Abnormal perinatal environment, commonly manifested as low birth weight, has been associated with birth defects as well as chronic disease. In particular, studies have shown low birth weight to have a deleterious association with type II diabetes, hyperlipidemia, hypertension, cardiovascular disease, renal disease, and renal failure. Of specific interest are the mechanistic details concerning low birth weight subjects’ altered excretion. Maternal low protein diet (LPD) during gestation and lactation results in offspring with significantly lower birth weight. Using the maternal LPD rat model, we determined renal excretion of select endogenous compounds by collecting blood and urine at various times of age. Clearance of creatinine and uric acid are indicators of glomerular filtration rate and reabsorption, respectively. High Performance Liquid Chromatography assays were used to simultaneously quantify creatinine and uric acid in the urine and in the blood of the rat. It was found that creatinine clearance and uric acid clearance decreased in LPD rats in an age-dependent and sex-specific fashion. Specifically, there were observed significant decreases in both creatinine clearance and uric acid clearance in LPD male offspring at both age’s day 90 and day 120 old. No differences were observed in both creatinine clearance and uric acid clearance in LPD female offspring until age day 120 old indicating an age-dependent programming of females as opposed to males. The results strongly correlate with decreased and altered excretion observed in low birth weight subjects and suggest that perinatal LPD has a long-term programming of renal excretion. These findings could partially explicate the underlying mechanisms that lead to higher incidence of renal disease/dysfunction in perinatally growth restricted subjects. Furthermore, altered renal function could affect the renal handling of therapeutic agents and thus affecting the overall treatment outcomes in low birth weight subjects.

Ubiquitin is a protein modifier that is essential for many eukaryotic cellular processes. Attachment of ubiquitin to substrate proteins (ubiquitination) occurs in a three-step cascade that involves a ubiquitin activase, ubiquitin conjugase, and ubiquitin ligase; it is the ligase that targets substrate proteins. One challenge for the ubiquitin field is identification of substrate cohorts for ubiquitin ligases. Although ubiquitination only occurs naturally in eukaryotic organisms, we previously demonstrated that it is possible to reconstitute the ubiqutination cascade in E. coli, allowing for analysis of ubiquitination outside of its natural environment. Our project consists of the construction of a protein complementation assay, using two fragments of dihydrofolate reductase (DHFR) to detect the ubiquitination of proteins in E. coli, outside of the natural eukaryotic environment of ubiquitin. To perform the assay, I transformed all of the essential components of the ubiquitination pathway, including individual fragments of DHFR fused to ubiquitin and a substrate protein of interest, into E. coli cells. The assay tests for the covalent attachment of ubiquitin to the substrate protein through growth on selective medium, which is possible only through the combination of the separated fragments of DHFR. After construction and transformation of vectors using a known yeast ubiquitin ligase and substrate partner, I have found that ubiquitin fused to a fragment of DHFR functionally attaches to other proteins via a known ubiquitin ligase. Transformation of entire cDNA libraries into the bacterial cell in place of a ubiquitin ligase will allow for high throughput screening of ligases with homologous function as well as discovery of new ubiquitin ligase substrates. As no functional screening mechanisms presently exist, our system to efficiently screen for ubiquitin ligases and substrates provides a novel tool to address a significant challenge faced by the ubiquitin field.

The amygdala, specifically the basolateral amygdala (BLA), has been implicated in fear-related processing and learning and is known to receive domaminergic input from the midbrain, although the specific nature of these connections is unclear. Mutations in functional glutamatergic NMDA receptors have previously been shown to impair phasic dopamine activity, leading to hindered fear-related learning and an onset of anxiety-like phenotypes following fear conditioning. To investigate how phasic dopamine activity modulates neuronal signaling in the BLA , I implanted mice with (controls) and without (KOs) a functional NMDA receptors in dopamine neurons with electrode microdrives bilaterally targeting the BLA and used in vivo electrophysiology to record neuronal activity during fear conditioning. The fear conditioning paradigm consists of 10 CS+ trials with a 10 second cue associated with a 0.3mA, 0.5 second foot shock and 10 CS- trials with a pulsed cue predicting the absence of a foot shock for a total of 20 trials in each session. Preliminary results show that the magnitude of the neuronal response to the unconditioned stimulus changes across days in the controls, while remaining constant in the KOs and that the KOs seem to show differences in tonic levels of activation during the trials compared to the controls. These results suggest that dopamine plays a role in modulating tonic activity in the BLA which may be implicated in influencing fear and anxiety related states. 

Quantitative mass spectrometry (MS)-based proteomics is a powerful approach to measure relative expression changes across thousands of proteins in a complex mixture. Commonly, stable isotopes like ¹³C and ¹⁵N are incorporated into proteins and peptides by metabolic labeling (ML) or chemical labeling (CL) to produce “heavy” labeled forms. Mixtures of light and heavy peptides can be identified and quantified by MS. In ML, proteins are labeled in vivo by growing cells with isotope labeled metabolic precursors, while peptides are chemically labeled in vitro in CL. These two labeling approaches have distinct workflows and their own strengths and limitations. In a typical proteomics experiment, complex mixtures of peptides are separated on reversed phase liquid chromatography (LC) and sampled in real-time by the mass spectrometer for peptide identification and quantification. We propose to analyze mixtures of chemically and metabolically labeled peptides in single LC-MS runs, comparing each method’s quantitative accuracy and precision, and other factors, such as analytical losses from sample processing without errors introduced by stochastic sampling. We compare stable isotope labeling by amino acids in cell culture (SILAC) and reductive dimethylation CL by mixing equal amounts of peptides from each approach.Preliminary results indicate minimal differences between ML and CL in quantifying proteins (median of >2 peptide ratios). With individual peptides, however, CL showed 13% larger variance than the ML. We observed signal losses in CL samples compared to ML samples, and fewer quantified peptides and proteins in CL (6005 peptides, 897 proteins) than in ML (7448 peptides, 1059 proteins). We attribute the differences to the extra labeling step in CL, and will perform further analyses with ratio mixtures of heavy and light labeled peptides to clarify the source of variance. Our comparison of CL and ML will guide future optimization and applications of both quantitative proteomics labeling methods.

The neurotransmitter dopamine is a key component of the limbic sysem, which plays a critical role in emotional processing, learning and memory, and reward processing.  Disruptions in dopamine synthesis, release, and reuptake underlie many neurological disorders, learning deficits, and diseases.  Previous work from our lab established the importance of the glutamatergic NMDA (N-methyl-D-aspartate) receptor in maintaining normal phasic activity in dopamine neurons of the ventral tegmental area (VTA) and showed that mutations of this receptor cause deficits in fear-related learning, highlighting the importance of dopamine in fear-related learning.  In this project we use in-vivo electrophysiology to record from dopamine neurons of the VTA during Pavlovian fear conditioning.  The paradigm consists of two types of trials where one cue (CS+) is paired with a mild footshock (0.3mA, 0.5 sec), while the other trials utilize a cue (CS-) which predicts the absence of a footshock.  Our preliminary data show plastic differences in neuronal activity patterns across training days associated with both conditioned and unconditioned stimuli, where initially there is increased activity in response to the fearful stimulus, but across days of conditioning, this response shifts to being associated with the conditioned stimulus.

Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy (SMEI), is a rare genetic form of epilepsy, caused by a mutation in the neuronal voltage-gated sodium channel, NaV1.1, encoded by the SCN1A gene. This form of epilepsy is characterized by febrile seizures at infancy that often change with age into afebrile seizures, which include generalized tonic-clonic and myoclonic seizures. In most cases, DS is a debilitating disorder that renders patients with decreased cognitive function and impaired development. To study this disorder, a mouse model of DS, with heterozygous deletion of the SCN1A gene (Scn1a +/-), was developed, demonstrating a phenocopy of the DS condition, with similar seizure patterns and atypical behaviors. In vitro work in the model demonstrates selective loss of sodium current and excitability in hippocampal GABAergic interneurons. However, it remains unknown if similar changes in neocortical GABAergic interneuron excitability occur. We hypothesize that fast spiking neocortical interneurons are responsible for control of cortical excitability via GABAergic inhibition and that reduced excitability of fast spiking interneurons associated with heterozygous loss of NaV1.1 contributes to hyperexcitability and seizures. To explore this hypothesis, we will compare neocortical interneuron excitability measured from single action potential parameters and repetitive action potential firing patterns in unaffected wild type mice and the DS mouse model, using pre-existing single-cell recordings. Excitability will be determined from the peak amplitude, width at half height, threshold for single action potentials, and from total number of action potentials in induced neuronal spike trains. If this hypothesis is correct, we expect that there will be atypical action potentials and firing patterns showing reduced excitability. This experiment will provide more insight into DS epilepsy and better therapeutic targets for the disorder.

Proteins typically adopt a three dimensional (3D) structure that allows for their specific activity and function. While proteins’ 3D structures are usually stable, they can be damaged by physical and/or chemical stresses. Such damage results in loss of structure, more commonly known as misfolding, which is often accompanied by exposure of hydrophobic residues normally buried in the interior of the protein. Exposed hydrophobicity can lead to the aggregation of misfolded proteins, and protein aggregation is associated with many prominent neurodegenerative diseases such as Alzheimer's and Parkinson's disease. To avoid these deleterious consequences, cells have evolved protein quality control (PQC) systems that degrade misfolded proteins. In eukaryotes, PQC utilizes ubiquitin ligases to flag proteins for proteosome degradation. How these PQC ligases recognize their misfolded substrates remains an open question. We have been addressing this question by studying the yeast PQC ubiquitin San1, which destroys misfolded proteins in the nucleus. We discovered that San1 recognizes exposed hydrophobicity minimally equal to 5 contiguous hydrophobic residues. Interestingly, we found that there is another PQC degradation system that recognizes exposed hydrophobicity at a lower threshold than San1. In this study we are trying to understand the mechanism by which this new pathway functions. By using degradation assays and western blots, we will take multiple constructs of varying degrees of hydrophocity to define how broad the pathway is. This allows us to determine whether the constructs are San1 dependent or independent, and if the San1-independent pathway is in fact a ubiquitin ligase. We know that a couple of the constructs are San1 independent, although they possess the hydrophibicity threshold required for San1 degradation. By exploring the mechanisms that ensure misfolded protein degradation, we hope to gain a better understanding of the PQC degradation systems within the cell and how they prevent severe pathologies caused by protein aggregation.

Author: Amanda Tanadinata, Karol Bomsztyk, Daniel Mar. Background: Apart from DNA mutations, alterations in chromatin structure, also referred to as epigenetic changes, have been shown to be among principal causes of tumor genesis that leads to cancer. However, unlike mutations, the usage of these chromatin alterations in gene expression is reversible. This feature, in fact, is what has nurtured the idea of creating an epigenetic-based treatment for cancer. One such treatment uses the compound 5-aza-2’-deoxycytidine (5-aza-dC), a DNA methyltrasferase inhibitor. DNA methylation can be crucial for transcriptional repression and activation, by altering recruitment of enzymes and transcription factors to chromatin . The goal of this study was to profile a set of pathways through the analysis of 5-aza-dC -mediated changes in DNA methylation, mRNA expression , RNA Polymerase II, histone marks, and enzymes. Method: Colorectal cancer cell line (HCT116) cells treated with or without 5-aza-dC were probed with different antibodies to see chromatin density differences at specific sites of the genome by Matrix Chromatin Immunoprecipitation (Matrix ChIP). Methylated DNA Immunoprecipitation (MeDIP), was used to assay DNA methylation sites at selective gene sites. The results were then quantified with real time PCR. In this study, SPARC promoter, H19, and UBE2b were examined specifically. Results: Treatment with 5-aza-dC increased SPARC mRNA levels, and reduced DNA methylation This was associated with changes in RNA polymerase II levels ,histone acetylation and methylation, and chromatin modifying enzymes at the SPARC but not the control H19 and UBE2b genes. Conclusion: These results imply that 5-aza-dC induced de-methylation of selective genomic sites alters enzyme binding and chromatin structure which then causes changes in rates of transcription.

Huntington’s disease (HD) is a devastating hereditary neurodegenerative disease characterized by motor and cognitive dysfunction. One of the earliest molecular events in HD pathogenesis is the down-regulation of the cannabinoid 1 receptor (CB₁) in medium spiny neurons (MSNs), cells that play a key role in movement and degenerate in HD patients. Our goal is to rescue the specific loss of CB₁ in striatal MSNs to understand the role of this event in HD pathology. To do this, we inserted flox-stop CB₁ (fsCB₁) into striatal MSNs of R6/2 mice, the best characterized HD mouse model, and confirmed its localization. To validate the functionality of the knock-in fsCB₁ protein, we crossed fsCB₁ mice into a CB₁-/- background, so that the resulting mice lacked endogenous CB₁ receptors, and crossed them with globally-expressed Cre, so that fsCB₁ receptors became globally-expressed. We then demonstrated that replacing endogenous CB₁ with fsCB₁ did not alter responses in four classic cannabinoid behaviors (analgesia, hypolocomotion, catalepsy, and hypothermia) by injecting these mice with cannabinoid agonists. Once the knocked-in mice were validated, we measured changes in motor phenotype and synaptic integrity between R6/2 and fsCB₁-R6/2 mice. Motor phenotype was measured using rotarod, open field, clasping behavior, and Catwalk assay biweekly from 4 to 12 weeks of age. Synaptic loss was measured in the striatum, globus pallidus and substantia nigra reticulata by costaining with synaptophysin and either PSD95 or neuroligin 2 to resolve excitatory and inhibitory synapses, respectively. Preliminary results indicate that CB₁ rescue did not improve motor phenotype, but did reverse loss of excitatory synapses in the dorsal-medial striatum. Our future goals are to identify the specific subset of excitatory synapses being lost. By understanding which synapses are lost and how CB₁ rescue improves this loss, we hope to obtain greater insight into the pathophysiology of HD.