L1 syndrome diagnosis complemented with functional analysis of L1CAM variants located to the two N-terminal Ig-like domains.

L1CAM gene mutations cause neurodevelopmental disorders collectively termed L1 syndrome. Insufficient information about L1CAM variants complicates clinical prognosis, genetic diagnosis and genetic counseling. We combined clinical data, in silico effect predictions and functional analysis of four L1CAM variants, p.I37N, p.T38M, p.M172I and p.D202Y, located to the two N-terminal Ig-like domains present in five families with symptoms of L1 syndrome. Software tools predicted destabilizing effects of p.I37N and p.D202Y but results for p.T38M and p.M172I were inconsistent. Cell surface expression of mutant proteins L1-T38M, L1-M172I and L1-D202Y was normal. Conversely, L1-I37N accumulated in the endoplasmic reticulum (ER) and showed temperature-sensitive protein maturation suggesting that p.I37N induces protein misfolding. L1CAM-mediated cell-cell aggregation was severely impaired by L1CAM variants p.I37N, p.M172I and p.D202Y but was preserved by the variant p.T38M. Our experimental data indicate that protein misfolding and accumulation in the ER affect function of the L1CAM variant p.I37N whereas the variants p.M172I and p.D202Y impair homophilic interaction at the cell surface.

Telomeric associations correlate with telomere length reduction and clonal chromosome aberrations in giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is characterized cytogenetically by frequent telomeric associations (tas). To explore the mechanisms behind the formation of tas in GCTB and to investigate their karyotypic consequences, the frequencies of tas and clonal aberrations other than tas in 20 GCTBs were compared to telomere length and status, as assessed by quantitative PCR, fluorescence in situ hybridization (FISH), and expression levels of four genes involved in telomere maintenance. Based on the G-banding results, the tumors were divided into two groups, one with a high frequency of tas and one with a low frequency. Clonal aberrations were found to be restricted to the group with a high level of tas, and the same group showed a significantly larger reduction in telomere length in tumor cells compared to peripheral blood cells. Furthermore, 65 out of 66 tas analyzed by FISH were negative for telomeric sequences. The expression levels of TERT, TERF1, TERF2, and POT1 did not correlate with telomere length or the frequency of tas. Thus, the present findings provide strong support for the notion that decreased telomere length is a prerequisite for tas in GCTBs and that the clonal changes occurring in GCTBs are derived from tas.

Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice.

The hypothesis that adenosine acting on adenosine A1 receptors (A1R) regulates several renal functions and mediates tubuloglomerular feedback (TGF) was examined using A1R knockout mice. We anesthetized knockout, wild-type, and heterozygous mice and measured glomerular filtration rate, TGF response using the stop-flow pressure (P(sf)) technique, and plasma renin concentration. The A1R knockout mice had an increased blood pressure compared with wild-type and heterozygote mice. Glomerular filtration rate was similar in all genotypes. Proximal tubular P(sf) was decreased from 36.7 +/- 1.2 to 25.3 +/- 1.6 mmHg in the A1R+/+ mice and from 38.1 +/- 1.0 to 27.4 +/- 1.1 mmHg in A1R+/- mice in response to an increase in tubular flow rate from 0 to 35 nl/min. This response was abolished in the homozygous A1R-/- mice (from 39.1 +/- 4.1 to 39.2 +/- 4.5 mmHg). Plasma renin activity was significantly greater in the A1R knockout mice [74.2 +/- 14.3 milli-Goldblatt units (mGU)/ml] mice compared with the wild-type and A1R+/- mice (36.3 +/- 8.5 and 34.1 +/- 9.6 mGU/ml), respectively. The results demonstrate that adenosine acting on A1R is required for TGF and modulates renin release.

Vascular endothelial growth factor-B-deficient mice display an atrial conduction defect.

BACKGROUND: Vascular endothelial growth factors (VEGFs) and their receptors are essential regulators of vasculogenesis and angiogenesis in both embryos and adults. One of the factors with a still unknown physiological function is VEGF-B, which is expressed in many tissues, including the heart. METHODS AND RESULTS: Mice carrying a targeted deletion in the VEGF-B gene were developed. In VEGF-B(-/-) animals, no gross abnormalities were observed in organs that normally show high expression of VEGF-B, such as the heart, muscle, and kidney. Analysis of heart function by ECG showed that adult VEGF-B(-/-) mice have an atrial conduction abnormality characterized by a prolonged PQ interval. VEGF- or basic fibroblast growth factor-induced corneal angiogenesis was similar in normal and VEGF-B(-/-) mice. CONCLUSIONS: VEGF-B seems to be required for normal heart function in adult animals but is not required for proper development of the cardiovascular system either during development or for angiogenesis in adults.

Pro islet amyloid polypeptide (ProIAPP) immunoreactivity in the islets of Langerhans.

Islet amyloid is typically found in type 2 diabetes mellitus and is believed to participate in the beta cell deterioration. The islet amyloid fibril consists of the 37-amino-acid islet amyloid polypeptide (IAPP) but its pathogenesis is only partly understood. We developed several different rabbit antisera against the flanking peptides of the IAPP precursor (proIAPP) and the proIAPP processing sites in order to study the possible occurrence of unprocessed proIAPP or parts thereof in islet amyloid. We applied these antisera in an immunohistochemical study on, islet amyloid deposits present in a newly generated mouse strain that over-expresses human IAPP but is devoid of mouse IAPP. Male mice of this strain develop severe islet amyloidosis when given a high fat diet. Generally, the antisera showed no immunoreactivity with the amyloid. However, in scattered single beta cells, where amyloid could be seen intracellularly, immunoreactivity with one or more of the antisera co-localized with the amyloid. Although virtually all amyloid in human islets of Langerhans is found extracellularly, we propose that the initial amyloid formation occurs intracellularly, perhaps by not fully processed or folded (pro)IAPP. This amyloid, which may develop rapidly under certain circumstances, probably leads to cell death. If not degraded these amyloid spots may then act as nidus for further amyloid formation from fully processed IAPP, secreted from surrounding beta cells.

Islet amyloid polypeptide in the islets of Langerhans: friend or foe?

Islet amyloid polypeptide (IAPP), or amylin, was originally discovered as the constituent peptide in amyloid occurring in human insulinomas and in pancreatic islets in human subjects with Type II (non-insulin-dependent) diabetes mellitus. Its normal expression in beta cells and its co-secretion with insulin in response to nutrient stimuli, suggest a metabolic function for the peptide. Specifically, IAPP has most frequently been shown to inhibit insulin secretion, implying that IAPP has a role in the regulation of islet hormone homeostasis. The physiological significance of IAPP in islets has been difficult to assess; very high IAPP concentrations are required to alter insulin secretion. Moreover, until recently, IAPP receptors have not been characterised at the molecular level, thus leaving the actual target cells for IAPP unidentified. Furthermore, in experimental diabetes in rodents, the ratio of IAPP expression to that of insulin invariably is increased. In view of the pleiotropic effects attributed to IAPP, such regulation could be both adverse and beneficial in diabetes. Metabolic characterisation of mice carrying a null mutation in the IAPP gene or which overexpress IAPP in beta cells have recently confirmed that IAPP is a physiological inhibitor of insulin secretion. Based on experiments in which IAPP-deficient mice develop a more severe form of alloxan-induced diabetes, we argue that the action of IAPP in the islets normally is beneficial for beta-cell function and survival; thus, the established up regulation of IAPP expression compared with that of insulin in experimental rodent diabetes could serve to protect islets under metabolically challenging circumstances.

Islet amyloid polypeptide (amylin)-deficient mice develop a more severe form of alloxan-induced diabetes.

To examine whether islet amyloid polypeptide (IAPP), other than through amyloid formation, may be of importance in diabetes pathogenesis, IAPP-deficient mice (IAPP(-/-)) were challenged with alloxan (day 0). Diabetes in IAPP(-/-) mice was more severe at day 35, indicated by greater weight loss; glucose levels were higher in alloxan-treated IAPP(-/-) mice, whereas insulin levels were lower, indicating a greater impairment of islet function. Accordingly, glucose levels upon intravenous glucose challenges at days 7 and 35 were consistently higher in alloxan-treated IAPP(-/-) mice. At day 35, insulin mRNA expression, but not beta-cell mass, was lower in untreated IAPP(-/-) mice. Yet, upon alloxan administration, beta-cell mass and numbers of beta-cell-containing islets were significantly more reduced in IAPP(-/-) mice. Furthermore, they displayed exaggerated beta-cell dysfunction, because in their remaining beta-cells, insulin mRNA expression was significantly more impaired and the localization of glucose transporter-2 was perturbed. Thus the lack of IAPP has allowed exaggerated beta-cell cytotoxic actions of alloxan, suggesting that there may be beneficial features of IAPP actions in situations of beta-cell damage.

Islet amyloid development in a mouse strain lacking endogenous islet amyloid polypeptide (IAPP) but expressing human IAPP.

BACKGROUND: Several mouse strains expressing human islet amyloid polypeptide (IAPP) have been created to study development of islet amyloid and its impact on islet cell function. The tendency to form islet amyloid has varied strongly among these strains by factors that have not been elucidated. Because some beta cell granule components are known to inhibit IAPP fibril formation in vitro, we wanted to determine whether a mouse strain expressing human IAPP but lacking the nonamyloidogenic mouse IAPP is more prone to develop islet amyloidosis. MATERIALS AND METHODS: Such a strain was created by cross-breeding a transgenic mouse strain and an IAPP null mouse strain. RESULTS: When fed a fat-enriched diet, male mice expressing only human IAPP developed islet amyloid earlier and to a higher extent than did mice expressing both human and mouse IAPP. Supporting these results, we found that mouse IAPP dose-dependently inhibits formation of fibrils from human IAPP. CONCLUSIONS: Female mice did not develop amyloid deposits, although small extracellular amorphous IAPP deposits were found in some islets. When cultivated in vitro, amyloid deposits occurred within 10 days in islets from either male or female mice expressing only human IAPP. The study shows that formation of islet amyloid may be dependent on the environment, including the presence or absence of fibril inhibitors or promoters.

Pituitary adenylate cyclase-activating polypeptide and islet amyloid polypeptide in primary sensory neurons: functional implications from plasticity in expression on nerve injury and inflammation.

Primary sensory neurons serve a dual role as afferent neurons, conveying sensory information from the periphery to the central nervous system, and as efferent effectors mediating, e.g., neurogenic inflammation. Neuropeptides are crucial for both these mechanisms in primary sensory neurons. In afferent functions, they act as messengers and modulators in addition to a principal transmitter; by release from peripheral terminals, they induce an efferent response, "neurogenic inflammation," which comprises vasodilatation, plasma extravasation, and recruitment of immune cells. In this article, we introduce two novel members of the sensory neuropeptide family: pituitary adenylate cyclase-activating polypeptide (PACAP) and islet amyloid polypeptide (IAPP). Whereas PACAP, a vasoactive intestinal polypeptide-resembling peptide, predominantly occurs in neuronal elements, IAPP, which is structurally related to calcitonin gene-related peptide, is most widely known as a pancreatic beta-cell peptide; as such, it has been recognized as a constituent of amyloid deposits in type 2 diabetes. In primary sensory neurons, under normal conditions, both peptides are predominantly expressed in small-sized nerve cell bodies, suggesting a role in nociception. On axotomy, the expression of PACAP is rapidly induced, whereas that of IAPP is reduced. Such a regulation of PACAP suggests that it serves a protective role during nerve injury, but that of IAPP may indicate that it is an excitatory messenger under normal conditions. In contrast, in localized adjuvant-induced inflammation, expression of both peptides is rapidly induced. For IAPP, studies in IAPP-deficient mice support the notion that IAPP is a pronociceptive peptide, because these mutant mice display a reduced nociceptive response when challenged with formalin.

Expression of non-classical islet hormone-like peptides during the embryonic development of the pancreas.

Understanding of islet embryogenesis may prove to be key in the design of future therapies for diabetes directed at re-initiating islet growth, with the goal to replace and/or replenish the impaired beta-cell mass in the disease. In this context, studies of islet neurohormonal peptides, known to play a role in the local regulation of islet function, and their expression during islet embryogenesis are important. Here we review our studies on the embryonic islet expression of islet amyloid polypeptide (IAPP) and the PP-fold peptides pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY). IAPP, which is constitutively expressed in beta- and delta-cells in the adult rat, was found to occur in the assumed pluripotent islet progenitor cell, together with PYY, glucagon, and to a lesser extent with insulin. As development proceeds, the insulin/IAPP phenotype is segregated from that of PYY/glucagon; with the formation of islet-like structures, insulin/IAPP-expressing cells primarily occupy their central portions, while PYY/glucagon-expressing cells are found in their periphery. At the time of formation of islet-like structures, expression of NPY is induced in the insulin/IAPP-containing cells. Whereas NPY-expression ceases at birth, PYY is constitutively expressed in non-beta-cells in the mature rat. Expression of PP is induced just prior to birth in a separate population of islet cells, occasionally co-expressed with PYY. Although a clear role for these peptides during embryogenesis has not been identified, they conceivably could play a role in the control of insulin secretion, islet growth and islet blood flow.

Reduced nociceptive behavior in islet amyloid polypeptide (amylin) knockout mice.

Islet amyloid polypeptide (IAPP or amylin) is predominantly expressed by insulin cells, but occurs also in primary sensory neurons in the rat. Here, using mice targeted for a null mutation in the IAPP gene, we establish murine expression of IAPP in sensory neurons; its distribution in a population of calcitonin gene-related peptide-containing neurons in the spinal cord and dorsal root ganglion is similar to that previously described in the rat. We also report the IAPP mutant mice display a reduced pain response in the paw formalin test. Adjuvant-induced joint inflammation was not altered in IAPP mutants, arguing against a peripheral inflammatory abnormality. These findings lead us to suggest that IAPP has a pro-nociceptive function in primary sensory neurons.

Increased insulin secretion and glucose tolerance in mice lacking islet amyloid polypeptide (amylin).

Islet amyloid polypeptide (IAPP or amylin) is costored and cosecreted with insulin and may regulate insulin secretion and blood glucose handling. However, the role and importance of endogenous IAPP in the regulation of insulin release and glucose homeostasis have been controversial. Here we report on the generation and phenotypic analysis of IAPP-deficient mice. These mice have normal, or near to normal, basal levels of circulating insulin and glucose. However, following glucose administration, IAPP-deficient males presented increased insulin responses paralleled with a more rapid blood glucose elimination compared to wild-type controls. Blood glucose elimination was also found to be enhanced in IAPP-deficient females, but the insulin response in this gender did not differ from controls. In a transgenic rescue experiment, using an insulin-promoter human-IAPP fusion gene, insulin responses and blood glucose elimination were reversed in IAPP-deficient males, whereas the female phenotype appeared unaffected. Our results provide the first firm evidence of a physiological role for endogenous IAPP and indicate that IAPP, apparently in a gender-dependent manner, limits the degree of glucose-induced insulin secretion and the rate of blood glucose elimination.

Targeted disruption of the mouse phospholipase C beta3 gene results in early embryonic lethality.

In order to investigate the biological function of phosphatidylinositol-specific phospholipase C (PLC) we generated mutant mice by gene targeting. Homozygous inactivation of PLCbeta3 is lethal at embryonic day 2.5. These mutants show poor embryonic organization as well as reduced numbers of cells. Identical phenotypes were recorded in homozygous mutants generated from two independently targeted embryonic stem cell clones. Heterozygous mutant mice, however, are viable and fertile for at least two generations. We also showed that mouse PLCbeta3 is expressed in unfertilized eggs, 3-cell and egg cylinder stages of embryos. In conclusion, these results indicate that PLCbeta3 expression is essential for early mouse embryonic development.

Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development.

PDGF-A(-/-) mice lack lung alveolar smooth muscle cells (SMC), exhibit reduced deposition of elastin fibres in the lung parenchyma, and develop lung emphysema due to complete failure of alveogenesis. We have mapped the expression of PDGF-A, PDGF receptor-alpha, tropoelastin, smooth muscle alpha-actin and desmin in developing lungs from wild type and PDGF-A(-/-) mice of pre- and postnatal ages in order to get insight into the mechanisms of PDGF-A-induced alveolar SMC formation and elastin deposition. PDGF-A was expressed by developing lung epithelium. Clusters of PDGF-Ralpha-positive (PDGF-Ralpha+) mesenchymal cells occurred at the distal epithelial branches until embryonic day (E) 15.5. Between E16.5 and E17.5, PDGF-Ralpha+ cells multiplied and spread to acquire positions as solitary cells in the terminal sac walls, where they remained until the onset of alveogenesis. In PDGF-A(-/-) lungs PDGF-Ralpha+ cells failed to multiply and spread and instead remained in prospective bronchiolar walls. Three phases of tropoelastin expression were seen in the developing lung, each phase characterized by a distinct pattern of expression. The third phase, tropoelastin expression by developing alveolar SMC in conjunction with alveogenesis, was specifically and completely absent in PDGF-A(-/-) lungs. We propose that lung PDGF-Ralpha+ cells are progenitors of the tropoelastin-positive alveolar SMC. We also propose that postnatal alveogenesis failure in PDGF-A(-/-) mice is due to a prenatal block in the distal spreading of PDGF-Ralpha+ cells along the tubular lung epithelium during the canalicular stage of lung development.

PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis.

A mouse platelet-derived growth factor A chain (PDGF-A) null allele is shown to be homozygous lethal, with two distinct restriction points, one prenatally before E10 and one postnatally. Postnatally surviving PDGF-A-deficient mice develop lung emphysema secondary to the failure of alveolar septation. This is apparently caused by the loss of alveolar myofibroblasts and associated elastin fiber deposits. PDGF alpha receptor-positive cells in the lung having the location of putative alveolar myofibroblast progenitors were specifically absent in PDGF-A null mutants. We conclude that PDGF-A is crucial for alveolar myofibroblast ontogeny. We have previously shown that PDGF-B is required in the ontogeny of kidney mesangial cells. The PDGFs therefore appear to regulate the generation of specific populations of myofibroblasts during mammalian development. The two PDGF null phenotypes also reveal analogous morphogenetic functions for myofibroblast-type cells in lung and kidney organogenesis.

Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities.

Platelet-derived growth factor (PDGF) affects the growth, migration, and function in vitro of mesenchymal cells, but little is known about its normal physiological functions in vivo. We show here that mice deficient for PDGF B die perinatally and display several anatomical and histological abnormalities. Kidney glomerular tufts do not form, apparently because of absence of mesangial cells. Instead, a single or a few distended capillary loops fill the glomerular space. The heart and some large arteries dilate in late-stage embryos. Most PDGF B mutant embryos develop fatal hemorrhages just prior to birth. Their hematological status includes erythroblastosis, macrocytic anemia, and thrombocytopenia. On the basis of these findings, we conclude that PDGF B has crucial roles in vivo in establishing certain renal and circulatory functions.

Islet amyloid polypeptide--hen or egg in type 2 diabetes pathogenesis?

Islet amyloid polypeptide (IAPP or amylin) was first identified as the major peptide constituent of amyloid deposited in the islets of Langerhans in patients with type-2 diabetes mellitus or in insulinomas. It was subsequently shown that IAPP is produced by the pancreatic beta-cells, co-stored and co-released with insulin. IAPP is homologous with the neuropeptide calcitonin gene-related peptide (CGRP) and has therefore been assumed to have a function as an endocrine, paracrine or autocrine hormone. This has prompted the search for its physiological function as well as a putative pathogenic role in type 2 diabetes mellitus.