Lothar Kussmaul

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD+, and the reduction potentials of the flavin and NAD+. Consequently, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD+ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.

Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition.

Synaptic dysfunction is an early event in Alzheimers disease patients and has also been detected in transgenic mouse models. In the present study, we analyzed proteomic changes in synaptosomal fractions from Tg2576 mice that overexpress mutant human amyloid precursor protein (K670N, M671L) and from their nontransgenic littermates. Cortical and hippocampal tissue was microdissected at the onset of cognitive impairment, but before deposition of amyloid plaques. Crude synaptosomal fractions were prepared by differential centrifugation, proteins were separated by 2‐D DIGE and identified by MS/MS. Significant alterations were detected in mitochondrial heat shock protein 70 pointing to a mitochondrial stress response. Subsequently, synaptosomal versus nonsynaptic mitochondria were purified from Tg2576 mice brains by density gradient centrifugation. Mitochondrial proteins were separated by IEF or Blue‐native gel electrophoresis in the first dimension and SDS‐PAGE in the second dimension. Numerous changes in the protein subunit composition of the respiratory chain complexes I and III were identified. Levels of corresponding mRNAs remain unchanged as shown by Affymetrix oligonucleotide array analysis. Functional examination revealed impaired state 3 respiration and uncoupled respiration in brain mitochondria from young Tg2576 mice. By immunoblotting, amyloid‐beta oligomers were detected in synaptosomal fractions from Tg2576 mice and reduced glucose metabolism was observed in Tg2576 mice brains by [14C]‐2‐deoxyglucose infusion. Taken together, we demonstrate alterations in the mitochondrial proteome and function that occur in Tg2576 mice brains before amyloid plaque deposition suggesting that mitochondria are early targets of amyloid‐beta aggregates.

Inhibition of CDK5 is protective in necrotic and apoptotic paradigms of neuronal cell death and prevents mitochondrial dysfunction

Previous studies suggested that pro-apoptotic stimuli may trigger a fatal reactivation of cell cycle elements in postmitotic neurons. Supporting this hypothesis, small molecule inhibitors of cyclin-dependent kinases (CDKs), which are known primarily as cell cycle regulators, are neuroprotective. However, available CDK inhibitors cannot discriminate between the different members of the CDK family and inhibit also CDK5, which is not involved in cell cycle control. Testing a new class of CDK inhibitors, we find that inhibitory activity against CDK5, but not cell cycle-relevant CDKs, confers neuroprotection. Moreover, we demonstrate that cleavage of the CDK5 activator protein p35 to p25 is associated with CDK5 overactivation after focal cerebral ischemia, but not in other models used in this study. We find that blocking CDK5 activity, but not caspase inhibition, protects mitochondrial integrity of lesioned neurons. Thus, in our models, CDK5, rather than cell cycle-relevant CDKs, activates neuronal cell death pathways upstream of mitochondrial dysfunction, and inhibition of CDK5 may promote functional long-term rescue of injured neurons. Moreover, we present the first CDK5-selective small molecule inhibitor, lacking unwanted cytostatic effects due to cross-inhibition of mitotic CDKs.

Globular and Protofibrillar Aβ Aggregates Impair Neurotransmission by Different Mechanisms

In Alzheimers disease, substantial evidence indicates the causative role of soluble amyloid β (Aβ) aggregates. Although a variety of Aβ assemblies have been described, the debate about their individual relevance is still ongoing. One critical issue hampering this debate is the use of different methods for the characterization of endogenous and synthetic peptide and their intrinsic limitations for distinguishing Aβ aggregates. Here, we used different protocols for the establishment of prefibrillar Aβ assemblies with varying morphologies and sizes and compared them in a head-to-head fashion. Aggregation was characterized via the monomeric peptide over time until spheroidal, protofibrillar, or fibrillar Aβ aggregates were predominant. It could be shown that a change in the ionic environment induced a structural rearrangement, which consequently confounds the delineation of a measured neurotoxicity toward a distinct Aβ assembly. Here, neuronal binding and hippocampal neurotransmission were found to be suitable to account for the synaptotoxicity to different Aβ assemblies, based on the stability of the applied Aβ aggregates in these settings. In contrast to monomeric or fibrillar Aβ, different prefibrillar Aβ aggregates targeted neurons and impaired hippocampal neurotransmission with nanomolar potency, albeit by different modalities. Spheroidal Aβ aggregates inhibited NMDAR-dependent long-term potentiation, as opposed to protofibrillar Aβ aggregates, which inhibited AMPAR-dominated basal neurotransmission. In addition, a provoked structural conversion of spheroidal to protofibrillar Aβ assemblies resulted in a time-dependent suppression of basal neurotransmission, indicative of a mechanistic switch in synaptic impairment. Thus, we emphasize the importance of addressing the metastability of prefacto characterized Aβ aggregates in assigning a biological effect.

Restoring long-term potentiation impaired by amyloid-beta oligomers: comparison of an acetylcholinesterase inhibitior and selective neuronal nicotinic receptor agonists.

As nicotinic acetylcholine receptor (nAChR) agonists directly address cholinergic neurotransmission with potential impact on glutamatergic function, they are considered as potential new symptomatic treatment options for Alzheimers disease compared to the indirectly operating acetylcholinesterase inhibitors such as the current gold standard donepezil. In order to evaluate the therapeutic value of nAChR activation to ameliorate cognitive dysfunction, a direct comparison between α4β2, α7 nAChR agonists, and donepezil was performed on the level of an ex vivo experimental model of impaired memory formation. First, we demonstrated that amyloid beta (Aβ)42 oligomers, which are believed to be the synaptotoxic Aβ-species causally involved in the pathophysiology of Alzheimers disease, have a detrimental effect on long-term potentiation (LTP) in the CA1 region of rat hippocampal slices, a widely used cellular model of learning and memory. Second, we investigated the potential of donepezil, the α4β2 nAChR agonist TC-1827 and the α7 nAChR partial agonist SSR180711 to reverse Aβ42 oligomer induced LTP impairment. Donepezil showed only a slight reversal of Aβ42 oligomer induced impairment of early LTP, and had no effect on Aβ42 oligomer induced impairment of late LTP. The same was demonstrated for the α4β2 nAChR agonist TC-1827. In contrast, the α7 nAChR partial agonist SSR180711 completely rescued early as well as late LTP impaired by Aβ42 oligomers. As activating α7 nAChRs was found to be most efficacious in restoring Aβ42 oligomer induced LTP deficits, targeting α7 nAChRs might represent a powerful alternative approach for symptomatic treatment of AD.

Lymphocytic Mitochondrial Aconitase Activity is Reduced in Alzheimer's Disease and Mild Cognitive Impairment

BACKGROUND Specific mechanisms behind the role of oxidative/nitrosative stress and mitochondrial dysfunction in Alzheimers disease (AD) pathogenesis remain elusive. Mitochondrial aconitase (ACO2) is a Krebs cycle enzyme sensitive to free radical-mediated damage. OBJECTIVE We assessed activity and expression of ACO2 extracted from blood lymphocytes of subjects with AD, mild cognitive impairment (MCI), older adults with normal cognition (OCN, age ≥65 years), and younger adults with normal cognition (YCN, age <65 years). Plasma levels and activities of antioxidants were also measured. METHODS Blood samples were collected from 28 subjects with AD, 22 with MCI, 21 OCN, and 19 YCN. ACO2 activity was evaluated in a subsample before and after in vitro exposure to free radicals. RESULTS ACO2 activity was significantly lower in AD and MCI cases than controls: ACO2 median activity was 0.64 ± 0.21 U/mg protein for AD, 0.93 ± 0.28 U/mg protein for MCI, 1.17 ± 0.78 U/mg protein for OCN subjects, and 1.23 ± 0.43 U/mg protein for YCN individuals. In subjects with AD and MCI, ACO2 expression was lower than OCN subjects, and ACO2 activity correlated with vitamin E plasma levels (rho: 0.64, p < 0.001) and Mini-Mental State Examination total score (rho: 0.82, p < 0.001). Furthermore, free radicals exposure reduced ACO2 activity more in individuals with AD than in OCN subjects. CONCLUSION Our results suggest that ACO2 activity is reduced in peripheral lymphocytes of subjects with AD and MCI and correlates with antioxidant protection. Further studies are warranted to verify the role of ACO2 in AD pathogenesis and its importance as a marker of AD progression.

Activation of the mitochondrial protein quality control system and actin cytoskeletal alterations in cells harbouring the MELAS mitochondrial DNA mutation

Point mutations in the mitochondrial genome are associated with a variety of metabolic disorders. The myopathy, encephalopathy, lactic acidosis, stroke-like episodes syndrome (MELAS), is most frequently associated with an A to G transition at position 3243 of the mitochondrial tRNA(Leu(UUR)) gene, and is characterized by biochemical and structural alterations of mitochondria. In the present study, we analyzed proteomic changes in an immortalized B-cell line harbouring the MELAS A3243G mutation by two-dimensional difference gel electrophoresis and immunoblot analysis. Although the cell line contained only 10% mutated mitochondrial genomes, we detected significant alterations in numerous proteins associated with the actin cytoskeleton and in nuclear-encoded subunits of mitochondrial respiratory chain complexes. Notably, mitochondrial Lon protease and Hsp60 were deregulated in MELAS cells, indicating an effect on the mitochondrial protein quality control system. By immunofluorescence microscopy, we detected mitochondrial Lon protease accumulation and changes in actin-binding proteins preferentially in MELAS cells containing numerous mitochondria with mutated genomes. Enzymatic assays revealed that Lon protease activity is increased in MELAS cell lysates. Although Lon protease has been shown to degrade misfolded proteins and to stabilize respiratory chain complexes within mitochondria, our MELAS cell line exhibited a higher sensitivity to mitochondrial stress. These findings provide novel insights into the cellular response to dysfunctional mitochondria containing mutated genomes.