CRAT missense variants cause abnormal carnitine acetyltransferase function in an early‐onset case of Leigh syndrome

Leigh syndrome, or subacute necrotizing encephalomyelopathy, is one of the most severe pediatric disorders of the mitochondrial energy metabolism. By performing whole‐exome sequencing in a girl affected by Leigh syndrome and her parents, we identified two heterozygous missense variants (p.Tyr110Cys and p.Val569Met) in the carnitine acetyltransferase (CRAT) gene, encoding an enzyme involved in the control of mitochondrial short‐chain acyl‐CoA concentrations. Biochemical assays revealed carnitine acetyltransferase deficiency in the proband‐derived fibroblasts. Functional analyses of recombinant‐purified CRAT proteins demonstrated that both missense variants, located in the acyl‐group binding site of the enzyme, severely impair its catalytic function toward acetyl‐CoA, and the p.Val569Met variant also toward propionyl‐CoA and octanoyl‐CoA. Although a single recessive variant in CRAT has been recently associated with neurodegeneration with brain iron accumulation (NBIA), this study reports the first kinetic analysis of naturally occurring CRAT variants and demonstrates the genetic basis of carnitine acetyltransferase deficiency in a case of mitochondrial encephalopathy.     

Neurotransmitter segregation: functional and plastic implications

Synaptic cotransmission is the ability of neurons to use more than one transmitter to convey synaptic signals. Cotransmission was originally described as the presence of a classic transmitter, which conveys main signal, along one or more cotransmitters that modulate transmission, later on, it was found cotransmission of classic transmitters. It has been generally accepted that neurons store and release the same set of transmitters in all their synaptic processes. However, some findings that show axon endings of individual neurons storing and releasing different sets of transmitters, are not in accordance with this assumption, and give support to the hypothesis that neurons can segregate transmitters to different synapses. Here, we review the studies showing segregation of transmitters in invertebrate and mammalian central nervous system neurons, and correlate them with our results obtained in sympathetic neurons. Our data show that these neurons segregate even classic transmitters to separated axons. Based on our data we suggest that segregation is a plastic phenomenon and responds to functional synaptic requirements, and to 'environmental' cues such as neurotrophins. We propose that neurons have the machinery to guide the different molecules required in synaptic transmission through axons and sort them to different axon endings. We believe that transmitter segregation improves neuron interactions during cotransmission and gives them selective and better control of synaptic plasticity.     

Intrinsic ruminal innervation in ruminants of different feeding types

According to their feeding habits, ruminants can be classified as grazers, concentrate selectors and those of intermediate type. The different feeding types are reflected in distinct anatomical properties of the forestomachs. The present study was designed to investigate whether the intrinsic innervation patterns of the rumen (the main part of the forestomach) differ between intermediate types and grazers. Myenteric plexus preparations from the rumen of goats (intermediate type), fallow deer (intermediate type), cattle (grazer) and sheep (grazer) were analysed by immunohistochemical detection of the following antigens: Hu-protein (HuC/D), choline acetyltransferase (ChAT), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), calbindin (CALB) and somatostatin (SOM). Myenteric ganglia of cattle contained 73 +/- 6 neurons per ganglion, whereas the ganglia of sheep were significantly smaller (45 +/- 18 neurons per ganglion). The ganglion density of the myenteric plexus was highest in fallow deer (15 +/- 3 ganglia per cm(2)) and lowest in cattle (6 +/- 1 ganglia per cm(2)). All myenteric neurons were either ChAT or NOS positive. The proportion of NOS-positive neurons was significantly lower in sheep (29.5 +/- 8.2% of all neurons) than in goats (44.2 +/- 9.8%). In all species, additional analysis of the different neuropeptides revealed the following subpopulations in descending order of percentile appearance: ChAT/SP > NOS/VIP/NPY > ChAT/- > NOS/NPY. Expression of CALB was detected in a minority of the ChAT-positive neurons in all species. Somatostatin immunoreactive somata were found only in preparations obtained from fallow deer and sheep. These data suggest that the rumen of grazers is under stronger cholinergic control than the rumen of species belonging to the intermediate type, although most subpopulations of neurons are present in all species. However, whether the strong mixing patterns of low quality roughage during digestion are enabled by the prominent excitatory input of the rumen of grazers requires elucidation in further studies.     

Attention and memory in aged rats: Impact of lifelong environmental enrichment

Aged rodents exhibit memory and attention dysfunctions. Environmental enrichment (EE) attenuates memory impairments. Whether it may reduce attention deficits is not known. At the age of 1 month, Long-Evans female rats were placed in standard or EE conditions and tested after 3 (young), 12 (middle-aged) or 24 (aged) months of differential housing. Spatial reference memory was assessed in a water-maze task. Attention performance was evaluated in the five-choice serial reaction time (5-CSRT) task. EE improved spatial memory at all ages, but did not ameliorate 5-CSRT performance in young and middle-aged rats; it prevented, however, the degradation of attention performances detected in aged rats. The number of ChAT (+30 to +64%)- and p75^NTR-positive (+35 to +44%) neurons was higher in the basal forebrain of aged enriched vs. standard rats, suggesting their EE-mediated protection. The weaker deficit of attention found in aged EE rats might be linked to a better survival in the very long term of neurons in the basalo-cortical system.     

The human carcinogen aristolochic acid i is activated to form DNA adducts by human NAD(P)H:quinone oxidoreductase without the contribution of acetyltransferases or sulfotransferases

Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with AA nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. We investigated the efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) to activate aristolochic acid I (AAI) and used in silico docking, using soft-soft (flexible) docking procedure, to study the interactions of AAI with the active site of human NQO1. AAI binds to the active site of NQO1 indicating that the binding orientation allows for direct hydride transfer (i.e., two electron reductions) to the nitro group of AAI. NQO1 activated AAI, generating DNA adduct patterns reproducing those found in urothelial tissues from humans exposed to AA. Because reduced aromatic nitro-compounds are often further activated by sulfotransferases (SULTs) or N,O-acetlytransferases (NATs), their roles in AAI activation were investigated. Our results indicate that phase II reactions do not play a major role in AAI bioactivation; neither native enzymes present in human hepatic or renal cytosols nor human SULT1A1, -1A2, -1A3, -1E, or -2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAI. Instead under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAI through the formation of a cyclic hydroxamic acid (N-hydroxyaristolactam I) favored by the carboxy group in peri position to the nitro group without additional conjugation. These results emphasize the major importance of NQO1 in the metabolic activation of AAI and provide the first evidence that initial nitroreduction is the rate limiting step in AAI activation.     

Clinical characteristics and biomarkers of breast cancer associated with choline concentration measured by 1H MRS

This study investigated the association between the total choline (tCho) concentration and the clinical characteristics and biomarker status of breast cancer. Sixty‐two patients with breast cancer, 1.5 cm or larger in size on MR images, were studied. The tCho concentration was correlated with the MRI features, contrast enhancement kinetics, clinical variables and biomarkers. Pairwise two‐tailed Spearman's nonparametric test was used for statistical analysis. The tCho concentration was higher in high‐grade than moderate‐/low‐grade tumors (p = 0.04) and in tumors with higher K_trans and k_ep (p < 0.001 for both). The association of tCho concentration with age (p = 0.05) and triple negative biomarker (p = 0.09) approached significance. tCho was not detected in 17 patients, including 15 with invasive ductal cancer and two with infiltrating lobular cancer. Fifteen of the 17 patients had moderate‐ to low‐grade cancers, and 11 had human epidermal growth factor‐2‐negative cancer, suggesting that these two factors might lead to false‐negative choline. Higher tCho concentration in high‐grade tumors and tumors with higher K_trans and k_ep indicates that choline is associated with cell proliferation and tumor angiogenesis. The higher choline level in younger women may be caused by their more aggressive tumor type. The results presented here may aid in the better interpretation of ¹H MRS for the diagnosis of breast lesions. Copyright © 2010 John Wiley & Sons, Ltd.     

Proton and phosphorus magnetic resonance spectroscopy of the healthy human breast at 7 T

In vivo water‐ and fat‐suppressed ¹H magnetic resonance spectroscopy (MRS) and ³¹P magnetic resonance adiabatic multi‐echo spectroscopic imaging were performed at 7 T in duplicate in healthy fibroglandular breast tissue of a group of eight volunteers. The transverse relaxation times of ³¹P metabolites were determined, and the reproducibility of ¹H and ³¹P MRS was investigated. The transverse relaxation times for phosphoethanolamine (PE) and phosphocholine (PC) were fitted bi‐exponentially, with an added short T₂ component of 20 ms for adenosine monophosphate, resulting in values of 199 ± 8 and 239 ± 14 ms, respectively. The transverse relaxation time for glycerophosphocholine (GPC) was also fitted bi‐exponentially, with an added short T₂ component of 20 ms for glycerophosphatidylethanolamine, which resonates at a similar frequency, resulting in a value of 177 ± 6 ms. Transverse relaxation times for inorganic phosphate, γ‐ATP and glycerophosphatidylcholine mobile phospholipid were fitted mono‐exponentially, resulting in values of 180 ± 4, 19 ± 3 and 20 ± 4 ms, respectively. Coefficients of variation for the duplicate determinations of ¹H total choline (tChol) and the ³¹P metabolites were calculated for the group of volunteers. The reproducibility of inorganic phosphate, the sum of phosphomonoesters and the sum of phosphodiesters with ³¹P MRS imaging was superior to the reproducibility of ¹H MRS for tChol. ¹H and ³¹P data were combined to calculate estimates of the absolute concentrations of PC, GPC and PE in healthy fibroglandular tissue, resulting in upper limits of 0.1, 0.1 and 0.2 mmol/kg of tissue, respectively.