Determination of thyroid hormones in placenta using isotope-dilution liquid chromatography quadrupole time-of-flight mass spectrometry

The transplacental passage of thyroid hormones (THs) is of great significance since the maternal THs are vitally important in ensuring the normal fetal development. In this paper, we determined the concentrations of seven THs, viz. L-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3), 3,3',5'-triiodo-l-thyronine (rT3), 3,3'-diiodo-l-thyronine (T2), 3,5-diiodo-l-thyronine (rT2), 3-iodo-l-thyronine (T1) and 3-iodothyronamine (T1AM), in placenta using isotope dilution liquid chromatography quadrupole time-of-flight mass spectrometry. We optimized the method using isotopically labeled quantification standards (13C6-T4, 13C6-T3, 13C6-rT3 and 13C6-T2) and recovery standard (13C12-T4) in combination with solid-liquid extraction, liquid-liquid extraction and solid phase extraction. The linearity was obtained in the range of 0.5-150 pg uL-1 with R2 values >0.99. The method detection limits (MDLs) ranged from 0.01 ng g-1 to 0.2 ng g-1, while the method quantification limits (MQLs) were between 0.04 ng g-1 and 0.7 ng g-1. The spike-recoveries for THs (except for T1 and T1AM) were in the range of 81.0%-112%, with a coefficient of variation (CV) of 0.5-6.2%. The intra-day CVs and inter-day CVs were 0.5%-10.3% and 1.19%-8.88%, respectively. Concentrations of the THs were 22.9-35.0 ng g-1 T4, 0.32-0.46 ng g-1 T3, 2.86-3.69 ng g-1 rT3, 0.16-0.26 ng g-1 T2, and < MDL for other THs in five human placentas, and 2.05-3.51 ng g-1 T4, 0.37-0.62 ng g-1 T3, 0.96-1.3 ng g-1 rT3, 0.07-0.13 ng g-1 T2 and < MDL for other THs in five mouse placentas. The presence of T2 was tracked in placenta for the first time. This method with improved selectivity and sensitivity allows comprehensive evaluation of TH homeostasis in research of metabolism and effects of environmental contaminant exposures

Expression analysis of <i>Lrrk2</i> mRNA in the forebrain and midbrain of postnatal mice.

<p>ISH for <i>Lrrk2</i> mRNA in coronal sections of forebrain (left part) and midbrain (right part) from postnatal day 7, postnatal day 21 and adult mice. Note that expression of <i>Lrrk2</i> is highly dynamic in the postnatal forebrain. While the expression level in the striatum and the olfactory tubercle seem to increase dramatically during development, the <i>Lrrk2</i> level in cortex remain rather unchanged (<b>A,C,E</b>). On the level of the midbrain, ISH signals for <i>Lrrk2</i> augment considerably in the hippocampus and cortex, while the level in midbrain structures like the Substantia nigra pars compacta (white arrows, SNc) remain quite low (<b>B,D,F</b>). Abbreviations: Cc, corpus callosum; Cx, cortex; Cp, choroid plexus (white arrowhead in B’); Cs, superior colliculus; Hi, hippocampus; Ot, olfactory tubercle; Pc, piriform cortex; Pn, parafascicular nucleus; Rn, red nucleus; Se, septum; SNc, SN pars compacta; SNr, SN pars reticulata; St, striatum. Scale bars represent 1 mm.</p

Comparative expression analysis of <i>Lrrk1</i> and <i>Lrrk2</i> mRNA in the brain of adult mice.

<p>ISH for <i>Lrrk1</i> (top part) and <i>Lrrk2</i> (bottom part) mRNA in sections from P21 (<b>A–D</b>) and adult mice (<b>E</b>). Note that <i>Lrrk1</i> mRNA is barely detectable in the adult mouse brain and only visible in the non-neuronal meninges (white arrow) and the olfactory bulb (white arrowhead) (<b>A</b>). A detailed view onto the adult olfactory bulb depicts the solely neuronal expression of <i>Lrrk1</i> in the mitral cell layer (<b>B</b>). Specificity of the <i>Lrrk1</i> signals were verified by using the corresponding sence-probe as negative control (<b>C</b>). In contrast, strong <i>Lrrk2</i> expression can be detected in various regions throughout the postnatal (<b>D</b>) and adult CNS (<b>E</b>). Abbreviations: An, anterior olfactory nucleus; Bs, brain stem; Cb, cerebellum; Cc, corpus callosum; Cm, motor cortex; Co, cortex; Cp, choroid plexus; Cs, somatosensory cortex; Cv, visual cortex; Gl, glomerular layer; Gr, granual layer; Hi, hippocampus; Me, meninges; Mi, mitral layer; Ob, olfactory bulb; Ot, olfactory tubercle; Pn, parafascicular nucleus; Pl, plexiform layer; Po, pons; SNc, substantia nigra pars compacta; St, striatum; Th, thalamus; Lv, lateral ventricle. Scale bars represent 1 mm (A, D, E) and 500 µm (B).</p

Comparative expression analysis of <i>Lrrk1</i> and <i>Lrrk2</i> mRNA during embryogenesis.

<p>ISH for <i>Lrrk1</i> (left part) and <i>Lrrk2</i> (right part) mRNA in sections from E10, E12.5 and E15.5 embryos. For each embryo, a brightfield image (e.g. A, for anatomical orientation) and a darkfield image (e.g. A’, ISH signals in white) are shown. (<b>A–D</b>) At E10, small hotspots of <i>Lrrk1</i> expression were observed only in the cephalic mesenchyme (Cm in A+B), the palate and the optic stalk (Oc in B), while <i>Lrrk2</i> mRNA was found basically in the urogenital ridge (Ur in C, arrow). Note that at this stage of gestation expression of <i>Lrrk1</i> and <i>Lrrk2</i> was virtually absent from the developing CNS (A–D). (<b>E,F</b>) At E12.5, additional spots for <i>Lrrk1</i> expression were visible in the epithelia of the nose and mouth (Nc in E) and for <i>Lrrk2</i> expression in the choroid plexus (Cp in F, arrow) and developing pituitary gland (Pi in F). (<b>G,H</b>) Around E15.5, <i>Lrrk1</i> and <i>Lrrk2</i> expression became stronger in several organs of the embryos (G+H) including liver (Li), kidney (Ki), lung (Lu) and heart (Pe) as well as in the choroid plexus (Cp, arrows). Ao, Aorta; Ba, branchial arch; Cm, cephalic mesenchyme; Cx, cortex; Cp, choroid plexus; Da, dorsal aorta; Dg, dorsal root ganglia; Fb, forebrain; Ge, ganglionic eminence; Gu, gut; Hb, hindbrain; Hl, hind limb; Id, intervertebral disc; In, incisive; Jl, lower jaw; Ki, kidney; Lb, limb but; Li, liver; Lu, lung; Mb, midbrain; Nc, nasal cavity; Oc, optic cup; Os, optic stalk; Pa, palate; Pe, pericardium; Rp, Rathke’s pouch; Sp, spinal cord; Ur, urogenital ridge; V4, fourth ventricle; Vl, lateral ventricle. Orientation of sections: A+C, coronal; B, horizontal; D–H, sagittal. Scale bars represent 500 µm in A–D, 1 mm in E–H.</p

Cellular expression analysis of <i>Lrrk2</i> mRNA in the striatum of adult mice.

<p>(<b>A</b>) High magnification of a representative brightfield ISH image using radioactive-labeled <i>Lrrk2</i>-specific riboprobes and counterstained by cresyl violet: <i>Lrrk2</i> mRNA is predominantly expressed in neurons (black arrows, <i>Lrrk2</i>-positive neurons; white arrows, <i>Lrrk2</i>-negative neurons; black arrowheads, <i>Lrrk2</i>-positive glia; white arrowheads, <i>Lrrk2</i>-negative glia). (<b>B, C</b>) Representative images of double <i>in situ</i>−/Immunohistochemistry (ISH/IHC) in the medial part of the putamen: ISH for <i>Lrrk2</i> using DIG-labeled riboprobes (non-fluorescent black precipitate) followed by IHC stainings (green) for the two main dopamine receptors D1 (DRD1a) and D2 (DRD2). (<b>D, E</b>) Quantification of the <i>Lrrk2</i>/DRD1a and <i>Lrrk2</i>/DRD2 ISH/IHC stainings in the striatum revealed 36% <i>Lrrk2</i>-positive, 25% DRD1a-positive and 39% double positive cells. In case of the DRD2 <i>in situ</i>−/Immunohistochemistry, 38% <i>Lrrk2</i>-positive, 24% DRD2-positive and 37% double positive cells could be detected. Scale bar represents 25 µm.</p

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains T

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that the deglycase DJ-1 (encoded by PARK7, a key familial Parkinson\u27s disease gene) is a pacemaker regulating PDH activity in CD

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains T(reg) homeostasis during ageing.

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that the deglycase DJ-1 (encoded by PARK7, a key familial Parkinson's disease gene) is a pacemaker regulating PDH activity in CD4(+) regulatory T cells (T(reg) cells). DJ-1 binds to PDHE1-β (PDHB), inhibiting phosphorylation of PDHE1-α (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Park7 (Dj-1) deletion impairs T(reg) survival starting in young mice and reduces T(reg) homeostatic proliferation and cellularity only in aged mice. This leads to increased severity in aged mice during the remission of experimental autoimmune encephalomyelitis (EAE). Dj-1 deletion also compromises differentiation of inducible T(reg) cells especially in aged mice, and the impairment occurs via regulation of PDHB. These findings provide unforeseen insight into the complicated regulatory machinery of the PDH complex. As T(reg) homeostasis is dysregulated in many complex diseases, the DJ-1-PDHB axis represents a potential target to maintain or re-establish T(reg) homeostasis

A patient-based model of RNA mis-splicing uncovers treatment targets in Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous neurodegenerative disorder with monogenic forms representing prototypes of the underlying molecular pathology and reproducing to variable degrees the sporadic forms of the disease. Using a patient-based in vitro model of PARK7-linked PD, we identified a U1-dependent splicing defect causing a drastic reduction in DJ-1 protein and, consequently, mitochondrial dysfunction. Targeting defective exon skipping with genetically engineered U1-snRNA recovered DJ-1 protein expression in neuronal precursor cells and differentiated neurons. After prioritization of candidate drugs, we identified and validated a combinatorial treatment with the small-molecule compounds rectifier of aberrant splicing (RECTAS) and phenylbutyric acid, which restored DJ-1 protein and mitochondrial dysfunction in patient-derived fibroblasts as well as dopaminergic neuronal cell loss in mutant midbrain organoids. Our analysis of a large number of exomes revealed that U1 splice-site mutations were enriched in sporadic PD patients. Therefore, our study suggests an alternative strategy to restore cellular abnormalities in in vitro models of PD and provides a proof of concept for neuroprotection based on precision medicine strategies in PD