Data_Sheet_1_Cerebrospinal fluid level of proNGF as potential diagnostic biomarker in patients with frontotemporal dementia.docx

IntroductionFrontotemporal dementia (FTD) is an extremely heterogeneous and complex neurodegenerative disease, exhibiting different phenotypes, genetic backgrounds, and pathological states. Due to these characteristics, and to the fact that clinical symptoms overlap with those of other neurodegenerative diseases or psychiatric disorders, the diagnosis based only on the clinical evaluation is very difficult. The currently used biomarkers help in the clinical diagnosis, but are insufficient and do not cover all the clinical needs.MethodsBy the means of a new immunoassay, we have measured and analyzed the proNGF levels in 43 cerebrospinal fluids (CSF) from FTD patients, and compared the results to those obtained in CSF from 84 Alzheimer’s disease (AD), 15 subjective memory complaints (SMC) and 13 control subjects.ResultsA statistically significant difference between proNGF levels in FTD compared to AD, SMC and controls subjects was found. The statistical models reveal that proNGF determination increases the accuracy of FTD diagnosis, if added to the clinically validated CSF biomarkers.DiscussionThese results suggest that proNGF could be included in a panel of biomarkers to improve the FTD diagnosis.</p

Measurement of nucleotides in the <i>e</i>PL kinase•PLP complexes^a.

a<p><i>e</i>PL kinase (130 µM) samples were incubated with different combinations of PL (0.5 mM), PLP (0.5 mM), MgATP (1 mM), MgADP (1 mM) and MgCl₂ (0.5 mM) for one hour at 37°C. These samples were then passed through a sizing column and the fractions containing the enzyme were collected and analyzed with respect to PLP and nucleotides content. The values reported in the table are percentages of saturation with respect to enzyme subunits.</p>b<p>n.d.: not detectable with the method employed (minimum detectable level 1.5 µM; see Experimental Procedures for details).</p>c<p>MgCl₂ was also omitted from the chromatography equilibration and elution buffer.</p

Vitamin B₆ metabolism.

<p>A) Reactions in B₆ metabolism. <i>Reaction 1</i>, enzymes involved in the <i>de novo</i> biosynthesis of pyridoxine 5′-phosphate (PNP); <i>Reaction 2</i>, PNP oxidase; <i>Reaction 3</i>, reaction of apo-B₆ enzymes with PLP to form active holo-B₆ enzymes; <i>Reaction 4</i>, degradation of holo-B₆ enzymes to amino acids and PLP; <i>Reaction 5</i>, PLP phosphatase; <i>Reaction 6</i>, PL kinase. B) Structures of B₆ vitamers.</p

Rate of formation of ePL kinase•PLP complex.

<p>To a series of 100 µl solutions in Eppendorf vials containing 0.4 mM MgATP, 0.20 mM MgCl₂ and either 0.150 mM PL or 0.150 mM PLP at 37°C was added 9 nmoles of <i>e</i>PL kinase (90 µM). After 2, 5, 15 and 25 min contents of vials were withdrawn and placed on small Sephadex G-50 columns at 4°C equilibrated with 1 mM MgATP and 0.2 mM MgCl₂ (see Experimental Procedures) to separate bound and free PLP. The eluate of each column was 400 µl. Spectra were recorded and the absorbance at 420 nm determined. Open circles, reactions initiated with PL, closed circles reactions initiated with PLP. The lines through the experimental points are those obtained from nonlinear least squares fittings of data to an exponential equation which gave rate constants of 0.4 min⁻¹ and 0.1 min⁻¹ and amplitudes of 100% and 83% for the experiments initiated with PL and PLP, respectively.</p

Kinetics of ePL kinase inhibition.

<p>The formation of PLP by ePL kinase was followed at 388 nm in 400 µl of reaction solution containing 1 mM MgATP, 0.2 mM MgCl₂, and 1 mM PL at 37°C. At the first arrow, <i>e</i>PL kinase was added to 0.9 µM and PLP formation followed for about 120 seconds. A second aliquot of <i>e</i>PL kinase was added at the second arrow.</p

Kinetic parameters for wild type and K229Q mutant <i>e</i>PL kinase.

<p>Kinetic parameters for wild type and K229Q mutant <i>e</i>PL kinase.</p

Rate of transfer of PLP from PL kinase•PLP to apo E. coli serine hydroxymethyltransferase.

<p>Fraction of apo-<i>e</i>SHMT (20 µM) being converted to holo-<i>e</i>SHMT with PLP (20 µM) (•–•). Fraction of apo-<i>e</i>SHMT (20 µM) being converted to holo-<i>e</i>SHMT with an equivalent amount of PL kinase•PLP (20 µM) (▴–▴). Repeat of the conversion of apo-<i>e</i>SHMT with free PLP (○–○) or PL kinase•PLP (Δ–Δ) to holo-<i>e</i>SHMT in the presence of 3 µM PLP phosphatase.</p

Mechanism of reaction between PLP and the active site K229.

<p>A) A scheme showing the structures of the carbinolamine intermediate and the enolimine form of the protonated PLP aldimine. B) Active site structure of the binary complex of <i>e</i>PL kinase and PL showing the position of K229.</p

Comparison of PLP formation with wild type and K229Q ePL kinases.

<p>The kinetics of PLP formation catalyzed by <i>e</i>PL kinase was followed at 388 nm upon addition (shown by the arrow) of 0.3 µM of wild type enzyme (continuous line) or 0.3 µM of K229Q enzyme (dotted line). Each reaction contained 1 mM MgATP, 0.2 mM MgCl₂ and 1 mM PL, at 37°C.</p

Rate of dissociation of PLP from the ePL kinase•PLP complex.

<p>The rate of dissociation of PLP from the <i>e</i>PL kinase•PLP complex was followed by observing the change in optical activity of the bound PLP at 37°C. Panel A: Spectra of the complex (60 µM) at time zero (curve a) and after 120 min in the presence of 10 µM PLP phosphatase (curve b). Panel B: measured decrease in optical activity after addition of PLP phosphatase at 415 nm with time and as an exponential process with rate constant of 0.012 min⁻¹.</p