Katja Rosenkranz

Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol

Peroxisomal import receptors bind their cargo proteins in the cytosol and target them to docking and translocation machinery at the peroxisomal membrane (reviewed in ref. 1). The receptors release the cargo proteins into the peroxisomal lumen and, according to the model of cycling receptors, they are supposed to shuttle back to the cytosol. This shuttling of the receptors has been assigned to peroxins including the AAA peroxins Pex1p and Pex6p, as well as the ubiquitin-conjugating enzyme Pex4p (reviewed in ref. 2). One possible target for Pex4p is the PTS1 receptor Pex5p, which has recently been shown to be ubiquitinated. Pex1p and Pex6p are both cytosolic and membrane-associated AAA ATPases of the peroxisomal protein import machinery, the exact function of which is still unknown. Here we demonstrate that the AAA peroxins mediate the ATP-dependent dislocation of the peroxisomal targeting signal-1 (PTS1) receptor from the peroxisomal membrane to the cytosol.

Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites

Tail-anchored proteins contain a single transmembrane domain (TMD) followed by a short C-terminal domain extending into the organellar lumen. Tail-anchored proteins are thought to target to the correct subcellular compartment by virtue of general physicochemical properties of their C-termini; however, the machineries that enable correct sorting remain largely elusive. Here we analyzed targeting of the human peroxisomal tail-anchored protein PEX26. Its C-terminal-targeting signal contains two binding sites for PEX19, the import receptor for several peroxisomal membrane proteins. One PEX19-binding site overlapped with the TMD, the other was contained within the luminal domain. Although the PEX19-binding site containing the TMD targeted to peroxisomes to some extent, the luminal site proved essential for correct targeting of the full-length protein, as it prevented PEX26 from mislocalization to mitochondria. Its function as a targeting motif was proved by its ability to insert a heterologous TMD-containing fragment into the peroxisomal membrane. Finally we show that PEX19 is essential for PEX26 import. Analysis of the yeast tail-anchored protein Pex15p revealed that it also harbors a luminal PEX19-binding site that acts as a peroxisomal-targeting motif. We conclude that C-terminal PEX19-binding sites mark tail-anchored proteins for delivery to peroxisomes.

The chemokine SDF-1/CXCL12 contributes to the 'homing' of umbilical cord blood cells to a hypoxic-ischemic lesion in the rat brain.

Previous studies have shown that transplanted human umbilical cord blood (hUCB)‐derived mononuclear cells exert therapeutic effects in various animal models of CNS impairments, including those of perinatal hypoxic‐ischemic brain injury. However, the mechanisms of how transplanted cells exert their beneficial effects on the damaged tissue are still unclear. As detection of hUCB cells at the lesion site coincides with the therapeutic effects observed in our model, we investigated the role of the chemokine stromal derived factor (SDF)‐1 (CXCL12) as a possible candidate for chemotaxis‐mediated ‘homing’ of transplanted hUCB cells to a hypoxic‐ischemic lesion in the perinatal rat brain. Following the hypoxic‐ischemic insult expression of SDF‐1 significantly increased in lesioned brain hemispheres and was mainly associated with astrocytes. Transplanted hUCB cells expressing the SDF‐1 receptor CXCR4 migrated to the lesion site within one day. Inhibition of SDF‐1 by application of neutralizing antibodies in vivo resulted in a significantly reduced number of hUCB cells at the lesioned area. The increase in glial SDF‐1 expression shortly after induction of the lesion and hUCB cells expressing the corresponding receptor makes SDF‐1 a potential chemotactic factor for hUCB cell migration. The reduction of hUCB cells present at the lesion site upon functional inhibition of SDF‐1 strengthens the view that the SDF‐1/CXCR4 axis is of major importance for cell ‘homing’.

Transplantation of human umbilical cord blood cells mediated beneficial effects on apoptosis, angiogenesis and neuronal survival after hypoxic-ischemic brain injury in rats

Transplantation of human umbilical cord blood (hucb) cells in a model of hypoxic-ischemic brain injury led to the amelioration of lesion-impaired neurological and motor functions. However, the mechanisms by which transplanted cells mediate functional recovery after brain injury are largely unknown. In this study, the effects of hucb cell transplantation were investigated in this experimental paradigm at the cellular and molecular level. As the pathological cascade in hypoxic-ischemic brain injury includes inflammation, reduced blood flow, and neuronal cell death, we analyzed the effects of peripherally administered hucb cells on these detrimental processes, investigating the expression of characteristic marker proteins. Application of hucb cells after perinatal hypoxic-ischemic brain injury correlated with an increased expression of the proteins Tie-2 and occludin, which are associated with angiogenesis. Lesion-induced apoptosis, determined by expression of cleaved caspase-3, decreased, whereas the number of vital neurons, identified by counting of NeuN-positive cells, increased. In addition, we observed an increase in the expression of neurotrophic and pro-angiogenic growth factors, namely BDNF and VEGF, in the lesioned brain upon hucb cell transplantation. The release of neurotrophic factors mediated by transplanted hucb cells might cause a lower number of neurons to undergo apoptosis and result in a higher number of living neurons. In parallel, the increase of VEGF might cause growth of blood vessels. Thus, hucb transplantation might contribute to functional recovery after brain injury mediated by systemic or local effects.

Structural and functional analysis of the interaction of the AAA‐peroxins Pex1p and Pex6p

The AAA‐peroxins Pex1p and Pex6p play a critical role in peroxisome biogenesis but their precise function remains to be established. These two peroxins consist of three distinct regions (N, D1, D2), two of which (D1, D2) contain a conserved ≈ 230 amino acid cassette, which is common to all ATPases associated with various cellular activities (AAA). Here we show that Pex1p and Pex6p from Saccharomyces cerevisiae do interact in vivo. We assigned their corresponding binding sites and elucidated the importance of ATP‐binding and ‐hydrolysis of Pex1p and Pex6p for their interaction. We show that the interaction of Pex1p and Pex6p involves their first AAA‐cassettes and demonstrate that ATP‐binding but not ATP‐hydrolysis in the second AAA‐cassette (D2) of Pex1p is required for the Pex1p–Pex6p interaction. Furthermore, we could prove that the second AAA‐cassettes (D2) of both Pex1p and Pex6p were essential for peroxisomal biogenesis and thus probably comprise the overall activity of the proteins.

Functional association of the AAA complex and the peroxisomal importomer

The AAA peroxins, Pex1p and Pex6p, are components of the peroxisomal protein import machinery required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We demonstrate that Pex1p and Pex6p form a stable complex in the cytosol, which associates at the peroxisomal membrane with their membrane anchor Pex15p and the peroxisomal importomer. The interconnection of Pex15p with the components of the importomer was independent of Pex1p and Pex6p, indicating that Pex15p is an incorporated component of the assembly. Further evidence suggests that the AAA peroxins shuttle between cytosol and peroxisome with proper binding of the Pex15p–AAA complex to the importomer and release of the AAA peroxins from the peroxisomal membrane depending on an operative peroxisomal protein import mechanism. Pex4p‐deficient cells exhibit a wild‐type‐like assembly of the importomer, which differs in that it is associated with increased amounts of Pex1p and Pex6p, in agreement with a function for Pex4p in the release of AAA peroxins from the peroxisomal membrane.

Umbilical cord blood cell transplantation after brain ischemia—From recovery of function to cellular mechanisms

Cell transplantation has been proposed as a potential approach to the treatment of neurological disorders. One cell population of interest consists of human umbilical cord blood (hUCB) cells, which have previously been shown to be useful for reparative medicine in haematological diseases. However, hUCB cells are also capable of differentiating into various non-haematopoietic cells, including those of the neural lineage. Moreover, hUCB cells can secrete numerous neurotrophic factors and modulate immune function and inflammatory reaction. Several studies on animal models of ischemic brain injury have demonstrated the potential of hUCB cells to minimize damage and promote recovery after ischemic brain injury.This review focuses on the treatment of both stroke and perinatal hypoxic-ischemic brain injury using hUCB cells. We discuss the therapeutic effects demonstrated after hUCB cell transplantation and emphasize possible mechanisms counteracting pathophysiological events of ischemia, thus leading to the generation of a regenerative environment that allows neural plasticity and functional recovery. The therapeutic functional effects of hUCB cells observed in animal models make the transplantation of hUCB cells a promising experimental approach in the treatment of ischemic brain injury. Together with its availability, low risk of transplantation, immaturity of cells, and simple route of application, hUCB transplantation may stand a good chance of being translated into a clinical setting for the therapy of ischemic brain injury.

Changes in Interleukin-1 alpha serum levels after transplantation of umbilical cord blood cells in a model of perinatal hypoxic-ischemic brain damage.

Transplantation of human umbilical cord blood (hUCB) cells is a potential approach for the treatment of perinatal hypoxic-ischemic brain injury. Neurological and motor deficits resulting from the brain lesion are ameliorated upon transplantation. The molecular mechanisms underlying these improvements are currently being unravelled. One parameter identified as part of the beneficial effects of hUCB cells is the reduction of brain inflammation. It is, however, unclear whether the modulation of brain inflammation is due to local or systemic effects of hUCB cells. In this study, the effects of hUCB cell transplantation in a model of perinatal hypoxic-ischemic brain injury were investigated at the systemic level by measurement of serum levels of pro-inflammatory cytokines by multiplex bead arrays. Two days after induction of the brain damage, levels of the pro-inflammatory cytokines Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), and Tumor necrosis factor α (TNFα) were increased in the serum of rats. Application of hUCB cells, in turn, correlated with a reduced elevation of serum levels of these pro-inflammatory cytokines. This decrease was accompanied by a reduced expression of CD68, a marker protein of activated microglia/macrophages in the brain. Therefore, systemic modulation of the immune response by hUCB cells could represent one possible mechanism of how these cells might mediate their beneficial effects. Creation of a regenerative environment with reduced inflammation might account for the functional regeneration observed upon hUCB cell treatment in lesioned animals.

Proteomic Analysis of Alterations Induced by Perinatal Hypoxic–Ischemic Brain Injury

Perinatal hypoxic-ischemic brain injury is an important cause of neurological deficits still causing mortality and morbidity in the early period of life. As efficient clinical or pharmaceutical strategies to prevent or reduce the outcome of perinatal hypoxic-ischemic brain damage are limited, the development of new therapies is of utmost importance. To evolve innovative therapeutic concepts, elucidation of the mechanisms contributing to the neurological impairments upon hypoxic-ischemic brain injury is necessary. Therefore, we aimed for the identification of proteins that are affected by hypoxic-ischemic brain injury in neonatal rats. To assess changes in protein expression two days after induction of brain damage, a 2D-DIGE based proteome analysis was performed. Among the proteins altered after hypoxic-ischemic brain injury, Calcineurin A, Coronin-1A, as well as GFAP were identified, showing higher expression in lesioned hemispheres. Validation of the changes in Calcineurin A expression by Western Blot analysis demonstrated several truncated forms of this protein generated by limited proteolysis after hypoxia-ischemia. Further analysis revealed activation of calpain, which is involved in the limited proteolysis of Calcineurin. Active forms of Calcineurin are associated with the dephosphorylation of Darpp-32, an effect that was also demonstrated in lesioned hemispheres after perinatal brain injury.

Cx43 expression and function in the nervous system—implications for stem cell mediated regeneration

Pathological conditions of the brain such as ischemia cause major sensorimotor and cognitive impairments. In novel therapeutic approaches to brain injury, stem cells have been applied to ameliorate the pathological outcome. In several experimental models, including hypoxia-ischemia and trauma, transplantation of stem cells correlated with an improved functional and structural outcome. At the cellular level, brain insults also change gap junction physiology and expression, leading to altered intercellular communication. Differences in expression in response to brain injury have been detected in particular in Cx43, the major astrocytic gap junction protein, and its overexpression or deletion was associated with the pathophysiological outcome. We here focus on Cx43 changes in host tissue mediated by stem cells. Stem cell-induced changes in connexin expression, and consecutively in gap junction channel or hemichannel function, might play a part in altered cell interaction, intercellular communication, and neural cell survival, and thereby contribute to the beneficial effects of transplanted stem cells.