Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis

"Delirium Day": A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 \ub1 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys

Loss of Sc65 results in dermal tears, abnormal collagen fibrils and skin fragility.

<p>a) H&E stained sections of WT and <i>Sc65KO</i> skin. Note the decreased density of collagen, the frayed dermis indicated by arrows and the reduced thickness of the muscle layer in the <i>Sc65-null</i> samples. b) Serial skin sections were stained with Sirius red. <i>Sc65-null</i> skin exhibits fewer large collagen fibers (red staining) and greater number of smaller collagen fibers stained in green compared to WT counterparts. c) Electron micrographs of 7 month-old mouse skin biopsy from WT and <i>Sc65KO</i> mice. Collagen fibrils, shown in cross-section, from <i>Sc65-null</i> skin tended to be smaller and have a decreased range of fibril diameter compared to WT fibrils. Loss of Sc65 also resulted in the presence of collagen fibrils with irregular profile and several large “cauliflower-like” fibrils (red arrow) which indicate abnormal fibrillogenesis (scale bar represents 500nm). d) Distribution of collagen fibril diameter in WT and <i>Sc65KO</i> mouse skin as measured from electron microscopy images. Measurements were collected from three different mice/genotype and >200 fibril/mouse. e) Skin EMs from <i>Sc65KO</i> mice also exhibited significantly more empty space among collagen fibrils compared to WT mice indicating a less densely packed collagen (*p = 0.01). Five electron micrograph images of non-overlapping areas were quantified from each mouse. f-h) Skin samples from WT and <i>Sc65KO</i> mice were subjected to a biomechanical skin loading test to measure tensile strength. Skin that lacks SC65 expression ruptured at a significantly lower peak load compared to WT skin indicating significant skin fragility (*p<0.01).</p

Schematic representation of a fibrillar collagen molecule in the ER.

<p>The long uninterrupted triple-helical domain is shown here already folded and without N- and C-propeptides. Depicted in different colors are some of the prolyl 3-hydroxylase enzymes (P3Hs), lysyl hydroxylase enzymes (LHs) and cyclophilin B (purple) with some of their known substrate residues. Our previous work identified the prolyl 3-hydroxylation complex that modifies P986; our current work suggests the existence of a new complex, possibly including CYPB, responsible for the hydroxylation of K87 and K930. Evidence indicates that CRTAP and SC65 act as unique orchestrators of essential molecular complexes for collagen post-translational modification. (LH2 is likely to also act within a protein complex but direct interactions have not yet been published).</p

<i>Sc65KO</i> mouse generation and confirmation of bone loss phenotype.

<p>a) Strategy for the creation of the <i>Sc65-null</i> allele. The schematic diagram shows the <i>Sc65</i> wild-type, targeted, floxed and excised allele. <i>Sc65</i> coding regions are in light blue while non-coding regions are in dark blue. Also note the proximity to the <i>Fkbp10</i> gene which is transcribed in the opposite orientation. b) PCR genotyping of <i>Sc65KO</i> mice (upper panel) and Western blot confirmation of SC65 protein (arrow) loss in multiple <i>Sc65KO</i> tissues from 3 day-old mice compared to WT controls (lower panel—Cal = calvaria, Kid = Kidney). c) Immunohistochemistry detection of SC65 in adult femur section from a WT mouse showing specific intracellular staining in bone forming cells (osteoblasts) aligned on the surface of a bone trabecula. <i>Sc65</i> expression is lost in a similar section from a <i>Sc65KO</i> mouse. Scale bars = 100μM (10x) or 20μM (63x). d) MicroCT analysis of long bones from 6 month-old WT and <i>Sc65KO</i> male mice (n = 9). Both femurs and tibias from <i>Sc65KO</i> mice exhibited decreased trabecular bone volume/tissue volume (BV/TV), connectivity density (Conn.D) and cortical thickness (Ct.Th) compared to WT controls (*p<0.05).</p

Increased electrophoretic mobility and altered cross-linking of type I collagen from <i>Sc65-null</i> skin.

<p>a) SDS-6%PAGE of type I collagen extracted from skin and decalcified bone of <i>Sc65KO</i> and WT mice shows increased mobility of α-chains and reduced ratio of cross-linked β to γ components in the <i>Sc65KO</i> skin extracts. An acetic acid extract from skin of the original Sc65-null mouse¹⁹ (1 mo.) created by gene-trap insertion is compared with that from the new <i>Sc65KO</i> (6 mo.) and their respective WT controls. Total heat denatured extracts of skin and bone collagens from new <i>Sc65KO</i> mice are shown on the right for comparison. Bone collagen from <i>Sc65KO</i> mice does not show the differences from WT in β/γ intensities evident for skin collagen. The strong (lower) γ band in SC65 skin extracts was identified as γ _112. Both original and new <i>Sc65KO</i> mice showed the same altered pattern of chain intensities from WT most pronounced in the acetic acid extracts of skin with an apparent increase in γ₁₁₂ at the expense of β_12. b) Densitometric analysis of collagen bands on SDS-PAGE. Densitometry was performed on bands 1–8 (counted from top to bottom) of acetic acid extracts from 1mo and 6mo skin samples of both original and new <i>Sc65KO</i> mice using NIH imageJ software. Values are means ± SD, n = 6; *p<0.01.</p

Characterization of a new SC65/LH1/P3H3 complex in the ER.

<p>a) Lysates of 714 mouse embryonic fibroblasts that were transiently transfected with an HA-tagged LH1 expression construct were immuno-precipitated with a HA antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 8% SDS-PAGE gel, blotted and probed with antibodies against HA and P3H3. Negative controls included non-transfected 714 cells incubated with the HA antibody (for non-specific binding of HA antibody, left lanes) and LH1-HA transfected cells incubated with no antibody (for non-specific proteins binding to beads, middle lanes). In both experiments, LH1-HA and P3H3 were found to interact (right lanes). b) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control and transiently transfected with a HA-tagged CYPB were used for IP utilizing an HA antibody. 10% of total input and immuno-precipitates were separated on a 12% SDS-PAGE gel, blotted and probed with antibodies against Flag and HA. The blot detecting SC65-Flag following IP with the HA antibody is shown over-exposed. c) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing similar content of CYPB protein in <i>Sc65KO</i> and WT samples. All experiments were performed at least 3 times.</p

SC65 directly interacts with prolyl 3-hydroxylase 3 (P3H3).

<p>a) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control were used for IP experiments utilizing a Flag antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 10% SDS-PAGE gel, blotted and probed with antibodies against FLAG and P3H3. The reciprocal interaction of SC65-Flag with P3H3 is confirmed in both experiments. b) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing significantly decreased levels of P3H3 protein in <i>Sc65KO</i> samples. Densitometric quantification of P3H3 protein normalized to β-actin from the western blot shown above (#p<0.01; *p<0.05; error bars represent SD). All experiments were performed at least 3 times.</p