NSC transplantation may provide a promising new approach to treat AD by elevating neurotrophin levels and enhancing endogenous synaptic connectivity . However, NSCs do not modify Aβ pathology; thus, the long-term benefit of NSC transplantation remains unclear. Because of their robust migratory capacity, NSCs provide a compelling approach to deliver therapeutic proteins to the brain. By combining the inherent benefits of NSC transplantation with ex vivo gene therapy, modified NSCs may provide a powerful combinatorial approach to treat AD. In this study, we examined whether NEP-expressing NSCs could target and reduce Aβ pathology in two well-established transgenic models of AD. We found that NSCs could be readily modified to express and secrete NEP without altering multipotentcy or differentiation potential. More importantly, sNEP-expressing NSCs dramatically reduced Aβ levels both in vitro and in vivo and elevated synaptic density in both transgenic models of AD.
We and others previously tested the effect of NEP overexpression in transgenic AD mice [7, 10, 21, 22]. Each of these studies observed significant reductions in Aβ plaque load in response to NEP. We also previously demonstrated improved cognitive function in AD mice following lentiviral delivery of sNEP . In contrast, Meilandt and colleagues found no improvement in cognition when hAPP-J20 mice were crossed to mice overexpressing membrane-bound NEP . Major differences in delivery approach and the use of secreted versus membrane-bound forms of NEP likely account for these differences. In the current study, we again utilized the secreted form of neprilysinNEP, but instead used NSCs as a delivery vehicle. Potential benefits of NSC-mediated delivery over viral methods include a greater distribution of NEP delivery. Whereas NSCs have previously been shown to migrate through the brain parenchema , viral-based approaches typically provide only a small radius (approximately 0.5 mm) of infectivity . As the human brain is approximately three thousand times larger than the mouse brain, scale-up of viral gene-therapy for clinical translation remains a considerable challenge. Peripheral delivery of NEP protein also appears to be ineffective, as a recent study found that intravenous delivery of a NEP fusion protein could reduce plasma Aβ, but failed to clear Aβ plaques within the brain . New approaches, such as stem cell mediated delivery, may therefore be needed to provide broader expression of therapeutic proteins in the brain. However, it remains to be determined whether the migratory capacity of NSCs would be sufficient to treat a widespread brain disease such as AD. Clearly NSC-based delivery provides a benefit over current gene therapy approaches, but multiple NSC injections would likely still be needed.
The unilateral transplantation design used in this study allowed us to directly compare plaque load and synaptic density within the same animals. Unfortunately, this design also precluded further biochemical analysis of soluble Aβ. It, therefore, remains possible that soluble Aβ levels are not altered by this approach. However, the observed sNEP-NSC mediated a 31.8% increase in synaptic density, suggesting that soluble Aβ oligomers are also likely reduced. Previous studies also support this notion, as lentiviral-mediated delivery of s-NEP reduces both soluble and insoluble Aβ . As most transgenic AD models, including those used, exhibit little or no neuronal loss we conclude that the effect of sNEP-NSCs on synaptic density is mediated via maintenance and/or enhancement of endogenous synaptic connectivity. It is important to note that synapse loss correlates strongly with cognitive dysfunction in AD patients . Quite notably in the current study we observed a 31.8% increase in hippocampal synaptic density three months after sNEP-NSC transplantation. The magnitude of this effect is similar to the approximately 38% loss of synapses that occurs in AD patients . We, therefore, conclude that the effect of sNEP-NSCs on synaptic density represents a meaningful functional outcome. In addition to Aβ plaques and synaptic loss, cerebral amyloid angiopathy (CAA) represents another important AD-associated pathology. However, neither the 3xTg-AD nor Thy1-APP models develop CAA at the ages studied. Future experiments are therefore needed to determine whether NSC-mediated NEP delivery can also influence CAA.
Other cell types may also be useful for delivering NEP to the AD brain. For example, induced pluripotent stem cells (iPSCs) offer an alternative and extremely promising new cell source that could be used to deliver NEP and personalized cell therapies. Two distinct advantages of iPSC-derived NSCs over allogeneic fetal-derived NSCs include the greatly increased capacity for scale-up and the potential ability to transplant a patient’s own cells, thereby reducing or eliminating the need for immune-suppression. Most recently, xeno-free clinical grade iPSCs have been generated, moving this approach several important steps closer to clinical reality .
Peripheral-derived cells are also worth considering. In support of this, Lebson and colleagues transfected CD11b + monocytes with NEP and infused these cells biweekly into AD transgenic mice . Some of these modified monocytes migrated into the brain and Aβ deposition was slowed. This promising approach further supports the notion that cell-based delivery of NEP can be used to target Aβ. However, the use of monocytes offers both advantages and disadvantages in comparison to NSC-based delivery. Monocytes, for example, have limited half-lives; thus, repeated injections are required. This potential drawback can also be viewed as a possible advantage by providing some protection against adverse events. Interestingly, upregulation of NEP, also known as CALLA (common acute lymphoblastic leukemia antigen), commonly occurs in acute leukemias . The overexpression of NEP in hematopoetic lineages is therefore concerning. It is also worth noting that without irradiation, concurrent stroke, or additional treatments, very few monocytes appear to migrate into the AD brain . Thus, monocyte mediated delivery of NEP may not provide as robust a therapeutic approach as NSC-based delivery.
In contrast, our data suggest that the disease-modifying effects of sNEP-NSCs can be extremely robust, modulating Aβ plaques in regions both adjacent to and interconnected with the grafted area. Reductions in local Aβ appear to occur via both NEP-mediated proteolysis and enhanced microglial degradation. The mechanism(s) by which sNEP-NSCs decrease plaque load in more distant regions, such as the amygdala and medial septum, however, remains unclear. Transplanted NSCs did not migrate into the amygdala or medial septum and we were unable to detect clear evidence of axonally-transported NEP. sNEP-NSCs may instead modulate the production and/or anterograde transport of Aβ from the hippocampus to these efferent targets. Intriguingly, recent studies point toward a network diffusion model for the transmission of neurodegenerative pathologies via synaptic networks [29, 30]. In light of this, our results could indicate that therapeutic proteins might also effectively modulate pathology via the same neural networks.