Alterations in the mitochondrial proteome of neuroblastoma cells 2 in response to complex 1 inhibition

Increasing evidence points to mitochondrial dysfunction in Parkinson's disease (PD) associated with complex I dysfunction, but the exact pathways which lead to cell death have not been resolved. 2D-gel electrophoresis profiles of isolated mitochondria from neuroblastoma cells treated with subcytotoxic concentrations of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), a well-characterized complex I inhibitor, were assessed to identify associated targets. Up to 27 differentially expressed proteins were observed, of which 16 were identified using peptide mass fingerprinting. Changes in protein levels were validated by immunoprobing ID blots, confirming increases in heat shock cognate 71 kDa (Hsc70), 60 kDa heat shock protein (Hsp60), fumarase, glutamate oxaloacetate transaminase 2, ATP synthase subunit d, and voltage-dependent anion-channel 1 (VDACl). Immunoprobing of 2D blots revealed isoform changes in Hsc70, Hsp60, and VDACl. Subcytoxic concentrations of MPTP modulated a host of mitochondrial proteins including chaperones, metabolic enzymes, oxidative phosphorylation-related proteins, an inner mitochondrial protein (mitofilin), and an outer mitochondrial membrane protein (VDACl). Early changes in chaperones suggest a regulated link between complex 1 inhibition and protein folding. VDACl, a multifunctional protein, may have a key role in signaling between mitochondria and the rest of the cell prior to cell death. Our work provides new important information of relevance to PD

The topology of connections between rat prefrontal and temporal cortices

Understanding the structural organization of the prefrontal cortex (PFC) is an important step toward determining its functional organization. Here we investigated the organization of PFC using different neuronal tracers. We injected retrograde (Fluoro-Gold, 100 nl) and anterograde [Biotinylated dextran amine (BDA) or Fluoro-Ruby, 100 nl] tracers into sites within PFC subdivisions (prelimbic, ventral orbital, ventrolateral orbital, dorsolateral orbital) along a coronal axis within PFC. At each injection site one injection was made of the anterograde tracer and one injection was made of the retrograde tracer. The projection locations of retrogradely labeled neurons and anterogradely labeled axon terminals were then analyzed in the temporal cortex: area Te, entorhinal and perirhinal cortex. We found evidence for an ordering of both the anterograde (anterior-posterior, dorsal-ventral, and medial-lateral axes: p < 0.001) and retrograde (anterior-posterior, dorsal-ventral, and medial-lateral axes: p < 0.001) connections of PFC. We observed that anterograde and retrograde labeling in ipsilateral temporal cortex (i.e., PFC inputs and outputs) often occurred reciprocally (i.e., the same brain region, such as area 35d in perirhinal cortex, contained anterograde and retrograde labeling). However, often the same specific columnar temporal cortex regions contained only either labeling of retrograde or anterograde tracer, indicating that PFC inputs and outputs are frequently non-matched

Assessment of the direct and indirect effects of MPP+ and dopamine on the human proteasome: implications for Parkinson's disease aetiology

Mitochondrial impairment, glutathione depletion and oxidative stress have been implicated in the pathogenesis of Parkinson’s disease (PD), linked recently to proteasomal dysfunction. Our study analysed how these factors influence the various activities of the proteasome in human SH-SY5Y neuroblastoma cells treated with the PD mimetics MPP+ (a complex 1 inhibitor) or dopamine. Treatment with these toxins led to dose- and time-dependent reductions in ATP and glutathione and also chymotrypsin-like and post-acidic like activities; trypsin-like activity was unaffected. Antioxidants blocked the effects of dopamine, but not MPP+, suggesting that oxidative stress was more important in the dopamine-mediated effects. With MPP+, ATP depletion was a prerequisite for loss of proteasomal activity. Thus in a dopaminergic neuron with complex 1 dysfunction both oxidative stress and ATP depletion will contribute independently to loss of proteasomal function. We show for the first time that addition of MPP+ or dopamine to purified samples of the human 20S proteasome also reduced proteasomal activities; with dopamine being most damaging. As with toxin-treated cells, chymotrypsin-like activity was most sensitive and trypsin-like activity the least sensitive. The observed differential sensitivity of the various proteasomal activities to PD mimetics is novel and its significance needs further study in human cells

SP1 and RARα regulate AGAP2 expression in cancer

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is considered a proto-oncogene, but not much is known about AGAP2 gene expression regulation. To get some insight into this process, AGAP2 proximal promoter was cloned and characterised using reporter assays. We have identified SP1 as a transcription factor bound to AGAP2 promoter and required for AGAP2 expression in two different types of cancer cells (KU812, a chronic myeloid leukaemia cell line; and DU145, a prostate cancer cell line): silencing SP1 decreased AGAP2 protein levels. We have also found that all-trans retinoic acid (ATRA) treatment increased AGAP2 protein levels in both cell lines whilst curcumin treatment reduced ATRA-mediated AGAP2 increase. Furthermore, chromatin immunoprecipitation studies revealed the presence of RARα, RXRα and the lysine acetyl transferase PCAF in AGAP2 promoter. Our results provide a novel understanding of AGAP2 expression regulation that could be beneficial to those patients with cancers where AGAP2 is overexpressed

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg

Evidence for widespread hydrated minerals on asteroid (101955) Bennu

Early spectral data from the Origins, Spectral Interpretation, Resource Identification, and Security-Regolith Explorer (OSIRIS-REx) mission reveal evidence for abundant hydrated minerals on the surface of near-Earth asteroid (101955) Bennu in the form of a near-infrared absorption near 2.7 µm and thermal infrared spectral features that are most similar to those of aqueously altered CM-type carbonaceous chondrites. We observe these spectral features across the surface of Bennu, and there is no evidence of substantial rotational variability at the spatial scales of tens to hundreds of metres observed to date. In the visible and near-infrared (0.4 to 2.4 µm) Bennu’s spectrum appears featureless and with a blue (negative) slope, confirming previous ground-based observations. Bennu may represent a class of objects that could have brought volatiles and organic chemistry to Earth

The dynamic geophysical environment of (101955) Bennu based on OSIRIS-REx measurements

The top-shaped morphology characteristic of asteroid (101955) Bennu, often found among fast-spinning asteroids and binary asteroid primaries, may have contributed substantially to binary asteroid formation. Yet a detailed geophysical analysis of this morphology for a fast-spinning asteroid has not been possible prior to the Origins, Spectral Interpretation, Resource Identification, and Security-Regolith Explorer (OSIRIS-REx) mission. Combining the measured Bennu mass and shape obtained during the Preliminary Survey phase of the OSIRIS-REx mission, we find a notable transition in Bennu’s surface slopes within its rotational Roche lobe, defined as the region where material is energetically trapped to the surface. As the intersection of the rotational Roche lobe with Bennu’s surface has been most recently migrating towards its equator (given Bennu’s increasing spin rate), we infer that Bennu’s surface slopes have been changing across its surface within the last million years. We also find evidence for substantial density heterogeneity within this body, suggesting that its interior is a mixture of voids and boulders. The presence of such heterogeneity and Bennu’s top shape are consistent with spin-induced failure at some point in its past, although the manner of its failure cannot yet be determined. Future measurements by the OSIRIS-REx spacecraft will provide insight into and may resolve questions regarding the formation and evolution of Bennu’s top-shape morphology and its link to the formation of binary asteroids