Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Additional file 1: of Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes

Figure S1. NO production in αSYN and TLR4-inhibitor treated astrocytes. Primary astrocytes were treated in OptiMEM (supplemented with penicillin G and streptomycin) with 5 µM of control peptide (ctrl pep; CP7: RNTISGNIYSARRRRRRRRR) or TLR4 inhibitor peptide (viper: KYSFKLILAEYRRRRRRRRR) (both from Imgenex/Biomol) two hours prior to the addition of recombinant αSYN (0.7 µM or 3.5 µM, as indicated) or LPS (0.1 µg/ml) as positive control or left untreated (Ø). 48 h later NO released to the media was measured using Griess reagent (Fluka/Sigma-Aldrich). The amount of NO was normalized to total protein amount in the sample well. The samples were incubated protected from light for 15 min, after which the optic absorption at 550 nm was measured (Model 680 Microplate Reader, Bio-Rad). A standard curve made from 0 to 50 µM sodium nitrite was used as reference. Measurements were normalized against the total protein content of the well. Error bars are standard deviation; n = 16

Additional file 3: of Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes

Figure S3. Low levels of contaminating endotoxins and proteins in the recombinant synuclein preparations. (A) Endotoxin levels in the purified protein samples were measured with the 0.06 EU/ml cut off using three dilutions of the samples (1:1, 1:10 and 1:25). Protein concentration was measured with nanodrop using the extinction factors ε(280, 0.1 %) 0.354 for αSYN and 0.417 for βSYN. The table indicates the concentrations of synuclein and endotoxin levels in the protein stocks used in this work. (B) Samples of purified recombinant αSYN and βSYN were tested for purity by a sensitive variant of Coomassie stained polyacrylamide gels. (C) Samples of purified synuclein proteins were immunoblotted using antibodies specific for αSYN and βSYN, respectively. (D) Samples that were cut out from Coomassie stained polyacrylamide gels (indicated by red dashed boxes in the upper right panel) were trypsinized and the peptides were analyzed by mass spectroscopy

Additional file 1: Table S2. of Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model

Primer sequences which were used for RT-PCR. (DOC 40 kb

Additional file 3: Figure S2. of Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model

Classification and scoring of phospho-α-synuclein and PK-PET Blot. (A) Classification of phospho-α-synuclein staining into four different scores. Representative pictures for scoring. Score 0: no p-α-synuclein staining, score 1: little staining in brainstem and DpMe, score 2: strong staining in brainstem and DpMe, score 3: strong p-α-synuclein staining in brainstem, DpMe, and cerebellum indicating severe disease progression. (B) Scoring to classify PK-PET Blot. Score 0: no PK resistant aggregates, score 1: light aggregates in brainstem and Deep Mesencepahlic nucleus (DpMe), score 2: clear PK resistant aggregates in brainstem and DpMe, score 3: dominant aggregates in brainstem and DpMe. Score 4: Prominent aggregates in brainstem, DpMe and cerebellum. (PDF 125 kb

Pogonia minor Makino

原著和名: ヤマトキサウ科名: ラン科 = Orchidaceae採集地: 熊本県 人吉市 田野 (肥後 人吉市 田野)採集日: 1974/8/9採集者: 萩庭丈壽整理番号: JH003082国立科学博物館整理番号: TNS-VS-95308