Carla J. Shatz - US grants

DESCRIPTION (provided by applicant): What enables a baby's brain to learn so rapidly during early developmental critical periods? What cell and molecular mechanisms cause the decline in extensive plasticity by adulthood? The goal here is to enhance synaptic plasticity by discovering and then blocking endogenous mechanisms that function to suppress plasticity and circuit change. Specifically, can manipulations of the neuronal receptor PirB (Paired Immunoglobulin-like receptor B; Lilrb3 in humans) release the brake on ocular dominance (OD) plasticity, a form of experience-dependent synaptic plasticity in visual cortex? In the immune system PirB is a receptor for Major Histocompatibility Class I molecules, famous ligands for T-cell receptors. This Lab made the unexpected discovery that neurons express PirB and MHCI molecules at synapses. OD plasticity is enhanced in visual cortex of mice with germline deletion of PirB, consistent with PirB acting to brake synaptic plasticity. Three specific aims are proposed: 1) Determine if acute deletion of PirB postnatally enhances OD plasticity: A conditional allele of PirB (PirB flox/flox) has been made, allowing acute temporal and cell-type disruption of PirB by crossing mice with tamoxifen-inducible Cre transgenic lines. Direct blockade of PirB with recombinant soluble truncated PirB protein or function-blocking antibodies will also be used. These experiments should reveal when and in what cell types PirB acts. 2) Link enhanced OD plasticity in PirB-/- mice to cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) will be studied in vitro in visual cortex slices using physiological methods. Dendritic spine density of YFP-labeled layer 5 pyramidal neurons will be measured in PirB-/- vs WT mice reared with normal visual experience or with monocular eye closure; spine stability will be examined using two-photon microscopy. These experiments should broaden understanding of how PirB acts at synaptic and structural levels to suppress plasticity. 3) Identify PirB signal transduction pathways in mouse visual cortex: Candidate signaling pathways downstream of PirB will be identified and evaluated by comparing visually-driven signaling in WT vs germline PirB-/- mouse visual cortex during and after the critical period. Changes in expression and phosphorylation levels will be assessed in candidate pathways including MAP Kinase, AKT and mTOR signaling. Studies here will employ genetic, biochemical, electrophysiological, imaging and anatomical methods in mice to assess OD plasticity at the systems level and to understand cellular and molecular mechanisms of PirB function. Together, experiments should elucidate how PirB normally acts in neurons to suppress synaptic-plasticity signaling pathways during and beyond the critical period, as well as test feasibility of restoring OD plasticity by acute PirB blockade. They represent key steps in understanding mechanisms of developmental critical periods, as well as for designing new ways to enhance CNS function and repair by engaging the brain's inherent capacity for neural plasticity.

This research project is dedicated to answering three basic questions about the development of the visual system: 1) how do nerve cells connect to each other; 2) how do nerve cells know where to connect to each other and 3) how does nerve cell activity influence the connections? The answers can be found by studying the prenatal and postnatal development of the cat visual system, which is remarkably similar to that in the human. During development the connections nerve cells make are not the same as those found in the adult. It is important, therefore, to ascertain what determines which connections are eliminated or preserved. Although it is known that activity influences which connections nerve cells make, it is not clear how it is done. This project will study the anatomy and physiology of developing nerve cells and whether certain types of "growth-associated proteins" (GAP) regulate this process. Abnormal nerve cell activity during development may lead to birth defects. The use of certain drugs which cross from mother to fetus may have this effect. Toxic substances could also affect the proper development of the visual system after birth by abnormally stimulating or inhibiting nerve cell activity. Studying the processes regulating normal development can further our understanding of defects of the visual system.

The Gordon Research Conference on Neural Plasticity has been held every alternate year since 1977 in July at Brewster Academy, Wolfeboro, New Hampshire. We are requesting partial support for the Conference to be held there on July 17-21, 1989. Gordon Research Conferences were established to stimulate ideas in an informal setting. Uninhibited discussion is fostered by GRC strictures on the publications, proceedings, or indeed the citation of presentations. The format has proved particularly useful in the Conference on Neural Plasticity- a highly interdisciplinary meeting in which the subject of modifiability of the nervous system is examined at the molecular, cellular and systems levels and in which the participants come from broadly different backgrounds (biochemical, pharmacological, anatomical, electrophysiological, behavioral). One evening is set aside for a keynote speaker and poster session. The remaining eight sessions will focus on specific issues, with 3 or 4 scheduled speakers so that significant time is preserved for discussion. The discussion tends to continue informally during the afternoon, when no formal sessions are scheduled. It is the experience of participants that these informal interactions are often more fruitful than the extended sessions characteristic of other meetings. The formal program includes sessions on: cellular and molecular models of learning, learning in the adult cerebral cortex, genetic and hormonal models of neural development and plasticity, regulation of neuronal receptors, oncogenes and neural plasticity, neural grafting, NMDA receptors and long term potentiation, and the role of ion channels in neural plasticity.

The major aim of this research is to understand the molecular mechanisms of how precise and orderly sets of synaptic connections in the adult mammalian central nervous system (CNS) are formed during development. Specifically, this proposal focuses on the role of neural activity in the prenatal development of connections in the mammalian visual system between retina and one of its central targets, the lateral geniculate nucleus (L:GN). When retinal ganglion cell axons initially grow into the LGN they are intermixed. The adult unmixed pattern emerges during a period in which axons from the two eyes segregate in a process known to be activity- dependent because it can be blocked by target application of tetrodotoxin (TTX). To identify genes encoding putative components of the cascade of molecular events underlying activity-dependent development of neuronal connectivity we propose to screen for candidate genes that satisfy two major criteria: regulation by neuronal activity and expression in retinal ganglion cells and/or LGN neurons during the formation of eye-specific layers. The results of these experiments should help break the impasse in moving from a systems to a molecular level of analysis of activity- dependent development of neuronal connectivity and thereby provide opportunities to design pharmacological treatments for developmental disorders of the CNS. Moreover, because it is thought that activity- dependent mechanisms are not only operational during development, but are also preserved at least in part during memory and learning in adulthood, these studies have broad significance beyond the developmental period.

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how experience during critical periods of brain development, mediated by the functioning of neural circuits, is translated into lasting structural change in synaptic connectivity. The specific hypothesis under study here is that MHC Class I genes (MHCI; HLA in human) expressed in neurons and at synapses act as a negative regulators to limit activity-dependent synaptic plasticity. The idea that there are molecules and signaling pathways normally working to oppose synaptic plasticity is novel and has significant therapeutic implications. MHCI genes are famous for their role in cell-mediated immunity, but here we study a novel role in neurons. Neuronal MHCI expression in the CNS was discovered unexpectedly in an unbiased screen for genes regulated by neural activity in development. Initial studies in mice provided indirect evidence for a role for these molecules in synaptic plasticity (Huh et al, 2000). Research during the past funding period has revealed that loss of function of just 2 of the 60+ MHCI genes, H2-Kb and/or H2-Db, alters synaptic plasticity rules, unexpectedly often enhancing plasticity and learning (McConnell et al, 2009). Three specific aims are planned: 1) Demonstrate requirement for H2- Db, H2-Kb in synaptic plasticity: Double mutant mice (KbDB-/-) will be studied to determine if these 2 genes can account for many of the changes in synaptic plasticity observed in initial studies of mice lacking surface expression of the majority of MHCI proteins. Mice overexpressing H2-Db will also be examined. Rescue experiments will be performed: Double transgenic mice (NSE-Db+/+; KbDb-/-) have been generated in which Db function is rescued only in neurons. GFP-tagged full-length cDNAs for H2-Kb or H2-Db will be expressed using Lentiviral vectors. 2) Determine if MHCI protein is located at synapses: A working model for neuronal MHCI suggests that MHCI protein located postsynaptically binds across the synapse to presynaptic receptors such as PirB. Immunostaining with MHCI antibodies will be used to examine synaptic distribution. Array Tomography (AT) will be used for higher resolution localization of MHCI protein in direct relation to multiple synaptic markers, as well as to PirB. Biochemical fractionation and Western Blotting will also be used to assess subcellular localization of MHCI and potential interacting partners. 3) Generate a conditional allele of H2-Db for studies of neuronal function: A transgenic mouse will be generated to obtain brain and neuronal cell-type specific knockouts for further study of requirement and specificity of H2-D in neurons. All experiments will make use of electrophysiological, anatomical and imaging studies of mouse visual system in vivo and in vitro to assess activity-dependent synapse development and plasticity. By studying mice with gain- or loss- of MHCI function, these experiments should help to establish whether and where H2-Kb and H2-Db are required normally in neurons and should permit a more systematic investigation of the function of specific MHCI molecules in the brain. 7. Project Narrative/Relevance 2-3 sentences Results from experiments proposed here will broaden understanding of how experience-dependent alterations at synapses, both during critical periods of learning in childhood and in memory formation throughout life, are ultimately encoded in the structure of neural circuits. Understanding molecules and mechanisms involved is crucial for addressing and ultimately curing disorders of learning and memory, from Dyslexia, Autism and other childhood neurological disorders, to Alzheimers' and other memory dysfunction in the aging brain. Moreover, neuronal MHCI is known to be modulated by inflammation and can be recognized by cells of the immune system, providing a direct means of communication between the nervous and the immune systems in both health and disease. PUBLIC HEALTH RELEVANCE: Results from experiments proposed here will broaden understanding of how experience- dependent alterations at synapses, both during critical periods of learning in childhood and in memory formation throughout life, are ultimately encoded in the structure of neural circuits. Understanding molecules and mechanisms involved is crucial for addressing and ultimately curing disorders of learning and memory, from Dyslexia, Autism and other childhood neurological disorders, to Alzheimers' and other memory dysfunction in the aging brain. Moreover, neuronal MHCI is known to be modulated by inflammation and can be recognized by cells of the immune system, providing a direct means of communication between the nervous and the immune systems in both health and disease.

DESCRIPTION (provided by applicant): What enables a baby's brain to learn so rapidly during early developmental critical periods? What cell and molecular mechanisms cause the decline in extensive plasticity by adulthood? The goal here is to enhance synaptic plasticity by discovering and then blocking endogenous mechanisms that function to suppress plasticity and circuit change. Specifically, can manipulations of the neuronal receptor PirB (Paired Immunoglobulin-like receptor B; Lilrb3 in humans) release the brake on ocular dominance (OD) plasticity, a form of experience-dependent synaptic plasticity in visual cortex? In the immune system PirB is a receptor for Major Histocompatibility Class I molecules, famous ligands for T-cell receptors. This Lab made the unexpected discovery that neurons express PirB and MHCI molecules at synapses. OD plasticity is enhanced in visual cortex of mice with germline deletion of PirB, consistent with PirB acting to brake synaptic plasticity. Three specific aims are proposed: 1) Determine if acute deletion of PirB postnatally enhances OD plasticity: A conditional allele of PirB (PirB flox/flox) has been made, allowing acute temporal and cell-type disruption of PirB by crossing mice with tamoxifen-inducible Cre transgenic lines. Direct blockade of PirB with recombinant soluble truncated PirB protein or function-blocking antibodies will also be used. These experiments should reveal when and in what cell types PirB acts. 2) Link enhanced OD plasticity in PirB-/- mice to cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) will be studied in vitro in visual cortex slices using physiological methods. Dendritic spine density of YFP-labeled layer 5 pyramidal neurons will be measured in PirB-/- vs WT mice reared with normal visual experience or with monocular eye closure; spine stability will be examined using two-photon microscopy. These experiments should broaden understanding of how PirB acts at synaptic and structural levels to suppress plasticity. 3) Identify PirB signal transduction pathways in mouse visual cortex: Candidate signaling pathways downstream of PirB will be identified and evaluated by comparing visually-driven signaling in WT vs germline PirB-/- mouse visual cortex during and after the critical period. Changes in expression and phosphorylation levels will be assessed in candidate pathways including MAP Kinase, AKT and mTOR signaling. Studies here will employ genetic, biochemical, electrophysiological, imaging and anatomical methods in mice to assess OD plasticity at the systems level and to understand cellular and molecular mechanisms of PirB function. Together, experiments should elucidate how PirB normally acts in neurons to suppress synaptic-plasticity signaling pathways during and beyond the critical period, as well as test feasibility of restoring OD plasticity by acute PirB blockade. They represent key steps in understanding mechanisms of developmental critical periods, as well as for designing new ways to enhance CNS function and repair by engaging the brain's inherent capacity for neural plasticity.

Pathological Alzheimer?s disease (AD) is a major cause of dementia characterized by memory loss and aggregation of insoluble beta amyloid plaques and tau tangles. Memories are stored at synapses, and it is thought that an early driver of dementia may be synapse pruning occurring even before plaque deposition. Extensive activity-dependent synaptic pruning also occurs during developmental critical periods when learning and experience strengthen and stabilize actively used synapses, while others weaken and are pruned. In an unbiased in vivo screen for genes regulated by neural activity during visual system development, my lab made the unexpected discovery that specific Major Histocompatibility Class I (MHCI) molecules, famous for their immune system roles, are expressed in neurons and at synapses. Next, we identified an innate immune MHCI receptor expressed in neurons: PirB (Paired immunoglobulin-like receptor B). Functional studies in mice reveal that the MHCI - PirB axis is required for synapse pruning during normal development. Genetic deletion of PirB selectively in cortical pyramidal neurons, or pharmacologic blockade using a recombinant protein, rapidly generates new spines and functional synapses even in adult cerebral cortex. In the APP/PS1 transgenic model of autosomal dominant AD, mice lacking PirB are protected from memory loss at 9 months of age despite high levels of beta amyloid. Remarkably, PirB is a receptor for soluble beta amyloid oligomers, with high affinity saturable binding. This interaction hyperactivates cofilin signaling which drives actin depolymerization and contributes to synapse pruning in the APP/PS1 AD mouse model. In human the LilrB (leukocyte immunoglobulin- like receptor B) family of 5 related molecules are PirB homologs. Similar to PirB, LilrB1 and LilrB2 are known to bind MHCI ligands, including HLA-A, B and C alleles, which are implicated in human GWAS and gene expression studies of AD. We discovered that LilrB2 binds soluble beta amyloid oligomers with nanomolar affinity, and LilrB2 protein is expressed in human frontal lobe. A crystal structure of the interaction between beta amyloid and LilrB2 has been solved, confirming genuine structural interactions and pointing to novel drug targets for AD. A major goal of this research is to test the hypothesis that innate immune signaling via MHCI-PirB/LilrB at the synapse is disrupted by pathological oAbeta, and to connect observations in mice to human neurobiology by (1) studying MHCI-PirB dependent signaling in neurons using RiboTag cell type- specific transcription profiling in AD model mice, and (2) by identifying and studying the function of human homologs, the HLA Class I and LilrB receptor families, in 3-dimensional forebrain organoids derived from human iPSCs, followed by validation in brain samples. Results from these studies will build a bridge between mouse models of AD and human neurons. They should also provide mechanistic information about how nervous and immune systems communicate at the synapse and open up new therapeutic avenues for treating synapse pruning disorders in development and in Alzheimer?s disease.