Differential Expression of A-Type and B-Type Lamins during Hair Cycling

Peer reviewe

A previously functional tetracycline-regulated transactivator fails to target gene expression to the bone

<p>Abstract</p> <p>Background</p> <p>The tetracycline-controlled transactivator system is a powerful tool to control gene expression <it>in vitro </it>and to generate consistent and conditional transgenic <it>in vivo </it>model organisms. It has been widely used to study gene function and to explore pathological mechanisms involved in human diseases. The system permits the regulation of the expression of a target gene, both temporally and quantitatively, by the application of tetracycline or its derivative, doxycycline. In addition, it offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator.</p> <p>Findings</p> <p>In this study, we report our problems using a reverse tetracycline-regulated transactivator (rtTA) in a transgenic mouse model system for the bone-specific expression of the Hutchinson-Gilford progeria syndrome mutation. Even though prior studies have been successful utilizing the same rtTA, expression analysis of the transactivator revealed insufficient activity for regulating the transgene expression in our system. The absence of transactivator could not be ascribed to differences in genetic background because mice in a mixed genetic background and in congenic mouse lines showed similar results.</p> <p>Conclusions</p> <p>The purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time.</p

The cytolinker plectin regulates nuclear mechanotransduction in keratinocytes.

The transmission of mechanical forces to the nucleus is important for intracellular positioning, mitosis and cell motility, yet the contribution of specific components of the cytoskeleton to nuclear mechanotransduction remains unclear. In this study, we examine how crosstalk between the cytolinker plectin and F-actin controls keratin network organisation and the 3D nuclear morphology of keratinocytes. Using micro-patterned surfaces to precisely manipulate cell shape, we find that cell adhesion and spreading regulate the size and shape of the nucleus. Disruption of the keratin cytoskeleton through loss of plectin facilitated greater nuclear deformation, which depended on acto-myosin contractility. Nuclear morphology did not depend on direct linkage of the keratin cytoskeleton with the nuclear membrane, rather loss of plectin reduced keratin filament density around the nucleus. We further demonstrate that keratinocytes have abnormal nuclear morphologies in the epidermis of plectin-deficient, epidermolysis bullosa simplex patients. Taken together, our data demonstrate that plectin is an essential regulator of nuclear morphology in vitro and in vivo and protects the nucleus from mechanical deformation.This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/J000914/1]; and by the Barts and the London Charity [grant number 442/1032]

Molecular studies of Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disease, with an incidence of 1 in 4-8 million live births, that causes segmental premature aging in children. The children look normal at birth but begin to develop symptoms of disease within the first years of life. The symptoms include growth retardation, scleroderma, osteoporosis and atherosclerosis of the coronary and cerebrovascular arteries. Myocardial infarction or stroke is the most common causes of death at a median age of 13 years. The aims of this work includes: to increase the understanding of the molecular mechanisms underlying progeria, to see if there is any possibility of disease reversal and to develop a specific method for analyzing LMNA transcripts during normal and in vitro aging. For these purposes, we developed an inducible tissue-specific transgenic mouse model system that included a minigene of human LMNA with either the wild-type sequence or the most common HGPS mutation, 1824C>T, and assays for absolute quantification of the LMNA transcripts in HGPS patient material and controls of different ages. PAPER I: Animal models are crucial to increase understanding of the ongoing molecular process during disease, especially for rare and severe diseases like HGPS. To get a better understanding of the HGPS skin phenotype, we developed an inducible and tissue specific model system with keratin 5- targeted transgenic expression. Bitransgenic animals with the HGPS mutation have a progressive phenotype. The phenotype is first characterized by an intermediate stage with varying degrees of hyperplasia of the interfollicular epidermis, mis-expression of keratins 5 and 6 and increased proliferation. The end stage is seen later, with loss of subcutaneous fat and fibrosis of the dermis, similar abnormalities to those seen in the skin of HGPS patients. The severity of the disease phenotype correlates with the level of transgenic expression (higher expression gives more severe disease phenotype). Animals expressing the wild-type allele had a normal appearance of the skin. PAPER II: To examine if expression of progerin affects the expression of lamin B or the progress of the hair cycle, we first characterized the normal expression of lamin A/C and B in mouse skin cell types during hair cycling. Immunohistochemical staining of the whole back skin of FVB/NCrl wild-type mice showed strong expression of lamin A/C and B in the basal layer of the epidermis, the outer root sheath of the hair follicle and the dermal papilla during all stages of the hair cycle. Lower expression was seen in the suprabasal cells of the epidermis, in the hypodermis and the bulb of catagen follicles. Analyzing the different phases of the first postnatal hair cycle and the expression of lamin B in our mouse model of HGPS did not reveal any shifts in the hair cycle or in the expression of lamin B. PAPER III: To examine if progeria disease is reversible and learn more about the possibility of treatment for the children with progeria who are already manifesting the disease, we used our inducible transgenic mouse model of HGPS. After disease development, transgenic expression was suppressed and the animals were observed for reversal in disease phenotype. The external phenotype of hair loss and skin crusting improved after only 1 week of suppression and after 6 or 13 weeks the external skin phenotype looked completely normal in most of the animals. The lower weights of bitransgenic animals increased after transgenic suppression and followed the trend of the wild-type curve. The disease pathology seen in the skin of bitransgenic animals was almost indistinguishable from wild-type after 6 and 13 weeks of suppressed transgenic expression. This shows that the expression of the HGPS mutationdoes not cause irreversible damage, at least in these tissues, which gives promise for future treatment for this disease. PAPER IV: To characterize the expression levels of the LMNA locus transcripts in HGPS patients and age-matched and parental controls and during in vitro aging, we developed a method for absolute quantification using real-time RT-PCR. Lamin A, C and lamin Adelta150 transcripts were quantified in HGPS and normal cells of different ages. Our results showed that lamin C is the most highly expressed transcript from the LMNA locus. The lamin Adelta150 transcript was present in unaffected controls but at >160-fold lower levels than in HGPS patient cells. While the levels of lamin A and C remained unchanged during in vitro aging, the lamin Adelta150 transcript increased in late passage cells from both HGPS and parental controls, which suggests that similar mechanisms exist in HGPS and normal aging cells

Effect of Virtual Reality training in people with Parkinson´s disease on balance and gait in comparison with traditional training : An overview of systematic reviews

Bakgrund: Virtual Reality (VR) är en teknik som skapar en simulerad miljö där användaren kan uppleva en virtuell värld. Effekten av VR-träning på gång och balansförmågan hos personer med Parkinsons sjukdom (PS) är ännu oklar trots flera systematiska studier. Sammanfattning av befintlig evidens kan ge en överblick samt kartlägga den kliniska nyttan av VR. Syfte: Syftet är att genomföra en översiktsstudie av systematiska litteraturstudier för att utvärdera behandlingseffekten av VR-träning jämfört med traditionell träning på gång- och balansförmåga hos personer med PS. Metod: Systematiska litteraturstudier i databaserna PubMed, CINAHL samt PEDro som publicerats mellan åren 2019-2022 har granskats. Kvalitetsgranskning utfördes med AMSTAR 2 och en narrativ syntes användes för att sammanfatta de rapporterade fynden av gång- och balansförmåga. Utfallsmåtten som använts är statisk och dynamisk balans samt gångparametrar. Resultat: Av totalt 298 systematiska litteraturstudier inkluderades sju studier med 128 randomiserade kontrollerade studier (4736 deltagare) som uppfyllde kriterier. Enligt AMSTAR 2 graderades fyra studier till kritiskt låg kvalitet. I fem av sju systematiska litteraturstudier rapporterades förbättrad statisk balans (MD=1.13-2.69 SMD=0.22-0.26) för gruppen med VR-träning jämfört med kontrollgrupp. I tre av sex studier rapporterades förbättring (MD –1.55 till -2.86) på dynamisk balans. Temporala gångparametrar rapporterade ingen skillnad mellan VR träning och kontrollgrupp. Spatiala gångparametrarna rapporterade blandade resultat. Slutsats: Studierna bedömdes övergripande ha låg metodologisk kvalitet. Resultaten visade att VR-träning kunde förbättra balansförmågan när det gäller statisk balans, men det fanns varierande resultat när det gällde dynamisk balans jämfört med kontrollgrupp. Ingen skillnad rapporterades mellan grupperna gällande temporala gångparametrar. Varierande resultat visades för spatiala gångparametrar. Ytterligare högkvalitativa systematiska översikter eftersträvas för att kunna dra mer tillförlitliga slutsatser.Background: Virtual Reality (VR) is a technology that creates a simulated environment where the user can experience a virtual world. The effect of VR training on gait and balance ability in people with Parkinson's disease (PS) remain unclear despite several systematic reviews. Summary of existing evidence can provide an overview of the current state of research and facilitate the mapping of the clinical benefit of VR. Aim: The aim is to conduct an overview of systematic reviews on gait and balance ability in people with PD using VR in comparison with traditional training. Method: A systematic search was performed including systematic reviews from PubMed, CINAHL and PEDro and published 2019-2022. Quality assessment was performed using AMSTAR 2.A narrative synthesis was conducted to synthesize the reported findings of gait and balance ability from the systematic reviews. The outcome measures used in this overview was staticand dynamic balance and gait parameters. Results: Of a total of 298 studies, seven systematic reviews with 128 randomized clinical trials (4736 participants) were included. Four studies had critically low quality after quality assessment. Static balance was improved (MD=1.13-2.69 SMD=0.22-0.26) in five of seven reviews with VR training compared to control group. Three of six reviews reported an improvement in dynamic balance (MD –1.55 till -2.86) with VR training. Temporal gait parameters reported no difference between VR training and control group. The spatial gait parameters reported mixed results among the systematic reviews. Conclusions: The included systematic reviews, in general, had low methodological quality. VR training were demonstrated to improve static balance but varied results regarding dynamic balance. Mixed findings were found regarding the spatial and temporal gait parameters. High-quality systematic reviews are warranted to general VR effect on gait and balance.Keyword: Balance, Gait, Overview, Parkinson´s disease, Virtual Realit

Effect of Virtual Reality training in people with Parkinson´s disease on balance and gait in comparison with traditional training : An overview of systematic reviews

Bakgrund: Virtual Reality (VR) är en teknik som skapar en simulerad miljö där användaren kan uppleva en virtuell värld. Effekten av VR-träning på gång och balansförmågan hos personer med Parkinsons sjukdom (PS) är ännu oklar trots flera systematiska studier. Sammanfattning av befintlig evidens kan ge en överblick samt kartlägga den kliniska nyttan av VR. Syfte: Syftet är att genomföra en översiktsstudie av systematiska litteraturstudier för att utvärdera behandlingseffekten av VR-träning jämfört med traditionell träning på gång- och balansförmåga hos personer med PS. Metod: Systematiska litteraturstudier i databaserna PubMed, CINAHL samt PEDro som publicerats mellan åren 2019-2022 har granskats. Kvalitetsgranskning utfördes med AMSTAR 2 och en narrativ syntes användes för att sammanfatta de rapporterade fynden av gång- och balansförmåga. Utfallsmåtten som använts är statisk och dynamisk balans samt gångparametrar. Resultat: Av totalt 298 systematiska litteraturstudier inkluderades sju studier med 128 randomiserade kontrollerade studier (4736 deltagare) som uppfyllde kriterier. Enligt AMSTAR 2 graderades fyra studier till kritiskt låg kvalitet. I fem av sju systematiska litteraturstudier rapporterades förbättrad statisk balans (MD=1.13-2.69 SMD=0.22-0.26) för gruppen med VR-träning jämfört med kontrollgrupp. I tre av sex studier rapporterades förbättring (MD –1.55 till -2.86) på dynamisk balans. Temporala gångparametrar rapporterade ingen skillnad mellan VR träning och kontrollgrupp. Spatiala gångparametrarna rapporterade blandade resultat. Slutsats: Studierna bedömdes övergripande ha låg metodologisk kvalitet. Resultaten visade att VR-träning kunde förbättra balansförmågan när det gäller statisk balans, men det fanns varierande resultat när det gällde dynamisk balans jämfört med kontrollgrupp. Ingen skillnad rapporterades mellan grupperna gällande temporala gångparametrar. Varierande resultat visades för spatiala gångparametrar. Ytterligare högkvalitativa systematiska översikter eftersträvas för att kunna dra mer tillförlitliga slutsatser.Background: Virtual Reality (VR) is a technology that creates a simulated environment where the user can experience a virtual world. The effect of VR training on gait and balance ability in people with Parkinson's disease (PS) remain unclear despite several systematic reviews. Summary of existing evidence can provide an overview of the current state of research and facilitate the mapping of the clinical benefit of VR. Aim: The aim is to conduct an overview of systematic reviews on gait and balance ability in people with PD using VR in comparison with traditional training. Method: A systematic search was performed including systematic reviews from PubMed, CINAHL and PEDro and published 2019-2022. Quality assessment was performed using AMSTAR 2.A narrative synthesis was conducted to synthesize the reported findings of gait and balance ability from the systematic reviews. The outcome measures used in this overview was staticand dynamic balance and gait parameters. Results: Of a total of 298 studies, seven systematic reviews with 128 randomized clinical trials (4736 participants) were included. Four studies had critically low quality after quality assessment. Static balance was improved (MD=1.13-2.69 SMD=0.22-0.26) in five of seven reviews with VR training compared to control group. Three of six reviews reported an improvement in dynamic balance (MD –1.55 till -2.86) with VR training. Temporal gait parameters reported no difference between VR training and control group. The spatial gait parameters reported mixed results among the systematic reviews. Conclusions: The included systematic reviews, in general, had low methodological quality. VR training were demonstrated to improve static balance but varied results regarding dynamic balance. Mixed findings were found regarding the spatial and temporal gait parameters. High-quality systematic reviews are warranted to general VR effect on gait and balance.Keyword: Balance, Gait, Overview, Parkinson´s disease, Virtual Realit

Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and “blinded” electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials

Differential expression of A-type and B-type lamins during hair cycling.

Multiple genetic disorders caused by mutations that affect the proteins lamin A and C show strong skin phenotypes. These disorders include the premature aging disorders Hutchinson-Gilford progeria syndrome and mandibuloacral dysplasia, as well as restrictive dermopathy. Prior studies have shown that the lamin A/C and B proteins are expressed in skin, but little is known about their normal expression in the different skin cell-types and during the hair cycle. Our immunohistochemical staining for lamins A/C and B in wild-type mice revealed strong expression in the basal cell layer of the epidermis, the outer root sheath, and the dermal papilla during all stages of the hair cycle. Lower expression of both lamins A/C and B was seen in suprabasal cells of the epidermis, in the hypodermis, and in the bulb of catagen follicles. In addition, we have utilized a previously described mouse model of Hutchinson-Gilford progeria syndrome and show here that the expression of progerin does not result in pronounced effects on hair cycling or the expression of lamin B