A Multifunctional Stem Cell Therapy With Improved Beta-amyloid Clearance And Neural Regeneration For Alzheimer’s Disease

Alzheimer’s disease(AD)is an incurable and most prevalent neurodegenerative disease,which is predominantly caused by amyloid-β(Aβ)protein depositions,tau-rich neurofibrillary tangles,neuroinflammation,and loss of neurons and synaptic connectivity in the brain.Current AD therapies(including anti-Aβ therapies,anti-tau therapies and immune modulation)all have been found to be only effective in preventing or delaying the development of AD in patients with very early pathology,while they were ineffective at slowing cognitive decline in patients who already have significant cognitive impairment.Given the high complexity of AD pathobiology,multifunctional strategies that are capable of simultaneously managing multiple AD pathologies,including Aβ clearance and neural regeneration,are urgently needed.Here,we developed a multifunctional neural stem cell(NSCs)therapy capable of simultaneous Aβ clearance and neural regeneration in an APPswe/PS1dE9 double transgenic mouse model.First,NSCs were genetically engineered to stably express the key Aβ-degrading protease,neprilysin(NEP).Enhancing the expression of the Aβ-degradation protease that is distinctly inhibited in AD pathology is considered to be a promising strategy for Aβ clearance and retarding amyloid pathology in AD.Therefore,we transfected primary NSCs with a mCherry-and NEP-containing lentivirus to obtain NEP overexpression NSCs(NEP-NSCs).To achieve this,lentiviral vector plasmid containing NEP gene was constructed.Lentivirus containing mCherry and NEP genes were prepared by transfection of HEK293 cells with pLVX-EF1α-NEP-lentiviral and packaging plasmids.Then,NSCs were infected with concentrated pLVX-EF1a-NEP-lentiviral stock for 48 h,positive NSCs expressing mCherry were screened by fluorescence-activated cell sorting(FACS).Further,RT-PCR,western blot,and immunofluorescence assays showed that both the expression level of NEP mRNA and the content of NEP protein were significantly enhanced in NEP-NSCs relative to the untransfected NSCs.Western blot and AβELISA assays showed that NEP content and Aβ degradation capability of NEP-NSCs or NEP-NSCs EVs were significantly enhanced relative to those of NSCs or the EVs derived from NSCs.Secondly,a highly efficient gene and drug delivery nanoformulation(PBAE-PLGA-Ag2S-RA-siSOX9,PPAR-siSOX9)containing SOX9 siRNA-expression plasmid,retinoic acid(RA),and Ag2S quantum dots(QDs)was developed to enhance the neuronal differentiation efficiency of NEP-NSCs via the regulation of the Wnt/β-catenin signaling and RA signaling pathways.The size distribution,zeta potential and TEM assays of the prepared PPAR showed that the nanoformulation had a spherical morphology with a diameter of approximately 150 nm and a zeta potential of+23.6 mV.And,the negatively charged plasmid genes could be effectively adsorbed to the positively charged surface.Then,the cellular delivery and gene transfection efficacy of the nanoformulation were accessed and optimized.RT-PCR results showed that PPAR carried the siSOX9 plasmid gene into neural stem cells and knocked down the expression of SOX9 gene.The neural differentiation of NSCs showed the astrocyte differentiation of NSCs was distinctly inhibited by the treatment of RA-containing nanoformulation.And,the siSOX9-expression plasmid in the nanoformulation knocked down the expression of SOX9 gene and further improved the neuronal differentiation of NSCs.In addition,WB and Aβ degradation assays showed that differentiated NEP-NSCs after the nanoformulation treatment were still able to express NEP at high levels and maintain their Aβ scavenging ability.These results suggested the efficacy of simultaneously Aβ clearance and neuronal regeneration can be achieved by treating NEP-NSCs with PPAR-siSOX9 nanoformulation,which laying a solid ground for AD treatment by the multifunctional NSCs.Finally,therapeutic efficacy of the multifunctional NSCs for the AD mice was tested.NSCs were stereotactically transplanted into the hippocampus,the most important lesion area of AD.Ag2S QD-based near-infrared fluorescence imaging(NIRFI)was used to guide NSC transplantation in real-time.Then,the short-term(30 days)and long-term(180 days)therapeutic efficacy of the multifunctional NSCs in Aβ clearance and neural regeneration in AD mice was systematically evaluated.It was shown that pathological Aβ aggregation in the brain was significantly decreased after the treatment by NEP-NSCs.And the neural regeneration in terms of improved neuronal viability and neuronal density was further enhanced in the brain of AD mice after the treatment by nanoformulation-NEP-NSCs.To further evaluate whether the multifunctional NSCs-based therapy can enhance the memory and cognitive functions of AD mice,the morris water maze(MWM)studies were performed.The mice in the nanoformulation-NEP-NSCs treatment group were shown to have best cognitive and memory levels,suggesting the strategy of multifunctional NSCs therapy has a good therapeutic efficacy on the treatment of AD.In summary,a nanoformulation-mediated multifunctional NSCs therapy was developed to simultaneously improving Aβ clearance and neural regeneration in AD mice.This strategy showed numerous benefits,including efficient and long-lasting Aβ degradation,improved neural regeneration,and NIRFI-guided accurate cell transplantation.This nanoformulation-mediated multifunctional treatment strategy is helpful for the further development of efficient AD therapies and has potential clinical application prospects.