Mitochondrial fusion/fission dynamics in neurodegeneration and neuronal plasticity.

Mitochondria are dynamic organelles that continually move, fuse and divide. The dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs, keeps mitochondria in good health by restoring or removing damaged organelles or precipitates cells in apoptosis in cases of severe defects. Mitochondrial fusion and fission are essential in mammals and their disturbances are associated with several diseases. However, while mitochondrial fusion/fission dynamics, and the proteins that control these processes, are ubiquitous, associated diseases are primarily neurological disorders. Accordingly, inactivation of the main actors of mitochondrial fusion/fission dynamics is associated with defects in neuronal development, plasticity and functioning, both ex vivo and in vivo. Here, we present the central actors of mitochondrial fusion and fission and review the role of mitochondrial dynamics in neuronal physiology and pathophysiology. Particular emphasis is placed on the three main actors of these processes i.e. DRP1,MFN1-2, and OPA1 as well as on GDAP1, a protein of the mitochondrial outer membrane preferentially expressed in neurons. This article is part of a Special Issue entitled: Mitochondria & Brain.

Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase.

CDC25 dual-specificity phosphatases are essential regulators that activate cyclin-dependent kinases (CDKs) at critical stages of the cell cycle. In human cells, CDC25A and C are involved in the control of G1/S and G2/M respectively, whereas CDC25B is proposed to act both in S phase and G2/M. Evidence for an interaction between CDC25 phosphatases and members of the 14-3-3 protein family has been obtained in vitro and in vivo in several organisms. On the basis of the work performed with CDC25C, it has been proposed that phosphorylation is required to mediate the interaction with 14-3-3. Here we have examined the molecular basis of the interaction between CDC25B phosphatases and 14-3-3 proteins. We show that in the two-hybrid assay all three splice variants of CDC25B interact similarly and strongly with 14-3-3eta, beta and zeta proteins, but poorly with epsilon and Theta. In vitro, CDC25B interacts at a low level with 14-3-3beta, epsilon, zeta, eta, and Theta isoforms. This interaction is not increased upon phosphorylation of CDC25B by CHK1 and is not abolished by dephosphorylation. In contrast, a specific, strong interaction between CDC25B and 14-3-3zeta and eta isoforms is revealed by a deletion of 288 residues in the amino-terminal region of CDC25B. This interaction requires the integrity of Ser 323, although it is independent of phosphorylation. Thus, interaction between 14-3-3 proteins and CDC25B is regulated in a manner that is different from that with CDC25C. We propose that, in addition to a low affinity binding site that is available for all 14-3-3 isoforms, post-translational modification of CDC25B in vivo exposes a high-affinity binding site that is specific for the zeta and eta14-3-3 isoforms.

The proto-oncogene c-fos increases the sensitivity of keratinocytes to apoptosis.

In human skin, most studies have suggested a role of c-fos or c-fos related genes in keratinocyte differentiation. The aim of our work was to more directly address this question by transfecting more or less differentiated keratinocyte cell lines (A431 and HaCaT) with constitutive expression vectors for c-Fos or c-Fos + c-Jun. Our results showed that c-Fos expression decreased keratinocyte growth, yet addition of c-Jun seemed to revert this c-Fos induced growth inhibition. Whereas no obvious differentiation program was turned on by c-Fos or c-Fos + c-Jun expression in our tissular model, apoptotic figures were observed and confirmed by in situ DNA fragmentation studies. These results do not rule out a role of c-Fos in keratinocyte differentiation but may indicate that the cell lines we used have reached an irreversible state of transformation so that they no longer respond to differentiation signals and rather die from apoptosis. These data add further evidence in favor of a role of c-Fos in epidermal homeostasis.

MDM-2 protein is expressed in different layers of normal human skin.

MDM-2 is one of the target genes of the p53 tumor suppressor protein. Its best characterized function is found in the inhibition of p53's ability to modulate transcription. Deregulated expression of MDM-2 could thus at least partially substitute for p53 mutation in the process of tumorigenesis. We show here that MDM-2 is highly expressed in biopsies of normal human skin or in vitro reconstituted human skin. The protein is detected in the nucleus of keratinocytes throughout the different layers of the epidermis and in reconstituted skin as early as the two to three cell layer stage. The 90 kiloDalton (kD) protein is one of the major forms detected in Western blot experiments. MDM-2 is detected in skin reconstituted from keratinocytes in which p53 is inactivated by mutation or degradation by E6 protein, providing evidence that MDM-2 expression in the skin can occur in the absence of wild type p53. Moreover, we found no correlation between the p53 status and MDM-2 expression levels in a series of basal and squamous cell carcinomas or Bowen diseases. Our data provide first evidence for the expression of MDM-2 in a differentiated adult tissue.

1,25-Dihydroxyvitamin D3 and its synthetic derivatives MC903 and EB1089 induce a partial tumoral phenotype reversal in a skin-equivalent system.

The antitumoral potency of 1,25-dihydroxyvitamin D3 and its synthetic derivatives MC903, EB1089, and KH1060 was investigated on a tumoral Bowen-like epidermis reconstructed from an immortalized human keratinocyte cell line transfected by expression vectors coding for E6 and E7 of human papillomavirus 16. Treatment of skin equivalents by vitamin D derivatives (10(-9) M or 10(-12) M) was performed during (from day 1 to day 15 of culture) or after tissue reconstruction (from day 15 to day 30). Pharmacologic effects were evaluated by morphologic and immunohistologic analysis and compared with those of controls (vehicle alone) and with treatment of skin equivalents derived from normal keratinocytes. When performed during epidermal reconstruction, treatment of tumoral skin equivalents induced only minor morphologic and immunohistologic changes. Conversely, when performed after epidermal reconstruction, treatment with 1,25-dihydroxyvitamin D3, MC903, and EB1089 clearly improved the phenotype of treated tissues. Morphologic analysis showed reorganization of epidermal layers with the appearance of a distinct basal layer and of stratified orthokeratotic stratum corneum. Immunohistochemical analysis demonstrated that the terminal differentiation markers profilaggrin and cytokeratin 10 were re-expressed in the treated tissues while absent in controls. Overall, the results indicate that 1,25-dihydroxyvitamin D3, MC903, and EB1089 can induce a partial reversion of the tumoral phenotype in this in vitro model.

Comparative analysis of normal and psoriatic skin both in vivo and in vitro.

The morphological and biochemical characteristics of psoriasis are well documented, but the pathogenesis of this disease is not clearly understood. A variety of in vitro models of psoriasis have been developed in attempts to identify the trigger factors, but no model so far reproduces the stable psoriatic phenotype accurately. In the present work, we initially checked the immunohistochemical distribution of proliferation/differentiation markers in psoriatic skin in vivo, and our results largely confirm previously reported data. However the study was performed using a new series of monoclonal antibodies to keratin. Subsequently we took normal or psoriatic skin biopsies, reconstructed skin equivalents using a recently described model and analysed the proliferation/differentiation status of the resulting epidermis. Dramatic morphological and antigenic differences were found between normal and psoriatic skin in vivo, but whatever the source of the initial biopsy, a unique in vitro phenotype was obtained in the reconstructed epidermis. This phenotype was marked by mild hyperproliferation and an altered distribution of differentiation-associated antigens suggesting a need for extracutaneous stimuli to maintain the psoriatic phenotype in vitro.

TP53 tumor suppressor gene and skin carcinogenesis.

The tumor suppressor gene TP53 encodes for a nuclear phosphoprotein involved in the control of cell proliferation, particularly in stressed cells. TP53 gene mutations are the most frequent genetic event found in human cancers. Most mutations locate in the highly conserved domains of the gene. Their localizations vary according to the tissue and tumor type, but define some hot spot regions that may have a certain degree of tissue specificity. In certain cases, the type of nucleotide substitutions observed can help to find the carcinogenic agent. In recent years, TP53 gene mutations have been frequently observed in human skin tumors. In epithelial carcinomas, they involve mainly exons 5, 7, and 8. Interestingly, many are C to T transitions at dipyrimidine sites; particularly, one can find CC to TT double-base changes that are known to be specific to ultraviolet radiation. These data confirm at the molecular level the role of ultraviolet radiation as an important etiologic factor in the genesis of these lesions. The high incidence of TP53 mutations suggest that they play a role in keratinocyte transformation. Nevertheless, this event has not yet been defined as an early or late event. In melanomas, most studies have shown the detection of the p53 protein by immunohistochemistry, suggestive of the presence of a mutation in the gene prolonging the protein half-life. Anti-p53 reactivity is frequent in these tumors and seems to correlate with tumor aggressiveness. Confirmation and characterization of TP53 gene mutation at the DNA level would help to precisely define the role of this gene in the development of these tumors.

C3d,g deposits in inflammatory skin diseases: use of psoriatic skin as a model of cutaneous inflammation.

Recent studies in our laboratories have shown that human keratinocytes synthesize and secrete complement components including C3. Moreover, human keratinocyte-derived C3 is regarded as a potential source of C3d,g, a recently described constituent of the sublamina densa region of normal epidermal basement membrane. Additionally, human keratinocyte-derived C3 may also contribute to epidermal basement membrane deposits of C3 in autoimmune or inflammatory skin disorders. To further our understanding of the specificity and origin of epidermal basement membrane C3 deposits in normal and diseased skin, we have characterized in situ deposits of C3 and C3 cleavage fragments in various inflammatory skin diseases and utilized a skin equivalent model to assess the deposition of C3 cleavage fragments in neo-basement membrane of epidermal outgrowths from normal or diseased human skin. C3d,g reactivity was found to be greater in all samples of inflamed skin, and typically associated with C3c reactivity at these sites. No immunoglobulins or other complement components were detected. When lesional psoriatic skin rich in epidermal basement membrane C3c was used in our organ culture system, C3 incorporation within neo-basement membrane was observed. These results show that human keratinocyte-derived C3 may contribute to inflammatory reactions in skin as well as account for deposits of C3d,g in normal epidermal basement membrane.

TP53 tumor-suppressor gene and human carcinogenesis.

The wild-type tumor-suppressor TP53 gene encodes for a nuclear protein which has been shown to act as a transcriptional modulator. The cellular role of the p53 protein is the control of cell proliferation, particularly important in stressed cells. The TP53 gene is frequently mutated in sporadic and familial human cancers. Most transforming mutations localize in highly conserved domains of the gene and define hot-spot regions that have a certain degree of tissue specificity. Moreover, most mutations are point mutations and the type and localization of the nucleotide substitution may sometimes help in recognizing the carcinogenic agent. This is the case for C to T transitions at dipyrimidine sites induced by UV radiation in cutaneous epitheliomas. Inactivation of p53 protein can also occur through mechanisms other than genetic alteration, such as binding to viral or cellular proteins. Loss of wild-type TP53 function seems therefore to play a crucial role in cell transformation in human cancers, either during carcinogenesis or later in tumor progression.

The expression of desmosomal and corneodesmosomal antigens shows specific variations during the terminal differentiation of epidermis and hair follicle epithelia.

Using five monoclonal antibodies (MAb), we studied by indirect immunofluorescence the desmosomes and a junctional structure specific to cornified layers, the corneodesmosome, in normal and plantar epidermis and in the various sheaths of the anagen hair follicle. The monoclonal antibodies DP1&2.2-15, PG5.1, and DG3.10, specific for desmoplakins I/II, plakoglobin, and desmoglein I, respectively, were used to study the desmosome antigens, and G36-19 and G20-21 to study the corneodesmosome antigens. The distribution and sequence of expression of the five antigens allowed the nine epithelial differentiation pathways studied to be merged into four distinct families: non-plantar epidermis, characterized by the absence of desmosome and corneodesmosome antigens in the stratum corneum; the outer root sheath of the hair follicle, which behaves like the viable layers of the epidermis with regard to the desmosome antigens but does not express the corneodesmosome antigens; plantar epidermis and the three components of the inner root sheath in which the corneodesmosome antigens are present up to the desquamating layer; and the three components of the hair shaft, which are characterized by the absence of expression of both the desmosome and the corneodesmosome antigens in its mature portion.

Identification of late differentiation antigens of human cornified epithelia, expressed in re-organized desmosomes and bound to cross-linked envelope.

Little is known about the process leading to desquamation in cornified epithelia. We describe late differentiation antigens (Ag) specific for human cornified squamous epithelia, defined by two murine monoclonal antibodies (MoAb), G36-19 and B17-21, produced after immunization with plantar stratum corneum (SC). Histologically, in epidermis both Ag are cytoplasmic in the lower stratum granulosum (SG), become pericellular in the upper SG, and progressively disappear in the lower SC. In contrast, they persist up to the desquamating corneocytes in the palmoplantar epidermis and hard palate epithelium, as well as in the three cornified epithelial components of the inner root sheath (IRS) of the hair follicle (HF). Cytologically, both Ag are expressed as surface spots only on rough corneocytes. They are largely preserved on cross-linked envelopes (CLE) of the fragile type. Ultrastructurally, both Ag appear in keratinosome-like cytoplasmic vesicles in the upper stratum spinosum (SS) and the SG keratinocytes, then are found in both the regular and reorganizing desmosomes of the SG keratinocytes, and lastly in the corneocyte-specific reorganized desmosomes we propose to name corneodesmosomes. On CLE, the Ag are located on fibrils gathered over the external side of the envelope. Immunochemically, the G36-19--defined epitope is sequential and shared by five non-cytokeratin protein antigens of molecular weight 33.5, 36.5, 40, 49, and 52 kD, the higher molecular weight polypeptides being possibly precursors of the 33.5-kD protein. In contrast, the B17-21 epitope, unaccessible by immunoblotting, is probably conformational. In long-term cultured keratinocytes, the Ag are only expressed when epidermal sheets are morphologically differentiated. The expression is enhanced in the absence of fetal calf serum (FCS) and of epidermal growth factor (EGF). G36-19 and B17-21 Ag participate in a corneodesmosome-CLE superstructure that is probably involved in corneocyte cohesiveness and partly responsible for the mechanical resistance of the SC. These Ag are relevant markers for studying desmosomal maturation during epidermal differentiation and desquamation.

Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.

Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.