Stem cells-derived exosomes alleviate neurodegeneration and Alzheimer’s pathogenesis by ameliorating neuroinflamation, and regulating the associated molecular pathways

Amyloid beta (Aβ) aggregation and tau hyper phosphorylation (p-tau) are key molecular factors in Alzheimer’s disease (AD). The abnormal formation and accumulation of Aβ and p-tau lead to the formation of amyloid plaques and neurofibrillary tangles (NFTs) which ultimately leads to neuroinflammation and neurodegeneration. β- and γ-secretases produce Aβ peptides via the amyloidogenic pathway, and several kinases are involved in tau phosphorylation. Exosomes, a recently developed method of intercellular communication, derived from neuronal stem cells (NSC-exos), are intriguing therapeutic options for AD. Exosomes have ability to cross the BBB hence highly recommended for brain related diseases and disorders. In the current study, we examined how NSC-exos could protect human neuroblastoma cells SH-SY5Y (ATCC CRL-2266). NSC-exos were derived from Human neural stem cells (ATCC-BYS012) by ultracentrifugation and the therapeutic effects of the NSC-exos were then investigated in vitro. NSC-exos controlled the associated molecular processes to drastically lower Aβ and p-tau. A dose dependent reduction in β- and γ-secretase, acetylcholinesterase, GSK3β, CDK5, and activated α-secretase activities was also seen. We further showed that BACE1, PSEN1, CDK5, and GSK-3β mRNA expression was suppressed and downregulated, while ADAM10 mRNA was increased. NSC- Exos downregulate NF-B/ERK/JNK-related signaling pathways in activated glial cells HMC3 (ATCC-CRL-3304) and reduce inflammatory mediators such iNOS, IL-1β, TNF-α, and IL-6, which are associated with neuronal inflammation. The NSC-exos therapy ameliorated the neurodegeneration of human neuroblastoma cells SH-SY5Y by enhancing viability. Overall, these findings support that exosomes produced from stem cells can be a neuro-protective therapy to alleviate AD pathology.

Alzheimer's disease (AD) is characterized by a progressive impairment of memory and cognitive abilities 1 .The parenchymal deposition of amyloid-β (Aβ) plaques, the development of tau neurofibrillary tangles (NFTs), and neuroinflammation are histological indicators of AD 2,3 .AD patients eventually experience synapse loss and neuronal death, and the buildup of these lesions in the brain results in cognitive decline 4 .AD can cause patients to struggle with swallowing, walking, and communication as the disease progresses.Evidence suggests that tau protein and amyloid are leading causes of AD.Current research aims to develop medications that target tau and Aβ for the treatment of AD.The leading concepts in the development of senile plaques and NFTs involve the accumulation of Aβ proteins and hyperphosphorylated tau (p-tau), which inhibit microglial cell proliferation and www.nature.com/scientificreports/activity was tested using the SensoLyte®520 kit appropriate to each enzyme.Six-well plates with cells were grown (0.3 × 10 6 cells/well) and incubated at 37 °C with 5% CO 2 .Cells were given NSC-exos treatment after attachment after 2 h and were kept under exosomes treatment for 24 h.Cells that were not treated served as the control.Each enzyme-specific assay required the preparation and usage of cell lysate.Using an ELISA microplate reader, absorbance was measured at 450 nm.The manufacturer's recommendations were followed while analyzing and comparing the data.Three duplicates of each experiment were conducted.
SH-SY5Y Human Neuroblastoma Cell Line (ATCC CRL-2266) were obtained from ATTC and were cultured in 6-well plates (0.3 × 10 6 cells/well) and exposed to various concentrations (5-15 μL) of NSC-exos treatment for 24 h to measure the activity of acetylcholinesterase.For the experiments, cells were collected, and lysate was added to 96-well microplates.Using a microplate reader, absorbance was measured at 450 nm.The manufacturer's recommendations were followed while analyzing and comparing the data.Acetylcholinesterase Activity Inhibitory Assay The manufacturer's instructions were followed while using an acetylcholinesterase activity assay kit (Sigma Aldrich) to measure the inhibition of acetylcholinesterase activity.
Determination of the levels of Aβ42 and phosphorylated tau (p-tau) using ELISA.The effects of NSC-exos on the expression levels of p-tau and Amyloid beta, which are linked to AD, were assessed using specific ELISA kits in accordance with the manufacturer's recommendations.NSC-exos were applied to SH-SY5Y cells that were grown in 6-well plates.For the experiments, cells were collected, and lysate was added to 96-well microplates.Using a microplate reader, absorbance was measured at 450 nm.In accordance with the manufacturer's recommendations, data were examined and contrasted.
RT-PCR analysis.SH-SY5Y cells were grown in 6-well plates with DMEM medium supplemented with 10% FBS and incubated at 37 °C in a CO 2 incubator.Following NSC -exos treatment, these cells were cultured for 24 h.Using an RNA isolation kit (Sigma Aldrich, Cat.No. 83913) and DNase treatment, total RNA was recovered from the cells.A NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) was used to quantify the extracted RNA.With the use of the Revert Aid Premium First Strand cDNA Synthesis Kit (Thermo Fisher Scientific No. K1621), the RNA (500 ng) was then reverse-transcribed into cDNA.With the use of a Thermo Scientific qPCR kit, cDNA was processed for real-time qPCR with gene-specific primers after being measured with the NanoDrop spectrophotometer.The list of primers used is mentioned in Table 1.
Western blotting.NSC-exos were administered to SH-SY5Y cells after they had been cultured and reached confluency.Cells were treated, then lysed on ice with radio immunoprecipitation assay buffer and rinsed with PBS buffer (Sigma-Aldrich).The supernatant, which contained the proteins, was collected after centrifuging and sonicating the lysed cells.Using the BCA protein assay (Pierce), protein content in the supernatant was determined.The electrophoresis of 10% sodium dodecyl sulfate polyacrylamide gel was used to separate 50 µg of each total protein.Proteins were transferred to nitrocellulose membranes (Bio-Rad).The membrane was blocked in 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS) for one hour at room temperature after being washed with double-distilled water.After that, the membranes were incubated with p-tau-specific primary antibodies diluted in TBS with 5% non-fat dried milk for two hours at 4 °C.Protein bands were visible through chemiluminescence after each membrane had been rinsed with double-distilled water and treated for two hours with a secondary antibody coupled to horseradish peroxidase (1:1000, Pierce Biotechnology).The protein bands Table 1.Primer sequences used in RT-PCR.BACE, β-secretase; PSEN1, presenilin 1 (γ-secretase); GSK-3β, glycogen synthase kinase-3; CDK5, cyclin-dependent kinase 5; ADAM10, A disintegrin and metalloproteinase domain-containing protein 10 (α-secretase); bp, base pairs.

Effects of exosomes on the activities of various enzymes. The in vitro activity of the enzymes
that produce A and p-tau, including acetylcholinesterase, β-secretase, γ-secretase, α-secretase, GSK-3, and CDK5, were shown to be regulated by exosomes.Different nontoxic concentrations of the recovered NSC-exos were applied to SH-SY5Y cells, and the cell lysate was then prepared for ELISA.The activity of the enzymes acetylcholinesterase, β-secretase, γ-secretase, GSK-3, and CDK5 were found to be dramatically reduced by exosomes in a dose-dependent manner at various significant levels (Fig. 3A-E).NCS-exos were also shown to improve the actions of α-secretase (Fig. 3F).When compared to the levels in the control, the ELISA showed that these proteins were present at dose-dependently higher levels.www.nature.com/scientificreports/RT-PCR analysis.qRT -PCR was used to measure the expression of genes involved in the Aβ pathway using gene-specific primers (Table 1).The results revealed a significant effect of exosome treatment on the expression of α-secretase (ADAM10).The expression level of ADAM10 was upregulated in a dose-dependent manner.Exosome treatment significantly decreased the expression of the β-secretase (BACE1), γ-secretase (PS1), GSK-3β, and CDK5 at various levels (Fig. 4).

Determination of Aβ and p-tau levels using ELISA.
After determining the effects of exosomes on the genes involved in AD alleviation and AD pathogenesis, the Aβ and p-tau levels in cells after NSC-exos treatment were measured using specific ELISA kits.p-tau were measured by WB (Full length uncroped images of WB gels are provided in Supplementary Files).The levels of p-tau and Aβ were significantly and dose-dependently reduced in treated cells compared to those in the non-treated cells (Fig. 5).These findings demonstrate the effects of NSC-exos on the inhibition of p-tau and Aβ accumulation-and promotion-associated genes and the enhanced expression levels of the AD alleviating genes.

Therapeutic effects of chromosomes on neurons degeneration.
The protective effects of NSCexos were determined by measuring the viability of the SH-SY5Y cells cultured and treated with various concentrations of NSC-exos for 72 h.The dead or degenerated cells were determined by viability using a live and dead cell detecting kit.It was found that NSC-exos treatment increased the viability of cells dose-dependently (Fig. 6).These therapeutic effects might be due to the regulation of the enzymes and genes in the SH-SY5Y cells by NSC-exos treatment.
Determination of Aβ and p-tau levels using ELISA.In order to check the effects of NSC-exo on neuroinflamation, glial cells HMC3 were culterd and treated with LPS to actvate and then treated with verious concentrations of exosomes.Proinflamtory cytokines were then quantified in traeted and control cells groups.
The results showed a good effects of the exosomes on the reduction of the verious inflamation causing cytokines e.g.iNOS, IL-1β, TNF-α, and IL-6 in dose dependent manner.The mRNA expression level of the MAPK/ERK and NF-kB were also downregulted after treatment with exosomes as compared to non-treated control in dose dpentdant manner (Fig. 7).

Discussion
The most prevalent neurodegenerative illness, AD, is defined by the death of neurons and the impairment of memory, cognition, and daily living skills.Death frequently results from impairment and the loss of fine motor abilities [40][41][42] .The formation of NFTs, which are made up of p-tau protein, and the accumulation of Aβ plaques are the primary pathological indicators of AD 43 .Early on, the medial temporal lobe is the primary site of these degenerative alterations, which thereafter disseminate across the neocortex 44 .Due to its ongoing breakdown of the neurotransmitter acetylcholine, acetylcholinesterase has been determined to be a progressive cause of AD.Due to the formation of A, α7 nAChR, the most prevalent subtype of acetylcholine receptor in the brain, is drastically decreased in AD patients.Neprilysin, acetylcholinesterase, Aβ generation, and α, β, and γ-secretases as well as other key molecular targets, are therefore essential for AD therapeutic drug screening.SH-SY5Y, as in vitro modules, are a promising choice for examining the therapeutic effects of medicines on modulation of APP processing and A-induced AD pathogenesis, but this model restricts the study of other hypotheses and molecular targets accountable for AD pathogenesis.Different kinases produce hyperphosphorylation of tau at diverse sites.In healthy individuals, tau kinase and tau phosphatase activity are in equilibrium.This event is crucial for decreasing tau's affinity for microtubules and boosting tau's resistance to calcium-activated neutral proteases and ubiquitin-proteosome-mediated tau degradation 47.Hyperphosphorylation, which results from an imbalance in tau phosphorylation, causes tau to fibrillize and aggregate, creating NFTs [45][46][47][48][49][50] .GSK3β, CDK5, calmodulin-dependent protein kinase II (CaMK II), and microtubule affinity regulating kinase (MARK) are a few of the most important tau kinases [51][52][53][54][55] .It is well known that AD and other associated illnesses alter tau kinase and tau phosphatase expression and activity 56,57.NSCs have neuroprotective paracrine actions that can stimulate neuronal development, proliferation, and survival in endogenous neurogenic niches in cellular models of AD 58,59 .NSC transplantation has been reported to minimize a deposition, induce neurogenesis, and ameliorate memory and spatial learning deficits in mouse AD models 60 .Since mass neuronal and synaptic loss characterizes AD as a neurodegenerative condition, regeneration of neuronal circuits by exogenous NSCs is a promising therapeutic approach 61 .However, there is still controversy around the hazards of tumor development, immunological rejection, and infusion toxicity in NSC transplantation.NSC-exos therapy has potential for treating Alzheimer's disease, but the possibility of off-target effects, adverse reactions, and long-term safety concerns must be considered.Immune reactions and inflammation may occur in response to the exosomes, and unintended targeting of healthy cells or tissues could lead to adverse effects.Additionally, the long-term safety of exosomes therapy is uncertain, so further research is needed to assess its potential risks and benefits.As a cell-free therapy, NSC-exos appear to be as successful as NSCs, and according to mounting evidence, NSC -exos are also less immunogenic than their parent cells, easier to generate and store, and do not possess tumorigenic properties 62,63 .
In the current study, we separated exosomes from stem cells and examined their impact on Aβ, the main contributor to neurodegeneration in AD.Our findings showed that NSC-exos dramatically reduced Aβ by stimulating APP processing by the non-amyloidogenic route vis raising ADAM10 (α-secretase) activities and by inhibiting BACE1 (β-secretase) and PESN1 (γ-secretase).NSC-exos also greatly reduced the amyloidogenic cleavage of APP.By reducing the expression and activity of BACE1 and PESN1, the synthesis of amyloidogenic Aβ have been prevented We discovered that, following NSC-exos treatment, BACE1 and PESN1 expression levels were all decreased in a dose-dependent manner whereas ADAM10 expression levels were increased.The increased non-amyloidogenic APP processing by ADAM10 and the decreased amyloidogenic processing by BACE1 and PSEN1, which is supported by the decline in Aβ level, may be the causes of the Aβ reduction.Overall, NSC-exos reduced the generation of Aβ while also activating a non-amyloidogenic route and suppressing the amyloidogenic pathway.For the prevention or reduction of neurodegeneration and AD, Aβ production must be inhibited or reduced.NCS-exo alleviate neuroinflamtion which is the major cause of neurodegeneration and Alzheimer pathogenesis by suppressing the expression of the proinflamatory cytokines and molecular pathways.The study has some limitations as it relies on in vitro models, further in vivo validation studies are needed to confirm the potential therapeutic effects of NSC-exos on AD pathology.

Conclusions
In the present study, we revealed that NSC-exos is a promising therapeutic for the treatment and prevention of AD.NSC-exos therapy reduced p-tau levels and Aβ formation via the suppression of kinase expression and activity, and AD pathology-promoting genes and proteins.Also the NC-Exo reduced neuroinflamation.Further studies and clinical trials are required to incorporate the NSC-exos in pharmaceutical formulations.

Figure 1 .
Figure1.Flowchart illustrating the experimental process.Cells were seeded in cell media containing exosomedepleted FBS for 48 h.After 48 h, cell-conditioned media was collected and centrifuged at 3000 g for 15 min to remove cell debris.NSC-exos were separated using centrifugation and separated NSC-exos were characterized for further analysis.Finally, NSC-exos were administered to the neurodegenerative disease cell culture model to analyze their effects.

Figure 2 .
Figure 2. Characterization of exosomes.(A) A flowchart indicated NSC-exos were separated using centrifugation and characterized for protein expression using WB, size distribution using NTA, and quantification using EXOCET.(B) Protein expression was measured using WB for exosome marker CD63 in control, only cell-free media, and in separated NSC-exos.GAPDH was used as an internal control.(C) Exosomes were quantified using an EXOCET assay in control (unprocessed cell-conditioned media), NSCexos, and negative control.(D) Concentration of separated exosomes was measured using NTA.The highest peak was observed at 105 nm.(E) Morphology of separated exosomes was visualized using AFM.The circular morphology was easily observed.

Figure 3 .
Figure 3. Inhibition of various enzymes involved in neurodegeneration leading to processing and production of p-tau and Aβ.Inhibition of the percentage of activities of (A) β-secretase (B) γ-secretase, (C) acetylcholinesterase, (D) CDK5, and (E) GSK-3 in untreated cells (control) or cells treated with 5, 10, or 15 µL NSC-exos.(F) The percentage activity of α-secretase increased in NSC-exos-treated conditions.Data is expressed as the mean ± standard error of the mean (SEM) from three independent experiments (n = 3).* = p < 0.01 and ** = p < 0.001.

Figure 4 .
Figure 4. mRNA expression level of various secretases and kinases involved in neurodegeneration and processing of Aβ and p-tau leading to plague and NFTs in the precence of various concentrations of NSC-exos.qRT-PCR results revealed the suppression of (A) BACE1, (B) PESN, (C) acetylcholinesterase, (D) GSK-3β, and (E) CDK5 mRNA expression, and the enchancment of (F) ADAM10 mRNA expression.Data is expressed as the mean ± standard error of the mean (SEM) from three independent experiments (n = 3).* = p < 0.01 and ** = p < 0.001.

Figure 5 .
Figure 5. Concentration level of Aβ and p-tau in non-treated and NSC-exos-treated SH-SY5Y cells determined by ELISA and WB.Concentration level of (A) Aβ, (B) P-tau, and (C) APP decreased in NSC-exos-treated cells in a dose-dependent manner.No effect was observed in non-treated cells.(D) Concentration of p-tau was measured in control and NSC-exos-treated cells.β-actin was used as an internal control.Data shown here as the mean ± standard error of the mean (SEM) from three independent experiments (n = 3).* = p < 0.01 and ** = p < 0.001.

Figure 6 .
Figure 6.Effect of NSC-exos on cell viability of SH-SY5Y cells.(A) Representative stained images and (B) quantification of NSC-exos treatment of cells.Cells were treated with Aβ42 and then with NSC-exos in a dosedependent manner.Cells showed increased viability with increased NSC-exos concentration, while more dead cells were observed in non-treated cells.Data is pressented as the mean ± standard error of the mean (SEM) from three independent experiments (n = 3).* = p < 0.01 and ** = p < 0.001.

Figure 7 .
Figure 7. Effect of NSC-exos on proinfmatory cytokines and mRNA exoression of the genes of kinases and transcriptional factors leading to neuroinflamtion in glial cells.Actvated Glial cells were treated with NSC--exos in and the proinflamatory cytokines were quantified and expression level of the AMPK/ERK and NF-kB were analysed.(A) Quantifification of iNOx, IL-1B,TNF-alpha and IL-6 in glial cells of control and treated groups.(B) mRNA expression level of NF-kB in verious groups cells (treated and controls) quantified by qRT-PCR.(C) mRNA expression level of NF-kB in verious groups cells (treated and controls) quantified by qRT-PCR.Data is pressented as the mean ± standard error of the mean (SEM) from thre independent experiments (n = 3).* = p < 0.01 and ** = p < 0.001.