C69 CHARACTERIZATION OF AMYOTROPHIC LATERAL SCLEROSIS‐LINKED PRO56SER MUTATION OF VESICLE‐ASSOCIATED MEMBRANE PROTEIN‐ASSOCIATED PROTEIN B (VAPB/ALS8)
Kanekura K, Nishimoto I, Aiso S, Matsuoka M
Keio University, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: The Pro56Ser (P56S) mutation in vesicle‐associated membrane protein‐associated protein B (VAPB) is the most recently identified ALS‐linked gene (ALS8) and causes autosomal‐dominant motoneuronal diseases (MND). VAPB is a homolog of aplysia VAP33, involved in secretion of neurotransmitter and known to localize in ER‐Golgi apparatus, but its physiological role remains unclear. It was reported that the P56S mutation induces a shift of VAPB from endoplasmic reticulum (ER) to non‐ER compartments; it still remains unclear how the P56S mutation causes MND.
Objectives: To characterize the properties of P56S‐VAPB that may be related to the onset of ALS.
Methods: To clarify how the P56S mutation affects the properties of the VAPB protein, we first investigated the interaction of P56S‐VAPB with its binding proteins by pull‐down analyses. Next, we compared solubility of wt‐VAPB and P56S‐VAPB in a 1% Triton‐X100 (Triton)‐containing buffer. For these purposes, wt‐VAPB or P56S‐VAPB, transiently overexpressed in motoneuronal NSC34 cells, were fractionated into Triton‐soluble or ‐insoluble fractions and subject to immunoblot analysis. We further investigated the abnormal localization of P56S‐VAPB by immunocytochemistry and sucrose density gradient centrifugation. For investigation of VAPB function, we used the unfolded protein response (UPR) assay because VAP and its yeast homolog SCS2 are suspected to be involved in UPR.
Results: Although the P56S mutation did not affect the homodimerization with VAPB or the heterodimerization between VAPB and VAPA or synaptobrevin (vesicle associated membrane protein‐1 and ‐2), it drastically decreased the solubility of VAPB protein and caused a shift in the localization of VAPB from ER to non‐ER compartment. Furthermore, P56S‐VAPB enhanced insolubility of co‐expressed wt‐VAPB, but not VAPA, VAMP1 or VAMP2. Enforced expression of wt‐VAPB triggered UPR, but P56S‐VAPB did not, indicating that P56S‐VAPB is a non‐functional mutant. Any tested P56X‐VAPB point‐mutant (X = A, K, and D) or VAPB with a deletion 56th proline showed the same characteristic as P56S‐VAPB, suggesting that the proline at the 56th position plays an important role in the proper structure of VAPB and its physiological functions.
Discussion and conclusions: We demonstrate that P56S‐VAPB is more insoluble in the Triton‐containing buffer than wt‐VAPB, indicating that it is misfolded. Accordingly, P56S‐VAPB loses the function to induce UPR, an ER reaction to ER stress that is triggered by overexpression of wt‐VAPB. P56S‐VAPB interferes with the folding of co‐expressed wt‐VAPB possibly because it still retains the ability to be firmly dimerized with wt‐VAPB despite its misfolding and mislocalization. As a result, co‐expression of P56S‐VAPB enhances the insolubility of wt‐VAPB and prevents wt‐VAPB from triggering UPR. We conclude that P56S‐VAPB is a loss‐of‐function mutant. We further conclude that it dominant‐negatively affects the function and localization of wt‐VAPB. These unique characteristics of P56S‐VAPB may play an important role in the pathomechanism underlying motoneuronal degeneration linked to ALS8.
C70 ALS2 IS LOCALIZED TO ENDOSOMES IN PRIMARY CULTURED HIPPOCAMPAL NEURONS AND IMPLICATED IN AXON ELONGATION
Otomo A, Kunita R, Suzuki‐Utsunomiya K, Mizumura H, Onoe K, Hadano S, Ikeda J‐E
Department of Molecular Neuroscience, The Institute of Medical Sciences, Tokai University, Kanagawa, Japan
E‐mail address for correspondence: [email protected]
Background: The ALS2 gene was identified as a causative gene for a number of juvenile recessive motor neuron diseases (MNDs), such as ALS2, PLSJ, HSP and IAHSP. It has been postulated that a functional loss of the ALS2 protein (ALS2/alsin) leads to selective motor neuron degeneration and MND. Recently, we have demonstrated that ALS2 acts as a guanine nucleotide exchange factor for small GTPase Rab5 and enhances early endosome fusion through the activation of Rab5. Thus, ALS2 may play an important role in the maintenance of motor neurons via regulating membrane trafficking. However, detailed distribution and functions of ALS2 in neurons are still unclear.
Objectives: The purpose of this study is to identify subcellular localization of neuronal ALS2 at different stages of neural development using primary cultured hippocampal neurons as a model for developing neurons. Further, we also examined whether levels of ALS2 expression affect axon outgrowth by comparing the length of axon in the ALS2 deficient, ALS2 overexpressing, and wild type neurons.
Methods: Hippocampal neurons were isolated from hippocampi dissected from post‐ neonatal day 1 mouse, and cultured for different periods of time to examine the ALS2 localization in the cells at different developmental stages. ALS2 was immunocytochemically detected and analysed by the Leica TCS‐NT system. Further, to measure the length of axon in stage 3 neurons, 2×104 cells were plated onto poly‐D‐lysine coated round glasses. After 36 h, the cells were fixed, permeabilized, and stained with anti‐MAP2 antibody and Alexa594‐Phalloidin. The longest neurite with attenuated MAP2 staining was defined as the axon. Captured cell images were analysed by ImageJ and axon length was determined.
Results: In stage 3 neurons, ALS2 was distributed to cytoplasm and patchy membrane structures. ALS2 was also enriched in membrane ruffles at the growth cone colocalizing with F‐actin. In stage 5 neurons, ALS2 was localized to endosomes both in dendrites and in the axon, which were partially labelled with Alexa‐594 transferrin. The ALS2‐positive axonal endosomes were also colocalized with synaptophysin prior to the synapse formation. Notably, at stage 3, although no significant differences in the outgrowth of MAP2‐positive dendrites among different genotypes were observed, ALS2 deficient neurons extended shorter axons than wild‐type ones, while overexpression of ALS2 promoted axon growth.
Discussion and conclusions: These observations suggest that ALS2 is implicated in axonal development in immature neurons. A unique endosomal localization of ALS2 in matured neurons implies that ALS2 might regulate membrane trafficking through the endosome fusion, thereby mediating the survival and maintenance of motor neurons. Further studies on the neuronal functions of ALS2 will lead to better understanding of the pathogenesis for ALS2‐linked, as well as other, MNDs.
C71 ALS2 IS A NOVEL RAC1‐REGULATED MACROPINOSOMAL RAB5GEF THAT MEDIATES INTERCONNECTION BETWEEN DISTINCT ENDOCYTIC PATHWAYS
Kunita R1, Otomo A1, Mizumura h2, Suzuki‐Utsunomiya K1, Hadano s1, Ikada j1
1Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan, 2SORST, JST, Saitama, Japan
E‐mail address for correspondence: [email protected]
Background: ALS2/alsin is a causative gene product for several juvenile recessive motor neuron diseases (MNDs). ALS2 acts as a guanine nucleotide exchange factor (GEF) for Rab5 (Rab5GEF) and is implicated in endosome fusion. It has been postulated that a perturbation of endosome dynamics caused by loss of the ALS2 function might underlie motor dysfunction. ALS2 is preferentially localized onto endosomal vesicles in cultured neurons, while it is sequestered in cytoplasm in most non‐neuronal cells. Therefore, intracellular distribution of ALS2 might be regulated by the upstream signal(s), thereby modulating endosome dynamics. However, the molecular mechanisms by which ALS2 is activated/redistributed in cells are still unknown.
Objectives: The purpose of this study is to identify the upstream activator(s) for ALS2. We also investigated cellular processes that ALS2 regulates. This study will lead to a better understanding of the pathogenesis underlying the ALS2‐linked MNDs.
Methods: We performed in vitro binding and co‐immunoprecipitation analyses to identify the small G protein that binds to ALS2. Subsequently, to investigate the physiological significance of the identified interaction, we conducted co‐transfection experiments of ALS2 with its interactor and/or activator in HeLa cells. The ALS2 localization and the ALS2‐positive endocytic vesicles were characterized using immunocytochemical methods. We also conducted uptake analyses of fluorescently‐labeled molecules, such as dextran and transferrin, in cultured cells, to identify the ALS2‐regulated endocytic pathways.
Results: Here we showed that ALS2 preferentially interacted with an activated form of Rac1 as a novel Rac1 effector. Interestingly, cytoplasmic ALS2 was recruited to membrane ruffles and then relocalized onto nascent macropinosomes via macropinocytosis, a mode of endocytosis, upon Rac1 signaling. Thus, Rac1 acts as an upstream activator for ALS2. ALS2 is the first Rab5GEF that is positively regulated by a Rho member, Rac1. At later endocytic stages, macropinosomal ALS2 regulated fusion between the ALS2‐localized macropinosomes and the CME‐derived endosomes, depending on the ALS2‐associated Rab5GEF activity. These findings reveal a fundamental role of ALS2 being implicated in the spatiotemporal regulation of Rac1‐Rab5 signaling.
Discussion and conclusions: We here demonstrate that the Rab5GEF ALS2/alsin mediates the interconnection between Rac1‐induced macropinosomes and CME‐derived endosomes. Endocytosis is crucial to numerous cellular processes and involves multiple internalization mechanisms, such as macropinocytosis, CME, and caveolin‐dependent endocytosis. Thus, we propose that both clathrin‐dependent and independent endocytic mechanisms that are coordinately interconnected by ALS2 are crucial for the survival of motor neurons. Future studies on the ALS2 functions in neurons will provide further insights into the pathogenesis for MNDs caused by the ALS2 mutations.
C72 DEREGULATION OF PKN BY GLUTAMATE AND ALS‐LINKED MUTANT SOD1 INDUCES CHANGES IN NEUROFILAMENT ORGANIZATION AND TRANSPORT
Stevenson AJ, Manser CF, Banner SI, Shaw CE, Mcloughlin DM, Miller CC
Institute of Psychiatry, King's College, London, UK.
E‐mail address for correspondence: [email protected]
Background and objectives: Protein kinase N (also known as Protein kinase C‐related kinase‐1; PRK1) is a serine/threonine kinase with a C‐terminal catalytic domain similar to that found in PKC and a unique N‐terminal regulatory region. Within neurons, it phosphorylates both neurofilaments and the microtubule‐associated protein tau, and this has led to the suggestion that abnormal PKN activity contributes to the pathogenesis of some neurodegenerative diseases. Indeed, PKN can be cleaved by caspase family proteases to release a deregulated C‐terminal fragment containing the catalytic domain devoid of N‐terminal regulatory moieties. Such a cleaved deregulated PKN fragment is generated in a number of experimental models of neurodegeneration. Here, we have investigated the role of PKN in ALS.
Method and results: We initially investigated the effect of glutamate on PKN activity. Treatment of cultured rat cortical neurons with 100 µM glutamate for up to 30 min led to a significant increase in PKN activity, as determined by in vitro kinase assays. This activation was shown to be mediated via NMDA‐type receptors. Extended treatment of neurons with glutamate induced proteolytic cleavage of PKN to release a predicted deregulated C‐terminal fragment. This cleavage was blocked by the caspase inhibitor ZVAD at 100 µM.
We next investigated whether cleavage of PKN to produce such a deregulated fragment also occurs in G93ASOD1 transgenic mice. Immunoblots of brain and spinal cord samples from G93ASOD1 and control mice revealed the presence of the cleaved PKN fragment specifically in spinal cords of G93ASOD1 mice. Additionally, immunohistochemical staining revealed that PKN was present in neurofilament accumulation containing motor neuron cell bodies of these mice.
To determine which region of neurofilament light chain (NFL) is phosphorylated by PKN, we performed in vitro kinase assays with different domains of NFL. PKN phosphorylated the head domain of NFL; such phosphorylation is believed to regulate the assembly properties of neurofilaments. We therefore examined neurofilament assembly and detergent solubility in SW13 cells' cortical neurons following modulation of PKN activity. The cleaved deregulated PKN induced subtle changes to neurofilament architecture. Finally, we studied whether PKN influenced axonal transport of neurofilaments in cultured neurons. Transfection of wild‐type PKN had no effect on neurofilament transport but transfection of the cleaved deregulated PKN induced a marked decrease in neurofilament transport.
Discussion and conclusions: We have shown that two insults associated with ALS, glutamate excitotoxicity and mutant SOD1, both induce changes in PKN activity. PKN phosphorylates the head domain of NFL and a deregulated PKN fragment induces changes in neurofilament assembly properties and axonal transport. Deregulation of PKN may thus be part of the pathogenic process in ALS. This work was supported by grants from the MND Association and Medical Research Council.
C73 REVERSIBLE DISRUPTION OF RETROGRADE AXONAL TRANSPORT IN SPINAL AND BULBAR MUSCULAR ATROPHY
Katsuno M, Adachi H, Minamiyama M, Waza M, Tokui K, Jiang YM, Banno H, Suzuki K, Tanaka F, Doyu M, Sobue G
Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan
E‐mail address for correspondence: [email protected]
Background: Spinal and bulbar muscular atrophy (SBMA) is a hereditary neurodegenerative disease caused by an expansion of a trinucleotide CAG repeat encoding the polyglutamine tract in the androgen receptor (AR) gene. As suggested in other polyglutamine diseases, pathogenic AR‐mediated transcriptional dysregulation has been implicated in the pathogenesis of SBMA. Mouse models of SBMA suggest that dysfunction of motor neurons precedes neuronal cell death in the process of neurodegeneration.
Objectives: The aim of this study is to elucidate the mechanism by which motor neuron function deteriorates prior to neuronal cell death in SBMA.
Methods: We used a transgenic mouse model of SBMA carrying the full‐length human AR gene harboring 24 (AR‐24Q) or 97 CAGs (AR‐97Q). Immunohistochemistry and immunofluorescent analysis were performed on mouse tissues. Axonal transport in the mice was investigated using retrograde neurotracer labelling and nerve ligation. The expression levels of axon motor proteins were determined using in situ hybridization and quantitative RT‐PCR.
Results: AR‐97Q mice demonstrated a striking accumulation of both phosphorylated and non‐phosphorylated NF‐H in the skeletal muscle, whereas this finding was not observed in AR‐24Q or wild‐types. Similar accumulation was also observed for middle molecular weight NF (NF‐M) and synaptophysin. Anti‐NF immunostaining demonstrated that intramuscular NF accumulation was detectable from as early as seven weeks old, prior to the onset of muscle weakness in this mouse model, and aggravated thereafter. To elucidate the molecular basis for abnormal distribution of NF, we studied axonal transport in this mouse model of SBMA. Retrograde neurotracer labeling and nerve ligation demonstrate that the retrograde transport is disrupted prior to the onset of neurological symptoms. To elucidate the molecular mechanism compromising retrograde axonal transport, we examined the expression level of axon motors and associated proteins. The spinal motor neurons demonstrated a lowered protein level of the largest subunit of dynactin (dynactin1) in spinal cord sections from AR‐97Q mice. In the ventral root, decrease in dynactin1 levels was significant before the onset of motor symptoms. Although the protein level of dynein heavy chain was slightly diminished in the advanced stage, this phenomenon was not observed before the onset of symptoms. In situ hybridization and quantitative RT‐PCR demonstrated that the mRNA level of dynactin1 was markedly repressed in the spinal cord of AR‐97Q mice in the pre‐onset stage.
Discussion and conclusions: The present study indicates that the accumulation of axonal components in distal motor axons appears to be a substantial pathology associated with degeneration of lower motor neurons. Although previous studies have suggested a direct inhibition of axonal transport by mutant AR protein within motor axons, our results demonstrate that polyglutamine‐mediated transcriptional dysregulation of dynactin1, the p150 subunit of dynactin, in affected neurons is a basis for perturbation of retrograde axonal transport in the SBMA mouse.
C74 SOD1 AGGREGATES GENERATED WITHIN MOTONEURONAL DENDRITES/CELL BODIES MOVE INTO AXONS BEFORE DISEASE ONSET IN A G93ASOD1 TRANSGENIC MOUSE MODEL
Takahashi R1, Tateno M2, Araki T2
1Kyoto University Graduate School of Medicine, Kyoto‐shi, Kyoto‐fu, Japan, 2National Center of Neurology and Psychiatry, Kodaira‐shi, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background and objectives: Aggregation of mutant SOD1 proteins is suggested to be responsible for the selective loss of motor neurons in SOD1‐related ALS, although the mechanisms underlying such aggregates' related toxicity have been elusive. Since the subcellular localization of the aggregates will provide an important clue in understanding the toxicity, we investigated the location of the SOD1 aggregates within motor neurons and how the localization changed during disease progression in a SOD1G93A‐Tg mouse model.
Methods: We analysed the localization of SOD1 aggregates by two different methods. First we performed subcellular fractionation of spinal cords producing five fractions in which typical organelles, as well as fragmented axons, were independently enriched. Secondly, we carried out an area‐specific isolation utilized laser‐assisted microdissection technique. From the frozen sections of non‐fixed spinal cords, we isolated several areas enriched with cell bodies of motor neurons, dendrites of motor neurons/neighbouring glial cells, etc. These fractions were subjected to SDS‐PAGE and Western blot analysis, and the properties of SOD1 aggregates were examined.
Results: The subcellular fractionation analysis revealed that SOD1 aggregates were first detected in the fractions enriched with mitochondria and axons a long time before disease onset, and then increasingly accumulated into the latter fraction by disease onset. With disease progression, the aggregates rapidly spread into other fractions, other than the fraction composed of cytosolic soluble proteins. The area‐specific isolation technique revealed that SOD1 aggregates were first detected within the areas including cell bodies and dendrites of motor neurons, followed by their detection within the area containing motoneuronal axons. Combined with these and other data, SOD1 aggregates are thought to be generated within dendrites/cell bodies and then move into the axons within motor neurons before disease onset.
Discussions and conclusions: We previously found that calcium‐permeable AMPA‐type glutamate receptors promote generation of SOD1 aggregates with an increased cellular oxidative stress in SOD1G93A‐Tg mice (1). AMPA receptors are localized to the dendrites of motor neurons and largely contribute to the excitotoxicity that generates oxidative stress. Since oxidative stress promotes the conversion of mutant SOD1 into aggregates, our findings imply a direct connection between excitotoxicity and generation of SOD1 aggregates within dendrites. Considering the remarkable accumulation of SOD1 aggregates within axon‐enriched fraction/area by disease onset, the toxicity of aggregates may be related to the translocation of the aggregates from dendrites/cell bodies to axons.
References
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