Sex-Specific and Traumatic Brain Injury Effects on Dopamine Receptor Expression in the Hippocampus.

Traumatic brain injury (TBI) is a major health concern. Each year, over 50 million individuals worldwide suffer from TBI, and this leads to a number of acute and chronic health issues. These include affective and cognitive impairment, as well as an increased risk of alcohol and drug use. The dopaminergic system, a key component of reward circuitry, has been linked to alcohol and other substance use disorders, and previous research indicates that TBI can induce plasticity within this system. Understanding how TBI modifies the dopaminergic system may offer insights into the heightened substance use and reward-seeking behavior following TBI. The hippocampus, a critical component of the reward circuit, is responsible for encoding and integrating the spatial and salient aspects of rewarding stimuli. This study explored TBI-related changes in neuronal D2 receptor expression within the hippocampus, examining the hypothesis that sex differences exist in both baseline hippocampal D2 receptor expression and its response to TBI. Utilizing D2-expressing tdTomato transgenic male and female mice, we implemented either a sham injury or the lateral fluid percussion injury (FPI) model of TBI and subsequently performed a region-specific quantification of D2 expression in the hippocampus. The results show that male mice exhibit higher baseline hippocampal D2 expression compared to female mice. Additionally, there was a significant interaction effect between sex and injury on the expression of D2 in the hippocampus, particularly in regions of the dentate gyrus. Furthermore, TBI led to significant reductions in hippocampal D2 expression in male mice, while female mice remained mostly unaffected. These results suggest that hippocampal D2 expression varies between male and female mice, with the female dopaminergic system demonstrating less susceptibility to TBI-induced plasticity.

Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View.

Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex.

Previous studies suggest that berberine, an isoquinoline alkaloid, has antiviral potential and is a possible therapeutic candidate against SARS-CoV-2. The molecular underpinnings of its action are still unknown. Potential targets include quadruplexes (G4Q) in the viral genome as they play a key role in modulating the biological activity of viruses. While several DNA-G4Q structures and their binding properties have been elucidated, RNA-G4Qs such as RG-1 of the N-gene of SARS-CoV-2 are less explored. Using biophysical techniques, the berberine binding thermodynamics and the associated conformational and hydration changes of RG-1 could be characterized and compared with human telomeric DNA-G4Q 22AG. Berberine can interact with both quadruplexes. Substantial changes were observed in the interaction of berberine with 22AG and RG-1, which adopt different topologies that can also change upon ligand binding. The strength of interaction and the thermodynamic signatures were found to dependent not only on the initial conformation of the quadruplex, but also on the type of salt present in solution. Since berberine has shown promise as a G-quadruplex stabilizer that can modulate viral gene expression, this study may also contribute to the development of optimized ligands that can discriminate between binding to DNA and RNA G-quadruplexes.

Structural Responses of Nucleic Acids to Mars-Relevant Salts at Deep Subsurface Conditions.

High pressure deep subsurface environments of Mars may harbor high concentrations of dissolved salts, such as perchlorates, yet we know little about how these salts influence the conditions for life, particularly in combination with high hydrostatic pressure. We investigated the effects of high magnesium perchlorate concentrations compared to sodium and magnesium chloride salts and high pressure on the conformational dynamics and stability of double-stranded B-DNA and, as a representative of a non-canonical DNA structure, a DNA-hairpin (HP), whose structure is known to be rather pressure-sensitive. To this end, fluorescence spectroscopies including single-molecule FRET methodology were applied. Our results show that the stability both of the B-DNA as well as the DNA-HP is largely preserved at high pressures and high salt concentrations, including the presence of chaotropic perchlorates. The perchlorate anion has a small destabilizing effect compared to chloride, however. These results show that high pressures at the kbar level and perchlorate anions can modify the stability of nucleic acids, but that they do not represent a barrier to the gross stability of such molecules in conditions associated with the deep subsurface of Mars.

Modulation of the Conformational Space of SARS-CoV-2 RNA Quadruplex RG-1 by Cellular Components and the Amyloidogenic Peptides α-Synuclein and hIAPP.

Given the emergence of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which particularly threatens older people with comorbidities such as diabetes mellitus and dementia, understanding the relationship between Covid-19 and other diseases is an important factor for treatment. Possible targets for medical intervention include G-quadruplexes (G4Qs) and their protein interaction partners. We investigated the stability and conformational space of the RG-1 RNA-G-quadruplex of the SARS-CoV-2 N-gene in the presence of salts, cosolutes, crowders and intrinsically disordered peptides, focusing on α-Synuclein and the human islet amyloid polypeptide, which are involved in Parkinson's disease (PD) and type-II diabetes mellitus (T2DM), respectively. We found that the conformational dynamics of the RG-1 G4Q is strongly affected by the various solution conditions. Further, the amyloidogenic peptides were found to strongly modulate the conformational equilibrium of the RG-1. Considerable changes are observed with respect to their interaction with human telomeric G4Qs, which adopt different topologies. These results may therefore shed more light on the relationship between PD as well as T2DM and the SARS-CoV-2 disease and their molecular underpinnings. Since dysregulation of G4Q formation by rationally designed targeting compounds affects the control of cellular processes, this study should contribute to the development of specific ligands for intervention.

Untangling the interaction of α-synuclein with DNA i-motifs and hairpins by volume-sensitive single-molecule FRET spectroscopy.

The intrinsically disordered protein α-synuclein causes Parkinson's disease by forming toxic oligomeric aggregates inside neurons. Single-molecule FRET experiments revealed conformational changes of noncanonical DNA structures, such as i-motifs and hairpins, in the presence of α-synuclein. Volumetric analyses revealed differences in binding mode, which is also affected by cellular osmolytes.

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.

Unraveling the binding characteristics of small ligands to telomeric DNA by pressure modulation.

Recently, non-canonical DNA structures, such as G-quadruplexes (GQs), were found to be highly pressure sensitive, suggesting that pressure modulation studies can provide additional mechanistic details of such biomolecular systems. Using FRET and CD spectroscopy as well as binding equilibrium measurements, we investigated the effect of pressure on the binding reaction of the ligand ThT to the quadruplex 22AG in solutions containing different ionic species and a crowding agent mimicking the intracellular milieu. Pressure modulation helped us to identify the different conformational substates adopted by the quadruplex at the different solution conditions and to determine the volumetric changes during complex formation and the conformational transitions involved. The magnitudes of the binding volumes are a hallmark of packing defects and hydrational changes upon ligand binding. The conformational substates of the GQ as well as the binding strength and the stoichiometry of complex formation depend strongly on the solution conditions as well as on pressure. High hydrostatic pressure can also impact GQs inside living cells and thus affect expression of genetic information in deep sea organisms. We show that sub-kbar pressures do not only affect the conformational dynamics and structures of GQs, but also their ligand binding reactions.

Harnessing Pressure Modulation for Exploring Ligand Binding Reactions in Cosolvent Solutions.

A comprehensive understanding of ligand-protein interactions requires information about all thermodynamic parameters that describe the complexation reaction, and they should be able to provide the necessary information to understand the molecular forces that drive complex formation. Usually, binding studies are performed at ambient pressure conditions. However, in addition to using temperature variation to reveal enthalpic and entropic contributions to ligand binding, complementary pressure-dependent studies providing volumetric properties of the reaction can be beneficial. Changes in partial molar volume can inform about changes in packing and hydration upon ligand binding. Here, after a general discussion of pressure effects on ligand binding reactions, we present a comprehensive study of the effect of pressure and a widely used organic cosolvent, dimethyl sulfoxide (DMSO), on the binding of a small aromatic ligand, proflavine, to the enzyme α-chymotrypsin. We found that DMSO, which acts as a competitive inhibitor for proflavine, has a strong impact on the interaction process, resulting in a decrease of the binding constant. While the reaction performed in neat buffer is basically pressure insensitive, the partial molar volume of the complex in the presence of DMSO is larger compared with the uncomplexed state, rendering the binding constant markedly smaller upon pressurization. We also show that the magnitude and sign of the binding volume provide valuable information about the interaction mechanism and hydration changes, which is of particular interest when cosolvents are present.

Antagonism of Macrophage Migration Inhibitory Factory (MIF) after Traumatic Brain Injury Ameliorates Astrocytosis and Peripheral Lymphocyte Activation and Expansion.

Traumatic brain injury (TBI) precedes the onset of epilepsy in up to 15-20% of symptomatic epilepsies and up to 5% of all epilepsy. Treatment of acquired epilepsies, including post-traumatic epilepsy (PTE), presents clinical challenges, including frequent resistance to anti-epileptic therapies. Considering that over 1.6 million Americans present with a TBI each year, PTE is an urgent clinical problem. Neuroinflammation is thought to play a major causative role in many of the post-traumatic syndromes, including PTE. Increasing evidence suggests that neuroinflammation facilitates and potentially contributes to seizure induction and propagation. The inflammatory cytokine, macrophage migration inhibitory factor (MIF), is elevated after TBI and higher levels of MIF correlate with worse post-traumatic outcomes. MIF was recently demonstrated to directly alter the firing dynamics of CA1 pyramidal neurons in the hippocampus, a structure critically involved in many types of seizures. We hypothesized that antagonizing MIF after TBI would be anti-inflammatory, anti-neuroinflammatory and neuroprotective. The results show that administering the MIF antagonist ISO1 at 30 min after TBI prevented astrocytosis but was not neuroprotective in the peri-lesion cortex. The results also show that ISO1 inhibited the TBI-induced increase in γδT cells in the gut, and the percent of B cells infiltrating into the brain. The ISO1 treatment also increased this population of B cells in the spleen. These findings are discussed with an eye towards their therapeutic potential for post-traumatic syndromes, including PTE.

Remodeling of the Conformational Dynamics of Noncanonical DNA Structures by Monomeric and Aggregated α-Synuclein.

Research on Parkinson's disease most often focuses on the ability of the protein α-synuclein (α-syn) to form oligomers and amyloid fibrils, and how such species promote brain death. However, there are indications that α-syn also plays a gene-regulatory role in the cell nucleus. Noncanonical tetrahelical nucleic acids, G-quadruplexes (G4Q), and i-motifs have been shown to play an important role in the control of genomic events. Using the conformation-sensitive single-molecule Förster resonance energy transfer technique we show that monomeric and oligomeric α-syn affect G4Qs and i-motifs in a different way and lead to remodeling of their conformational substates. Aggregated α-syn destabilizes the G4Q leading to unfolding. In contrast, both monomeric and aggregated α-syn enhance folding of the i-motif sequence of telomeric DNA. Importantly, macromolecular crowding is able to partially rescue G4Q from unfolding.

Perturbation of liquid droplets of P-granule protein LAF-1 by the antimicrobial peptide LL-III.

In recent years, liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization. But there is rapidly growing evidence that LLPS may also be associated with a number of medical conditions, including neurodegenerative diseases, by acting as a modulator of pathological protein aggregation. Here we show how LLPS formed by the P-granule protein LAF-1 and RNA can be affected by antimicrobial peptides, such as LL-III, leading to enhanced formation of amorphous protein aggregates and the loss of droplet function as an efficient reaction center and organizational hub.

Neuroinflammatory mechanisms of post-traumatic epilepsy.

BACKGROUND: Traumatic brain injury (TBI) occurs in as many as 64-74 million people worldwide each year and often results in one or more post-traumatic syndromes, including depression, cognitive, emotional, and behavioral deficits. TBI can also increase seizure susceptibility, as well as increase the incidence of epilepsy, a phenomenon known as post-traumatic epilepsy (PTE). Injury type and severity appear to partially predict PTE susceptibility. However, a complete mechanistic understanding of risk factors for PTE is incomplete. MAIN BODY: From the earliest days of modern neuroscience, to the present day, accumulating evidence supports a significant role for neuroinflammation in the post-traumatic epileptogenic progression. Notably, substantial evidence indicates a role for astrocytes, microglia, chemokines, and cytokines in PTE progression. Although each of these mechanistic components is discussed in separate sections, it is highly likely that it is the totality of cellular and neuroinflammatory interactions that ultimately contribute to the epileptogenic progression following TBI. CONCLUSION: This comprehensive review focuses on the neuroinflammatory milieu and explores putative mechanisms involved in the epileptogenic progression from TBI to increased seizure-susceptibility and the development of PTE.

Influence of crowding agents on the dynamics of a multidomain protein in its denatured state: a solvation approach.

It is now well appreciated that the crowded intracellular environment significantly modulates an array of physiological processes including protein folding-unfolding, aggregation, and dynamics to name a few. In this work we have studied the dynamics of domain I of the protein human serum albumin (HSA) in its urea-induced denatured states, in the presence of a series of commonly used macromolecular crowding agents. HSA was labeled at Cys-34 (a free cysteine) in domain I with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) to act as a solvation probe. In partially denatured states (2-6 M urea), lower crowder concentrations (~ < 125 g/L) induced faster dynamics, while the dynamics became slower beyond 150 g/L of crowders. We propose that this apparent switch in dynamics is an evidence of a crossover from soft (enthalpic) to hard-core (entropic) interactions between the protein and crowder molecules. That soft interactions are also important for the crowders used here was further confirmed by the appreciable shift in the wavelength of the emission maximum of BADAN, in particular for PEG8000 and Ficoll 70 at concentrations where the excluded volume effect is not dominant.

Alteration of the Conformational Dynamics of a DNA Hairpin by α-Synuclein in the Presence of Aqueous Two-Phase Systems.

The effect of an amyloidogenic intrinsically disordered protein, α-synuclein, which is associated with Parkinson's disease (PD), on the conformational dynamics of a DNA hairpin (DNA-HP) was studied by employing the single-molecule Förster resonance energy transfer method. The open-to-closed conformational equilibrium of the DNA-HP is drastically affected by binding of monomeric α-synuclein to the loop region of the DNA-HP. Formation of a protein-bound intermediate conformation is fostered in the presence of an aqueous two-phase system mimicking intracellular liquid-liquid phase separation. Using pressure modulation, additional mechanistic information about the binding complex could be retrieved. Hence, in addition to toxic amyloid formation, α-synuclein may alter expression profiles of disease-modifying genes in PD. Furthermore, these findings might also have significant bearings on the understanding of the physiology of organisms thriving at high pressures in the deep sea.

SMARCB1 Gene Mutation Predisposes to Earlier Development of Glioblastoma: A Case Report of Familial GBM.

Glioblastoma (GBM) is the most aggressive adult brain tumor. While GBM typically occurs sporadically, familial GBM can be associated with certain hereditary disorders and isolated familial GBMs in the absence of syndrome are rare. Relevant hereditary factors have remained elusive in these cases. Understanding specific genetic abnormality may potentially lead to better treatment strategies in these patients. Here, we analyzed GBM tissue from our patient and 2 afflicted family members, with next generation sequencing to better understand the genetic alterations associated with this disease development. DNA was extracted and sequenced and the data were then analyzed. Results revealed 2 common mutations in afflicted family members; PDGFRA and HRAS. In addition, both siblings showed a mutation of the SMARCB1 gene. The sister of our patient exhibited a homozygous mutation, while our patient had heterozygous mutation of this gene in the tumor tissue. This result suggests that mutation of SMARCB1, either alone or in the presence of PDGFRA and HRAS mutations, is associated with earlier onset GBM.

Crowder induced structural modulation of a multi-domain protein during its early stages of aggregation: A FRET-based and protein solvation study.

Protein aggregation has been known for long to be the prime cause for several neurological disorders in human beings. While protein aggregation is itself a complex process, understanding the same in the context of a crowded cellular medium remains a challenge. In this work, using Förster resonance energy transfer (FRET) and solvation dynamics, we have tried to gain important insights into the structural rearrangements, during the early stages of aggregation of the multidomain protein bovine serum albumin (BSA) in presence of a range of synthetic macromolecular crowding agents. FRET studies show that there is an initial compaction in the domain size (domain I) at the early time points of incubation followed by an increase in the distance between the donor-acceptor pair. Analyses of the solvent correlation traces of BADAN (labeled at free Cys-34 in domain I of BSA) reveal that the same domain becomes rigid during the initial phase of the aggregation process subsequent to which there was a gradual increase in flexibility, the latter we propose being a necessary step that allows facile addition of more protein units.

Gulf War agents pyridostigmine bromide and permethrin cause hypersensitive nociception that is restored after vagus nerve stimulation.

Gulf war illness (GWI) is a chronic multi-symptom disease that afflicts 25-33% of troops that were deployed in the 1990-1991 Gulf War. GWI symptoms include cognitive, behavioral and emotional deficits, as well as migraines and pain. It is possible that exposure to Gulf War agents and prophylactics contributed to the reported symptomology. Pyridostigmine bromide (PB) and permethrin (PER) were given to protect from nerve gas attacks and insect vector born disease, respectively. Previous studies have demonstrated that 10 days of exposure to these chemicals can cause symptoms analogous to those observed in GWI, including impairment of long-term memory in mice. Other studies using this model have shown chronic neuroinflammation, and chronic neuroinflammation can lead to altered nociceptive sensitivity. At 10-weeks after the 10-day PB and PER exposure paradigm, we observed lowered nociceptive threshold on the Von Frey test that was no longer evident at 28 weeks and 38 weeks post-exposure. We further determined that vagus nerve stimulation, initiated at 38 weeks after exposure, restores the lowered nociceptive sensitivity. Therefore, stimulating the vagus nerve appears to influence nociception. Future studies are need to elucidate possible mechanisms of this effect.

Mixed Macromolecular Crowding: A Protein and Solvent Perspective.

In the living cell, biomolecules perform their respective functions in the presence of not only one type of macromolecules but rather in the presence of various macromolecules with different shapes and sizes. In this study, we have investigated the effects of five single macromolecular crowding agents, Dextran 6, Dextran 40, Dextran 70, Ficoll 70, and PEG 8000 and their binary mixtures on the modulation in the domain separation of human serum albumin using a Förster resonance energy transfer-based approach and the translational mobility of a small fluorescent probe fluorescein isothiocyanate (FITC) using fluorescence correlation spectroscopy (FCS). Our observations suggest that mixed crowding induces greater cooperativity in the domain movement as compared to the components of the mixtures. Thermodynamic analyses of the same provide evidence of crossovers from enthalpy-based interactions to effects dominated by hard-sphere potential. When compared with those obtained for individual crowders, both domain movements and FITC diffusion studies show significant deviations from ideality, with an ideal solution being considered to be that arising from the sum of the contributions of those obtained in the presence of individual crowding agents. Considering the fact that domain movements are local (on the order of a few angstroms) in nature while translational movements span much larger lengthscales, our results imply that the observed deviation from simple additivity exists at several possible levels or lengthscales in such mixtures. Moreover, the nature and the type of deviation not only depend on the identities of the components of the crowder mixtures but are also influenced by the particular face of the serum protein (either the domain I-II or the domain II-III face) that the crowders interact with, thus providing further insights into the possible existence of microheterogeneities in such solutions.