Journal: Journal of neuropathology and experimental neurology

Article Title: Hereditary ferritinopathy: a novel mutation, its cellular pathology, and pathogenetic insights.

doi: 10.1093/jnen/64.4.280

Figure Lengend Snippet: FIGURE 9. Hyaline deposits. Pallidum. (A) Pleomorphic hyaline deposits, some containing nuclei, within the same area of GPe as in Figure 6A. (B) Prussian-blue reactivity. (C) PTAH reactivity. (D) Polyclonal anti-ferritin immunoreactivity. (E) Rare association of hyaline deposits with GFAP-immunoreactive astrocyte. (F) HO-2-immunoreactivity. Original magnifications: (A) Hematoxylin and eosin, 1503; (B) Perl’s stain, 2203; (C) 2203; (D) 2503; (E) 2203; (F) 2503.

Article Snippet: In order to identify the cell(s) of origin of the unique vacuolated nuclei, antibodies to S-100 (polyclonal, 1:20k, Lot #941; Innovation Foundation, Toronto, Ontario, Canada), GFAP, CD68 (monoclonal, 1:200, Lot #120201; DAKO), and carbonic anhydrase II (CAII) (polyclonal, 100-401-136/4383, 1:15k with retrieval; Rockland, Gilbertsville, PA) were applied to sections of basal ganglia and cerebellum, while antibodies to synaptophysin (monoclonal, 1:100 with retrieval, #M0363-UC/0403; Biogenex, San Ramon, CA), PGP 9.5 (polyclonal, 1:1,000 with retrieval, Lot #11830; Biogenesis, Handsown, NH), neurofilament protein 2F11 (monoclonal, 1:350, #M0762/117 DAKO) (101), and amyloid precursor protein (APP A4; monoclonal, 1:20K with heat retrieval, #23080547, Chemicon) were applied to the section of basal ganglia.

Techniques: Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Plasmid map for GDNF production

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Bone-marrow derived macrophages were transfected by electroporation using four different conditions described in Experimental section. Then, cells were washed and cultured in complete media for up to 6 days. The GDNF expression levels in cells (solid bars), and EVs collected from conditioned media (stripped bars) was assessed by ELISA on day 1 (white bars), day 4 (grey bars), and day 6 (black bars). Successful transfection was accomplished with three conditions (#2 - #4). N = 4, *p < 0.05, compared to sham-transfected macrophages (dashed line, condition #1).

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Derivative Assay, Transfection, Electroporation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Primary macrophages were transfected with GDNF-encoding p DNA by electroporation (condition #4), and EV-GDNF were collected from conditioned media on day 6. EV-GDNF were characterized for size, zeta potential, and morphology by ZetaView QUATT Nanoparticle Tracking Microscope PMX-420 ( A ), and AFM ( B ). The presence of EV-specific membrane proteins was EV-GDNF was confirmed by Wes ( C ) and quantified using Compass SW software ( D ). The bar: 1 µm.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Electroporation, Zeta Potential Analyzer, Microscopy, Membrane, Software

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: EVs samples from sham-transfected (white bars), and GDNF-transfected (black bars) macrophages were digested ( N =3) with trypsin and examined by nano-liquid chromatography tandem MS (nanoLC–MS/MS) with multiple reaction monitoring (MRM). Samples of 20 µg total protein were used, and 0.08 µg (0.4 % of the sample) was injected. No significant differences in specific proteins expression were found between sham EVs and EV-GDNF (t-tests, p < 0.05). Peptide identification is shown in . A CD81 peptide employed in other studies was not detected in these analyses. Values are means ± SD.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Liquid Chromatography, Tandem Mass Spectroscopy, Targeted Proteomics, Injection, Expressing

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Macrophages were transfected with GDNF-encoding p DNA by electroporation, and the levels of GDNF-DNA in the cells ( A ) and EVs released by these cells ( B ) were assessed. A significant amount of GDNF-DNA was detected in parent cells, as well as in the EVs. Statistical significance was assessed by One Way ANOVA corrected for multiple comparisons using the FDR. ** p < 0.01, or **** p < 0.0001.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Electroporation

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: The effect of EV-GDNF on motor functions and activity was assessed in Wire hanging test, and Rotarod test ( A, B ), as well as in OFA tests ( C, D ). ( A, B ) Transgenic mice were i.n . injected with EV-GDNF (triangles, 3×10 9 particles/10 µL/mouse), or sham EVs (empty circles, 3×10 9 particles/10 µL/mouse), or saline (filled circles, 10 µL/mouse). Wild type mice were i.n . injected with saline (filled squares, 10 µL/mouse) were used as controls. Wire hanging test ( A ), and Rotarod test ( B ) demonstrated significant improvements in motor functions upon treatment with EV-GDNF. ( C, D ) OFA tests at 12 mo. demonstrated improved behavior in EV-GDNF treated PD mice (striped bars) compared to PD mice treated with saline (white bars) that was similar as in healthy WT mice (black bars) including decreases in the hyperactivity and anxiety-like behavior. The differences between sham EVs and saline in PD mice were inconclusive. Values are means ± SEM ( N = 10), * p < 0.05, ** p < 0.005, and # p < 0.05, as compared to WT control.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Activity Assay, Transgenic Assay, Injection, Saline, Control

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old, N = 10) were i.n . injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, and brain slides were stained with TH, a marker for dopaminergic neurons ( A ); or Ab to CD11b for activated microglia ( B ). The images indicate significant preservation of TH-positive neurons and decrease in microglial activation in Parkin Q311(X)A mice upon EV-GDNF treatment compared to PD mice treated with saline. The administration of sham EVs did not cause significant therapeutic effects.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining, Marker, Preserving, Activation Assay

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brains were removed post-mortem, and homogenized in cell lysis buffer. Elevated cytokine levels in the brain, were recorded in PD mice treated with saline and Sham EVs. Administration of EV-GDNF significantly decreased pro-inflammatory molecules in the brain compared with PD mice treated with saline. N = 4, # p < 0.05 compared to healthy WT animals; * p < 0.05 compared to PD mice treated with saline, $ p <0.05 compared to PD mice treated with saline and sham EVs.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Lysis

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or ( 3 ) EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining ( A – D ) and H&E staining ( E – H ). The obtained bright light images show lower number of Nissl bodies with neuronal shrinkage ( B ) and damages tissues with degeneration in the neurons ( F ) in PD mice treated with saline when compared to WT mice ( A, E ). Histological analysis indicate neuroprotective effects in the brain of PD mice treated with EV-GDNF with healthy morphology in tissue structure and high integrity of neurons ( C, G ) when comparted to PD mice treated with saline ( B, F ). The administration of sham EVs did not have significant therapeutic effect in PD mice ( D, H ). Black arrows, degenerated neurons; blue arrows, elongated irregular nuclear morphology.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse) weekly three times. Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining. The obtained bright light images show lower number of Nissl bodies with neuronal shrinkage and damages tissues with degeneration in the neurons in PD mice treated with saline when compared to WT mice. Histological analysis indicates neuroprotective effects in the brain of PD mice treated with GDNF-EVs with healthy morphology in tissue structure and high integrity of neurons when comparted to PD mice treated with saline. The administration of sham EVs did not have significant therapeutic effect in PD mice.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse) weekly three times. Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining. The obtained bright light images show damaged tissues with degeneration in the neurons in PD mice treated with saline when compared to WT mice. Histological analysis indicates neuroprotective effects in the brain of PD mice treated with GDNF-EVs with healthy morphology in tissue structure when comparted to PD mice treated with saline. The administration of sham EVs did not have significant therapeutic effect in PD mice.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. of age) were i.n . injected with saline, or EV-GDNF, or sham EVs (3×10 9 particles/10 µL/mouse, once a week, 3x weeks). At 16 mo. of age total weigh of the animals was recorded. No gross toxicity manifested in the losing weight was detected in mice injected with EV-GDNF and well as sham EVs.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline