Leon Reijmers - US grants

DESCRIPTION (Provided by the applicant) Abstract: A functional neural circuit consists of a group of connected neurons that collaborate to execute a specific function of the brain. The understanding of the molecular mechanisms that underlie the development, maintenance and experience-dependent modification of functional neural circuits is incomplete due to limitations of existing methods. I propose to generate a transgenic mouse that addresses these limitations by exploiting two recent methodological advances. The first advance is the TetTag mouse, which is a transgenic mouse that can be used to genetically tag a single functional neural circuit. This tag enables the selective molecular analysis of neurons that have a shared function. This method is more sensitive to changes within a single functional neural circuit than other available methods, which have to rely on spatial criteria and thereby include neurons that do not participate in the circuit of interest. The second advance is the Translating Ribosome Affinity Purification (TRAP) method, which enables purification of actively translated messenger RNA from a genetically defined group of neurons. TRAP analysis reflects changes at both the transcriptional and translational level, while other available methods only detect transcriptional changes. I will combine TetTag and TRAP within a single transgenic mouse to generate the first tool that enables the comprehensive analysis of all translational events within a single functional neural circuit. Neurons tagged with the TetTag mouse during fear conditioning provide a stable neural correlate of the fear memory. I will use the TetTag/TRAP mouse to purify actively translated messenger RNA from these tagged neurons in order to detect the protein synthesis events that underlie the storage of a memory. The TetTag/TRAP mouse can be used for the molecular analysis of various functional neural circuits, including those involved in memory, addiction, epilepsy, circadian rhythms, spinal cord regeneration, pain, brain development, and neuronal cell death. Public Health Relevance: The proposed research will lead to a better understanding of how the synthesis of new proteins helps neurons in the brain to work together and execute the functions of the brain. This increased understanding can be used to develop therapies for various brain diseases, including memory impairment, addiction and epilepsy.

DESCRIPTION (provided by applicant): The brain processes and stores information by constantly adjusting the strength of connections between its neurons. In order to adjust the strength of these connections, called synapses, the neurons have to synthesize new proteins. Proteins can be synthesized in the soma of the neuron and then travel through dendrites to reach the synapses. Alternatively, proteins can be synthesized within the dendrites close to the synapses. This dendritic protein synthesis provides a rapid and efficient way of modifying the strength of single synaptic connections. Dendritic protein synthesis is important for normal brain development and cognitive functions. When dendritic protein synthesis is impaired, for example in Fragile X Syndrome, it causes severe neurodevelopmental and cognitive deficits. Despite its importance, there is an incomplete understanding of dendritic protein synthesis. A major remaining question is: which proteins can be synthesized in dendrites? This project will test and apply novel tools that can answer this question. The tools allow for the selective isolation of ribosome-bound messenger RNA (mRNA) from in-vivo dendrites. Since ribosomes bind to mRNA in order to synthesize proteins through a process called mRNA translation, the ribosome-bound translated mRNA directly reflects which proteins are being synthesized. In order to completely characterize dendritic translated mRNA, this project will use next-generation sequencing in order to sequence each mRNA molecule within the sample (RNA-Seq). Knowing the sequence of each dendritic translated mRNA molecule not only answers the question which proteins can be synthesized in dendrites, but also provides insights into the specific protein isoforms that are synthesized. The sequence data generated by this project can also be used to discover RNA motifs that are shared by groups of dendritic mRNA. Some of these motifs could regulate the transport of mRNA into the dendrite or the translation of mRNA within the dendrite. The insights into dendritic protein synthesis generated by this project will aid the development of treatments for brain disorders associated with impaired dendritic protein synthesis. PUBLIC HEALTH RELEVANCE: The brain constantly adjusts the strength of neuronal connections called synapses through a process that requires the synthesis of new proteins. Much of this protein synthesis takes place close to the synaptic connections in a part of the neuron called the dendrite, and impairments in this dendritic protein synthesis can cause impairments in brain development and cognitive function. This project will develop and use novel tools for determining which proteins are synthesized in dendrites, and will thereby generate knowledge that can be used to develop treatments for certain brain disorders like Fragile X Syndrome.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by a severe loss of memories that causes great emotional suffering for patients and caregivers. There is currently no treatment for AD associated memory loss, because of insufficient understanding of the molecular mechanism that causes this memory loss. Memory loss is present during early stages of AD. There is growing evidence that memory loss during the early stages of AD is caused by effects of soluble amyloid-beta (Abeta) oligomers on synapses. Since protein synthesis is essential for both memory storage and normal synapse physiology, altered protein synthesis provides a plausible molecular mechanism for mediating the effects of soluble Abeta oligomers on memories and synapses. This project will determine if soluble Abeta oligomers impair protein synthesis associated with memory storage. To achieve this, an AD mouse model based on the intracerebroventricular injection of Abeta oligomers will be used. Intracerebroventricular injection of Abeta oligomers acutely impairs the formation of a contextual fear memory. In addition, a transgenic AD mouse model will be studied. These AD mouse models will be combined with a unique transgenic mouse that was designed for the explicit purpose of measuring protein synthesis during memory storage. This transgenic mouse enables the isolation of translated mRNA from neurons in the hippocampus by expressing tagged ribosomes. By sequencing the mRNA associated with the tagged ribosomes, the synthesis rate of all neuronal proteins can be determined. By using this unbiased genome-wide approach, new unanticipated therapeutic targets can be discovered. Since the molecular pathway responsible for AD associated memory loss might also contribute to the progression of AD, these therapeutic targets might slow down or even prevent the progression of AD.

? DESCRIPTION (provided by applicant): Fragile X syndrome is a genetic disorder that causes intellectual disability and autistic symptoms. Fragile X is caused by epigenetic silencing of the Fmr1 gene, which results in the loss of Fragile X Mental Retardation Protein (FMRP). FMRP is highly expressed in neurons, where it can bind to hundreds of different mRNAs and thereby decrease their rate of translation. In addition, loss of FMRP causes a general increase in mRNA translation through secondary effects that act on the translational machinery. The effects of FMRP loss on neuronal translation contribute to structural and functional deficits in synapses and impaired cognition in the Fragile X mouse model, and are therefore considered a central component of Fragile X pathophysiology. In addition to their presence in neuronal soma, FMRP and many of its target mRNAs also localize in neuronal dendrites. This raises the question whether the increased translation resulting from FMRP loss preferentially impacts dendritic or somatic translation. As the effects of FMRP loss on mRNA translation should profoundly alter the ribosome-association of affected mRNAs, we propose a genome-wide analysis of the in-vivo ribosome- association of dendritic and somatic mRNA in the absence of FMRP. For this we will use a novel method for the separate collection of in-vivo ribosome-associated mRNA from the dendrites and soma of CA1 pyramidal neurons. This exploratory proposal will make a number of contributions. First, it will perform the first analysis of in-vivo subcellular changes in translaton in a Fragile X model. Second, it will identify which dendritic and somatic mRNAs are affected at the translational level in the absence of FMRP, thereby identifying potentially novel therapeutic targets and pathways. Third, it might establish altered ribosome-association as a molecular endophenotype of the Fragile X mouse model. Fourth, it will set the stage for future studies that analyze dendritic and somatic ribosome-bound mRNA in mouse models for other types of autism and intellectual disability, which might lead to the discovery of shared molecular mechanisms among these brain disorders.

PROJECT SUMMARY Exposure therapy is the most widely used treatment for excessive fear caused by post-traumatic stress disorder and phobias. During exposure therapy the patient repeatedly confronts the fear-inducing situation or the memory of a traumatic event in a safe environment, which over time results in decreased fear in most patients. However, exposure therapy in its current form rarely leads to a permanent suppression of fear. A better understanding of how exposure therapy, also known as fear extinction, works is therefore needed. A brain region called the basolateral amygdala (BLA) can cause increased fear in both humans and other mammals. We found that the BLA of mice undergoes changes during fear extinction that might help to suppress fear. Specifically, fear extinction silenced BLA fear neurons, while changing the inhibitory synapses that are located around these fear neurons. To test if these changes in perisomatic inhibitory synapses contribute to fear suppression, we silenced the parvalbumin-positive (PV+) interneurons that make these perisomatic inhibitory synapses. This increased the activation of BLA fear neurons and the expression of fear. Furthermore, it changed the activation of neurons in a brain region outside of the BLA called the medial prefrontal cortex (mPFC). Finally, silencing PV+ interneurons in the BLA altered the frequency distribution of local field potential (LFP) oscillations in both the BLA and mPFC, indicating broad changes in the activation of neuronal circuits that connect these two brain regions. Based on these results, we formulated a model in which extinction decreases fear by increasing the ability of PV+ perisomatic synapses to inhibit BLA fear neurons, thereby giving fear-suppressing circuits a competitive advantage over fear-promoting circuits. The three aims of this proposal will test if this model is correct. Aim 1 is to determine the contribution of PV+ perisomatic synapse remodeling in the BLA to extinction-induced fear suppression. To achieve this, we will monitor the strength of PV+ perisomatic synapses under conditions when fear suppression stops working, and by manipulating brain-derived neurotrophic factor signaling during fear extinction, which is predicted to interfere with extinction-induced perisomatic synapse remodeling. Aim 2 is to localize and manipulate functionally opposed LFP oscillations in the BLA during extinction-induced fear suppression. To achieve this, we will manipulate the activation state of three types of BLA neurons (PV+ interneurons, fear neurons, extinction neurons), while measuring both LFP oscillations and fear behavior. Aim 3 is to identify downstream neural circuits that mediate the contribution of BLA PV+ interneurons to extinction-induced fear suppression. This will be achieved by analyzing and manipulating BLA projection pathways to two subdivisions of the mPFC. Completion of this proposal can identify a critical role for BLA PV+ interneurons in tuning the balance between a fear-promoting circuit and a fear-suppressing circuit following fear extinction, which would aid the rationale design of new and more effective treatments for patients suffering from excessive fear.