Subcellular localization of alpha-synuclein in primary neuronal cultures: effect of missense mutations

Numerous recent observations have implicated alpha-synuclein in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies and multiple-system atrophy. Two missense mutations in the gene for alpha-synuclein have been identified in some cases of familial Parkinson's disease and it is thought that these may disrupt the normal structure of the protein and thus promote aggregation into Lewy body filaments. Here, we examine the subcellular localization of alpha-synuclein in primary cortical neurons maintained in a monolayer culture. The protein has widespread expression throughout neurons, including the nucleus, and has a discete localization in the neurites of more mature neurons, reminiscent of synaptic specializations. Interestingly, in a subpopulation of cortical neurons transfected at 13 days in vitro, we find that alpha-synuclein appears to aggregate into distinct punctate inclusions in the cytoplasm and proximal neurites. Unlike Lewy bodies, these structures are not ubiquitin positive. These regions of alpha-synuclein accumulation are observed following transfections with wild-type, Ala30Pro or Ala53Thr alpha-synuclein; neither mutation alters their frequency.     

Histochemical features of stress-induced aggregates in alpha-synuclein overexpressing cells

alpha-Synuclein is a major component of intracytoplasmic inclusions including Lewy bodies (LB), Lewy neurites (LN) and glial cytoplasmic inclusions, and plays a key role in neurodegenerative processes in Parkinson's disease (PD) and other synucleinopathies. Although the molecular mechanisms of the disease process still remain to be elucidated, recent studies have suggested that an interaction between reactive oxygen species (ROS) and alpha-synuclein may be closely associated with the initiation and/or the progression of synucleinopathies. In this study, we established human dopaminergic SH-SY5Y cell lines overexpressing wild-type or mutant alpha-synuclein and exposed them to various ROS generators. After the exposure to ROS, alpha-synuclein aggregates were formed in the cytoplasm of these cells, and these were immunopositive for ubiquitin, nitrotyrosine and dityrosine, and positive for thioflavin S staining. Thus, the obtained cytoplasmic aggregates shared many features with inclusion bodies in synucleinopathies. The gamma-tubulin and molecular chaperones coexisted as well, suggesting that the aggregate formation is associated with the intracellular transport along microtubules and may reflect protective responses against neuronal insults. This cellular model not only will be informative for our understanding of the pathophysiological process in synucleinopathies, but also can be applied to the screening of neuroprotective molecules with therapeutic potential.     

Subcellular localization of neural-specific NPDC-1 protein

NPDC-1 is a gene specifically expressed in neural cells when they stop to divide and begin to differentiate. Immunocytochemical study analysis of differentiated PC12 cells transfected with NPDC-tag vectors showed that NPDC-1 is transported in vesicles from the Golgi apparatus to the cell membrane and is then likely internalized into endosomes. The protein colocalized, at least partially, with synaptic vesicle proteins: synaptophysin, synaptobrevin 2, and Rab3 GEP (Rab3 GTP/GDP exchange protein). Moreover, subcellular fractionation of rat brain showed that crude synaptic membrane and crude synaptic vesicle fractions were enriched in NPDC-1. Although NPDC-1 bound Rab3 GEP in vitro, it seems unlikely to be involved in Ca2+-dependent exocytosis and, thus, in synaptic vesicle trafficking.     

Lewy bodies in the amygdala: increase of alpha-synuclein aggregates in neurodegenerative diseases with tau-based inclusions

BACKGROUND: Increased attention has been given to alpha-synuclein aggregation in nonsynucleinopathies because alpha-synuclein-containing Lewy bodies (LBs) influence symptoms. However, the spectrum of disorders in which secondary inclusions are likely to occur has not been defined. Amygdala neurons commonly develop large numbers of secondary LBs, making it a practical region for studying this phenomenon. OBJECTIVE: To characterize the spectrum of diseases associated with LB formation in the amygdala of neurodegenerative disease and control cases. DESIGN: An autopsy series of 101 neurodegenerative disease and 34 aged control cases. Using immunohistochemistry studies, we examined the amygdala for alpha-synuclein aggregates. RESULTS: Lewy bodies were often abundant in classic Pick disease, argyrophilic grain disease, Alzheimer disease, and dementia with LBs but not in cases with amygdala degeneration lacking tau-based inclusions, control cases, preclinical disease carriers, or degenerative diseases lacking pathologic involvement of the amygdala. The exposed alpha-synuclein epitopes were similar in all cases containing LBs. CONCLUSIONS: Abnormal alpha-synuclein aggregation in the amygdala is disease selective, but not restricted to disorders of alpha-synuclein and beta-amyloid. Our data are compatible with the notion that tau aggregates predispose neurons to develop secondary LBs.     

Multiple ligand interaction of alpha-synuclein produced various forms of protein aggregates in the presence of Abeta25-35, copper, and eosin.

Various protein aggregates of alpha-synuclein developed by way of the common protein self-oligomerization in the presence of Abeta25-35, copper, and eosin were examined. All the aggregates exhibited congo red birefringence although the actual amounts of the aggregates were varied as determined by thioflavin T binding fluorescence. When their morphologies were analyzed in relation to in vitro cytotoxicity, the smallest granular aggregates obtained with copper exhibited the highest cytotoxicity, while the fibrous structures by eosin did not affect the cell.     

Subcellular localization of epitope-tagged neurotrophins in neuroendocrine cells

A growing body of evidence suggests that neurotrophins (NTs) play a critical role in synaptic plasticity and other activity dependent processes in the CNS. Release of these growth factors by neurons and neuroendocrine cells was recently shown to occur via the regulated secretory pathway, representing a possible mechanism for preferentially supplying NTs locally to active synapses. However, the identity and characteristics of the intracellular storage compartment for NTs undergoing stimulus-coupled secretion remains controversial. As a step towards addressing these issues we have investigated the subcellular localization of epitope-tagged nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in neuroendocrine cells. Placement of the myc-epitope tag at the neurotrophin carboxy terminus did not affect essential properties of the NTs such as their ability to induce Trk tyrosine phosphorylation or their sorting into the regulated secretory pathway in PC12 and AtT-20 neuroendocrine cells. Epitope-tagged NTs colocalize with dense core vesicle (DCV)-markers at the light microscopic level in both cell lines investigated. Furthermore, at an EM level immunoreactivity (IR) for myc-tagged NGF was found over dense core granules (DCGs) in PC12 cells. These data provide evidence that NTs can be stored in DCVs in neuronal model cell lines and, potentially, in neurons as well. J. Neurosci. Res. 51:463–472, 1998. © 1998 Wiley-Liss, Inc.     

Expression pattern and localization of alpha-synuclein in the human enteric nervous system

BACKGROUND: Alpha-synuclein (α-syn) is abundantly expressed in the central nervous system and involved in the regulation of neurotransmission. Insoluble fibrils of phosphorylated α-synuclein (p-α-syn) have been implicated in several neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease). The aim of the study was to determine the gene expression pattern and localization of α-syn/p-α-syn in the human enteric nervous system (ENS). METHODS: Human colonic specimens (n=13, 15-83years) were processed for α-syn and p-α-syn immunohistochemistry. Colocalization of α-syn was assessed by dual-labeling with pan-neuronal markers (PGP 9.5, HuC/D). For qPCR studies, tissue was obtained from full-thickness sections, tunica muscularis, submucosa, mucosa, and laser-microdissected (LMD) enteric ganglia. RESULTS: Highest α-syn levels were detectable within the tunica muscularis and submucosa. Ganglia isolated by LMD showed high expression of α-syn mRNA. All myenteric and submucosal ganglia and nerve fibers were immunoreactive for α-syn. Dual-labeling revealed colocalization of α-syn with both pan-neuronal markers. p-α-syn immunoreactivity was consistently observed in specimens from adults with increasing age. CONCLUSIONS: α-syn is abundantly expressed in all nerve plexus of the human ENS including both neuronal somata and processes. The presence of p-α-syn within the ENS is a regular finding in adults with increasing age and may not be regarded as pathological correlate. The data provide a basis to unravel the functions of α-syn and to evaluate altered α-syn in enteric neuropathies and α-synucleinopathies of the CNS with gastrointestinal manifestations.     

Tubular aggregates: their association with myalgia.

Three thousand consecutive muscle biopsies were reviewed for the presence of tubular aggregates and their association with clinical symptomatology. Tubular aggregates were detected in 19 patients (0.6%). Twelve of these nineteen patients had severe myalgia, and the most abundant tubular aggregates were found in biopsies of patients with myalgia. Seven patients had only myalgia as their clinical symptomatology with normal physical examination. An additional five patients with tubular aggregates and myalgia had concomitant amyotrophic lateral sclerosis (2) or neuropathy (3). The high incidence of myalgia associated with tubular aggregates in our patients and the fact that tubular aggregates originate from sarcoplasmic reticulum suggest a role played by this structure in the pathogenesis of myalgia.     

Subcellular localization of calcium-permeable AMPA receptors in spinal motoneurons

Activation of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been linked to potent effects on survival and dendritic outgrowth of spinal motoneurons. Ca(2+) permeability of AMPA receptors is controlled by the GluR2 subunit. Whole-cell electrophysiological studies have suggested that GluR2-containing and GluR2-lacking AMPA receptors may coexist in individual motoneurons. However, there has not been a direct demonstration of heterogeneity in AMPA receptor subunit composition in single motoneurons, nor of distinct subcellular distributions of GluR2-containing and GluR2-lacking receptors. In the present study, we have used confocal microscopy, immunocytochemistry and Ca(2+) imaging to characterize the subcellular localization of AMPA receptors in cultured rat spinal motoneurons. Immunoreactivity for GluR2 and GluR4 was concentrated in clusters, the vast majority of which were found in dendrites at synapses. Double-labelling for GluR2 and GluR4 revealed variability in relative expression of GluR2 and GluR4 between clusters within individual motoneurons; most AMPA receptor clusters were immunoreactive for both GluR2 and GluR4, but a significant minority of clusters were immunoreactive for GluR2 only or for GluR4 only. The majority of GluR2-immunonegative AMPA receptor clusters was present in dendrites, but the relative proportion of GluR2-immunonegative and GluR2-immunopositive clusters was similar in dendrites and soma. Imaging of [Ca(2+)](i) rises triggered by AMPA receptor activation confirmed Ca(2+) influx in motoneuron dendrites. These findings strongly support a model in which GluR2-containing and GluR2-lacking AMPA receptors coexist in motoneurons, clustered at synapses, and mixed in a relative proportion that varies considerably between cell membrane microdomains.     

Subcellular localization of presenilin 2 endoproteolytic C-terminal fragments.

Mutations in the genes that encode the presenilin 1 and 2 (PS1 and PS2) proteins cause the majority of familial Alzheimer's disease (FAD). Differential cleavage of the presenilins results in a generation of at least two C-terminal fragments (CTFs). An increase in the smaller of these two CTFs is one of the few changes in presenilin processing associated with FAD mutations in both PS1 and PS2. Interestingly, the phosphorylation of PS2 modulates the production of the smaller, caspase-derived PS2 CTF, which indicates that the generation of this fragment is a regulated, physiologic event. To date, there is no data concerning the subcellular distribution of the caspase-derived PS2 CTF. Because this fragment is normally present at levels that are difficult to detect, we have used cell lines in which the production of wild-type or N141I mutant PS2 is controlled by a tetracycline-regulated promoter in order to assess the subcellular localization of the caspase CTF in relation to the larger, constitutive PS2 CTF and to PS2 holoprotein. We have found that when levels of PS2 are low, the constitutive CTF colocalizes with markers consistent with localization in the early Golgi-ER-Golgi intermediate compartment (ERGIC) while the caspase CTF colocalizes with markers for the endoplasmic reticulum (ER). Following induction of wild-type or mutant PS2, when the levels of PS2 are high, the primary localization of the constitutive CTF appears to shift from the early Golgi-ERGIC in addition to the ER. Interestingly, while the induction of wild-type PS2 resulted in the localization of the caspase CTF primarily in the ER, the induction of mutant PS2 resulted in the localization of the caspase CTF to both the ER and the early Golgi-ERGIC. In summary, these data suggest that the two presenilin 2 CTFs have different patterns of subcellular localization and that the N141I PS2 mutation alters the localization pattern of the PS2 caspase fragment.     

Subcellular localization of GABAB receptor subunits in rat globus pallidus

The inhibitory amino acid gamma-aminobutyric acid (GABA) is the major neurotransmitter in the globus pallidus. Although electrophysiological studies have indicated that functional GABA(B) receptors exist in rat globus pallidus, the subcellular localization of GABA(B) receptor subunits and their spatial relationship to glutamatergic and GABAergic synapses are unknown. Here, we use pre-embedding immunogold labeling to study the subcellular localization of GABA(B) receptor subunits, GABA(B1) and GABA(B2), in globus pallidus neurons and identified populations of afferent terminals. Immunolabeling for GABA(B1) and GABA(B2) was observed throughout the globus pallidus, with GABA(B1) more strongly expressed in perikarya and GABA(B2) mainly expressed in the neuropil. Electron microscopic analysis revealed that the majority of GABA(B1) labeling was localized within the cytoplasm, whereas most of GABA(B2) labeling was associated with the plasma membrane. At the subcellular level, both the GABA(B1) and GABA(B2) immunogold labeling was localized at pre- and postsynaptic sites. At asymmetric, putative excitatory, synapses, GABA(B1) and GABA(B2) immunogold labeling was found at perisynaptic sites of both pre- and postsynaptic specializations. Double immunolabeling, using the vesicular glutamate transporter 2 (VGLUT2), revealed the glutamatergic nature of most immunogold-labeled asymmetric synapses. At symmetric, putative GABAergic, synapses, including those formed by anterogradely labeled striatopallidal terminals, GABA(B1) and GABA(B2) immunogold labeling was found in the main body of both pre- and postsynaptic specializations. These results demonstrate the existence of presynaptic GABA(B) auto- and heteroreceptors and postsynaptic GABA(B) receptors, which may be involved in modulating synaptic transmission in the globus pallidus.     

Subcellular localization of presenilins: association with a unique membrane pool in cultured cells

We have investigated the subcellular distribution of presenilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol-based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, Golgi SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast to PS1, PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.     

Glycerol oxidation in rat brain: Subcellular localization and kinetic characteristics

The oxidation of [1,3-¹⁴C]glycerol to ¹⁴CO was measured in slices, whole homogenates, and subcellular fractions of rat brain. In all of these tissue preparations, the Lineweaver-Burk plots of glycerol oxidation were biphasic, yielding two apparent K_m and V values. Similar kinetic characteristics were obtained with brain homogenates from guinea pig, mouse, rabbit, monkey, and pig. In other tissues of the rat, including heart, kidney, liver, and skeletal muscle, the Lineweaver-Burk plots for glycerol oxidation were not biphasic but were linear. Heating the brain homogenates for five minutes at 5°C caused a 50% decrease in the rate of oxidation of glycerol without a change in the biphasic double reciprocal plot. The addition of purified glycerol kinase (EC 2.7.1.30) to the homogenate caused an increase in the rate of oxidation and resulted in a linear Lineweaver-Burk plot. Brain mitochondria were prepared by two different methods, both of which yielded an enrichment of glycerol oxidation. In contrast, the rate of glucose oxidation was higher in homogenates than in mitochondria, and glucose competed with glycerol as substrate only extramitochondrially. The effects of various metabolic inhibitors suggested the participation of intact, coupled mitochondria, of glycolytic enzymes, and of electron transport in the oxidation of glycerol. The data support the primary localization of glycerol oxidation in nonsynaptic mitochondria in brain and the presence in that organelle of enzymes of the Embden-Meyerhoff pathway or an as yet unidentified system for oxidizing this compound.     

Subcellular localization of dopamine beta-hydroxylase and endogenous norepinephrine in the rat hypothalamus

The subcellular distribution of endogenous norepinephrine, dopamine β-hydroxylase (EC 1.14.2.1) and endogenous inhibitors to dopamine β-hydroxylase have been examined in the rat hypothalamus. Sixty percent of the recovered norepinephrine and activity of dopamine β-hydroxylase sedimented in the crude mitochondrial (P₂) fraction; fractionation on a linear sucrose gradient revealed that the particulate-bound amine and enzyme were localized in the synaptosomal fractions. After osmotic shock of the P₂ fraction the released storage vesicles were purified by differential centrifugation or centrifugation on a sucrose gradient; 40–70% of the dopamine β-hydroxylase activity released from the P₂ fraction was localized in the fractions reported to contain storage vesicles. In contrast, less than 10% of P₂-norephinephrine was found in these fractions. The fraction from the gradient that had the highest specific activity of dopamine β-hydroxylase was shown by electron microscopy to be markedly enriched with large, dense-core vesicles. Endogenous inhibitors to dopamine β-hydroxylase were found in all subcellular fractions with only a 2.5-fold difference between the least and most inhibitory fractions.     

福山型先天性肌营养不良和肌-眼-脑病的基因产物在细胞内的定位

目的 探讨福山型先天性肌营养不良（Fukuyama congenital muscular dystrophy，FCMD）和肌-眼-脑病（muscle-eye-braindisease，MEB）的基因产物在细胞内的定位及其可能的功能。方法 构建FCMD的致病基因fukutin，MEB的致病基因N-乙酰氨基葡萄糖．甘露糖转移酶1（protein．O．1inkedmannose1β1，2-N-acetylglucosaminyl-transferasel，POMGnTl）的真核表达载体，脂质体介导转染fukufin—nag（pcDNA3，0）、POMGnTl一V5（pEFl）至小鼠骨骼肌成肌细胞系C2C12细胞，利用高尔基体和内质网的标记物（GM130，KDEL）进行免疫荧光双标染色，荧光显微镜下观察基因产物fukufin蛋白和POMGnT1在细胞内的定位。并构建不同长度的fukutin缺失体如fukufin跨膜结构域、跨膜结构域缺失体及突变体Y371C，观察fukutin蛋白是否与POMGnT1蛋白相结合以及突变型fukutin蛋白在细胞内的定位。结果fukutin蛋白和POMGnT1蛋白在细胞内定位于高尔基体，属于高尔基体膜蛋白。fukutin蛋白通过其跨膜结构域定位于高尔基体并与POMGnTl蛋白相结合，突变体Y371c定位于内质网。结论 FCMD和MEB均属于先天性肌营养不良中的仅一抗肌萎缩相关糖蛋白病（α-dystroglycanopathy），POMGnTl已被证实为参与仅-抗肌萎缩相关糖蛋白的O-连接糖基化的糖基转移酶，fukufin和POMGnT1定位于高尔基体并互相结合表明它们在高尔基体内参与α-抗肌萎缩相关糖蛋白的糖基化，并可能通过同一酶活性通路起作用。突变体Y371C导致了fukutin的定位错误。     

Subcellular, regional and immunohistochemical localization of adenosine deaminase in various species

Immunohistochemical and subcellular fractionation techniques were employed to compare the cellular and subcellular localization of adenosine deaminase (ADA) in various brain regions of several mammalian species. A relatively restricted distribution of ADA-immunoreactive neurons in rat brain was previously reported. Mouse brain exhibited a pattern similar in many respects to rat and, in addition, contained intensely immunostained neurons in lateral habenula and hippocampus. Glial immunostaining was absent or very light in rat but evident in mouse. Prominent immunoreactive fibers and neurons were observed in hamster spinal cord and anterior hypothalamus, respectively. ADA-immunostaining in guinea-pig was localized to presumptive fibers in the superficial layers of spinal cord dorsal horn and to glial cells throughout the brain. Demonstration of specific immunostaining in rabbit was not possible. ADA activity was far more heterogeneously distributed in rat and most brain areas in guinea-pig and rabbit contained up to 5-fold and 10-fold higher levels of activity, respectively, compared with rat. Crude synaptosomal (P2) fractions of rat cortex contained a greater proportion of ADA activity than those of rabbit cortex. Within rat, relatively high activity was found in P2 fractions of whole hypothalamus, cerebellum, and hippocampus. ADA activity was greater in P2 fractions of rat anterior compared with whole hypothalamus and the greatest proportion of the enzyme in this fraction was localized to purified synaptosomes. The large variations in the activity and cellular location of ADA in the animals examined suggest species differences in mechanisms governing adenosine metabolism in brain and possible differences in the relationships between cellular metabolism, ADA and the neuroregulatory role of adenosine in the CNS.     

Lack of direct role of parkin in the steady-state level and aggregation of alpha-synuclein and the clearance of pre-formed aggregates

Mutations in parkin and alpha-synuclein (alpha-syn) are linked to heritable forms of Parkinson's disease (PD). Recently, it has been shown that parkin mitigates alpha-syn-induced neuronal cell death in animal and tissue culture models, suggesting that there is a functional relationship between these two proteins. Although the mechanism by which parkin protects cells from alpha-syn-induced cytotoxicity remains elusive, it is tempting to speculate that parkin might directly regulate the normal metabolism and aggregation of alpha-syn. In the current study, we show that neither the suppression of endogenous parkin expression nor ectopic overexpression affects the steady-state levels of endogenous alpha-syn expression, overall aggregation of this protein, or breakdown of pre-formed aggregates in human neuroblastoma cells. These results suggest that parkin is not directly involved in the metabolism of alpha-syn, its aggregation, or the clearance of pre-formed aggregates.