Studies on the functions of calpain as a plectin isoform 1a-degrading protease involved in keratinocyte differentiation and Epidermolysis Bullosa Simplex Ogna

Plectin ist ein ungewöhnlich großes (500 kDa) und vielseitiges cytoskelettäres Vernetzungsprotein. Es interagiert mit einer Vielzahl von Zytoskelettkomponen-ten, unter anderem Aktin Filamenten, Mikrotubuli und Intermediärfilamenten. Plectin kommt in unterschiedlichen Isoformen vor. Die in der Epidermis expre-mierte Isoform 1a (P1a) bildet gemeinsam mit Integrin β4 (ITGβ4) Hemidesmo-somen (HD) aus. HD sind trans-membranöse Adhesionskomplexe, welche die basalen Keratinozyten der Epidermis an der darunterliegenden Basallamina ver-ankern und so der Haut Stabilität verleihen. Eine autosomal-dominante Mutation einer einzelnen Base im Plectin Gen verursacht die Hautkrankheit Epidermolysis Bullosa Simplex-Ogna (EBS-Ogna). In der Epidermis von EBS-Ogna Modell-mäusen sind die Proteinniveaus von P1a, sowie die entsprechende Anzahl von HDen stark reduziert. Calpaine sind weit verbreitete Cystein Proteasen welche unter anderm ITGβ4 proteolytisch abbauen können. Der proteolytische Abbau von HD-Proteinen könnte einen essenziellen Schritt im Zuge der terminalen Differen-zierung von Keratinozyten darstellen. Weiters besteht die Möglichkeit, dass die gezielte Proteolyse von HD-Proteinen zum Pathomechanimsus von EBS-Ogna beiträgt. Im Zuge dieser Diplomarbeit wurde daher die Rolle von Calpainen in der Pathogenese von EBS-Ogna, sowie der terminalen Differenzierung von Keratino-zyten untersucht. Mithilfe einer enzymatischen in vitro Messmethode konnte ich die Aktivität von Calpainen in immortalisierten Mauskeratinozyten nachweisen. Weiters konnte ich mittels subzellulärer Fraktionierung zeigen, dass Calpaine sowohl an der Plasma-membran lokalisiert als auch mit dem Cytoskellett assoziiert sind. Aktivierung von Calpainen in immortalisierten Keratinozyten zeigte, dass Calpaine P1a und ITGβ4 abbauen können, und dass sich dieser Abbau durch Inaktivierung von Calpainen mittels spezifischer Peptid-Inhibitoren verhindern ließ. In terminal diffe-renzierenden primären Mauskeratinozyten erhöhte die Inhibierung von Calpainen die Proportion an inaktiven Calpainen und gab somit Hinweise darauf, dass Cal-paine auch im Zuge der terminalen Differenzierung aktiv sind. Mithilfe einer EBS-Ogna Mauslinie konnte ich des Weiteren die Beteiligung von Calpainen im Zuge der Pathogenese von EBS-Ogna zeigen. In primären Keratinozyten von EBS-Ogna Mäusen führte die Inhibierung von Calpainen zu einer Erhöhung der P1a Proteinspiegel und Anzahl an HD-Proteinen angereicherten Komplexen. Zu-sätzlich wurde eine klonale Zelllinie von Keratinozyten etabliert, welche die EBS-Ogna Mutation heterozygot exprimieren (Plecwt/Ogna). Im Zuge der Etablierung zeigten die Plecwt/Ogna Klone ein langsameres Wachstum verglichen mit den Zellen des Wildtyps. Diese Beobachtung könnte eventuell einen Hinweis auf eine bisher nicht beschriebene Funktion von HDen (und HPCs) als Zellproliferationregulatoren liefern.Plectin is a large (~500 kDa) and versatile member of a family of proteins called plakins or cytolinkers. It is able to interact with a multitude of cytoskeletal pro-teins including components of all major cytoskeletal filament systems of the cy-toskeleton, and is spliced into various isoforms. Plectin isoform 1a (P1a), which is predominantly expressed in skin, is an essential component of hemidesmosomes (HDs) where it binds to integrin β4 (ITGβ4). HDs are transmembrane adhesion complexes, which anchor basal keratinocytes of the epidermis to the underlying basal lamina, thus conferring mechanical stability to the skin. A single autosomal dominant missense mutation in the plectin gene causes the skin blistering disease Epidermolysis Bullosa Simplex-Ogna (EBS-Ogna), which manifests with dra-matically diminished P1a protein levels and reduced numbers of HDs. Calpains, cysteine proteases expressed in several tissues including the epidermis, have been demonstrated to degrade ITGβ4. Proteolytic degradation of HD components could be an important prerequisite for terminal differentiation of keratinocytes and could also be involved in the pathomechanism underlying EBS-Ogna. For these reasons, I assessed the role of calpains as possible candidates for P1a proteolysis at the onset of terminal differentiation and in the pathogenesis of EBS-Ogna. Performing an enzymatic activity assay, I could demonstrate that calpains are ac-tive in immortalized mouse keratinocytes. I also found that in immortalized mouse keratinocytes a fraction of calpains is localized at the plasma membrane and asso-ciated with the cytoskeleton. Furthermore, hyperactivation of calpains in immorta-lized mouse keratinocytes led to P1a and ITGβ4 degradation, which could be blocked by treatment with a cell permeable peptide inhibitor of calpains. Results with the analysis of primary mouse keratinocytes undergoing differentiation sug-gested that a fraction of calpains must be active during terminal differentiation since treatment of the cells with a pharmacological inhibitor of cysteine proteases increased the protein levels of the inactive full length form of calpains. Further-more, using a mouse model mimicking EBS-Ogna I found that calpains are in-volved in the proteolysis of P1a in primary keratinocytes derived from mutant animals. Upon inhibition of calpains in primary EBS-Ogna keratinocytes, elevated P1a protein levels could be detected along with increased numbers of HD-like protein complexes (HPCs). Finally, I established clonal keratinocyte cell lines carrying the heterozygous EBS-Ogna mutation (Plecwt/Ogna). Compared to wild-type clones, Plecwt/Ogna keratinocytes showed signs of proliferation defects pointing towards a hitherto undetected role of HDs as regulators of cell proliferation

Targeted Proteolysis of Plectin Isoform 1a Accounts for Hemidesmosome Dysfunction in Mice Mimicking the Dominant Skin Blistering Disease EBS-Ogna

Autosomal recessive mutations in the cytolinker protein plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS) associated with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and congenital myasthenia (EBS-CMS). In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated plectin isoform 1a. We found that plectin 1a, in contrast to plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased plectin 1a protein expression levels. Moreover, we report that plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline) polymers. We propose focal self-association of plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization

The future of mental health nursing: are we barking up the wrong tree?

This commentary has been prompted by a degree of disquiet among the UK mental health nursing community in response to the Shape of Caring Review on the future of nurse education in England (Willis 2015). Proposals for the structure of nurse education have been interpreted as emphasizing generic at the expense of field-specific (e.g. mental health) education, with much specialist training beyond the scope of pre-registration courses (Lintern 2014, Middleton 2015). Specifically, there is a suggestion that student nurses will not enter their specialized field until completing two years of more generalist learning; reminiscent of the previous Project 2000 approach, criticized for supposed inadequate preparation of mental health students because general adult nursing dominated curriculum and teaching (UKCC 1999)

Drug Attitude and Adherence to Anti-Glaucoma Medication

PURPOSE: The purpose of this study is to assess patient attitudes towards anti-glaucoma medication and their association with adherence, visual quality of life, and personality traits. MATERIALS AND METHODS: One hundred and forty-seven glaucoma patients were enrolled this study. The participants were divided into 'pharmacophobic' and 'pharmacophilic' groups according to their scores on the Modified Glaucoma Drug Attitude Inventory (MG-DAI). To establish a correlation with patient drug attitude, each group had their subjective drug adherence, visual quality of life, and personality traits examined. For personality traits, the Myers-Briggs Type Indicator (MBTI) was used to sub-classify each group. RESULTS: Among the patients analyzed, 91 (72.80%) patients showed a 'pharmacophobic' attitude and 34 (27.20%) patients showed a 'pharmacophilic' attitude. The pharmacophobic group tended to have worse adherence than the pharmacophilic group. Personality dichotomies from the MBTI also showed different patterns for each group. CONCLUSION: In glaucoma patients, pharmacological adherence was influenced by their attitude towards drugs; an association might exist between drug attitude and underlying personality traits.ope

Stigma and social support in substance abuse: Implications for mental health and well-being

Individuals with substance abuse may suffer from severe public and internalized stigma. Little is known about how social support can reduce stigma and improve mental health and well-being for them. This research examined how perceived stigma influences individuals in treatment for substance abuse, and whether internalized stigma and shame are mechanisms which link social support with better mental health and well-being. Sixty-four participants in treatment for substance abuse (alcohol, drugs), aged between 18 and 64, completed an online survey measuring perceived stigma, internalized stigma, shame, perceived social support, and mental health and well-being (self-esteem, depression and anxiety, sleep). We found that perceived stigma was associated with lower self-esteem, higher depression and anxiety, and poorer sleep. Furthermore, perceived social support followed the opposite pattern, and was associated with higher self-esteem, lower depression and anxiety, and better sleep. The effects of perceived stigma and of perceived social support on our outcome measures were mediated by internalized stigma and by internalized shame. Helping individuals with substance abuse to utilize their social support may be fruitful for combatting the negative impact of internalized stigma and shame on mental health and well-being

Improving community health networks for people with severe mental illness : a case study investigation

Introduction Policy drivers in mental health to address personal recovery, stigma and poor physical health indicate that new service solutions are required. This study aimed to understand how connections to people, places and activities were utilised by individuals with severe mental illness (SMI) to benefit health and wellbeing. Methods A five-module mixed-methods design was undertaken in two study sites. Data were collected from 150 network-mapping interviews and 41 in-depth follow-up interviews with people with SMI; in-depth interviews with 30 organisation stakeholders and 12 organisation leaders; and 44 telephone interviews with practitioners. We undertook a three-stage synthesis process including independent lived experience feedback, and a patient and public involvement team participated in tool design, data collection, analysis and write-up. Results Three personal network types were found in our study using the community health network approach: diverse and active; family and stable; formal and sparse. Controlled for other factors we found only four variables significantly associated with which network type a participant had: living alone or not; housing status; formal education; long-term sickness or disability. Diagnosis was not a factor. These variables are challenging to address but they do point to potential for network change. The qualitative interviews with people with SMI provided further understanding of connection-building and resource utilisation. We explored individual agency across each network type, and identified recognition of the importance and value of social support and active connection management alongside the risks of isolation, even for those most affected by mental illness. We identified tensions in personal networks, be that relationships with practitioners or families, dealing with the impact of stigma, or frustrations of not being in employment, which all impact on network resources and well-being. The value of connectedness within personal networks of people, place and activity for supporting recovery was evident in shaping identity, providing meaning to life and sense of belonging, gaining access to new resources, structuring routines and helping individuals ‘move on’ in their recovery journey. Health-care practitioners recognised that social factors were important in recovery but reported system-level barriers (workload, administrative bureaucracy, limited contact time with clients) in addressing these issues fully. Even practitioners working in third-sector services whose remit involved increasing clients’ social connection faced restrictions due to being evaluated by outcome criteria that limited holistic recovery-focused practices. Service providers were keen to promote recovery-focused approaches. We found contrasts between recovery ideology within mental health policy and recovery practice on the ground. In particular, the social aspects of supporting people with SMI are often underprioritised in the health-care system. In a demanding and changing context, strategic multiagency working was seen as crucial but we found few examples of embedded multisector organisation partnerships. Conclusion While our exploratory study has limitations, findings suggest potential for people with SMI to be supported to become more active managers of their personal networks to support well-being regardless of current network type. The health and social care system does not currently deliver multiagency integrated solutions to support SMI and social recovery