Burnstock and the legacy of the inhibitory junction potential and P2Y1 receptors

The synaptic event called the inhibitory junction potential (IJP) was arguably one of the more important discoveries made by Burnstock and arguably one of his finer legacies. The discovery of the IJP fundamentally changed how electromechanical coupling was visualised in gastrointestinal smooth muscle. Its discovery also set in motion the search for novel inhibitory neurotransmitters in the enteric nervous system, eventually leading to proposal that ATP or a related nucleotide was a major inhibitory transmitter. The subsequent development of purinergic signalling gave impetus to expanding the classification of surface receptors for extracellular ATP, not only in the GI tract but beyond, and then led to successive phases of medicinal chemistry as the P2 receptor field developed. Ultimately, the discovery of the IJP led to the successful cloning of the first P2Y receptor (chick P2Y1) and expansion of mammalian ATP receptors into two classes: metabotropic P2Y receptors (encompassing P2Y1, P2Y2, P2Y4, P2Y6, P2Y11–14 receptors) and ionotropic P2X receptors (encompassing homomeric P2X1–P2X7 receptors). Here, the causal relationship between the IJP and P2Y1 is explored, setting out the milestones reached and achievements made by Burnstock and his colleagues.

Mechanism of efficient double-strand break repair by a long non-coding RNA

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1’s ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA–RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

MOD: A novel machine-learning optimal-filtering method for accurate and efficient detection of subthreshold synaptic events in vivo

To understand the mechanisms of information coding in single neurons, it is necessary to analyze subthreshold synaptic events, action potentials (APs), and the interrelation between these two forms of activity in different behavioral states. However, detecting excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) in awake, behaving animals remains challenging, because of unfavorable signal-to-noise ratio, high frequency, fluctuating amplitude, and variable time course of synaptic events. Here, we developed a new method for synaptic event detection, termed MOD (Machine-learning Optimal-filtering Detection-procedure), which combines concepts of supervised machine learning and optimal Wiener filtering. First, experts were asked to manually score short epochs of data. Second, the algorithm was trained to obtain the optimal filter coefficients of a Wiener filter and the optimal detection threshold. Third, scored and unscored data were processed with the optimal filter, and events were detected as peaks above threshold. Finally, the area under the curve (AUC) of the receiver operating characteristics (ROC) curve was used to quantify accuracy and efficiency of detection. Additionally, cross-validation was performed to exclude overfitting of the scored data, a potential concern with machine-learning approaches. We then challenged the new detection method with EPSP traces in vivo in mice during spatial navigation and EPSC traces in vitro in slices under conditions of enhanced transmitter release. When benchmarked using a (1−AUC)−1 metric, MOD outperformed previous methods (template-fit and deconvolution) by a factor of up to 3. Thus, MOD may become an important tool for large-scale analysis of synaptic activity in vivo and in vitro.HighlightsA new method for detection of synaptic events, termed MOD, is describedThe method combines the concepts of supervised machine learning and optimal filteringThe method is useful for analysis of both in vitro and in vivo data setsMOD outperforms previously published methods for synaptic event detection by a factor of up to 3

Axonal ER Ca2+ Release Enhances Miniature, but Reduces Activity-Dependent Glutamate Release in a Huntington Disease Model

Action potential-independent (miniature) neurotransmission occurs at all chemical synapses, but remains poorly understood, particularly in pathological contexts. Spontaneous release of Ca2+ from the axonal endoplasmic reticulum (ER) is thought to facilitated miniature neurotransmission, and aberrant ER Ca2+ handling is notably implicated in the progression of Huntington’s disease (HD) and other neurodegenerative diseases. Here, we report elevated glutamate-mediated miniature synaptic event frequencies in YAC128 (HD-model) cortical neurons, which pharmacological experiments suggest is mediated by enhanced spontaneous ER Ca2+ release. Calcium imaging using an axon-localized sensor revealed slow action potential (AP)-independent axonal Ca2+ waves, which were more common in YAC128 cortical neurons. Conversely, spontaneous axonal ER Ca2+ release was associated with reduced AP-dependent axonal Ca2+ events and consequent glutamate release. Together, our results suggest spontaneous release of axonal ER Ca2+ stores oppositely regulates activity-dependent and -independent neurotransmitter release in HD, with potential implications for the fidelity and plasticity of cortical excitatory signaling.

Hyperexcitable phenotypes in iPSC-derived neurons from patients with 15q11-q13 duplication syndrome, a genetic form of autism

Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, Dup15q patients commonly meet the criteria for autism spectrum disorder (ASD) and have a high prevalence of seizures. Here, we explored mechanisms of hyperexcitability in neurons derived from induced pluripotent stem cell (iPSC) lines from Dup15q patients. Maturation of resting membrane potential in Dup15q-derived neurons was similar to neurons from unaffected control subjects, but Dup15q neurons had delayed action potential maturation and increased synaptic event frequency and amplitude. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared to control neurons, in part due to disruption of KCNQ2 channels. Together these data point to multiple mechanisms underlying hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.