Generation and characterization of a mouse model for the achalasia, alacrima and mental retardation (AAMR) syndrome

The AAMR syndrome is characterized by mental retardation and gait abnormalities as well as achalasia and alacrima. This disorder is inherited as an autosomal recessive trait and is caused by mutations in the Guanosine-diphosphate-(GDP)-mannose-pyrophosphorylase A (GMPPA) gene. GMPPA encodes the 420 aa protein GMPPA. Its homolog, the GDP-mannose-pyrophosphorylase B (GMPPB), converts mannose-1-phosphate and guanosine triphosphate (GTP) to GDP-mannose, which is an essential substrate for glycosylation. Up to date, 18 patients with inactivating mutations in the GMPPA gene have been reported worldwide. To elucidate the function of GMPPA in more detail we generated a Gmppa knockout (KO) mouse model. Importantly, these mice recapitulate many features of human AAMR syndrome patients, e.g. homozygous Gmppa KO mice show structural brain alterations. Moreover, Gmppa KO mice show a progressive gait disorder with muscle weakness accompanied by centralization of nuclei, alterations of the mean fiber diameter, the distribution of extracellular matrix (ECM) proteins and of the Z-disc related protein α-Actinin. In immunoblot analysis we found hyperglycosylation of proteins, especially hyperglycosylation of alpha-Dystroglycan (α-DG). Hyperglycosylated α-DG shows decreased protein stability and an increased binding to ECM proteins. Suggesting that the observed muscle phenotype is muscle intrinsic, sciatic nerve structure and nerve conduction velocities are normal in Gmppa KO mice. Mechanistically, elevated GDP-mannose levels and a direct interaction of GMPPA with GMPPB support a role of GMPPA as an allosteric feedback inhibitor of GMPPB. Gmppa knockdown studies in myoblasts revealed an increased α-DG turnover and activation of ERK signaling. In mice, a mannose-depleted diet dramatically improved the motor phenotype and almost normalized glycosylation of α-DG and ERK signaling. Thus, we propose that AAMR syndrome caused by GMPPA mutations is at least in part a treatable condition.Beim AAMR-Syndrom leiden Betroffene unter einer geistigen Behinderung, Gangstörungen sowie Achalasie und Alakrimie. Diese autosomal-rezessiv vererbbare Erkrankung wird durch Mutationen im Guanosin-Diphosphat-(GDP)-Mannose-Pyrophosphorylase A (GMPPA) Gen hervorgerufen, welches das 420 Aminosäuren lange Protein GMPPA kodiert. Das Homolog von GMPPA, die GDP-Mannose-Pyrophosphorylase B (GMPPB), konvertiert Mannose-1-Phosphat und Guanosin-Triphosphat (GTP) zu GDP-Mannose, welches ein essentielles Substrat der Glykosylierungskette darstellt. Bis heute sind nur 18 Patienten mit GMPPA-Mutationen beschrieben worden. Um die Funktion von GMPPA näher zu beleuchten, haben wir ein Gmppa-Knockout-(KO)-Mausmodell generiert. Diese Mäuse zeigen ein Krankheitsbild das viele Aspekte der humanen Patienten wiederspiegelt, so zeigen homozygote Gmppa-KO-Mäuse kognitive Defekte sowie strukturelle Veränderungen des Gehirns. Des Weiteren zeigen Gmppa-KO-Mäuse eine progressive Gangstörung mit zunehmender Muskelschwäche sowie Veränderungen extrazellulärer Matrix-(ECM) und Z-Disk-Proteine. Wir konnten an Gewebe-Lysaten zeigen, dass Proteine von Gmppa-KO-Mäusen, insbesondere auch alpha-Dystroglykan (α-DG), hyperglykosyliert sind. Die Hyperglykosylierung von α-DG führt zu einer verminderten Proteinstabilität und einer erhöhten Bindung an ECM-Proteine. Da in Gmppa-KO-Mäusen der Ischiasnerv morphologisch unverändert war und die elektrophysiologischen Eigenschaften unauffällig waren, gehen wir von einem muskelintrinsischen Defekt aus. Da die GDP-Mannose-Spiegel im Gewebe erhöht waren und eine direkte Interaktion von GMPPA mit GMPPB gezeigt werden konnte, vermuten wir, dass GMPPA als allosterischer Inhibitor von GMPPB fungiert. Gmppa-knockdown-Studien in Myoblasten lassen auf eine Aktivierung des ERK-Signalweges schließen. Wir konnten durch Gabe einer Mannose-freien Diät eine erhebliche Verbesserung der motorischen Auffälligkeiten sowie eine fast vollständige Normalisierung der Glykosylierung und der ERK-Aktivierung erreichen. Möglicherweise ist eine Mannose-freie Diät auch eine therapeutische Option für Patienten

Altered Glycosylation in the Aging Heart

Cardiovascular disease is one of the leading causes of death in developed countries. Because the incidence increases exponentially in the aging population, aging is a major risk factor for cardiovascular disease. Cardiac hypertrophy, fibrosis and inflammation are typical hallmarks of the aged heart. The molecular mechanisms, however, are poorly understood. Because glycosylation is one of the most common post-translational protein modifications and can affect biological properties and functions of proteins, we here provide the first analysis of the cardiac glycoproteome of mice at different ages. Western blot as well as MALDI-TOF based glycome analysis suggest that high-mannose N -glycans increase with age. In agreement, we found an age-related regulation of GMPPB, the enzyme, which facilitates the supply of the sugar-donor GDP-mannose. Glycoprotein pull-downs from heart lysates of young, middle-aged and old mice in combination with quantitative mass spectrometry bolster widespread alterations of the cardiac glycoproteome. Major hits are glycoproteins related to the extracellular matrix and Ca 2+ -binding proteins of the endoplasmic reticulum. We propose that changes in the heart glycoproteome likely contribute to the age-related functional decline of the cardiovascular system

Knockdown of INPP5K compromises the differentiation of N2A cells

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation

Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild typ

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.</p

Consequences of GMPPB deficiency for neuromuscular development and maintenance

Guanosine diphosphate-mannose pyrophosphorylase B (GMPPB) catalyzes the conversion of mannose-1-phosphate and GTP to GDP-mannose, which is required as a mannose donor for the biosynthesis of glycan structures necessary for proper cellular functions. Mutations in GMPPB have been associated with various neuromuscular disorders such as muscular dystrophy and myasthenic syndromes. Here, we report that GMPPB protein abundance increases during brain and skeletal muscle development, which is accompanied by an increase in overall protein mannosylation. To model the human disorder in mice, we generated heterozygous GMPPB KO mice using CIRSPR/Cas9. While we were able to obtain homozygous KO mice from heterozygous matings at the blastocyst stage, homozygous KO embryos were absent beyond embryonic day E8.5, suggesting that the homozygous loss of GMPPB results in early embryonic lethality. Since patients with GMPPB loss-of-function manifest with neuromuscular disorders, we investigated the role of GMPPB in vitro. Thereby, we found that the siRNA-mediated knockdown of Gmppb in either primary myoblasts or the myoblast cell line C2C12 impaired myoblast differentiation and resulted in myotube degeneration. siRNA-mediated knockdown of Gmppb also impaired the neuron-like differentiation of N2A cells. Taken together, our data highlight the essential role of GMPPB during development and differentiation, especially in myogenic and neuronal cell types

Presentation_1_Knockdown of INPP5K compromises the differentiation of N2A cells.pdf

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation.</p

Data_Sheet_1_Knockdown of INPP5K compromises the differentiation of N2A cells.docx

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation.</p

Altered mannose metabolism in chronic stress and depression is rapidly reversed by vitamin B12.

GDP-Mannose Pyrophosphorylase B (GMPPB) is a key enzyme for glycosylation. Previous studies suggested a dysregulation of GMPBB and mannose in depression. Evidence, however, was sporadic and interventions to reverse these changes are unknown. Here, we show that GMPPB protein, but not RNA abundance is increased in the postmortem prefrontal cortex (PFC) of depressed patients and the chronic variable stress (CVS) mouse-model. This is accompanied by higher plasma mannose levels. Importantly, a single dose of intraperitoneally administered vitamin B12, which has previously been shown to rapidly reverse behavioral symptoms and molecular signatures of chronic stress in mice, normalized GMPPB plasma mannose levels and elevated GDP-mannose abundance. In summary, these data underline metabolic dysregulation in chronic stress and depression and provide further support for rapid effects of vitamin B12 on chronic stress

Impact of Hypermannosylation on the Structure and Functionality of the ER and the Golgi Complex

Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway