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1.
Int J Mol Sci ; 23(23)2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36499143

RESUMO

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models.


Assuntos
Aprendizado Profundo , Encefalomielite Autoimune Experimental , Esclerose Múltipla , Doenças Neurodegenerativas , Neurite Óptica , Camundongos , Animais , Camundongos Endogâmicos C57BL , Neurite Óptica/patologia , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/patologia , Degeneração Neural , Inflamação , Modelos Animais de Doenças
2.
WIREs Mech Dis ; 15(2): e1594, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36600404

RESUMO

Central nervous system (CNS) inflammation is a key factor in multiple sclerosis (MS). Invasion of peripheral immune cells into the CNS resulting from an unknown signal or combination of signals results in activation of resident immune cells and the hallmark feature of the disease: demyelinating lesions. These lesion sites are an amalgam of reactive peripheral and central immune cells, astrocytes, damaged and dying oligodendrocytes, and injured neurons and axons. Sustained inflammation affects cells directly located within the lesion site and further abnormalities are apparent diffusely throughout normal-appearing white matter and grey matter. It is only relatively recently, using animal models, new tissue sampling techniques, and next-generation sequencing, that molecular changes occurring in CNS resident cells have been broadly captured. Advances in cell isolation through Fluorescence Activated Cell Sorting (FACS) and laser-capture microdissection together with the emergence of single-cell sequencing have enabled researchers to investigate changes in gene expression in astrocytes, microglia, and oligodendrocytes derived from animal models of MS as well as from primary patient tissue. The contribution of some dysregulated pathways has been followed up in individual studies; however, corroborating results often go unreported between sequencing studies. To this end, we have consolidated results from numerous RNA-sequencing studies to identify and review novel patterns of differentially regulated genes and pathways occurring within CNS glial cells in MS. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology.


Assuntos
Esclerose Múltipla , Substância Branca , Animais , Esclerose Múltipla/genética , Microglia/metabolismo , Astrócitos/metabolismo , Substância Branca/metabolismo , Inflamação/genética , RNA/metabolismo , Oligodendroglia/metabolismo
3.
Cell Chem Biol ; 27(6): 657-667.e6, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32220335

RESUMO

Targeting protein-protein interactions (PPIs) is a promising approach in the development of drugs for many indications. 14-3-3 proteins are a family of phosphoprotein-binding molecules with critical functions in dozens of cell signaling networks. 14-3-3s are abundant in the central nervous system, and the small molecule fusicoccin-A (FC-A), a tool compound that can be used to manipulate 14-3-3 PPIs, enhances neurite outgrowth in cultured neurons. New semisynthetic FC-A derivatives with improved binding affinity for 14-3-3 complexes have recently been developed. Here, we use a series of screens that identify these compounds as potent inducers of neurite outgrowth through a polypharmacological mechanism. Using proteomics and X-ray crystallography, we discover that these compounds extensively regulate the 14-3-3 interactome by stabilizing specific PPIs, while disrupting others. These results provide new insights into the development of drugs to target 14-3-3 PPIs, a potential therapeutic strategy for CNS diseases.


Assuntos
Proteínas 14-3-3/antagonistas & inibidores , Glicosídeos/farmacologia , Neuritos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas 14-3-3/isolamento & purificação , Proteínas 14-3-3/metabolismo , Animais , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Feminino , Glicosídeos/química , Masculino , Modelos Moleculares , Conformação Molecular , Neuritos/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/química
4.
eNeuro ; 7(2)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32001550

RESUMO

In contrast to neurons in the CNS, damaged neurons from the peripheral nervous system (PNS) regenerate, but this process can be slow and imperfect. Successful regeneration is orchestrated by cytoskeletal reorganization at the tip of the proximal axon segment and cytoskeletal disassembly of the distal segment. Collapsin response mediator protein 4 (CRMP4) is a cytosolic phospho-protein that regulates the actin and microtubule cytoskeleton. During development, CRMP4 promotes growth cone formation and dendrite development. Paradoxically, in the adult CNS, CRMP4 impedes axon regeneration. Here, we investigated the involvement of CRMP4 in peripheral nerve injury in male and female Crmp4-/- mice following sciatic nerve injury. We find that sensory axon regeneration and Wallerian degeneration are impaired in Crmp4-/- mice following sciatic nerve injury. In vitro analysis of dissociated dorsal root ganglion (DRG) neurons from Crmp4-/- mice revealed that CRMP4 functions in the proximal axon segment to promote the regrowth of severed DRG neurons and in the distal axon segment where it facilitates Wallerian degeneration through calpain-dependent formation of harmful CRMP4 fragments. These findings reveal an interesting dual role for CRMP4 in proximal and distal axon segments of injured sensory neurons that coordinately facilitate PNS axon regeneration.


Assuntos
Traumatismos dos Nervos Periféricos , Degeneração Walleriana , Animais , Axônios , Feminino , Gânglios Espinais , Masculino , Camundongos , Proteínas Musculares , Regeneração Nervosa , Nervo Isquiático , Semaforina-3A
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