NETosis induction reflects COVID-19 severity and long COVID: insights from a 2-center patient cohort study in Israel.

BACKGROUND: COVID-19 severity and its late complications continue to be poorly understood. Neutrophil extracellular traps (NETs) form in acute COVID-19, likely contributing to morbidity and mortality. OBJECTIVES: This study evaluated immunothrombosis markers in a comprehensive cohort of acute and recovered COVID-19 patients, including the association of NETs with long COVID. METHODS: One-hundred-seventy-seven patients were recruited from clinical cohorts at 2 Israeli centers: acute COVID-19 (mild/moderate, severe/critical), convalescent COVID-19 (recovered and long COVID), along with 54 non-COVID controls. Plasma was examined for markers of platelet activation, coagulation, and NETs. Ex vivo NETosis induction capability was evaluated after neutrophil incubation with patient plasma. RESULTS: Soluble P-selectin, factor VIII, von Willebrand factor, and platelet factor 4 were significantly elevated in patients with COVID-19 versus controls. Myeloperoxidase (MPO)-DNA complex levels were increased only in severe COVID-19 and did not differentiate between COVID-19 severities or correlate with thrombotic markers. NETosis induction levels strongly correlated with illness severity/duration, platelet activation markers, and coagulation factors, and were significantly reduced upon dexamethasone treatment and recovery. Patients with long COVID maintained higher NETosis induction, but not NET fragments, compared to recovered convalescent patients. CONCLUSIONS: Increased NETosis induction can be detected in patients with long COVID. NETosis induction appears to be a more sensitive NET measurement than MPO-DNA levels in COVID-19, differentiating between disease severity and patients with long COVID. Ongoing NETosis induction capability in long COVID may provide insights into pathogenesis and serve as a surrogate marker for persistent pathology. This study emphasizes the need to explore neutrophil-targeted therapies in acute and chronic COVID-19.

Olfactory bulb-derived cells seeded on 3D scaffolds exhibit neurotrophic factor expression and pro-angiogenic properties.

Olfactory bulb (OB)-derived cells include fibroblasts, astrocytes, and olfactory ensheathing cells (OECs). OECs are a distinctive type of glia that secrete neurotrophic factors and form myelin sheaths around axons projecting from the olfactory mucosa into the OB of the central nervous system. Their unique properties make them candidates for cell therapy of spinal cord injury (SCI). Current SCI cellular repair techniques suffer from massive cell loss post implantation. To overcome this difficulty, we propose to seed and propagate OB-derived cells on biodegradable scaffolds to form a stable tissue construct. Upon implantation, scaffolds may serve as carriers to introduce the tissue into the lesion site. In this study, we characterized OB-derived cells cultured on biodegradable poly-l-lactic acid/polylactic-co-glycolic acid scaffolds in vitro. We showed that cells remained viable and proliferative for up to 2 weeks on the scaffolds. We have shown that OB-derived cells induce neuronal differentiation of pheochromocytoma cells (PC12) on scaffolds, and that a purified population of OECs is sufficient for the differentiation. Selective inhibition of nerve growth factor (NGF) on PC12 cells blocks the differentiation. We have shown that the expression of BDNF and NGF genes in OB-derived cells grown on 3D scaffolds compared to 2D monolayer cultures was significantly upregulated. In addition, OB-derived cells stimulated network formation of endothelial cells grown on the same scaffolds. Taken together, these results clearly demonstrate the vast potential of 3D scaffolds in maintaining and strengthening the unique therapeutic properties of embedded OB-derived cells. This strategy will enable more efficient therapeutic usage of OB-derived cells for treatment of SCI.

A novel role for protein synthesis in long-term neuronal plasticity: maintaining reduced postburst afterhyperpolarization.

Memory consolidation, the process of transformation of short-term to long-term memory, has been shown to be protein synthesis dependent in a variety of different learning paradigms, brain structures, and species. At the cellular level, protein synthesis was shown to be crucial for induction of long-term synaptic plasticity; application of protein synthesis inhibitors prevents the transformation of early long-term potentiation (LTP) to late LTP. Thus, protein synthesis has been traditionally thought to affect long-term memory consolidation by stabilizing synaptic transmission. However, long-term memory is not supported only by modulation of synaptic strength; modifications in intrinsic neuronal properties also subserve learning-related behavioral changes. Learning-induced reduction in the postburst afterhyperpolarization (AHP), which results with enhanced neuronal excitability and decreased spike frequency adaptation, is apparent in hippocampal and cortical pyramidal neurons. Such postburst AHP reduction lasts for days after training completion and is implicated in maintaining learned skills. Short-term modulation of intrinsic neuronal excitability can be also induced in vitro. Intense synaptic activation induces AHP reduction and enhanced neuronal excitability in hippocampal pyramidal neurons. Here, we show that synaptic activation-induced short-term postburst AHP reduction can be transformed to long-term AHP reduction, such that persists for prolonged time periods. This long-lasting AHP reduction is protein synthesis dependent for up to 1 h after induction. We suggest that, much like synaptic plasticity, activity-induced long-lasting modulation of intrinsic neuronal excitability requires molecular consolidation. It would appear that both synaptic and intrinsic modifications and maintenance are activated jointly to enable long-lasting memories.

Persistent ERK activation maintains learning-induced long-lasting modulation of synaptic connectivity.

Pyramidal neurons in the piriform cortex from olfactory-discrimination (OD) trained rats undergo synaptic modifications that last for days after learning. A particularly intriguing modification is reduced paired-pulse facilitation (PPF) in the synapses interconnecting these cells; a phenomenon thought to reflect enhanced synaptic release. The molecular machinery underlying this prolonged physiological modulation of synaptic connectivity is yet to be described. We have recently shown that extracellular regulated kinase (ERK) pathway and protein kinase C (PKC) are also required for learning-induced enhancement of intrinsic neuronal excitability. Here we examine whether these signal-transduction cascades are instrumental for the learning-induced, long-lasting PPF reduction. Days after learning completion, PD98059, a selective inhibitor of MEK, the upstream kinase of ERK, increased PPF in neurons from trained, but not in neurons from naïve and pseudo-trained rats. Consequently, the differences in PPF between neurons from trained rats and controls were abolished. The level of activated ERK in synaptoneurosomes was significantly higher in piriform cortex samples prepared from trained rats. Notably, ERK activation revealed that PPF reduction lags behind ERK activation by 2 d. Similarly, the PKC blocker, GF-109203X, enhanced PPF in neurons from trained rats only, thus abolishing the differences between groups. Interestingly, the PKC activator, OAG, had no effect, indicating that PKC activation is required, but not sufficient for long-lasting PPF reduction. Our data show that persistent ERK activation has a key role in maintaining learning-induced PPF reduction for days. This time frame of compartmental ERK-dependent synaptic modulation suggests a novel role for ERK in cortical function.

A novel role for extracellular signal-regulated kinase in maintaining long-term memory-relevant excitability changes.

Pyramidal neurons in the piriform cortex from olfactory-discrimination-trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the postburst afterhyperpolarization (AHP), which is generated by repetitive spike firing. AHP reduction is attributable to decreased conductance of a calcium-dependent potassium current, the sI(AHP). We have previously shown that such learning-induced AHP reduction is maintained by PKC activation. However, the molecular machinery underlying such long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the extracellular signal-regulated kinase I/II (ERKI/II) pathway, which is known to be crucial in learning, memory, and synaptic plasticity processes, is instrumental for the long-term maintenance of learning-induced AHP reduction. PD98059 or UO126, which selectively block MEK, the upstream kinase of ERK, increased the AHP in neurons from trained rats but not in neurons from naive and pseudo-trained rats. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls were abolished. This effect was not mediated by modulation of basic membrane properties. In accordance with its effect on neuronal excitability, the level of activated ERK in the membranal fraction was significantly higher in piriform cortex samples taken from trained rats. In addition, the PKC activator OAG (1-oleoyl-20acety-sn-glycerol), which was shown to reduce the AHP in neurons from control rats, had no effect on these neurons in the presence of PD98059. Our data show that ERK has a key role in maintaining long-lasting learning-induced enhancement of neuronal excitability.