• Laboratory-Observed Behavioral Disinhibition in the Young Offspring of Parents With Bipolar Disorder: A High-Risk Pilot Study

      Hirshfeld-Becker, Dina R.; Biederman, Joseph; Henin, Aude; Faraone, Stephen V.; Cayton, Gabrielle A.; Rosenbaum, Jerrold F. (American Psychiatric Association Publishing, 2006-02)
      Objective: This study tested whether behavioral disinhibition is more prevalent among offspring of parents with bipolar disorder than among offspring of parents without bipolar disorder. Method: The authors conducted a secondary analysis of data from a preexisting high-risk study of offspring at risk for panic disorder and depression (N=278) that had included some children with parents who had bipolar disorder (N=34). Children (ages 2–6) had been classified as behaviorally inhibited, disinhibited, or neither in laboratory assessments. Results: Offspring of bipolar parents had significantly higher rates of behavioral disinhibition than offspring of parents without bipolar disorder. Behavioral inhibition did not differ between groups. Differences were not accounted for by parental panic disorder or major depression or by parental history of attention deficit hyperactivity disorder, conduct disorder, antisocial personality, or substance use disorders. Conclusions: Results suggest a familial link between bipolar disorder in parents and behavioral disinhibition in their offspring. Behavioral disinhibition may be a familially transmitted predisposing factor for dysregulatory distress later in life.
    • Lack of Association Between Behavioral Inhibition and Psychosocial Adversity Factors in Children at Risk for Anxiety Disorders

      Hirshfeld-Becker, Dina R.; Biederman, Joseph; Faraone, Stephen V.; Segool, Natasha; Buchwald, Jennifer; Rosenbaum, Jerrold F. (American Psychiatric Association Publishing, 2004-03)
      Objective: In a previous controlled study of offspring at risk for anxiety disorders, the authors found that parental panic disorder with comorbid major depression was associated with child behavioral inhibition, the temperamental tendency to be quiet and restrained in unfamiliar situations. To explore whether this association was mediated by environmental factors, the authors examined associations between psychosocial adversity variables and behavioral inhibition in this group of children. Method: Subjects included 200 offspring of parents with panic disorder and/or major depression and 84 comparison children of parents without mood or anxiety disorders. Behavioral inhibition was assessed through laboratory observations. The associations between behavioral inhibition and the following psychosocial factors were examined: socioeconomic status; an index of adversity factors found in previous studies to be additively associated with child psychopathology; family intactness, conflict, expressiveness, and cohesiveness; exposure to parental psychopathology; sibship size; birth order; and gender. Results: The results showed no associations between behavioral inhibition and any of the psychosocial factors in the study group as a whole, despite adequate power to detect medium effect sizes. Among low-risk comparison children only, some definitions of behavioral inhibition were associated with low socioeconomic status, low family cohesion, and female gender. Conclusions: The results suggest that the psychosocial adversity factors examined in this study do not explain the previous finding that offspring of parents with panic disorder are at high risk for behavioral inhibition.
    • Lack of Association Between Parental Alcohol or Drug Addiction and Behavioral Inhibition in Children

      Biederman, Joseph; Hirshfeld-Becker, Dina R.; Rosenbaum, Jerrold F.; Perenick, Sarah G.; Wood, Julia; Faraone, Stephen V. (American Psychiatric Association Publishing, 2001-10)
      Objective: “Behavioral inhibition to the unfamiliar” has been proposed as a precursor to anxiety. A recent study proposed that it may also be a precursor to alcoholism. The authors sought to replicate the latter finding through a secondary analysis of data from a large study of young children (age 2–6 years)—offspring of parents with panic and depressive disorders—who had been assessed for behavioral inhibition through laboratory-based observations. Method: The offspring were stratified on the basis of presence or absence of parental lifetime history of DSM-III-R alcohol dependence (N=115 versus N=166, respectively) or drug dependence (N=78 versus N=203). The rates of behavioral inhibition were then compared between groups. Results: Despite adequate power to detect associations, neither parental alcohol dependence nor drug dependence was associated with a higher risk for behavioral inhibition in the offspring. Conclusions: These results are not consistent with the hypothesis linking behavioral inhibition to addictions
    • Laminins regulate retinal angiogenesis

      Brunken, William J.; Biswas,Saptarshi (2017)
      Vascular pathologies are the leading causes of acquired blindness in the developed world. While many studies sought to unravel cell-intrinsic and growth factor-mediated regulations of angiogenesis, it is only recently that the role of the basement membrane (BM) components in angiogenesis began to be explored. Several diseases with ocular manifestations are known to alter vascular BM compositions. Therefore, a detailed knowledge of the BM-mediated signals that regulate angiogenesis is of great importance. Laminins, a critical component of the BM, have been shown to regulate several aspects of angiogenesis in the retina. Our laboratory previously demonstrated that the laminin composition of the inner limiting membrane (ILM) regulates astrocyte migration, and consequently vascular expansion along the retinal surface. Here, I examined the role of γ3- and β2-containing laminins in two specific aspects of angiogenesis: 1) vascular branching and endothelial cell proliferation in the nascent vascular plexus, and 2) arterial morphogenesis in the remodeling zone. Results presented in Chapter 2 and Appendix 1 demonstrate that laminin composition of the BM is a critical regulator of microglial recruitment to the growing nascent plexus, where microglia facilitate vascular branching. Furthermore, microglia interact with the astrocyte-derived layer of the vascular BM, and that this interaction regulates iii microglial activation. The activation state of microglia, in turn, regulates endothelial cell proliferation. Results presented in Chapter 3 and Appendix 2 demonstrate that vascular BM laminins are critical regulators of arterial morphogenesis. Specifically, my results reveal a novel mechanism where γ3- containing laminins signal through dystroglycan to induce Dll4/Notch signaling in arterial endothelial cells, regulating proper arterial morphogenesis. Finally, in Appendix 3, I examined the coordinated expression of different laminin chains in the vascular BM. My preliminary results suggest that expressions of laminin α2-, α5- and γ3-chains in the retinal vascular BM are coordinately regulated with the expression of laminin β2-chain. In conclusion, this study sheds light on hitherto unexplored mechanisms by which BM laminins regulate retinal vascular development.
    • Linkage of chromosome 13q32 to schizophrenia in a large veterans affairs cooperative study sample

      Faraone, Stephen V.; Skol, Andrew D.; Tsuang, Debby W.; Bingham, Stephen; Young, Keith A.; Prabhudesai, Sarita; Haverstock, Susan L.; Mena, Felicitas; Menon, Aerath Sri Kumar; Bisset, Darren; et al. (Wiley, 2002-07-29)
      Several prior reports have suggested that chromosomal region 13q32 may harbor a schizophrenia susceptibility gene. In an attempt to replicate this finding, we assessed linkage between chromosome 13 markers and schizophrenia in 166 families, each with two or more affected members. The families, assembled from multiple centers by the Department of Veterans Affairs Cooperative Studies Program, included 392 sampled affected subjects and 216 affected sib pairs. By DSM-III-R criteria, 360 subjects (91.8%) had a diagnosis of schizophrenia and 32 (8.2%) were classified as schizoaffective disorder, depressed. The families had mixed ethnic backgrounds. The majority were northern European-American families (n = 62, 37%), but a substantial proportion were African-American kindreds (n = 60, 36%). Chromosome 13 markers, spaced at intervals of approximately 10 cM over the entire chromosome and 2-5 cM for the 13q32 region were genotyped and the data analyzed using semi-parametric affected only linkage analysis. For the combined sample (with race broadly defined and schizophrenia narrowly defined) the maximum LOD score was 1.43 (Z-score of 2.57; P = 0.01) at 79.0 cM between markers D13S1241 (76.3 cM) and D13S159 (79.5 cM). Both ethnic groups showed a peak in this region. The peak is within 3 cM of the peak reported by Brzustowicz et al.
    • Liver-specific glucocorticoid action in alcoholic liver disease

      Lu, Hong; Wang, Yazheng (2021)
      The number of deaths due to alcoholic liver disease is increasing every year. Glucocorticoids (GCs) are the only first-line drugs for alcoholic hepatitis (AH) treatment but have limited efficacy. Long-term high-dose GC use can cause various side effects on extrahepatic tissues, such as immunosuppression and neuromuscular side effects, which may be a limiting factor for GC treatment of AH. Therefore, liver-specific GC-targeted therapy may have multiple advantages compared with systemic GC for AH. This research explored the role of liver-specific deficiency of glucocorticoid receptor (GR) in AH induced by a high-fat diet (HFD) plus ethanol binge. Females are less prone to AH induced by HFD plus acute binge drinking, likely due to sex differences in estrogen (E2) signaling. We found that hepatic GR deficiency worsened steatosis in both genders of AH mice but only aggravated the liver injury in male AH mice. Multiple signaling pathways were dysregulated in GR knockout AH mice. Interestingly, hepatic expression of estrogen receptor (ERα) was induced, and the E2-inactivating enzyme was markedly down-regulated in GR knockout AH mice, suggesting enhanced E2 signaling in these mice. Our data mining found marked dysregulation of many GR-target genes important for lipid catabolism, cytoprotection, and inflammation in patients with severe AH. These key GR-target genes were similarly induced or down-regulated by our liver-targeting GC prodrugs and the parent drug at 1μM in primary human hepatocytes. In contrast, GC prodrugs had much weaker inhibitory effects than the parent drug on LPS-induction of IL-1B in mouse macrophages, suggesting a good liver selectivity of our liver-targeting GC prodrugs. The ultimate goal of this study is to determine the mechanistic role of GR in alcoholic fatty liver disease and develop targeted drug therapies to treat alcoholic hepatitis.
    • Localization of sarcomeric proteins during muscle assembly in mouse cardiomyocytes and skeletal myotubes

      Sanger, Jean; Sanger, Joseph; Welchons, Matthew J (2017)
      This study seeks to investigate the role of contractions in myofibrillogenesis, and structure of nascent myofibrils. The model system employed in these experiments was cultured quail myotubes. In order to determine the role of contractions in myofibrillogensis, contractions were indirectly inhibited with elevated KCl, and directly inhibited with 2,3 butanedione monoxime (BDM), a small cell-permeable inhibitor of actin & muscle myosin interactions. Myotubes were treated with contraction inhibitors at 2½days –soon after the main fusion event. On the 6thculture day, there was significant delay in myofibrillogensis in myotubes exposed to elevated KCl. This delay was characterized by the expansion of nascent myofibrils at the spreading edges of myotubes. This delay in myofibrillogenesis was not accompanied by diminished accumulations of muscle myosin. On the 4thculture day, there was complete arrest of myofibrillogenesis at the nascent step with the treatment of BDM. As a result, it is concluded that contractions are necessary for the progression of nascent myofibrils to mature myofibrils. The structure of nascent myofibrils was further investigated with super resolution microscopy. Structured illumination microscopy (SIM) and stimulated emission depletion (STED) microscopy revealed mini-A-bands –shorter than 1.5 μmin length –associated with nascent myofibrils. These structures were spaced at varying intervals, and oriented at angles deviating the from the actin superstructure of the nascent myofibril. Mini-A-bands were also observed to progressively in expand in length distal to the spreading edges of myotubes. STED imaging indicates that some mini-A-band are adjacent to, and not integrated within, the actin superstructure of nascent myofibrils.
    • Long-term outcome of pediatric obsessive-compulsive disorder: a meta-analysis and qualitative review of the literature

      Stewart, S. E.; Geller, D. A.; Jenike, M.; Pauls, D.; Shaw, D.; Mullin, B.; Faraone, Stephen V. (Wiley, 2004-07)
      Objective: To review the extant literature on the long-term outcome of child/adolescent-onset obsessive–compulsive disorder (OCD). Method: Medline and Psychlit databases were systematically searched for articles regarding long-term outcomes of child/adolescent-onset OCD. Meta-analysis regression was applied to evaluate predictors and persistence of OCD. Results: Sixteen study samples (n ¼ 6–132; total ¼ 521 participants) in 22 studies had follow-up periods ranging between 1 and 15.6 years. Pooled mean persistence rates were 41% for full OCD and 60% for full or subthreshold OCD. Earlier age of OCD onset (z ¼ )3.26, P ¼ 0.001), increased OCD duration (z ¼ 2.22, P ¼ 0.027) and inpatient vs. out-patient status (z ¼ 2.94, P ¼ 0.003) predicted greater persistence. Comorbid psychiatric illness and poor initial treatment response were poor prognostic factors. Although psychosocial function was frequently compromised, most studies lacked comprehensive outcome measures. Conclusion: Long-term persistence of pediatric OCD may be lower than believed. Future studies should include broader measures of outcome including symptomatic persistence and functional impairment in multiple domains.
    • Machine Learning And MRI-Based Diagnostic Models For ADHD: Are We There Yet?

      Zhang-James, Yanli; Hoogman, Martine; Franke, Barbara; Faraone, Stephen V. (Cold Spring Harbor Laboratory, 2020-10-23)
      Machine learning (ML) has been applied to develop magnetic resonance imaging (MRI)-based diagnostic classifiers for attention-deficit/hyperactivity disorder (ADHD). This systematic review examines this literature to clarify its clinical significance and to assess the implications of the various analytic methods applied. We found that, although most of studies reported the classification accuracies, they varied in choice of MRI modalities, ML models, cross-validation and testing methods, and sample sizes. We found that the accuracies of cross-validation methods inflated the performance estimation compared with those of a held-out test, compromising the model generalizability. Test accuracies have increased with publication year but were not associated with training sample sizes. Improved test accuracy over time was likely due to the use of better ML methods along with strategies to deal with data imbalances. Ultimately, large multi-modal imaging datasets, and potentially the combination with other types of data, like cognitive data and/or genetics, will be essential to achieve the goal of developing clinically useful imaging classification tools for ADHD in the future.
    • Machine-Learning Prediction of Comorbid Substance Use Disorders in ADHD Youth Using Swedish Registry Data

      Zhang-James, Yanli; Chen, Qi; Kuja-Halkola, Ralf; Lichtenstein, Paul; Larsson, Henrik; Faraone, Stephen V. (Cold Spring Harbor Laboratory, 2019-06-06)
      Background: Children with attention-deficit/hyperactivity disorder (ADHD) have a high risk for substance use disorders (SUDs). Early identification of at-risk youth would help allocate scarce resources for prevention programs. Methods: Psychiatric and somatic diagnoses, family history of these disorders, measures of socioeconomic distress, and information about birth complications were obtained from the national registers in Sweden for 19,787 children with ADHD born between 1989 and 1993. We trained (a) a cross-sectional random forest (RF) model using data available by age 17 to predict SUD diagnosis between ages 18 and 19; and (b) a longitudinal recurrent neural network (RNN) model with the Long Short-Term Memory (LSTM) architecture to predict new diagnoses at each age. Results: The area under the receiver operating characteristic curve (AUC) was 0.73(95%CI 0.70–0.76) for the random forest model (RF). Removing prior diagnosis from the predictors, the RF model was still able to achieve significant AUCs when predicting all SUD diagnoses (0.69, 95%CI 0.66–0.72) or new diagnoses (0.67, 95%CI: 0.64, 0.71) during age 18–19. For the model predicting new diagnoses, model calibration was good with a low Brier score of 0.086. Longitudinal LSTM model was able to predict later SUD risks at as early as 2 years age, 10 years before the earliest diagnosis. The average AUC from longitudinal models predicting new diagnoses 1, 2, 5 and 10 years in the future was 0.63. Conclusions: Population registry data can be used to predict at-risk comorbid SUDs in individuals with ADHD. Such predictions can be made many years prior to age of the onset, and their SUD risks can be monitored using longitudinal models over years during child development. Nevertheless, more work is needed to create prediction models based on electronic health records or linked population registers that are sufficiently accurate for use in the clinic.
    • A matter of life and death: human cytomegalovirus induction of monocyte survival and differentiation into macrophages through manipulation of the PI3K/Akt pathway

      Chan, Gary; Cojohari, Olesea (2017)
      Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus infecting up to 80% of the US population and reaching 100% seroprevalence in many parts of the world. In mostindividuals HCMV infection is usually asymptomatic. In contrast, in immunodeficient or immunonaive people, such as transplant recipients and the developing fetus, the virus is a major cause of morbidity and mortality. During a primary infection, HCMVcan spread very effectively in the body infecting many organ types and monocytes are believed to be the principal cell type responsible for HCMV dissemination throughout the body. Monocytes, however, are naturally programmed to undergo apoptosis after 48h in the circulation and are not permissive for viral replication. Our lab has shown that in order to combat these biological hurdles, HCMV promotes survival of these short-lived cells past their 48h “viability gate”. Besides inducing survival, the virus also mediates the differentiation of monocytes into macrophages skewed towards an M1 pro-inflammatory phenotype with select M2 anti-inflammatory features, which are long-lived cells, permissive for viral replication. However, the mechanisms used by HCMV to concomitantly induce survival and macrophage differentiation -two linked but separate processes, are not fully understood. The studies in this thesis reveal that upon binding and entry, HCMV initiates a survival program in monocytes by inducing a rapid and sustained activation of the PI3K/Akt pathway, which isdifferent from that induced by myeloid growth factors. Moreover, after inducing cellular survival across the 48-h viability gate, the virus also employsthe PI3K/Akt pathway to regulate caspase 3 activation which mediatesthe atypical M1/M2 polarization. Our work suggests that virus not only makes use of the PI3K/Akt pathway, but manipulates it at multiple levels toallow for viral-specific downstream functional changes.Deciphering how the virus uniquely maneuvers signaling pathways in monocytes to drive their survival and differentiation might allow us to develop new treatments targeting HCMV-infected monocytes and preventing viral spread and disease.
    • MECHANISM OF ANT1-INDUCED HUMAN DISEASES AND STRESS SIGNALING

      Liu, Yaxin (2016)
      Adenine nucleotidetranslocase(Ant) is a mitochondrial inner membraneprotein, the primaryfunction of which is to mediate the ADP/ATP exchange acrossthe inner membrane. Missense mutations in Ant1, the skeletal muscle-and heart-specificisoform, induce human disordersincluding autosomal dominant ProgressiveExternalOpthalmoplegia(adPEO), cardiomyopathyand myopathy. Several models were proposed to interpret the pathogenesis of mutant Ant1-induced diseases, but no consensus has been reached.Our lab has previouslyfound that mutant Aac2, the homologof Ant1 in yeast, causes cell death due to the mitochondrialbiogenesis defect. In the present study, we provided biochemical evidencesupporting the idea that themutant Aac2 proteins are misfolded, which derailsthe proteostasis on the inner membrane.We found that the assemblyand stability of multipleprotein complexeson the inner membrane are affected, including those involved in mitochondrialrespirationand protein transport. In human cells, the mutant Ant1 proteinshave reduced steady-state levels and increased degradation, consistent with misfolding and increased susceptibility to protein quality control machineries. In the second part of thework, we sought to identify the cellularsignalingpathways that respond to Ant1-induced proteostatic stress on the mitochondrialinner membrane. We generatedANT1alleles that have enhanced toxicity and are able to induce proteostatic stress on the inner membrane in human. Although these mutant Ant1 proteins only mildly affectmitochondrialrespiration, they trigger robust transcriptional responses in the nucleus to remodel cellular signaling. The upregulationof the cytosolic chaperone/ubiquitin-proteasome systems, and the downregulation of mTOR signaling may serveas adaptiveresponses to mitigate mitochondrial PrecursorOver-AccumulationStress (mPOS), a novel pathway of cell death recently discovered in yeast. We also found that humancells respond to mitochondrialinner membrane stress by increasingEgr1 signaling and the alternative splicing of many genes important for cell survival/deathcontrol. Insummary, the data suggested that protein misfoldingcauses Ant1-induced pathogenesisin human diseases. Our study provided support for mitochondria-induced proteostatic stress in the cytosol (or mPOS) in human cells. We also identified several novel mitochondria-to-nucleus signaling pathways, which may help in developingtherapeutic interventionsfor the treatmentof mitochondria-induced pathologies.
    • Mechanism of gene regulation of HNF4α

      Lu, Hong; Guo, Shangdong (2018)
      Hepatocyte Nuclear Factor 4 alpha(HNF4α)is a masterregulatorthat modulatesthe liver development andfunction. The dysfunction of HNF4αcauses multiplehumandiseases, such as hepatocellular carcinoma(HCC) and maturity onset diabetes of the young1 (MODY1). Incontrast,the restoration of HNF4α can inhibit the development of liver cancer and improve the liver function simultaneously.However, HNF4αis anorphan nuclear receptor whose activating ligand remains elusive. Therefore, an alternative approach to enhance the HNF4αactivity is to up-regulate the proteinexpression.While a great progress has been made on the functional study of HNF4α,the mechanistic details regarding the gene regulationofHNF4αare still a vast knowledge gap. The present study was aimed to investigatethe mechanism of gene regulation ofHNF4αsystematically. In chapter 2 and chapter 3, we reported the strong translational inhibition of both humanand mouse HNF4αinduced by the nucleicacid secondary structuretermed “G-quadruplex”(G4)within the 5` untranslated region(UTR).By performing the deletion/mutation studies, we determined the compositionof the G4in HNF4A 5`UTR. We further speculated thatthis G4 required the adjacent cis-elements, such as the RNA-binding proteins and other secondary structures, to form a conjunction for the strong translational inhibition. We for the first time reported the RNA-G4 induced translational repression within the 5`UTR of a tumor suppressor and highlighted the significanceof the “biostability”of G4s in exerting their biological functions. In chapter 4, we conductedacomprehensivestudy that coveredthe auto-regulation, transcriptionalregulation and transactivation activity of HNF4α. By creatingvariousreporter constructs, we were able to validate the self-stimulation of HNF4αand discovered the strong correlation between HNF4αand its corresponding anti-sense RNA, HNF4A-AS1. Additionally, we identified novel HNF4αmutations such as Q277X that may affect the crosstalk of HNF4αwith other transcriptionfactors.Overall, the novel findings from our study shedlight on the gene regulation of HNF4αand providefurther insights into ourultimate goal that is to up-regulate HNF4αprotein expression/activity to treat human diseases.
    • Mechanisms of aseptic loosening in total knee arthroplasty

      Cyndari, Karen (2017)
      Introduction: Cemented Total Knee Arthroplasty (TKA) is the gold standard of care for end-stage, multi-etiologic arthritis. While the longevity of these devices may now reach or even surpass 15 years in service, a minority (~10%) will fail prematurely due to a process called aseptic loosening. Historically, this process has been attributed to an inflammatory reaction against wear debris from the TKA polyethylene (PE) insert. However, we have previously estimated supraphysiologic fluid shear stress (FSS) (exceeding 900 Pa) at the cement-bone interface of cemented joint replacements, and examined this as a possible alternative cause of increased osteoclast activity. Methods: We analyzed the cement-bone interlock of tibial and femoral components from en-bloc, postmortem-retrieved, non-revised TKAs to explore the process of loss of fixation, prior to any clinical loosening. For the tibial components, we used a novel protocol wherein whole undecalcified bone+PMMA cement segments from the proximal tibia were embedded in Spurr’s resin and thinly sectioned. Polarized light microscopy was used to identify and quantify co-located PE debris. Using a novel bioreactor developed by our lab called the Multi-Well Fluid Loading (MFL) System, we examined static, subphysiologic, physiologic, and supraphysiologic FSS on RAW 264.7 osteoclast activity and morphology, with and without PE particle treatment. Results: We found no association been the amount or presence of PE debris and the amount or location of loss of interlock in retrieved TKAs. FSS up to 17 Pa increased the ability of osteoclasts to resorb mineral, and FSS up to 4.4 Pa induced the formation of larger osteoclasts. FSS and fluid shear rate interacted together to increase the area of actin rings, while PE treatment increased the number of actin rings and TRAP production. FSS up to 4.4 Pa decreased expression of Ctsk and Il1a, but PE co-treatment abolished this effect. Conclusions: These results indicate there may be alternative factors leading to aseptic loosening apart from PE debris. We demonstrated that osteoclasts are mechanosensitive and able to adjust activity, morphology, and gene expression based on FSS. Further, PE interferes with osteoclast gene downregulation in response to FSS, indicating PE could be a potentiator of osteoclast activity or differentiation.
    • MECHANISMS OF WISKOTT ALDRICH SYNDROME PROTEIN WSP1 POSITIONING AND REGULATION AT SITES OF ENDOCYTOSIS IN S. POMBE

      Sirotkin, Vladimir; Macquarrie, Cameron Dale (2020-12-30)
      Branched actin networks nucleated by the Arp2/3 complex provide force needed to carry out endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including Wiskott-Aldrich Syndrome protein WASp. The WASp Interacting Protein WIP binds WASp, protecting it from degradation. Humans with mutations disrupting this interaction develop a serious immune disorder, Wiskott-Aldrich Syndrome. However, in the fission yeast S. pombe WASp homolog Wsp1 remains stable in the absence of WIP homolog Vrp1, providing an ideal environment to study additional WASp and WIP functions. In fission yeast, Wsp1, Vrp1, and the class-1 myosin Myo1 localize to sites of endocytosis, known as actin patches. Wsp1 and Myo1 play an important role in activating the Arp2/3 complex and initiating the actin network needed to internalize endocytic vesicles. S. pombe endocytosis is a rapid, reproducible event involving over 40 proteins. While several of these proteins are throught to regulate branched actin assembly, many still have poorly defined functions. Importantly, how WASp proteins are regulated at sites of endocytosis remains unclear. The following studies explore mechanisms of Wsp1 regulation using quantitative live cell imaging. In Chapter 2, we observed Wsp1, Vrp1, and Myo1 forming a transient complex near the membrane, positioning branched filaments in a way that optimizes force generation. In Chapter 3, we explored the role of WIP homolog Vrp1 in actin assembly and discovered the Vrp1-Wsp1 interaction is essential for Wsp1-mediated branched actin assembly. In Chapter 4, we examined how a separate module of endocytic coat proteins contributes to actin patch assembly and discovered the coat protein Sla1 inhibits Wsp1 NPF activity, after the endocytic vesicle begins to internalize. In Chapter 5, we examined how Wsp1 and Myo1 impact additional endocytic modules and discovered Wsp1 plays an important role in expediating endocytosis and Myo1 contributes to the localization of several proteins. Lastly, in Chapter 6, we observed that blocking the fastgrowing end of actin filaments does not impact actin assembly in patches. These studies provide key insight into how WASp family proteins are regulated in vivo.
    • Memory and Effector B cell Responses to Viral and Intracellular Bacterial Infection

      Gary M. Winslow; Newell, Krista (2021)
      Infection with an intracellular pathogen presents the host with the immunological challenge of intracellular access and of clearing infection without excessive damage to host tissues. This challenge was long thought to be addressed primarily by cell-mediated immunity, but is now known to include a significant humoral component. To better understand the B cell-mediated contribution to intracellular pathogen control, we investigated memory and effector B cell responses to the intracellular bacterial pathogen Ehrlichia muris, and SARS-CoV-2 infection. B cells expressing the transcription factor T-bet were identified in both mice and humans, and T-bet played an important role in directing antibody class switch recombination, but not in the generation of memory B cells during E. muris infection. T-bet expression was identified in cells resembling murine B-1 B cells, an innate-like subset of B cells important for early T cell-independent B cell responses. These results suggest that T-bet expression in B-1 B cells may contribute to their participation in the early B cell response to murine intracellular bacterial infection. Following human SARS-CoV-2 infection, we revealed that in addition to the canonical class-switched B cell memory response, the presence of a substantial pool of peripheral blood unswitched IgM+ memory B cells was correlated with reduced symptom duration and enhanced generation of antigen-specific antibody. These IgM+ memory B cells were stable, unlike the contracting plasmablast response. These studies underscore the importance of innate and unswitched B cell subsets to the functional plasticity of the humoral response and contribute to our understanding of correlates of innate protection and adaptive immunity.
    • Meta-Analysis of Alzheimer's Disease Risk with Obesity, Diabetes, and Related Disorders

      Profenno, Louis A.; Porsteinsson, Anton P.; Faraone, Stephen V. (Elsevier BV, 2010-03)
      Background: Late-onset Alzheimer’s disease (AD) is a multifactorial and heterogeneous disorder with major risk factors including advanced age, presence of an apolipoprotein E 4 (APOE4) allele, and family history of AD. Other risk factors may be obesity and diabetes and related disorders, which are highly prevalent. Methods: We reviewed longitudinal epidemiological studies of body mass, diabetes, metabolic syndrome, and glucose and insulin levels on risk for AD. We conducted meta-analyses of the results from these studies. Results: For obesity assessed by body mass index, the pooled effect size for AD was 1.59 (95% confidence interval [CI] 1.02–2.5; z 2.0; p .042), and for diabetes, the pooled effect size for AD was 1.54 (95% CI 1.33–1.79; z 5.7; p .001). Egger’s test did not find significant evidence for publication bias in the meta-analysis for obesity (t 1.4, p .21) or for diabetes (t .86, p .42). Since these disorders are highly comorbid, we conducted a meta-analysis combining all studies of obesity, diabetes, and abnormal glucose or insulin levels, which yielded a highly significant pooled effect size for AD of 1.63 (95% CI 1.39 –1.92; z 5.9; p .001). Conclusions: Obesity and diabetes significantly and independently increase risk for AD. Though the level of risk is less than that with the APOE4 allele, the high prevalence of these disorders may result in substantial increases in future incidence of AD. Physiological changes common to obesity and diabetes plausibly promote AD.
    • Meta-Analysis of the Association Between the 7-Repeat Allele of the Dopamine D4Receptor Gene and Attention Deficit Hyperactivity Disorder

      Faraone, Stephen V.; Doyle, Alysa E.; Mick, Eric; Biederman, Joseph (American Psychiatric Association Publishing, 2001-07)
      Objective: Family, twin, and adoption studies show attention deficit hyperactivity disorder (ADHD) to have a substantial genetic component. Although several studies have shown an association between ADHD and the 7-repeat allele of the dopamine D4 receptor gene (DRD4), several studies have not. Thus, the status of the ADHD-DRD4 association is uncertain. Method: Meta-analysis was applied to case-control and family-based studies of the association between ADHD and DRD4 to assess the joint evidence for the association, the influence of individual studies, and evidence for publication bias. Results: For both the case-control and family-based studies, the authors found 1) support for the association between ADHD and DRD4, 2) no evidence that this association was accounted for by any one study, and 3) no evidence for publication bias. Conclusions: Although the association between ADHD and DRD4 is small, these results suggest that it is real. Further studies are needed to clarify what variant of DRD4 (or some nearby gene) accounts for this association.
    • Metabolic Control of Autoimmunity in the Liver

      Perl, Andras; Oaks, Zachary (2016)
      Autophagy,literally meaning “self-eating,” is an integral part of cellularturnover of damaged organelles and proteins.This process is inextricably linked to mitochondrial function and turnover. Mitochondria can be degraded viaautophagy, known as mitophagy, as well as donate lipid membraneto generate autophagosomes fordigestingother organelles and proteins. On a larger scale, autophagy is essential for organ homeostasis. In the liver, autophagy ensures the turnover of damaged mitochondria that may otherwise increase oxidative stress which modifies DNA, proteins, and lipids resulting in the production of autoantigens or neoplasia. We investigated the role of autophagy and mitochondrial dysfunction prior to disease onset in mouse models of systemic lupus erythematosus (SLE). Patients and mice with SLE exhibit overexpression of transaldolase (TAL) and show predisposition to anti-phospholipid antibody production and associated liver diseases, including hepatocellular carcinoma. Wediscovered deficient mitophagy in the liver of lupus-prone mice prior to disease onset. Furthermore, these mice had increased mitochondrial respirationwith concomitant inner membrane hyperpolarization. These changes were coupled to overexpression of Rab4A, which depletes Drp1and thus inhibitsmitophagy.In addition,activation of complex I of the mechanistic target of rapamycin (mTORC1)was noted along with enhanced production of autoantibodies against mitochondrial phospholipids in lupus-prone mice. These changes were reversed by blockade of mTORC1 by rapamycin treatment in vivo. We then examined the role of TAL, a key enzyme of the pentose phosphate pathway (PPP) in mitochondrial dysfunction and oxidative stress. TAL-deficientmice showedincreased mitochondrial electron transport chain (ETC) activity and mTORC1 activation andreduced autophagy.Since inactivation of TAL caused oxidative stress via depletion of NADPH, we tested the hypothesis that aldose reductase(AR), a NADPH dependent enzymecan correct this metabolic defect without reversing the accumulation of TAL-specific substrates, sedoheptulose 7-phosphate and erythrose 4-phosphate. Moreover, deletion of AR reversed mTORC1 activation without affecting enhanced mitochondrial ETC activity or diminished autophagy. On a more global scale, predisposition to neoplasia and acetaminophen-induced liver failurewere reversed, while anti-phospholipid autoantibody production and liver fibrosis persisted in TAL/AR double-knockout mice indicatingthat the PPPmay act as a metabolic rheostat of organ-specific disease pathogenesis.
    • Metabolic Control of Autoimmunity Through Autophagy

      Choudhary, Gourav (2019)
      Metabolism plays a key role in immune cell activation and differentiation. Immune cell activation depending on their biosynthetic and bioenergetic needs leads to profound metabolic reprograming. Proinflammatory subsets of immune system cells such as effector T cells show dependency on glycolysis, whereas, regulatory T cells rely on oxidative phosphorylation. Under metabolic stress, immune cells utilize autophagy to overcome nutrient scarcity, an alternate method of recycling amino acids and other metabolic precursors. Limitation of nutrients such as amino acids activates mechanistic target of rapamycin (mTOR) in the immune cells. mTOR acts as a metabolic mediator, associated with mitochondria and metabolic needs of the immune cells. Homeostasis between mTOR activation and autophagy decides the fate and functionality of specific immune cells. The activation of mTOR is widely acknowledged in the pathogenesis of SLE, whereas, autophagy has been linked with antigen processing, presentation, and immunoregulation. In this study, we focused on Rab4A, an endosomal GTPase and Transaldolase, a rate limiting enzyme of the pentose phosphate pathway (PPP). Rab4A is over expressed in SLE T cells and facilitates lysosomal degradation of CD4 and CD3. Transaldolase is also overexpressed in T cells from SLE patients and SLE prone mice. First, we examined the role of Rab4A in a pristane-induced mouse model of SLE. Since Rab4A protects from pristane-induced alveolar lung hemorrhage, we tested the hypothesis that Rab4A will also protect from pristane-induced lupus nephritis. We found that overexpression of a constitutively active form of Rab4A limits antinuclear antibody production. Further, we found that Rab4A protects from pristane-induced renal injury by restricting immune complex depositions in the kidney. In additions, we found that Rab4A abrogates kidney-infiltration by lymphocytes and protects from podocyte injury. Furthermore, Rab4A facilitates the lysosomal mediated activation of mTOR. Possibly, the Rab4A mediated activation of mTOR in regulatory T cells leads to suppression of pristane-induced pro-inflammation signaling. In the second part, we investigated if aldose reductase (AR) deficiency can protect from Transaldolase mediated pathogenesis of liver disease. We found a coordinated regulation between AR and TAL, leading to the disease progression.