The role of Neuropilin-2 in excitatory and inhibitory neuron development, morphogenesis and function
Creators: name: Eisenberg, Carol Bakke
Publisher: name: No Publisher Supplied
Year: 2022

description: Maintaining a balance between excitatory and inhibitory activity in the brainis an essential feature of neural networks in the mammalian neocortex. This is achieved and maintained through an abundance of developmental processes including cell differentiation, neuronal migration, dendritic development, synapse formation and synaptic refinement/plasticity. When any part of a developmental process is impaired, dysfunction at the cellular, circuit or organism level is likely to occur. Genetic mutations resulting in loss or reduction in neuronal inhibition, greater excitation or both are risk factors for neurological disorders including autism spectrum disorders (ASD) and epilepsy.There are a myriad of gene mutations associated with autism. But the individual genes primarily regulate either excitatory or inhibitory neuron development and/or function. Neuropilin-2 (Nrp2), which encodes a cell surface receptor that can form a holoreceptor complex with the Plexin-A3 (PlxnA3) receptor and its obligate binding partner, the secreted Semaphorin 3F ligand (Sema3F), has been shown to be a key molecular player in controlling both excitatory and inhibitory processes during development. Nrp2 is involved in excitatory neuron dendritic spine morphogenesis and inhibitory interneuron migration during development. Furthermore, polymorphisms in the Nrp2 locus have been found in individuals with autism and Nrp2 knockout (KO) mutant animals exhibit increased susceptibility to seizures. Therefore, Nrp2 is a member of a limited class of molecules involved in both excitatory and inhibitory brain circuit development.Previous studies showed that Nrp2 deficient mice have fewer inhibitory interneurons in the hippocampus, increased dendritic spine densities, are susceptible to chemically induced seizures and have deficits in tests of motor, learning and memory and social and emotional behaviors. Additionally, Sema3F deletion in interneurons has been linked to decreased numbers of interneurons in the hippocampus and somatosensory cortex, increased susceptibility to seizures and autistic behaviors. Taken together, previous results suggest that Nrp2 may play distinct roles in different neuronal populations to regulate neuronal development and function at various stages of the animal’s life. However, the specific developmental timeframes impacting excitatory or inhibitory neurons remain unclear. Additionally, the role of Nrp2 in specific interneuron subtypes found in hippocampal subregions and the cortex has not been fully studied. My hypothesis is that Nrp2 is required in a spatiotemporal specific manner to regulate distinct cellular processes of excitatory and inhibitory neuron development and function.To address this hypothesis, I first analyzed specific interneuron populations in both the hippocampus and cortex of Nrp2 global knockout animals and assessed the cognitive flexibility of these animals. Then, I conditionally deleted Nrp2 within specific inhibitory and excitatory neural populations at specific developmental timepoints. Using this approach, I asked 1) whether the deletion of Nrp2 in inhibitory neuron progenitors during early development will result in fewer numbers of interneurons reaching their final destinations in the cortex and hippocampus and what functional and behavioral consequences result from this manipulation. When Nrp2 is absent during development, migration of interneurons from the MGE to the cortex is disturbed resulting in fewer interneurons in the cortex (Marin et al., 2001). However, the quantification of cortical interneuron subsets from Nrp2-/- mice is not known. It has also been shown that during migration, interneurons travel through the cortex to arrive at the hippocampus (Pleasure et al., 2000). So, it is possible that hippocampal interneuron migration is also disturbed when Nrp2 is absent in developing MGE progenitors. Hippocampal interneuron subsets from Nrp2-/- mice have been quantified previously except for the CA2 region (Gant et al., 2009). However, the previous interneuron quantifications in the hippocampus were not complete (Gant et al., 2009), and did not capture the entire anterior to posterior hippocampus. Furthermore, the results from the previous study were obtained only in the Nrp2 global KO animals. I additionally asked 2) whether loss of Nrp2 in excitatory cortical projection neurons modulates excitatory synapse formation and function, leading to the regulation of behaviors such as motor, learning and memory or social and emotional. Previous studies have shown that Nrp2-/- mice have increased cortical and hippocampal spine density (Tran et al., 2009) and altered behaviors compared to controls (Shiflett et al., 2005; Assous et al., 2019). Specifically, Nrp2-/- mice were shown to have deficiencies in novel object recognition, social novelty, rotarod, grooming and goal directed tests. However, mice with acute deletion of Nrp2 in only excitatory neurons specifically during development or adulthood have not been studied. Nrp2 expression continues through adulthood (Marin et al, 2001; Wang et al, 2017; Ng et al, 2013; Giger et al., 2000; Assous et al., 2019) and spine turnover can also occur through adulthood (Yuste and Bonhoeffer, 2001; Runge et al, 2020; Wang and Zhou, 2010). Therefore, it is important to understand Nrp2’s functions at various timepoints.Following the above approach, I first analyzed the developmental inhibitory functions of Nrp2 by determining parvalbumin (PV), neuropeptide Y (NPY) and somatostatin (SOM) expressing interneuron density and distribution within the hippocampus and somatosensory cortex of Nrp2 global knockout mice. In order to examine if the density and distribution are cell autonomous, I also examined PV+, NPY+ and SOM+ numbers within the hippocampus of mice with Nrp2 deletion in interneurons originating in the medial ganglionic eminence and specific to peak interneuron migration periods. To accomplish this, I crossed a Nrp2 floxed mouse with an Nkx-2.1CreERT2 mouse line (Nrp2f/f;NkxCreERT2+). Transcription factor Nkx-2.1 is found in all interneurons migrating from the medial ganglionic eminence. Then, I analyzed behaviors associated with ASD in these mice after they reach adulthood to determine which impairments seen in the global knockout are attributable to Nrp2 deletion in inhibitory interneurons only.I found that PV+, NPY+ and SOM+ hippocampal populations are reduced in the Nrp2 global knockout mouse. In the somatosensory cortex, the three populations had three different outcomes. PV+ neurons were increased in the Nrp2 global knockout compared to wild-type mice. NPY+ neuron density was statistically unchanged between knockout and wild-type mice and SOM+ neurons were reduced in the Nrp2 global knockout mice compared to wild-types. These mice also exhibited cognitive inflexibility as seen in an operant chamber reversal learning test. Similar to the Nrp2 global KO, Nrp2f/f;NkxCreERT2+ mice had reduced PV+, NPY+ and SOM+ neurons in the hippocampus, indicating this finding is cell autonomous. These animals also exhibited social and goal directed behavior impairments resulting directly from Nrp2 deletion in inhibitory interneurons.I then investigated the cell autonomous requirement of Nrp2 in restraining dendritic spine density in layer V pyramidal neurons in vivo and analyzed behaviors associated with ASD in adult mice with Nrp2 specific deletion in layer V pyramidal neurons prior to peak dendritic spine development. To accomplish this, I crossed the Nrp2 flox mouse with an Etv1-CreERT2 mouse line (Nrp2f/f;Etv1+/CreERT2) and further crossed the progeny with the Thy1-EGFP reporter line that expresses EGFP specifically in layer V pyramidal neurons and assessed the spine density on the apical dendrites of these neuron following Nrp2 deletion during peak spinogenesis in the neocortex. Using this mouse line, I then determined the requirement of Nrp2 in dendritic spine density maintenance in layer V pyramidal neurons in adult mice with acute Nrp2 deletion during adulthood in vivo and analyzed behaviors associated with those mice.As previously shown in the global Nrp2 knockout animal, Nrp2f/f;Etv1+/CreERT2 mice had significantly more cortical layer V dendritic spines compared to littermate controls, verifying the cell autonomous nature of the finding. This finding was true regardless of whether Nrp2 was deleted at early postnatal (during peak spinogenesis) or in adult stages. Nrp2f/f;Etv1+/CreERT2 mice with postnatal Nrp2 deletion showed impairments in motor, learning and memory and social and emotional behaviors, while Nrp2f/f;Etv1+/CreERT2 mice with Nrp2 adult deletion exhibited only motor deficits.Taken together the results of my thesis provide novel insights for global Nrp2 and, importantly, cell autonomous functions in both inhibitory interneurons and excitatory cortical layer V neurons. By examining the cell autonomous Nrp2 deletions, I can distinguish behavior by either excitatory or inhibitory functional origin. These results give insights into the molecular and behavioral pathogenesis in autism and epilepsy. The findings from this thesis may aid future research into novel treatments for both disorders.