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.

A mouse model for SPG48 reveals a block of autophagic flux upon disruption of adaptor protein complex five.

Hereditary spastic paraplegia is a spastic gait disorder that arises from degeneration of corticospinal axons. The subtype SPG48 is associated with mutations in the zeta subunit of the adaptor protein complex five (AP5). AP5 function and the pathophysiology of SPG48 are only poorly understood. Here, we report an AP5 zeta knockout mouse, which shows an age-dependent degeneration of corticospinal axons. Our analysis of knockout fibroblasts supports a trafficking defect from late endosomes to the transGolgi network and reveals a structural defect of the Golgi. We further show that both autophagic flux and the recycling of lysosomes from autolysosomes were impaired in knockout cells. In vivo, we observe an increase of autophagosomes and autolysosomes and, at later stages, the accumulation of intracellular waste in neurons. Taken together, we propose that loss of AP5 function blocks autophagy and thus leads to the aberrant accumulation of autophagic cargo, which finally results in axon degeneration.

In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

A novel mutation in GMPPA in siblings with apparent intellectual disability, epilepsy, dysmorphism, and autonomic dysfunction.

GMPPA encodes the GDP-mannose pyrophosphorylase A protein (GMPPA). The function of GMPPA is not well defined, however it is a homolog of GMPPB which catalyzes the reaction that converts mannose-1-phosphate and guanosine-5'-triphosphate to GDP-mannose. Previously, biallelic mutations in GMPPA were reported to cause a disorder characterized by achalasia, alacrima, neurological deficits, and intellectual disability. In this study, we report a female proband with achalasia, alacrima, hypohydrosis, apparent intellectual disability, seizures, microcephaly, esotropia, and craniofacial dysmorphism. Exome sequencing identified a previously unreported homozygous c.853+1G>A variant in GMPPA in the proband and her affected sister. Their unaffected parents were heterozygous, and unaffected brother homozygous wild type for this variant. Lymphoblast cells from the affected sisters showed complete loss of the GMPPA protein by Western blotting, and increased levels of GDP-mannose in lymphoblasts on high performance liquid chromatography. Based on our findings and the previous report describing patients with an overlapping phenotype, we conclude that this novel variant in GMPPA, identified by exome sequencing in the proband and her affected sister, is the genetic cause of their phenotype and may expand the known phenotype of this recently described glycosylation disorder.

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.

Ubiquitination contributes to the regulation of GDP-mannose pyrophosphorylase B activity.

GDP-mannose pyrophosphorylase B (GMPPB) loss-of-function is associated with muscular dystrophy and variable additional neurological symptoms. GMPPB facilitates the catalytic conversion of mannose-1-phosphate and GTP to GDP-mannose, which serves as a mannose donor for glycosylation. The activity of GMPPB is regulated by its non-catalytic paralogue GMPPA, which can bind GDP-mannose and interact with GMPPB, thereby acting as an allosteric feedback inhibitor of GMPPB. Using pulldown, immunoprecipitation, turnover experiments as well as immunolabeling and enzyme activity assays, we provide first direct evidence that GMPPB activity is regulated by ubiquitination. We further show that the E3 ubiquitin ligase TRIM67 interacts with GMPPB and that knockdown of TRM67 reduces ubiquitination of GMPPB, thus reflecting a candidate E3 ligase for the ubiquitination of GMPPB. While the inhibition of GMPPB ubiquitination decreases its enzymatic activity, its ubiquitination neither affects its interaction with GMPPA nor its turnover. Taken together, we show that the ubiquitination of GMPPB represents another level of regulation of GDP-mannose supply.

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.

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.

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.

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 Ca2+-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.

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 type.

GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation.

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.