Deletion of Munc18-1 in 5-HT Neurons Results in Rapid Degeneration of the 5-HT System and Early Postnatal Lethality

The serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. The majority of mutant mice died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival

Genetic and phenotypic heterogeneity in sporadic and familial forms of paroxysmal dyskinesia

<p>Paroxysmal dyskinesia (PxD) is a group of movement disorders characterized by recurrent episodes of involuntary movements. Familial paroxysmal kinesigenic dyskinesia (PKD) is caused by PRRT2 mutations, but a distinct etiology has been suggested for sporadic PKD. Here we describe a cohort of patients collected from our movement disorders outpatient clinic in the period 1996-2011. Fifteen patients with sporadic PxD and 23 subjects from three pedigrees with familial PKD were screened for mutations in candidate genes. PRRT2 mutations co-segregated with PKD in two families and occurred in two sporadic cases of PKD. No mutations were detected in patients with non-kinesigenic or exertion-induced dyskinesia, and none in other candidate genes including PNKD1 (MR-1) and SLC2A1 (GLUT1). Thus, PRRT2 mutations also cause sporadic PKD as might be expected given the variable expressivity and reduced penetrance observed in familial PKD. Further genetic heterogeneity is suggested by the absence of candidate gene mutations in both sporadic and familial PKD suggesting a contribution of other genes or non-coding regions.</p>

Recent insights into the structure and functions of heparan sulfate proteoglycans in the human glomerular basement membrane

As the first barrier to be crossed on the way to urinary space, the glomerular basement membrane (GBM) plays a key role in renal function. The permeability of the GBM for a given molecule is highly dependent on its size, shape and charge. As early as 1980, the charge-selective permeability was demonstrated to relate to the electrostatic properties of covalently bound heparan sulfates (HS) within the GBM. Since the identification of perlecan as a heparan sulfate proteoglycan (HSPG) of basement membranes, the hypothesis that perlecan could be a crucial determinant of GBM permselectivity received considerable attention. In addition to perlecan, the GBM also contains other HSPG species, one of which was identified as agrin. The high local expression of agrin in the GBM, together with the presence of agrin receptors at the cell-matrix interface, suggests that this HSPG contributes to glomerular function in multiple ways. Here, we review the current knowledge regarding the structure and functions of HSPGs in the GBM, and discuss how these molecules could be involved in various glomerular diseases. Possible directions for future investigation are suggested

Postnatal lethality in SERT-Cre^cre/wt Munc18-1^lox/lox mice.

<p>(A) Genotype of the conditional knockout mice. Crossing SERT-Cre with Munc18-1^lox/lox mice results in deletion of Munc18-1 only and specifically in SERT expressing neurons, whereas all other neurons still express Munc18-1. (B) Genotyping mice after weaning at three weeks of age revealed that only few SERT-Cre^cre/wt Munc18-1^lox/lox mice survived up to three weeks.</p

In SERT-Cre^cre/wt Munc18-1^lox/wt mice number of 5-HT cell bodies and 5-HT innervations are not affected.

<p>(A-D) Midbrain sections containing the DRN from control (A) and hz (B) mice shows that 5-HT neurons are distributed in the characteristic DRN topology. Zooming in on 5-HT cell bodies show that both in control (C) and hz sections (D) these neurons have a fusiform or ovoid morphology and grow out several neurites. (E–H) 5-HT projection density is not different between control and hz mice in the midbrain containing the DRN (E and F respectively) or in cortical sections from control and hz mice (G and H respectively). (I) Number of 5-HT cell bodies per section does not differ between control and hz. (J) 5-HT projection density in midbrain containing DRN and cortical sections is not different between control and hz. Scale bars: 200 µm in B, 50 µm in D and H.</p

Degeneration of 5-HT projections in DRN and cortex.

<p>(A<b>–</b>F) At E16 in the midbrain containing the DRN, there was no difference in 5-HT projection density between control (A) and SERT-Cre^cre/wt Munc18-1^lox/lox (lox/lox) (B) mice. In control sections at E18 (C) and P2 (E) several 5-HT projections were present. However, in SERT-Cre^cre/wt Munc18-1^lox/lox sections at E18 (D) and P2 (F) only very few 5-HT projections are left. (G–L) In the cortex already at E16 there is a reduction in 5-HT projection density in sections from SERT-Cre^cre/wt Munc18-1^lox/lox (H) mice compared to control (G). At E18 (J) and P2 (L), in cortical sections from SERT-Cre^cre/wt Munc18-1^lox/lox mice only very few remaining 5-HT projections are present, in contrast to E18 (I) and P2 (K) control cortical sections. (M) Quantification of the 5-HT projection density in the DRN revealed that at E16 there is no difference, but at E18 and P2 5-HT projection density is reduced to ∼20%. (N) In cortical sections, at E16 and E18 the 5-HT projection density is reduced to ∼20% and at P2 the 5-HT projection density is reduced to ∼10%. Scale bar 50 µm in L. Data shown are mean±SEM * p<0.05.</p

Rapid degeneration of 5-HT neurons in SERT-Cre^cre/wt Munc18-1^lox/lox mice.

<p>We compared the number of 5-HT cell bodies in the DRN in control and SERT-Cre^cre/wt Munc18-1^lox/lox mice at E16, E18 and P2. (A-F) At E16, in both control mice (A) and SERT-Cre^cre/wt Munc18-1^lox/lox (lox/lox) mice (B) the 5-HT cell bodies are distributed in the DRN topology. At E18 and P2, however, in midbrain sections from SERT-Cre^cre/wt Munc18-1^lox/lox brains (D, F respectively) only few 5-HT neurons are present compared to control sections (C, E respectively). (G-L) Zooming in on 5-HT cell bodies shows that in control sections at E16 (G), E18 (I) and P2 (K) the cell bodies grow out several neurites and have a fusiform or ovoid morphology. However, remaining 5-HT cell bodies in SERT-Cre^cre/wt Munc18-1^lox/lox sections at E16 (H), E18 (J) and P2 (L) have a round morphology with hardly any neurites. (M,N) Blow up of some 5-HT cell bodies in control and SERT-Cre^cre/wt Munc18-1^lox/lox mice sections shows the differences in 5-HT cell body morphology. (O) Analysis of the number of 5-HT cell bodies in the sections showed that there is no difference at E16, but at E18 and P2 there is a decrease of 80% to 90% in the number of 5-HT cell bodies in SERT-Cre^cre/wt Munc18-1^lox/lox sections. Scale bars: 200 µm in F, 50 µm in L and 20 µm in N. Data shown are mean ± standard error of the mean (SEM). * p<0.05.</p

Expression and characterization of human perlecan domains I and II synthesized by baculovirus-infected insect cells

We present the in vitro expression and purification of N-terminal fragments of human perlecan in insect cells. Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus expression vectors (BEVs) encoding amino acids 1-196 (domain I), 1-404 (domain I + IIa) and 1-506 (domain I + IIab). The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restriction enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [35S]methionine showed that expression of the perlecan-(1-404)- and the -(1-506)-peptide was successful, but in the case of the perlecan-(1-196)-peptide no recombinant protein was produced. Immunoblotting showed that both the (1-404)-peptide and (1-506)-peptide are recognized by 95J10, a monoclonal antibody that was previously raised against perlecan-(24-404)-peptide expressed in Escherichia coli. Gel permeation and anion-exchange chromatography were applied to purify the recombinant proteins. Glycosaminoglycans were demonstrated to be present. Deglycosylation with chondroitinase ABC showed that the perlecan-(1-404)-peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated from the perlecan-(1-404)-peptide were identified as chondroitin sulfate by agarose gel electrophoresis. Furthermore the perlecan-(1-404)-peptide showed affinity to immobilized basic fibroblast growth factor. The availability of baculovirusderived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future