University Of Massachusetts Amherst

NIH Research Projects · FY 2024 · 2015-09

PROJECT SUMMARY Streamline assessment of early lethal phenotypes in the mouse Although the generation of a loss of function allele at every locus in the mouse genome is well underway, there is a gap in established pipelines for assessment of early lethal phenotypes (as stated in PAR-17-005). Here we propose to continue our efforts to characterize up to 150 early lethal phenotypes occurring between fertilization and organogenesis. As described within, we have instituted an efficient strategy to analyze early lethal phenotypes and have already analyzed more than 100 novel phenotypes. We will provide a tremendous amount of novel data to the scientific community and foster collaborative efforts towards functional annotation of the mammalian genome. The proposed work capitalizes on techniques that our groups perform and publish routinely, maximizing the data generation by eliminating training/troubleshooting steps as well as boosting our individual research programs by providing novel phenotypes of interest. Characterization of knock-out alleles will be invaluable towards understanding genetic pathways and predicting mechanisms of diseases/phenotypes found in adults – in both heterozygotes and homozygous knockouts of genes in gene/protein networks or pathways. We will provide detailed morphogenetic characterization for each mutant phenotype. Characterization of novel gastrulation and preimplantation phenotypes will complement and extend our current morphogenetic understanding of early developmental events. Our proposal dovetails perfectly with existing phenotyping efforts and fills an essential need to characterize early lethal phenotypes towards functional annotation of the genome.

NIH Research Projects · FY 2025 · 2015-07

Although generation of loss of function alleles at every locus in the mouse genome is underway, there is a gap in established pipelines for assessment of early lethal phenotypes (as stated in PAR-23-074). Here we continue our efforts and characterize an additional 10-15 early lethal phenotypes occurring between fertilization and organogenesis. As described within, we have instituted an efficient strategy and have already analyzed more than 250 alleles. We will provide a tremendous amount of novel data to the scientific community and foster collaborative efforts towards functional annotation of the mammalian genome. The proposed work capitalizes on techniques that our group performs routinely, maximizing the data generation by eliminating training/troubleshooting steps as well as in depth analysis of select phenotypes of interest. Phenotyping of knock-out alleles is invaluable towards understanding genetic pathways and predicting mechanisms of adult diseases/phenotypes. Characterization of newly generated alleles with gastrulation and preimplantation phenotypes will complement and extend our current morphogenetic understanding of early developmental events. Our proposal dovetails perfectly with existing KOMP/IMPC efforts and fills an essential need to characterize early lethal phenotypes towards functional annotation of the genome.

NIH Research Projects · FY 2026 · 2014-04

PROJECT SUMMARY Neuromodulators like oxytocin, dopamine, serotonin, and catecholamines regulate the activity of forebrain neurons during sensory and cognitive tasks. This scale of neuromodulation allows neural circuits to dynamically encode and respond to sensory stimuli depending on contexts like parenting, aggression, mating, and stress. Neuromodulators classically enhance stimulus extraction from noisy backgrounds, and this regulation is crucial for learning and memory. A recently-discovered neuromodulatory system - the synthesis and action of ‘neuroestrogens’ by specific neuronal cell types – holds a great deal of promise for sensory perception, memory, and cognitive function. In human patients, estrogens can be beneficial treatments for a variety of neurological disorders, including Parkinson’s disease, Alzheimer’s disease, and epilepsy. Yet because the neuromodulatory perspective of brain estrogen synthesis is relatively new, the therapeutic potential of neuroestrogen signaling itself is untapped. This unmet potential is also hampered by our poor understanding of the cellular and circuit mechanisms for neuroestrogen signaling, and their implications for complex behavior. The research program in this proposal will specifically study how neuroestrogens regulate the cellular, microcircuit, and network interactions between excitatory and inhibitory neurons, and situate these outcomes in ethologically-relevant behaviors. Our proposed experiments will clarify the neural circuit mechanisms for neuroestrogen signaling by unraveling the specific contribution of inhibitory and excitatory neurons, including their chemical and electrical synaptic interactions. The proposed projects will take advantage of recent molecular and technological advances that allow us to genetically target fast-spiking inhibitory neurons and principal excitatory neurons in the songbird forebrain. This work will therefore address a fundamental gap in our understanding of how estrogen production within the brain guides complex behavior, and could ultimately inform the development of highly-targeted estrogen therapies for cognitive and neurological disorders.

NIH Research Projects · FY 2025 · 1999-04

Summary The vast majority of the ~7,000 proteins that traffic through the mammalian secretory are modified by one or more glycans. These alterations include the modifications of Asn (N-linked) and Ser/Thr (O-linked) residues. Carbohydrates appended to proteins can assist with protein folding, quality control and trafficking, or control their activity and function. In this proposal, we will study the mechanism and role of the addition of the hexose epimers of glucose and mannose in the endoplasmic reticulum (ER). Protein maturation is monitored by a quality control process that evaluates the structural integrity of maturing nascent chains, and permits the passage of properly folded proteins. Alternatively, non-native proteins are marked for ER retention so that the defect can be repaired, or if irreparable, targeted for degradation. Calnexin and calreticulin are ER carbohydrate binding molecular chaperones that direct the folding and trafficking of secretory pathway cargo by selectively binding to monoglucosylated side chains on maturing proteins. Therefore, to a large extent the glucosylation state of a glycoprotein controls its flow proteins in the secretory pathway. The glucosylation state is controlled by the UGGT family members UGGT1 and UGGT2 that appear to selectively modify immature or non-native clients to support persistent chaperone binding. These soluble UGGTs transfer glucose from UDP-glucose to maturing cargo in the early secretory pathway. In the first two aims of this proposal, we will test the hypothesis that the UGGTs are central quality control gatekeepers that control the flux of proteins through the ER by examining their specificity and the role of reglucosylation in the cell. The hexose epimer mannose is also added to proteins in the ER. In contrast to reglucosylation, mannosylation involves the transfer of mannose by a membrane embedded transferase from a dolichol- P-precursor directly to Ser/Thr residues to form an O-glycosidic bond. There are two families of putative O-mannosyltransferases that reside in the ER membrane in mammalian cells, the POMTs (POMT1 and 2) and the recently discovered TMTCs (TMTC1-4). Little is known about their mechanisms of action and the function of adding a mannose to maturing proteins in the ER. We have recently found that TMTC3 is involved in the O-mannosylation of E-cadherin and that E-cadherin’s O-mannosylation aids in cell adhesion and neurodevelopment. Mutations in TMTC3 are associated with the neurodevelopment diseases, underscoring the biological significance of this post-translational modification. Specific aim 3 is to understand the mechanism and significance of O-mannosylation in the ER. The long-term goal of this project is to understand the process of hexose addition including the substrate selection and modification steps, and how these post-translational modifications control the trafficking and functions of proteins.