Thomas Abbott

L-Histidine Decarboxylase and Tourette's Syndrome

Tourettes syndrome is a common developmental neuropsychiatric disorder characterized by chronic motor and vocal tics. Despite a strong genetic contribution, inheritance is complex, and risk alleles have proven difficult to identify. Here, we describe an analysis of linkage in a two-generation pedigree leading to the identification of a rare functional mutation in the HDC gene encoding L-histidine decarboxylase, the rate-limiting enzyme in histamine biosynthesis. Our findings, together with previously published data from model systems, point to a role for histaminergic neurotransmission in the mechanism and modulation of Tourettes syndrome and tics.

Functional Genomic and Proteomic Analysis Reveals Disruption of Myelin-Related Genes and Translation in a Mouse Model of Early Life Neglect

Early life neglect is an important public health problem which can lead to lasting psychological dysfunction. Good animal models are necessary to understand the mechanisms responsible for the behavioral and anatomical pathology that results. We recently described a novel model of early life neglect, maternal separation with early weaning (MSEW), that produces behavioral changes in the mouse that persist into adulthood. To begin to understand the mechanism by which MSEW leads to these changes we applied cDNA microarray, next-generation RNA-sequencing (RNA-seq), label-free proteomics, multiple reaction monitoring (MRM) proteomics, and methylation analysis to tissue samples obtained from medial prefrontal cortex to determine the molecular changes induced by MSEW that persist into adulthood. The results show that MSEW leads to dysregulation of markers of mature oligodendrocytes and genes involved in protein translation and other categories, an apparent downward biasing of translation, and methylation changes in the promoter regions of selected dysregulated genes. These findings are likely to prove useful in understanding the mechanism by which early life neglect affects brain structure, cognition, and behavior.

Early-Life Stress Perturbs Key Cellular Programs in the Developing Mouse Hippocampus

Conflicting reports are available with regard to the effects of childhood abuse and neglect on hippocampal function in children. While earlier imaging studies and some animal work have suggested that the effects of early-life stress (ELS) manifest only in adulthood, more recent studies have documented impaired hippocampal function in maltreated children and adolescents. Additional work using animal modes is needed to clarify the effects of ELS on hippocampal development. In this regard, genomic, proteomic, and molecular tools uniquely available in the mouse make it a particularly attractive model system to study this issue. However, very little work has been done so far to characterize the effects of ELS on hippocampal development in the mouse. To address this issue, we examined the effects of brief daily separation (BDS), a mouse model of ELS that impairs hippocampal-dependent memory in adulthood, on hippocampal development in 28-day-old juvenile mice. This age was chosen because it corresponds to the developmental period in which human imaging studies have revealed abnormal hippocampal development in maltreated children. Exposure to BDS caused a significant decrease in the total protein content of synaptosomes harvested from the hippocampus of 28-day-old male and female mice, suggesting that BDS impairs normal synaptic development in the juvenile hippocampus. Using a novel liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) assay, we found decreased expression of many synaptic proteins, as well as proteins involved in axonal growth, myelination, and mitochondrial activity. Golgi staining in 28-day-old BDS mice showed an increase in the number of immature and abnormally shaped spines and a decrease in the number of mature spines in CA1 neurons, consistent with defects in synaptic maturation and synaptic pruning at this age. In 14-day-old pups, BDS deceased the expression of proteins involved in axonal growth and myelination, but did not affect the total protein content of synaptosomes harvested from the hippocampus, or protein levels of other synaptic markers. These results add two important findings to previous work in the field. First, our findings demonstrate that in 28-day-old juvenile mice, BDS impairs synaptic maturation and reduces the expression of proteins that are necessary for axonal growth, myelination, and mitochondrial function. Second, the results suggest a sequential model in which BDS impairs normal axonal growth and myelination before it disrupts synaptic maturation in the juvenile hippocampus.

Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

Several Rho GDP/GTP exchange factors include a Sec14 domain and spectrin repeats. The Sec14 domain of Kalirin 7 is a determinant of spine length. Together the noncatalytic Sec14 domain and spectrin repeats of Kalirin 7 support spine formation, localize to the postsynaptic density, and attract presynaptic endings.

Phosphoproteomic Analysis Reveals a Novel Mechanism of CaMKIIα Regulation Inversely Induced by Cocaine Memory Extinction versus Reconsolidation

Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance current strategies for addiction treatment.

Development of a Targeted Urine Proteome Assay for kidney diseases

Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases.

Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins

We present a comprehensive workflow for large scale (>1000 transitions/run) label‐free LC‐MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling that improves S/N by twofold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, normalized group area ratio, MLR normalization, weighted regression analysis, and data dissemination through the Yale protein expression database. As a proof of principle we developed a robust 90 min LC‐MRM assay for mouse/rat postsynaptic density fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label‐free and SIS analyses. Overall, our method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC‐MRM assay.

Use of a Targeted Urine Proteome Assay (TUPA) to identify protein biomarkers of delayed recovery after kidney transplant

Development of delayed graft function (DGF) following kidney transplant is associated with poor outcomes. An ability to rapidly identify patients with DGF versus those with immediate graft function (IGF) may facilitate the treatment of DGF and the research needed to improve prognosis. The purpose of this study was to use a Targeted Urine Proteome Assay to identify protein biomarkers of delayed recovery from kidney transplant.

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long-term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multisite phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity, and reduced GEF activity.