ALS2, a novel guanine nucleotide exchange factor for the small GTPase Rab5, is implicated in endosomal dynamics

ALS2 mutations account for a number of recessive motor neuron diseases including forms of amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. Although computational predictions suggest that ALS2 encodes a protein containing multiple guanine nucleotide exchange factor (GEF) domains [RCC1-like domain (RLD), the Dbl homology and pleckstrin homology (DH/PH), and the vacuolar protein sorting 9 (VPS9)], the functions of the ALS2 protein have not been revealed as yet. Here we show that the ALS2 protein specifically binds to small GTPase Rab5 and functions as a GEF for Rab5. Ectopically expressed ALS2 protein localizes with Rab5 and early endosome antigen-1 (EEA1) onto early endosomal compartments and stimulates the enlargement of endosomes in cultured cortical neurons. The carboxy-terminus of ALS2 protein carrying a VPS9 domain mediates not only the activation of Rab5 via a guanine-nucleotide exchanging reaction but also the endosomal localization of the ALS2 protein, while the amino-terminal half containing RLD acts suppressive in its membranous localization. Further, the DH/PH domain in the middle portion of ALS2 protein enhances the VPS9 domain-mediated endosome fusions. Taken together, the ALS2 protein as a novel Rab5-GEF, ALS2rab5GEF seems to be implicated in the endosomal dynamics in vivo. Notably, a feature common to eight reported ALS2 mutations among motor neuron diseases is the loss of VPS9 domain, resulting in the failure of Rab5 activation. Thus, a perturbation of endosomal dynamics caused by loss of ALS2 rab5GEF activity might underlie neuronal dysfunction and degeneration in a number of motor neuron diseases.     

DLK is a novel immunohistochemical marker for adrenal gland tumors

Delta-like protein (DLK) is expressed in fetal and adult adrenal glands. We have investigated if this expression is maintained in adrenal gland-derived tumors. All the studied 37 cortical tumors, including five carcinomas, stained positively as well as the 13 examined pheochromocytomas. Thus, DLK is a very sensitive marker for adrenal tumors of cortical and medullary origin. Renal cell carcinomas, presenting the major differential diagnostic problem for cortical tumors, were all negative, as well as melanomas, which are similar to high portion of adrenocortical tumors that react with melan-A. However, all paragangliomas, some carcinoids, and thyroid medullary carcinomas were also positive for DLK. Therefore, this novel immunohistochemical marker seems useful for the identification of adrenocortical tumors while it has limited value for the distinction of pheochromocytomas from diagnostically related neuroendocrine tumors.     

Caveolin-3 is a sensitive and specific marker for rhabdomyosarcoma.

Caveolin-3 (Cav-3) is a principal structural protein of caveolae membrane domains. Animal studies have revealed that Cav-3 is expressed in skeletal and cardiac myocytes but absent in other types of cells. Recent studies have shown that abnormalities in the Cav-3 gene are associated with some forms of muscular dystrophy, while skeletal muscle abnormalities have been observed in Cav-3 transgenic and knockout mice. In this study the authors evaluated the distribution of Cav-3 in normal human tissues and compared the expression of Cav-3 with that of myogenin and myoD1 in rhabdomyosarcoma (RMS), malignant mixed mullerian tumor (MMMT), and an array of neoplasms that mimic RMS to assess the utility of Cav-3 as a diagnostic marker for tumors with skeletal muscle differentiation. In nonneoplastic human tissues, crisp membrane staining for Cav-3 was present in cardiac and skeletal myocytes and occasionally in arterial smooth muscle cells and prostatic stromal cells, while other cell types were negative for Cav-3. Eighty-eight percent (21/24) of RMS studied were positive for Cav-3. Positive staining was generally observed in the more maturely differentiated tumor cells but not the primitive tumor cells. Eight of nine cases of MMMT stained strongly with Cav-3 in their rhabdomyosarcomatous component but not in other components. Fifty-four other neoplasms (13 leiomyosarcomas, 8 neuroblastomas, 5 lymphomas, 6 Wilms tumors without skeletal muscle differentiation, 5 Ewing sarcomas, 4 malignant fibrous histiocytomas, 4 angiosarcomas, 6 malignant melanomas, and 3 synovial sarcomas) were negative for Cav-3 expression. Nearly all (96% [23/24]) cases of RMS were positive for myogenin, while 88% (21/24) were positive for myoD1. Primitive tumor cells showed significantly increased expression of myoD1 and myogenin; conversely, more differentiated tumor cells were negative or weakly stained. The rhabdomyosarcomatous component of MMMT stained focally with myogenin and myoD1, in contrast to the strong Cav-3 labeling in these cells. These results demonstrate that Cav-3 is specifically expressed in human cardiac and skeletal myocytes. Furthermore, its high specificity and relatively high sensitivity (88%) for tumors with skeletal muscle differentiation suggest that Cav-3 is a valuable marker for these tumors and may be used to assess the degree of differentiation of RMS and to identify residual tumor cells in post-chemotherapy specimens.     

K(ATP) channel activity is required for hatching in Xenopus embryos.

A growing body of work suggests that the activity of ion channels and pumps is an important regulatory factor in embryonic development. We are beginning to identify functional roles for proteins suggested by a survey of expression of ion channel and pump genes in Xenopus and chick embryos (Rutenberg et al. [2002] Dev Dyn 225, this issue). Here, we report that the ATP-sensitive K(+) channel protein is present in the hatching gland of Xenopus embryos; moreover, we show that its activity is necessary for hatching in Xenopus. Pharmacologic inhibition of K(ATP) channels not only specifically prevents the hatching process but also greatly reduces the endogenous expression of Connexin-30 in the hatching gland. Based on recent work which showed that gap-junctional communication mediated by Cx30 in the hatching gland was required for secretion of the hatching enzyme, we propose that K(ATP) channel activity is upstream of Cx30 expression and represents a necessary endogenous step in the hatching of the Xenopus embryo.     

Bcr/Abl P190 interaction with Spa-1, a GTPase activating protein for the small GTPase Rap1

The Bcr/Abl oncogene is responsible for the development of Ph-chromosome positive acute lymphoblastic leukemia and chronic myelogenous leukemia in humans. Previous studies demonstrated that Bcr/Abl expression is associated with elevated levels of activated Rap1, a small GTPase. Levels of activated Rap1 are determined by a balance between GTPase activating and G-nucleotide exchange factor activity. We show that Bcr/Abl forms a protein-protein complex with Spa-1, a GTPase activating protein for Rap1, both in COS-1 cells as well as in primary lymphoblastic leukemia cells from a transgenic P190 BCR/ABL mouse model. The interaction between Spa-1 and P190 did not affect the tyrosine kinase activity of P190, nor did Spa-1 become phosphorylated on tyrosine as a result of the interaction. P190 and Spa-1 co-localized to peripheral actin structures in primary lymphoblasts and expression of Spa-1 in the leukemic lymphoblasts decreased the migration of these cells. The binding of Bcr/Abl to Spa-1 may cause aberrant subcellular location of Spa-1 and affect migration of these cells.     

Caveolin-3是诊断横纹肌肉瘤敏感和特异性标记

Caveolin-3是细胞膜穴样凹陷膜结构域的主要结构蛋白。动物研究显示caveolin-3可在心肌细胞和骨骼肌细胞表达，但在其他细胞不表达。最近研究发现caveolin-3基因的异常与某些类型肌营养不良密切相关，而在caveolin-3转基因或敲除鼠中可见骨骼肌异常。这些结果表明caveolin-3在骨骼肌分化和功能中发挥重要的作用。     

Microglial activation is a pharmacologically specific marker for the neurotoxic amphetamines

Neurotoxic amphetamines cause damage to monoamine nerve terminals of the striatum by unknown mechanisms. Microglial activation contributes to the neuronal damage that accompanies injury, disease, and inflammation, but a role for these cells in amphetamine-induced neurotoxicity has received little attention. We show presently that D-methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), D-amphetamine, and p-chloroamphetamine, each of which has been linked to dopamine (DA) or serotonin nerve terminal damage, result in microglial activation in the striatum. The non-neurotoxic amphetamines l-methamphetamine, fenfluramine, and DOI do not have this effect. All drugs that cause microglial activation also increase expression of glial fibrillary acidic protein (GFAP). At a minimum, microglial activation serves as a pharmacologically specific marker for striatal nerve terminal damage resulting only from those amphetamines that exert neurotoxicity. Because microglia are known to produce many of the reactive species (e.g., nitric oxide, superoxide, cytokines) that mediate the neurotoxicity of the amphetamine-class of drugs, their activation could represent an early and essential event in the neurotoxic cascade associated with high-dose amphetamine intoxication.     

ERK activation in endothelial cells is a novel marker during neovasculogenesis

Vasculogenesis is essential during early development to construct networks transporting oxygen, blood and nutrients. Tip and stalk cells are specialized endothelial cells involved in novel vessel formation because of their behavior such as sprouting as a leading cell and following tip cell. However, the spatiotemporal details determining the emergence of these cells are unknown. Here, we first show that the ERK activity in endothelial cells represents the precursor of tip and stalk cells for vasculogenesis in zebrafish. We identified that tip and stalk cells for intersegmental vessel (ISV) formation were already specialized in the dorsal aorta (DA) before sprouting. Furthermore, similar specialization was observed in tip cells during parachordal vessel (PAV) formation in lymphangiogenesis. We also identified that the ERK activity was required for specialized cells to emerge from existing blood vessels. Our data show that the ERK activity is a novel marker for determining the emergence of cells in both angiogenesis and lymphangiogenesis.     

Desmin is a specific marker for rhabdomyosarcomas of human and rat origin.

Putative human rhabdomyosarcoma (RMS) has been divided into two groups according to desmin content. Twenty-five tumors with histologic features consistent with but not necessarily sufficient to prove a diagnosis of RMS were desmin-positive. More than 95% of the tumor cells were desmin-positive, suggesting a muscle origin and supporting the diagnosis of RMS. Nine tumors for which the preferred first histologic diagnosis was also RMS were desmin-negative. Reexamination of the original histologic slides together with results from intermediate filament typing resulted in a diagnosis other than RMS for all tumors in this second group, and in several instances other tests were used to prove the correctness of the final diagnosis. The results on human material were extended to a rat model system in which RMS was induced by nickel sulfide. Again, all 24 tumors tested were desmin-positive. Vimentin was coexpressed in a varying percentage of tumor cells in RMS of human and rat origin. The results show that desmin is an excellent marker for rhabdomyosarcoma, yielding few if any false-positive or false-negative results in frozen or alcohol-fixed material.     

PLEXIN-D1, a novel plexin family member, is expressed in vascular endothelium and the central nervous system during mouse embryogenesis.

The genetic defect in M?bius syndrome 2 (MBS2, MIM 601471), a dominantly inherited disorder characterised by paralysis of the facial nerve, is situated at chromosome 3q21-q22. We characterised the cDNA and predicted protein, and examined the expression pattern during mouse embryogenesis of a positional candidate gene, PLEXIN-D1 (PLXND1). The cDNA for PLXND1 is 7095 base pairs in length, coding for a predicted protein of 1925 amino acids. The protein features all known domains of plexin family members, with the exception of the third Met-related sequence. Northern analysis revealed a very low expression of PLXND1 in adult mouse and adult human tissues. To investigate the expression of PlxnD1 during embryogenesis, RNA in situ hybridisation was performed on mouse embryos from various stages. This investigation revealed expression of PlxnD1 in cells from the central nervous system (CNS) and in vascular endothelium. Early expression in the CNS is located in the ganglia, cortical plate of the cortex, and striatum. At later embryologic stages, neural expression was also seen in the external granular layer of the cerebellum and several nerve nuclei. The expression in the vascular system resides solely in the endothelial cells of developing blood vessels. Based on our results, we suggest that this expression of a member of the plexin family in vascular endothelium could point toward a role in embryonic vasculogenesis.     

Polo-like kinase, a novel marker for cellular proliferation.

PLK (polo-like kinase) belongs to a family of serine/threonine kinases and represents the human counterpart of polo in Drosophila melanogaster and of CDC5 in Saccharomyces cerevisiae. It is strongly involved in spindle formation and chromosome segregation during mitosis. We have shown previously that PLK mRNA expression correlates with the mitotic activity of cells and the prognosis of lung cancer patients. In this report, the level of PLK protein was analyzed using immunohistochemical techniques. PLK protein was found expressed in the nuclei of tumor cells from lung and breast cancer as well as in several tumor cell lines. Furthermore, in peripheral lymphocytes treated with phytohemagglutinin, elevated proliferative activity of the cells correlated with the up-regulation of PLK protein expression. In contrast, in U937 and HL-60 cells after induction of differentiation with phorbol ester, PLK immunostaining disappeared under conditions of terminal differentiation. Most of the PLK protein was found in the nucleus of proliferating cells with diffuse but distinct staining also in the cytoplasm. Taken together, high levels of PLK protein are associated with cellular proliferation. Combined with other proliferative and oncogene markers, PLK may be useful for improved prediction of the clinical prognosis of cancer patients and for early cancer diagnosis. Due to its activity late in the cell cycle, it may be a target for cancer chemotherapy.     

Neurofibromin, a predominantly neuronal GTPase activating protein in the adult, is ubiquitously expressed during development.

The onset of manifestations of the common, autosomal dominantly inherited disease type 1 neurofibromatosis (NF1) is usually in childhood. To begin to understand the pathogenesis of NF1, we analyzed the developmental pattern of expression of the protein product of the NF1 gene, neurofibromin, by Western blotting and immunohistochemistry using the rat as a model system. Neurofibromin is uniformly distributed throughout embryonic day 10 and 12 rat embryos. By embryonic day 16, neurofibromin immunoreactivity is enriched in neurons of the cortical plate, in peripheral ganglia, and in developing CNS and PNS fiber tracts, but remains detectable outside the nervous system. Expression decreases in nonneural tissues by postnatal day 6, and neurofibromin is greatly decreased (lung, adrenal cortex, skin) or absent (skeletal muscle, cartilage) in adult tissues except for brain, spinal cord, peripheral nerve, and adrenal medulla. Transient expression of neurofibromin during development in many tissues suggests the importance of this GTPase-activating protein in morphogenesis and organ growth. A separate role is proposed for neurofibromin in growing axons and in the mature nervous system.     

Myogenin is a specific marker for rhabdomyosarcoma: an immunohistochemical study in paraffin-embedded tissues.

Myogenin belongs to a group of myogenic regulatory proteins whose expression determines commitment and differentiation of primitive mesenchymal cells into skeletal muscle. The expression of myogenin has been demonstrated to be extremely specific for rhabdomyoblastic differentiation, which makes it a useful marker in the differential diagnosis of rhabdomyosarcomas (RMS) from other malignant small round cell tumors of childhood. Commercially available antibodies capable of detecting myogenin in routinely processed formalin-fixed paraffin-embedded (FFPE) tissue are now available. In this study, we evaluated myogenin expression using the monoclonal myf-4 antibody (Novocastra Labs) on FFPE in a large number of pediatric tumors in order to define the clinical utility of this marker. A total of 119 tumors were studied. These included 48 alveolar RMS (ARMS), 20 embryonal RMS (ERMS), one spindle cell RMS, 16 Ewing's sarcomas (ES), six nephroblastomas, two ectomesenchymomas, seven precursor hematopoietic neoplasms, five olfactory neuroblastomas, three neuroblastomas, six desmoplastic small round cell tumors, and five rhabdoid tumors. Distinct nuclear staining for myogenin was noted in all 69 RMS. Notably, the number of positive tumor cells differed between the ARMS and ERMS. In ARMS, the majority of tumor cells (75 to 100%) were positive, in contrast to ERMS, in which the positivity ranged from rare + to 25% in all but three tumors. Additionally, myogenin positivity was seen in two of two ectomesenchymomas and in two nephroblastomas with myogenous differentiation. All other tumors were clearly negative. Our results indicate that staining for myogenin is an extremely reliable and specific marker for rhabdomyoblastic differentiation. It gives consistent and easily interpretable results in routinely fixed tissues.     

Docking protein SNT1 is a critical mediator of fibroblast growth factor signaling during Xenopus embryonic development.

The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from several growth factor receptors to the mitogen-activated protein (MAP) kinase signaling cascade, but its biological function during development is not well characterized. Here, we show that the Xenopus homolog of mammalian SNT1/FRS-2 (XSNT1) plays a critical role in the appropriate formation of mesoderm-derived tissue during embryogenesis. XSNT1 has an expression pattern that is quite similar to the fibroblast growth factor receptor-1 (FGFR1) during Xenopus development. Ectopic expression of XSNT1 markedly enhanced the embryonic defects induced by an activated FGF receptor, and increased the MAP kinase activity as well as the expression of a mesodermal marker in response to FGF receptor signaling. A loss-of-function study using antisense XSNT1 morpholino oligonucleotides (XSNT-AS) shows severe malformation of trunk and posterior structures. Moreover, XSNT-AS disrupts muscle and notochord formation, and inhibits FGFR-induced MAP kinase activation. In ectodermal explants, XSNT-AS blocks FGFR-mediated induction of mesoderm and the accompanying elongation movements. Our results indicate that XSNT1 is a critical mediator of FGF signaling and is required for early Xenopus development.