Thomas Ciossek

Control of triglyceride storage by a WD40/TPR-domain protein

Obesity is a metabolic disorder related to improper control of energy uptake and expenditure, which results in excessive accumulation of body fat. Initial insights into the genetic pathways that regulate energy metabolism have been provided by a discrete number of obesity‐related genes that have been identified in mammals. Here, we report the identification of the adipose (adp) gene, the mutation of which causes obesity in Drosophila. Loss of adp activity promotes increased fat storage, which extends the lifespan of mutant flies under starvation conditions. By contrast, adp gain‐of‐function causes a specific reduction of the fat body in Drosophila. adp encodes an evolutionarily conserved WD40/tetratricopeptide‐repeat‐domain protein that is likely to represent an intermediate in a novel signalling pathway.

Autoproteolytic Fragments Are Intermediates in the Oligomerization/Aggregation of the Parkinson's Disease Protein Alpha-Synuclein as Revealed by Ion Mobility Mass Spectrometry

Gas-phase protein separation by ion mobility: With its ability to separate the Parkinsons disease protein α-synuclein and its autoproteolytic products-despite the small concentrations of the latter-ion-mobility MS has enabled the characterization of intermediate fragments in in vitro oligomerization-aggregation. In particular, a possible key fragment, the highly aggregating C-terminal fragment, αSyn(72-140), has been revealed.

Lower affinity of isradipine for L-type Ca2+ channels during substantia nigra dopamine neuron-like activity: implications for neuroprotection in Parkinson's disease

Ca2+-influx through L-type Ca2+-channels (LTCCs) is associated with activity-related stressful oscillations of Ca2+ levels within dopaminergic (DA) neurons in the substantia nigra (SN), which may contribute to their selective degeneration in Parkinsons disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by in vivo pretreatment with therapeutically relevant isradipine plasma levels, or Cav1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Cav1.2 α1-subunit, long and short Cav1.3 α1-subunit splice variants; β3/α2δ1). During SN DA-like pacemaking, only Cav1.3 variants conducted Ca2+ current (ICa) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated ICa during (Cav1.2 plus Cav1.3) and after (Cav1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Cav1.3 variants during SN DA-like pacemaking compared with Cav1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca2+ oscillations. Our data predict that Cav1.2 and Cav1.3 splice variants contribute differentially to Ca2+ load in SN DA neurons, with prominent Cav1.3-mediated ICa between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Cav1.2. SIGNIFICANCE STATEMENT The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinsons disease (PD). Ca2+ influx through voltage-gated L-type Ca2+ channels (LTCCs), in particular Cav1.3, appears to contribute to this vulnerability, and the LTCC inhibitor isradipine is currently being tested as a neuroprotective agent for PD in a phase III clinical trial. However, in our study isradipine plasma concentrations approved for therapy were not neuroprotective in a PD mouse model. We provide an explanation for this observation by demonstrating that during SN DA-like neuronal activity LTCCs are less sensitive to isradipine than Cav1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca2+ oscillations of SN DA neurons.

Identification and Affinity-Quantification of ß-Amyloid and α-Synuclein Polypeptides Using On-Line SAW-Biosensor-Mass Spectrometry

AbstractBioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS- acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (KD) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer’s disease and Parkinson’s disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions. Figureᅟ

Increased Oxidative Stress Exacerbates α-Synuclein Aggregation In Vivo

Increasing evidence suggests a relationship between oxidative stress and α-synuclein aggregation, the primary pathological hallmark of Parkinson disease (PD). However, a direct causal relationship has not yet been established in vivo in mouse models of PD. Superoxide dismutase 2 (SOD2) is rate limiting in the antioxidant machinery of the mitochondria and even its partial deficiency elevates oxidative stress in mice. Therefore, in order to investigate a possible interaction between oxidative stress and α-synuclein aggregation in vivo, a transgenic model of PD with haplodeficiency for SOD2 was generated on the basis of the well-characterized murine (Thy-1)-h[A30P]-α-synuclein transgenic line. In comparison with littermate controls with full SOD2 capacity, α-synuclein transgenic mice with partial SOD2 deficiency exhibited a significantly more advanced stage of synucleinopathy at 16 months, as demonstrated by higher median PK-PET blot scores (p < 0.01) and a greater amount of truncated α-synuclein in the insoluble fraction of homogenized brains (p < 0.05). These results show that compromising the capacity to scavenge free radicals can exacerbate α-synuclein aggregation, indicating that elevated levels of oxidative stress could modulate the progression of PD.