Mutant profilin1 transgenic mice recapitulate cardinal features of motor neuron disease

The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeletonregulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis

Enhancement of cutaneous wound healing by Dsg2 augmentation of uPAR secretion

In addition to playing a role in adhesion, desmoglein 2 (Dsg2) is an important regulator of growth and survival signaling pathways, cell proliferation, migration and invasion, and oncogenesis. While low-level Dsg2 expression is observed in basal keratinocytes and is downregulated in non-healing venous ulcers, overexpression has been observed in both melanomas and non-melanoma malignancies. Here, we show that transgenic mice overexpressing Dsg2 in basal keratinocytes primed the activation of mitogenic pathways, but did not induce dramatic epidermal changes or susceptibility to chemical-induced tumor development. Interestingly, acceleration of full-thickness wound closure and increased wound-adjacent keratinocyte proliferation was observed in these mice. As epidermal cytokines and their receptors play critical roles in wound healing, Dsg2-induced secretome alterations were assessed with an antibody profiler array and revealed increased release and proteolytic processing of the urokinase-type plasminogen activator receptor (uPAR). Dsg2 induced uPAR expression in the skin of transgenic compared to wild-type mice. Wound healing further enhanced uPAR in both epidermis and dermis with concomitant increase in the pro-healing laminin-332, a major component of the basement membrane zone, in transgenic mice. This study demonstrates that Dsg2 induces epidermal activation of various signaling cascades and accelerates cutaneous wound healing, in part, through uPAR-related signaling cascades

Abstract LB-32: The unique region of a desmosomal cadherin, desmoglein 2, regulates internalization via tail-tail interactions

Abstract Desmogleins and desmocollins are desmosomal cadherins that make up the adhesive core of desmosomes, intercellular junctions specialized for helping cells withstand mechanical stress. Desmoglein 2 (Dsg2) is the most widely expressed member of the Dsg subfamily and misregulation of Dsg2 has been reported in many types of cancer. While endocytosis is known to be a critical determinant in regulating cell surface levels of classic cadherins during tissue remodeling and cancer progression, less is known about the regulation of desmosomal cadherins. The trafficking of transmembane proteins is frequently dictated by the presence of specific sequences in their cytoplasmic tails. The cytoplasmic tail of Dsg contains a membrane proximal region, which is conserved in classic cadherins, and a desmoglein unique region (DUR), which is only present in the Dsg family. To determine the contribution of the Dsg2 tail to its endocytosis, we generated a series of mutants with progressive truncations of the cytoplasmic tail. A time course to track the internalized pool of Dsg2 mutants following cell surface biotinylation demonstrated that the DUR attenuates Dsg2 internalization. Analysis of a series of chimeric molecules in which variable lengths of the Dsg2 tail were fused with interleukin-2 ecto- and transmembrane domains showed that this function of the DUR is independent of the status of the Dsg2 ectodomain. With or without the DUR, internalization of the Dsg2 mutants exhibited the same dependence on cholesterol and dynamin, suggesting that the DUR inhibits Dsg2's entry into a common pathway(s). Detergent solubility and sucrose gradient fractionation assays indicated that the presence of the DUR alters the Dsg2 protein complex profile, even though there was no change in the interaction of Dsg2 mutants with known desmoglein binding partners, plakoglobin and plakophilin 3. To test whether the DUR controls the formation of Dsg2 complexes by affecting the oligomeric status of the Dsg2 cytoplasmic domain, we carried out in situ proximity ligation and yeast two hybrid analyses. The data suggest that Dsg2 inter- but not intra-molecular interactions occur, and that these are mediated through the DUR. To investigate whether DUR-dependent inhibition of internalization and mediation of tail-tail interactions are mechanistically linked, one copy of a dimerization motif was fused to the C-terminus of a Dsg2 construct lacking the DUR. Forced dimerization of this mutant using a cell-permeable bivalent compound led to its decreased internalization. Collectively, our studies demonstrate for the first time that the Dsg2 DUR mediates intermolecular interactions between the Dsg2 cytoplasmic domains, and that this function is linked to the inhibition of Dsg2 internalization. This discovery has important implications for normal tissue remodeling and homeostasis, in addition to the pathogenesis of diseases such as cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-32. doi:1538-7445.AM2012-LB-32</jats:p

Plakophilin 2 Couples Actomyosin Remodeling to Desmosomal Plaque Assembly via RhoA

The desmosomal armadillo protein plakophilin 2 (PKP2) regulates cell contact-initiated cortical actin remodeling through the regulation of RhoA localization and activity to couple adherens junction maturation with desmosomal plaque assembly

Validated quantitative trait loci for eggshell quality in experimental and commercial laying hens

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Absence of UCHL 1 function leads to selective motor neuropathy.

OBJECTIVE: The aim of this study was to investigate the role of ubiquitin C-terminal hydrolase-L1 (UCHL1) for motor neuron circuitry and especially in spinal motor neuron (SMN) health, function, and connectivity. METHODS: Since mutations in UCHL1 gene leads to motor dysfunction in patients, we investigated the role of UCHL1 on SMN survival, axon health, and connectivity with the muscle, by employing molecular and cellular marker expression analysis and electrophysiological recordings, in healthy wild-type and Uchl1 (nm3419) (UCHL1-/-) mice, which lack all UCHL1 function. RESULTS: There is pure motor neuropathy with selective degeneration of the motor, but not sensory axons in the absence of UCHL1 function. Neuromuscular junctions (NMJ) are impaired in muscle groups that are innervated by slow-twitch or fast-twitch SMN. However, unlike corticospinal motor neurons, SMN cell bodies remain intact with no signs of elevated endoplasmic reticulum (ER) stress. INTERPRETATION: Presence of NMJ defects and progressive retrograde axonal degeneration in the absence of major SMN soma loss suggest that defining pathology as a function of neuron number is misleading and that upper and lower motor neurons utilize UCHL1 function in different cellular events. In line with findings in patients with mutations in UCHL1 gene, our results suggest a unique role of UCHL1, especially for motor neuron circuitry. SMN require UCHL1 to maintain NMJ and motor axon health, and that observed motor dysfunction in the absence of UCHL1 is not due to SMN loss, but mostly due to disintegrated circuitry. Ann Clin Transl Neurol 2016 Mar 7; 3(5):331-45

The C-terminal unique region of desmoglein 2 inhibits its internalization via tail–tail interactions

Desmosomal cadherins, desmogleins (Dsgs) and desmocollins, make up the adhesive core of intercellular junctions called desmosomes. A critical determinant of epithelial adhesive strength is the level and organization of desmosomal cadherins on the cell surface. The Dsg subclass of desmosomal cadherins contains a C-terminal unique region (Dsg unique region [DUR]) with unknown function. In this paper, we show that the DUR of Dsg2 stabilized Dsg2 at the cell surface by inhibiting its internalization and promoted strong intercellular adhesion. DUR also facilitated Dsg tail–tail interactions. Forced dimerization of a Dsg2 tail lacking the DUR led to decreased internalization, supporting the conclusion that these two functions of the DUR are mechanistically linked. We also show that a Dsg2 mutant, V977fsX1006, identified in arrhythmogenic right ventricular cardiomyopathy patients, led to a loss of Dsg2 tail self-association and underwent rapid endocytosis in cardiac muscle cells. Our observations illustrate a new mechanism desmosomal cadherins use to control their surface levels, a key factor in determining their adhesion and signaling roles

A Pathophysiologic Role for Epidermal Growth Factor Receptor in Pemphigus Acantholysis

The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus