Neuroprotection by noble gases and other drugs in models of trauma

Traumatic Brain Injury (TBI) is a debilitating and complex condition that consists of a ‘primary’ injury followed by a ‘secondary’ injury, which develops over time. Much of the morbidity occurs due to the secondary injury, for which there are currently no approved pharmacotherapies. This thesis is structured as a study of models of TBI and an investigation of drugs to treat the condition. First, an injury quantification methodology was developed in an in vitro model of TBI using organotypic hippocampal slice cultures and propidium iodide fluorescence. Second, ‘combination therapies’ with the noble gases xenon and argon, progesterone and meloxicam to modulate multiple secondary injury mechanisms were explored. 25% argon and 3μM, 10μM and 25μM progesterone are additively protective at 72 hours. Co-administering 25μM progesterone with 25% argon (29±6% protective) abolished injury. Co-administering 50% argon (41±5% protective) synergistically abolished injury with 3μM progesterone and additively abolished injury with 10μM and 25μM progesterone. 25% xenon and 3μM, 10μM and 25μM progesterone were additively protective at 72 hours. Co-administering 25μM progesterone with 25% xenon (44±5% protective) abolished injury. 25% argon in combination with 0.1μM and 1μM meloxicam works additively. Neuroprotection by 25% argon doubled when co-administered with 1μM meloxicam. 25% xenon and 1μM meloxicam are additive, abolishing injury at 72 hours unlike sub-maximally effective 25% xenon. Third, experiments to calibrate a controlled cortical impact (CCI) model of in vivo TBI were conducted. Contusion volume was measured and the expression of inflammatory cytokines was quantified in blood and brain samples at different time points. Finally, a Drosophila melanogaster model of polytrauma was developed as a high throughput assay. Drosophila are more sensitive to trauma in the early phase of their circadian day. Hypothermia treatment (40C) increased survival to the level of uninjured shams at 24 hours and more than doubled survival over 14 days.Open Acces

A cysteine-based molecular code informs collagen C-propeptide assembly

Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that the presence or absence of a single cysteine in type-I collagen’s C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca2+ in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.National Science Foundation (U.S.) (Grant NSF-0070319)National Science Foundation (U.S.). Center for Science of Information (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant R03AR067503)National Institutes of Health (U.S.) (Grant 1R01AR071443)National Institutes of Health (U.S.). Ruth Kirschstein Predoctoral Fellowship (1F31AR067615

Distinct subnetworks of the thalamic reticular nucleus

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition1–5. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders6–9. However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN–thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.NIH/NIMH (Grants R01NS098505, R01NS113245)NIH (Grants R01NS098505, R01MH107680