Mineralocorticoid Receptor Signaling in Peripheral Blood Cells in Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is associated with alterations in neuroendocrine function, primarily the hypothalamic-pituitary-adrenal axis, including lower expression of the glucocorticoid receptor (GR) and its target genes in peripheral blood mononuclear cells (PBMC) or full blood. We previously found reduced mineralocorticoid receptor (MR) expression in MS patients' peripheral blood. MS is being treated with a widening variety of disease-modifying treatments (DMT), some of which have similar efficacy but different mechanisms of action; body-fluid biomarkers to support the choice of the optimal initial DMT and/or to indicate an unsatisfactory response before clinical activity are unavailable. Using cell culture of volunteers' PBMCs and subsequent gene expression analysis (microarray and qPCR validation), we identified the mRNA expression of OTUD1 to represent MR signaling. The MR and MR target gene expression levels were then measured in full blood samples. In 119 MS (or CIS) patients, the expression of both MR and OTUD1 was lower than in 42 controls. The expression pattern was related to treatment, with the MR expression being particularly low in patients treated with fingolimod. While MR signaling may be involved in the therapeutic effects of some disease-modifying treatments, MR and OTUD1 expression can complement the neuroendocrine assessment of MS disease course. If confirmed, such assessment may support clinical decision-making.

Adaptive gene regulation in the Striatum of RGS9-deficient mice.

BACKGROUND: RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions. RESULTS: Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient mice. CONCLUSION: Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchallenged conditions. SIGNIFICANCE: Identified signaling components may represent novel targets in antidyskinetic therapy. The long splice variant of the regulator of G-protein signaling 9 (RGS9-2) is enriched in striatal medium spiny neurons and dampens dopamine D2 receptor signaling. Lack of RGS9-2 can promote while its overexpression prevents drug-induced dyskinesia. Other animal models of drug-induced dyskinesia rather pointed towards overactivity of dopamine receptor-mediated signaling. To evaluate changes in signaling pathways mRNA expression levels were determined and compared in wild-type and RGS9-deficient mice. Unexpectedly, expression levels of dopamine receptors were unchanged in RGS9-deficient mice, while several genes related to Ca2+ signaling and long-term depression were differentially expressed when compared to wild type animals. Detailed investigations at the protein level revealed hyperphosphorylation of DARPP32 at Thr34 and of ERK1/2 in striata of RGS9-deficient mice. Whole cell patch clamp recordings showed that spontaneous synaptic events are increased (frequency and size) in RGS9-deficient mice while long-term depression is reduced in acute brain slices. These changes are compatible with a Ca2+-induced potentiation of dopamine receptor signaling which may contribute to the drug-induced dyskinesia in RGS9-deficient mice.

Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2ß subunits.

BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2ß subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro.

Human midbrain-derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson's disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole-cell patch-clamp recordings, l-glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration-response plots of differentiated NPCs yielded an EC(50) of 2.2 microM for glutamate and an EC(50) of 36 microM for NMDA. Glutamate-induced currents were markedly inhibited by memantine in contrast to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggesting a higher density of functional NMDA than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. NMDA-evoked currents and calcium signals were blocked by the NR2B-subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage-gated calcium channels by verapamil abolished AMPA-induced calcium responses but only partially reduced NMDA-evoked transients suggesting the expression of calcium-impermeable, GluR2-containing AMPA receptors. Quantitative real-time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 microM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase-immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.

Functional and molecular analysis of GABA receptors in human midbrain-derived neural progenitor cells.

GABA(A) receptor function is involved in regulating proliferation, migration, and differentiation of rodent neural progenitor cells (NPCs). However, little is known about the molecular composition and functional relevance of GABA(A) receptors in human neural progenitors. Here, we investigated human fetal midbrain-derived NPCs in respect to their GABA(A) receptor function and subunit expression using electrophysiology, calcium imaging, and quantitative real-time PCR. Whole-cell recordings of ligand- and voltage-gated ion channels demonstrate the ability of NPCs to generate action potentials and to express functional GABA(A) receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular characterizations indicate a predominance of GABA(A) receptor heteromers containing subunits alpha2, beta1, and/or beta3, and gamma. Intracellular Ca(2+) measurements and the expression profile of the Na(+)-K(+)-Cl(-) co-transporter 1 and the K(+)-Cl(-) co-transporter 2 in differentiated NPCs suggest that GABA evokes depolarizations mediated by GABA(A) receptors. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential GABA(A) receptor properties during neuronal maturation in vitro.

V2 vasopressin receptor deficiency causes changes in expression and function of renal and hypothalamic components involved in electrolyte and water homeostasis.

Polyuria, hypernatremia, and hypovolemia are the major clinical signs of inherited nephrogenic diabetes insipidus (NDI). Hypernatremia is commonly considered a secondary sign caused by the net loss of water due to insufficient insertion of aquaporin-2 water channels into the apical membrane of the collecting duct cells. In the present study, we employed transcriptome-wide expression analysis to study gene expression in V2 vasopressin receptor (Avpr2)-deficient mice, an animal model for X-linked NDI. Gene expression changes in NDI mice indicate increased proximal tubular sodium reabsorption. Expression of several key genes including Na+-K+-ATPase and carbonic anhydrases was increased at the mRNA levels and accompanied by enhanced enzyme activities. In addition, altered expression was also observed for components of the eicosanoid and thyroid hormone pathways, including cyclooxygenases and deiodinases, in both kidney and hypothalamus. These effects are likely to contribute to the clinical NDI phenotype. Finally, our data highlight the involvement of the renin-angiotensin-aldosterone system in NDI pathophysiology and provide clues to explain the effectiveness of diuretics and indomethacin in the treatment of NDI.

Linking the functions of unrelated proteins using a novel directed evolution domain insertion method.

We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 beta-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b(562) with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of alpha-helices and beta-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.

Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx).

Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeq(NNN). The bla gene encoding TEM-1 beta-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.

Investigating protein structural plasticity by surveying the consequence of an amino acid deletion from TEM-1 beta-lactamase.

While the deletion of an amino acid is a common mutation observed in nature, it is generally thought to be disruptive to protein structure. Using a directed evolution approach, we find that the enzyme TEM-1 beta-lactamase was broadly tolerant to the deletion mutations sampled. Circa 73% of the variants analysed retained activity towards ampicillin, with deletion mutations observed in helices and strands as well as regions important for structure and function. Several deletion variants had enhanced activity towards ceftazidime compared to the wild-type TEM-1 demonstrating that removal of an amino acid can have a beneficial outcome.