Glycolytic Dysregulation in Amyotrophic Lateral Sclerosis

Qi Lab

Abstract:Amyotrophic Lateral Sclerosis is a late-onset neurodegenerative disease characterized by upper and lower motor neuron loss [1]. Clinically, ALS patients have documented an increased resting energy expenditure, defined as hypermetabolism, creating an imbalance between energy supply and demand [2]. Since the brain consumes about 20% of the body’s total energy to maintain regulatory functions [3], a drop in energy supply could be fatal. The primary source of this energy, glucose, is broken down through glycolysis and oxidative phosphorylation. Alterations in glycolytic output via lactate production and transcription levels of rate limiting glycolytic enzymes have been found in ALS patients and patient derived IPSC motor neurons, respectively [4-6]. Preliminary data from our lab corroborates previous data with increased lactate production in post mortem ALS patient spinal cord samples. Despite the evidence showing a dysregulation of glycolysis in ALS, the role of glycolysis in motor neurodegeneration and the impact of glycolysis impairment on the development of ALS are unknown. In this study, I will be investigating the role of glycolysis and rate-limiting enzymes functions in ALS. Based on preliminary data, a significant increase in the mRNA level of Hexokinase 1 (HK1), the first enzyme of the glycolytic pathway, in ALS patient postmortem spinal cord samples. Either an increase or decrease in HK1 level has been shown to associate with cell death and neuroinflammation [7, 8]. However, whether the increase in HK1 and lactate are contributing factors of motor neurodegeneration in ALS or the consequences of disease progression remains to be determined.

Glutamate is the primary excitatory neurotransmitter in the brain and is required for proper brain function

Tajima Lab

Abstract:Glutamate is the primary excitatory neurotransmitter in the brain and is required for proper brain function. Excitatory transmission affects the sympathetic and parasympathetic nervous system thereby directly modulating cardiovascular activity. Kainate receptors (KARs) are a type of glutamate receptor that are involved in the genesis of epilepsy thereby contributing to an increase in heart rate. KARs are expressed in the brain and therefore have the added effect of contributing to processes associated with memory and learning. Neuropilin-and-tolloid- like (NETO) proteins are single-transmembrane proteins that will interact with KARs and affect their desensitization and deactivation rates. NETO proteins also play role in localizing glutamate receptors to the synapse. However, little is known about the structure of KARs in complex with NETO proteins. Understanding this structure will help us understand how the amino acid residues play key roles in modulating KAR activity. With this understanding, we will be able to develop therapeutics that are aimed to treat various mental disorders including epilepsy and schizophrenia as well as potentially target tachycardia. There are two types of NETO proteins; NETO1 and NETO2, of which, NETO1 is less understood. Structural studies using cryogenic electron microscopy (CryoEM) have elucidated the structure of NETO2 but not NETO1 and have shown how NETO2 forms a complex with GluK2. Therefore, the structure of NETO1 has yet to be determined as well as the structure of it bound with GluK1. The overall hypothesis of this proposal is that NETO1 is structurally different from NETO2 thereby increasing the rate of desensitization and deactivation of the GluK1 homomer. We will address this hypothesis and achieve the goals that are necessary to prove or disprove this hypothesis by purifying NETO1 and the GluK1 homomer and subsequently using CryoEM to determine the structure of the complex. We will plan on doing the same procedure with NETO2 and GluK1 to compare the relevant interactions that take place. This will help us determine the amino acids in the GluK1 homomer and NETO1 protein that are important in modulating the desensitization and deactivation rates of these glutamate receptors. The findings of this proposal will provide a complete molecular basis for excitatory neurotransmission.

Heart failure (HF) represents one of the largest global health concerns of the modern world

Stelzer Lab

Abstract:Heart failure (HF) represents one of the largest global health concerns of the modern world. Unfortunately, current HF therapies do little to improve prognosis or quality of life. Given that health experts forecast that HF's prevalence and economic burden will only continue to rise in the coming decade, effective HF therapies are in high demand. Despite extensive research and testing of several potential therapeutic strategies, none have proven clinically viable. A recent approach to treating HF uses small molecules to activate myosin motors within the heart to improve force-generating capabilities. Omecamtiv mecarbil (OM) represents the first of such molecules to pass clinical trials and obtain FDA approval. While many had high hopes for OM, recent large-scale clinical trials reveal its therapeutic limitations, suggesting that OM may not be the silver bullet HF needs. Furthermore, the lack of in vivo mechanistic studies dissecting how OM interacts with the complex pathophysiology of HF may underscore our misunderstanding of OM’s clinical behavior. Since drug companies continue to pursue small molecule myosin activators as an HF therapy, this talk outlines how rodent models of HF can improve our preclinical understanding of myosin activation in the failing heart and better assess its therapeutic potential.

Progesterone Receptor Pathways in Preterm Birth

Chakravarti Lab

Abstract:Progesterone is the most important hormone for the maintenance of pregnancy, and its withdrawal, whether functional or systemic, initiates parturition. There are two main progesterone receptors (PRs) found in human myometrium, PR-A and PR-B. Previous research has indicated that PR-B has an anti-inflammatory function and favors pregnancy, while PR-A has an inflammatory function and favors parturition. However, gaps in our understanding of these highly complex progesterone signaling pathways remain. Due the potency of progesterone and the consequences of its withdrawal during pregnancy, understanding these pathways is integral to the prevention and mitigation of preterm birth. Targeting factors that lead to inflammatory events within PR pathways may help keep a fetus at risk for preterm birth in the uterus longer, increasing chances of survival and decreasing potential negative consequences. Emerging research shows that the phosphorylated conformation of PR-A may be responsible for the inflammatory action associated with it, while the non-phosphorylated and inactivated (unliganded) states of PR-A may have other undetermined actions. The goal of this study is to determine if there are indeed three separate PR-A pathways; the phosphorylated state (with canonical inflammatory effects), the non-phosphorylated state (with anti-inflammatory effects comparable to PR-B), and the inactive state (in which PR-B actions dominate). Preliminary data generated using immortalized cell lines, western blots, PCR, and Elisa assays support this hypothesis, demonstrating the anti-inflammatory effects of PR-B alone and the modulated effects with phospho-PR-A.

Glycolytic Dysregulation in Amyotrophic Lateral Sclerosis

Chakrapani Lab

Abstract:Type-3 serotonin receptors (5-HT3Rs) are pentameric ligand gated ion channels that mediate fast synaptic signaling in response to binding of the neurotransmitter serotonin. 5-HT3Rs play a major role in regulating gut motility, secretion, and visceral perception. Hyperactivity of 5-HT3Rs underlies pathologies such as chemotherapy-induced nausea and vomiting, irritable bowel syndrome, depression, anxiety, bipolar disorder, and excessive visceral pain, making it an important drug target. Previous research has resulted in the structures of the 5-HT3AR in complex with the agonist serotonin and setrons, 5-HT3R antagonists. Recently, 5-HT3AR partial agonists have been proposed to treat 5-HT3R pathologies with less severe side effects than full antagonists. However, the molecular mechanisms of partial agonism by orthosteric ligands of the 5-HT3R are still poorly understood. Here we present structures of the 5-HT3AR in complex with two novel orthosteric partial agonists generated from cryo-electron microscopy imaging. Structural stability of the ligand binding poses was assessed by molecular dynamic simulations, and ligand function was assessed by two-electrode voltage clamp in wild type and mutant receptors. Using our ligand-bound models of the 5-HT3AR, we propose structure-based mechanisms for the functional differences between orthosteric partial agonists, full agonist, and antagonists.

Glycolytic Dysregulation in Amyotrophic Lateral Sclerosis

Surewicz Lab

Abstract:Dysregulation of TDP-43 liquid-liquid phase separation (LLPS) is implicated in the pathogenesis of proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies indicate that the segment encompassing residues ~320-~340 within TDP-43’s low complexity domain (LCD) forms a transient α-helix that is essential for LLPS. The above region contains Ser333, which has been postulated to be one of TDP-43’s phosphorylation sites. Here we investigated the effect of Ser333 phosphorylation on TDP-43 LCD LLPS using a strategy of phosphomimetic substitution. We also examined sporadic ALS and FTD-ALS cases to determine whether TDP-43 phosphorylated at Ser333 is indeed present in disease.

Glycolytic Dysregulation in Amyotrophic Lateral Sclerosis

Boron Lab

Abstract:Receptor protein tyrosine phosphatases ζ (RPTPζ) is transmembrane signaling proteins that, as monomers, catalyze dephosphorylation of tyrosine residues on cytosolic targets. RPTPζ are unusual in having an extracellular carbonic-anhydrase like domain (CALD). Car-bonic anhydrases (CAs) catalyze the reaction, CO₂ + H₂O ? HCO₃– + H⁺, but CALDs are enzymatically inactive. Previous studies on renal proximal tubules (PTs) strongly suggest that RPTPγ is a dual extracellular CO₂/HCO₃^- sensor. RPTPγ expressed on the PT membrane is essential for modulating acid-base transport in response to changes in basolateral [CO₂] and [HCO₃^-]. RPTPζ is also present in the central nervous system (CNS), including mouse hippocampus (HC). Here, we investigate RPTPζ in brain, like RPTPγ in kidney, play roles in sensing extracellular [CO₂]₀ and [HCO₃^-]₀ and altering acid-base transport. We hypothesize that activation/termination of the intracellular phosphatase activity of these RPTPζ regulates initial steps in signaling cascades that defend cells against intracellular pH (pHi) decreases triggered by metabolic acidosis (MAc, caused by ↓ [HCO₃^-]₀) or respiratory acidosis (RAc, caused by ↑ [CO₂]₀).We propose that the RPTPζ respond to ↑[CO₂]₀ or ↓[HCO₃^-]₀ by promoting a shift from dimers to monomers, thereby disinhibiting the activity of the intracellular phosphatase domains. The ultimate result of the RPTPζ? disinhibition would be increased acid-extrusion rate (JE) or decreased acid-loading rate (JL), both of which would minimize the fall of pHi during a first exposure to MAc or RAc, and may promote adaptation during a second exposure. Immunocytochemistry of mixed HC neuron-astrocyte cultures from wild-type (WT) mice using a novel primary antibody against the RPTPζ shows that RPTPζ colocalizes with MAP2 in HC neurons but not with GFAP in HC astrocytes in mixed culture of neuron and astrocyte and also in the pup and adult tissue. RT-PCR cloning from mixed HC cultures and also pup and adult tissue identifies specific RPTPζ variants. Three RPTPζ variants (V) have previously been validated in mice: VR3 (NM_001081306), VR4 (NM_001311064), and VR5 (NM_001361349). Three others were previously only reported as hypothetical assemblies: X2 (XM_006505013), X3 (XM_006505014), and X4 (XM_006505015). These data represent the first known detection of the expression of these 3 variant in the neuronal culture and tissues. In primary RPTPζ KO neuronal/astrocyte co-cultures, paired RAc and MAc exposures produced unexpected effects on astrocytes (which do not express RPTPζ), arguing for a novel form of RPTPζ dependent crosstalk between neurons and astrocytes.

Characterizing the Signaling Mechanism of IL-1? Secretion during Lysosomal Membrane Permeabilization in Neutrophils

Dubyak Lab

Interleukin-1 beta (IL-????) is a pro-inflammatory cytokine that can be secreted by leukocytes in response to infection or inflammation. Canonically, IL-1???? is associated with the activation of Nod-like receptor pyrin-domain containing 3 protein (NLRP3) and oligomerization of the inflammasome. The NLRP3 inflammasome causes the autocatalytic cleavage of caspase-1. Cleaved caspase-1 is then able to cleave and activate IL-1????, as well as remove the autoinhibitory C-terminus of Gasdermin D (GSDMD) to allow N-GSDMD to form pores in the plasma membrane to act as a conduit for IL-1???? release. The NLRP3 inflammasome can be activated by a large variety of stimuli, though the commonality between all agonists is potassium ion (K+) efflux. Lysosomal membrane permeabilization has been identified to activate the NLRP3 inflammasome and IL-1???? release in macrophages and dendritic cells. Neutrophils are another leukocyte that is able to assemble the NLRP3 inflammasome and are an important source of IL-1????. However, the ability of neutrophils to respond to lysosomal disruption is not well understood. Therefore, we sought to identify the mechanism of IL-1? secretion in response to lysosomal membrane permeabilization in neutrophils. We hypothesized that neutrophils would secrete IL-1???? in an NLRP3 and N-GSDMD dependent manner. Surprisingly, preliminary data suggests that upon lysosomal disruption with a lysosomotropic agent, L-Leucyl L-Leucine methyl ester (LLME), neutrophils secrete IL-1???? in an NLRP3-independent manner. Likewise, further findings suggest that this process is also independent of caspase-1, and only partially dependent on GSDMD. These preliminary findings point towards a novel neutrophil-specific signaling mechanism. As neutrophils are characterized by an abundance of enzyme-containing granules that are known to interact with NLRP3 inflammasome components, we believe it is likely that the canonical pathway is being perturbed and the observed novel mechanism is driven by the serine proteases held within the azurophilic granules. Future studies will seek to determine the necessity of serine proteases and their effect on IL-1???? secretion.

Parkinson’s Disease is a neurodegenerative disease marked by progressive motor impairment that arises as a result of reduced dopamine release

Qi Lab

Parkinson’s Disease is a neurodegenerative disease marked by progressive motor impairment that arises as a result of reduced dopamine release. It is characterized by aggregation of a- synuclein, which is thought to play a role in the disruption of various cell processes. However, much remains to be understood about the mechanisms of PD development and progression. Mitochondrial dysfunction is widely implicated as a contributing factor in neuronal cell death owing to its importance in cell energetics, though the variety of processes in which it is involved and their relative complexity makes it difficult to determine the exact part it plays in PD. In this study, we will be investigating branched chain amino acid (BCAA) metabolism, a catabolic pathway taking place within mitochondria that appears to be involved in development of parkinsonism, but has not been widely studied in this context. Our study focuses mainly on branched chain ketoacid dehydrogenase kinase (BCKDK), which, through phosphorylation of its substrate complex, inhibits branched chain amino acid metabolism. We link Parkinson’s Disease with BCAA metabolic dysregulation and explore the effects of BCKDK on metabolism and cellular function.

Structural basis of NINJ1-mediated plasma membrane rupture

Dai Lab

NINJ1, a 16 kDa plasma membrane protein, came to limelight recently as a mediator of plasma membrane rupture (PMR) in lytic cell death, a process previously thought to be a passive event. PMR is the final cataclysmic step during lytic cell death processes (e.g., pyroptosis) and is key to host-defense against pathogen infections. However, dysregulation of this process is also implicated in many inflammatory diseases and pathological conditions. Our goal is to understand the structural basis of NINJ1-mediated PMR. To achieve this, we started our purification optimizations on NINJ2 and extended the knowledge to purify NINJ1 later. NINJ2 is another protein that is highly similar with ~50% sequence identity to NINJ1, but it does not induce PMR. We purified and used the NINJ1/2 oligomers/filaments for structure studies using cryo-EM. Our results indicate that NINJ1/2 protomers in the filaments have a N-terminal amphipathic helix followed by two transmembrane helices. This amphipathic helix has likely undergone dramatic conformational rearrangements to pack adjacent to the other two TM regions seen in NINJ1/2 filaments. While this amphipathic helix in NINJ1 facilitates oligomerization necessary for PMR, similar arrangements in NINJ2 oligomers doesn’t induce PMR. Both NINJ2 and NINJ1 filaments have two antiparallel layers of respective protein chains. Interestingly, NINJ2 filaments are slightly curved and have a thin lipid bilayer trapped in between while NINJ1 filaments are relatively straight and contain only patches of lipid clusters from the outer leaflet of plasma membrane. Based on these details, we hypothesized that in PMR, a long sheet of NINJ1 “protofilament” assembled around a membrane bleb, or several shorter ones acting together, would make the membrane bleb become hydrophilic on all edges (i.e., “soluble”) and then fall off the plasma membrane. Indeed, we have seen many small membrane blebs in the negative staining of the NINJ1 sample that seem to fit this scenario. In contrast, no single such bleb was seen in the NINJ2 sample. A possible explanation is that the curvature of the NINJ2 oligomerization would prevent its assembly into a long filament on the relatively flat plasma membrane. Surprisingly, the cyto-protective effect of Glycine is also recently linked to inhibition of NINJ1 polymerization preventing PMR. We are currently investigating the structural basis of glycine inhibition of NINJ1 mediated PMR. Together, outcomes of the study would provide a complete, mechanistic understanding of this fundamentally important process, and might enable future development of therapeutics for various inflammatory diseases.

NMR identification of dynamin pleckstrin homology domain (PHD)- PIP2 interactions reveals mechanisms of membrane remodeling during endocytic vesicle scission

Buck Lab

The mechanoenzymatic GTPase dynamin catalyzes rapid vesicle scission during endocytosis. However, the mechanisms are incompletely understood. The cryo-EM resolution revolution widely expected to resolve dynamin-membrane interactions has failed due to inherent dynamics at the protein-membrane interface. Here, using NMR, we have identified the interactions of the dynamin pleckstrin homology domain (PHD) with its target phospholipid, phosphatidylinositol-4,5- bisphosphate (PIP2), under various conditions. Surprisingly, we find that PIP2 headgroup interactions and membrane insertion (curvature sensing) employ distinct structural elements in the PHD, occur independently of each other, and are acutely sensitive to membrane curvature. Our data reveal that with the progressively increasing curvature of the invaginated membrane neck that connects the nascent vesicle to the plasma membrane, the dynamin PHD dynamically switches orientation on the membrane surface eventually inserting into the hydrocarbon core of the bilayer to stably anchor the PHD. Stable PHD-membrane insertion is necessary to alleviate the auto-inhibition imposed by the PHD on dynamin helical self-assembly, and further couple force-generating conformational rearrangements originating at the distal G domain-BSE interface to the membrane to enable neck constriction and fission.

Fine-Tuning of Gi/o Signaling by RGS2 and 5 is Critical to Ensuring Normal Heart and Kidney Function

Osei-Owusu Lab

Essential hypertension is marked by functional changes in primary determinants that control arterial blood pressure, augmented peripheral vascular resistance and/or increased cardiac output. Increased cardiac output can be caused by defects in water and electrolyte homeostasis in the kidney parenchyma, thereby leading to sodium and water retention and blood volume expansion. RGS proteins act as GTPase activating proteins (GAPs) to fine-tune G protein signaling. Previous studies have shown that G protein dysregulation due to the absence of Rgs2, results in the impairment of cardiac structure and function, increasing susceptibility to hypertrophy and arrhythmia. To study the function and mechanism of RGS2 under various physiological and pathological conditions we generated Rgs2 KO and Rgsflag/flag mouse. In heart we found Echocardiographic assessment showed no difference in baseline cardiac structure between wild type (WT) and Rgs2 KO mice. At a low frequency of electrical field stimulation, ventricular myocytes from Rgs2 developed more arrhythmias than WT cells, and the difference was resolved by Gi/o blockade with pertussis. Cardiomyocytes Rgs2 also developed irregular excitation- contraction coupling and death in response to increasing concentrations of ?-adrenergic receptors with isoproterenol. cAMP production was decrease significantly in Rgs2 KO group. In addition, we found that Rgs deficiency impairs renal function and autoregulation by increasing renal vascular resistance and reducing renal blood flow. Western blotting showed the expression of RGS2 protein level was highly variable in different tissues. Moreover, the proteolytically unstable RGS2 mutant were also observed and the expression varies across tissues. Accordingly, these results suggest that RGS2 acts an essential role in the development of hypertension in both heart and kidney, suggesting that RGS2 is potential therapeutic target for hypertension and arrhythmia.

Roles of EphA2-ephrin Signaling in Prostate Development and Cancer Progression

Wang Lab

Prostate cancer (PCa) is the most common cancer in the US men. While usually indolent or benign, a small fraction (~5%) rapidly progress to malignant diseases. PCa is initially responsive to androgen deprivation therapy (ADT) or castration. However, aggressive forms of the disease inevitably become resistant to the therapy, leading progressively to metastatic castration resistant PCa (mCRPC), a fraction of which further progress to neuroendocrine prostate cancer (NEPC) and double negative PCa (DNPC). A major goal of PCa research is to broadly identify molecular and cellular mechanisms aiding nearly inevitable progression to identify vulnerabilities that could be targeted. Multiple receptor tyrosine kinases (RTKs) have been implicated in PCa. A significant body of literature points to an important role of EphA2, a member of the Eph subfamily of RTKs, in PCa. Notably as first reported by Chinnaiyan lab, EphA2 RTK is overexpressed in metastatic CRPC, but not early localized PCa tumors. In tumors where EphA2 is overexpressed, there is loss of the cognate ligand EphrinA1. In fact, Colm Morrissey was the first to discover that EphrinA1 is one of the top three genes whose expression is lost in metastatic PCa, particularly in bone metastases. The Wang lab, a leading group in studying Eph/Ephrin system in cancer biology, discovered that EphA2 has dual opposed roles during tumor development and progression, i.e., a ligand dependent tumor suppressor in the early stage of tumorigenesis and a ligand- independent oncogenic protein in the late stage tumor progression in several cancer types. Our preliminary data indicate that in PCa, EphA2-EphrinA signaling also has tumor suppressor role in early-stage PCa, and pro-oncogenic functions in metastatic CRPC. Excitingly, ongoing studies in our lab show potential for regulation of EphA2 expression by androgen receptor (AR) function. Our current hypothesis is that EphA2-ephrinA interaction plays a multifaceted regulatory role in prostate cancer (PCa) development and malignant progression toward late stage PCa. The outstanding questions are addressed by modeling PCa initiation and progression in the context of EphA2/EphrinA signaling using a murine prostate organoid system, a viral, in vivo spontaneous initiation of PCa termed RapidCap, and common PCa cell lines that have been previously characterized in the literature.

Receptor Protein Tyrosine Phosphatase localization in mouse renal proximal tubule, thick ascending limb and distal convoluted tubule

Boron Lab

Intracellular pH (pHi) regulation depends on extracellular pH (pHo), which is contingent on arterial pH (pHa). pHa is determined by the arterial [CO₂]/[HCO₃^-] ratio. Changes in the CO₂/HCO₃^- ratio alter pHa, directly affecting pHi and therefore cellular function. This ratio is regulated primarily by the lungs, where ventilation and gas exchange modulate [CO₂], and the kidneys, which adjust HCO₃^- transport and H+ secretion across membranes. The renal proximal tubule (PT) handles ~80% of acid secretion, which not only reabsorbs virtually all of the filtered HCO3?, but also excretes extra H+ that leads to generation of “new” HCO₃^-. Isolated PTs from mice lacking the receptor protein tyrosine phosphatase (RPTP -KO) are insensitive to acute changes in the basolateral [CO₂] or [HCO₃^-]. Furthermore, RPTP -KO mice have a deficiency in defending against whole-body metabolic acidosis. RPTP structural components also implicate it as a major [CO₂] or [HCO₃^-] sensor. RPTP possesses an extracellular region with high homology to canonical carbonic anhydrases, but which is catalytically inactive. We hypothesize that CO₂ or HCO₃^- binds to this carbonic-anhydrase-like domain (CALD) to elicit conformational changes in the RPTP protein that dictate RPTP dimerization state in the membrane. The signal effectors are the intracellular D1 and D2 phosphatase domains, with the inactive D2 domain from one protomer binding to the catalytically active D1 phosphatase domain of the other RPTP protomer to switch off phosphatase activity when RPTP dimerizes. We hypothesize that RPTP is localized in nephron segments important for regulating HCO3? transport/reabsorption, in the basal membranes that face the blood. To determine RPTP localization in WT mouse kidney cryosections, we use a chicken IgY anti-RPTP antibody (Ab) raised against an epitope within the extracellular FNIII domain, and co-stain with rabbit polyclonal Abs against the Na/HCO₃ cotransporter (NBCe1-A) to define PTs, the Na/K/Cl cotransporter 2 (NKCC2) to define thick ascending limb (TAL), and the Na/Cl cotransporter (NCC) as a distal convoluted tubule (DCT) marker. We further differentiate the classic DCT into early DCT (DCT1), late DCT (DCT2) and connecting tubule (CNT) using parvalbumin (PVB; exclusive to DCT1), and calbindin (CB), to denote CNTs. Confocal images of cryosections from at least four WT mice show RPTP and NBCe1A co-localization on PT basal membranes. Additionally, 56% of PTs also exhibit apical RPTP staining. We also observe both apical and basal RPTP staining in Bowman’s capsules. We observe RPTP staining in a minority of TALs, and at much lower intensity than observed in the PT. RPTP shows similar amount of apical staining in DCT1 and DCT2; however, a higher percentage of DCT1 tubules exhibit RPTP staining in their basal membranes compared to DCT2. In the CNT, RPTP primarily localizes in the apical membrane but is also present, to a lesser extent, in the basal membrane. The specificity of the anti-RPTP Abs is validated on RPTP -KO kidney sections. In conclusion, these data confirm RPTP expression in PT basal membranes, but also describe the first examples of apical RPTP expression in the PT and other renal epithelia.

Biophysical characterization of Raf RBD-CRD-KD interactions in the autoinhibited state

Buck Lab

The Raf family of signaling proteins is involved in cellular differentiation, proliferation, and growth, and are known to be mutated in certain cancers. They are activated by up-stream binding of Ras, an oncogenic protein mutated in more than 30% cancers. Raf is usually held in an inhibited state, either in conjunction with other proteins, such as 14-3-3, or via intramolecular interactions. These inhibitory interactions are released when Ras binds Raf domains. In particular, contacts with two Raf domains, the Ras-binding domain (RBD) and cysteine-rich domain (CRD), have been well characterized by NMR spectroscopy, X-ray crystallography, and recently by cryo-electron microscopy. However, possible interactions of the RBD-CRD with the Raf kinase domain (KD) in absence of the 14-3-3 protein are not understood. Using solution NMR, molecular modeling, and dynamics simulations, we have begun to study the possible interactions. We also utilize Alphafold2, a machine learning program which is revolutionizing structural biology due to the accuracy of its predictions. Microscale thermophoresis (MST) and surface plasmon resonance (SPR) are used to verify the regions of cRaf that bind to each other in conjunction with site directed mutagenesis. The ultimate goal is to better understand the regulatory mechanism of Raf, especially in the absence of its 14-3-3 binding partners and to find ways to stabilize the inhibited state.

Characterization of Human ‘AE4’ as an Electrogenic Na+-coupled HCO – Transporter

Boron Lab

Human ‘AE4’ (Anion Exchanger 4; GenBank ID: AAK16733) has 84% sequence identity to the previously cloned rabbit AE4 at the amino-acid level (Tsuganezawa et al, JBC 2001 and Parker et al, BBRC 2001). Although rabbit AE4 has been originally reported to be a Na+- independent Cl-HCO3 exchanger (i.e., AE), both human and rabbit orthologs have more sequence similarity to the Na+-coupled HCO3– transporter family (i.e., NBC)—particularly to the electrogenic NBCe1—than to the AE family. Many groups have tried to characterize this protein but they have reached discordant conclusions in terms of DIDS (well-known blocker of AEs and NBCs) sensitivity, cation dependency, and stoichiometry. On the other hand, they all concluded that AE4 is electroneutral. Although some studies (Tsuganezawa et al, Ko et al, AJP 2002, and Xu et al, AJP 2003) showed that this protein is cation-independent, more recent ones (Chambrey et al, PNAS 2012 and Peña-Münzenmayer et al, JBC 2015) reported that AE4 is Na+-dependent. Because this discrepancy may originate from use of different expression systems (e.g., recombinant vs. native) we decided to test human AE4 in a recombinant system. Using Xenopus oocytes, we investigated surface expression of AE4 as eGFP-fusion protein at the COOH terminus. When compared with NBCe1-A, which is well known to traffic to the plasma membrane, AE4 appeared to have serious defects in surface trafficking and glycosylation. However, we repeatedly observed ~10 mV hyperpolarization when we exposed oocytes to 5% CO2/33mM HCO3–. Because the extracellular loop 3 (EL3) of NBCe1-A is well known to have functional glycosylation sites, we engineered an AE4 clone with its EL3 replaced by that of NBCe1-A. Interestingly, this maneuver elicited greater expression of AE4 in both lysate and plasma membrane. In addition, we observed ~40 mV hyperpolarization upon exposure to CO₂/HCO₃^– and an increase in HCO₃^–-dependent slope conductance. All together these data suggest that AE4 may be an electrogenic Na+-coupled HCO₃^– transporter belonging to a new NBC family, namely NBCe3. To further investigate these data, we cloned 19 new variants of AE4 from human kidney cDNA and are currently testing if any of these variants have significantly better surface expression than the original AE4.

Structural Characterization of Novel Plexin–GTPase interactions

Buck Lab

Plexins along with their ligands semaphorins and co-receptors play crucial role in neuronal development, particularly in axon guidance, cytoskeletal re-arrangement and signal transduction. Plexin-mediated cell signaling is also critical to other processes involving cell migration, pathogenic angiogenesis, and immune responses. In the earlier work, Buck lab discovered a unique Rho GTPase binding domain (RBD) in plexins. The Rho-family regulatory GTPases interact with RBD carrying out plexin regulation in the intracellular environment. More recently the lab discovered another intracellular region in plexins–the juxtamembrane (JM) region which appears to interact tightly with Rho GTPases and mediate inhibition of GAP functions. The structural mechanism of the effects of these Rho GTPases on plexin signaling, however, remains elusive. In this study we used JM peptide constructs with GCN4 tag to probe their molecular interactions with Rho GTPases using an array of biophysical techniques–microscale thermophoresis (MST), and nuclear magnetic resonance (NMR) spectroscopy. We also carried out AlphaFold 2 (AF2) based predictions of these complexes. Our results indicate that JM peptides uniquely interact with Rho GTPases. Furthermore, we will obtain a detailed structural and sequence specific insights into these plexin JM-Rho GTPase interactions.

UNDERSTANDING THE STRUCTURAL INSIGHTS INTO EPHA2-HOMODIMERIZATION AND-MEMBRANE INTERACTIONS BY MOLECULAR DYNAMICS SIMULATIONs

Buck Lab

EphA2 plays a critical role in cellular growth, differentiation and motility. In line with EphA2 mRNA expression in multiple tissues and organs, its overexpression is reported in several different cancer types, and even in cancer-derived cell lines. EphA2 overexpression may have significance and could be used as a biomarker in the clinical management of cancer. Besides this, the differential EphA2 expression in normal versus cancer cells makes it a key therapeutic target. In EphA2, ligand binding regulates the monomer-dimer equilibrium through stabilization of the dimeric state by inducing a conformational change in the extracellular domain. Thus, dimerization is a key regulatory step in the activity and signaling process of EphA2. In EphA2, the juxtamembrane (JM) region follows the TM domain at one end and connects to a catalytic domain at the other. The JM domain functions in synergy with the TM domain for signal transduction. The JM domain has several basic residues (K/R) that are closely positioned to the membrane surface. Other signaling molecules specifically PIP2 and PIP3 utilize these basic amino acids for binding and occlude the nearby region from phosphorylation. Therefore, studying the structural mechanism of EphA2 dimerization and membrane interactions will help us better understand the activity and signaling of this receptor in different cancer types. Importantly, working with membrane proteins is extremely challenging. However, the recent developments in molecular dynamics simulation have provided a powerful tool to study membrane proteins and their interactions. Here, we modeled the relevant domains of the EphA2 receptor and studying its structural mechanism of activation for signaling by harnessing the power of advanced in-silico techniques. Our objective is to understand the structural basis of EphA2 dimerization and the role of the transmembrane region in signal transduction of EphA2 and also the characterization of protein-lipid bilayer interaction and their role in EphA2 regulation.

Glycine Receptors (GlyR) are chloride conducting ligand-gated ion channels that provide inhibitory input to the spinal cord.

Chakrapani Lab

Glycine Receptors (GlyR) are chloride conducting ligand-gated ion channels that provide inhibitory input to the spinal cord. They are composed of five subunits that come in two varieties, alpha and beta. Alpha subunit expression is necessary and sufficient for functional channels, but synaptic GlyR requires a beta subunit as it binds the scaffold protein gephyrin. Recent GlyR structures from our group and others revealed channel stoichiometry, subunit-specific structural features and the effects of a channel agonist (glycine), antagonist (strychnine), and allosteric modulator (ivermectin). Our work found ivermectin shows preferential binding to the alpha/beta interface, inducing an asymmetric pore conformation and molecular dynamic simulations suggest this state may be along the pathway of an open conformation. Together, these results expand our understanding and guide new hypotheses for this important neurotransmitter-gated ion channel.

N-methyl-D-aspartate receptors (NMDARs) are gated by the primary excitatory neurotransmitter, glutamate

Mu Lab

N-methyl-D-aspartate receptors (NMDARs) are gated by the primary excitatory neurotransmitter, glutamate. They play essential roles in neuronal formation, maturation, as well as central nervous system function. As such, the GRIN genes that encode the GluN subunits of NMDARs are highly intolerant to genetic variation and are thereby more likely to result in disease states. In this study, 25 mutations within the GluN2B subunit classified as pathogenic or likely pathogenic in the literature were generated for screening to determine their impacts on receptor homeostasis. In order for these receptors to gain their physiological function, they must first be properly folded and assembled within the endoplasmic reticulum and trafficked to the plasma membrane. Here, we expressed NMDARs containing select variants within the GluN2B subunits in HEK293T cells. Cells were harvested, lysed, and subjected to SDS-PAGE and Western blotting to determine the impact of variants on the expression and aggregation propensity of GluN2B subunits. After initial screening, variants that demonstrated decreased expression were transfected into HEK293T cells which were then treated with novel proteostasis regulators to determine if they could influence the expression of NMDARs. It was found, that variants within the GluN2B subunit differentially impact the expression of the receptors and select variants are more prone to forming aggregates within the cell. Further, treatment with proteostasis regulators showed significant increases in receptor expression containing loss-of-function variants. Together, these results indicate that disease-associated variants within the GluN2B subunit impair the overall expression and stability of these receptors. Additionally, the effects on receptor expression observed after treatment with the proteostasis regulators provide a potential targeted pharmacological therapeutic approach to restoring the expression and signaling of NMDARs containing GluN2B subunits harboring disease-associated variants.

Understanding the Structural Mechanisms of Glycine Receptors Expressed During Neurodevelopment

Chakrapani Lab

The glycine receptor alpha 2 (GlyRα2), an anionic-selective pentameric ligand gated ion channel, is involved in neurodevelopment and mediates tonic inhibition in the adult brain. Mutations in GlyRα2 have been linked to autism spectrum disorder and epilepsy. Functional studies of homomeric GlyRα2 have shown slow activation and desensitization kinetics compared to other GlyR subtypes, corresponding to their distinct role in the nervous system. However, the mechanistic differences between GlyRα2 and other GlyR subtypes is not understood. Furthermore, there is a lack of information regarding the diversity of orthosteric and allosteric ligands that alter the function of GlyRα2. We are investigating unexplored GlyRα2 structure-function relationship using cryogenic electron microscopy (Cryo-EM) and electrophysiology. Specifically, we are using Cryo-EM to analyze the extracellular domain (ECD) orthosteric binding pocket, coupling region to the transmembrane domains (TMD), channel pore, and TMD allosteric binding pockets in the presence of diverse ligands. These findings will inform the mechanisms of activation and desensitization and distinct pharmacology. These observations will be validated using patch-clamp electrophysiology of WT GlyRα2 and GlyRα2 channels with structurally relevant mutations. The recombinant full-length human GlyRα2 gene (GLRA2) has been expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus expression vector system. The protein has been solubilized in detergent and reconstituted in MSP1E3D1 lipid nanodiscs. Preliminary structural results have been obtained with detergent solubilized samples on graphene oxide coated grids in the presence of strychnine, a GlyR antagonist. Iterative rounds of 2D classification reveal a distribution of top, side, and tilted views of in-tact particles, which give rise to reconstructions at moderate resolution. Automated patch-clamp electrophysiology analysis of a similar recombinant GlyR exhibited glycine-induced currents in a dose-dependent manner. From this structure-function analysis and pharmacologic characterization, we can better understand the role of GlyRα2 in neurogenesis and neuropathology and set the stage for future therapeutic development.

Remodeling the endoplasmic reticulum proteostasis network rescues the pathogenic NMDA receptors

Mu Lab

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors, which are composed of two obligatory GluN1 subunits and two GluN2 subunits. Pathogenic NMDAR subunits result in their misfolding and inefficient assembly, limiting receptor expression and activity at the plasma membrane. Consequently, this leads to loss of their function and corresponding disease phenotypes. Genetic NMDAR dysfunction often causes impairments in synaptic plasticity and cognitive deficits in animal models. Importantly, genes encoding NMDAR subunits are remarkably intolerant to mutations and have been implicated in the onset of multiple neurological disorders such as epilepsy, autism, intellectual disability, and schizophrenia. We concentrate on the proteostasis maintenance of GluN2A-containing NMDARs, which is largely unknown in the literature. Proteostasis deficiency in NMDARs causes loss of their surface expression and thus function on the plasma membrane, leading to epilepsy and other neurological diseases. Well-characterized epilepsy-causing variants include the M705V and A727T mutations in the GluN2A subunit, which causes extensive protein misfolding in the endoplasmic reticulum (ER) and expedited degradation. We aimed to correct the misfolding of the GluN2A variants in the ER as an approach to restore their functional surface expression. Our results showed that the application of BIX, a specific and potent ER resident HSP70 family protein BiP activator, significantly increased the surface expression of the mutant receptors in human HEK293T cells. BIX attenuates the degradation of NMDARs and enhances their forward trafficking and function. Furthermore, we demonstrated that modest activations of the IRE1 pathway rather than ATF6 pathway of the unfolded protein response genetically enhance the plasma membrane trafficking of the NMDARs protein in HEK293T cells since BiP is one major target of IRE1 and ATF6. Our results showed that regulating the ER proteostasis network can restore the function of NMDAR variants, indicating that enhancing the folding, assembly, and trafficking of loss-of-function NMDAR variants has a potential for alleviating pathology associated with genetic epilepsy.

Investigating the role of phosphorylation on transition states of the 5-HT3A receptor

Chakrapani Lab

The 5-hydroxytryptamine type 3 receptor (5-HT₃R) is a pentameric ligand-gated ion channel with important roles in gut modulation and gut-brain communication. 5-HT₃Rs have also been linked to a variety of diseases, such as irritable bowel syndrome, obsessive compulsive disorder, and depression. This receptor can form homomeric receptors, made up of the 5-HT₃ A subunit, or heteromeric receptors, which must always contain 5-HT₃ A but will also contain 5-HT₃ B, C, D, and/or E subunits. The A subunit of the guinea pig 5-HT₃ receptor has been found to be phosphorylated at a serine conserved within the intracellular M3-M4 loop of both murine and human 5-HT₃ A subunits. This conserved serine is within a strong protein kinase A (PKA) consensus site (RXS). Potential upstream regulators of PKA activation, such as forskolin and cAMP, have been shown to increase rates of murine 5-HT₃R desensitization. Thus, PKA may be a direct phosphorylating agent of the 5-HT₃ A subunit and influence activation kinetics. Addition or removal of this phosphorylation within the 5-HT₃ A subunit may help shift the equilibrium of receptor transition states toward or away from a desensitized state. Through the use of cryogenic electron microscopy (cryo-EM), altering phosphorylation of the murine homomeric 5- HT₃AR and solving serotonin-bound structures will provide insight into the role of this post- translational modification on inducing a 5-HT₃R desensitized state.

Qi Lab

INTRODUCTION: Our previous findings established CAST-Drp1 as a druggable signaling axis in Huntington’s disease (HD) pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead molecule for developing HD therapies.

METHODS: Performing chemical optimization of CHIR99021 and cell-based functional screening, we identified A36 as a leading analogue of CHIR99201. To determine its therapeutic potential in vivo, we intraperitoneally injected A36 in R62 mouse model of HD, and examined the effect on HD-related neuropathology and motor deficits.

RESULTS: HD-related motor dysfunction in R62 mice was significantly rescued upon A36 treatment. We observed that the levels of medium spiny neuron and myelin volume in the brain significantly increased in the R62 mouse brain after A36 treatment. Moreover, A36 treatment significantly reduced mutant huntingtin inclusions and microglial activation.

DISCUSSION: We demonstrated the protective effect of A36 in HD pathogenesis. A36 may be a promising lead compound to treat HD.

ATAD3A is a critical protein for mitochondria quality control, however its role in the neural retina is poorly understood.

Qi Lab

ATAD3A is a critical protein for mitochondria quality control, however its role in the neural retina is poorly understood. Accumulating evidence has suggested that mutations in the ATAD3A gene cause vision loss. In this study, we explored the function of ATAD3A and how it regulates autophagy of mitochondria (mitophagy). Via immunohistochemistry we explored the impact of conditional knockout of ATAD3A in the projection neurons of the retina in a mouse model. Our preliminary results suggest cellular stress in the ATAD3A cKO retina, as microglia markers as well as mitochondrial biogenesis markers were increased. Additionally, TUNEL staining revealed rampant cell death in the inner nuclear layer of the cKO retina. In summary, ATAD3a is necessary for retinal neuron health and maintenance and its absence may lead to cell death and optic neuropathy.

Gasdermin D (GSDMD) plays a role in pyroptosis, a form of lytic cell death, and IL-1? release during inflammasome signaling which has been characterized previously in macrophages and other mononuclear myeloid leukocytes.

Dubyak Lab

Gasdermin D (GSDMD) plays a role in pyroptosis, a form of lytic cell death, and IL-1β release during inflammasome signaling which has been characterized previously in macrophages and other mononuclear myeloid leukocytes. This involves the cleavage of GSDMD and IL-1β by Caspase-1. The cleaved GSDMD then migrates to the plasma membrane (PM) where it can oligomerize, eventually forming a pore that allows the release of the cleaved IL-1β. Notably, granulocytic leukocytes, such as neutrophils and eosinophils, are also an important source of IL-1β and play a role in a number of innate immune responses and inflammatory diseases. However, there are only a few studies on GSDM- family roles in these types of cells. Our eosinophil studies so far have shown that they have the previously described inflammasome components, such as ASC and NLRP3, as well as pro-GSDMD, a precursor of the pore forming N-GSDMD. Furthermore we have seen that when stimulated with bacterial LPS, primary mouse eosinophils can express pro-IL-1β, which has been previously seen in other granulocytes, like neutrophils when undergoing the same stimulation. Our most recent data has shown that during inflammasome activation, primary murine eosinophils express, process and release IL-1β without the need of GSDM protein processing. Interestingly, even though these cells release IL-1β without GSDMD cleavage, our data shows that it is dependent on GSDMD expression for its release. Our data demonstrates that eosinophil’s inflammasome signaling and consequent IL-1β release follows a different mechanism of those seen previously in macrophages and neutrophils.

Gasdermin D (GSDMD) plays a role in pyroptosis, a form of lytic cell death, and IL-1? release during inflammasome signaling which has been characterized previously in macrophages and other mononuclear myeloid leukocytesA CHCHD6-APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

Qi Lab

The mechanistic relationship between amyloid-beta precursor protein (APP) processing and mitochondrial dysfunction in Alzheimer’s disease (AD) has long eluded the field. Here, we report that coiled-coil-helix-coiled-coil-helix domain containing 6 (CHCHD6), a core protein of the mammalian mitochondrial contact site and cristae organizing system, mechanistically connects these AD features through a circular feedback loop that lowers CHCHD6 and raises APP processing. In cellular and animal AD models and human AD brains, the APP intracellular domain fragment inhibits CHCHD6 transcription by binding its promoter. CHCHD6 and APP bind and stabilize one another. Reduced CHCHD6 enhances APP accumulation on mitochondria- associated ER membranes and accelerates APP processing, and induces mitochondrial dysfunction and neuronal cholesterol accumulation, promoting amyloid pathology. Compensation for CHCHD6 loss in an AD mouse model reduces AD-associated neuropathology and cognitive impairment. Thus, CHCHD6 connects APP processing and mitochondrial dysfunction in AD. This provides a potential new therapeutic target for patients.

TAD3A Knockout in Neurons Induces pathological changes of TDP-43 and Triggers Mitochondrial DNA Release to Contributes Motor Neurons Degeneration

Qi Lab

ATAD3A is a nuclear-encoded mitochondrial protein in the ATPase family. It is involved in diverse cellular processes, including mitochondrial dynamics, cell death and cholesterol metabolism. We have recently reported that ATAD3A plays a key role in neurodegeneration by linking Drp1- induced mitochondrial fragmentation to defective mtDNA maintenance. To study the neuron- specific function of ATAD3A, we generated inducible, neuron-specific Atad3a-deficient mice (Slick-Cre; ATAD3Af/f) and motor neuron-specific Atad3a-deficient mice (HB9-Cre; ATAD3Af/f). Both of these conditional knockout mice showed decrease bodyweight, developed behavioral abnormalities and have diminished life span. Moreover, immunostaining of the brain and spinal cord showed that astrocytes and microglials of Atad3a-deficient mice were hyper-activated in projection neuron and motor neuron. In vitro model, Atad3a deletion induced cytosolic release of mtDNA and activation of the cGAS/STING pathway. We also found that ATAD3A deficiency can induced the Pathobiology of TDP-43 CTF35 expression level. These results were also validate in ALS patient samples. Our findings reveal that neuronal ATAD3A plays an essential role in motor function and survival of mice, suggesting that ATAD3A is a potential therapeutic target for degenerative motor neuron disease.

TAD3A Knockout in Neurons Induces pathological changes of TDP-43 and Triggers Mitochondrial DNA Release to Contributes Motor Neurons Degeneration

Dubyak Lab

Pancreatic ductal adenocarcinoma (PDAC) is a deadly form of cancer, complicated by late diagnosis and difficulty penetrating tissue stroma with chemotherapeutics. Ferroptosis is a form of cell death of which the mechanisms are not well-characterized, but has been shown to propagate in a wave-like pattern through affected tissues. The purpose of this study is to characterize ferroptosis as a lytic form of cell death, initiated by the inhibition of glutathione peroxidase 4, a key enzyme in reducing reactive oxygen species. Additionally, this study aimed to investigate the role of ninjurin-1, a known mediator of plasma membrane rupture, in the ferroptotic death of PDAC cell lines. These cancerous epithelial cells were treated with known initiators of ferroptosis, erastin and RSL3, at increasing concentrations. A difference in sensitivity to ferroptotic initiators was noted between the PDAC cell lines MIA PaCa-2 and PANC-1, using the CellTiterGlo assay. Measured by GAPDH release into cellular supernatant, this form of cell death was determined to be lytic in nature. Treatment of the same cell lines with ninjurin-1 siRNA yielded no change in cell viability in response to initiators of ferroptosis.

Alzheimer’s disease (AD) is the most common form of dementia defined by the pathological deposition of amyloid-? plaques and neurofibrillary tangles containing hyperphosphorylated tau.

Qi Lab

Alzheimer’s disease (AD) is the most common form of dementia defined by the pathological deposition of amyloid-? plaques and neurofibrillary tangles containing hyperphosphorylated tau. Protein sorting is a subtly orchestrated process, critical for cell and tissue physiology, while on the other hand dysregulated protein sorting is also associated with AD pathologies. The coat protein complex II (COPII), which mediates anterograde trafficking from the endoplasmic reticulum to Golgi apparatus, is a highly conserved, key control point for protein sorting. However, whether it is functionally implicated in AD pathologies remains unknown. In this work, we detected transcriptional levels of different COPII components genes in brains samples from AD patients and AD mouse model, finding several genes in COPII system are dysregulated. Our results provide evidence on potential dysfunction of COPII system in the pathogenesis of AD.

The role of phosphorylation in LLPS of tau protein in vitro

Surewicz Lab

Objectives. Hyperphosphorylation is a key feature of tau isolated from the brains of patients with Alzheimer’s disease and other tauopathies. Recent reports demonstrated that tau can undergo liquid-liquid phase separation (LLPS). Here we aim to determine the relationship between tau phosphorylation and LLPS and the effect of phosphorylation on tau aggregation within liquid droplets.

Methods.The role of phosphomimetic substitutions within different regions of tau on protein’s capacity to undergo LLPS and aggregate in vitro was evaluated by using a combination of several methods including turbidity measurements, optical microscopy, fluorescence recovery after photobleaching, Thioflavin T fluorescence assay, and atomic force microscopy.

Results. We assessed the capacity of different phosphomimetic tau441 variants to undergo LLPS by comparing their saturation concentrations. Our data show that phosphomimetic substitutions within the proline-rich domain of tau441 inhibit LLPS, whereas substitutions within the C-terminal domain promote LLPS. Furthermore, we found that phosphomimetic substitutions influence the material properties of tau441 within the condensed phase, with those in the proline-rich region and the repeat domain increasing dynamic properties of the droplets and slowing down their maturation into more rigid structures. This correlates with the aggregation kinetics of phosphomonetic tau variants within the droplets.

Conclusions. This data indicates that the phosphorylation patterns that increase the polarity of charge distribution in tau441 promote protein LLPS, whereas those that decrease charge polarity has an opposite effect. Overall, this study further supports the notion that tau LLPS is driven by attractive electrostatic interactions between oppositely charged domains.

Cryo-EM structure of disease-related prion fibrils provides insights into seeding barriers

Surewicz Lab

Abstract: One of the least understood aspects of prion diseases is the structure of infectious prion protein aggregates. Here we report a high-resolution cryo-EM structure of amyloid fibrils formed by human prion protein with the Y145Stop mutation that is associated with a familial prion disease. This structural insight allows us not only to explain previous biochemical findings, but also provides direct support for the conformational adaptability model of prion transmissibility barriers.

Elastin Insufficiency Promotes Structural remodeling of the Renal Microvasculature Leading to Accelerated Age-related Impairment of Renal Hemodynamics

Osei-Owusu Lab

Gradual loss of functional elastin fibers during aging leads to increased arterial stiffness and decreased compliance. Elastin insufficiency in mice and humans has been shown to be associated with the development of hypertension and arteriopathy of large arteries, such as the aorta. However, it is unclear whether elastin insufficiency in the resistance vasculature, such as those of the kidney, contributes to age-related decline in renal function. Therefore, this project aims to determine whether elastin insufficiency is involved in age-related changes in the structure of the renal microvasculature and whether such changes contribute to impaired renal hemodynamics and function. We hypothesize that elastin insufficiency exacerbates age-induced impairment of renal hemodynamics by accelerating functional and structural changes to the renal microvasculature. To test this hypothesis, we assessed renal hemodynamics under anesthesia in young (4-6-month-old) and old (14–16-month-old) female wild-type (WT) and elastin heterozygous (Eln+/-) mice. Renal autoregulation was assessed by a stepwise increase in renal perfusion pressure (RPP) by simultaneously occluding the superior mesenteric and celiac arteries. Autonomic activity was clamped by continuous infusion of a cocktail containing and vasoactive hormones. Baseline systolic blood pressure (SBP; 91.1 ± 4.2 vs 90.1 ±3.1mmHg) and renal vascular resistance (RVR; 13.1 ± 2.4 vs 10.0 ± 1.5 mmHg/μL/min/g left kidney weight) was more elevated in old versus young WT mice. Additionally, Renal blood flow (RBF; 7.3 ± 1.7 ± 9.8 ± 1.4 μL/min/g left kidney weight), renal plasma flow (RPF; 5 ± 1.2 vs 6 ± 1 mL/min/g left kidney weight) as well as glomerular filtration rate (GFR), all trended lower in old WT mice compared to young WT mice. However, in Eln+/- mice, while SBP was lower in old mice (78.2 ± 9.4 vs 104.6 ± 4.9mmHg), RVR (13.5± 2.6 vs 14.4±1.1), RBF (6.7 ±1.2 vs 7.5 ± 0.6), RPF (4.2 ± 0.7 vs 4.5 ± 0.3) and GFR were all similar between young and old mice. An increase in RPP caused a sharp increase in RVR and decrease in RBF and RPF, with the response being more robust in young vs old WT mice. While GFR and filtration fraction (FF) increased after the occlusion in WT mice, old mice exhibited a greater response. GFR. Subsequently, the maximal changes in RBF (5.8 ± 0.6 vs 5.5 ± 1 μL/min/g left kidney weight), RVR (23.8 ± 2.5 vs 21.8 ± 7.0 mmHg/μL/min/g left kidney weight), urine flow rate, GFR, and FF were less robust and similar between young and old Eln+/- mice. Autoregulatory index analysis indicated greater renal dysfunction in young and old Eln+/- mice as well as old WT mice. This was further supported by correlation analysis which showed that a high pulse pressure had a more positive correlation to high RBF. There was no difference in kidney weight/tibia length ratio among the different groups. Therefore, our data demonstrate renal hemodynamics declines with age and that elastin insufficiency accelerates the age-induced impairment, possibly through functional and structural remodeling of the renal microvasculature. These findings provide some insights as to how the loss of elastin due to aging impacts the resistance arteries that play a critical role in blood pressure regulation.

Principles of scaffold interactions in multiphase liquid droplets: an evaluation of tau and TDP-43

Surewicz Lab Lab

Amyloid fibrils are a common pathological feature of several neurodegenerative diseases. They are formed by protein aggregation and their composition has been used to classify diseases such as tauopathies and TDP-43 proteinopathies. However, co-localization of tau and TDP-43 aggregates have been reported in Alzheimer’s disease. Interestingly, both proteins are able to undergo liquid-liquid phase separation (LLPS), a phenomena associated with protein aggregation. LLPS of tau is driven by electrostatic interactions, while TDP-43 LLPS is driven by hydrophobic interactions. Nevertheless, no information is available about the phase behavior of the mixture of these proteins, which we explore through fluorescence microscopy. Molecules driven LLPS are called scaffolds, while recruited components are called clients. We hypothesize that any of these two proteins could behave as client or scaffold, depending on protein concentration or inhibition of the interactions leading to their LLPS. We use high salt concentration to inhibit electrostatic interactions of tau, and hexanediol to prevent hydrophobic interactions of TDP-43. In conditions where only tau undergoes LLPS, TDP-43 is recruited into tau droplets. When only TDP-43 undergoes LLPS, tau is recruited into TDP-43 droplets. Similar arrangements are obtained by prevention of phase separation using low protein concentration or inhibiting specific interactions, which is the first evidence of intermolecular interactions tuning the scaffold-client behavior. Mixtures of proteins in conditions where both undergo LLPS lead to the formation of immiscible droplets, where TDP-43 droplets are engulfed by tau droplets due to differences in droplet surface tension. Lastly, we show that the recruitment of TDP-43 into tau droplets is mediated by electrostatic interactions. Altogether, we uncover principles of multilayered droplets that may be regulated in a cellular environment through post-translational modifications, such as phosphorylation and acetylation.

Maladaptation of renal hemodynamics contributes to kidney dysfunction following thoracic spinal cord injury in mice

Osei-Owusu Lab Lab

Abstract: Renal dysfunction is a major consequence of spinal cord injury (SCI). However, the mechanisms underlying renal dysfunction after injury are unclear, for which renal hemodynamics remain overlooked. In this study, we show that T3 (T3Tx) or T10 (T10Tx) complete thoracic spinal cord transection results in hypotension, bradycardia, and hypothermia immediately after injury. While SCI-induced hypotension in T3Tx mice (measured via radiotelemetry) gradually recovered to levels comparable to T10Tx and uninjured controls, while bradycardia and hypothermia did not. When assessing renal hemodynamic changes after SCI, we observed that during chronic injury, renal vascular resistance (RVR) response to a step increase in renal perfusion pressure or a bolus of angiotensin II or norepinephrine was almost completely lost after T3Tx SCI. Histological analysis showed renal interstitial and vascular elastin fragmentation after SCI increased and was most severe during chronic T3Tx SCI. Additionally, bulk RNA-sequencing showed enrichment of genes involved in chemokine signaling and extracellular matrix (ECM) remodeling in the kidneys from T3Tx SCI animals. Additionally, serum amyloid A1 levels were significantly increased during acute and chronic injury phases in T3Tx animals as well. In summary, tissue fibrosis and hemodynamic impairment contribute to renal dysfunction following thoracic SCI, being most severe during chronic high-thoracic SCI; our data indicate these alterations are at least partially mediated by remodeling of the renal microvascular ECM. Additional studies are required to further probe pathological mechanisms underlying renal microvascular impairment following thoracic SCI that is injury-level dependent.

Maladaptation of renal hemodynamics contributes to kidney dysfunction following thoracic spinal cord injury in mice

Tajima Lab Lab

Kainate glutamate receptors (KARs) facilitate fast excitatory neurotransmission in the mammalian brain by coupling the binding of pre-synaptically released L-glutamate to the opening of a transmembrane cation channel. KARs play an important role in vertebrate cognition, and their dysfunction is at the foundation of many disorders of the nervous system, such as schizophrenia, epilepsy, mood disorders, memory formation, and abnormal pain regulation. These receptors assemble in multiple homo- and hetero-tetramers within the post-synaptic density, resulting in a receptor complex with highly variable gating kinetics, pharmacology, and pore properties. The previous structural studies reported assemblies of GluK1, GluK2, GluK3 and GluK2/5 in resting, desensitized, and antagonized states, however the structural information pertaining to fully- and partially-active gating modalities is still lacking. The described pore architecture remains merely indicative of its’ functional state due to use of ligands with fast gating kinetics and the limited resolution, thus the cooperation between these epitopes still remains largely unresolved. In our work we aim to overcome these limitations by using domoate (DOQ), a powerful toxin and a partial agonist known to reduce KAR desensitization and slow gating kinetics. We combine cryo-EM and electrophysiology to derive the structures of the GluK2 complex in resting and DOQ-bound states, and we explain the unique conformations of their ligand-binding domains (LBD). We correlate these findings to the pore response to demonstrate how DOQ alters the gating ring and suspends the receptor in a semi-conductive state. By resolving multiple high- resolution structures ranging from 2.9 to 3.8 Å, we propose hot epitopes as potential therapeutic targets to disrupt detrimental attenuated desensitization and restore the affected receptors.

Gas Permeation through human AQP5

Boron Lab Lab

Movement of water across membranes in cells is facilitated by membrane channels named aquaporins (AQPs). They are ubiquitous and are involved in many crucial physiological roles. AQPs are homo-tetramers consisting of four monomeric or water pores (one pore per monomer for water conductance). Furthermore, tetrameric association generates an additional hydrophobic pore in the center (central pore). Besides water movement, gas transport through membranes plays an essential role in human physiology as well. Boron and coworkers published for the first time that the water channel aquaporin AQP1 plays an essential role in CO2 conductance through membranes. There has still been an ongoing debate, whether CO2 conductance occurs through the monomeric (water pores) or the central pore of AQPs. To get more insight into possible gas conductance through the central pore of human AQP5, residues T41 or/and L43 at the extracellular mouth of the central pore were mutated to H. Physiological data of human AQP5 T41H, L43H and T41H/L43H mutants in the presence of Ni²⁺ and Zn²⁺, as well as crystallographic and Molecular Dynamic (MD) Simulations data of the T41H mutant with Ni²⁺ prove that the four/eight histidine residues of the tetrameric AQP5 T41H, L43H and T41H/L43H mutants bind those divalent cations, leading to an occlusion of the central pore and, consequently, reducing/preventing gas (CO₂) permeation through it. Our structure of AQP5 T41H in complex with Ni²⁺ is the first example of an inhibitor blocking the central pore.

Gas Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling

Osei-Owusu Lab Lab

Abstract: G protein signaling is involved in many cardiovascular mechanisms, including the “fight or flight” response. Initiation of these signaling cascades must be tightly regulated to ensure the correct length and magnitude of the response to stimulation. Loss or impairment of this regulation has been implicated in disorders like hypertension and cardiac arrhythmia. Regulators of G protein Signaling (RGS) accelerate the termination of G protein signaling. The R4 subfamily of small RGS proteins are highly expressed throughout the cardiovascular system. These proteins accelerate termination of Gαi and Gα_q/11 signaling. A new area of study in regards to RGS proteins is identifying whether the cardiac expression of multiple isoforms of RGS proteins is functionally redundant, or if multiple isoforms coordinate their regulation in a synergistic or additive manner. To elucidate this, we generated Rgs2^-/-/Rgs5^-/- mice (double knock out (dbKO). Using isolated left ventricular cardiomyocytes (LVCM) from wild type (WT), Rgs2 KO, Rgs5 KO, and dbKO male mice we examined the role of R4 proteins in protection from β- adrenergic stimulation induced arrhythmia. Isoproterenol induced arrhythmia in a similar percentage of CMs from Rgs2 and Rgs5 KO mice, while dbKO CMs displayed the highest rate of arrhythmia. This is likely partially due to the high level of basal intracellular Ca²⁺ seen only in the dbKO LVCMs. We found that forskolin-induced cAMP production was suppressed in LVCMs from Rgs5 KO males to a lesser extent than from Rgs2 KO and dbKO males. From these results we conclude that RGS2 and RGS5 display unique regulatory roles in regards to Gαi signaling.