The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses.

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.

AMPA Induces NO-Dependent cGMP Signals in Hippocampal and Cortical Neurons via L-Type Voltage-Gated Calcium Channels.

The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100 nM and 3 µM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.

Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.

The choroid plexus sodium-bicarbonate cotransporter NBCe2 regulates mouse cerebrospinal fluid pH.

KEY POINTS: Normal pH is crucial for proper functioning of the brain, and disorders increasing the level of CO2 in the blood lead to a decrease in brain pH. CO2 can easily cross the barriers of the brain and will activate chemoreceptors leading to an increased exhalation of CO2 . The low pH, however, is harmful and bases such as HCO3- are imported across the brain barriers in order to normalize brain pH. We show that the HCO3- transporter NBCe2 in the choroid plexus of the blood-cerebrospinal fluid barrier is absolutely necessary for normalizing CSF pH during high levels of CO2 . This discovery represents a significant step in understanding the molecular mechanisms behind regulation of CSF pH during acid-base disturbances, such as chronic lung disease. ABSTRACT: The choroid plexus epithelium (CPE) is located in the brain ventricles where it produces the majority of the cerebrospinal fluid (CSF). The hypothesis that normal brain function is sustained by CPE-mediated CSF pH regulation by extrusion of acid-base equivalents was tested by determining the contribution of the electrogenic Na+ -HCO3- cotransporter NBCe2 to CSF pH regulation. A novel strain of NBCe2 (Slc4a5) knockout (KO) mice was generated and validated. The base extrusion rate after intracellular alkalization was reduced by 77% in NBCe2 KO mouse CPE cells compared to control mice. NBCe2 KO mice and mice with CPE-targeted NBCe2 siRNA knockdown displayed a reduction in CSF pH recovery during hypercapnia-induced acidosis of approximately 85% and 90%, respectively, compared to control mice. NBCe2 KO did not affect baseline respiration rate or tidal volume, and the NBCe2 KO and wild-type (WT) mice displayed similar ventilatory responses to 5% CO2 exposure. NBCe2 KO mice were not protected against pharmacological or heating-induced seizure development. In conclusion, we establish the concept that the CPE is involved in the regulation of CSF pH by demonstrating that NBCe2 is necessary for proper CSF pH recovery after hypercapnia-induced acidosis.

High folic acid diet enhances tumour growth in PyMT-induced breast cancer.

BACKGROUND: The B-vitamin folate is among the most studied bioactive food compound, and a dietary intake meeting the daily requirements has been found to reduce the risk of cancer and cardiovascular diseases as well as preventing neural tube defects during fetal development. Several countries have therefore introduced dietary fortification with folic acid. However, clinical and animal studies suggest that folic acid has a dual role in cancer development. METHODS: During the period of initial tumour progression, MMTV-PyMT (MMTV-polyoma virus middle T) transgenic mice were fed with normal diet and high folic acid diet. RESULTS: We found that PyMT-induced breast tumours highly express the cancer-specific folate receptor (FR), a feature they share with several human epithelial cancers in which expression of FRα correlates with tumour grade. Mice receiving a high folic acid diet displayed a significantly increased tumour volume compared with mice receiving normal diet. In the largest tumours, only found in mice on high folic acid diet, STAT3 was activated. In primary cells from PyMT tumours, STAT3 was activated upon treatment with folic acid in culture. CONCLUSIONS: Our results offer a novel molecular explanation for folic acid-induced growth of existing tumours.

MFAP4 Promotes Vascular Smooth Muscle Migration, Proliferation and Accelerates Neointima Formation.

OBJECTIVE: Arterial injury stimulates remodeling responses that, when excessive, lead to stenosis. These responses are influenced by integrin signaling in vascular smooth muscle cells (VSMCs). Microfibrillar-associated protein 4 (MFAP4) is an integrin ligand localized to extracellular matrix fibers in the vascular wall. The role of MFAP4 in vascular biology is unknown. We aimed to test the hypothesis that MFAP4 would enhance integrin-dependent VSMC activation. APPROACH AND RESULTS: We produced Mfap4-deficient (Mfap4(-/-)) mice and performed carotid artery ligation to explore the role of MFAP4 in vascular biology in vivo. Furthermore, we investigated the effects of MFAP4 in neointimal formation ex vivo and in primary VSMC and monocyte cultures in vitro. When challenged with carotid artery ligation, Mfap4(-/-) mice exhibited delayed neointimal formation, accompanied by early reduction in the number of proliferating medial and neointimal cells, as well as infiltrating leukocytes. Delayed neointimal formation was associated with decreased cross-sectional area of ligated Mfap4(-/-) carotid arteries resulting in lumen narrowing 28 days after ligation. MFAP4 blockade prohibited the formation of neointimal hyperplasia ex vivo. Moreover, we demonstrated that MFAP4 is a ligand for integrin αVß3 and mediates VSMC phosphorylation of focal adhesion kinase, migration, and proliferation in vitro. MFAP4-dependent VSMC activation was reversible by treatment with MFAP4-blocking antibodies and inhibitors of focal adhesion kinase and downstream kinases. In addition, we showed that MFAP4 promotes monocyte chemotaxis in integrin αVß3-dependent manner. CONCLUSIONS: MFAP4 regulates integrin αVß3-induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.

Distinct Functions for Anterograde and Retrograde Sorting of SORLA in Amyloidogenic Processes in the Brain.

SORLA is a neuronal sorting receptor implicated both in sporadic and familial forms of AD. SORLA reduces the amyloidogenic burden by two mechanisms, either by rerouting internalized APP molecules from endosomes to the trans-Golgi network (TGN) to prevent proteolytic processing or by directing newly produced Aß to lysosomes for catabolism. Studies in cell lines suggested that the interaction of SORLA with cytosolic adaptors retromer and GGA is required for receptor sorting to and from the TGN. However, the relevance of anterograde or retrograde trafficking for SORLA activity in vivo remained largely unexplored. Here, we generated mouse models expressing SORLA variants lacking binding sites for GGA or retromer to query this concept in the brain. Disruption of retromer binding resulted in a retrograde-sorting defect with accumulation of SORLA in endosomes and depletion from the TGN, and in an overall enhanced APP processing. In contrast, disruption of the GGA interaction did not impact APP processing but caused increased brain Aß levels, a mechanism attributed to a defect in anterograde lysosomal targeting of Aß. Our findings substantiated the significance of adaptor-mediated sorting for SORLA activities in vivo, and they uncovered that anterograde and retrograde sorting paths may serve discrete receptor functions in amyloidogenic processes. SIGNIFICANCE STATEMENT: SORLA is a sorting receptor that directs target proteins to distinct intracellular compartments in neurons. SORLA has been identified as a genetic risk factor for sporadic, but recently also for familial forms of AD. To confirm the relevance of SORLA sorting for AD processes in the brain, we generated mouse lines, which express trafficking mutants instead of the wild-type form of this receptor. Studying neuronal activities in these mutant mice, we dissected distinct trafficking routes for SORLA guided by two cytosolic adaptors termed GGA and retromer. We show that these sorting pathways serve discrete functions in control of amyloidogenic processes and may represent unique therapeutic targets to interfere with specific aspects of neurodegenerative processes in the diseased brain.

Stanniocalcin-2 inhibits mammalian growth by proteolytic inhibition of the insulin-like growth factor axis.

Mammalian stanniocalcin-2 (STC2) is a secreted polypeptide widely expressed in developing and adult tissues. However, although transgenic expression in mice is known to cause severe dwarfism, and targeted deletion of STC2 causes increased postnatal growth, its precise biological role is still unknown. We found that STC2 potently inhibits the proteolytic activity of the growth-promoting metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). Proteolytic inhibition requires covalent binding of STC2 to PAPP-A and is mediated by a disulfide bond, which involves Cys-120 of STC2. Binding of STC2 prevents PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF, required for normal growth. Concordantly, we show that STC2 efficiently inhibits PAPP-A-mediated IGF receptor signaling in vitro and that transgenic mice expressing a mutated variant of STC2, STC2(C120A), which is unable to inhibit PAPP-A, grow like wild-type mice. Our work identifies STC2 as a novel proteinase inhibitor and a previously unrecognized extracellular component of the IGF system.

Lack of the serum- and glucocorticoid-inducible kinase SGK1 improves muscle force characteristics and attenuates fibrosis in dystrophic mdx mouse muscle.

Duchenne muscular dystrophy (DMD) is a human genetic disease characterized by fibrosis and severe muscle weakness. Currently, there is no effective treatment available to prevent progressive fibrosis in skeletal muscles. The serum- and glucocorticoid-inducible kinase SGK1 regulates a variety of physiological functions and participates in fibrosis stimulation. Here, we investigated whether SGK1 influences structure, function and/or fibrosis of the muscles from the mdx mouse, an animal model for DMD. As expected, mdx muscles showed the typical pathological features of muscular dystrophy including fiber size variations, central nuclei of muscle fibers, fibrosis in the diaphragm, and force reduction by 30-50 %. Muscles from sgk1 (-/-) mice were histologically overall intact and specific force was only slightly reduced compared to wild-type muscles. Surprisingly, soleus and diaphragm muscles of mdx/sgk1 (-/-) mice displayed forces close to wild-type levels. Most muscle fibers of the double mutants contained central nuclei, but fibrosis was not observed in any of the tested limb and diaphragm muscles. We conclude that the sole lack of SGK1 in mouse muscle does not lead to pronounced changes in muscle structure and function. However, dystrophin-deficient mdx muscle seems to benefit from SGK1 deficiency. SGK1 appears to be an important enzyme in the process of fibrotic remodeling and subsequent weakness of dystrophin-deficient mouse muscle.

Ptf1a-mediated control of Dll1 reveals an alternative to the lateral inhibition mechanism.

Neurog3-induced Dll1 expression in pancreatic endocrine progenitors ostensibly activates Hes1 expression via Notch and thereby represses Neurog3 and endocrine differentiation in neighboring cells by lateral inhibition. Here we show in mouse that Dll1 and Hes1 expression deviate during regionalization of early endoderm, and later during early pancreas morphogenesis. At that time, Ptf1a activates Dll1 in multipotent pancreatic progenitor cells (MPCs), and Hes1 expression becomes Dll1 dependent over a brief time window. Moreover, Dll1, Hes1 and Dll1/Hes1 mutant phenotypes diverge during organ regionalization, become congruent at early bud stages, and then diverge again at late bud stages. Persistent pancreatic hypoplasia in Dll1 mutants after eliminating Neurog3 expression and endocrine development, together with reduced proliferation of MPCs in both Dll1 and Hes1 mutants, reveals that the hypoplasia is caused by a growth defect rather than by progenitor depletion. Unexpectedly, we find that Hes1 is required to sustain Ptf1a expression, and in turn Dll1 expression in early MPCs. Our results show that Ptf1a-induced Dll1 expression stimulates MPC proliferation and pancreatic growth by maintaining Hes1 expression and Ptf1a protein levels.

An eEF1A1 truncation encoded by PTI-1 exerts its oncogenic effect inside the nucleus.

BACKGROUND: The oncogene PTI-1 was originally isolated from a prostate cancer cell line by its capability to transform rat fibroblasts. The PTI-1 mRNA has a very eccentric structure as the 5'UTR is similar to prokaryotic 23S rRNA, while the major open reading frame and the 3'UTR corresponds to a part of the mRNA encoding human translation elongation factor eEF1A1. Thus, the largest open reading frame encodes a truncated version of eEF1A1 lacking the first 67 amino acids, while having three unique N-terminal amino acids. Previously, the UTRs were shown to be a prerequisite for the transforming capacity of the PTI-1 transcript. In this study, we have investigated the possible role of the UTRs in regulating protein expression and localization. METHODS: The protein expression profiles of a number of PTI-1 mRNA variants were studied in vitro and in vivo. Furthermore, the oncogenic potentials of the same PTI-1 mRNAs were determined by monitoring the capacities of stably transfected cells expressing these mRNAs to induce tumors in nude mice and form foci in cell culture. Finally, the cellular localizations of PTI-1 proteins expressed from these mRNAs were determined by fluorescence microscopy. RESULTS: The PTI-1 mRNA was found to give rise to multiple protein products that potentially originate from translation initiation at downstream, inframe AUGs within the major open reading frame. At least one of the truncated protein variants was also found to be oncogenic. However, the UTRs did not appear to influence the amount and identities of these truncated protein products. In contrast, our localization studies showed that the UTRs of the transcript promote a nuclear localization of the encoded protein(s). CONCLUSIONS: Translation of the PTI-1 mRNA results in multiple protein products of which (a) truncated variant(s) may play a predominant role during cellular transformation. The PTI-1 UTRs did not seem to play a role in translation regulation, but appeared to contribute to a nuclear localization of the PTI-1 protein(s). This indicates that the PTI-1 protein(s) exert(s) its/their oncogenic function inside the nucleus.

The Rag2⁻Il2rb⁻Dmd⁻ mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.

The development of innovative therapeutic strategies for muscular dystrophies, particularly cell-based approaches, is still a developing field. Although positive results have been obtained in animal models, they have rarely been confirmed in patients and resulted in very limited clinical improvements, suggesting some specificity in humans. These findings emphasized the need for an appropriate animal model (i.e., immunodeficient and dystrophic) to investigate in vivo the behavior of transplanted human myogenic stem cells. We report a new model, the Rag2(-)Il2rb(-)Dmd(-) mouse, which lacks T, B, and NK cells, and also carries a mutant Dmd allele that prevents the production of any dystrophin isoform. The dystrophic features of this new model are comparable with those of the classically used mdx mouse, but with the total absence of any revertant dystrophin positive fiber. We show that Rag2(-)Il2rb(-)Dmd(-) mice allow long-term xenografts of human myogenic cells. Altogether, our findings indicate that the Rag2(-)Il2rb(-)Dmd(-) mouse represents an ideal model to gain further insights into the behavior of human myogenic stem cells in a dystrophic context, and can be used to assess innovative therapeutic strategies for muscular dystrophies.

Kidney derived apolipoprotein M and its role in acute kidney injury.

Aim: Apolipoprotein M (apoM) is mainly expressed in liver and in proximal tubular epithelial cells in the kidney. In plasma, apoM associates with HDL particles via a retained signal peptide and carries sphingosine-1-phosphate (S1P), a small bioactive lipid. ApoM is undetectable in urine from healthy individuals but lack of megalin receptors in proximal tubuli cells induces loss of apoM into the urine. Besides this, very little is known about kidney-derived apoM. The aim of this study was to address the role of apoM in kidney biology and in acute kidney injury. Methods: A novel kidney-specific human apoM transgenic mouse model (RPTEC-hapoMTG) was generated and subjected to either cisplatin or ischemia/reperfusion injury. Further, a stable transfection of HK-2 cells overexpressing human apoM (HK-2-hapoMTG) was developed to study the pattern of apoM secretion in proximal tubuli cells. Results: Human apoM was present in plasma from RPTEC-hapoMTG mice (mean 0.18 µM), with a significant increase in plasma S1P levels. In vitro apoM was secreted to both the apical (urine) and basolateral (blood) compartment from proximal tubular epithelial cells. However, no differences in kidney injury score was seen between RPTEC-hapoMTG and wild type (WT) mice upon kidney injury. Further, gene expression of inflammatory markers (i.e., IL6, MCP-1) was similar upon ischemia/reperfusion injury. Conclusion: Our study suggests that kidney-derived apoM is secreted to plasma, supporting a role for apoM in sequestering molecules from excretion in urine. However, overexpression of human apoM in the kidney did not protect against acute kidney injury.

Improving laboratory animal genetic reporting: LAG-R guidelines.

The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework. LAG-R aims to document animals' genetic makeup in scientific publications, providing essential details for replication and appropriate model use. While verifying complete genetic compositions may be impractical, better reporting and validation efforts enhance reliability of research. LAG-R standardization will bolster reproducibility, peer review, and overall scientific rigor.

Liver-specific Aquaporin 11 knockout mice show rapid vacuolization of the rough endoplasmic reticulum in periportal hepatocytes after amino acid feeding.

Aquaporin 11 (AQP11) is a protein channel expressed intracellularly in multiple organs, yet its physiological function is unclear. Aqp11 knockout (KO) mice die early due to malfunction of the kidney, a result of hydropic degeneration of proximal tubule cells. Here we report the generation of liver-specific Aqp11 KO mice, allowing us to study the role of AQP11 protein in liver of mice with normal kidney function. The unchallenged liver-specific Aqp11 KO mice have normal longevity, their livers appeared normal, and the plasma biochemistries revealed only a minor defect in lipid handling. Fasting of the mice (24 h) induced modest dilatation of the rough endoplasmic reticulum (RER) in the periportal hepatocytes. Refeeding with standard mouse chow induced rapid generation of large RER-derived vacuoles in Aqp11 KO mice hepatocytes. Similar effects were observed following oral administration of pure protein or larger doses of various amino acids. The fasting/refeeding challenge is associated with increased expression of markers of ER stress Grp78 and GADD153 and decreased glutathione levels, suggesting that ER stress may play role in the development of vacuoles in the AQP11-deficient hepatocytes. NMR-based metabolome analysis of livers from mice subject to amino acid challenge showed decreased amount of extractable metabolites in the AQP11-deficient livers and particularly a decrease in glucose levels. In conclusion, in the liver, deletion of AQP11 results in disrupted RER homeostasis and increased sensitivity to RER injury upon metabolic challenge with amino acids.

Septin9 is involved in T-cell development and CD8+ T-cell homeostasis.

SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8(+) T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8(+) central memory T-cells.

Loss of vps54 function leads to vesicle traffic impairment, protein mis-sorting and embryonic lethality.

The identification of the mutation causing the phenotype of the amyotrophic lateral sclerosis (ALS) model mouse, wobbler, has linked motor neuron degeneration with retrograde vesicle traffic. The wobbler mutation affects protein stability of Vps54, a ubiquitously expressed vesicle-tethering factor and leads to partial loss of Vps54 function. Moreover, the Vps54 null mutation causes embryonic lethality, which is associated with extensive membrane blebbing in the neural tube and is most likely a consequence of impaired vesicle transport. Investigation of cells derived from wobbler and Vps54 null mutant embryos demonstrates impaired retrograde transport of the Cholera-toxin B subunit to the trans-Golgi network and mis-sorting of mannose-6-phosphate receptors and cargo proteins dependent on retrograde vesicle transport. Endocytosis assays demonstrate no difference between wobbler and wild type cells, indicating that the retrograde vesicle traffic to the trans-Golgi network, but not endocytosis, is affected in Vps54 mutant cells. The results obtained on wobbler cells were extended to test the use of cultured skin fibroblasts from human ALS patients to investigate the retrograde vesicle traffic. Analysis of skin fibroblasts of ALS patients will support the investigation of the critical role of the retrograde vesicle transport in ALS pathogenesis and might yield a diagnostic prospect.