Transcriptional Landscape of Repetitive Elements in Psoriatic Skin from Large Cohort Studies: Relevance to Psoriasis Pathophysiology.

While studies demonstrating the expression of repetitive elements (REs) in psoriatic skin using RNA-seq have been published before, not many studies have focused on the genome-wide expression patterns using larger cohorts. This study investigated the transcriptional landscape of differentially expressed REs in lesional and non-lesional skin from two previously published large datasets. We observed significant differential expression of REs in lesional psoriatic skin as well as the skin of healthy controls. Significant downregulation of several ERVs, HERVs (including HERV-K) and LINEs was observed in lesional psoriatic skin from both datasets. The upregulation of a small subset of HERV-Ks and Alus in lesional psoriatic skin was also reported. An interesting finding from this expression data was the significant upregulation and overlapping of tRNA repetitive elements in lesional and non-lesional psoriatic skin. The data from this study indicate the potential role of REs in the immunopathogenesis of psoriasis. The expression data from the two independent large study cohorts are powerful enough to confidently verify the differential expression of REs in relation to psoriatic skin pathology. Further studies are warranted to understand the functional impact of these repetitive elements in psoriasis pathogenesis, thereby expanding their significance as a potential targeting pathway for the disease treatment of psoriasis and other inflammatory diseases.

Transcriptional Basis of Psoriasis from Large Scale Gene Expression Studies: The Importance of Moving towards a Precision Medicine Approach.

Transcriptome profiling techniques, such as microarrays and RNA sequencing (RNA-seq), are valuable tools for deciphering the regulatory network underlying psoriasis and have revealed large number of differentially expressed genes in lesional and non-lesional skin. Such approaches provide a more precise measurement of transcript levels and their isoforms than any other methods. Large cohort transcriptomic analyses have greatly improved our understanding of the physiological and molecular mechanisms underlying disease pathogenesis and progression. Here, we mostly review the findings of some important large scale psoriatic transcriptomic studies, and the benefits of such studies in elucidating potential therapeutic targets and biomarkers for psoriasis treatment. We also emphasised the importance of looking into the alternatively spliced RNA isoforms/transcripts in psoriasis, rather than focussing only on the gene-level annotation. The neutrophil and blood transcriptome signature in psoriasis is also briefly reviewed, as it provides the immune status information of patients and is a less invasive platform. The application of precision medicine in current management of psoriasis, by combining transcriptomic data, improves the clinical response outcome in individual patients. Drugs tailored to individual patient's genetic profile will greatly improve patient outcome and cost savings for the healthcare system.

Dystrophic Dmdmdx rats show early neuronal changes (increased S100ß and Tau5) at 8 months, supporting severe dystropathology in this rodent model of Duchenne muscular dystrophy.

The intrinsic necrosis of skeletal muscles in animal models of Duchenne muscular dystrophy (DMD) damages neuromuscular junctions (NMJs) with progressively altered NMJs associated with denervation and premature changes in dystrophic nerves. In the mdx mouse model of DMD, the proteins S100ß and Tau5 are significantly increased in sciatic nerves by 13 months (M) of age, far earlier (by 9 M) than in normal wildtype (WT) nerves. Since dystrophic Dmdmdx rats are reported to have a more severe dystropathology than mdx mice, we hypothesised that Dmdmdx rat nerves would show earlier neuronal changes compared with mdx nerves. We quantified levels of 8 proteins (by immunoblotting) in sciatic and radial nerves from young adult Dmdmdx rats (aged 8 M) and mdx mice (9 M), plus levels of 7 mRNAs (by qPCR) in rat nerves only. Sciatic nerves of 8 M Dmdmdx rats had more consistently increased levels of S100ß and Tau5 proteins, compared with 9 M mdx mice, supporting pronounced dystropathology in the rat model. There were no differences for mRNA levels, apart from higher gelsolin mRNA in Dmdmdx sciatic nerves. The pronounced protein changes in Dmdmdx nerves indicate a severe ongoing myonecrosis, and likely consequent myofibre denervation, for the dystrophic rat model. These data support increased neuronal proteins in dystrophic nerves as a novel pre-clinical readout of ongoing myonecrosis for DMD research. In older DMD boys, such progressive neuronal changes over many years are likely to contribute to loss of muscle function, and may complicate evaluation of late-onset clinical therapies.

Regeneration of adult rat sensory and motor neuron axons through chimeric peroneal nerve grafts containing donor Schwann cells engineered to express different neurotrophic factors.

Large peripheral nerve (PN) defects require bridging substrates to restore tissue continuity and permit the regrowth of sensory and motor axons. We previously showed that cell-free PN segments repopulated ex vivo with Schwann cells (SCs) transduced with lentiviral vectors (LV) to express different growth factors (BDNF, CNTF or NT-3) supported the regeneration of axons across a 1 cm peroneal nerve defect (Godinho et al., 2013). Graft morphology, the number of regrown axons, the ratio of myelinated to unmyelinated axons, and hindlimb locomotor function differed depending on the growth factor engineered into SCs. Here we extend these observations, adding more LVs (expressing GDNF or NGF) and characterising regenerating sensory and motor neurons after injection of the retrograde tracer Fluorogold (FG) into peroneal nerve distal to grafts, 10 weeks after surgery. Counts were also made in rats with intact nerves and in animals receiving autografts, acellular grafts, or grafts containing LV-GFP transduced SCs. Counts and analysis of FG positive (+) DRG neurons were made from lumbar (L5) ganglia. Graft groups contained fewer labeled sensory neurons than non-operated controls, but this decrease was only significant in the LV-GDNF group. These grafts had a complex fascicular morphology that may have resulted in axon trapping. The proportion of FG+ sensory neurons immunopositive for calcitonin-gene related peptide (CGRP) varied between groups, there being a significantly higher percentage in autografts and most neurotrophic factor groups compared to the LV-CNTF, LV-GFP and acellular groups. Furthermore, the proportion of regenerating isolectin B4+ neurons was significantly greater in the LV-NT-3 group compared to other groups, including autografts and non-lesion controls. Immunohistochemical analysis of longitudinal graft sections revealed that all grafts contained a reduced number of choline acetyltransferase (ChAT) positive axons, but this decrease was significant only in the GDNF and NT-3 graft groups. We also assessed the number and phenotype of regrowing lumbar FG+ motor neurons in non-lesioned animals, and in rats with autografts, acellular grafts, or in grafts containing SCs expressing GFP, CNTF, NGF or NT-3. The overall number of FG+ motor neurons per section was similar in all groups; however in tissue immunostained for NeuN (expressed in α- but not γ-motor neurons) the proportion of NeuN negative FG+ neurons ranged from about 40-50% in all groups except the NT-3 group, where the percentage was 82%, significantly more than the SC-GFP group. Immunostaining for the vesicular glutamate transporter VGLUT-1 revealed occasional proprioceptive terminals in 'contact' with regenerating FG+ α-motor neurons in PN grafted animals, the acellular group having the lowest counts. In sum, while all graft types supported sensory and motor axon regrowth, there appeared to be axon trapping in SC-GDNF grafts, and data from the SC-NT-3 group revealed greater regeneration of sensory CGRP+ and IB4+ neurons, preferential regeneration of γ-motor neurons and perhaps partial restoration of monosynaptic sensorimotor relays.

Implications of increased S100ß and Tau5 proteins in dystrophic nerves of two mdx mouse models for Duchenne muscular dystrophy.

This study investigates changes with respect to increasing protein levels in dystrophic nerves of two mdx mouse models of Duchenne muscular dystrophy (DMD). We propose that these nerve changes result from progressive ongoing damage to neuromuscular junctions (NMJs) due to repeated intrinsic bouts of necrosis in dystrophic muscles. We compared sciatic nerves from classic mdx mice aged 13, 15 and 18 months (M), with D2.mdx mice (on DBA2 background) aged 9 and 13 M, using immunoblotting to quantify levels of 7 proteins. The neuronal proteins S100ß and Tau5 were increased by 13 M in mdx nerves (compared with WT), indicating ongoing myonecrosis in this strain. In striking contrast there was no difference in levels of these neuronal proteins for D2.mdx and D2.WT sciatic nerves at 13 M, indicating reduced myonecrosis over this time in D2.mdx mice compared with mdx. These novel changes in mdx sciatic nerves by 13 M, suggest early denervation or neurodegeneration of dystrophic nerves that is likely irreversible and progressive. This neuronal readout of persistent myonecrosis may provide a useful new long-term biomarker for preclinical studies that aim to reduce myonecrosis, plus such neuronal changes present potential new drug targets to help maintain the function of DMD muscles.

Age-related loss of VGLUT1 excitatory, but not VGAT inhibitory, immunoreactive terminals on motor neurons in spinal cords of old sarcopenic male mice.

Age-related changes in ventral lumbar spinal cord (L3-L5) were compared in young [3 month, (M)] and old (27 M) C57BL/6J male mice. The aged mice had previously been shown to exhibit sarcopenia and changes to peripheral nerve morphology. The putative connectivity of ß-III tubulin positive α-motor neurons was compared in immunostained transverse sections using excitatory and inhibitory terminal markers vesicular glutamate transporter-1 (VGLUT1) and vesicular GABA transporter (VGAT). Glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) immunostaining was used to monitor changes in astrocyte and microglial phenotype respectively. For a given motor neuron, the neuronal perimeter was outlined and terminals immunoreactive for VGLUT1 or VGAT in close apposition to the soma were identified. By 27 M, the percentage coverage and total number of VGLUT1 immunoreactive terminals immediately adjacent to the soma of α-motor neurons was significantly decreased compared with young mice. However, percentage coverage of immunoreactive VGAT inhibitory terminals did not change significantly with age. The gray matter of 27 M spinal cords showed increased astrocytic and microglial activity. The loss of VGLUT1 terminals on α-motor neurons, terminals known to be derived from proprioceptive muscle afferents, may further impair sensorimotor control of hind limb skeletal muscle function in old mice.

Resistance wheel exercise from mid-life has minimal effect on sciatic nerves from old mice in which sarcopenia was prevented.

The ability of resistance exercise, initiated from mid-life, to prevent age-related changes in old sciatic nerves, was investigated in male and female C57BL/6J mice. Aging is associated with cellular changes in old sciatic nerves and also loss of skeletal muscle mass and function (sarcopenia). Mature adult mice aged 15 months (M) were subjected to increasing voluntary resistance wheel exercise (RWE) over a period of 8 M until 23 M of age. This prevented sarcopenia in the old 23 M aged male and female mice. Nerves of control sedentary (SED) males at 3, 15 and 23 M of age, showed a decrease in the myelinated axon numbers at 15 and 23 M, a decreased g-ratio and a significantly increased proportion of myelinated nerves containing electron-dense aggregates at 23 M. Myelinated axon and nerve diameter, and axonal area, were increased at 15 M compared with 3 and 23 M. Exercise increased myelinated nerve profiles containing aggregates at 23 M. S100 protein, detected with immunoblotting was increased in sciatic nerves of 23 M old SED females, but not males, compared with 15 M, with no effect of exercise. Other neuronal proteins showed no significant alterations with age, gender or exercise. Overall the RWE had no cellular impact on the aging nerves, apart from an increased number of old nerves containing aggregates. Thus the relationship between cellular changes in aging nerves, and their sustained capacity for stimulation of old skeletal muscles to help maintain healthy muscle mass in response to exercise remains unclear.

A Neurogenic Perspective of Sarcopenia: Time Course Study of Sciatic Nerves From Aging Mice.

To elucidate the neural basis for age-related sarcopenia, we quantified morphologic and molecular changes within sciatic nerves of aging male and female C57BL/6J mice aged between 3 and 27 months using immunoblotting, immunohistochemistry, and electron microscopy. Protein analyses by immunoblotting of nerves of male mice aged 4, 15, 18, 22, and 24 months showed increased levels of heavy chain SMI-32-positive neurofilaments, vimentin, tau5, choline acetyltransferase (ChAT), and p62 by 18-22 months. Similar protein increases were seen in 26-month-old compared with 3-month-old female mice. Immunostaining of longitudinal sections of old (27-month-old) male sciatic nerves revealed intense staining for tau5 and p62 that was increased compared with that at 3 months, but there were decreased numbers of axon profiles stained for ChAT or isolectin B4 (motor and sensory axons, respectively). Ultrastructural analysis revealed electron-dense aggregates within axons in peripheral nerves of old male mice; the proportion of axons that contained aggregates more than doubled between 15 and 27 months. Overall, the observed age-related accumulation of many proteins from about 18 months of age onward suggests impaired mechanisms for axonal transport and protein turnover. These peripheral nerve changes may contribute to the morphological and functional muscle deficits associated with sarcopenia.

Development of a System and Method for Automated Isolation of Stromal Vascular Fraction from Adipose Tissue Lipoaspirate.

Autologous fat grafting for soft tissue reconstruction is challenged by unpredictable long-term graft survival. Fat derived stromal vascular fraction (SVF) is gaining popularity in tissue reconstruction as SVF-enriched fat grafts demonstrate improved engraftment. SVF also has potential in regenerative medicine for remodeling of ischemic tissues by promoting angiogenesis. Since SVF cells do not require culture expansion, attempts are being made to develop automated devices to isolate SVF at the point of care. We report development of a closed, automated system to process up to 500 mL lipoaspirate using cell size-dependent filtration technology. The yield of SVF obtained by automated tissue digestion and filtration (1.17 ± 0.5 × 10(5) cells/gram) was equivalent to that obtained by manual isolation (1.15 ± 0.3 × 10(5); p = 0.8), and the viability of the cells isolated by both methods was greater than 90%. Cell composition included CD34+CD31- adipose stromal cells, CD34+CD31+ endothelial progenitor cells, and CD34-CD31+ endothelial cells, and their relative percentages were equivalent to SVF isolated by the manual method. CFU-F capacity and expression of angiogenic factors were also comparable with the manual method, establishing proof-of-concept for fully automated SVF isolation, suitable for use in reconstructive surgeries and regenerative medicine applications.

Goat endometrial heat shock protein-90 (Hsp-90): development of an expedient method for its purification and observations on its intracellular movement.

An expedient method has been developed by which goat uterine Hsp-90 could be isolated and purified to homogeneity in less than 1day. The yield is roughly 1mg from 60g tissue. This method takes into advantage three of our earlier observation that (a) Hsp-90 gets linked to the non-activated estrogen receptor (naER) in the presence of 10mM sodium molybdate; (b) naER, but not Hsp-90 binds to phosphocellulose and (c) exposure to estradiol facilitates dissociation of Hsp-90 from naER through estradiol binding to naER and the possible change in naER conformation. Intracellular movement of Hsp-90 and naER was monitored in goat endometrial cells in culture following exposure of the cells to estradiol. Confocal microscopic analysis revealed a clear presence of both proteins within the nucleus within 3h after exposure to estradiol. Whether Hsp-90 has its own nuclear-transport machinery is debatable. Being an actin-binding protein, there is a distinct possibility that the nuclear entry of Hsp-90 is actin dependent. The functional significance of the nuclear entry of Hsp-90, along with naER, remains to be determined; it may, however, be speculated that the Hsp-90 might be directly involved in the naER to nER II transformation by functioning as a molecular chaperone and helping the protein in re-orienting its structural organization.

Cytoskeletal elements and intracellular transport.

Recent advances in the understanding of the functions of various components of the cytoskeleton indicate that, besides serving a structural role, the cytoskeletal elements may regulate the transport of several proteins in the cell. Studies reveal that there are co-operative interactions between the actin and microtubule cytoskeletons including functional overlap in the transport influenced by different motor families. Multiple motors are probably involved in the control of the dynamics of many proteins and intriguing hints about how these motors are co-ordinated are appearing. It has been shown that some of the intermediate elements also participate in selected intracellular transport mechanisms. In view of the author's preoccupation with the steroid receptor systems, special attention has been given to the role of the cytoskeletal elements, particularly actin, in the intracellular transport of steroid receptors and receptor-related proteins.