Heterogeneous brain region-specific responses to astrocytic mitochondrial DNA damage in mice.

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.

Validation of terminal Schwann cell gene marker expression by fluorescent in situ hybridization using RNAscope.

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.

Nerve sprouting capacity in a pharmacologically induced mouse model of spinal muscular atrophy.

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.

Defective Acetylcholine Receptor Subunit Switch Precedes Atrophy of Slow-Twitch Skeletal Muscle Fibers Lacking ERK1/2 Kinases in Soleus Muscle.

To test the role of extracellular-signal regulated kinases 1 and 2 (ERK1/2) in slow-twitch, type 1 skeletal muscle fibers, we studied the soleus muscle in mice genetically deficient for myofiber ERK1/2. Young adult mutant soleus was drastically wasted, with highly atrophied type 1 fibers, denervation at most synaptic sites, induction of "fetal" acetylcholine receptor gamma subunit (AChRγ), reduction of "adult" AChRε, and impaired mitochondrial biogenesis and function. In weanlings, fiber morphology and mitochondrial markers were mostly normal, yet AChRγ upregulation and AChRε downregulation were observed. Synaptic sites with fetal AChRs in weanling muscle were ~3% in control and ~40% in mutants, with most of the latter on type 1 fibers. These results suggest that: (1) ERK1/2 are critical for slow-twitch fiber growth; (2) a defective γ/ε-AChR subunit switch, preferentially at synapses on slow fibers, precedes wasting of mutant soleus; (3) denervation is likely to drive this wasting, and (4) the neuromuscular synapse is a primary subcellular target for muscle ERK1/2 function in vivo.

Muscle-derived extracellular signal-regulated kinases 1 and 2 are required for the maintenance of adult myofibers and their neuromuscular junctions.

The Ras-extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice.

Improvement of neuromuscular synaptic phenotypes without enhanced survival and motor function in severe spinal muscular atrophy mice selectively rescued in motor neurons.

In the inherited childhood neuromuscular disease spinal muscular atrophy (SMA), lower motor neuron death and severe muscle weakness result from the reduction of the ubiquitously expressed protein survival of motor neuron (SMN). Although SMA mice recapitulate many features of the human disease, it has remained unclear if their short lifespan and motor weakness are primarily due to cell-autonomous defects in motor neurons. Using Hb9(Cre) as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChAT(Cre+) ) as a driver on the same mice, and another report that used Hb9(Cre) as a driver on a different line of conditional SMA mice, we found no improvement in survival, weight, motor behavior and presynaptic neurofilament accumulation. However, like in ChAT(Cre+) mice, we detected rescue of endplate size and mitigation of neuromuscular junction (NMJ) denervation status. The rescue of endplate size occurred in the absence of an increase in myofiber size, suggesting endplate size is determined by the motor neuron in these animals. Real time-PCR showed that the expression of spinal cord SMN transcript was sharply reduced in Hb9(Cre+) SMA mice relative to ChAT(Cre+) SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9(Cre) . Nonetheless, the low levels of SMN were sufficient to rescue two NMJ structural parameters indicating that these motor neuron cell autonomous phenotypes are very sensitive to changes in motoneuronal SMN levels. Our results directly suggest that even those therapeutic interventions with very modest effects in raising SMN in motor neurons may provide mitigation of neuromuscular phenotypes in SMA patients.

Host impact on the stability of a plant virus gene vector as measured by a new fluorescent local lesion passaging assay.

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.