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1.
bioRxiv ; 2024 May 30.
Article in English | MEDLINE | ID: mdl-38853966

ABSTRACT

Astrocytes use Ca 2+ signals to regulate multiple aspects of normal and pathological brain function. Astrocytes display context-specific diversity in their functions, and in their response to noxious stimuli between brain regions. Indeed, astrocytic mitochondria have emerged as key players in governing astrocytic functional heterogeneity, given their ability to dynamically adapt their morphology to regional demands on their ATP generation and Ca 2+ buffering functions. Although there is reciprocal regulation between mitochondrial dynamics and mitochondrial Ca 2+ signaling in astrocytes, the extent of this regulation into the rich diversity of astrocytes in different brain regions remains largely unexplored. Brain-wide, experimentally induced mitochondrial DNA (mtDNA) loss in astrocytes showed that mtDNA integrity is critical for proper astrocyte function, however, few insights into possible diverse responses to this noxious stimulus from astrocytes in different brain areas were reported in these experiments. To selectively damage mtDNA in astrocytes in a brain-region-specific manner, we developed a novel adeno-associated virus (AAV)-based tool, Mito-PstI, which expresses the restriction enzyme PstI, specifically in astrocytic mitochondria. Here, we applied Mito-PstI to two distinct brain regions, the dorsolateral striatum, and the hippocampal dentate gyrus, and we show that Mito-PstI can induce astrocytic mtDNA loss in vivo , but with remarkable brain-region-dependent differences on mitochondrial dynamics, spontaneous Ca 2+ fluxes and astrocytic as well as microglial reactivity. Thus, AAV-Mito-PstI is a novel tool to explore the relationship between astrocytic mitochondrial network dynamics and astrocytic mitochondrial Ca 2+ signaling in a brain-region-selective manner.

2.
Neurosci Lett ; 771: 136468, 2022 02 06.
Article in English | MEDLINE | ID: mdl-35065247

ABSTRACT

Recent RNA-seq studies have generated a new crop of putative gene markers for terminal Schwann cells (tSCs), non-myelinating glia that cap axon terminals at the vertebrate neuromuscular junction (NMJ). While compelling, these studies did not validate the expression of the novel markers using in situ hybridization techniques. Here, we use RNAscope technology to study the expression of top candidates from recent tSC and non-myelinating Schwann cell marker RNA-seq studies. Our results validate the expression of these markers at tSCs but also demonstrate that they are present at other sites in the muscle tissue, specifically, at muscle spindles and along intramuscular nerves.


Subject(s)
Nerve Tissue Proteins/genetics , RNA-Seq/methods , Schwann Cells/metabolism , Animals , Female , In Situ Hybridization, Fluorescence/methods , In Situ Hybridization, Fluorescence/standards , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Neuromuscular Junction/metabolism , RNA-Seq/standards , Reference Standards
3.
Sci Rep ; 9(1): 7799, 2019 05 24.
Article in English | MEDLINE | ID: mdl-31127156

ABSTRACT

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the survival of motoneuron gene 1 (SMN1). SMA is characterized by motoneuron death, skeletal muscle denervation and atrophy. Disease severity inversely correlates with copy number of a second gene (SMN2), which harbors a splicing defect that causes the production of inadequate levels of functional SMN protein. Small molecules that modify SMN2 splicing towards increased production of functional SMN significantly ameliorate SMA phenotypes in mouse models of severe SMA. At suboptimal doses, splicing modifiers, such as SMN-C1, have served to generate mice that model milder SMA, referred to as pharmacological SMA mice, which survive into early adulthood. Nerve sprouting at endplates, known as terminal sprouting, is key to normal muscle fiber reinnervation following nerve injury and its promotion might mitigate neuromuscular symptoms in mild SMA. Sprouting has been difficult to study in severe SMA mice due to their short lifespan. Here, we show that pharmacological SMA mice are capable of terminal sprouting following reinnervation that is largely SMN-C1 dose-independent, but that they display a reinnervation delay that is critically SMN-C1 dose-dependent. Data also suggest that SMN-C1 can induce by itself a limited terminal sprouting response in SMA and wild-type normally-innervated endplates.


Subject(s)
Muscle, Skeletal/innervation , Muscular Atrophy, Spinal/physiopathology , Neuromuscular Junction/physiopathology , Animals , Disease Models, Animal , Humans , Mice , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Muscular Atrophy, Spinal/chemically induced , Muscular Atrophy, Spinal/pathology , Nerve Regeneration , Neuromuscular Junction/pathology , Schwann Cells/pathology
4.
J Virol Methods ; 179(2): 289-94, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22119627

ABSTRACT

Viruses can be used as vectors for transient expression of proteins in plants but frequently foreign gene inserts are not maintained stably over time due to recombination events. In this study the hypothesis was that the choice of plant host affects the foreign gene retention level by a Tomato bushy stunt virus (TBSV) vector expressing green fluorescent protein (GFP). To accomplish this, a novel virus vector integrity bioassay was developed based on an old concept, whereby RNA transcripts of the TBSV-GFP vector were rub-inoculated onto leaves of test plants, and at 3 days post inoculation (dpi), these leaves were used as inoculum for passage to cowpea (Vigna unguiculata), a local lesion host. Chlorotic lesions at points of virus infection were counted on cowpea at 4dpi and then the leaves were exposed to ultraviolet light to count green fluorescent foci. These tests with seven different plant species covering five families showed that the percentage of green fluorescent lesions varied on the cowpea indicator plants in a host-dependent manner. For instance, the vector was relatively unstable in Nicotiana benthamiana, tomato, bean, and spinach, but compared to those its stability in lettuce was significantly improved (~3-fold). This host-dependent effect suggests that some plants may present a more suitable environment than others to support or maintain optimum levels of virus vector-mediated foreign gene expression.


Subject(s)
Genetic Vectors , Genomic Instability , Plants/virology , Tombusvirus/genetics , Fluorescence , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Plant Leaves/virology
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