RANBP9 inhibitor (NC-9) mitigates reduction in spine density and development of neurofibrillary tangle-mediated neurodegeneration in a model of Alzheimer’s disease
Background: Neuronal spine stability is compromised during the progression of neurogenerative diseases. Tau phosphorylation, Neurofibrillary tangle (NFT) formation. RanBP9 is the scaffolding protein responsible for stabilizing the protein-protein interaction between cofilin and SlingShot1 to facilitate the activation of cofilin.
The hallmarks of AD involve the development of pathological markers including neurofibrillary tangles via tau hyper-phosphorylation and amyloid plaque formation which lead to neurodegeneration. Dendritic spines are protrusions from the dendrite membranes, where contact with neighboring axons form in order to receive synaptic input. Alterations in synaptic spines are associated with learning and memory, however they are also centrally involved with the progression of neurodegeneration in AD. Due to the preeminent importance of protecting synaptic spines, there have been major efforts into developing techniques that enable visualization and analysis of dendritic spines in neurons. Actin dynamics are centrally involved in spine formation and reduction and shape. Postsynaptic density is a characteristic feature of excitatory spines and consists of densely packed ion channels, receptors and kinase and phosphatases anchored by scaffolding proteins. The two most important morphological characteristics of neurons are dendritic arbor structure and dendritic spine density. The shape, size, and complexity of dendritic trees can modulate action potential propagation and influence the firing pattern of a neuron. Similarly, the shape and the number of dendritic spines play important roles in synaptic plasticity. Increasing evidence indicates that deficient structural neuronal network connectivity is a major, if not primary, cause of several neurodegenerative disorders including Alzheimer’s disease (AD), Huntington’s disease (HD) and Parkinson’s disease (PD). Moreover, changes in the structure and function of dendritic spines contribute to several physiological processes including synaptic transmission and learning and memory. Therefore identification of novel pharmacological agents that prevent the loss of dendritic arbor and spine density is crucial in understanding their role in neurodegenerative diseases.
RanBP9 is a multi-domain scaffolding protein known to integrate extracellular signaling with intracellular targets. It has been demonstrated that RanBP9 enhances Aβ generation and amyloid plaque burden which results in loss of specific pre- and postsynaptic proteins in vivo in a transgenic mouse model. Additionally, they have shown that the levels of spinophilin, a marker of dendritic spines were inversely proportional to the RanBP9 protein levels within the synaptosomes isolated from AD brains. They observed reduced dendritic intersections within the layer 6 pyramidal neurons of the cortex as well as hippocampus of RanBP9 over expressing mice compared to age-matched wild-type (WT) controls. Dendritic arbor and spine density are directly correlated to the levels of phosphorylated form of cofilin in the RanBP9 transgenic mice. Similarly, cortical synaptosomes showed a 20% reduction in the levels of spinophilin in the RanBP9 transgenic mice. These results provided the physical basis for the loss of synaptic proteins by RanBP9 and most importantly it also explains the impaired spatial learning and memory skills previously observed in the RanBP9 transgenic mice.
Figure 1 Activation of Cofilin via oxidative stress induces Cofilin rods and F-actin (synaptic damage), mitochondrial damage by cofilin accumulation and microtubule instability. All these lead to actin depolymerization, Tau tangles and neurofibrillary tangles (NFTs).
Figure 2 RanBP9 a membrane scaffold protein induces an increase in Aβ formation as well as Tau related pathology.
Figure 3 Structure of RanBP9 indicates physical structure between domains.
Figure 4 Activation of Cofilin by SSH1 phosphatase activity. Cofilin also interacts with LisH to cleave actin filaments and produce NFTs.
Figure 5 In silico image showing the binding pose of Compound 9 with the Spry and LisH domains of RanBP9.
RanBPM has been shown to play a pathogenic role in AD as it acts as a scaffold bringing together amyloid precursor protein (APP), low-density lipoprotein receptor-related protein (LRP) and β-site APP-cleaving enzyme 1 (BACE1) (Figure 2). This complex promotes the cleavage of APP to Aβ, the toxic fragment resulting in Aβ pathological plaques, a major hallmark of AD, at the expense of the non-pathogenic APP processing by α-secretase cleavage. Knockdown of RanBPM resulted in a decreased secretion of Aβ and RanBPM overexpression decreased the amount of APP at the cell surface due to increased internalization, which is necessary for its pathogenic cleavage. An in vivo mouse study mirrored these results, further confirming that RanBPM overexpression significantly increased the generation of Aβ in an AD mouse model. A comparison of human AD brains to those of age-matched controls found increased levels of a RanBPM proteolytic fragment, N60 RanBPM. This N60 fragment was actually found to be more potent than full-length RanBPM in enhancing β-secretase processing of APP. It remains unclear how this proteolytic fragment is generated, but it has been shown to be dependent on cell density, as cells at low density express higher levels of the N60 fragment compared with cells at a higher confluency.
RanBP9 is the structural protein responsible for stabilizing the protein-protein docking between cofilin and SSH1 to facilitate the dephosphorylation of cofilin (Figure 1 and 2). AD is known to be associated with dendritic spine reduction and elimination in the hippocampal and cortexes areas, prior to clinical evidence of the disease including decline in memory.
Published reports suggest that RanBP9 may serve as a potential therapeutic target for pharmaceutical intervention to prevent the docking between cofilin and SSH1. Within RanBP9, the SPRY domain is the location where SSH1 and cofilin dock to dephosphorylate the cofilin whereas the LisH domain is involved in actin binding (Figure 3). The importance of the SPRY and LisH domains is derived from our hypothesis that the LisH domain folds back on the SPRY domain to cut the actin filament. Further that a compound that is bound to the SPRY domain, can induce a conformational change that results in preventing RanBP9 mediating the docking between cofilin and SSH1. Our recent in-silico studies suggest several ligands as potential leads for RanBP9, however there is room for improvement for improving specificity to RanBP9 and the overall polarity of the proposed ligands. Thus, we have developed novel compounds in-silico for RanBP9 to interact with the SPRY and LisH domains to prevent the docking between SSH1 and cofilin. Targeting overactive cofilin is a crucial component for managing AD because cofilin can form cofilin-actin rods that lead to mitochondrial dysfunction, excessive actin dynamics, and interactions that induce cofilin-tau microtubule instability. Our studies aim to reduce the amount of overactive dephosphorylated cofilin by intervening with the RanBP9 structural protein to reduce cofilin-actin rods and the interactions between cofilin and tau. Experimentally, we intend to use primary neurons to determine the co-localization of tau and cofilin. Then examine the effect that current SSH1, PLD1, and RanBP9 inhibitors have on p-cofilin/cofilin.