Georg Haase

Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity.

Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies. They represent ways for discarding proteins and metabolites and also for intercellular transfer of proteins and RNAs. In the nervous system, it has been hypothesized that exosomes might be involved in the normal physiology of the synapse and possibly allow the trans-synaptic propagation of pathogenic proteins throughout the tissue. As a first step to validate this concept, we used biochemical and morphological approaches to demonstrate that mature cortical neurons in culture do indeed secrete exosomes. Using electron microscopy, we observed exosomes being released from somato-dendritic compartments. The endosomal origin of exosomes was demonstrated by showing that the C-terminal domain of tetanus toxin specifically endocytosed by neurons and accumulating inside multivesicular bodies, is released in the extracellular medium in association with exosomes. Finally, we found that exosomal release is modulated by glutamatergic synaptic activity, suggesting that this process might be part of normal synaptic physiology. Thus, our study paves the way towards the demonstration that exosomes take part in the physiology of the normal and pathological nervous system.

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2

In healthy adults, activation of γ-aminobutyric acid (GABA)A and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl–]i), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), EIPSP, in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT2A receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT2AR agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

Signaling by death receptors in the nervous system.

Cell death plays an important role both in shaping the developing nervous system and in neurological disease and traumatic injury. In spite of their name, death receptors can trigger either cell death or survival and growth. Recent studies implicate five death receptors--Fas/CD95, TNFR1 (tumor necrosis factor receptor-1), p75NTR (p75 neurotrophin receptor), DR4, and DR5 (death receptors-4 and -5)--in different aspects of neural development or degeneration. Their roles may be neuroprotective in models of Parkinsons disease, or pro-apoptotic in ALS and stroke. Such different outcomes probably reflect the diversity of transcriptional and posttranslational signaling pathways downstream of death receptors in neurons and glia.

Adenoviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy

Cardiotrophin-1 (CT-1), an IL-6-related cytokine, causes hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types, including motoneurons. Here, we analyzed systemic CT-1 effects in progressive motor neuronopathy (pmn) mice that suffer from progressive motoneuronal degeneration, muscle paralysis, and premature death. Administration of an adenoviral CT-1 vector to newborn pmn mice leads to sustained CT-1 expression in the injected muscles and bloodstream, prolonged survival of animals, and improved motor functions. CT-1-treated pmn mice showed a significantly reduced degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. The terminal innervation of skeletal muscle, grossly disturbed in untreated pmn mice, was almost completely preserved in CT-1-treated pmn mice. The remarkable neuroprotection conferred by CT-1 might become clinically relevant if CT-1 side effects, including cardiotoxicity, could be circumvented by a more targeted delivery of this cytokine to the nervous system.

Progressive Motor Neuronopathy: A Critical Role of the Tubulin Chaperone TBCE in Axonal Tubulin Routing from the Golgi Apparatus

Axonal degeneration represents one of the earliest pathological features in motor neuron diseases. We here studied the underlying molecular mechanisms in progressive motor neuronopathy (pmn) mice mutated in the tubulin-specific chaperone TBCE. We demonstrate that TBCE is a peripheral membrane-associated protein that accumulates at the Golgi apparatus. In pmn mice, TBCE is destabilized and disappears from the Golgi apparatus of motor neurons, and microtubules are lost in distal axons. The axonal microtubule loss proceeds retrogradely in parallel with the axonal dying back process. These degenerative changes are inhibited in a dose-dependent manner by transgenic TBCE complementation that restores TBCE expression at the Golgi apparatus. In cultured motor neurons, the pmn mutation, interference RNA-mediated TBCE depletion, and brefeldin A-mediated Golgi disruption all compromise axonal tubulin routing. We conclude that motor axons critically depend on axonal tubulin routing from the Golgi apparatus, a process that involves TBCE and possibly other tubulin chaperones.

Golgi fragmentation in pmn mice is due to a defective ARF1/TBCE cross-talk that coordinates COPI vesicle formation and tubulin polymerization

Golgi fragmentation is an early hallmark of many neurodegenerative diseases but its pathophysiological relevance and molecular mechanisms are unclear. We here demonstrate severe and progressive Golgi fragmentation in motor neurons of progressive motor neuronopathy (pmn) mice due to loss of the Golgi-localized tubulin-binding cofactor E (TBCE). Loss of TBCE in mutant pmn and TBCE-depleted motor neuron cultures causes defects in Golgi-derived microtubules, as expected, but surprisingly also reduced levels of COPI subunits, decreased recruitment of tethering factors p115/GM130 and impaired Golgi SNARE-mediated vesicle fusion. Conversely, ARF1, which stimulates COPI vesicle formation, enhances the recruitment of TBCE to the Golgi, increases polymerization of Golgi-derived microtubules and rescues TBCE-linked Golgi fragmentation. These data indicate an ARF1/TBCE-mediated cross-talk that coordinates COPI formation and tubulin polymerization at the Golgi. We conclude that interruption of this cross-talk causes Golgi fragmentation in pmn mice and hypothesize that similar mechanisms operate in human amyotrophic lateral sclerosis and spinal muscular atrophy.

Golgi Fragmentation in ALS Motor Neurons. New Mechanisms Targeting Microtubules, Tethers, and Transport Vesicles

Pathological alterations of the Golgi apparatus, such as its fragmentation represent an early pre-clinical feature of many neurodegenerative diseases and have been widely studied in the motor neuron disease amyotrophic lateral sclerosis (ALS). Yet, the underlying molecular mechanisms have remained cryptic. In principle, Golgi fragmentation may result from defects in three major classes of proteins: structural Golgi proteins, cytoskeletal proteins and molecular motors, as well as proteins mediating transport to and through the Golgi. Here, we present the different mechanisms that may underlie Golgi fragmentation in animal and cellular models of ALS linked to mutations in SOD1, TARDBP (TDP-43), VAPB, and C9Orf72 and we propose a novel one based on findings in progressive motor neuronopathy (pmn) mice. These mice are mutated in the TBCE gene encoding the cis-Golgi localized tubulin-binding cofactor E, one of five chaperones that assist in tubulin folding and microtubule polymerization. Loss of TBCE leads to alterations in Golgi microtubules, which in turn impedes on the maintenance of the Golgi architecture. This is due to down-regulation of COPI coat components, dispersion of Golgi tethers and strong accumulation of ER-Golgi SNAREs. These effects are partially rescued by the GTPase ARF1 through recruitment of TBCE to the Golgi. We hypothesize that defects in COPI vesicles, microtubules and their interaction may also underlie Golgi fragmentation in human ALS linked to other mutations, spinal muscular atrophy (SMA), and related motor neuron diseases. We also discuss the functional relevance of pathological Golgi alterations, in particular their potential causative, contributory, or compensatory role in the degeneration of motor neuron cell bodies, axons and synapses.

TBCE Mutations Cause Early-Onset Progressive Encephalopathy with Distal Spinal Muscular Atrophy

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration.

Editorial: Golgi Pathology in Neurodegenerative Diseases

The Golgi apparatus is a central organelle that lies at the heart of the secretory pathway sustaining the delivery of proteins from their site of synthesis in the endoplasmic reticulum to their final destination, the extracellular medium, the plasma membrane, and the endo-lysosomal system. It ensures post-translational protein modifications such as glycosylation and proteolytic cleavage and processing and acts as a sorting device including to neuronal axons and dendrites (Horton and Ehlers, 2003; Ye et al., 2007). n nThe mammalian Golgi apparatus was first described by Camillo Golgi in 1998 as “apparato reticolare interno,” “a fine and elegant network within the cell body … completely internal in the nerve cells” (Golgi, 1898a,b). This large reticulum comprises stacks of flattened membrane bound compartments called cisternae which are laterally linked to form the so-called Golgi ribbon. n nStructural and functional alterations of the Golgi apparatus, which are here collectively termed Golgi pathology, are now recognized as a constant pathological hallmark of various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Parkinson, Alzheimer, Huntington, and prion diseases (Fan et al., 2008). In ALS, structural Golgi alterations have been revealed by the pioneering work of Gonatas and colleagues (Mourelatos et al., 1990; Gonatas et al., 1992; Fujita et al., 2002). They manifest as fragmentation—transformation of the Golgi ribbon into disconnected stacks, cisternae, tubules and vesicles, and as atrophy—loss of Golgi membrane material. n nThese morphological changes are often accompanied by functional Golgi alterations, such as those affecting the anterograde and retrograde transport in the early secretory pathway, both in cellular models of Parkinson (Cooper et al., 2006; Cho et al., 2014), Huntington (Caviston et al., 2007; Pardo et al., 2010), and Alzheimer (Annaert et al., 1999; Joshi et al., 2014) diseases as well as in ALS (Stieber et al., 2004; Soo et al., 2015). n nAt least in ALS, Golgi pathology manifests as an early pre-clinical feature in degenerating neurons both in affected patients and in animal models (Mourelatos et al., 1996), suggesting that it may be relevant to the disease process instead of just representing an epiphenomenon. Yet, neither the molecular mechanisms underlying the changes in the functional organization of the Golgi apparatus nor their precise relevance to neurodegeneration have yet been completely elucidated. n nThese important questions got a new boost by the discovery of mutations in genes encoding Golgi-related proteins as direct causes of neurodegeneration. For instance, mutations in Optineurin (Maruyama et al., 2010), VPS54/wobbler (Schmitt-John et al., 2005), and TBCE/pmn (Martin et al., 2002) have been identified in ALS and related motor neuron diseases. Furthermore, mutations in the Parkinson disease-associated proteins α-Synuclein (Cooper et al., 2006; Thayanidhi et al., 2010), LRRK2 (Lin et al., 2009; Cho et al., 2014), Parkin (Shimura et al., 1999; Kubo et al., 2001), and VPS35 (McGough et al., 2014; Zavodszky et al., 2014; Malik et al., 2015) have been shown to affect Golgi structure or transport processes to and from the Golgi. n nFurthermore, the recognition of Golgi-derived microtubules and their specific functions, the better understanding of Golgi transport processes, the recognition of the Golgi apparatus as a sensor of cellular stress and as trigger of Golgi-specific cell death pathways provide new hints to the molecular mechanisms underlying Golgi pathology. n nTo cover these emerging themes, this Frontiers Research Topic is organized as follows. The issue starts with a summary on Golgi functional organization in neurons (Valenzuela and Perez) and the relation of this organelle with microtubules (Sanders and Kaverina). n nThis is followed by pathological, genetic, and mechanistic descriptions of the major neurodegenerative diseases including Parkinson disease (Wang and Hay), Alzheimer disease by Wang and colleagues (Joshi et al.) and ALS by Atkin and colleagues (Sundaramoorthy et al.). The Research topic then focuses on Golgi fragmentation brought about by defects in vesicle biogenesis and dynamics to and through the Golgi by Lupashin and colleagues (Climer et al.) and by Schmitt-John, including those caused by defects in Golgi-derived microtubules in ALS (Haase and Rabouille) and microtubule-dependent motors in proximal SMA (Jaarsma and Hoogenraad; Wirth and Martinez-Carrera). n nThe third part of this issue starts by posing the hypothesis of Farhan and colleagues that cellular stress can be the cause of Golgi fragmentation, which in turn amplifies cellular stress and leads to neurodegeneration (Alvarez-Miranda et al.). This is argued by reviews on the effect of DNA damage on the Golgi by Field and colleagues (Buschman et al.) and on the role of the Golgi as a cell death trigger (Machamer). n nFuture studies on Golgi pathology in neurodegenerative diseases will continue to benefit not only from conceptual advances but also from new technical developements that have been gigantic since Golgis original description of the black reaction (tissue hardening with potassium dichromate and cell staining by silver impregnation) (Mazzarello et al., 2009). n nElectron microscopy has been used to unravel Golgi fragmentation (Mourelatos et al., 1996) and in particular the Golgi fragmentation into tubules and vesicles observed in degenerating motor neurons (Bellouze et al., 2014), and its resolution may be further improved in tissues prepared by high pressure freezing (Walther et al., 2013). 3D reconstructions of the Golgi and its microtubules (Marsh et al., 2001; Efimov et al., 2007) may illustrate pathological changes in their intricate connections. n nGolgi fragmentation can also be monitored by live imaging (Altan-Bonnet et al., 2006), and super resolution microscopy (Betzig et al., 2006; Lippincott-Schwartz and Manley, 2009) may help refining the process. Last, system biology approches may shed light on new pathways connecting Golgi fragmentation to neurodegeneration by identifying novel gene networks (Alvarez-Miranda et al.). n nHowever, this field faces further challenges. It will be crucial to determine whether Golgi pathology is contributory, causative or homeostatic in neurodegeneration. In particular, it is important to understand whether Golgi alterations are linked to axonal degeneration and synapse loss or dysfunction. n nIt will also be crucial to analyze whether Golgi pathology in each neurodegenerative disease is restricted to the neuron types that are specifically affected, i.e., motor neurons in ALS, dopaminergic neurons in PD, striatal neurons in Huntington. If so, what may be the corresponding mechanisms of vulnerability and resistance? n nFurthermore, we will need to determine whether Golgi alterations in degenerating neurons impact on the function of their non-neuronal cellular neighbors, including astrocytes, microglia and Schwann cells. Can this provide a potential explanation for the non-cell autonomous disease spread observed in numerous neurodegenerative diseases? n nFinally and most importantly, can our burgeoning knowledge on the molecular mechanisms of Golgi pathology in neurodegenerative diseases be translated into earlier disease diagnosis and new therapies for these severe and hitherto untreatable disorders?

Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique

Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.