tissue. We report that naked mole-rat brain mitochondria buffer >2-fold more exogenous Ca2+ than mouse brain mitochondria, and that the half-maximal inhibitory concentration (IC50 ) at which Ca2+ inhibits aerobic oxidative phosphorylation is >2-fold higher in naked mole-rat brain. The primary driving force of Ca2+ uptake is the mitochondrial membrane potential (Δψm ), and the IC50 at which Ca2+ decreases Δψm is ∼4-fold higher in naked mole-rat than mouse brain. The ability of naked mole-rat brain mitochondria to safely retain large volumes of Ca2+ may be due to ultrastructural differences that support the uptake and physical storage of Ca2+ in mitochondria. Specifically, and relative to mouse brain, naked mole-rat brain mitochondria are larger and have higher crista density and increased physical interactions between adjacent mitochondrial membranes, all of which are associated with improved energetic homeostasis and Ca2+ management. We propose that excessive Ca2+ influx into naked mole-rat brain is buffered by physical storage in large mitochondria, which would reduce deleterious Ca2+ overload and may thus contribute to the hypoxia and ischaemia-tolerance of naked mole-rat brain. KEY POINTS: Unregulated Ca2+ influx is a hallmark of hypoxic brain death; however, hypoxia-mediated Ca2+ influx into naked mole-rat brain is markedly reduced relative to mice. This is important because naked mole-rat brain is robustly tolerant against in vitro hypoxia, and because Ca2+ is a key driver of hypoxic cell death in brain. We show that in hypoxic naked mole-rat brain, oxidative capacity and mitochondrial membrane integrity are better preserved following exogenous Ca2+ stress. This is due to mitochondrial buffering of exogenous Ca2+ and is driven by a mitochondrial membrane potential-dependant mechanism. The unique ultrastructure of naked mole-rat brain mitochondria, as a large physical storage space, may support increased Ca2+ buffering and thus hypoxia-tolerance.

17.
Elife ; 122023 07 14.
Article in English | MEDLINE | ID: mdl-37449984

ABSTRACT

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or 'discs', located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called 'incisures'. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.


Subject(s)
Rhodopsin , Rod Cell Outer Segment , Animals , Mice , Rhodopsin/metabolism , Peripherins/metabolism , Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Vision, Ocular
18.
eNeuro ; 10(8)2023 08.
Article in English | MEDLINE | ID: mdl-37500494

ABSTRACT

The hypothalamic suprachiasmatic nucleus (SCN) is the central circadian pacemaker in vertebrates. The SCN receives photic information exclusively through melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize circadian rhythms with the environmental light cycles. The SCN is composed of two major peptidergic neuron types in the core and shell regions of the SCN. Determining how mRGCs interact with the network of synaptic connections onto and between SCN neurons is key to understand how light regulates the circadian clock and to elucidate the relevant local circuits within the SCN. To map these connections, we used a newly developed Cre-dependent electron microscopy (EM) reporter, APEX2, to label the mitochondria of mRGC axons. Serial blockface scanning electron microscopy was then used to resolve the fine 3D structure of mRGC axons and synaptic boutons in the SCN of a male mouse. The resulting maps reveal patterns of connectomic organization in the core and shell of the SCN. We show that these regions are composed of different neuronal subtypes and differ with regard to the pattern of mRGC input, as the shell receives denser mRGC synaptic input compared with the core. This finding challenges the present view that photic information coming directly from the retina is received primarily by the core region of the SCN.


Subject(s)
Circadian Clocks , Suprachiasmatic Nucleus , Male , Mice , Animals , Circadian Rhythm/physiology , Retinal Ganglion Cells/physiology , Microscopy, Electron
19.
J Neurosci ; 43(30): 5468-5482, 2023 07 26.
Article in English | MEDLINE | ID: mdl-37414561

ABSTRACT

The rod photoreceptor synapse is the first synapse of dim-light vision and one of the most complex in the mammalian CNS. The components of its unique structure, a presynaptic ribbon and a single synaptic invagination enclosing several postsynaptic processes, have been identified, but disagreements about their organization remain. Here, we have used EM tomography to generate high-resolution images of 3-D volumes of the rod synapse from the female domestic cat. We have resolved the synaptic ribbon as a single structure, with a single arciform density, indicating the presence of one long site of transmitter release. The organization of the postsynaptic processes, which has been difficult to resolve with past methods, appears as a tetrad arrangement of two horizontal cell and two rod bipolar cell processes. Retinal detachment severely disrupts this organization. After 7 d, EM tomography reveals withdrawal of rod bipolar dendrites from most spherules; fragmentation of synaptic ribbons, which lose their tight association with the presynaptic membrane; and loss of the highly branched telodendria of the horizontal cell axon terminals. After detachment, the hilus, the opening through which postsynaptic processes enter the invagination, enlarges, exposing the normally sequestered environment within the invagination to the extracellular space of the outer plexiform layer. Our use of EM tomography provides the most accurate description to date of the complex rod synapse and details changes it undergoes during outer segment degeneration. These changes would be expected to disrupt the flow of information in the rod pathway.SIGNIFICANCE STATEMENT Ribbon-type synapses transmit the first electrical signals of vision and hearing. Despite their crucial role in sensory physiology, the three-dimensional ultrastructure of these synapses, especially the complex organization of the rod photoreceptor synapse, is not well understood. We used EM tomography to obtain 3-D imaging at nanoscale resolution to help resolve the organization of rod synapses in normal and detached retinas. This approach has enabled us to show that in the normal retina a single ribbon and arciform density oppose a tetrad of postsynaptic processes. In addition, it enabled us to provide a 3-D perspective of the ultrastructural changes that occur in response to retinal detachment.


Subject(s)
Retinal Detachment , Female , Animals , Cats , Microscopy, Electron , Synapses/metabolism , Retina/ultrastructure , Retinal Bipolar Cells , Retinal Rod Photoreceptor Cells/ultrastructure , Mammals
20.
Nat Commun ; 14(1): 4159, 2023 07 13.
Article in English | MEDLINE | ID: mdl-37443171

ABSTRACT

Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication, and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis.


Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Humans , Ebolavirus/genetics , Viral Replication Compartments , Transcription, Genetic , Virus Replication , Nucleotidyltransferases/genetics
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