Altered acylcarnitine metabolism and inflexible mitochondrial fuel utilization characterize the loss of neonatal myocardial regeneration capacity.

Myocardial regeneration capacity declines during the first week after birth, and this decline is linked to adaptation to oxidative metabolism. Utilizing this regenerative window, we characterized the metabolic changes in myocardial injury in 1-day-old regeneration-competent and 7-day-old regeneration-compromised mice. The mice were either sham-operated or received left anterior descending coronary artery ligation to induce myocardial infarction (MI) and acute ischemic heart failure. Myocardial samples were collected 21 days after operations for metabolomic, transcriptomic and proteomic analyses. Phenotypic characterizations were carried out using echocardiography, histology and mitochondrial structural and functional assessments. In both groups, MI induced an early decline in cardiac function that persisted in the regeneration-compromised mice over time. By integrating the findings from metabolomic, transcriptomic and proteomic examinations, we linked regeneration failure to the accumulation of long-chain acylcarnitines and insufficient metabolic capacity for fatty acid beta-oxidation. Decreased expression of the redox-sensitive mitochondrial Slc25a20 carnitine-acylcarnitine translocase together with a decreased reduced:oxidized glutathione ratio in the myocardium in the regeneration-compromised mice pointed to a defect in the redox-sensitive acylcarnitine transport to the mitochondrial matrix. Rather than a forced shift from the preferred adult myocardial oxidative fuel source, our results suggest the facilitation of mitochondrial fatty acid transport and improvement of the beta-oxidation pathway as a means to overcome the metabolic barrier for repair and regeneration in adult mammals after MI and heart failure.

Muscle NAD+ depletion and Serpina3n as molecular determinants of murine cancer cachexia-the effects of blocking myostatin and activins.

OBJECTIVE: Cancer cachexia and muscle loss are associated with increased morbidity and mortality. In preclinical animal models, blocking activin receptor (ACVR) ligands has improved survival and prevented muscle wasting in cancer cachexia without an effect on tumour growth. However, the underlying mechanisms are poorly understood. This study aimed to identify cancer cachexia and soluble ACVR (sACVR) administration-evoked changes in muscle proteome. METHODS: Healthy and C26 tumour-bearing (TB) mice were treated with recombinant sACVR. The sACVR or PBS control were administered either prior to the tumour formation or by continued administration before and after tumour formation. Muscles were analysed by quantitative proteomics with further examination of mitochondria and nicotinamide adenine dinucleotide (NAD+) metabolism. To complement the first prophylactic experiment, sACVR (or PBS) was injected as a treatment after tumour cell inoculation. RESULTS: Muscle proteomics in TB cachectic mice revealed downregulated signatures for mitochondrial oxidative phosphorylation (OXPHOS) and increased acute phase response (APR). These were accompanied by muscle NAD+ deficiency, alterations in NAD+ biosynthesis including downregulation of nicotinamide riboside kinase 2 (Nrk2), and decreased muscle protein synthesis. The disturbances in NAD+ metabolism and protein synthesis were rescued by treatment with sACVR. Across the whole proteome and APR, in particular, Serpina3n represented the most upregulated protein and the strongest predictor of cachexia. However, the increase in Serpina3n expression was associated with increased inflammation rather than decreased muscle mass and/or protein synthesis. CONCLUSIONS: We present evidence implicating disturbed muscle mitochondrial OXPHOS proteome and NAD+ homeostasis in experimental cancer cachexia. Treatment of TB mice with a blocker of activin receptor ligands restores depleted muscle NAD+ and Nrk2, as well as decreased muscle protein synthesis. These results indicate putative new treatment therapies for cachexia and that although acute phase protein Serpina3n may serve as a predictor of cachexia, it more likely reflects a condition of elevated inflammation.

Characterization of human palladin, a microfilament-associated protein.

Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, alpha-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the alpha-helical domain of ezrin and by Ig-domains 2-3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.

Parkin interacts with the proteasome subunit alpha4.

Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.

HB-GAM is a cytokine present in Alzheimer's and Down's syndrome lesions.

The distribution of heparin binding growth associated molecule (HB-GAM) in the cerebral amyloidoses of Alzheimer's disease (AD) and Down's syndrome (DS), conditions characterized by the deposition of amyloid beta (A beta), was investigated immunohistochemically. Antibodies to HB-GAM, a cytokine which plays an important role in brain development and maturation, showed strong immunoreactivity with senile plaques in both AD and DS. Anti-HB-GAM reacted with pre-amyloid lesions, but only when markers of dystrophic neurites were present. The presence of HB-GAM in AD brains, but not in control brains, was confirmed by Western blotting. We suggest that the presence of HB-GAM in A beta lesions is a marker of neuronal injury.

Amyloid beta binding proteins in vitro and in normal human cerebrospinal fluid.

A major neuropathological feature of Alzheimer's disease (AD) is the deposition of amyloid beta (A beta) in the form of senile plaques. The A beta peptide exists both in a beta-pleated sheet fibrillar form in amyloid deposits and as a normal soluble protein in biological fluids. Numerous proteins have been identified immunohistochemically to be associated with senile plaques, where A beta is the major constituent. Some of the latter have also been suggested to be carriers of the normal soluble A beta (sA beta) including apolipoprotein J (apoJ), apolipoprotein E (apoE) and transthyretin (TTR). We have found, using several different methods, that numerous proteins can bind synthetic A beta peptides when high concentrations are used; however, using an affinity anti-sA beta column we confirm that apoJ is the major binding protein in pooled human cerebrospinal fluid. On the other hand it is known that apoE co-purifies with A beta biochemically extracted from senile plaques. In AD tissue there may be a change in the major apolipoprotein binding A beta from apoJ to apoE.

Lipid metabolism parameters in patients with Alzheimer's disease and their first degree relatives.

Recently, it was suggested that the presence of total cholesterol (TC), age and sex interaction in Alzheimer's type dementia (AD) is linked with the apolipoprotein E (APOE) genotype. Our objective was to determine whether the serum lipid profile in AD patients and their first degree non-demented relatives of a certain age (NDR) was dependent on APOE genotype. We included 28 mild to moderate AD and 30 NDR according to DSM-III-R and NINCDS-ADRDA criteria. NDR individuals were investigated in an age group similar to the AD group (brother-sister relationship) and in a group including younger individuals (AD patients-children relationship). Our data indicate significant differences between decreased total cholesterol and low density lipoprotein cholesterol ratio in the group of AD patients versus NDR individuals of similar age, independent of APOE genotype, and an increased total cholesterol and low density lipoprotein cholesterol ratio in a group of AD patients versus their children of the same genotype. There was no significant correlation between triglycerides and high density lipoprotein levels with APOE genotype in any of the tested groups. In conclusion, there was a decreased selected lipid serum profile parameters in AD compared to age matched non demented first degree relatives.

ApoE polymorphism in Polish patients with Alzheimer's disease.

Alzheimer's disease is a genetically heterogeneous disorder of CNS. The presence of APOE-epsilon 4 allele is known to increase the risk of early and late onset sporadic and late onset familial forms of AD. In various Western European countries, USA, Canada, Japan and Australia the allelic frequency ranges between 0.1-0.18 in controls, and between 0.24-0.52 in AD patients. In the present study on Polish population, we analyzed the frequency of APOE-epsilon 4 allele in persons with Alzheimer's disease (AD). APOE genotypes were determined in 30 mild to moderate AD (83%) and mixed dementia (MIX, 17%), as well as in 11 nondemented first-degree relatives of AD (NDR), recruited from AD patient registry in Warsaw. Among the AD and MIX patients the APOE-epsilon 4, epsilon 3, epsilon 2 allele frequency was 0.333, 0.65 and 0.017 respectively.

[Patterns of cognitive impairment and apolipoprotein genotype in persons with Alzheimer's disease]. / Wzorce aktywnosci poznawczej a polimorfizm apolipoproteiny E w chorobie Alzheimera.

The aim of this study was to test the possible relationship between patterns of cognitive deficits--especially impairment of memory processes--and ApoE genotype in patients with AD. Fifty seven right-handed subjects (31 males and 26 females) were tested in this study. The age of subjects ranged from 50 to 79, the education lasted from 11 to 16 years. All subjects were diagnosed as probable AD patients on the basis of DSM IV and NINCDS-ADRDA criteria. Each subject was examined for: 1) ApoE genotype, 2) general level of activity (GDS and MMSE), 3) neuropsychological evaluation of cognitive processes, using full test battery. 37 patients had at least one of ApoE epsilon 4 allele (e2/4, 3 and 4/4) and 20 patients had none of ApoE 4 allele (e 2/3 and 3/3). The group of tested subjects were subdivided into 2 groups. The first group was comprised by 31 patients with 3-rd stage (according to GDS) of mental activity. Twenty six patients with 4-th stage were included into the second group. Those subgroups did not significantly differ if age, education, gender or ApoE allele were considered. Experimental data were normalized and then analyzed using a statistical package SPSS/PC+. The analysis of variance showed that the type of test, stage of disease and two-way interaction ApoE x type of test were highly significant (P < 0.0001). Some results were obvious and not surprising (e.g. that results of patients with 4-th stage were much worse than the results of patients with stage 3-rd). It turned out that the best results were obtained by our patients in naming tests, the worst--in learning test with distraction. Patients with ApoE epsilon 4 performed better than patients with none ApoE epsilon 4 in the Rey's test, in the similarity test and in the test which required repeating numbers starting from the last one. The differences between the subgroups of patients with different ApoE alleles were confirmed by different distributions of correlations. All statistical analyses were repeated for more homogenous group of patients (only with stage 3-rd). The pattern of results resembled the previous one (i.e. better performance in the same tests) with one exception: additionally, in delayed recall test patients with none ApoE epsilon 4 performed much better that ApoE epsilon 4. Our results showed that some cognitive processes depended on ApoE genotype. Patients with none ApoE epsilon 4 genotype had less severe deficits in delayed recall of new information. On the other hand, working memory appeared to be less affected in patients with ApoE epsilon 4 genotype. Independent of genotype, both group showed similar impairment of learning ability without deficits in remote memory.

Huntingtin interacts with the receptor sorting family protein GASP2.

Protein interaction networks are useful resources for the functional annotation of proteins. Recently, we have generated a highly connected protein-protein interaction network for Huntington's disease (HD) by automated yeast two-hybrid (Y2H) screening (Goehler et al., 2004). The network included several novel direct interaction partners for the disease protein huntingtin (htt). Some of these interactions, however, have not been validated by independent methods. Here we describe the verification of the interaction between htt and GASP2 (G protein-coupled receptor associated sorting protein 2), a protein involved in membrane receptor degradation. Using membrane-based and classical coimmunoprecipitation assays we demonstrate that htt and GASP2 form a complex in cotransfected mammalian cells. Moreover, we show that the two proteins colocalize in SH-SY5Y cells, raising the possibility that htt and GASP2 interact in neurons. As the GASP protein family plays a role in G protein-coupled receptor sorting, our data suggest that htt might influence receptor trafficking via the interaction with GASP2.

Micropreparative gel electrophoresis of low-molecular-weight peptides: purification of highly insoluble amyloid peptide fragments.

We have used the continuous-elution micropreparative gel electrophoresis device described by Baumann and Lauraeus (Anal. Biochem. 214, 142-148, 1993) to purify low-molecular-weight peptide fragments from in-gel digested standard proteins as well as highly in-soluble amyloid peptides. Alzheimer's amyloid beta-peptide, gelsolin-derived amyloid peptide of the Finnish type, and a novel amyloid of the British type were purified from either homogenized brain or kidney tissue material to a high degree of purity in a single run. Using the high resolving capacity of the Tris-Tricine-SDS buffer system of Schaegger and von Jagow (Anal. Biochem. 166, 368-379, 1978) we were able to isolate two synthetic peptides with M(r)4329 and 3284, differing only by 1045 in mass. The total peptide recovery, as determined by amino acid sequence analysis and scanning densitometry, ranged between 60 and 80%. In order to demonstrate the utility of this technique we subjected some of the purified peptides to direct N-terminal amino acid sequence analysis, mass spectrometry, microbore high-performance liquid chromatography, and immunochemical studies. Our results show that micropreparative gel electrophoresis is an effective tool for the isolation of not only larger polypeptides but also small peptide fragments in a form suitable for further biological use.

Is Alzheimer's disease an apolipoprotein E amyloidosis?

The presence of the apolipoprotein E4 allele has been identified as a major risk factor for late-onset Alzheimer's disease. Apolipoprotein E has also been found immunohistochemically in Alzheimer's disease lesions. We biochemically isolated amyloid beta from senile plaques and found that a carboxyl-terminal fragment (residues 216-299) of apolipoprotein E co-purified. In vitro this fragment from recombinant apolipoprotein E could form amyloid-like fibrils, which were Congo-red positive. Thus senile plaques may contain both amyloid beta and apolipoprotein E amyloid fibrils.

Amyloid beta 1-42 deposits do not lead to Alzheimer's neuritic plaques in aged dogs.

In alzheimer's disease, amyloid beta (A beta) is deposited in senile plaques and amyloid angiopathy. Longer A beta peptides, which extend to residue 42 (A beta 42), have been suggested to be critical for the seeding of amyloid. Aged dogs develop cerebral vessel amyloid and parenchymal preamyloid lesions. Preamyloid in humans is related to senile plaques, whereas in dogs such progression is rare. We evaluated the composition of aged canine vessel amyloid and preamyloid both biochemically and immunohistochemically. The vessel amyloid extended mainly to residue 40 (A beta 40), while preamyloid contained a mixture of A beta 17-42 and A beta 42, with minimal A beta 40. Our results suggest other factors besides A beta 42 are important for neuritic plaque formation.

Meningocerebrovascular amyloidosis associated with a novel transthyretin mis-sense mutation at codon 18 (TTRD 18G)

We describe a novel transthyretin mutation at codon 18 where Asp is replaced by Gly (D18G) in a Hungarian kindred. This mutation is associated with meningocerebrovascular amyloidosis, producing dementia, ataxia, and spasticity. Fifty different transthyretin mutations are related to amyloid deposition, typically producing a peripheral neuropathy or cardiac dysfunction. These symptoms are absent in this family. Up to now, amyloid-beta (A beta), cystatin C, and prion proteins have been known to be deposited as amyloid in the brain, leading to stroke or dementia. With this report we establish that transthyretin amyloid deposition can also produce central nervous system dysfunction as the major clinical symptom.

The "nonamyloidogenic" p3 fragment (amyloid beta17-42) is a major constituent of Down's syndrome cerebellar preamyloid.

Down's syndrome (DS) patients show accelerated Alzheimer's disease (AD) neuropathology, which consists of preamyloid lesions followed by the development of neuritic plaques and neurofibrillary tangles. The major constituents of preamyloid and neuritic plaques are amyloid beta (Abeta) peptides. Preamyloid lesions are defined as being Abeta immunoreactive lesions, which unlike neuritic plaque amyloid are Congo red-negative and largely nonfibrillar ultrastructurally. DS patients can develop extensive preamyloid deposits in the cerebellum, without neuritic plaques; hence, DS cerebellums are a source of relatively pure preamyloid. We biochemically characterized the composition of DS preamyloid and compared it to amyloid in the neuritic plaques and leptomeninges in the same patients. We found that Abeta17-42 or p3 is a major Abeta peptide of DS cerebellar preamyloid. This 26-residue peptide is also present in low quantities in neuritic plaques. We suggest that preamyloid can now be defined biochemically as lesions in which a major Abeta peptide is p3.