Leader: Fiorenzo Conti (INRCA); Other collaborator(s):
Neurofilaments (NFs) are heteropolymers that include four known subunits, NFL, NFM, NFH and α-internexin (INT). They have long been considered as structural proteins required for the radial growth of axons and to support the dendrites of large motor neurons. Recently, however, they have been shown to be expressed and to play a functional role at synapses (Yuan et al., 2015; Bragina and Conti, 2018), and their elimination disrupts synaptic plasticity and impairs social memory, with important implications for some neuropsychiatric diseases.
The novelty is that our study will focus on an analysis of “synaptic” NFs rather than on NFs, as has been done so far. The idea behind the proposal is that NFs may contribute to alter synaptic function, thus triggering a synaptopathy that may underlie the cognitive decline. The idea of monitoring changes in expression and function of NFs at different time points (from adulthood [P30] to 30 weeks-old animals] is, to the best of our knowledge, highly original. The second strength of the project is that it will be based on a strong interdisciplinary approach: we shall indeed use confocal and electron microscopy, western blotting, electrophysiology, and behavioral methods to correlate morfo-functional data on hippocampal and cortical synapses with cognitive abilities in wild-type and NFs KO rodents.
Brief description of the activities and of the intermediate results
We have almost completed phase 1, i.e., the analysis of the synaptic expression of NF-L, NF-M, NF-H, and alpha-internexin in the hippocampus and cerebral of the four groups of animals (Young, 6months old; Adult, 12 months old; Late Adult, 20 months old; and Old, 24 months old) and of their behavior at the Morris water maze test . To complete this part of the analysis, we only need to increase the n of Old animals. This will be achieved in the next few months. The results obtained so far, and analyzed with stringent statistical analysis indicates a region specific age-dependent difference in the xpression of NF-L that correlates with changes in learning and memory.
Main policy, industrial and scientific implications
The preliminary results suggest that NF-L might contribute to synaptic plasticity, and thereby they might open new therapeutic strategies.
Besides terminating the analysis of data of phase 1, we have studied the effects of environmental enrichment on NF-L expression. Male and female rats, 9 months old, were housed in an enriched environment for 3 months. At the end of the protocol, the animals (adults) were either immediately sacrificed or sacrificed after being evaluated using the Morris Water Maze test. For both groups, the cerebral cortex and hippocampus were extracted (cryopreserved after synaptosome separation). Statistical analyses of hippocampal synaptic neurofilament levels at the four time points (young, adult, late adult, and old) were performed, as well as their involvement in spatial learning (also at the four time points). Finally, a cognitive/behavioral phenotyping of the NF-L knockout mouse model was initiated, which will be followed by electrophysiological investigations.
The analysis of the expression of NFs at all time points has been completed, and so that relative to the effects of environmental enrichment. Furthermore, we have completed the phenotyping of the NF-L KO and we are performing the analysis of the electrophysiological and molecular effects.
Electrophysiological and molecular studies show that, in NF-L KO, glutamatergic transmission is altered both pre- and post-synaptically. We are now studying the effects of environmental enrichment in WT and KO mice to verify whether there are differences in the number and distribution of spines in dendrites of cortical pyramidal neurons, We are assembling the data, and plan to be able to submit a paper in three months.
In parallel, we are completing the analysis of molecular and behavioral studies in adult and old animals, including NF-l KO mice. These studies will be completed by early fall, and the manuscript (already in preparation) will ready for submission by November.
Coming soon