Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS.

Protein misfolding and mislocalization are common themes in neurodegenerative disorders, including motor neuron disease, and amyotrophic lateral sclerosis (ALS). Maintaining proteostasis is a crosscutting therapeutic target, including the upregulation of heat shock proteins (HSP) to increase chaperoning capacity. Motor neurons have a high threshold for upregulating stress-inducible HSPA1A, but constitutively express high levels of HSPA8. This study compared the expression of these HSPs in cultured motor neurons expressing three variants linked to familial ALS: TAR DNA binding protein 43 kDa (TDP-43)G348C, fused in sarcoma (FUS)R521G, or superoxide dismutase I (SOD1)G93A. All variants were poor inducers of Hspa1a, and reduced levels of Hspa8 mRNA and protein, indicating multiple compromises in chaperoning capacity. To promote HSP expression, cultures were treated with the putative HSP coinducer, arimoclomol, and class I histone deacetylase inhibitors, to promote active chromatin for transcription, and with the combination. Treatments had variable, often different effects on the expression of Hspa1a and Hspa8, depending on the ALS variant expressed, mRNA distribution (somata and dendrites), and biomarker of toxicity measured (histone acetylation, maintaining nuclear TDP-43 and the neuronal Brm/Brg-associated factor chromatin remodeling complex component Brg1, mitochondrial transport, FUS aggregation). Overall, histone deacetylase inhibition alone was effective on more measures than arimoclomol. As in the FUS model, arimoclomol failed to induce HSPA1A or preserve Hspa8 mRNA in the TDP-43 model, despite preserving nuclear TDP-43 and Brg1, indicating neuroprotective properties other than HSP induction. The data speak to the complexity of drug mechanisms against multiple biomarkers of ALS pathogenesis, as well as to the importance of HSPA8 for neuronal proteostasis in both somata and dendrites.

Improved Human Muscle Biopsy Method To Study Neuromuscular Junction Structure and Functions with Aging.

Reduced mobility and physical independence of elders has emerged as a major clinical and public health priority with extended life expectancy. The impact of the neuromuscular function on muscle activity and properties has emerged as a critical factor influencing the progress and outcome of muscle changes with aging. However, very little is known about the neuromuscular junctions (NMJs) in humans, in part due to technical constraints limiting the access to healthy, fresh neuromuscular tissue. Here, we describe a method, called Biopsy using Electrostimulation for Enhanced NMJ Sampling (BeeNMJs) that improves the outcome of muscle biopsies. We used local cutaneous stimulation to identify the area enriched with NMJs for each participant at the right Vastus lateralis (VL). The needle biopsy was then performed in proximity of that point. The BeeNMJs procedure was safe for the participants. We observed NMJs in 53.3% of biopsies in comparison with only 16.7% using the traditional method. Furthermore, we observed an average of 30.13 NMJs per sample compared to only 2.33 for the traditional method. Importantly, high-quality neuromuscular material was obtained whereby pre-, postsynaptic, and glial elements were routinely labeled, simultaneously with myosin heavy chain type I. The BeeNMJs approach will facilitate studies of NMJs, particularly in human disease or aging process.

Purinergic-Dependent Glial Regulation of Synaptic Plasticity of Competing Terminals and Synapse Elimination at the Neuromuscular Junction.

The precise wiring of synaptic connections requires the elimination of supernumerary inputs competing for innervation of the same target cell. This competition is activity-dependent, strengthening some inputs whereas others are eliminated. Although glial cells are required for the elimination and clearance of terminals, their involvement in activity-dependent synaptic competition remains ill-defined. Here, we used the developing neuromuscular junctions of mice to show that perisynaptic glial cells, through 2Y1 purinergic receptors (P2Y1Rs), decode synaptic efficacy of competing terminals in a Ca2+-dependent manner. This glial activity induces long-lasting synaptic potentiation of strong but not weak terminals via presynaptic adenosine 2A receptors. Blockade of glial activity by intracellular Ca2+ chelation or blockade of P2Y1Rs prevents this plasticity. In addition, blockade of P2Y1Rs delays synapse elimination in vivo. Hence, P2Y1Rs drive glial cell regulation of strong synaptic inputs and influence synapse competition and elimination.

Astrocytes detect and upregulate transmission at inhibitory synapses of somatostatin interneurons onto pyramidal cells.

Astrocytes are important regulators of excitatory synaptic networks. However, astrocytes regulation of inhibitory synaptic systems remains ill defined. This is particularly relevant since GABAergic interneurons regulate the activity of excitatory cells and shape network function. To address this issue, we combined optogenetics and pharmacological approaches, two-photon confocal imaging and whole-cell recordings to specifically activate hippocampal somatostatin or paravalbumin-expressing interneurons (SOM-INs or PV-INs), while monitoring inhibitory synaptic currents in pyramidal cells and Ca2+ responses in astrocytes. We found that astrocytes detect SOM-IN synaptic activity via GABABR and GAT-3-dependent Ca2+ signaling mechanisms, the latter triggering the release of ATP. In turn, ATP is converted into adenosine, activating A1Rs and upregulating SOM-IN synaptic inhibition of pyramidal cells, but not PV-IN inhibition. Our findings uncover functional interactions between a specific subpopulation of interneurons, astrocytes and pyramidal cells, involved in positive feedback autoregulation of dendritic inhibition of pyramidal cells.

Astrocytes are endogenous regulators of basal transmission at central synapses.

Basal synaptic transmission involves the release of neurotransmitters at individual synapses in response to a single action potential. Recent discoveries show that astrocytes modulate the activity of neuronal networks upon sustained and intense synaptic activity. However, their ability to regulate basal synaptic transmission remains ill defined and controversial. Here, we show that astrocytes in the hippocampal CA1 region detect synaptic activity induced by single-synaptic stimulation. Astrocyte activation occurs at functional compartments found along astrocytic processes and involves metabotropic glutamate subtype 5 receptors. In response, astrocytes increase basal synaptic transmission, as revealed by the blockade of their activity with a Ca(2+) chelator. Astrocytic modulation of basal synaptic transmission is mediated by the release of purines and the activation of presynaptic A(2A) receptors by adenosine. Our work uncovers an essential role for astrocytes in the regulation of elementary synaptic communication and provides insight into fundamental aspects of brain function.

Perisynaptic glia discriminate patterns of motor nerve activity and influence plasticity at the neuromuscular junction.

In the nervous system, the induction of plasticity is coded by patterns of synaptic activity. Glial cells are now recognized as dynamic partners in a wide variety of brain functions, including the induction and modulation of various forms of synaptic plasticity. However, it appears that glial cells are usually activated by stereotyped, sustained neuronal activity, and little attention has been given to more subtle changes in the patterns of synaptic activation. To this end, we used the mouse neuromuscular junction as a simple and useful model to study glial modulation of synaptic plasticity. We used two patterns of motor nerve stimulation that mimic endogenous motor-neuronal activity. A continuous stimulation induced a post-tetanic potentiation and a phasic Ca(2+) response in perisynaptic Schwann cells (PSCs), glial cells at this synapse. A bursting pattern of activity induced a post-tetanic depression and oscillatory Ca(2+) responses in PSCs. The different Ca(2+) responses in PSCs indicate that they decode the pattern of synaptic activity. Furthermore, the chelation of glial Ca(2+) impaired the production of the sustained plasticity events indicating that PSCs govern the outcome of synaptic plasticity. The mechanisms involved were studied using direct photo-activation of PSCs with caged Ca(2+) that mimicked endogenous plasticity. Using specific pharmacology and transgenic knock-out animals for adenosine receptors, we showed that the sustained depression was mediated by A1 receptors while the sustained potentiation is mediated by A(2A) receptors. These results demonstrate that glial cells decode the pattern of synaptic activity and subsequently provide bidirectional feedback to synapses.

In vivo long-term synaptic plasticity of glial cells.

Evidence showing the ability of glial cells to detect, respond to and modulate synaptic transmission and plasticity has contributed to the notion of glial cells as active synaptic partners. However, synaptically induced plasticity of glia themselves remains ill defined. Here we used the amphibian neuromuscular junction (NMJ) to study plasticity of perisynaptic Schwann cells (PSCs), glial cells at this synapse, following long-term in vivo modifications of synaptic activity. We used two models that altered synaptic activity in different manners. First, chronic blockade of postsynaptic nicotinic receptors using alpha-bungarotoxin (alpha-BTx) decreased facilitation, increased synaptic depression and decreased post-tetanic potentiation (PTP). Second, chronic nerve stimulation increased facilitation and resistance to synaptic depression, while leaving PTP unaltered. Our results indicate that there is no direct relationship between transmitter release and PSC calcium responses. Indeed, despite changes in transmitter release and plasticity in stimulated NMJs, nerve-evoked PSC calcium responses were similar to control. Similarly, PSC calcium responses in alpha-BTx treated NMJs were delayed and smaller in amplitude, even though basal level of transmitter release was increased. Also, when isolating purinergic and muscarinic components of PSC calcium responses, we found an increased sensitivity to ATP and a decreased sensitivity to muscarine in chronically stimulated NMJs. Conversely, in alpha-BTx treated NMJs, PSC sensitivity remained unaffected, but ATP- and muscarine-induced calcium responses were prolonged. Thus, our results reveal complex modifications of PSC properties, with differential modulation of signalling pathways that might underlie receptor regulation or changes in Ca(2+) handling. Importantly, similar to neurons, perisynaptic glial cells undergo plastic changes induced by altered synaptic activity.

Neurotrophins modulate neuron-glia interactions at a vertebrate synapse.

Neurotrophins are important modulators of synaptic function at both developing and mature synapses in the CNS and PNS. At the neuromuscular junction (NMJ), neurotrophins, as well as perisynaptic Schwann cells (PSCs) are critical for the long-term maintenance and stability of the synapse. Considering this correlation and the acute interactions that occur at the synapse between PSCs and the nerve terminal, we wondered if neurotrophins could also be involved in neuron-glia signalling. To test if neurotrophins were able to signal to PSCs we used brief applications of neurotrophin-3 (NT-3), brain-derived neurotophic factor (BDNF) or nerve growth factor (NGF; 100 ng/mL). Soleus muscles of mice were incubated with the Ca(2+) indicator Fluo-4AM and Ca(2+) responses in PSCs were elicited through nerve stimulation (50 Hz, 30 s). Our results indicate that acute application of both NT-3 and BDNF, but not NGF, increased PSC Ca(2+) responses. Investigation of the mechanisms involved in these increases revealed distinct pathways for BDNF and NT-3. BDNF increased PSC responsiveness through potentiation of ATP responses while NT-3 modulated muscarinic acetylcholine receptor signalling. Using local applications of the neurotrophins, we found that both neurotrophins were able to elicit Ca(2+) responses in PSCs where BDNF used a phospholipase C-inositol 1,4,5-triphosphate (PLC-IP(3)) mechanism, while NT-3 required extracellular Ca(2+). Our results demonstrate a neurotrophin-dependent modulation of neuron-glia signalling through differential mechanisms employed by NT-3 and BDNF. Hence, neurotrophins precisely and differentially regulate PSC functions through modulation of either purinergic or cholinergic signalling pathways.

Neuron-glia interactions at the neuromuscular synapse.

The contribution of glial cells in the regulation of the transfer of information in CNS and PNS is now increasingly recognized. Perisynaptic Schwann cells (PSCs), glial cells at the neuromuscular junction (NMJ), have proven to be an exceptionally important model for studying these roles. PSCs surround nerve terminals at the NMJ and are activated by transmitter release in a frequency-dependent manner. All of these receptors, except one type, are coupled to G proteins and can be regrouped into two categories: activators and modulators of PSCs. In the former category are muscarinic (unknown subtype) and purinergic receptors (P2X and P2Y). In the latter category are adenosine (A1), Substance P (NK-1) and CGRP receptors. All receptors coupled to G proteins induce the release of Ca2+ from internal stores. In return for this activation, PSCs modulate synaptic activity and short-term plasticity. In this review, we will focus on the role of purines in the induction of glial cell activity and their possible involvement in the modulation of synaptic transmission as a result of the synaptic-induced glial activity.

GABAergic network activation of glial cells underlies hippocampal heterosynaptic depression.

Tetanus-induced heterosynaptic depression in the hippocampus is a key cellular mechanism in neural networks implicated in learning and memory. A growing body of evidence indicates that glial cells are important modulators of synaptic functions, but very little is known about their role in heterosynaptic plasticity. We examined the role of glial cells in heterosynaptic depression, knowing that tetanization and NMDA application caused depression of synaptic field responses (fEPSPs) and induced Ca2+ rise in glial cells. Here we report that chelating Ca2+ in a glial syncytium interfered with heterosynaptic depression and NMDA-induced fEPSP depression, suggesting that Ca2+ activation of glial cells is necessary for heterosynaptic depression. The NMDA-induced Ca2+ rise in glial cells was sensitive to tetrodotoxin and reduced by the GABAB antagonist CGP55845. Both heterosynaptic depression and simultaneous Ca2+ activation of glial cells were prevented by CGP55845, suggesting an involvement of the GABAergic network in glial activation and heterosynaptic depression. Also, the GABAB agonist baclofen caused both a Ca2+ rise in glial cells and fEPSP depression. Heterosynaptic depression, as well as NMDA- and baclofen-induced depression, were attenuated by an A1 antagonist, cyclopentyl-theophylline, whereas glial cell activation was not, indicating a role of adenosine downstream of glial activation. Finally, heterosynaptic depression requires ATP degradation because ectonucleotidase inhibitors reduced this plasticity. Our work indicates that Ca2+ activation of glial cells is necessary for heterosynaptic depression, which involves the sequential interaction of Schaffer collaterals, the GABAergic network, and glia. Thus, glial and neuronal networks are functionally associated during the genesis of heterosynaptic plasticity at mammalian central excitatory synapses.

Purinergic modulation of synaptic signalling at the neuromuscular junction.

Purines have physiologically important functions throughout the nervous system. In both the central (CNS) and peripheral nervous systems (PNS), purines in the form of adenosine triphosphate and adenosine can play a number of roles in neuronal activation and inhibition. In addition, purines are known to be important for glial cell signaling in both the CNS and PNS. In the PNS, the neuromuscular junction (NMJ) is an excellent model for studying simple synaptic interactions. It is well suited to investigations of neuron-glia interactions because synaptic properties are well defined and perisynaptic Schwann cells (PSCs), glial cells at the NMJ, dynamically interact with the pre- and postsynaptic elements. At the NMJ, purines are critical for presynaptic modulation but also for neuron-glia interactions. Purines signal to PSCs through metabotropic and ionotropic receptors and activation of these receptors can have both modulatory and activating functions. This review will discuss recent developments in our understanding of purinergic modulation of the NMJ with an emphasis on the involvement of purines in neuron-glia interactions at this synapse.