The striking differences in the bioenergetics of brain and liver mitochondria are enhanced in mitochondrial disease.

Mitochondrial disorders are hallmarked by the dysfunction of oxidative phosphorylation (OXPHOS) yet are highly heterogeneous at the clinical and genetic levels. Striking tissue-specific pathological manifestations are a poorly understood feature of these conditions, even if the disease-causing genes are ubiquitously expressed. To investigate the functional basis of this phenomenon, we analyzed several OXPHOS-related bioenergetic parameters, including oxygen consumption rates, electron transfer system (ETS)-related coenzyme Q (mtCoQ) redox state and production of reactive oxygen species (ROS) in mouse brain and liver mitochondria fueled by different substrates. In addition, we determined how these functional parameters are affected by ETS impairment in a tissue-specific manner using pathologically relevant mouse models lacking either Ndufs4 or Ttc19, leading to Complex I (CI) or Complex III (CIII) deficiency, respectively. Detailed OXPHOS analysis revealed striking differences between brain and liver mitochondria in the capacity of the different metabolic substrates to fuel the ETS, reduce the ETS-related mtCoQ, and to induce ROS production. In addition, ETS deficiency due to either CI or CIII dysfunction had a much greater impact on the intrinsic bioenergetic parameters of brain compared with liver mitochondria. These findings are discussed in terms of the still rather mysterious tissue-specific manifestations of mitochondrial disease.

Double administration of self-complementary AAV9NDUFS4 prevents Leigh disease in Ndufs4-/- mice.

Leigh disease, or subacute necrotizing encephalomyelopathy, a genetically heterogeneous condition consistently characterized by defective mitochondrial bioenergetics, is the most common oxidative-phosphorylation related disease in infancy. Both neurological signs and pathological lesions of Leigh disease are mimicked by the ablation of the mouse mitochondrial respiratory chain subunit Ndufs4-/-, which is part of, and crucial for, normal Complex I activity and assembly, particularly in the brains of both children and mice. We previously conveyed the human NDUFS4 gene to the mouse brain using either single-stranded adeno-associated viral 9 recombinant vectors or the PHP.B adeno-associated viral vector. Both these approaches significantly prolonged the lifespan of the Ndufs4-/- mouse model but the extension of the survival was limited to a few weeks by the former approach, whereas the latter was applicable to a limited number of mouse strains, but not to primates. Here, we exploited the recent development of new, self-complementary adeno-associated viral 9 vectors, in which the transcription rate of the recombinant gene is markedly increased compared with the single-stranded adeno-associated viral 9 and can be applied to all mammals, including humans. Either single intra-vascular or double intra-vascular and intra-cerebro-ventricular injections were performed at post-natal Day 1. The first strategy ubiquitously conveyed the human NDUFS4 gene product in Ndufs4-/- mice, doubling the lifespan from 45 to ≈100 days after birth, when the mice developed rapidly progressive neurological failure. However, the double, contemporary intra-vascular and intra-cerebroventricular administration of self-complementary-adeno-associated viral NDUFS4 prolonged healthy lifespan up to 9 months of age. These mice were well and active at euthanization, at 6, 7, 8 and 9 months of age, to investigate the brain and other organs post-mortem. Robust expression of hNDUFS4 was detected in different cerebral areas preserving normal morphology and restoring Complex I activity and assembly. Our results warrant further investigation on the translatability of self-complementary-adeno-associated viral 9 NDUFS4-based therapy in the prodromal phase of the disease in mice and eventually humans.

Activation of Akt-mTORC1 signalling reverts cancer-dependent muscle wasting.

BACKGROUND: Cancer-related muscle wasting occurs in most cancer patients. An important regulator of adult muscle mass and function is the Akt-mTORC1 pathway. While Akt-mTORC1 signalling is important for adult muscle homeostasis, it is also a major target of numerous cancer treatments. Which role Akt-mTORC1 signalling plays during cancer cachexia in muscle is currently not known. Here, we aimed to determine how activation or inactivation of the pathway affects skeletal muscle during cancer cachexia. METHODS: We used inducible, muscle-specific Raptor ko (mTORC1) mice to determine the effect of reduced mTOR signalling during cancer cachexia. On the contrary, in order to understand if skeletal muscles maintain their anabolic capacity and if activation of Akt-mTORC1 signalling can reverse cancer cachexia, we generated mice in which we can inducibly activate Akt specifically in skeletal muscles. RESULTS: We found that mTORC1 signalling is impaired during cancer cachexia, using the Lewis lung carcinoma and C26 colon cancer model, and is accompanied by a reduction in protein synthesis rates of 57% (P < 0.01). Further reduction of mTOR signalling, as seen in Raptor ko animals, leads to a 1.5-fold increase in autophagic flux (P > 0.001), but does not further increase muscle wasting. On the other hand, activation of Akt-mTORC1 signalling in already cachectic animals completely reverses the 15-20% loss in muscle mass and force (P < 0.001). Interestingly, Akt activation only in skeletal muscle completely normalizes the transcriptional deregulation observed in cachectic muscle, despite having no effect on tumour size or spleen mass. In addition to stimulating muscle growth, it is also sufficient to prevent the increase in protein degradation normally observed in muscles from tumour-bearing animals. CONCLUSIONS: Here, we show that activation of Akt-mTORC1 signalling is sufficient to completely revert cancer-dependent muscle wasting. Intriguingly, these results show that skeletal muscle maintains its anabolic capacities also during cancer cachexia, possibly giving a rationale behind some of the beneficial effects observed in exercise in cancer patients.

The ß-amyloid peptide compromises Reelin signaling in Alzheimer's disease.

Reelin is a signaling protein that plays a crucial role in synaptic function, which expression is influenced by ß-amyloid (Aß). We show that Reelin and Aß oligomers co-immunoprecipitated in human brain extracts and were present in the same size-exclusion chromatography fractions. Aß treatment of cells led to increase expression of Reelin, but secreted Reelin results trapped together with Aß aggregates. In frontal cortex extracts an increase in Reelin mRNA, and in soluble and insoluble (guanidine-extractable) Reelin protein, was associated with late Braak stages of Alzheimer's disease (AD), while expression of its receptor, ApoER2, did not change. However, Reelin-dependent induction of Dab1 phosphorylation appeared reduced in AD. In cells, Aß reduced the capacity of Reelin to induce internalization of biotinylated ApoER2 and ApoER2 processing. Soluble proteolytic fragments of ApoER2 generated after Reelin binding can be detected in cerebrospinal fluid (CSF). Quantification of these soluble fragments in CSF could be a tool to evaluate the efficiency of Reelin signaling in the brain. These CSF-ApoER2 fragments correlated with Reelin levels only in control subjects, not in AD, where these fragments diminished. We conclude that while Reelin expression is enhanced in the Alzheimer's brain, the interaction of Reelin with Aß hinders its biological activity.

Reelin in Alzheimer's Disease, Increased Levels but Impaired Signaling: When More is Less.

In the continuing search for proteins that play a role in Alzheimer's disease (AD) and that are related to the pathological hallmarks, those that influence cognitive function and that constitute potential therapeutic targets deserve special interest. Reelin is a signaling protein that is involved in a cascade of cytoplasmic events that control tau phosphorylation and that regulate synaptic neurotransmission, plasticity, and memory. Both Reelin expression and glycosylation are modulated by amyloid-ß (Aß), suggesting that the activity of Reelin could be affected in AD and hence, its possible influence on this pathology should be taken into consideration. The levels of Reelin in the brain of AD patients appear to be altered and interestingly, disrupted Reelin signaling is associated with increased tau phosphorylation as well as with amyloid-ß protein precursor processing. We discuss here the somewhat contradictory data regarding Reelin levels in AD and we evaluate the processing of the Reelin receptor, ApoER2, and other downstream events, such as the phosphorylation of the intracellular adapter Dab1. Together with brain Reelin levels, these changes may represent a relevant read-out of Reelin signaling in the human brain.

ApoER2 processing by presenilin-1 modulates reelin expression.

The reelin signaling protein and its downstream components have been associated with synaptic plasticity and neurotransmission. The reelin signaling pathway begins with the binding of reelin to the transmembrane lipoprotein receptor apolipoprotein E receptor 2 (ApoER2), which in turns induces the sequential cleavage of ApoER2 by the sequential action of α- and γ-secretases. Using conditional-knockout mice of the catalytic component of the γ-secretase complex, presenilin 1 (PS1), we demonstrated increased brain ApoER2 and reelin protein and transcript levels, with no changes in the number of reelin-positive cells. Using the human SH-SY5Y neuroblastoma cell line, we showed that ApoER2 processing occurs in the presence of PS1, producing an intracellular ApoER2 C-terminal fragment. In addition, the pharmacologic inhibition of γ-secretase in SH-SY5Y cells led to increased reelin levels. Overexpression of ApoER2 decreased reelin mRNA levels in these cells. A luciferase reporter gene assay and nuclear fractionation confirmed that increased amounts of intracellular fragment of ApoER2 suppressed reelin expression at a transcriptional level. Chromatin immunoprecipitation experiments corroborated that the intracellular fragment of ApoER2 bound to the RELN promoter region. Our study suggests that PS1/γ-secretase-dependent processing of the reelin receptor ApoER2 inhibits reelin expression and may regulate its signaling.

Beta-amyloid impairs reelin signaling.

Reelin is a signaling protein increasingly associated with the pathogenesis of Alzheimer's disease that relevantly modulates tau phosphorylation. We have previously demonstrated that ß-amyloid peptide (Aß) alters reelin expression. We have now attempted to determine whether abnormal reelin triggered by Aß will result in signaling malfunction, contributing to the pathogenic process. Here, we show that reelin forms induced by ß-amyloid are less capable of down-regulating tau phosphorylation via disabled-1 and GSK3ß kinase. We also demonstrate that the scaffold protein 14-3-3 that increases tau phosphorylation by modulating GSK3ß activity, is up-regulated during defective reelin signaling. Binding of reelin to its receptor, mainly ApoER2 in the brain, relays the signal into the cell. We associate the impaired reelin signaling with inefficiency of reelin in forming active homodimers and decreased ability to bind efficiently to its receptor, ApoER2. More remarkably, reelin from Alzheimer cortex shows a tendency to form large complexes instead of homodimers, the active form for signaling. Our results suggest that reelin expression is altered by Aß leading to impaired reelin signaling.

Organ-specific changes in the expression of mannose-6-phosphate receptors during postnatal development in rats.

The significance of the coexistence of 2 mannose-6-phosphate receptors (MPRs) in most cell types still remains poorly understood. In this study, expression of the cation-dependent MPR (CD-MPR) and the cation-independent MPR (CI-MPR) was measured by Western blot in rat organs at 3 ages, i.e. in newborn and 10- and 90-day-old rats. It was observed that expression of the CI-MPR tends to diminish from newborns to adults in 5 of the 6 organs studied, whereas the CD-MPR did not show a clear tendency over time. In pancreas, conversely, both MPRs increased progressively from newborns to adults. The activity of 2 acid hydrolases was also measured at the different ages, and a low correlation was found with the expression of the 2 MPRs. With the exception of the pancreas, it is possible that the CI-MPR is mostly occupied with the clearance of insulin-like growth factor-II at early stages of development, and that later both MPRs may participate in the maturation of the lysosomal apparatus. We propose that in the pancreas, both receptors may be involved in increasing the proteolytic activity of this exocrine gland during postnatal development.

Inhibition of early endosome fusion by Rab5-binding defective Ras interference 1 mutants.

Rin1 has been shown to play an important role in endocytosis. In this study we demonstrated that depletion of Rin1 from the cytosol blocked the fusion reaction. More importantly, endosome fusion was rescued by the addition of Rin1 proteins depending on the presence of Rab5, and its effector EEA1. Furthermore, we found that Syntaxin 13, but not Syntaxin 7, was required by Rin1 to support endosome fusion. We also identified six mutations on the Vps9 domain of Rin1 that failed to rescue the fusion reaction. Two of them, Rin1: D537A and Rin1: Y561F mutants showed dramatic inhibitory effect on the fusion reaction, which correlate with their inability to properly activate Rab5 or to bind endosomal membranes. Taken together, our results suggest that specific residues on the Vsp9 domain of Rin1 are required for its interaction with Rab5, binding to the endosomal membranes and subsequent regulation of the fusion reaction.