Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-ß hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-ß hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

Localization of D1a dopamine receptors on cell bodies and axonal endings in the substantia nigra pars reticulata of the rat.

In the present study, we analyzed the localization of D1a receptors within the rat substantia nigra pars reticulata (SNr) using specific D1a immunochemistry at the ultrastructural level and RT-PCR. At the electron microscopic level, D1a receptors were strongly associated with axons and axonal endings in the SNr, but also with numerous glutamic acid decarboxylase-positive dendrites and neuronal cell bodies. This neuronal expression of D1a receptors was confirmed using RT-PCR. G(alphaolf) protein-specific immunostaining displayed a similar distribution in dendrites and cell bodies to that of D1a receptors. The localization of D1a receptors in both GABAergic cell bodies and terminals is in accordance with the well known complex action of dopamine in the SNr. Moreover, the intracytoplasmic localization of D1a receptors in cell bodies and dendrites that we observed suggests that these receptors are only effective in specific conditions, or are transported to different nigral targets where they may have a presynaptic function.

Mutations in the SPG3A gene encoding the GTPase atlastin interfere with vesicle trafficking in the ER/Golgi interface and Golgi morphogenesis.

Mutations in SPG3A causing autosomal dominant pure spastic paraplegia led to identification of atlastin, a new dynamin-like large GTPase. Atlastin is localized in the endoplasmic reticulum, the Golgi, neurites and growth cones and has been implicated in neurite outgrowth. To investigate whether it exerts its activity in the early secretory system, we expressed normal and mutant atlastin in cell culture. Pathogenic mutations in the GTPase domain interfered with the maturation of Golgi complexes by preventing the budding of vesicles from the endoplasmic reticulum, whereas mutations in other regions of the protein disrupted fission of endoplasmic reticulum-derived vesicles or their migration to their Golgi target. Atlastin, therefore, plays a role in vesicle trafficking in the ER/Golgi interface. Furthermore, atlastin partially co-localized with proteins of the p24/emp/gp25L family that regulate vesicle budding and trafficking in the early secretory pathway, and co-immunoprecipitated p24, suggesting a functional relationship that should be further explored.

Ultrastructural localization of parkin in the rat brainstem, thalamus and basal ganglia.

The parkin gene encodes a 52 kd putative E3 ubiquitin-protein ligase involved in an autosomal recessive form of early onset parkinsonism. Parkin ultrastructural localization was studied by immunohistochemistry in the adult rat brain and in a parkin inducible PC12 cell line (HS22). In the rat brain, parkin immunoreactivity was detected in neuronal and glial cell bodies and in nerve processes. In the neurons, it was mostly localized on the periphery of large vesicles, some rare mitochondria and endoplasmic reticulum in the cell bodies, and on the periphery of large vesicles in the dendrites and terminals of the neurons. In addition, parkin immunoreactivity was also found around synaptic vesicles in the presynaptic elements of some axons. In HS22 cells over-expressing parkin, the distribution of the protein was similar to that observed in the perikarya of the labeled neurons.

Plasticity of afferent fibers to striatal neurons bearing D1 dopamine receptors in Parkinson's disease.

The loss of dopaminergic neurons in the substantia nigra provokes a plasticity of corticostriatal synapses in Parkinson's disease (PD). The corticostriatal pathway nevertheless makes synapses with neurons bearing D1 dopamine receptors (D1R) and/or D2 dopamine receptors. At the ultrastructural level, we analyzed the morphological characteristics of synapses formed by afferent fibers making asymmetric contacts with the dendritic spines of neurons identified by D1R immunoreactivity, in the striatum of control subjects and PD patients. A quantitative analysis of the morphological characteristics of the synapses and of the number of perforated synapses (considered to be very active) was performed. In PD, a 50% increase in the number of perforated synapses making contact with D1R dendritic spines was observed, whereas no change in the number of perforated synapses on non-D1R spines was observed. The change in the number of perforated synapses on D1R dendrites was associated with a slight but nonsignificant increase in the surface area of the corticostriatal afferent fibers and the surface of the mitochondria in these fibers (+29.0% and +34.6%, respectively). This suggests a hyperactivity of corticostriatal fibers in contact with D1R-bearing neurons of the direct pathway in the basal ganglia circuitry. Since stimulation of the direct pathway is thought to alleviate the clinical symptoms of PD, this suggests that the differences observed may be involved in compensatory mechanisms.

Parkin immunoreactivity in the brain of human and non-human primates: an immunohistochemical analysis in normal conditions and in Parkinsonian syndromes.

The etiology of Parkinson's disease is unknown, but the gene involved in an autosomic recessive form of the disease with early onset has recently been identified. It codes for a protein with an unknown function called parkin. In the present study we produced a specific polyclonal antiserum against human parkin. Immunohistochemical analysis showed that parkin is expressed in neuronal perikarya and processes but also in glial and blood vessels in the primate brain (human and monkey). Electron microscopy indicated that parkin immunoreactivity is mostly located in large cytoplasmic vesicles and at the level of the endoplasmic reticulum. Parkin was expressed heterogeneously in various structures of the brain. It was detectable in the dopaminergic systems at the level of the perikarya in the mesencephalon but also in the striatum. However, parkin was also expressed by numerous nondopaminergic neurons. The staining intensity of parkin was particularly high in the hippocampal formation, the pallidal complex, the red nucleus, and the cerebellum. Comparison of control subjects with patients with Parkinson's disease and control animals with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated animals revealed a loss of parkin-immunoreactive neurons only in the substantia nigra pars compacta. Furthermore, the surviving dopaminergic neurons in the parkinsonian state continued to express parkin at a level similar to that observed in the control situation. These data indicate that parkin is a widely expressed protein. Thus, the degeneration of dopaminergic neurons in familial cases of Parkinson's disease with autosomal recessive transmission cannot be explained solely in terms of an alteration of this protein.

Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.

Mitochondrial free calcium levels measured by Rhod-2 fluorescence and ultrastructure were examined during cell death in nerve growth factor (NGF)-differentiated PC12 cells that were 1) exposed to C2-ceramide, 2) deprived of serum to induce endogenous ceramide production, or 3) treated with calcium ionophore A23187. Rhod-2 fluorescence in mitochondria and also in the nucleolus increased to a maximum within 3 hours after C2-ceramide treatment or serum withdrawal. In A23187-treated cells, Rhod-2 fluorescence remained at baseline levels. In all three models, enlargement of the endoplasmic reticulum was the first ultrastructural alteration, followed by mitochondrial shrinkage in ionophore-treated cells, but by mitochondrial swelling in the ceramide-dependent models, in which rupture of the outer mitochondrial membrane and unfolding of the inner membrane were frequently seen. Dihydro-C2-ceramide, which did not cause cell death, had no effect on cellular ultrastructure. NGF, which inhibits ceramide-dependent cell death, prevented the effects of serum deprivation on mitochondrial ultrastructure but not on endoplasmic reticulum morphology or Rhod-2 fluorescence. Nuclear shrinkage with loss of nuclear membrane integrity, characterized by nuclear pores, free or surrounded by electron-dense filaments, was a late event in ceramide-dependent cell death. Chromatin condensation and other morphological features associated with apoptosis were seen in only a few atypical cells. Ceramide-mediated cell death, therefore, did not involve classical apoptosis but was mediated by a reproducible series of events beginning in the endoplasmic reticulum, followed by the mitochondria, and then the nucleus. NGF-dependent cell death inhibition intervenes at the mitochondrial level, not by blocking the increase in Rhod-2 fluorescence but by preventing the ultrastructural changes that follow.

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin-1, galectin-3, galectin-10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well-known lectin, galectin-3, a lactose-binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin-3 interacts in the cytoplasm with Bcl-2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N-acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur?

Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease.

Caspase-3 is an effector of apoptosis in experimental models of Parkinson's disease (PD). However, its potential role in the human pathology remains to be demonstrated. Using caspase-3 immunohistochemistry on the postmortem human brain, we observed a positive correlation between the degree of neuronal loss in dopaminergic (DA) cell groups affected in the mesencephalon of PD patients and the percentage of caspase-3-positive neurons in these cell groups in control subjects and a significant decrease of caspase-3-positive pigmented neurons in the substantia nigra pars compacta of PD patients compared with controls that also could be observed in an animal model of PD. This suggests that neurons expressing caspase-3 are more sensitive to the pathological process than those that do not express the protein. In addition, using an antibody raised against activated caspase-3, the percentage of active caspase-3-positive neurons among DA neurons was significantly higher in PD patients than in controls. Finally, electron microscopy analysis in the human brain and in vitro data suggest that caspase-3 activation precedes and is not a consequence of apoptotic cell death in PD.

Levodopa induces a cytoplasmic localization of D1 dopamine receptors in striatal neurons in Parkinson's disease.

Parkinson's disease is characterized by a massive loss of nigral dopamine neurons that results in a reduction of dopamine concentrations in the striatum. The most commonly used treatment for this disease is levodopa therapy to restore striatal dopamine. This treatment is mediated by dopamine receptors, but the effect of treatment and the disease on receptor distribution is unknown. In this study, the distribution of D1 dopamine receptors was analyzed at the cellular and subcellular level in the striatum of 5 patients with Parkinson's disease (all treated with levodopa) and 4 control subjects. In the control brains, D1 dopamine receptors were mostly detected on the plasma membrane of medium-sized spiny neurons. The quantitative analysis performed at the ultrastructural level in patients with Parkinson's disease revealed an increase in immunostaining in the cytoplasm of medium-sized neurons. This effect was likely the result of the treatment rather than the dopaminergic denervation, as such changes were not observed in the striatum of rats with a unilateral 6-hydroxydopamine nigrostriatal lesion, but were present in normal or lesioned rats treated with a D1 dopamine agonist. Altered localization of D1 dopamine receptors may participate in the occurrence of side effects of levodopa therapy such as dyskinesia and fluctuations in motor performances.

Atypical microtubule organization in undifferentiated human colon cancer cells.

We previously reported that undifferentiated colonic cancer HT-29 cells, unlike the differentiated ones, exhibit unusual organelle distributions and atypical vesicle trafficking patterns, which are microtubule-independent and microfilament-dependent. In the present study, we have analyzed the microtubule network in both phenotypes, using confocal microscopy, and determined the expression levels of some microtubule-associated proteins by quantitative immunoblotting. Differentiated cells exhibited the microtubular organization of polarized epithelial cells. Non-polarized undifferentiated cells presented an atypical microtubule organization as microtubules were localized mainly at the cell 'top'. Immunoblot analysis indicated the absence or low content of several structural and motor microtubule-associated proteins in undifferentiated cells, compared to differentiated cells. This may explain in part their atypical microtubular organization. This study agrees with a crucial role for microfilaments in the intracellular organization of undifferentiated HT-29 cancer cells, while differentiated HT-29 cells exhibit intracellular organization similar to that of normal enterocytic cells, although they are also tumoral.

Nuclear translocation of NF-kappaB is increased in dopaminergic neurons of patients with parkinson disease.

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-kappaB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-kappaB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-kappaB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-kappaB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-kappaB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

Barrier function of reconstructed epidermis at the air-liquid interface: influence of dermal cells and extracellular components.

To evaluate the epidermal barrier function of in vitro reconstructed epidermis, we measured the penetration of estradiol and water across human keratinocytes cultured in defined medium (DM), in the presence of proliferative fibroblasts (pF) or conditioned medium derived from pF, at the air-liquid interface on synthetic porous membrane, noncoated or coated with laminin, fibronectin, type I collagen or type IV collagen. Ultrastructural analysis showed a well-developed stratum corneum whatever the culture conditions. The permeability of reconstructed epidermis in DM on a noncoated porous membrane was 5- to 10-fold higher than human native epidermis, with both tracers. No significant change in barrier function was observed whatever the culture conditions.

Human keratinocyte models: Assessment of cell adhesion and dermotoxicity using fluorescent probes.

To assess the molecular and cellular events that occur in the skin during biological and pharmaco-toxicological processes, we developed different in vitro models. Two major systems are described: (1) relatively simple ones such as normal human keratinocytes (NHK) grown in monolayer or continuous culture of spontaneously immortalized keratinocyte cells, the HaCaT cell line. This cell line forms a monolayer and displays the same phenotypic morphology, pattern of differentiation markers as NHK. (2) More complex models such as NHK multilayers differentiated on a synthetic porous membrane. Indeed, NHK grown at the air-liquid interface of culture inserts may undergo epidermal differentiation in 21 days (Noël-Hudson et al., 1995a). Under the same culture conditions, no stratification of the HaCaT cell line was obtained. NHK and/or HaCaT monolayers were used to study the cell surface molecules involved in heterologous cell interactions, and to estimate the cytotoxic effects of different compounds through a sensitive fluorimetric microtitration assay. When cell adhesion was measured with calcein-AM labelled lymphocytes, it appeared that lymphocytes display the same behaviour towards NHK or HaCaT cells. The importance of the activation status of each cell and the involvement of alpha(2) and alpha(3)beta(1) integrins in lymphocyte-keratinocyte interactions were demonstrated. Likewise cytotoxicity of SDS and DNP was easily and rapidly detected with calcein-AM and Alamar blue probes. Skin models in combination with fluorescent probes offer promising alternatives for assessing cell interactions as well as cytotoxic effects.

Organization of the endoplasmic reticulum-Golgi system is related to the state of enterocytic differentiation of human HT-29 cells.

The organization of the endoplasmic reticulum (ER)-intermediate compartment (IC)-Golgi system was studied in tumoral HT-29 cells. Depending on the culture conditions, these cells are either undifferentiated or exhibit enterocytic differentiation after reaching confluence. In differentiated HT-29 cells, these organelles were organized as in most cell types. They displayed a classical structure and appeared associated with microtubules, as nocodazole altered both their structure and intracellular localization. Likewise, membrane dynamics of the Golgi appeared normal: as in many other cells, brefeldin A (BFA) induced retrograde transport from the Golgi to the ER, demonstrated by tubulation of the Golgi elements and shift of the galactosyltransferase activity from the Golgi- to the RER-enriched fraction, isolated by subcellular fractionation. In contrast, atypical features were observed in undifferentiated HT-29 cells: the Golgi structure exhibited abnormal swellings; the IC elements were very rare. Only cytochalasin D altered the structure and intracellular localization of the three organelles, suggesting that they were associated with microfilaments instead of microtubules. The membrane dynamics were unusual: brefeldin A led to a vesiculation of the Golgi elements with a slowed-down retrograde transport of galactosyltransferase. HT-29 cells engaged in the differentiation process, but which were still undifferentiated, showed mainly the features of undifferentiated cells, with a few characteristics of differentiated cells. These results indicate that the structure of the Golgi apparatus, IC and ER, their relationships to cytoskeletal elements and membrane dynamics depend on the state of differentiation of HT-29 cells. Although they are tumoral, differentiated HT-29 cells exhibit features observed in non-tumoral polarized epithelial cells. On the contrary, undifferentiated HT-29 cells display important abnormalities that may be related to their metastatic properties.

Ricin toxicity and intracellular routing in tumoral HT-29 cells. I. Ricin routing and toxicity are related to the state of differentiation of HT-29 cells.

We previously showed that ricin, which is more cytotoxic to undifferentiated than to differentiated tumoral HT-29 cells, enters these cells by different routes. The final steps of ricin endocytosis were investigated in order to identify the translocation site from which ricin exerts its toxicity. Toxicity measurements and kinetic experiments followed by subcellular fractionation were run in parallel. In differentiated cells, from 20 min of internalization, radiolabeled ricin was found in a Golgi-enriched fraction. At 60 min, which corresponds to the lag time for ricin toxicity, the amount of radioactivity located in this fraction decreased without any concomitant increase in the other fractions. In undifferentiated cells, from 20 min of incubation, radiolabeled ricin was detected in the ER-enriched fractions. At 30 min, the lag time for ricin toxicity, the amount of radioactivity detected in these fractions decreased without any concomitant increase in the Golgi-enriched fraction. Monensin, which was used to confirm the passage of ricin through the Golgi, greatly increased ricin toxicity and diminished the lag time only in differentiated cells. Brefeldin A inhibited ricin toxicity when added before the end of the lag time in both cell populations and reduced the amount of ricin detected, respectively, in the Golgi- and ER-enriched fractions in differentiated and undifferentiated cells. We propose that ricin enters the cytosol from the Golgi apparatus and essentially from the ER in differentiated and undifferentiated HT-29 cells, respectively, and that these different intracellular routings might explain the differential toxicity of ricin.

Ricin toxicity and intracellular routing in tumoral HT-29 cells. II. Differential ricin toxicity from the apical and basolateral surfaces of differentiated HT-29 cells.

We previously showed that ricin, a toxin commonly used in the construction of immunotoxins, was more toxic to undifferentiated than to differentiated HT-29 tumoral cells. This results from differences in the intracellular routings of the toxin. As these studies concerned the entry through the apical pole of differentiated polarized HT-29 cells, we investigated and compared the intracellular routing of ricin from the apical and basolateral membranes of differentiated HT-29 cells and the toxicity of ricin depending on the pole of administration. For this purpose, we developed the culture of polarized HT-29 cells on porous membrane filters and demonstrated that differentiated HT-29 cells can establish a leakproof monolayer. Ricin is 2.5-fold less toxic when it is added at the basolateral than at the apical pole of the cells, which may result from different observations: (1) less ricin is bound at the basolateral membrane than at the apical one, leading to a lesser internalization of the toxin; (2) ricin sorting in the apical and basolateral endocytic compartments of HT-29 cells differs: apically internalized ricin is targeted intracellularly while basolaterally internalized ricin uses mainly the transcytotic pathway; using NH4Cl and monensin, we observed that ricin follows the same pathway from both sides of the cells, namely the endosomal system, to reach the Golgi apparatus from which toxin intoxication occurs; (3) kinetics studies showed that a delay exists before an efficient intoxication by the basolateral pole is observed. The use of monensin at low concentration in order to perturb only the Golgi functions indicated that this delay could account for a different presentation of the toxin toward the membrane of the apical and basolateral endocytic compartments. Together, our results showed that, in differentiated HT-29 cells, if the pathways carrying ricin from the apical and basolateral membranes to the Golgi apparatus appear identical, ricin exerts differentially its toxicity depending upon the surface of administration, i.e., the apical or the basolateral surface of the cells.

Human epidermis reconstructed on synthetic membrane: influence of experimental conditions on terminal differentiation.

Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no proper stratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)