Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.

Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

Genes implicated in translation control have been associated with autism spectrum disorders (ASDs). However, some important genetic causes of autism, including the 16p11.2 microdeletion, bear no obvious connection to translation. Here, we use proteomics, genetics, and translation assays in cultured cells and mouse brain to reveal altered translation mediated by loss of the kinase TAOK2 in 16p11.2 deletion models. We show that TAOK2 associates with the translational machinery and functions as a translational brake by phosphorylating eukaryotic elongation factor 2 (eEF2). Previously, all signal-mediated regulation of translation elongation via eEF2 phosphorylation was believed to be mediated by a single kinase, eEF2K. However, we show that TAOK2 can directly phosphorylate eEF2 on the same regulatory site, but functions independently of eEF2K signaling. Collectively, our results reveal an eEF2K-independent signaling pathway for control of translation elongation and suggest altered translation as a molecular component in the etiology of some forms of ASD.

Mice heterozygous for an osteogenesis imperfecta-linked MBTPS2 variant display a compromised subchondral osteocyte lacunocanalicular network associated with abnormal articular cartilage.

Missense variants in the MBTPS2 gene, located on the X chromosome, have been associated with an X-linked recessive form of osteogenesis imperfecta (X-OI), an inherited bone dysplasia characterized by multiple and recurrent bone fractures, short stature, and various skeletal deformities in affected individuals. The role of site-2 protease, encoded by MBTPS2, and the molecular pathomechanism underlying the disease are to date elusive. This study is the first to report on the generation of two Mbtps2 mouse models, a knock-in mouse carrying one of the disease-causative MBTPS2 variants (N455S) and a Mbtps2 knock-out (ko) mouse. Because both loss-of-function variants lead to embryonic lethality in hemizygous male mutant mice, we performed a comprehensive skeletal analysis of heterozygous Mbtps2+/N455S and Mbtps2+/ko female mice. Both models displayed osteochondral abnormalities such as thinned subchondral bone, altered subchondral osteocyte interconnectivity as well as thickened articular cartilage with chondrocyte clustering, altogether resembling an early osteoarthritis (OA) phenotype. However, distant from the joints, no alterations in the bone mass and turnover could be detected in either of the mutant mice. Based on our findings we conclude that MBTPS2 haploinsufficiency results in early OA-like alterations in the articular cartilage and underlying subchondral bone, which likely precede the development of typical OI phenotype in bone. Our study provides first evidence for a potential role of site-2 protease for maintaining homeostasis of both bone and cartilage.

hGFAP-mediated GLI2 overexpression leads to early death and severe cerebellar malformations with rare tumor formation.

The zinc-finger transcription factor GLI2 is frequently amplified in childhood medulloblastoma of the Sonic-hedgehog type (SHH-MB), with or without amplification of NMYC or deletion of TP53. Despite the aggressive tumor behavior, tumorigenesis is not well understood, and adequate mouse models are lacking. Therefore, we generated mice with a GLI2 overexpression under control of the hGFAP-promoter. These mice died within 150 days. The majority only survived until postnatal day 40. They displayed severe cerebellar hypoplasia, cortical malformations, but no brain tumors, except for one out of 23 animals with an undifferentiated hindbrain lesion. Additional loss of p53 did not result in cerebellar tumors, but partially rescued the cerebellar phenotype induced by GLI2 overexpression. Similarly, the combination of GLI2 and NMYC was neither sufficient for the development of SHH-MB. We therefore assume that the development of childhood SHH-MB in mice is either occurring in cellular origins outside the hGFAP-positive lineage or needs additional genetic drivers.

Introduction of a novel chimeric active immunization mouse model of PLA2R1-associated membranous nephropathy.

The phospholipase A2 receptor 1 (PLA2R1) is the major target antigen in patients with membranous nephropathy (MN), an antibody-mediated autoimmune glomerular disease. Investigation of MN pathogenesis has been hampered by the lack of reliable animal models. Here, we overcome this issue by generating a transgenic mouse line expressing a chimeric PLA2R1 (chPLA2R1) consisting of three human PLA2R1 domains (cysteine-rich, fibronectin type-II and CTLD1) and seven murine PLA2R1 domains (CTLD2-8) specifically in podocytes. Mice expressing the chPLA2R1 were healthy at birth and showed no major glomerular alterations when compared to mice with a wild-type PLA2R1 status. Upon active immunization with human PLA2R1 (hPLA2R1), chPLA2R1-positive mice developed anti-hPLA2R1 antibodies, a nephrotic syndrome, and all major histological features of MN, including granular deposition of mouse IgG and complement components in immunofluorescence and subepithelial electron-dense deposits and podocyte foot process effacement in electron microscopy. In order to investigate the role of the complement system in this model, we further crossed chPLA2R1-positive mice with mice lacking the central complement component C3 (C3-/- mice). Upon immunization with hPLA2R1, chPLA2R1-positive C3-/- mice had substantially less severe albuminuria and nephrotic syndrome when compared to chPLA2R1-positive mice with a wild-type C3 status. In conclusion, we introduce a novel active immunization model of PLA2R1-associated MN and demonstrate a pathogenic role of the complement system in this model.

Non-functional ubiquitin C-terminal hydrolase L1 drives podocyte injury through impairing proteasomes in autoimmune glomerulonephritis.

Little is known about the mechanistic significance of the ubiquitin proteasome system (UPS) in a kidney autoimmune environment. In membranous nephropathy (MN), autoantibodies target podocytes of the glomerular filter resulting in proteinuria. Converging biochemical, structural, mouse pathomechanistic, and clinical information we report that the deubiquitinase Ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced by oxidative stress in podocytes and is directly involved in proteasome substrate accumulation. Mechanistically, this toxic gain-of-function is mediated by non-functional UCH-L1, which interacts with and thereby impairs proteasomes. In experimental MN, UCH-L1 becomes non-functional and MN patients with poor outcome exhibit autoantibodies with preferential reactivity to non-functional UCH-L1. Podocyte-specific deletion of UCH-L1 protects from experimental MN, whereas overexpression of non-functional UCH-L1 impairs podocyte proteostasis and drives injury in mice. In conclusion, the UPS is pathomechanistically linked to podocyte disease by aberrant proteasomal interactions of non-functional UCH-L1.

Podocyte expression of human phospholipase A2 receptor 1 causes immune-mediated membranous nephropathy in mice.

Antibody-mediated autoimmune pathologies like membranous nephropathy are difficult to model, particularly in the absence of local target antigen expression in model organisms such as mice and rats; as is the case for phospholipase A2 receptor 1 (PLA2R1), the major autoantigen in membranous nephropathy. Here, we generated a transgenic mouse line expressing the full-length human PLA2R1 in podocytes, which has no kidney impairment after birth. Beginning from the age of three weeks, these mice spontaneously developed anti-human PLA2R1 antibodies, a nephrotic syndrome with progressive albuminuria and hyperlipidemia, and the typical morphological signs of membranous nephropathy with granular glomerular deposition of murine IgG in immunofluorescence and subepithelial electron-dense deposits by electron microscopy. Importantly, human PLA2R1-expressing Rag2-/- mice, which lack mature and functioning B and T lymphocytes, developed neither anti-PLA2R1 antibodies nor proteinuria. Thus, our work demonstrates that podocyte expression of human PLA2R1 can induce membranous nephropathy with an underlying antibody-mediated pathogenesis in mice. Importantly, this antibody-mediated model enables proof-of-concept evaluations of antigen-specific treatment strategies, e.g., targeting autoantibodies or autoantibody-producing cells, and may further help understand the autoimmune pathogenesis of membranous nephropathy.

Disruption of tubulin-alpha4a polyglutamylation prevents aggregation of hyper-phosphorylated tau and microglia activation in mice.

Dissociation of hyper-phosphorylated Tau from neuronal microtubules and its pathological aggregates, are hallmarks in the etiology of tauopathies. The Tau-microtubule interface is subject to polyglutamylation, a reversible posttranslational modification, increasing negative charge at tubulin C-terminal tails. Here, we asked whether tubulin polyglutamylation may contribute to Tau pathology in vivo. Since polyglutamylases modify various proteins other than tubulin, we generated a knock-in mouse carrying gene mutations to abolish Tuba4a polyglutamylation in a substrate-specific manner. We found that Tuba4a lacking C-terminal polyglutamylation prevents the binding of Tau and GSK3 kinase to neuronal microtubules, thereby strongly reducing phospho-Tau levels. Notably, crossbreeding of the Tuba4a knock-in mouse with the hTau tauopathy model, expressing a human Tau transgene, reversed hyper-phosphorylation and oligomerization of Tau and normalized microglia activation in brain. Our data highlight tubulin polyglutamylation as a potential therapeutic strategy in fighting tauopathies.

The calcium-sensing receptor stabilizes podocyte function in proteinuric humans and mice.

Calcimimetic agents allosterically increase the calcium ion sensitivity of the calcium-sensing receptor (CaSR), which is expressed in the tubular system and to a lesser extent in podocytes. Activation of this receptor can reduce glomerular proteinuria and structural damage in proteinuric animal models. However, the precise role of the podocyte CaSR remains unclear. Here, a CaSR knockdown in cultured murine podocytes and a podocyte-specific CaSR knockout in BALB/c mice were generated to study its role in proteinuria and kidney function. Podocyte CaSR knockdown abolished the calcimimetic R-568 mediated calcium ion-influx, disrupted the actin cytoskeleton, and reduced cellular attachment and migration velocity. Adriamycin-induced proteinuria enhanced glomerular CaSR expression in wild-type mice. Albuminuria, podocyte foot process effacement, podocyte loss and glomerular sclerosis were significantly more pronounced in adriamycin-treated podocyte-specific CaSR knockout mice compared to wild-type littermates. Co-treatment of wild-type mice with adriamycin and the calcimimetic cinacalcet reduced proteinuria in wild-type, but not in podocyte-specific CaSR knockout mice. Additionally, four children with nephrotic syndrome, whose parents objected to glucocorticoid therapy, were treated with cinacalcet for one to 33 days. Proteinuria declined transiently by up to 96%, serum albumin increased, and edema resolved. Thus, activation of podocyte CaSR regulates key podocyte functions in vitro and reduced toxin-induced proteinuria and glomerular damage in mice. Hence, our findings suggest a potential novel role of CaSR signaling in control of glomerular disease.

Amelioration of the abnormal phenotype of a new L1 syndrome mouse mutation with L1 mimetics.

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.

Revisiting the proteolytic processing of cell adhesion molecule L1.

The important functions of cell adhesion molecule L1 in the nervous system depend on diverse proteolytic enzymes which generate different L1 fragments. It has been reported that cleavage in the third fibronectin type III (FNIII) homologous domain generates the fragments L1-80 and L1-140, while cleavage in the first FNIII domain yields the fragments L1-70 and L1-135. These results raised questions concerning the L1 cleavage sites. We thus generated gene-edited mice expressing L1 with mutations of the cleavage sites either in the first or third FNIII domain. By immunoprecipitations and immunoblot analyses using brain homogenates and different L1 antibodies, we show that L1-70 and L1-135 are generated in wild-type mice, but not or only to a low extent in L1 mutant mice. L1-80 and L1-140 were not detected in wild-type or mutant mice. Mass spectrometry confirmed the results from immunoprecipitations and immunoblot analyses. Based on these observations, we propose that L1-70 and L1-135 are the predominant fragments in the mouse nervous system and that the third FNIII domain is decisive for generating these fragments. Treatment of cultured cerebellar neurons with trypsin or plasmin, which were both proposed to generate L1-80 and L1-140 by cleaving in the third FNIII domain, showed by immunoprecipitations and immunoblot analyses that both proteases lead to the generation of L1-70 and L1-135, but not L1-80 and L1-140. We discuss previous observations on the basis of our new results and propose a novel view on the molecular features that render previous and present observations compatible.

Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits.

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

Mice Carrying a Ubiquitous R235W Mutation of Wnt1 Display a Bone-Specific Phenotype.

Since a key function of Wnt1 in brain development was established early on through the generation of non-viable Wnt1-deficient mice, it was initially surprising that WNT1 mutations were found to cause either early-onset osteoporosis (EOOP) or osteogenesis imperfecta type XV (OI-XV). The deduced function of Wnt1 as an osteoanabolic factor has been confirmed in various mouse models with bone-specific inactivation or overexpression, but mice carrying disease-causing Wnt1 mutations have not yet been described. Triggered by the clinical analysis of EOOP patients carrying a heterozygous WNT1 mutation (p.R235W), we introduced this mutation into the murine Wnt1 gene to address the question of whether this would cause a skeletal phenotype. We observed that Wnt1+/R235W and Wnt1R235W/R235W mice were born at the expected Mendelian ratio and that they did not display postnatal lethality or obvious nonskeletal phenotypes. At 12 weeks of age, the homozygous presence of the Wnt1 mutation was associated with reduced trabecular and cortical bone mass, explained by a lower bone formation rate compared with wild-type littermates. At 52 weeks of age, we also observed a moderate bone mass reduction in heterozygous Wnt1+/R235W mice, thereby underscoring their value as a model of WNT1-dependent EOOP. Importantly, when we treated wild-type and Wnt1+/R235W mice by daily injection of parathyroid hormone (PTH), we detected the same osteoanabolic influence in both groups, together with an increased cortical thickness in the mutant mice. Our data demonstrate the pathogenicity of the WNT1-R235W mutation, confirm that controlling skeletal integrity is the primary physiological function of Wnt1, and suggest that osteoanabolic treatment with teriparatide should be applicable for individuals with WNT1-dependent EOOP. © 2020 American Society for Bone and Mineral Research.

A novel mouse model of phospholipase A2 receptor 1-associated membranous nephropathy mimics podocyte injury in patients.

The phospholipase A2 receptor 1 (PLA2R1) is the major autoantigen in patients suffering from membranous nephropathy. To date, the lack of endogenous glomerular expression of PLA2R1 in mice and rats has impeded the establishment of PLA2R1-dependent animal models of this disease. Here, we generated a transgenic mouse line expressing murine full-length PLA2R1 in podocytes. Furthermore, expression of murine PLA2R1 did not result in any morphological disturbance as high-resolution confocal microscopy demonstrated an intact nephrin distribution with normal foot processes. Transfer of rabbit anti-mPLA2R1 antibodies to these mice induced nephrotic range proteinuria, hypercholesterolemia, and histomorphological signs of membranous nephropathy. Immunohistochemical and immunofluorescence analyses revealed enhanced staining for murine PLA2R1 in the presence of unaffected staining for murine thrombospondin type-1 domain-containing 7A in the diseased mice, resembling what is classically found in patients with PLA2R1-associated membranous nephropathy Thus, our mouse model of membranous nephropathy will allow investigation of PLA2R1-specific pathomechanisms and may help to develop and assess antigen-specific treatments in vivo.

Segregated Expression of ENaC Subunits in Taste Cells.

Salt taste is one of the 5 basic taste qualities. Depending on the concentration, table salt is perceived either as appetitive or aversive, suggesting the contribution of several mechanisms to salt taste, distinguishable by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. A taste-specific knockout of the α-subunit of the ENaC revealed the relevance of this polypeptide for low-salt transduction, whereas the response to other taste qualities remained normal. The fully functional ENaC is composed of α-, ß-, and γ-subunits. In taste tissue, however, the precise constitution of the channel and the cell population responsible for detecting table salt remain uncertain. In order to examine the cells and subunits building the ENaC, we generated mice carrying modified alleles allowing the synthesis of green and red fluorescent proteins in cells expressing the α- and ß-subunit, respectively. Fluorescence signals were detected in all types of taste papillae and in taste buds of the soft palate and naso-incisor duct. However, the lingual expression patterns of the reporters differed depending on tongue topography. Additionally, immunohistochemistry for the γ-subunit of the ENaC revealed a lack of overlap between all potential subunits. The data suggest that amiloride-sensitive recognition of table salt is unlikely to depend on the classical ENaCs formed by α-, ß-, and γ-subunits and ask for a careful investigation of the channel composition.

Sox9 overexpression exerts multiple stage-dependent effects on mouse spinal cord development.

The high-mobility-group (HMG)-domain protein Sox9 is one of few transcription factors implicated in gliogenesis in the vertebrate central nervous system. To further study the role of Sox9 in early spinal cord development, we generated a mouse that allows expression of Sox9 in a temporally and spatially controlled manner. Using this mouse, we show that premature Sox9 expression in neural precursor cells disrupted the neuroepithelium of the ventricular zone. Sox9 also compromised development and survival of neuronal precursors and neurons. Additionally, we observed in these mice substantial increases in oligodendroglial and astroglial cells. Reversing the normal order of appearance of essential transcriptional regulators during oligodendrogenesis, Sox10 preceded Olig2. Our study reinforces the notion that Sox9 has a strong gliogenic activity. It also argues that Sox9 expression has to be tightly controlled to prevent negative effects on early spinal cord structure and neuronal development.

The Microtubule Severing Protein Katanin Regulates Proliferation of Neuronal Progenitors in Embryonic and Adult Neurogenesis.

Microtubule severing regulates cytoskeletal rearrangement underlying various cellular functions. Katanin, a heterodimer, consisting of catalytic (p60) and regulatory (p80) subunits severs dynamic microtubules to modulate several stages of cell division. The role of p60 katanin in the mammalian brain with respect to embryonic and adult neurogenesis is poorly understood. Here, we generated a Katna1 knockout mouse and found that consistent with a critical role of katanin in mitosis, constitutive homozygous Katna1 depletion is lethal. Katanin p60 haploinsufficiency induced an accumulation of neuronal progenitors in the subventricular zone during corticogenesis, and impaired their proliferation in the adult hippocampus dentate gyrus (DG) subgranular zone. This did not compromise DG plasticity or spatial and contextual learning and memory tasks employed in our study, consistent with the interpretation that adult neurogenesis may be associated with selective forms of hippocampal-dependent cognitive processes. Our data identify a critical role for the microtubule-severing protein katanin p60 in regulating neuronal progenitor proliferation in vivo during embryonic development and adult neurogenesis.

Myosin XVI Regulates Actin Cytoskeleton Dynamics in Dendritic Spines of Purkinje Cells and Affects Presynaptic Organization.

The actin cytoskeleton is crucial for function and morphology of neuronal synapses. Moreover, altered regulation of the neuronal actin cytoskeleton has been implicated in neuropsychiatric diseases such as autism spectrum disorder (ASD). Myosin XVI is a neuronally expressed unconventional myosin known to bind the WAVE regulatory complex (WRC), a regulator of filamentous actin (F-actin) polymerization. Notably, the gene encoding the myosin's heavy chain (MYO16) shows genetic association with neuropsychiatric disorders including ASD. Here, we investigated whether myosin XVI plays a role for actin cytoskeleton regulation in the dendritic spines of cerebellar Purkinje cells (PCs), a neuronal cell type crucial for motor learning, social cognition and vocalization. We provide evidence that both myosin XVI and the WRC component WAVE1 localize to PC spines. Fluorescence recovery after photobleaching (FRAP) analysis of GFP-actin in cultured PCs shows that Myo16 knockout as well as PC-specific Myo16 knockdown, lead to faster F-actin turnover in the dendritic spines of PCs. We also detect accelerated F-actin turnover upon interference with the WRC, and upon inhibition of Arp2/3 that drives formation of branched F-actin downstream of the WRC. In contrast, inhibition of formins that are responsible for polymerization of linear actin filaments does not cause faster F-actin turnover. Together, our data establish myosin XVI as a regulator of the postsynaptic actin cytoskeleton and suggest that it is an upstream activator of the WRC-Arp2/3 pathway in PC spines. Furthermore, ultra-structural and electrophysiological analyses of Myo16 knockout cerebellum reveals the presence of reduced numbers of synaptic vesicles at presynaptic terminals in the absence of the myosin. Therefore, we here define myosin XVI as an F-actin regulator important for presynaptic organization in the cerebellum.

A Mouse Model of Creatine Transporter Deficiency Reveals Impaired Motor Function and Muscle Energy Metabolism.

Creatine serves as fast energy buffer in organs of high-energy demand such as brain and skeletal muscle. L-Arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase are responsible for endogenous creatine synthesis. Subsequent uptake into target organs like skeletal muscle, heart and brain is mediated by the creatine transporter (CT1, SLC6A8). Creatine deficiency syndromes are caused by defects of endogenous creatine synthesis or transport and are mainly characterized by intellectual disability, behavioral abnormalities, poorly developed muscle mass, and in some cases also muscle weakness. CT1-deficiency is estimated to be among the most common causes of X-linked intellectual disability and therefore the brain phenotype was the main focus of recent research. Unfortunately, very limited data concerning muscle creatine levels and functions are available from patients with CT1 deficiency. Furthermore, different CT1-deficient mouse models yielded conflicting results and detailed analyses of their muscular phenotype are lacking. Here, we report the generation of a novel CT1-deficient mouse model and characterized the effects of creatine depletion in skeletal muscle. HPLC-analysis showed strongly reduced total creatine levels in skeletal muscle and heart. MR-spectroscopy revealed an almost complete absence of phosphocreatine in skeletal muscle. Increased AGAT expression in skeletal muscle was not sufficient to compensate for insufficient creatine transport. CT1-deficient mice displayed profound impairment of skeletal muscle function and morphology (i.e., reduced strength, reduced endurance, and muscle atrophy). Furthermore, severely altered energy homeostasis was evident on magnetic resonance spectroscopy. Strongly reduced phosphocreatine resulted in decreased ATP/Pi levels despite an increased inorganic phosphate to ATP flux. Concerning glucose metabolism, we show increased glucose transporter type 4 expression in muscle and improved glucose clearance in CT1-deficient mice. These metabolic changes were associated with activation of AMP-activated protein kinase - a central regulator of energy homeostasis. In summary, creatine transporter deficiency resulted in a severe muscle weakness and atrophy despite different compensatory mechanisms.