Early SARS-CoV-2 dynamics and immune responses in unvaccinated participants of an intensely sampled longitudinal surveillance study.

Background: A comprehensive understanding of the SARS-CoV-2 infection dynamics and the ensuing host immune responses is needed to explain the pathogenesis as it relates to viral transmission. Knowledge gaps exist surrounding SARS-CoV-2 in vivo kinetics, particularly in the earliest stages after exposure. Methods: An ongoing, workplace clinical surveillance study was used to intensely sample a small cohort longitudinally. Nine study participants who developed COVID-19 between November, 2020 and March, 2021 were monitored at high temporal resolution for three months in terms of viral loads as well as associated inflammatory biomarker and antibody responses. CD8 + T cells targeting SARS-CoV-2 in blood samples from study participants were evaluated. Results: Here we show that the resulting datasets, supported by Bayesian modeling, allowed the underlying kinetic processes to be described, yielding a number of unexpected findings. Early viral replication is rapid (median doubling time, 3.1 h), providing a narrow window between exposure and viral shedding, while the clearance phase is slow and heterogeneous. Host immune responses different widely across participants. Conclusions: Results from our small study give a rare insight into the life-cycle of COVID-19 infection and hold a number of important biological, clinical, and public health implications.

Botulinum neurotoxin accurately separates tonic vs. phasic transmission and reveals heterosynaptic plasticity rules in Drosophila.

In developing and mature nervous systems, diverse neuronal subtypes innervate common targets to establish, maintain, and modify neural circuit function. A major challenge towards understanding the structural and functional architecture of neural circuits is to separate these inputs and determine their intrinsic and heterosynaptic relationships. The Drosophila larval neuromuscular junction is a powerful model system to study these questions, where two glutamatergic motor neurons, the strong phasic-like Is and weak tonic-like Ib, co-innervate individual muscle targets to coordinate locomotor behavior. However, complete neurotransmission from each input has never been electrophysiologically separated. We have employed a botulinum neurotoxin, BoNT-C, that eliminates both spontaneous and evoked neurotransmission without perturbing synaptic growth or structure, enabling the first approach that accurately isolates input-specific neurotransmission. Selective expression of BoNT-C in Is or Ib motor neurons disambiguates the functional properties of each input. Importantly, the blended values of Is+Ib neurotransmission can be fully recapitulated by isolated physiology from each input. Finally, selective silencing by BoNT-C does not induce heterosynaptic structural or functional plasticity at the convergent input. Thus, BoNT-C establishes the first approach to accurately separate neurotransmission between tonic vs. phasic neurons and defines heterosynaptic plasticity rules in a powerful model glutamatergic circuit.

Minibrain kinase and calcineurin coordinate activity-dependent bulk endocytosis through synaptojanin.

Neurons use multiple modes of endocytosis, including clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE), during mild and intense neuronal activity, respectively, to maintain stable neurotransmission. While molecular players modulating CME are well characterized, factors regulating ADBE and mechanisms coordinating CME and ADBE activations remain poorly understood. Here we report that Minibrain/DYRK1A (Mnb), a kinase mutated in autism and up-regulated in Down's syndrome, plays a novel role in suppressing ADBE. We demonstrate that Mnb, together with calcineurin, delicately coordinates CME and ADBE by controlling the phosphoinositol phosphatase activity of synaptojanin (Synj) during varying synaptic demands. Functional domain analyses reveal that Synj's 5'-phosphoinositol phosphatase activity suppresses ADBE, while SAC1 activity is required for efficient ADBE. Consequently, Parkinson's disease mutation in Synj's SAC1 domain impairs ADBE. These data identify Mnb and Synj as novel regulators of ADBE and further indicate that CME and ADBE are differentially governed by Synj's dual phosphatase domains.

Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration.

Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. We have investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions in which Drosophila larvae are terminally arrested, we have characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). Although wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which time NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.