Journal: Alzheimer's & Dementia

Article Title: Nerve growth factor (NGF) pathway biomarkers in Down syndrome prior to and after the onset of clinical Alzheimer's disease: A paired CSF and plasma study

doi: 10.1002/alz.12229

Figure Lengend Snippet: Upregulation of proNGF (nerve growth factor) in plasma and cerebrospinal fluid (CSF) from people with Down syndrome (DS). A, The 27 kDa form of proNGF, measured by Western blotting, is upregulated in plasma from Alzheimer's disease (AD)‐asymptomatic people with Down syndrome (aDS) and in Down syndrome with symptomatic AD (DSAD) compared to non‐trisomic controls, one‐way analysis of variance (ANOVA), F = 5.03, P = .01 and Bonferroni post hoc correction; * P < .05. B, ProNGF (27 kDa) is also upregulated in CSF from people with aDS and DSAD compared to non‐trisomic controls, Kruskal Wallis test P = .0002 H = 16.83 and Dunn's post hoc correction; ** P < .01; *** P < .001. C, The 50 kDa form of proNGF is upregulated in plasma from people with aDS and DSAD compared to non‐trisomic controls, one‐way ANOVA, F = 5.62, P = .006 and Bonferroni post hoc correction; * P < .05. D, 50 kDa proNGF is upregulated in the CSF of people with aDS compared to non‐trisomic controls, and further upregulated in people with DSAD, one‐way ANOVA, F = 21.17, P < .0001 and Bonferroni post‐hoc correction; *** P < .001. Data are displayed in box and whisker plots, in which the median is represented by the horizontal line and the whiskers go from each quartile to the minimum or maximum value. HC, Healthy controls: n = 16, aDS: n = 14, DSAD: n = 22; IOD, integrated optical density

Article Snippet: Quantifiability across physiologically relevant concentrations/integrated optical density (IOD) values was established by reconstitution experiments in which plasma/CSF was spiked with 2, 5, 10, or 20 ng of human recombinant proNGF protein (Alomone Labs, Israel), and subsequently analyzed by Western blotting as described above.

Techniques: Western Blot, Whisker Assay

Journal: Alzheimer's & Dementia

Article Title: Nerve growth factor (NGF) pathway biomarkers in Down syndrome prior to and after the onset of clinical Alzheimer's disease: A paired CSF and plasma study

doi: 10.1002/alz.12229

Figure Lengend Snippet: Cerebrospinal fluid (CSF) matrix metalloproteinase (MMP)‐9 and proNGF (precursor of nerve growth factor) discriminate effectively between Alzheimer's disease (AD) symptomatic and asymptomatic people with Down syndrome (DS). A, CSF levels of the 50 kDa proNGF form effectively identify people with Down syndrome with symptomatic Alzheimer's disease (DSAD) from within the total DS sample group, with an area under the receiver operator characteristic (ROC) curve of 0.86. B, CSF MMP‐9 levels effectively identify people with DSAD from within the total DS sample group, with an area under the ROC curve of 0.87. C, ROC curves for the core CSF biomarkers of AD demonstrate that MMP‐9 and proNGF (50 kDa) measured in CSF outperformed CSF pTau‐181 (AUC = 0.85) and total tau (AUC = 0.75), while being outperformed by the Aβ42/40 ratio (AUC = 0.88)

Article Snippet: Quantifiability across physiologically relevant concentrations/integrated optical density (IOD) values was established by reconstitution experiments in which plasma/CSF was spiked with 2, 5, 10, or 20 ng of human recombinant proNGF protein (Alomone Labs, Israel), and subsequently analyzed by Western blotting as described above.

Techniques:

Journal: eNeuro

Article Title: Cortical Brain Injury Causes Retrograde Degeneration of Afferent Basal Forebrain Cholinergic Neurons via the p75NTR

doi: 10.1523/ENEURO.0067-23.2023

Figure Lengend Snippet: Proneurotrophins are induced in the ipsilateral cortex but not the basal forebrain after cortical FPI. a–c , Brain tissue lysates from naive, sham, and injured (2 atm) wild-type adult mice were obtained 1DPI, 3DPI, and 7DPI to determine levels of proBDNF ( a , b ) and proNGF ( c ) in the injured versus uninjured side. Cortical tissue lysate ( a ) harvested for Western blot was probed for proBDNF (32 kDa) in the ipsilateral and contralateral cortex at 1DPI, 3DPI, and 7DPI in naive, sham, and injured mice. Basal forebrain tissue lysate ( b ) harvested for Western blot was probed for proBDNF (32 kDa) in the ipsilateral versus contralateral basal forebrain at 1DPI, 3DPI, and 7DPI in naive, sham, and injured mice. Cortex and basal forebrain tissue lysates ( c ) harvested for Western blot were probed for proNGF (37 kDa) at 3DPI after FPI in the ipsilateral versus contralateral side of the cortex and the basal forebrain; n = 4 (naive), n = 4 (sham 1DPI), n = 4 (injured 1DPI), n = 4 (sham 3DPI), n = 4 (injured 3DPI), n = 3 (sham 7DPI), n = 3 (injured 7DPI; a , b ); n = 3 (naive), n = 4 (sham 3DPI), n = 4 (injured 3DPI; c ). The established size of proBDNF is 32 kDa; however, a prominent band of 25 kDa was also recognized by the BDNF antibody that appeared to be regulated by injury, but the identity of that band is unclear.

Article Snippet: Recombinant human proNGF (cleavage resistant) protein (catalog #N-285) and recombinant mouse proBDNF (cleavage resistant) protein (catalog #B-243) were purchased from Alomone Labs. Poly- d -lysine, glucose, transferrin, insulin, putrescine, selenium, progesterone, penicillin, and streptomycin were purchased from Sigma-Aldrich.

Techniques: Western Blot

Journal: BMC Endocrine Disorders

Article Title: The precursor for nerve growth factor (proNGF) is not a serum or biopsy-rinse biomarker for thyroid cancer diagnosis

doi: 10.1186/s12902-019-0457-1

Figure Lengend Snippet: ProNGF serum levels after thyroidectomy and half-life. a Change in serum proNGF following total thyroidectomy . Pre- and post- thyroidectomy serum samples were available for 11 cases where pre-operative serum proNGF was detectable. No significant difference was detected between pre- and post- thyroidectomy levels of proNGF. b ProNGF in vitro half-life. Aliquots of serum negative for proNGF was spiked with 20 ng/mL recombinant proNGF dissolved in Assay Diluent A (Biosensis, Australia) in a 1:1 ratio, then incubated at 37 °C for increments of 24 h, then assayed at 1:20 dilution with Heterophilic Blocking Antibody (BL-003-1000). An exponential decay curve was fitted, giving an estimated in-vitro half-life in serum of 1.5 h. Similar results were obtained using phosphate-buffered-saline as diluent

Article Snippet: Samples were run in triplicate at 1:20 dilution (to minimize matrix effects, as recommended by the manufacturer), with positive results confirmed on a second plate; and run with an in-house quality control (QC) samples of serum spiked with recombinant human proNGF (Biosensis Pty Ltd., Adelaide, Australia).

Techniques: In Vitro, Recombinant, Incubation, Blocking Assay, Saline

Journal: PLoS ONE

Article Title: LPS-Induced proNGF Synthesis and Release in the N9 and BV2 Microglial Cells: A New Pathway Underling Microglial Toxicity in Neuroinflammation

doi: 10.1371/journal.pone.0073768

Figure Lengend Snippet: Characterization of rhproNGF is shown by western blot (A). Positive expression of p75NTR and sortilin is detected in SH-SY5Y cells in both control and LPS stimulation conditions (B). MTT shows no significant change of SH-SY5Y cell viability after proNGF treatment at different concentrations (C). Hoechst/PI staining indicates cell damage of SH-SY5Y cells resulted from proNGF treatment at 10ng/ml and 40ng/ml (D). E, Immunoblot shows a slight increase of cleaved caspase-3 expression (mainly 19kDa) following proNGF treatment (E). TUNEL assay shows appearance and increase of apoptotic cell death in SH-SY5Y cells with proNGF treatment (F).

Article Snippet: When cells were at 80% confluence, stock solution of recombinant human proNGF (rhproNGF, SinoBio, C078, Shanghai, China) was added the free serum cultural medium at final concentration of 0,1, 2.5, 5, 10, 20, 40ng/ml and kept for 24 h. Cell samples were respectively processed for MTT assay, Hoechst/PI and TUNEL staining to detect cell viability and cell death or cell apoptosis.

Techniques: Western Blot, Expressing, Control, Staining, TUNEL Assay