Secreted semaphorin signaling regulates aging-associated dendritic modifications and cognitive function
Creators: name: Baek, Jiyeon
Publisher: name: No Publisher Supplied
Year: 2024

description: The age-associated loss of neuronal dendritic complexity may account for the synaptic loss and cognitive decline seen in aged mammals, including humans. Identifying the molecular players involved in these changes is necessary to understand the causes of the neurological phenotypes of normal aging. Some of the molecules involved in dendritic morphogenesis are the class 3 secreted semaphorins and their signaling receptors. In development, semaphorin 3A (Sema3A) signals through its receptors Neuropilin-1/Plexin(Plxn)-A4 to induce dendritic elaboration in layer V cortical neurons, while Sema3F signals through Neuropilin-2/Plexin-A3 to induce spine pruning on the apical dendrite of the same neurons. Importantly, Sema3A and Sema3F and their receptors are also expressed in adult and aged mammalian cortices, but their role in age-associated dendritic remodeling is unclear. Thus, I wondered if the presence of these class 3 secreted semaphorins (Sema3s) in the aged brain might maintain their developmental roles in regulating and/or maintaining the dendritic arbor of cortical neurons in the aged animals. Therefore, my hypothesis is: Altered expression of secreted semaphorins and their receptors in the aging brain drives changes in cortical neuron dendritic morphology and cognition. To address this hypothesis, I first established an in vitro model of aging in order to manipulate the direct effects of Sema3A and Sema3F on dendritic elaboration of primary cortical neurons grown at different length of time (age) in culture, which is more difficult to execute in vivo. By growing mouse primary cortical neurons up to 30 days in vitro (DIV), I demonstrated that aging-associated β-galactosidase levels also increase with age in culture. Then using this in vitro aged-neuron model, I found that treating with either Sema3A or Sema3F induces significantly more dendritic branching at 5DIV and 15DIV, but not at 30DIV, at which only simultaneous treatment with both Sema3A and Sema3F increased dendritic branching, suggesting there are changes in the cellular response to semaphorins with age in culture. Brain-Derived Neurotrophic Factor (BDNF) is another molecule known to increase dendritic branching and is also expressed in the mammalian cortex. BDNF gene therapy is currently being used as a treatment to build synapses and improve memory in patients of AD. Therefore, I wondered if BDNF and Sema3s might have crosstalk in their abilities to promote dendritic elaboration. I examined the effect of BDNF on dendritic branching at 5 and 30DIV and treated cultured neurons simultaneously with BDNF and Sema3A or Sema3F to test for any additive effects on dendritic branching. I found that BDNF alone was able to increase dendritic branching at both 5 and 30DIV, but that Sema3F suppressed BDNF-induced dendritic branching at both 5DIV and 30DIV. Sema3A suppressed BDNF-induced dendritic branching in more distal dendrites at 5DIV, but increased perisomatic dendritic branching compared to the BDNF single treatment at 30DIV. These results suggest that the interaction between Sema3A and BDNF is altered with age in vitro, and that Sema3A and Sema3F may be signaling through distinct mechanisms to alter dendritic growth at different distances from the soma. I hypothesized that these changes in the response to semaphorin treatment in cultured neurons with time in culture may be driven by a decrease in the expression of Sema3A receptors in culture. Surprisingly, Plexin-A4 and Nrp1 expression levels increased with time in culture, which is counterintuitive to what is expected as aged 30DIV neurons no longer respond to Sema3A-dependent dendritic branching in vitro. This demonstrated a need to look in vivo to better understand endogenous mechanisms and changes to dendritic morphology in the animal as it ages. When I assessed the age-associated endogenous changes to the Nrp1 and Plexin-A4 receptor expression levels in vivo, I found an age-associated decrease in Nrp1 and Plexin-A4 expression in cortices of wildtype mice from 6 to 18 months of age, along with an associated decrease in dendritic elaboration in cortical layer V pyramidal neurons comparable to the decreased branching seen in adult PlxnA4-/- cortices. Layer V cortical pyramidal neurons from aged PlxnA4-/- mice also had decreased dendritic elaboration compared to adult and aged wildtype animals. Additionally, I examined the activity of RhoA-GTPase, a cytoskeletal regulator downstream of Plexin-A4 whose activity was previously found to inhibit dendrite outgrowth and found that RhoA activity is elevated in aged wildtype and adult PlxnA4-/- mouse cortices as compared to adult wildtype mouse cortices. Aged PlxnA4-/- mouse cortices did not have increased RhoA activity. Adult wildtype and adult PlxnA4+/- mice had similar levels of dendritic branching as well as similar levels of Plexin-A4 expression and RhoA activity. However, in aged PlxnA4+/- mice, there was no age-associated impairment in dendritic elaboration and no age-associated decrease in Plexin-A4 expression or increase in RhoA activity from 6 to 18 months of age. Due to the changes in the dendritic arbor seen in the aged 18-month-old wildtype and adult 6-month-old PlxnA4-/- animals, in association with decreased Plexin-A4 expression levels in the aged mice, I wondered if there might be changes in cognitive function in these animals. I examined the learning ability, memory, and cognitive flexibility of these mice in the reversal learning behavioral test that requires the corticostriatal pathway, which involves the layer V cortical pyramidal neurons. I found that both adult PlxnA4-/- and aged wildtype mice have impaired learning and memory compared to adult wildtype mice, while aged PlxnA4+/- mice did not show any impairments in learning or memory, consistent with the levels of dendritic branching and molecular changes seen in those animals. Additionally, the aged PlxnA4-/- mice, the only group with decreased dendritic branching but no elevated RhoA activity, also behaved uniquely, as they did not have defects in memory compared to adult wildtype mice, as measured by winstay lever presses, but did have impairments in learning over sessions, and defects in cognitive flexibility. These results suggest that a precise level of Plexin-A4 expression is required in development for maintenance of proper cognition in aged animals, though acute deletion of Plexin-A4 in adulthood must be performed in order to confirm the role of Plexin-A4 in the aging brain. Collectively, my findings provide novel insights into the involvement of semaphorin-plexin signaling and changes in its downstream molecular effectors, which may account for the age-associated decline of dendritic complexity and cognitive function.