Disease Models and Mechanisms

Published by Company of Biologists
Online ISSN: 1754-8411
Print ISSN: 1754-8403
Seventy-five percent of lung adenocarcinomas with epidermal growth factor receptor (EGFR) mutations respond to treatment with the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib; however, drug-resistant tumors eventually emerge. In 60% of cases, resistant tumors carry a secondary mutation in EGFR (T790M), amplification of MET, or both. Here, we describe the establishment of erlotinib resistance in lung tumors, which were induced by mutant EGFR, in transgenic mice after multiple cycles of drug treatment; we detect the T790M mutation in five out of 24 tumors or Met amplification in one out of 11 tumors in these mice. This preclinical mouse model, therefore, recapitulates the molecular changes responsible for resistance to TKIs in human tumors and holds promise for the discovery of additional mechanisms of drug resistance in lung cancer.
Most spinal cord injuries lead to permanent paralysis in mammals. By contrast, the remarkable regenerative abilities of salamanders enable full functional recovery even from complete spinal cord transections. The molecular differences underlying this evolutionary divergence between mammals and amphibians are poorly understood. We focused on upstream regulators of gene expression as primary entry points into this question. We identified a group of miRNAs that are conserved between the Mexican axolotl salamander and mammals, but show marked cross-species differences in regulation patterns following spinal cord injury. We found that precise post-injury levels of one of these miRNAs (miR-125b) is essential for functional recovery, and guides correct regeneration of axons through the lesion site in a process involving the direct downstream target Sema4D in axolotls. Translating these results to a mammalian model, we increased miR-125b levels in the rat through mimic treatments following spinal cord transection. These treatments down-regulated Sema4D and other glial-scar related genes, and enhanced the animal's functional recovery. Our study identifies a key regulatory molecule conserved between salamander and mammal, and shows that the levels of miR-125b and its target gene Sema4D must be carefully controlled in the right cells at the correct level to promote regeneration. We also show that this molecular component of the salamander's regeneration-permissive environment can be experimentally harnessed to improve treatment outcomes for mammalian spinal cord injuries.
Spinocerebellar ataxia type 13 (SCA13) is an autosomal dominant disease caused by mutations in the Kv3.3 voltage-gated potassium (K(+)) channel. SCA13 exists in two forms: infant onset is characterized by severe cerebellar atrophy, persistent motor deficits and intellectual disability, whereas adult onset is characterized by progressive ataxia and progressive cerebellar degeneration. To test the hypothesis that infant- and adult-onset mutations have differential effects on neuronal development that contribute to the age at which SCA13 emerges, we expressed wild-type Kv3.3 or infant- or adult-onset mutant proteins in motor neurons in the zebrafish spinal cord. We characterized the development of CaP (caudal primary) motor neurons at ~36 and ~48 hours post-fertilization using confocal microscopy and 3D digital reconstruction. Exogenous expression of wild-type Kv3.3 had no significant effect on CaP development. In contrast, CaP neurons expressing the infant-onset mutation made frequent pathfinding errors, sending long, abnormal axon collaterals into muscle territories that are normally innervated exclusively by RoP (rostral primary) or MiP (middle primary) motor neurons. This phenotype might be directly relevant to infant-onset SCA13 because interaction with inappropriate synaptic partners might trigger cell death during brain development. Importantly, pathfinding errors were not detected in CaP neurons expressing the adult-onset mutation. However, the adult-onset mutation tended to increase the complexity of the distal axonal arbor. From these results, we speculate that infant-onset SCA13 is associated with marked changes in the development of Kv3.3-expressing cerebellar neurons, reducing their health and viability early in life and resulting in the withered cerebellum seen in affected children.
Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders that manifest deficits in social interaction, and verbal and non-verbal communication, in addition to restrictive interests and repetitive behaviors. Recent reports indicate that ASDs may occur in as many as
Constitutively active, 'oncogenic' H-RAS can drive proliferation and transformation in human cancer, or be a potent inducer of cellular senescence. Moreover, aberrant activation of the Ras pathway owing to germline mutations can cause severe developmental disorders. In this study we have generated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observed that fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activating mutations in the gene H-RAS, and can be used as a model for the disease. In Costello-like fish, low levels of oncogenic H-RAS expression are associated with both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together with activated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activation of the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes to the development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively active H-RAS expression.
Figure S1
Figure S1. 16p11.2 homologs are expressed during development and in the brain. (A) RNA expression analysis by RT-PCR. Total embryo RNA was analyzed for expression of 16p11.2 homologs at 2.5 hpf (blastula), 6 hpf (early gastrula), 11 hpf (late gastrula), 18 hpf (mid-somitogenesis), 24 hpf and 48 hpf. Most genes are expressed maternally, at 2.5 hpf, and expression persists zygotically (6 hpf and later). asphd1, doc2a, prrt2 and sez6L2 show zygotic expression only, suggesting that these genes act later in development. tbx24 expression declines sharply by 48 hpf, suggesting a narrow temporal activity window. Further, this gene is not expressed in brain. (B) Spatial expression analysis by whole mount in situ hybridization. Gene expression was analyzed at 24 hpf (see Methods and Table S4). (a-t) dorsal view, (a'-t') lateral view. All genes except tbx24 were expressed in the brain.
Figure S2
Figure S3. Pigmentation is abnormal in LOF embryos. Pigment cells were observed after LOF, at 48 hpf, by brightfield imaging. Rostral and midline cells were specifically scored as a measure of normal melanocyte migration. (A-I) dorsal views. Genes targeted for LOF by MO injection are indicated above each set of panels. Red arrowhead, no rostral pigment cells; red arrow, no midline pigment cells; scale bar, 150 µM. Average of 35 embryos assayed per gene, over 2-4 independent experiments, 70-100% affected compared to control MO-injected embryos.
Deletion or duplication of one copy of the human 16p11.2 interval is tightly associated with impaired brain function, including autism spectrum disorders (ASDs), intellectual disability disorder (IDD) and other phenotypes, indicating the importance of gene dosage in this copy number variant region (CNV). The core of this CNV includes 25 genes; however, the number of genes that contribute to these phenotypes is not known. Furthermore, genes whose functional levels change with deletion or duplication (termed 'dosage sensors'), which can associate the CNV with pathologies, have not been identified in this region. Using the zebrafish as a tool, a set of 16p11.2 homologs was identified, primarily on chromosomes 3 and 12. Use of 11 phenotypic assays, spanning the first 5 days of development, demonstrated that this set of genes is highly active, such that 21 out of the 22 homologs tested showed loss-of-function phenotypes. Most genes in this region were required for nervous system development - impacting brain morphology, eye development, axonal density or organization, and motor response. In general, human genes were able to substitute for the fish homolog, demonstrating orthology and suggesting conserved molecular pathways. In a screen for 16p11.2 genes whose function is sensitive to hemizygosity, the aldolase a (aldoaa) and kinesin family member 22 (kif22) genes were identified as giving clear phenotypes when RNA levels were reduced by ~50%, suggesting that these genes are deletion dosage sensors. This study leads to two major findings. The first is that the 16p11.2 region comprises a highly active set of genes, which could present a large genetic target and might explain why multiple brain function, and other, phenotypes are associated with this interval. The second major finding is that there are (at least) two genes with deletion dosage sensor properties among the 16p11.2 set, and these could link this CNV to brain disorders such as ASD and IDD.
Scheme of the construct expressing dnBmpr1a and changes in the downstream effector pathways of Bmpr1a-mediated signaling in the nasal processes of the double-tg embryos. (A)Construct pCAG-XstopXdnBmpr1a-IRES-NLLacZ-polyA used for generating Tg(CAG-flox-dnBmpr1aNLLacZ)1Nobs mice. (B-D)Whole-mount views of LacZ-stained control (B), double-tg (C) and Tg(CAG-cat-lacZ)11Miya tg/+ ;Tg(Mpz-cre)94Imeg tg/+ (D) embryos at 10.5 d.p.c. (E)RT-PCR analysis of Msx 1, Msx 2 and dnBmpr1A expression in the nasal processes of 10.5 d.p.c. control (n4) and double-tg (n4) embryos. (F)Relative Msx 1 and Msx 2 expression levels. (G)Western blot analysis of phosphorylated Smad (p-Smad)1/5/8, p-Smad2, p-Smad3, and Bmpr1a in the upper jaws of 15.5 d.p.c. control (n3) and double-tg (n5) embryos. (H)Relative p-Smad1/5/8 expression levels. Error bars indicate mean ± s.e.m. *P<0.05, **P<0.01. Scale bars: 1.0 mm. 
Facial abnormalities of double-tg mice at later stages of development. (A-F)Lateral (A-C) and frontal (D-F) views of the control (A,D) and double-tg mice (B,C,E,F) without cleft face (B,E) and with cleft face (C,F) at birth. (G,H)Whole-mount view of a normal closed palate (G) and view of cleft palate (arrowheads) in double-tg mice (H) with facial midline cleft indicated by asterisks (F,H). (I-L)Lateral (I,J) and superior (K,L) views of control (I,K) and double-tg mice (J,L) at 3 weeks after birth. Facial length and distance between the eyes were measured and designated as FL and DE, respectively, as indicated. (M)FLs of control (n16) and double-tg (n11) mice. (N)DEs of control (n16) and doubletg (n11) mice. Error bars indicate mean ± s.e.m. *P<0.05, **P<0.001. Scale bars: 5.0 mm (A-C), 2.5 mm (D-F), 1.0 mm (G,H) and 1.0 cm (I-L). 
Histomorphogenesis of frontonasal processes and mesenchymal apoptosis and proliferation in double-tg embryos at 9.5 and 10.5 d.p.c. (A,B)HEstained sagittal section views of 9.5 d.p.c. control (A) and double-tg (B) embryos. (C,D)Whole mount in situ hybridization of 9.5 d.p.c. control (C) and double-tg (D) embryos. using a probe for Tfap2a. (E,F)Whole-mount frontal views of 10.5 d.p.c. control (E) and double-tg embryos (F). The arrowheads indicate the right and left frontonasal processes. (G)Frontonasal mesenchymal cell densities of control (n4) and double-tg (n4) embryos at 9.5 and 10.5 d.p.c. (H,I)Transverse sections of the nasal processes of 10.5 d.p.c. control (H) and double-tg (I) embryos stained for TUNEL reaction (black) and with anti-p53 antibody (orange); magnification of boxed areas are shown in each inset. The arrowheads and arrows in H and I indicate the TUNEL and anti-P53 immunostaining double-positive cells and anti-P53 immunostaining single-positive cells, respectively. (J)Ratio between TUNEL single-positive cells and frontonasal mesenchymal cells, ratio between TUNEL/P53 double-positive cells and frontonasal mesenchymal cells, and ratio between P53 single-positive cells and frontonasal mesenchymal cells in control (n3) and the double-tg (n3) embryos. (K)RT-PCR analysis of Bcl-XL and c-Myc expression in the nasal processes of 10.5 d.p.c. control (n4) and double-tg (n4) embryos. (L)Relative Bcl-XL and c-Myc expression levels. (M,N)Transverse sections of the nasal processes of 10.5 d.p.c. control (M) and double-tg (N) embryos stained with anti-PCNA antibody (brown) and counterstained with Kernechtrot; magnification of boxed areas are shown in each inset. (O)Ratio between PCNA-positive cells and frontonasal mesenchymal cells in control (n4) and double-tg (n4) embryos. (P,Q)Whole-mount frontal views of 11.5 d.p.c. LacZ-stained control (P) and double-tg (Q) embryos. Normally fused (n2/9; data not shown) and abnormally unfused (n7/9; P) nasal processes of double-tg embryos. The arrowheads in P and Q indicate the midlines of embryo faces. Max, maxillary process; man, mandibular process; ln, lateral nasal process; mn, medial nasal process. Error bars indicate mean ± s.e.m. *P<0.05, **P<0.01. Scale bars: 100m (A,B), 200m (C,D), 300m (E,F), 50m (H,I,M,N), 100m (inset in H,I,M,N) and 500m (P,Q). 
Analysis of skulls and frontal bone primordia of double-tg mice. (A-D)Lateral (A,B) and frontal (C,D) CT images of the skulls of control (A,C) and double-tg (B,D) mice at 3 weeks after birth. The arrowhead in D indicates calvarial foramina. (EJ)Lateral (E-G) and superior (H-J) views of Alizarin Red-(bone) and Alcian Blue-stained (cartilage) skulls of control (E,H) and doubletg without cleft face (F,I) and with cleft face (G,J) at birth. (K,L)Transverse sections of the frontal primordium of 10.5 d.p.c. control (K) and double-tg (L) embryos stained with anti-PCNA antibody (brown) and counterstained with Kernechtrot (red). (M,N)Transverse sections of the frontal primordium of 10.5 d.p.c. control (M) and double-tg (N) embryos stained for TUNEL reaction (brown) and counterstained with Kernechtrot. (O,P)Transverse sections of the frontal primordium of 10.5 d.p.c. control (O) and double-tg (P) embryos stained with anti-P53 antibody (brown) and counterstained with Kernechtrot. P53-positive cells were not detectable in the controls (n3). (Q)Ratio between PCNA-positive cells and frontal primordial mesenchymal cells in control (n3) and double-tg (n3) embryos. (R)Ratio between TUNEL reactionpositive cells and frontal primordial mesenchymal cells in control (n3) and double-tg (n3) embryos at 10.5 d.p.c. (S)Ratio between p53-positive cells and frontal primordial mesenchymal cells in control (n3) and double-tg (n3) embryos. The error bars indicate mean ± s.e.m. *P<0.05. (T,U)Transverse sections of the frontal primordium of 12.5 d.p.c. control (T) and double-tg (U) embryos stained for alkaline phosphatase (blue) and counterstained with Kernechtrot. (V,W)HE-stained transverse sections of the frontal primordium of control (V) and double-tg (W) embryos at 15.5 d.p.c. (X,Y)Alizarin Red-and Alcian Bluestained transverse sections of the frontal primordium of control (X) and double-tg (Y) embryos at 15.5 d.p.c. ch, cerebral hemisphere; e, eye; eo, exoccipital; fr, frontal; ip, interparietal; ma, mandible; mx, maxilla; na, nasal; nc, nasal cartilage; pmx, premaxilla; pr, parietal; so, supraoccipital. Scale bars: 2.0 mm (A-F), 1.0 cm (G-J), 25m (K-P), 100m (T,U) and 200m (V-Y). 
Bone morphogenetic protein (BMP) receptor type 1A (BMPR1A) mutations are associated with facial dysmorphism, which is one of the main clinical signs in both juvenile polyposis and chromosome 10q23 deletion syndromes. Craniofacial development requires reciprocal epithelial/neural crest (NC)-derived mesenchymal interactions mediated by signaling factors, such as BMP, in both cell populations. To address the role of mesenchymal BMP signaling in craniofacial development, we generated a conditional knockdown mouse by expressing the dominant-negative Bmpr1a in NC-derived cells expressing the myelin protein zero (Mpz)-Cre transgene. At birth, 100% of the conditional mutant mice had wide-open anterior fontanelles, and 80% of them died because of cleft face and cleft palate soon after birth. The other 20% survived and developed short faces, hypertelorism and calvarial foramina. Analysis of the NC-derived craniofacial mesenchyme of mutant embryos revealed an activation of the P53 apoptosis pathway, downregulation of both c-Myc and Bcl-XL, a normal growth rate but an incomplete expansion of mesenchymal cells. These findings provide genetic evidence indicating that optimal Bmpr1a-mediated signaling is essential for NC-derived mesenchymal cell survival in both normal nasal and frontal bone development and suggest that our model is useful for studying some aspects of the molecular etiology of human craniofacial dysmorphism.
Recent studies revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the mechanisms underlying the disease were discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S(-/-)), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S(-/-) and Ltbp4 null (Ltbp4(-/-)) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival with distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a novel interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for ECM incorporation of fibulin-4. Overall, our results contribute to the current understanding of elastogenesis and provide of an animal model of ARCL1C.
Parkin regulates the proteasomal degradation of PARIS and PGC-1-dependent mitochondrial biogenesis. (A)Normal physiological conditions that maintain mitochondrial homeostasis: PINK1 recruits parkin to the mitochondria, where these two proteins interact to eliminate abnormal mitochondria through mitophagy. Alterations in mitochondrial membrane potential (m; a key indicator of mitochondrial physiology and cell viability) initiate the PINK1-parkin cascade of events that lead to mitophagy. Furthermore, parkin ubiquitylates and thereby promotes proteasomal degradation of PARIS. Because PARIS represses the expression of PGC-1, degradation of PARIS by parkin allows PGC-1-dependent gene expression and enables mitochondrial biogenesis. Parkin seems to be an integral regulator of mitochondrial homeostasis, controlling both degradation and biogenesis. (B) Loss of parkin function as a result of familial mutations (in the case of AR-PD) or aging, environmental or cellular stress (in the case of sporadic PD) leads to the accumulation of abnormal mitochondria, owing to faulty mitophagy. In addition, PARIS accumulates and represses PGC-1, preventing mitochondrial biogenesis. Loss of parkin function does not tip the balance between mitochondrial biogenesis and degradation to either side, but leads to a general breakdown of mitochondrial homeostasis that can ultimately lead to PD.  
Summary and comment on a recent Cell paper entitled ‘PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson’s disease’ (Shin et al., 2011).
Summary and comment on a recent Nature paper entitled ‘A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis’ (Boström et al., 2012).
Altered palate and jaw morphology in LgDel mice. Top: quantitative real time PCR (qPCR) analysis of a subset of 22q11 deleted genes in microdissected rudiments of key orofacial structures for feeding and swallowing: BA1A, which includes the maxilla, which gives rise to the upper jaw, mouth structures and some muscles of mastication, and BA1B/BA2, which includes the mandible and hyoid, which gives rise to the lower jaw and some pharyngeal structures. These 22q11 genes were chosen based upon expression above a threshold level-0.01% of Gapdh expression measured in the same sample-in a previous analysis of developing E10.5 embryos or the brain from E12.5 onward (Maynard et al., 2003). Expression was quantified. Middle: sections through the anterior and posterior palatal region in E13.5 WT and LgDel embryos demonstrating failure of palatal elevation in the LgDel (compare arrows). The palate has not fused in either the WT or LgDel at this age. WT palatal shelves are more frequently elevated (left arrows) than those in the LgDel (right arrows). Bottom left: lateral view of the mandibular process from a WT mouse, with reference points for measurements indicated as numbers 1-6. Bottom right: morphometry of point-to-point distances between cardinal locations shown at left indicates that mandibular growth is diminished in LgDel versus WT littermate mice (*P≤0.5; **P≤0.01, t-test; n=48 LgDel, 46 WT). 
Heterozygous inactivation of Raldh2 rescues CN V, but not CN IX/X, phenotypes in LgDel embryos. Top left: (first row) comparison of CN V differentiation in E10.5 Raldh2 +/− , which resembles the WT (see Fig. 5), Raldh2 +/− :LgDel, which also resembles the WT, and LgDel, which shows clear disruption of fasciculation and differentiation of all major branches of CN V. The ophthalmic (op, arrow), maxillary (mx, bracket) and mandibular (md, arrows) branches of CN V are shown. (Second row) Comparison of CN IX/X differentiation in E10.5 Raldh2 +/− , Raldh2 +/− :LgDel and LgDel embryos. CNs in Raldh2 +/− embryos resemble the WT, whereas Raldh2 +/− :LgDel and LgDel have similar ganglion fusions (arrows) and disrupted axon trajectories. Top right: frequency of phenotypes in 12 individual CN V and CN IX/X from six E10.5 Raldh2 +/− :LgDel, or from six LgDel embryos. Statistical comparisons made using Fisher's exact analysis. Middle: A-P rhombomere selective genes, many of which are RA regulated (see Fig. 4), return to WT levels in E9.5 LgDel:Raldh2 +/− hindbrains, based upon qPCR analysis of microdissected hindbrain samples from n=8 Raldh2 +/− ; 7 LgDel:Raldh2 +/− ; 13 WT; and 12 LgDel embryos. Ø: genes for which LgDel:Raldh2 +/− levels are statistically indistinguishable from WT and Raldh2 +/− levels; *: genes for which LgDel levels are significantly increased over both Raldh2 +/− and WT. Bottom left: scatterplots showing the ranges of individual hindbrain expression values for four 'rescued' genes in WT, LgDel (LD) and LgDel:Raldh2 +/− (LD:Ra). The minimum and maximum values in the WT and LgDel:Raldh2 +/− are similar, and the minimum and maximum values for the LgDel are consistently increased. Bottom right: Cyp26b1 patterns in the Raldh2 +/− hindbrain are similar to WT (compare to WT panels in Figs 4 and 7); those in LgDel:Raldh2 +/− hindbrain resemble the Raldh2 +/− and WT as well; in the LgDel, intensity increases in r6, r5, r4 and r3, the barely detectable expression in r2 is more robust, and, in this case, apparently extends into r1. Right hand brackets show expansion of r5/6 in LgDel but not Raldh2 +/− or LgDel:Raldh2 +/− hindbrain; left brackets show that r2-r4 are apparently compressed in the LgDel but not Raldh2 +/− or LgDel:Raldh2 +/− hindbrain. 
We assessed feeding-related developmental anomalies in the LgDel mouse model of Chromosome 22q11 Deletion Syndrome (22q11DS), a common developmental disorder that frequently includes perinatal dysphagia - debilitating feeding, swallowing and nutrition difficulties from birth onward - within its phenotypic spectrum. LgDel pups gain significantly less weight during the first postnatal weeks, and have several signs of respiratory infections due to food aspiration. Most 22q11 genes are expressed in anlagen of craniofacial and brainstem regions critical for feeding and swallowing, and diminished expression in LgDel embryos apparently compromises development of these regions. Palate and jaw anomalies indicate divergent oro-facial morphogenesis. Altered expression and patterning of hindbrain transcriptional regulators, especially those related to retinoic acid (RA) signaling prefigures these disruptions. Subsequently, gene expression, axon growth and sensory ganglion formation in the trigeminal (V), glossopharyngeal (IX), or vagus (X) cranial nerves (CN) that innervate targets essential for feeding, swallowing and digestion are disrupted. Posterior CN IX and X ganglia anomalies primarily reflect diminished dosage of the 22q11DS candidate gene Tbx1. Genetic modification of RA signaling in LgDel embryos rescues the anterior CN V phenotype and returns expression levels or pattern of RA-sensitive genes to that in wild type embryos. Thus, diminished 22q11 gene dosage, including but not limited to Tbx1, disrupts oro-facial and cranial nerve development by modifying RA-modulated anterior-posterior hindbrain differentiation. These disruptions likely contribute to dysphagia in infants and young children with 22q11DS.
Duplication of the gene encoding lamin B1 (LMNB1) with increased mRNA and protein levels has been shown to cause severe myelin loss in the brains of adult-onset autosomal dominant leukodystrophy patients. Similar to many neurodegenerative disorders, patients with adult-onset autosomal dominant leukodystrophy are phenotypically normal until adulthood and the defect is specific to the central nervous system despite the ubiquitous expression pattern of lamin B1. We set out to dissect the molecular mechanisms underlying this demyelinating phenotype. Increased lamin B1 expression results in disturbances of inner nuclear membrane proteins, chromatin organization and nuclear pore transport in vitro. It also leads to premature arrest of oligodendrocyte differentiation, which might be caused by reduced transcription of myelin genes and by mislocalization of myelin proteins. We identified the microRNA miR-23 as a negative regulator of lamin B1 that can ameliorate the consequences of excessive lamin B1 at the cellular level. Our results indicate that regulation of lamin B1 is important for myelin maintenance and that miR-23 contributes to this process, at least in part, by downregulating lamin B1, therefore establishing novel functions of lamin B1 and miR-23 in the regulation of oligodendroglia development and myelin formation in vitro.
Cancer cachexia describes the progressive skeletal muscle wasting and weakness that is associated with many cancers. It impairs quality of life and accounts for >20% of all cancer-related deaths. The main outcome that affects quality of life and mortality is loss of skeletal muscle function and so preclinical models should exhibit similar functional impairments in order to maximize translational outcomes. Mice bearing colon-26 (C-26) tumors are commonly used in cancer cachexia studies but few studies have provided comprehensive assessments of physiological and metabolic impairment, especially those factors that impact quality of life. Our aim was to characterize functional impairments in mildly and severely affected cachectic mice, and determine the suitability of these mice as a preclinical model. Metabolic abnormalities are also evident in cachectic patients and we investigated whether C-26-tumor-bearing mice had similar metabolic aberrations. Twelve-week-old CD2F1 mice received a subcutaneous injection of PBS (control) or C-26 tumor cells. After 18-20 days, assessments were made of grip strength, rotarod performance, locomotor activity, whole body metabolism, and contractile properties of tibialis anterior (TA) muscles (in situ) and diaphragm muscle strips (in vitro). Injection of C-26 cells reduced body and muscle mass, and epididymal fat mass. C-26-tumor-bearing mice exhibited lower grip strength and rotarod performance. Locomotor activity was impaired following C-26 injection, with reductions in movement distance, duration and speed compared with controls. TA muscles from C-26-tumor-bearing mice had lower maximum force (-27%) and were more susceptible to fatigue. Maximum specific (normalized) force of diaphragm muscle strips was reduced (-10%) with C-26 injection, and force during fatiguing stimulation was also lower. C-26-tumor-bearing mice had reduced carbohydrate oxidation and increased fat oxidation compared with controls. The range and consistency of functional and metabolic impairments in C-26-tumor-bearing mice confirm their suitability as a preclinical model for cancer cachexia. We recommend the use of these comprehensive functional assessments to maximize the translation of findings to more accurately identify effective treatments for cancer cachexia.
Complex I deficiencies are the most common causes of mitochondrial disorders. They can result from mutations not only in the structural subunits but also in a growing number of assembly factors. A branch-site mutation in the human gene encoding assembly factor NUBPL has recently been associated with mitochondrial encephalopathy and complex I deficiency in seven independent cases. Moreover, the mutation is present in 1.2% of European haplotypes. To investigate its pathogenicity, we have reconstructed the altered C-terminus resulting from the branch-site mutation and frameshift in the homologous Ind1 protein in the respiratory yeast Yarrowia lipolytica. We demonstrate that the altered sequence did not affect IND1 mRNA stability, yet it led to a decrease in Ind1 protein level. The instability of mutant Ind1 resulted in a strong decrease in complex I activity and slow growth resembling the phenotype of the IND1 deletion strain. The presented data confirms the deleterious impact of the altered C-terminus resulting from the branch-site mutation. Furthermore, our approach demonstrates the great potential of Y. lipolytica as a model to investigate complex I deficiencies, especially in cases with genetic complexity.
Testing conduction velocity and amplitude. (A) Schematic representation of the setup for nerve excitability testing of the saphenous nerve. (B) Stimulus paradigm for recording the recovery cycle. A single supramaximal conditioning stimulus or a train of seven conditioning stimuli is followed by the test stimulus at a variable latency of 0.7 to 200 milliseconds. (C) In animals carrying the Gars C201R mutation, the magnitude of the superexcitability is lower and later in the recovery cycle, and the relative refractory period is longer compared with in wildtype controls. Using a single conditioning stimulus, superexcitability is defined as the decrease of the threshold below the line of neutrality (0 on the ordinate). The magnitude of the maximal threshold reduction is marked for wild-type (red arrow) and Gars C201R/+ (blue arrow) by vertical arrows. The relative refractory period is defined as the time between zero and the point at which the excitability threshold crosses the line of neutrality. The horizontal arrows show this time period: wild-type (red arrow) and Gars C201R/+ (blue arrow). (D) The magnitude (vertical arrows) and peak time (horizontal arrows) of the late subexcitability is not significantly different. 
Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, Gars(C201R), with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The Gars(C201R) mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.
Respiratory distress syndrome (RDS) caused by preterm delivery is a major clinical problem with limited mechanistic insight. Late stage embryonic lung development is driven by hypoxia and hypoxia inducible transcription factors Hif-1α and Hif-2α, which act as important regulators for lung development. Expression of BTB-kelch protein KLEIP (Kelch-like ECT2 interacting protein; also named Klhl20) is controlled by two hypoxia response elements and KLEIP regulates stabilization and transcriptional activation of Hif-2α. Based on the data, we hypothesised an essential role for KLEIP in murine lung development and function. Therefore, we have performed a functional, histological, mechanistic and interventional study in embryonic and neonatal KLEIP(-/-) mice. Here we show that half of the KLEIP(-/-) neonates die due to respiratory failure that is caused by insufficient aeration, septal thickness, reduced glycogenolysis, type II pneumocyte immaturity and reduced surfactant production. Expression analyses in E18.5 lungs identified KLEIP in lung capillaries and strongly reduced mRNA and protein levels for Hif-2α and VEGF, which is associated with embryonic endothelial cell apoptosis and lung bleedings. Betamethasone injection in pregnant females prevented respiratory failure in KLEIP(-/-) neonates, normalized lung maturation, aeration and function and increased neonatal Hif-2α expression. Thus, the experimental study shows that respiratory failure in KLEIP(-/-) neonates is determined by insufficient angiocrine Hif-2α/VEGF signaling and that betamethasone activates this new identified signaling cascade in late stage embryonic lung development.
Steps to aGvHD in mice. The progression of events occurring in the development of aGvHD in the mouse is illustrated. The five crucial steps of immune priming (A), activation (B), T-cell expansion (C), T-cell trafficking (D) and host tissue injury (E) are outlined. DC, dendritic cell; XRT, radiation conditioning. See main text for full details.
Steps to cGvHD in mice. The progression of events occurring in the development of cGvHD in the mouse is illustrated. The five steps illustrate immune priming (A), activation of T and B cells (B), T-cell expansion and B-cell autoantibody production (C), trafficking to sites of tissue damage (D), and end organ damage through chronic inflammation and fibrosis (E). See main text for full details.
Mouse models of cGVHD
The limiting factor for successful hematopoietic stem cell transplantation (HSCT) is graft-versus-host disease (GvHD), a post-transplant disorder that results from immune-mediated attack of recipient tissue by donor T cells contained in the transplant. Mouse models of GvHD have provided important insights into the pathophysiology of this disease, which have helped to improve the success rate of HSCT in humans. The kinetics with which GvHD develops distinguishes acute from chronic GvHD, and it is clear from studies of mouse models of GvHD (and studies of human HSCT) that the pathophysiology of these two forms is also distinct. Mouse models also further the basic understanding of the immunological responses involved in GvHD pathology, such as antigen recognition and presentation, the involvement of the thymus and immune reconstitution after transplantation. In this Perspective, we provide an overview of currently available mouse models of acute and chronic GvHD, highlighting their benefits and limitations, and discuss research and clinical opportunities for the future.
Ischemia/reperfusion injury and tissue hypoxia are of high clinical relevance, as they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms of the ischemia/reperfusion induced cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. Aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions (pO2<10mmHg) in the culture medium. Hydrogen peroxide assays, glucose measurements and westernblotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium (pO2 0 minutes: 107.57±0.99mmHg, 70 minutes: 9.00±0.58mmHg, 120 minutes: 5.00±0.00mmHg, 170 minutes: 2.00±0.00mmHg, 360 minutes: 2.00±0.00mmHg). Glucose concentrations gradually decreased ([Glc] 0 minutes: 4.50±0.02g/l, 360 minutes: 1.22±0.07g/l) while levels of hydrogen peroxide were not altered ([H2O2] 0 minutes: 9.57±0.00µM, 360 minutes: 7.96±0.67µM). Moreover, a rapid and reversible (on/off) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of LDH as a biochemical marker of cell damage (hypoxia: 0.50±0.08a.u., normoxia: 0.20±0.05a.u.; P<0.05). Hypoxic conditions also increased the amounts of cellular procaspase-3 (hypoxia: 1.45±0.19a.u., normoxia: 0.98±0.01a.u.; P<0.05) and catalase (hypoxia: 1.71±0.55a.u., normoxia: 0.61±0.09a.u.; P<0.05) as well as phosphorylation of the prosurvival kinase Akt (hypoxia: 0.65±0.14a.u., normoxia: 0.05±0.01a.u.; P<0.05), but not Erk1/2 or STAT5. In summary, we present a novel framework in investigating hypoxia mediated mechanisms on cellular level. We claim that the model, the first of its kind, enables researches to rapidly and reversibly induce hypoxic conditions in-vitro without interference of the hypoxia inducing agent with the cultured cells. The system may help to further unravel hypoxia associated mechanisms which are clinically relevant in various tissues and organs.
The epidemics of obesity and diabetes have aroused great interest in the analysis of energy balance, with the use of organisms ranging from nematode worms to humans. Although generating energy-intake or -expenditure data is relatively straightforward, the most appropriate way to analyse the data has been an issue of contention for many decades. In the last few years, a consensus has been reached regarding the best methods for analysing such data. To facilitate using these best-practice methods, we present here an algorithm that provides a step-by-step guide for analysing energy-intake or -expenditure data. The algorithm can be used to analyse data from either humans or experimental animals, such as small mammals or invertebrates. It can be used in combination with any commercial statistics package; however, to assist with analysis, we have included detailed instructions for performing each step for three popular statistics packages (SPSS, MINITAB and R). We also provide interpretations of the results obtained at each step. We hope that this algorithm will assist in the statistically appropriate analysis of such data, a field in which there has been much confusion and some controversy.
Core body temperature and activity in 4-and 6-month-old 3xTgAD mice. (A-L)Core body temperature (°C) and activity (counts/minute) were monitored continuously by remote radiotelemetry over a 7-day period in separate groups of individually housed 3xTgAD and Non-Tg mice at 4 (temperature, AC; activity, G-I) and 6 (temperature, D-F; activity J-L) months of age. The mean 24-hour profiles over the 7 days (expressed as change, ) are illustrated in A and D for temperature and G and J for activity. White bars on the abscissa represent the light, inactive phase of the day; black bars represent the dark, active phase. The peak-trough amplitude in temperature rhythms was increased in 6-month-old 3xTgAD mice (E) but not in 4-month-old mice (B). A phase advance in the temperature rhythms was observed in both 4-(C) and 6-(F) month-old 3xTgAD mice. No change in the peak-trough amplitude or phase onset for activity rhythms was observed at 4 (H,I) and 6 (K,L) months of age. Data are expressed as mean ± s.d. and individual values for each animal are represented; n4-6 per group. **P<0.01, ***P<0.001 versus age-matched Non-Tg control mice using Student's t-test.
Effect of ibuprofen on mean daily body temperature in 3xTgAD mice
Ibuprofen does not affect the changes in core body temperature in 3xTgAD mice. (A,B)Core body temperature was monitored continuously by remote radiotelemetry in individually housed 3xTgAD and Non-Tg mice at 10.5 months of age. Mice were maintained on either a control diet or a diet containing ibuprofen (375 ppm) for 12 weeks. The mean daily peak-trough amplitude (°C; A) and phase (in ZT; B) of temperature rhythms over a 7-day period were calculated after 1, 4, 8 and 12 weeks of treatment. Data are mean ± s.d. for n3-8 per group. *P<0.05, **P<0.01, ***P<0.001 versus respective Non-Tg mice.
A plaques and neurofibrillary tangles are not present in the hypothalamus of 3xTgAD mice. Immunohistochemistry to detect A plaques (top row) and neurofibrillary tangles (bottom row; hyperphosphorylated tau) was performed using 6E10 and AT8 antibodies, respectively, on brain sections from 12-month-old 3xTgAD mice. No A plaques or neurofibrillary tangles were detected in the hypothalamus of 3xTgAD mice. A plaques and neurofibrillary tangles were present in the hippocampus of 12-month-old 3xTgAD mice. 3V, third ventricle of the hypothalamus. Scale bar: 100m.
Alzheimer's disease (AD) is characterised, not only by cognitive deficits and neuropathological changes, but also by several non-cognitive behavioural symptoms that can lead to a poorer quality of life. Circadian disturbances in core body temperature and physical activity are reported in AD patients, although the cause and consequences of these changes are unknown. We therefore characterised circadian patterns of body temperature and activity in male triple transgenic AD mice (3xTgAD) and non-transgenic (Non-Tg) control mice by remote radiotelemetry. At 4 months of age daily temperature rhythms were phase advanced and by 6 months of age an increase in mean core body temperature and amplitude of temperature rhythms were observed in 3xTgAD mice. No differences in daily activity rhythms were seen in 4-9-month-old 3xTgAD mice, but by 10 months of age an increase in mean daily activity and the amplitude of activity profiles for 3xTgAD mice were detected. At all ages (4-10 months), 3xTgAD mice exhibited greater food intake compared to Non-Tg mice. The changes in temperature did not appear to be solely due to increased food intake and were not cyclooxygenase dependent, since the temperature rise was not abolished by chronic ibuprofen treatment. No beta amyloid (Aβ) plaques or neurofibrillary tangles were noted in the hypothalamus of 3xTgAD mice, a key area involved in temperature regulation, although these pathological features were observed in the hippocampus and amygdala of 3xTgAD mice from 10 months of age. These data demonstrate age-dependent changes in core body temperature and activity in 3xTgAD mice that are present before significant AD-related neuropathology and are analogous to those observed in AD patients. The 3xTgAD mouse might therefore be an appropriate model to study the underlying mechanisms involved in non-cognitive behavioural changes in AD.
Primary growth assay screen. Yeast expressing Ab-MRF were inoculated into-Ade or +Ade media in microplate wells with 12,800 compounds at 22mM. Negative [Ab-MRF (Ab)] and positive [Abm2-MRF (m2)] controls, both lacking compounds, showed a clear easily scored difference in growth in-Ade. All Ab control wells had diffuse growth throughout the well; all m2 control wells showed absence of growth (blackness) in some position of each well, although small colony 'spots' , indicative of genetic revertants, were seen. Candidates in wells marked with a circle look like the m2 growthinhibited controls in-Ade, but do not inhibit growth in +Ade, and were thus considered to be hits. Although compounds from wells marked with arrows were able to inhibit growth in-Ade, they were eliminated because they were toxic even in +Ade. *Uninoculated (blank) wells. 
Recent reports point to small soluble oligomers, rather than insoluble fibrils, of amyloid β (Aβ), as the primary toxic species in Alzheimer's disease. Previously, we developed a low-throughput assay in yeast that is capable of detecting small Aβ(42) oligomer formation. Specifically, Aβ(42) fused to the functional release factor domain of yeast translational termination factor, Sup35p, formed sodium dodecyl sulfate (SDS)-stable low-n oligomers in living yeast, which impaired release factor activity. As a result, the assay for oligomer formation uses yeast growth to indicate restored release factor activity and presumably reduced oligomer formation. We now describe our translation of this assay into a high-throughput screen (HTS) for anti-oligomeric compounds. By doing so, we also identified two presumptive anti-oligomeric compounds from a sub-library of 12,800 drug-like small molecules. Subsequent biochemical analysis confirmed their anti-oligomeric activity, suggesting that this form of HTS is an efficient, sensitive and cost-effective approach to identify new inhibitors of Aβ(42) oligomerization.
Cohort demographics and PROG levels 
Effect of PROG treatment on survival time and locomotor control of TDP43 A315T mutant mice. (A)Levels of PROG in brain tissue of treated and untreated wild-type mice. (B)Survival time from day 0, with PROG pellet implantation at approximately week 6, to week 25 (at 100% mortality of entire cohort). Untreated TDP43 A315T mutant mice (green line, n=14 animals per group) were all deceased at week 17 compared with week 21 for treated mutant mice (pink line, n=13 animals per group; P>0.05). (C)Overall rate of change in locomotor control with increasing age was modelled as quadratic regression curves. The slope of the curve, which measures the decrease in time spent on the rotarod as the mice aged, was significantly decreased by PROG treatment of TDP43 A315T mutant mice (pink triangles) compared with untreated mutant (green triangles) mice. Significances of P<0.05 (*) and P<0.001 (**) are indicated. 
Frontotemporal dementia (FTD) is associated with motor neurone disease (FTD-MND), corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP). Together, this group of disorders constitutes a major cause of young-onset dementia. One of the three clinical variants of FTD is progressive nonfluent aphasia (PNFA), which is focused on in this study. The steroid hormone progesterone (PROG) is known to have an important role as a neurosteroid with potent neuroprotective and promyelination properties. In a case-control study of serum samples (39 FTD, 91 controls), low serum PROG was associated with FTD overall. In subgroup analysis, low PROG levels were significantly associated with FTD-MND and CBS, but not with PSP or PNFA. PROG levels of >195 pg/ml were significantly correlated with lower disease severity (frontotemporal dementia rating scale) for individuals with CBS. In the human neuroblastoma SK-N-MC cell line, exogenous PROG (9300-93,000 pg/ml) had a significant effect on overall Tau and nuclear TDP-43 levels, reducing total Tau levels by ~1.5-fold and increasing nuclear TDP-43 by 1.7- to 2.0-fold. Finally, elevation of plasma PROG to a mean concentration of 5870 pg/ml in an Ala315Thr (A315T) TARDBP transgenic mouse model significantly reduced the rate of loss of locomotor control in PROG-treated, compared with placebo, mice. The PROG treatment did not significantly increase survival of the mice, which might be due to the limitation of the transgenic mouse to accurately model TDP-43-mediated neurodegeneration. Together, our clinical, cellular and animal data provide strong evidence that PROG could be a valid therapy for specific related disorders of FTD.
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by complex neuronal and glial phenotypes. Recently, RNA-based mechanisms have been linked to ALS via RNA-binding proteins such as TDP-43, which has been studied in vivo using models ranging from yeast to rodents. We have developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of pathology, including motor neuron loss, locomotor dysfunction and reduced survival. Here we report the phenotypic consequences of expressing wild-type and four different ALS-linked TDP-43 mutations in neurons and glia. We show that TDP-43-driven neurodegeneration phenotypes are dose- and age-dependent. In motor neurons, TDP-43 appears restricted to nuclei, which are significantly misshapen due to mutant but not wild-type protein expression. In glia and in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta. TDP-43-containing RNA granules are motile in cultured motor neurons, although wild-type and mutant variants exhibit different kinetic properties. At the neuromuscular junction, the expression of TDP-43 in motor neurons versus glia leads to seemingly opposite synaptic phenotypes that, surprisingly, translate into comparable locomotor defects. Finally, we explore sleep as a behavioral readout of TDP-43 expression and find evidence of sleep fragmentation consistent with hyperexcitability, a suggested mechanism in ALS. These findings support the notion that although motor neurons and glia are both involved in ALS pathology, at the cellular level they can exhibit different responses to TDP-43. In addition, our data suggest that individual TDP-43 alleles utilize distinct molecular mechanisms, which will be important for developing therapeutic strategies.
A significant decline in human male reproductive function has been reported for the past 20 years but the molecular mechanisms remain poorly understood. However, recent studies showed that the gap junction protein connexin-43 (CX43; also known as GJA1) might be involved. CX43 is the predominant testicular connexin (CX) in most species, including in humans. Alterations of its expression are associated with different forms of spermatogenic disorders and infertility. Men with impaired spermatogenesis often exhibit a reduction or loss of CX43 expression in germ cells (GCs) and Sertoli cells (SCs). Adult male transgenic mice with a conditional knockout (KO) of the Gja1 gene [referred to here as connexin-43 (Cx43)] in SCs (SCCx43KO) show a comparable testicular phenotype to humans and are infertile. To detect possible signaling pathways and molecular mechanisms leading to the testicular phenotype in adult SCCx43KO mice and to their failure to initiate spermatogenesis, the testicular gene expression of 8-day-old SCCx43KO and wild-type (WT) mice was compared. Microarray analysis revealed that 658 genes were significantly regulated in testes of SCCx43KO mice. Of these genes, 135 were upregulated, whereas 523 genes were downregulated. For selected genes the results of the microarray analysis were confirmed using quantitative real-time PCR and immunostaining. The majority of the downregulated genes are GC-specific and are essential for mitotic and meiotic progression of spermatogenesis, including Stra8, Dazl and members of the DM (dsx and map-3) gene family. Other altered genes can be associated with transcription, metabolism, cell migration and cytoskeleton organization. Our data show that deletion of Cx43 in SCs leads to multiple alterations of gene expression in prepubertal mice and primarily affects GCs. The candidate genes could represent helpful markers for investigators exploring human testicular biopsies from patients showing corresponding spermatogenic deficiencies and for studying the molecular mechanisms of human male sterility.
Since its first splash 30 years ago, the use of the zebrafish model has been extended from a tool for genetic dissection of early vertebrate development to the functional interrogation of organogenesis and disease processes such as infection and cancer. In particular, there is recent and growing attention in the scientific community directed at the immune systems of zebrafish. This development is based on the ability to image cell movements and organogenesis in an entire vertebrate organism, complemented by increasing recognition that zebrafish and vertebrate immunity have many aspects in common. Here, we review zebrafish immunity with a particular focus on recent studies that exploit the unique genetic and in vivo imaging advantages available for this organism. These unique advantages are driving forward our study of vertebrate immunity in general, with important consequences for the understanding of mammalian immune function and its role in disease pathogenesis.
The small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed 'pathological airway remodeling', affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6-30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease.
Conservation of the AcP10 enhancer in terrestrial vertebrates and geographical distribution of the rs6983267 SNP. (A)We used software available on the University of California Santa Cruz (UCSC) genome browser to assess the degree of DNA sequence conservation in the region of the AcP10 enhancer. Shown is the Placental Mammal Basewise Conservation assessed by PhyloP (blue vertical lines), and vertebrate Multiz alignment of sequences from rhesus monkey, mouse, dog, elephant, opossum, platypus, chicken, lizard, Xenopus tropicalis and stickelback (black vertical lines). The 1.5 kb fragment containing the AcP10 enhancer is indicated by the horizontal line above. The nucleotide sequence of the region surrounding the rs6983267 functional SNP is shown below. Note that all species shown have the G allele. (B)Global distribution of the high-risk G allele and lower risk T allele of rs6983267 as assessed by the UCSC genome browser. Note the prevalence of the G allele in Africa, suggesting that it might have originated there.  
AcP10 directs reporter gene expression to cells of the prostate, mammary gland, colon and skin. We produced transgenic mouse lines carrying the AcP10-hsp68-lacZ construct. We examined three such lines for transgene expression in mouse tissues (A-H). Tissues were sectioned and stained for -gal activity (blue) and counterstained with nuclear fast red. (A)Sections of prostate from a control, nontransgenic mouse. (B)Sections of prostate from lines 1, 2 and 3. Note -gal-positive cells in lumenal epithelium in each line. Line 2 was further examined for  galactosidase expression in the colon (C), mammary gland (D), skin (E), liver (F), lung (G) and spleen (H). Note expression in colon, mammary gland and skin. AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate. Arrows indicate lacZexpressing cells.  
Expression of AcP10-hsp68-lacZ transgenic line 2 in different regions of the prostate. Positive cells were present in the anterior prostate (AP; A,D,G), the dorsolateral prostate (DLP; B,E,H) and the ventral prostate (VP; C,F,I). Higher magnification images show that virtually all positive cells were located in the lumenal epithelium (G-I).  
AcP10 is active in prostate lumenal epithelial cells. Immunostaining with the basal cell marker CK5 (A,E; green fluorescence) and the lumenal cell marker CK8 (B,F; green fluorescence) showed that -gal (C-F; red fluorescence) was found exclusively in lumenal cells (compare overlapped images in E and F).  
AcP10 is active in prostate tumors. We produced mice with the genotype AcP10-lacZ; Pten cko/cko ; probasin-Cre. We examined the prostates of these mice after 5 months when virtually 100% had prostate tumors. We carried out immunostaining for the basal cell marker CK5 (A), the lumenal cell marker CK8 (B) and -gal (C,D). Overlapped images are shown in E and F. Note clusters of -gal-positive cells in areas of the prostate that have a tumor phenotype (C,D). Note also that the -gal signal is found exclusively in cells positive for the lumenal cell marker CK8 (F).  
Regions in the 8q24 gene desert contribute significantly to the risk of prostate cancer and other adult cancers. This region contains several DNA regions with enhancer activity in cultured cells. One such segment, histone acetylation peak 10 (AcP10), contains a risk single nucleotide polymorphism (SNP) that is significantly associated with the pathogenesis of colorectal, prostate and other cancers. The mechanism by which AcP10 influences cancer risk remains unknown. Here we show that AcP10 contains a sequence that is highly conserved across terrestrial vertebrates and is capable in transgenic mice of directing reporter gene expression to a subset of prostate lumenal epithelial cells. These cells include a small population of Nkx3.1-positive cells that persist even after androgen ablation. Castration-resistant Nkx3.1-positive (CARN) cells were shown by others to function both as stem cells and cells of origin of prostate cancer. Our results thus provide a mechanism by which AcP10 could influence prostate cancer risk.
Endocochlear potential at P19-P21 
Myo7a is expressed in intermediate cells of stria vascularis in Atp6v0a4 mutant mice. (A-F)Expression of Myo7a in sections of Atp6v0a4 wild-type and homozygous mutant mice at P0, as shown by immunohistochemistry. (A,D)Mid-modiolar sections of the cochlea. (B,E)Higher magnification of the boxed regions in A and D, respectively. (C,F)Higher magnifications of the stria vascularis from B and E, respectively. In both wild types and mutants, Myo7a is strongly expressed in the hair cells of the organ of Corti (B,E) and in the melanocyte-like cells within the stria vascularis (B,E; arrowheads in C,F). HC, hair cells; SV, stria vascularis. Scale bars: 200 μm (A,D); 400 μm (B,E); 20 μm (C,F). 
Kcnj10 and Kcnq1 are expressed in the stria vascularis of Atp6v0a4 mutant mice. (A,C)Mid-modiolar sections of the inner ear of control (A) and mutant (C) mice at P20. Regions in boxes in A and C are magnified in B and D, respectively. In wild types and mutants, a similar Kcnj10 expression pattern is observed. Especially strong expression is found in the intermediate cells of the stria vascularis (B,D, black arrowheads), whereas the marginal cells are negative (B,D, red arrowheads). It is also noticeable that mutants examined (n5) did not seem to show Reissner's membrane so much distended towards the scala vestibuli as at P0 and P5. We think this might be due to the decalcification process used to facilitate the sectioning of these samples, or to the resolution of any pressure difference across Reissner's membrane by rupture. (E,F)Expression of Kcnq1 is specifically found in the marginal cells of the stria vascularis at P20 in both wild types and mutants (E,F, red arrowheads). RM, Reissner's membrane; SV, stria vascularis. Scale bars: 100 μm (A,C); 20 μm (B,D-F). 
Expression of Atp6v0a4 in inner ear. (A-F)Sections of controls (A-C) and mutants (D-F) at P0 (A,B,D,E) and P20 (C,F) stained by immunohistochemistry. In wild-type mice at both ages, the expression of a4 subunit was specifically found in a subpopulation of cells within the ES epithelium (B,C, arrowheads). No expression was observed anywhere else within the inner ear (A). In Atp6v0a4 knockouts, no expression of this protein was detected (E,F). CO, cochlea. Scale bars: 300 μm (A,D); (B,E) 200 μm; 50 μm (C,F). 
Foxi1 and Pds are expressed in the ES of Atp6v0a4 mutants. (A-D)Expression of Foxi1 in the endolymphatic duct and sac of wild-type and mutant mice at P0. (B,D)Higher magnification of the boxed regions in A and C, respectively. In control mice, Foxi1 is strongly expressed in a subpopulation of cells within the infolded epithelium of the ES (B, arrowheads). A similar pattern is observed in the flattened epithelium of the ES in mutants (D, arrowheads). (E-H)Expression of Pds in the ES of wild-type and mutant mice at P0. (F,H)Higher magnification of the boxed regions in E and G, respectively. In wild types, Pds expression is found in a few scattered cells within the ES (F, arrowheads). A similar pattern is found in the flattened ES seen in mutants (H, arrowheads). ED, endolymphatic duct. Scale bars: 100 μm (A,C,E,G); 20 μm (B,D,F,H). 
Mutations in the ATP6V0A4 gene lead to autosomal recessive distal renal tubular acidosis in patients who often show sensorineural hearing impairment. A first Atp6v0a4 knockout mouse model that recapitulates the loss of H(+)-ATPase function seen in humans has been generated and recently published (Norgett et al., 2012). Here we present the first detailed analysis of the structure and function of the auditory system in Atp6v0a4(-/-) knockout mice. Measurements of the auditory brainstem response (ABR) showed significantly elevated thresholds in homozygous mutant mice, which indicate severe hearing impairment. Heterozygote thresholds were normal. Analysis of paint-filled inner ears and sections from E16.5 embryos revealed a marked expansion of cochlear and endolymphatic ducts in Atp6v0a4(-/-) mice. A regulatory link between Atp6v0a4, Foxi1 and Pds has been reported and we found that the endolymphatic sac of Atp6v0a4(-/-) mice expresses both Foxi1 and Pds, which suggests a downstream position of Atp6v0a4. These mutants also showed a lack of endocochlear potential, suggesting a functional defect of the stria vascularis on the lateral wall of the cochlear duct. However, the main K(+) channels involved in the generation of endocochlear potential, Kcnj10 and Kcnq1, are strongly expressed in Atp6v0a4(-/-) mice. Our results lead to a better understanding of the role of this proton pump in hearing function.
Generation of NET A457P knock-in mice. (A)Schematic of the wild-type (WT) SLC6A2 (NET) locus, targeting vector and NET locus following recombination. 'WT NET' shows the location of the targeting arms, and BglII sites and 3 probe used in Southern confirmation. White arrows depict the PCR primers used to genotype A457P mice. 'A457P Construct' depicts the position of the floxed Neo/CRE cassette and the PGK cassette. Neo is under the control of the RNA polymerase II promoter and CRE is driven by the testis-specific angiotensin converting enzyme promoter, producing excision of the floxed region upon germline targeting. A457P in exon 9 in the 5 arm is depicted. 'Targeted A457P Allele' illustrates the construct targeted to the NET locus. The BglII sites and 3 probe location illustrate the shift in size of the BglII fragment, produced by insertion of the construct, to 11.6 kb (from 7.8 kb in WT NET). Arrows indicate PCR primers used to confirm correct insertion at both the 5 (white arrows) and 3 (black arrows) end of NET. The gray arrow shows a forward primer used with the 5 arm reverse primer (white arrow) to genotype the A457P mutation through PCR and dideoxy sequencing. 'Targeted A457P Allele Floxed Region Excised' shows the final status of the NET allele, with the Neo/CRE cassette excised and one loxP site remaining in intron 11. White arrows depict the PCR primers used to genotype A457P mice, as shown above in WT NET. (B) Southern analysis of BglII-digested DNA from NET +/+ and NET +/P mice. (C) PCR genotype analysis of NET +/+ , NET +/P and NET P/P mouse DNA to detect the residual loxP site.  
[ 3 H]NE transport and expression in A457P knock-in mice. Data are expressed as mean percent ± s.e.m. of NET +/+ mice (A-C,E,F) or as raw values ± s.e.m. (D). *P<0.05, **P<0.01 and ***P<0.001 compared with NET +/+ mice (twotailed t-test). (A)[ 3 H]NE transport in the SNS, n=5-6. Vent., ventricle; VD, vas deferens. (B,C)Single point [ 3 H]NE transport in brain, n=6-7. (D)Saturation kinetics of [ 3 H]NE transport in brain, n=8. Cortex NET +/+ (black squares), V MAX =6.6±0.40 moles×10 −16 /μg/minute, K M =87.8±20.9 nM; cortex NET +/P (white squares), V MAX =4.6±0.50 moles×10 −16 /μg/minute, K M =96.4±38.1 nM; hippocampus NET +/+ (black triangles), V MAX =8.2±0.77 moles×10 −16 /μg/minute, K M =118.3±40.6 nM; hippocampus NET +/P (white triangles), V MAX =5.5±0.54 moles×10 −16 /μg/minute, K M =136.8±46.3 nM. (E,F)NET expression in total and surface fractions of cortex (CTX) and hippocampus (HPC) synaptosomes in NET +/P (n=7; E) and NET P/P (n=7; F) mice compared with NET +/+ mice.  
Radiotelemetry in NET +/+ versus NET +/P mice. (A)3-day collection of HR measures; data were analyzed and graphed in 30-minute increments. Traces shown start at lights off in the colony room. (B)HR during 3 days of data collection, expressed as means ± s.e.m. averaged over the 24-hour period and during 12-hour day and night periods. Significance was determined by twoway ANOVA. Genotype: F(1,27)=15.59, P<0.001. Time of day: F(2,27)=18.26, P<0.0001. *P<0.05, post-hoc t-test, n=5. (C)Mean arterial BP measured during the same 3-day period as in A and B (n=5).  
Baroreflex sensitivity in NET +/+ versus NET +/P mice. (A,B)Dose response of systolic BP (SBP) to PHE (A) and NTP (B). Two-way ANOVA revealed effects of dose for both PHE and NTP [F(4,50)=61.78, P<0.01, n=3-6 for NTP and F(5,60)=23.53, P<0.01, n=3-6 for PHE]. (C,D)Baroreceptor sensitivities [the ratio of the change in HR to the changes in SBP at 20 μg/kg PHE (C) and 30 μg/kg NTP (D)] were calculated. BRS, baroreflex sensitivity; PHE, phenylephrine; NTP, nitroprusside.  
Catecholamines and metabolites in A457P knock-in mice. Plasma and urinary NE and DHPG (A,C), and DA and EPI (B,D) in male (A,B) and female (C,D) A457P mice. Data are expressed as the mean percent ± s.e.m. compared with NET +/+ mice. Significance was determined by two-way ANOVA. Male plasma genotype, F(2,135)=50.77, P<0.001; male urine genotype, F(2,215)=10.82; P<0.001; female plasma genotype, F(2,110)=20.21, P<0.001; female urine interaction, F(8,85)=2.88, P<0.01. *P<0.05, **P<0.01 and ***P<0.001 compared with NET +/+ . n=6-12 (male plasma), 9-20 (male urine), 8-9 (female plasma) and 6-7 (male urine).  
Postural orthostatic tachycardia syndrome (POTS) is a common autonomic disorder of largely unknown etiology that presents with sustained tachycardia on standing, syncope, and elevated norepinephrine spillover. Some POTS patients experience anxiety, depression and cognitive dysfunction. Previously, we identified a mutation, A457P, in the norepinephrine (NE) transporter (NET, SLC6A2) in POTS patients. NET is expressed at presynaptic sites in NE neurons and plays a critical role in regulating NE signaling and homeostasis through NE reuptake into noradrenergic nerve terminals. Our in vitro studies demonstrate that A457P reduces both NET surface trafficking and NE transport and exerts a dominant-negative impact on wild-type NET proteins. Here we report the generation and characterization of NET A457P mice, demonstrating the ability of A457P to drive the POTS phenotype and behaviors consistent with reported comorbidities. Mice carrying one A457P allele (NET(+/P)) exhibited reduced brain and sympathetic NE transport levels compared to wild-type (NET(+/+)) mice, whereas transport activity in mice carrying two A457P alleles (NET(P/P)) was nearly abolished. NET(+/P) and NET(P/P) mice exhibited elevations in plasma and urine NE levels, reduced DHPG, and reduced DHPG/NE ratios, consistent with a decrease in sympathetic nerve terminal NE reuptake. Radiotelemetry in unanesthetized mice revealed tachycardia in NET(+/P) mice without a change in blood pressure or baroreceptor sensitivity, consistent with studies of human NET A457P carriers. NET(+/P) mice also demonstrated behavioral changes consistent with CNS NET dysfunction. Our findings support that NET dysfunction is sufficient to produce a POTS phenotype and introduces the first genetic model suitable for more detailed mechanistic studies of the disorder and its comorbidities.
The F508 mutation post-transcriptionally destabilizes the abundance and apical membrane localization of C. elegans PGP-3. (A)Schematic of the Mos excision strategy used to create the single-copy PGP-3 wt and PGP-3 F508 transgenes. (B)Quantitative PCR of the pgp-3–mCherry transgenic mRNA. As shown in A, the primers only amplify the transgenic pgp-3 and not the native pgp-3. n4 samples, P>0.05. (C)In vivo localization of the PGP-3 wt ::mCherry and PGP-3 F508 ::mCherry protein in intestinal epithelial cells. Arrows indicate the apical membrane of the intestine. Dotted lines denote the basolateral membranes of the intestine. Imaging settings for both lines are identical. Inset image has been adjusted for intensity and contrast to show the presence of a weak signal in the PGP-3 F508 animals. Scale bars: 10m. (D)Quantification of total and apical intestinal mCherry fluorescence from PGP-3 wt ::mCherry or PGP-3 F508 ::mCherry single-copy transgenes. ***P<0.001. n12 (PGP-3 wt ) or 13 (PGP- 3 F508 ). (E)Western blot against transgenic PGP-3 (mCherry and FLAG antibodies), a positive control membrane protein (VHA-5) and a positive control cytosolic protein (HSP-90). Membrane and cytosolic fractions were prepared from single-copy transgenic animals as described in the Methods. '-Tg' indicates animals that lack the single copy transgene.  
C. elegans ERAD homologs and effect of their gene knockdown on PGP-3 F508 expression
Physiological activation of the osmotic stress response posttranscriptionally stabilizes PGP-3 F508 and PGP-3 wt. (A)Genetics of the Osr pathway. Arrows indicate positive regulation and blocked lines indicate negative regulation, based on previous research (Solomon et al., 2004; Lamitina et al., 2006; Wheeler and Thomas, 2006; Rohlfing et al., 2010; Rohlfing et al., 2011); loss-of-function mutations in the Osr genes result in constitutive activation of the pathway even under isotonic conditions. (B)Wide-field fluorescence images of PGP-3-mCherry grown on standard NGM plates (control) or high salt plates (200 mM NaCl, 24-hour exposure). Exposure and imaging settings were identical across all conditions. Arrows indicate sites of increased intestine expression in high-salt-treated PGP-3 F508 animals. Scale bars: 10m. (C)Quantification of total and apical membrane fluorescence from animals grown on 50 mM NaCl or 200 mM NaCl growth plates. Data for both PGP-3 wt and PGP-3 F508 are normalized relative to the isotonic (50 mM NaCl) control. **P<0.01, ***P<0.001, one-way ANOVA. (D)Representative western blot of synchronized young adult PGP-3 wt or PGP-3 F508 grown on either control (50 mM NaCl) or +NaCl (200 mM NaCl) NGM media. PGP-3 was detected with an anti-mCherry antibody. The numbers beneath each lane represent the normalized PGP-3:actin ratio for the experiment shown. One of four representative experiments is shown.
Genetic activation of the Osr pathway stabilizes PGP-3 F508 and PGP-3 wt . (A)DIC and wide-field fluorescence images of PGP-3 F508 in the indicated Osr mutant backgrounds grown on standard (50 mM NaCl) NGM plates. Arrows point to sites of PGP-3 F508 apical membrane localization. Scale bars: 10m. (B)Quantification of total and apical membrane fluorescence from animals expressing PGP-3 F508 or PGP-3 wt in the indicated genetic background. Data are normalized relative to the wild-type control. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA. (C)Representative western blot of PGP-3 F508 and PGP-3 wt in the indicated genetic background. The numbers beneath each set of images represent the normalized PGP-3:actin ratio for the experiment shown. One of two representative experiments is shown.  
ERAD homologs are required to efficiently destabilize PGP-3 F508 but not PGP-3 wt . (A)PGP-3 wt ::mCherry fluorescence following RNAi inhibition of R151.6 (Derlin 2) or cdc-48.2 (p97). EV, empty vector. Scale bars: 10m. (B)PGP-3 F508 ::mCherry fluorescence following RNAi inhibition of R151.6 or cdc-48.2. Scale bars: 10m. Arrows point to sites of enriched apical mCherry expression. (C)Quantification of apical and total PGP-3 wt (young adults) or PGP-3 F508 (L4 stage) mCherry fluorescence following the indicated RNAi treatments. Data are normalized to the empty vector control and exposure settings within the PGP wt and PGP-3 F508 test groups were identical. Data shown represent the mean ± s.d. of one representative experiment. *P<0.05, **P<0.01, one-way ANOVA. n9-16 animals per condition.  
Membrane proteins make up ~30% of the proteome. During the early stages of maturation, this class of proteins can experience localized misfolding in distinct cellular compartments, such as the cytoplasm, endoplasmic reticulum (ER) lumen and ER membrane. ER quality control (ERQC) mechanisms monitor folding and determine whether a membrane protein is appropriately folded or is misfolded and warrants degradation. ERQC plays crucial roles in human diseases, such as cystic fibrosis, in which deletion of a single amino acid (F508) results in the misfolding and degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We introduced the ΔF508 mutation into Caenorhabditis elegans PGP-3, a 12-transmembrane ABC transporter with 15% identity to CFTR. When expressed in intestinal epithelial cells, PGP-3(wt) was stable and efficiently trafficked to the apical plasma membrane through a COPII-dependent mechanism. However, PGP-3(ΔF508) was post-transcriptionally destabilized, resulting in reduced total and apical membrane protein levels. Genetic or physiological activation of the osmotic stress response pathway, which causes accumulation of the chemical chaperone glycerol, stabilized PGP-3(ΔF508). Efficient degradation of PGP-3(ΔF508) required the function of several C. elegans ER-associated degradation (ERAD) homologs, suggesting that destabilization occurs through an ERAD-type mechanism. Our studies show that the ΔF508 mutation causes post-transcriptional destabilization and degradation of PGP-3 in C. elegans epithelial cells. This model, combined with the power of C. elegans genetics, provides a new opportunity to genetically dissect metazoan ERQC.
Zebrafish (Danio rerio) can serve as a model system to study heritable skin diseases. The skin is rapidly developed during the first 5-6 days of embryonic growth, accompanied by expression of skin-specific genes. Transmission electron microscopy (TEM) of wild-type zebrafish at day 5 reveals a two-cell-layer epidermis separated from the underlying collagenous stroma by a basement membrane with fully developed hemidesmosomes. Scanning electron microscopy (SEM) reveals an ordered surface contour of keratinocytes with discrete microridges. To gain insight into epidermal morphogenesis, we have employed morpholino-mediated knockdown of the abca12 and snap29 genes, which are crucial for secretion of lipids and intracellular trafficking of lamellar granules, respectively. Morpholinos, when placed on exon-intron junctions, were >90% effective in preventing the corresponding gene expression when injected into one- to four-cell-stage embryos. By day 3, TEM of abca12 morphants showed accumulation of lipid-containing electron-dense lamellar granules, whereas snap29 morphants showed the presence of apparently empty vesicles in the epidermis. Evaluation of epidermal morphogenesis by SEM revealed similar perturbations in both cases in the microridge architecture and the development of spicule-like protrusions on the surface of keratinocytes. These morphological findings are akin to epidermal changes in harlequin ichthyosis and CEDNIK syndrome, autosomal recessive keratinization disorders due to mutations in the ABCA12 and SNAP29 genes, respectively. The results indicate that interference of independent pathways involving lipid transport in the epidermis can result in phenotypically similar perturbations in epidermal morphogenesis, and that these fish mutants can serve as a model to study the pathomechanisms of these keratinization disorders.
Mutations associated with ion-channel-trafficking diseases a 
Mechanisms of HSC/HSP70 functional interaction with substrate polypeptide. (A) The HSP70 ATPase cycle. Clockwise, starting from left: in the ATP-bound state, HSP70 does not bind substrate polypeptide. A substrate-binding DNAJ co-chaperone contacts HSP70 to stimulate ATP hydrolysis. In the ADP-bound state, HSP70 binds substrate tightly, and the DNAJ dissociates. An NEF co-chaperone promotes the exchange of ADP for ATP and dissociates, returning HSP70 to the ATP-bound state. (B) Chaperone-assisted folding and ER-associated degradation (ERAD) of CFTR. CFTR follows chaperone-mediated pathways for degradation (top) as well as folding (bottom). In the folding pathway, starting from the left, DNAJA1 (red) activates the binding of HSC/HSP70 (yellow) to CFTR to initiate folding; co-chaperone HOP (light green) transfers CFTR from HSC/HSP70 to HSP90 (purple) and its co-chaperones (dark green) to complete folding and allow trafficking to the cell surface. In the degradation pathway, the HSC/HSP70 co-chaperone CHIP (orange) is an E3 ubiquitin ligase that promotes degradation of misfolded CFTR. CHIP functions in parallel to membrane-anchored E3 ubiquitin ligases gp78 and RMA1 and associated components (brown), which do not depend on HSC/HSP70. The DNAJ co-chaperones (red) CSPα and DNAJB12 promote CFTR degradation by CHIP, and by gp78 and RMA1, respectively. The NEFs (blue) BAG2, BAG1 and HSPBP1 interfere with CHIP activity, BAG1 by causing HSC/HSP70 to release substrate, BAG2 and HSPBP1 by binding directly to CHIP. The NEF HSP110 also promotes CFTR degradation. HSP110 is homologous to HSC/HSP70 in the ATP-bound state.
Topologies of channels, oligomeric arrangement and trafficking-disease mutations. Known or predicted transmembrane, cytosolic and extracellular domains are shown schematically, and the position of disease mutations discussed in this Review are marked with stars. CFTR contains two membrane-spanning domains (MSD1 and MSD2), two cytosolic nucleotide-binding domains (NBD1 and NBD2) and a regulatory region (R, to give NBD1-R). hERG, KCNQ4 and the Kv1.5 α-subunit KCNA5 have a conserved transmembrane region containing voltage-sensor and pore segments, and all form tetramers. In the top views, one subunit is shown in a color matching the domains, the other subunits in gray. hERG has a Per-Arnt-Sim (PAS) domain in its N-terminus and a cyclic-nucleotide-binding domain (CNBD) in its C-terminus, which likely also tetramerizes; the arrangement of the PAS domain is unclear and not shown in the top view. The KCNQ4 C-terminus contains a tetramerization domain. The KCNA5 N-terminus is the binding site for various β-subunits, which also form tetramers. The ENaC and ASIC subunits have a conserved structure including the ENaC/Deg (Degenerin) extracellular domain. The bottom view of the ENaC channel shows the expected α2βγ arrangement. The bottom view of the glioma-specific channel shows a hypothetical arrangement based on comparisons with the ENaC channel. The vascular-smooth-muscle-cell ASIC channels are heterogenous and not represented here. The ASIC2 subunit causes trafficking defects in the glioma-specific and vascular-smooth-muscle-cell ASIC channels.
Mutations associated with ion-channel-trafficking diseasesa
Chaperones and co-chaperones that interact with ion channels in trafficking/misfolding diseasesa
Protein-folding diseases are an ongoing medical challenge. Many diseases within this group are genetically determined, and have no known cure. Among the examples in which the underlying cellular and molecular mechanisms are well understood are diseases driven by misfolding of transmembrane proteins that normally function as cell-surface ion channels. Wild-type forms are synthesized and integrated into the endoplasmic reticulum (ER) membrane system and, upon correct folding, are trafficked by the secretory pathway to the cell surface. Misfolded mutant forms traffic poorly, if at all, and are instead degraded by the ER-associated proteasomal degradation (ERAD) system. Molecular chaperones can assist the folding of the cytosolic domains of these transmembrane proteins; however, these chaperones are also involved in selecting misfolded forms for ERAD. Given this dual role of chaperones, diseases caused by the misfolding and aberrant trafficking of ion channels (referred to here as ion-channel-misfolding diseases) can be regarded as a consequence of insufficiency of the pro-folding chaperone activity and/or overefficiency of the chaperone ERAD role. An attractive idea is that manipulation of the chaperones might allow increased folding and trafficking of the mutant proteins, and thereby partial restoration of function. This Review outlines the roles of the cytosolic HSP70 chaperone system in the best-studied paradigms of ion-channel-misfolding disease - the CFTR chloride channel in cystic fibrosis and the hERG potassium channel in cardiac long QT syndrome type 2. In addition, other ion channels implicated in ion-channel-misfolding diseases are discussed.
Among the myriad of alterations present in cancer cells are an abundance of aberrant mRNA transcripts. Whether abnormal gene transcription is a by-product of cellular transformation or whether it represents an inherent element that contributes to the properties of cancer cells is not yet clear. Here, we present growing evidence that in many cases, aberrant mRNA transcripts contribute to essential phenotypes associated with transformed cells, suggesting that alterations in the splicing machinery are common and functionally important for cancer development. The proteins encoded by these abnormal transcripts are often truncated or missing domains, thereby altering protein function or conferring new functions altogether. Thus, aberrant splicing regulation has genome-wide effects, potentially altering gene expression in many cancer-associated pathways.
Traditional “Top Down” Hypothesis of Necrotizing Enterocolitis 
Paneth Cells (PCs) – Normal Anatomy and Function 
Paneth Cells -- The “Bottom Up” Hypothesis of Necrotizing Enterocolitis 
Selective ablation of Paneth cells followed by enteric infection with Klebsiella induces a NEC-like injury in 14- to 16-day-old mice 
Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. During NEC pathogenesis, bacteria are able to penetrate innate immune defenses and invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Normally, Paneth cells appear in the intestinal crypts during the first trimester of human pregnancy. Paneth cells constitute a major component of the innate immune system by producing multiple antimicrobial peptides and proinflammatory mediators. To better understand the possible role of Paneth cell disruption in NEC, we quantified the number of Paneth cells present in infants with NEC and found that they were significantly decreased compared with age-matched controls. We were able to model this loss in the intestine of postnatal day (P)14-P16 (immature) mice by treating them with the zinc chelator dithizone. Intestines from dithizone-treated animals retained approximately half the number of Paneth cells compared with controls. Furthermore, by combining dithizone treatment with exposure to Klebsiella pneumoniae, we were able to induce intestinal injury and inflammatory induction that resembles human NEC. Additionally, this novel Paneth cell ablation model produces NEC-like pathology that is consistent with other currently used animal models, but this technique is simpler to use, can be used in older animals that have been dam fed, and represents a novel line of investigation to study NEC pathogenesis and treatment.
Summary of liver phenotypes after ATG morpholino knockdown of 14 zebrafish candidate genes 
Genome-wide association studies (GWAS) have revealed numerous associations between many phenotypes and gene candidates. Frequently, however, further elucidation of gene function has not been achieved. A recent GWAS identified 69 candidate genes associated with liver enzyme concentrations, which are clinical liver disease markers. To investigate their role in liver homeostasis, we narrowed down this list to 12 genes based on zebrafish orthology, zebrafish liver expression, and disease correlation. To assess the function of gene candidates during liver development, we assayed hepatic progenitors at 48 hours post fertilization (hpf) and hepatocytes at 72 hpf using in situ hybridization following morpholino knockdown in zebrafish embryos. Knockdown of three genes (pnpla3, pklr, and mapk10) decreased expression of hepatic progenitor cells, while knockdown of eight genes (pnpla3, cpn1, trib1, fads2, slc2a2, pklr, mapk10, and samm50) decreased cell-specific hepatocyte expression. We then induced liver injury in zebrafish embryos using acetaminophen exposure and observed changes in liver toxicity incidence in morphants. Prioritization of GWAS candidates and morpholino knockdown expedites the study of novel genes impacting liver development and represents a feasible method for initial assessment of candidate genes to instruct further mechanistic analyses. Our analysis can be extended to GWAS for additional disease-associated phenotypes.
Mutations in subunits of Succinyl-CoA Synthetase/Ligase (SCS), a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA), and mitochondrial DNA (mtDNA) depletion. While performing a FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES) cells for abnormal mitochondrial phenotypes, a gene trap allele of Sucla2 (Sucla2(SAβgeo)) has been isolated in mouse embryonic stem (ES) cells and used to generate transgenic animals. Sucla2 encodes the ADP-specific β subunit isoform of SCS. Sucla2(SAβgeo) homozygotes exhibit recessive lethality, with most mutants dying late in gestation (e18.5). Mutant placenta and embryonic (e17.5) brain, heart and muscle show varying degrees of mtDNA depletion (20-60%), while there is no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA are observed in embryonic brain. SCS deficient mouse embryonic fibroblasts (MEFs) demonstrate a 50% reduction in mtDNA content compared to wild type MEFs. The mtDNA depletion results in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies, while mtDNA content can be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.
Delayed cerebellar development is a hallmark of Zellweger syndrome (ZS), a severe neonatal neurodegenerative disorder. ZS is caused by mutations in PEX genes, such as PEX13, which encodes a protein required for import of proteins into the peroxisome. The molecular basis of ZS pathogenesis is not known. We have created a conditional mouse mutant with brain-restricted deficiency of PEX13 that exhibits cerebellar morphological defects. PEX13 brain mutants survive into the postnatal period, with the majority dying by 35 days, and with survival inversely related to litter size and weaning body weight. The impact on peroxisomal metabolism in the mutant brain is mixed: plasmalogen content is reduced, but very-long-chain fatty acids are normal. PEX13 brain mutants exhibit defects in reflex and motor development that correlate with impaired cerebellar fissure and cortical layer formation, granule cell migration and Purkinje cell layer development. Astrogliosis and microgliosis are prominent features of the mutant cerebellum. At the molecular level, cultured cerebellar neurons from E19 PEX13-null mice exhibit elevated levels of reactive oxygen species and mitochondrial superoxide dismutase-2 (MnSOD), and show enhanced apoptosis together with mitochondrial dysfunction. PEX13 brain mutants show increased levels of MnSOD in cerebellum. Our findings suggest that PEX13 deficiency leads to mitochondria-mediated oxidative stress, neuronal cell death and impairment of cerebellar development. Thus, PEX13-deficient mice provide a valuable animal model for investigating the molecular basis and treatment of ZS cerebellar pathology.
Myotonic Dystrophy Type I (DM1) is a multi-system, autosomal dominant disorder due to expansion of a CTG repeat sequence in the 3'UTR of the DMPK gene. Repeat size correlates with age of onset and disease severity, with large repeats leading to congenital forms of DM1 associated with hypotonia and intellectual disability. In models of adult DM1, expanded CUG repeats lead to an RNA toxic gain of function at least in part mediated by sequestering specific RNA splicing proteins, most notably muscleblind related proteins (MBNL). However, the impact of CUG RNA repeat expression on early developmental processes is not well understood. To better understand early developmental processes in DM1, we utilized the zebrafish, Danio rerio, as a model system. Direct injection of (CUG)91 repeat-ontaining mRNA into single cell embryos induces toxicity in the nervous system and muscle during early development. These effects manifest as abnormal morphology, behavioral abnormalities, and broad transcriptional changes based on cDNA microarray analysis. Co-injection of zebrafish mbnl2 RNA suppresses (CUG)91 RNA toxicity and reverses the associated behavioral and transcriptional abnormalities. Taken together, these findings suggest that early expression of exogenously transcribed CUG repeat RNA can disrupt normal muscle and nervous system development and provides a new model for DM1 research that is amenable to small molecule therapeutic development.
Biphasic ambulatory activity and reduced avoidance in Id2 −/− mice. (A-C)Spontaneous locomotor activity in wild-type (WT) and Id2 −/− mice (n=8 mice per group) was analyzed using the openfield test at 1, 3 and 6 months of age (videotracking , analyzed using Anymaze software). Changes in distance travelled (A), average speed (B) and total body rotations (C) were analyzed using two-way repeated measure ANOVA. Changes at individual time points were analyzed using post-hoc Bonferronicorrected t-test for multiple comparisons; number of comparisons, 3. (D)Representative track plot tracings generated by Anymaze software demonstrate ambulatory activity in wildtype and Id2 −/− mice at 1, 3 and 6 months of age. (E,F)Crossings into, and time spent in, an identically defined center zone were quantified from the same cohort of animals analyzed in A-C. Total entries (E) and time spent in the center zone (F) were analyzed as for A-C. (G,H)Avoidance behaviors were analyzed in a naïve cohort of wild-type and Id2 −/− mice (n=6 mice per group) using the elevated plus maze paradigm. Entries (G) and time spent (H) in open arms (OA) and closed arms (CA) were calculated from track plots using Anymaze software. All data are expressed as the average of each group; error bars represent s.e.m. *P<0.05 analyzed using a two-tailed t-test for effect resulting from genotype in each measure.  
Age-dependent histological alterations in Id2 −/− mice. (A)Protein extracts obtained from micro-dissected midbrain tissue from wild-type (WT) and Id2 −/− littermates at e12.5. SDS-PAGE and immunoblotting demonstrate Id2 expression in the developing midbrain, absent in Id2 −/− animals. (B)Histological sections obtained from e12.5 embryos cut on the sagittal plane and stained for TH (brown) and counterstained with hematoxylin. Left: low magnification image of wild-type TH-positive progenitors in the mesencephalic flexure. Right: maximized images of boxed zone show TH-positive progenitors in wild-type and Id2 −/− mice. (C)Representative coronal sections obtained from 6-day-old wild-type and Id2 −/− mice stained for TH (brown) and counterstained with hematoxylin. (D)Morphometrically aligned sections from wild-type and Id2 −/− mice at 6 months of age stained with anti-TH antibody (brown) and counterstained with hematoxylin. TH staining of the SNpC is indicated by black arrows (original magnification 10×). (E)Total SNpC cell number manually counted in serial histological sections (6 sections per animal) in 6, 30 and 180-day-old mice stained for TH. Serial sections were cut at 5 μM and counted sections were spaced at 25 μM apart; only nucleated cells were counted; n=8 animals per group; *P=0.023, analyzed using a two-tailed t-test. (F)Sagittal sections obtained from 6-month-old wildtype and Id2 −/− mice stained with anti-TH antibodies (brown). Arrows indicate TH-staining intensity within the caudate putamen (CPu). Black line designates the dorsal boundary of the ventral striatum (Vst) (original magnification 10×). (G)Representative brightfield photomicrographs of the TH-stained (brown) histological sections of the CPu and ventral striatum obtained from wild-type and Id2 −/− mice counterstained with hematoxylin (original magnification 40×). (H)Densitometric measurements of TH-positive fiber density obtained from the CPu and the ventral striatum (vst) of wild-type and Id2 −/− mice. Photomicrographs obtained at 40× magnification were batch converted to binary, grayscale images. TH-positive fiber density was calculated using ImageJ software. Average density was normalized to background staining in the cortex (n=3 animals per group, three representative areas sampled per individual) *P=0.004, analyzed using a two-tailed t-test. Error bars represent s.e.m.  
Histological evidence of neurodegeneration in the SNpC of Id2 −/− mice. (A)TH-stained histological sections of the SNpC in wild-type and Id2 −/− mice at 6 months of age. Black arrows indicate neurons of the SNpC with increased staining intensity and reduced somal volume in Id2 −/− mice. (B)Average TH-stained somal area, excluding nuclei, was calculated from 100 neurons per section, three sections per animal (n=3 animals per group). Values indicate means and s.e.m.; *P<0.05, calculated using a two-tailed t-test. (C)Adjacent histological SNpC sections to A, stained for cleaved caspase-3 (brown) and counterstained with hematoxylin. Arrows indicate cleaved caspase-3-positive cells visible in the SNpC of Id2 −/− but not wild-type mice. Inset: maximized image of a cleaved caspase-3-positive cell (asterisk) within the SNpC of a representative Id2 −/− mouse. (D)Adjacent histological sections to A of the SNpC in wild-type and Id2 −/− mice, stained for Th (green) and GFAP (red). Images indicate increased glial fibers present within degenerating tissues of the SNpC in Id2 −/− mice.  
Id2 is required for normal levels of DAT expression. (A)Total mRNA was obtained from micro-dissected midbrain tissues of wild-type and Id2 −/− mice at e12.5. Real-time PCR analysis was performed in triplicate to determine the expression levels of the DA markers En1, Lmx1a, Pitx3, Th, AADC and DAT. Data was normalized to the expression of β-actin and expressed as relative fold change compared with the average wild-type expression level (n=4 per group); *P<0.05 analyzed using a Bonferroni-corrected t-test for multiple comparisons. (B,C)Representative images of striatal cryosections from wild-type (B) and Id2 −/− (C) mice stained with anti-DAT antibody. Fluorescent conjugated secondary antibodies were used for visualization and the images examined by confocal microscopy. The images are representative of an evaluation of 6 animals of each genotype. (D,E)Id2 (D) and DAT (E) expression was examined in wild-type, Id2 +/− and Id2 −/− NPCs at 0, 5 and 10 days after the induction of DA-directed differentiation. The values shown are from a representative experiment conducted in triplicate; *P<0.04 analyzed using a Bonferroni-corrected t-test for multiple comparisons. (F)DA uptake was assessed in wild-type and Id2 −/− cells immediately prior to and following DA-directed differentiation (day 10) as described for D and E. Black bars represent [ 3 H]DA uptake and white bars represent identical experiments conducted in the presence of unlabeled DA competitor. Data were normalized to data obtained from identical assays conducted at 4°C. Three independent experiments were conducted in triplicate; *P<0.05 analyzed using a two-tailed t-test. (G,H)Confocal images of histological sections of the SNpC of 1-month-old wild-type (F) and Id2 −/− (G) mice co-immunolabeled for TH (green) and DAT (red) and counterstained with Hoechst 33342 dye (blue). All values indicate means ± s.e.m.  
Characterizing dopaminergic neuronal development and function in novel genetic animal models may uncover strategies for researchers to develop disease-modifying treatments for neurologic disorders. Id2 is a transcription factor expressed in the developing central nervous system. Id2(-/-) mice have fewer dopaminergic neurons in the olfactory bulb and reduced olfactory discrimination, a pre-clinical marker of Parkinson's disease. Here, we summarize behavioral, histologic, and in vitro molecular biological analyses to determine if midbrain dopaminergic neurons were affected by Id2 loss. Id2(-/-) mice were hyperactive at 1 and 3 months of age, but by 6 months, showed reduced activity. Id2(-/-) mice showed age-dependent histologic alterations in dopaminergic neurons of the substantia nigra pars compacta (SNpC) associated with changes in locomotor activity. Reduced dopamine transporter (DAT) expression was observed at early ages in Id2(-/-) mice and DAT expression was dependent on Id2 expression in an in vitro dopaminergic differentiation model. Evidence of neurodegeneration including activated caspase-3 and glial infiltration were noted in the SNpC of older Id2(-/-) mice. These findings document a novel role for Id2 in the maintenance of midbrain dopamine neurons. The Id2(-/-) mouse should provide unique opportunities to study the progression of neurodegenerative disorders involving the dopamine system.
Early born neuronal populations are overproduced in Sall1-/-mice. (A,B)Tuj1 (green) staining identified an increase in preplate size at E12.5 in Sall1deficient animals (B) compared with controls (A). (C)Quantification of cortical cell number at E11.5 and E12.5. *, P<0.05; **, P<0.01. (D,E)CSPG (red) immunostaining of the subplate (bracketed region) and marginal zone identified an increase in these structures in Sall1-/-mice (E) compared with controls (D) at E14.5. Note: CSPG is also expressed by the meninges at the pial surface. (F,G)Reelin (red) immunostaining identified an increase in Cajal-Retzius cells in Sall1-/mice (G) compared with controls (F) at E14.5. (A,B,D-G) Sections are counterstained with DAPI (blue). PP, preplate; PC, progenitor population; IZ, intermediate zone; SP, subplate; CP, cortical plate; MZ, marginal zone; P, pial surface. Scale bar: 100m (A,B); 200m (D,E); 400m (F,G). 
Sall1 regulates the number of cells committed to an early born fate. (A-C)Quantification of the proportion of total cells born (BrdU+) on E11.5 (A), E12.5 (B) or E14.5 (C) in defined cortical layers at E18.5. (D,E)Quantification of cell number in molecularly defined [Tbr1+: subplate and layer VI (LVI); Cux1+: layers II/III, IV (LII/III,IV)] cortical laminae at E18.5 (D) and the proportion of cells in cortical laminae as a percentage of total cortical plate cell number at E18.5 (E). *, P<0.05; **, P<0.01. PC, progenitor population; IZ, intermediate zone; SP, subplate; CP, cortical plate; MZ, marginal zone. 
Progenitor cells in the cerebral cortex undergo dynamic cellular and molecular changes during development. Sall1 is a putative transcription factor that is highly expressed in progenitor cells during development. In humans, the autosomal dominant developmental disorder Townes-Brocks syndrome (TBS) is associated with mutations of the SALL1 gene. TBS is characterized by renal, anal, limb and auditory abnormalities. Although neural deficits have not been recognized as a diagnostic characteristic of the disease, ~10% of patients exhibit neural or behavioral abnormalities. We demonstrate that, in addition to being expressed in peripheral organs, Sall1 is robustly expressed in progenitor cells of the central nervous system in mice. Both classical- and conditional-knockout mouse studies indicate that the cerebral cortex is particularly sensitive to loss of Sall1. In the absence of Sall1, both the surface area and depth of the cerebral cortex were decreased at embryonic day 18.5 (E18.5). These deficiencies are associated with changes in progenitor cell properties during development. In early cortical progenitor cells, Sall1 promotes proliferative over neurogenic division, whereas, at later developmental stages, Sall1 regulates the production and differentiation of intermediate progenitor cells. Furthermore, Sall1 influences the temporal specification of cortical laminae. These findings present novel insights into the function of Sall1 in the developing mouse cortex and provide avenues for future research into potential neural deficits in individuals with TBS.
GALT enzyme activity levels detected in adult flies 
GALT-null D. melanogaster are defective in climbing but not startle response. (A)The proportion of flies in each indicated cohort that reached above a designated mark in 20 seconds. (B)The average number of seconds it took all of the flies in each cohort to regain a standing posture after vigorous agitation for 10 seconds. All values were modeled in a one-way ANOVA and mean ± s.e.m. are plotted. n, number of cohorts tested. 
Despite neonatal diagnosis and life-long dietary restriction of galactose, many patients with classic galactosemia grow to experience significant long-term complications. Among the more common are speech, cognitive, behavioral, ovarian and neurological/movement difficulties. Despite decades of research, the pathophysiology of these long-term complications remains obscure, hindering prognosis and attempts at improved intervention. As a first step to overcome this roadblock we have begun to explore long-term outcomes in our previously reported GALT-null Drosophila melanogaster model of classic galactosemia. Here we describe the first of these studies. Using a countercurrent device, a simple climbing assay, and a startle response test to characterize and quantify an apparent movement abnormality, we explored the impact of cryptic GALT expression on phenotype, tested the role of sublethal galactose exposure and galactose-1-phosphate (gal-1P) accumulation, tested the impact of age, and searched for potential anatomical defects in brain and muscle. We found that about 2.5% residual GALT activity was sufficient to reduce outcome severity. Surprisingly, sublethal galactose exposure and gal-1P accumulation during development showed no effect on the adult phenotype. Finally, despite the apparent neurological or neuromuscular nature of the complication we found no clear morphological differences between mutants and controls in brain or muscle, suggesting that the defect is subtle and/or is physiologic rather than structural. Combined, our results confirm that, like human patients, GALT-null Drosophila experience significant long-term complications that occur independently of galactose exposure, and serve as a proof of principle demonstrating utility of the GALT-null Drosophila model as a tool for exploring genetic and environmental modifiers of long-term outcome in GALT deficiency.
The matrix metalloproteinase (MMP) family of extracellular proteases performs crucial roles in development and repair of the skeleton owing to their ability to remodel the extracellular matrix (ECM) and release bioactive molecules. Most MMP-null skeletal phenotypes that have been previously described are mild, thus permitting the assessment of their functions during bone repair in the adult. In humans and mice, MMP2 deficiency causes a musculoskeletal phenotype. In this study, we assessed the role of MMP2 during mouse fracture repair and compared it with the roles of MMP9 and MMP13. Mmp2 was expressed at low levels in the normal skeleton and was broadly expressed in the fracture callus. Treatment of wild-type mice with a general MMP inhibitor, GM6001, caused delayed cartilage remodeling and bone formation during fracture repair, which resembles the defect observed in Mmp9(-/-) mice. Unlike Mmp9- and Mmp13-null mutations, which affect both cartilage and bone in the callus, the Mmp2-null mutation delayed bone remodeling but not cartilage remodeling. This remodeling defect occurred without changes in either osteoclast recruitment or vascular invasion of the fracture callus compared with wild type. However, we did not detect changes in expression of Mmp9, Mmp13 or Mt1-Mmp (Mmp14) in the calluses of Mmp2-null mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13-null mice. In keeping with the skeletal phenotype of Mmp2-null mice, MMP2 plays a role in the remodeling of new bone within the fracture callus and impacts later stages of bone repair compared with MMP9 and MMP13. Taken together, our results indicate that MMPs play unique and distinct roles in regulating skeletal tissue deposition and remodeling during fracture repair.
Social behavior in Shank3-deficient mice. (A,B) Three-chambered social approach test. Normal sociability, characterized by significantly more time spent with another mouse compared with an object, was found for mice from all genotypes and all strains in the sociability test (A). C57 and FVB mice spent significantly more time with the unfamiliar mouse in the social novelty and recognition test. The 129SVE (129) knockout (KO) mice showed a trend in their preference (B). (C) Nest building. No differences between the genotypes were found for any of the mouse strains in the nest building assay. The graph on the left shows the average score for the nests built for each genotype, the three graphs on the right shows more detailed analysis of this data. Data represents means±s.e.m. *P<0.05, **P<0.01, ***P<0.001. Het, heterozygous; WT, wild type. 
Learning and memory in Shank3-deficient mice. (A) Fear conditioning. Training (left column) was altered in Shank3 heterozygous and knockout mice on the 129SVE (129) background, but no differences between the genotypes were found in either contextual (middle column) or cued recall (right column) phases. No differences between the genotypes were observed for C57 mice. All FVB mice were strongly impaired in all phases of the fear conditioning test. During cued recall, the percentage of mice that exhibited freezing behavior immediately after the first tone was lower in FVB Shank3deficient animals than in wild-type. The x-axes indicate the time (minutes) for which the mice were recorded. (B) Y-maze spontaneous alternation. No differences were noted for the genotypes of 129SVE, C57 or FVB mice. The data represents means±s.e.m. o P<0.05, oo P<0.01, ooo P<0.001 for wild-type (WT) versus heterozygous (Het) mice; *P<0.05, **P<0.01, ***P<0.001 for WT versus knockout (KO) mice; # P<0.05, 
Stereotypies, repetitive behavior and perseverance in Shank3-deficient mice. (A,B) Repetitive behaviors. No significant differences were observed between the genotypes for (A) the number of revolutions in the open-field (the left panel shows the total number of revolutions, and the right panel shows the percentage of the total revolutions that were performed in a clockwise direction) or (B) perseveration (the number of times an animal choose the same arm of the maze for three consecutive times) in the Y-maze. (C,D) Grooming and barbering. No significant differences between the genotypes were observed for (C) the number of grooming bouts (left panel) or time (right panel) and (D) the percentage of animals with barbering. The data represent means±s.e.m. 129, 129SVE. 
Haploinsufficiency of SHANK3, caused by chromosomal abnormalities or mutations that disrupt one copy of the gene, leads to a neurodevelopmental syndrome called Phelan-McDermid Syndrome that can include absent or delayed speech, intellectual disability, neurological changes, and autism spectrum disorders. The SHANK3 protein forms a key structural part of the post-synaptic density. We previously generated and characterized mice with a targeted disruption of Shank3 in which exons coding for the ankyrin repeat domain were deleted and expression of full-length Shank3 was disrupted. We documented specific deficits in synaptic function and plasticity, along with reduced reciprocal social interactions in Shank3 heterozygous mice. Changes in phenotype due to a mutation at a single locus are quite frequently modulated by other loci, most dramatically when the entire genetic background is changed. In mice, each strain of laboratory mouse represents a distinct genetic background and alterations in phenotype due to gene knockout or transgenesis are frequently different across strains, which can lead to the identification of important modifier loci. We have investigated the effect of genetic background on phenotypes of Shank3-heterozygous, knock-out and wild-type mice, using C57BL/6, 129SVE, and FVB/Ntac strain backgrounds. We focused on observable behaviors with the goal of carrying out subsequent analyses to identify modifier loci. Surprisingly, there were very modest strain effects over a large battery of analyses. These results indicate that behavioral phenotypes associated with Shank3 haploinsufficiency are largely strain independent.
Age-dependent progression of apoptotic cell death. Representative examples show TUNEL-positive nuclei in neurons (Elav positive; A-A") and glia (Repo positive; B-B") in p35 mutant brains. Note the weaker Elav signal in the apoptotic neuron in comparison with surrounding cells (white arrow in A-A") and the weaker Repo signal in dying glial cell (white arrowhead in B-B"). Single confocal plan, optic slice: 1.6m. Scale bar: 10m. (C,D)The absolute number of TUNEL-positive cells per hemisphere is presented on the y axis (n≥6; mean ± s.e.m.). (C)Apoptosis in neurons. The pan-neuronal marker Elav was used to identify neurons (n≥6; mean ± s.e.m.) in three independent experiments. c, control. (D)Apoptosis in glia. The pan-glial marker Repo was used to identify glia (n≥6; mean ± s.e.m.). Time points under the chart show absolute age in days and refer to the position on the mortality curve; results are grouped according to the chronological points. For instance: 45-day-old p35 mutant flies reached 80% of mortality in population and were analyzed simultaneously with 45-day-old wild-type controls that reached only 20% in mortality. 
Progressive increase in necrotic cell death. Necrotic cells were identified as propidium iodide (PI)-and Hoechst (HO)-positive nuclei. (A)Z-stack of confocal images through the MB area near the top of the brain (12m). Small trachea tubes surround the calyx (ca) of the MB and stain positive for PI (yellow arrow). Note the significant number of PI-positive cells due to physical damage: this portion of the brain was excluded from analysis. (B-B") Example of a moreinternal 5-m-thick area included in analysis (outer white dotted line). PI-positive nuclei also exhibit bright HO signal and often have a larger diameter (hollow white arrow). Many cells have very low HO signal (white arrowheads). Necrotic cells were often clustered in both controls and mutants (dashed rectangles). Abundance and position of PI-positive cells implied that the majority of necrotic cells were neurons. Scale bar: 20m. (C)Increase in the percentage of necrotic cells with age; comparison of PI-and HO-positive versus HO-positive cells (n≥6; mean ± s.e.m.). Age-dependent time points are described in the legend to Fig. 1A and Fig. 2C,D. c, control. 
Accumulation of autophagosomal organelles in old p35 mutant brains. Electron micrographs of MB neurons from control wild-type and p35 mutant brains at different time points. (A,B)Representative images of a middle-aged wild-type neuron (A) and young p35 mutant neuron (B). Boundaries of individual cells are highlighted by colored dotted lines. N, nucleus. (C,D)Age-dependent accumulation of autophagosomes (arrow) in both controls (C) and p35 mutants (D). Note the clusters of adjacent cells containing multiple ATG organelles. C'-F' are high-magnification images of organelles highlighted in C-F, respectively. (E,F)Characteristic appearance of age-dependent multi-molecular depositions (arrowhead) in controls (E) and p35 mutants (F). Depositions with hexagonal symmetry were often located in close proximity to multilamellar structures. Scale bar: 500 nm. (G)Increase in the average number of autophagic organelles (AO) per neuron in old p35 mutants. Because APGs containing multilamellar structures represent the vast majority of all autophagic organelles that we observed in this project, we combined data for APGs, MLBs and APGLs. A minimum of 20 neurons per brain were analyzed. Values are mean ± s.e.m. (n≥3 brains). c, control. 
Age-related neuronal degeneration. MARCM clones were analyzed in old flies above 80% of mortality of population. Images are Z-stack projections of sequential confocal slices covering the whole MB area. Brain midline is on the left, dorsal side is up. (A,A) The first sign of degeneration in controls is when the axonal medial (m) lobe of -neuron appears thin and partially fragmented (arrowhead). (B,B) Control neuron with breaks in the proximal neurite (white dashed arrow) and completely missing axonal lobes (white dotted line). Note the end of the existing axon correlated with distal peduncle border (white hollow arrow). (C,C) p35 mutant MB neuron has multiple breaks in the proximal neurite (dashed arrow), and the dorsal and medial axonal lobes (arrows). Note that the peduncle part of the proximal axon exhibits a reduced amount of fragmentation. Present parts of the proximal neurite (pn), peduncle (p), dorsal (d) and medial (m) axonal lobes are highlighted with a single dashed line. Missing links of neurites are highlighted with fine white dotted line. Other non-MB neuronal types labeled with GFP are marked with a star. Scale bar: 30m. 
Age-dependent changes in morphology of p35 mutant neurons. MARCM clones were analyzed in young (5-day old) and old flies above 80% of mortality in population. Images are Z-stack projections of the whole MB area. For orientation and labeling details, see legend to Fig. 5. Young (A) and old (B) controls and young p35 mutants (C) have normal morphology. Old p35 mutants (D) develop swelling (white arrow and bracket) in the area of AIS compartment (white rectangle). Scale bar: 30m. (D,E) Higher magnification showing details of swellings. (D) Boxed area in D; note bright GFP clog. (E) Example of axonal swelling from another brain. Position of the axon is highlighted by a white dotted line; dendrites are marked as 'de'. Scale bar: 10m. 
Altered function of Cdk5 kinase is associated with many forms of neurodegenerative disease in humans. We show here that inactivating the Drosophila Cdk5 ortholog, by mutation of its activating subunit, p35, causes adult-onset neurodegeneration in the fly. In the mutants, a vacuolar neuropathology is observed in a specific structure of the central brain, the 'mushroom body', which is the seat of olfactory learning and memory. Analysis of cellular phenotypes in the mutant brains reveals some phenotypes that resemble natural aging in control flies, including an increase in apoptotic and necrotic cell death, axonal fragmentation, and accumulation of autophagosomes packed with crystalline-like depositions. Other phenotypes are unique to the mutants, notably age-dependent swellings of the proximal axon of mushroom body neurons. Many of these phenotypes are also characteristic of mammalian neurodegenerative disease, suggesting a close relationship between the mechanisms of Cdk5-associated neurodegeneration in fly and human. Together, these results identify the cellular processes that are unleashed in the absence of Cdk5 to initiate the neurodegenerative program, and they provide a model that can be used to determine what part each process plays in the progression to ultimate degeneration.
Human Menkes disease is a lethal neurodegenerative disorder of copper metabolism that is caused by mutations in the ATP7A copper-transporting gene. In the present study, we attempted to construct a Drosophila model of Menkes disease by RNA interference (RNAi)-induced silencing of DmATP7, the Drosophila orthologue of mammalian ATP7A, in the digestive tract. Here, we show that a lowered level of DmATP7 mRNA in the digestive tract results in a reduced copper content in the head and the rest of the body of surviving adults, presumably owing to copper entrapment in the gut. Similar to Menkes patients, a majority of flies exhibit an impaired neurological development during metamorphosis and die before eclosion. In addition, we show that survival to the adult stage is highly dependent on the copper content of the food and that overexpression of the copper homeostasis gene, metal-responsive transcription factor-1 (MTF-1), enhances survival to the adulthood stage. Taken together, these results highlight the role of DmATP7-mediated copper uptake in the neurodevelopment of Drosophila melanogaster and provide a framework for the analysis of potential gene interactions influencing Menkes disease.
Disease phenotype is unaffected by testosterone treatment. (A)Testosterone treatment resulted in elevated serum testosterone levels in transgenic (tg) T-treated (n8) and non-transgenic (ntg) T-treated (n10) animals when compared with tg sham-treated (n6) mice (P<0.05), with the exception of months three and six. Although, testosterone levels dropped at the end of pellet life, testosterone levels were still significantly elevated in ntg T-treated mice in month six and there was a trend towards elevated testosterone in tg T-treated animals. (B)Ability to remain on a rotarod was impaired in AR112Q T-treated (n10) and AR112Q sham-treated (n12) males when compared with T-treated non-transgenics (n14) (P<0.05), but rotarod performance was similar between T-treated AR112Q males and sham-treated AR112Q males (P>0.05). (C-E)Grip strength [using forepaws (C) or all paws (D)] and vertical activity (E) of all AR112Q males was reduced in comparison to T-treated non-transgenic males (P<0.05), but no significant decrease in any of these measures was observed upon T-treatment of AR112Q males (P>0.05). (F)Clasping behavior increased with age in all AR112Q males and was not affected by T-treatment; non-transgenic males rarely showed clasping behavior. 
Testosterone treatment does not alter cellular disease markers. NIIs were detected with AR-H280 antibody in spinal cord of all AR112Q males (A, top); no differences in NII frequency were detected upon T-treatment (B; n2). Nuclei of non-transgenic (ntg) T-treated males show diffuse nuclear AR and no NIIs (A). Unphosphorylated NF-H, as detected by SMI32 immunostaining (A, bottom), was abundant in neuronal soma of the non-transgenic spinal cord but was reduced in AR112Q tissue. (C)Quantification of SMI32 intensity did not reveal any differences between transgenic (tg) T-treated and tg sham-treated males (n2). Panels showing NIIs (A, top) and SMI32 (A, bottom) are not from the same fields. Insets in A represent the location within the anterior horn from which higher-magnification images were taken (yellow box). The red oval outlines the central canal, with the dorsal aspect to the left and the ventral aspect to the right. 
Evidence from multiple animal models demonstrates that testosterone plays a crucial role in the progression of symptoms in spinal and bulbar muscular atrophy (SBMA), a condition that results in neurodegeneration and muscle atrophy in affected men. Mice bearing a transgene encoding a human androgen receptor (AR) that contains a stretch of 112 glutamines (expanded polyglutamine tract; AR112Q mice) reproduce several aspects of the human disease. We treated transgenic male AR112Q mice with testosterone for 6 months. Surprisingly, testosterone treatment of AR112Q males did not exacerbate the disease. Although transgenic AR112Q males exhibited functional deficits when compared with non-transgenics, long-term testosterone treatment had no effect on motor function. Testosterone treatment also failed to affect cellular markers of disease, including inclusion formation (the accumulation of large nuclear aggregates of mutant AR protein) and levels of unphosphorylated neurofilament heavy chain. These data suggest that the mechanism of disease in SBMA saturates at close to endogenous hormone levels and that individuals with SBMA who take, or have taken, testosterone for its putative therapeutic properties are unlikely to suffer adverse effects.
Individuals with metabolic syndrome are at high risk of developing chronic kidney disease (CKD) through unclear pathogenic mechanisms. Obesity and diabetes are known to induce glucolipotoxic effects in metabolically relevant organs. However, the pathogenic role of glucolipotoxicity in the aetiology of diabetic nephropathy is debated. We generated a murine model, the POKO mouse, obtained by crossing the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) knockout (KO) mouse into a genetically obese ob/ob background. We have previously shown that the POKO mice showed: hyperphagia, insulin resistance, hyperglycaemia and dyslipidaemia as early as 4 weeks of age, and developed a complete loss of normal β-cell function by 16 weeks of age. Metabolic phenotyping of the POKO model has led to investigation of the structural and functional changes in the kidney and changes in blood pressure in these mice. Here we demonstrate that the POKO mouse is a model of renal disease that is accelerated by high levels of glucose and lipid accumulation. Similar to ob/ob mice, at 4 weeks of age these animals exhibited an increased urinary albumin:creatinine ratio and significantly increased blood pressure, but in contrast showed a significant increase in the renal hypertrophy index and an associated increase in p27(Kip1) expression compared with their obese littermates. Moreover, at 4 weeks of age POKO mice showed insulin resistance, an alteration of lipid metabolism and glomeruli damage associated with increased transforming growth factor beta (TGFβ) and parathyroid hormone-related protein (PTHrP) expression. At this age, levels of proinflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1), and fibrotic factors were also increased at the glomerular level compared with levels in ob/ob mice. At 12 weeks of age, renal damage was fully established. These data suggest an accelerated lesion through glucolipotoxic effects in the renal pathogenesis in POKO mice.
Chloride/proton exchange by the lysosomal anion transporter ClC-7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue Cattle with a severe symptomatology including peri-natal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.
Jim Smith has made fundamental contributions to our understanding of early embryonic development. Here, in conversation with DMM Consulting Editor Kathy Weston, he discusses his stutter, how he became interested in developmental biology, and his role in helping establish what will be the UK’s biggest multidisciplinary research laboratory, the UK Centre for Medical Research and Innovation (UKCMRI).
Top-cited authors
Paul Huang
  • Massachusetts General Hospital
Janine T Erler
  • University of Copenhagen
Thomas R Cox
  • Garvan Institute of Medical Research
Paolo Bonaldo
  • University of Padova
Marco Sandri
  • Telethon Institute of Genetics and Medicine