Andrew J. Sweatt

Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats.

Summary.  The vitamin K‐dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein‐2 (BMP‐2). Here we present additional evidence that the Ca2+‐induced conformer of the vitamin K‐dependent Gla region in MGP is involved in BMP‐2 binding. Recombinant BMP‐2 binds to the Gla‐containing region of MGP in the presence of Ca2+. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly γ‐carboxylated and did not bind BMP‐2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP‐2/MGP complex exists in vivo, consistent with a role for MGP as a BMP‐2 inhibitor. Age‐related arterial calcification may be a consequence of under‐γ‐carboxylation of MGP, allowing unopposed BMP‐2 activity.

Mitochondrial transport proteins of the brain.

In this study, cellular distribution and activity of glutamate and γ‐aminobutyric acid (GABA) transport as well as oxoglutarate transport across brain mitochondrial membranes were investigated. A goal was to establish cell‐type‐specific expression of key transporters and enzymes involved in neurotransmitter metabolism in order to estimate neurotransmitter and metabolite traffic between neurons and astrocytes. Two methods were used to isolate brain mitochondria. One method excludes synaptosomes and the organelles may therefore be enriched in astrocytic mitochondria. The other method isolates mitochondria derived from all regions of the brain. Immunological and enzymatic methods were used to measure enzymes and carriers in the different preparations, in addition to studying transport kinetics. Immunohistochemistry was also employed using brain slices to confirm cell type specificity of enzymes and carriers. The data suggest that the aspartate/glutamate carriers (AGC) are expressed predominantly in neurons, not astrocytes, and that one of two glutamate/hydroxyl carriers is expressed predominantly in astrocytes. The GABA carrier and the oxoglutarate carrier appear to be equally distributed in astrocytes and neurons. As expected, pyruvate carboxylase and branched‐chain aminotransferase were predominantly astrocytic. Insofar as the aspartate/glutamate exchange carriers are required for the malate/aspartate shuttle and for reoxidation of cytosolic NADH, the data suggest a compartmentation of glucose metabolism in which astrocytes catalyze glycolytic conversion of glucose to lactate, whereas neurons are capable of oxidizing both lactate and glucose to CO2 + H2O.

A molecular mechanism for genetic warfarin resistance in the rat

Warfarin targets vitamin K 2,3‐epoxide reductase (VKOR), the enzyme that produces reduced vitamin K, a required cofactor for γ‐carboxylation of vitamin K‐dependent proteins. To identify VKOR, we used 4′‐azido‐warfarin‐3H‐alcohol as an affinity label. When added to a partially purified preparation of VKOR, two proteins were identified by mass spectrometry as calumenin and cytochrome B5. Rat calumenin was cloned and sequenced and the recombinant protein was produced. When added to an in vitro test system, the 47 kDa recombinant protein was found to inhibit VKOR activity and to protect the enzyme from warfarin inhibition. Calumenin was also shown to inhibit the overall activity of the complete vitamin K‐dependent γ‐carboxylation system. The results were repeated in COS‐1 cells overexpressing recombinant calumenin. By comparing calumenin mRNA levels in various tissues from normal rats and warfarin‐resistant rats, only the livers from resistant rats were different from normal rats by showing increased levels. Partially purified VKOR from resistant and normal rat livers showed no differences in Km‐values, specific activity, and sensitivity to warfarin. A novel model for genetic warfarin resistance in the rat is proposed, whereby the concentration of calumenin in liver determines resistance.

Functional impact of high protein intake on healthy elderly people

Decline in muscle mass, protein synthesis, and mitochondrial function occurs with age, and amino acids are reported to enhance both muscle protein synthesis and mitochondrial function. It is unclear whether increasing dietary protein intake corrects postabsorptive muscle changes in aging. We determined whether a 10-day diet of high [HP; 3.0 g protein x kg fat-free mass (FFM)(-1) x day(-1)] vs. usual protein intake (UP; 1.5 g protein x kg FFM(-1) x day(-1)) favorably affects mitochondrial function, protein metabolism, and nitrogen balance or adversely affects insulin sensitivity and glomerular filtration rate (GFR) in 10 healthy younger (24+/-1 yr) and 9 older (70+/-2 yr) participants in a randomized crossover study. Net daily nitrogen balance increased equally in young and older participants, but postabsorptive catabolic state also increased, as indicated by higher whole body protein turnover and leucine oxidation with no change in protein synthesis. Maximal muscle mitochondrial ATP production rate was lower in older people, with no change occurring in diet. GFR was lower in older people, and response to HP was significantly different between the two groups, with a significant increase occurring only in younger people, thus widening the differences in GFR between the young and older participants. In conclusion, a short-term high-protein diet increased net daily nitrogen balance but increased the postabsorptive use of protein as a fuel. HP did not enhance protein synthesis or muscle mitochondrial function in either young or older participants. Additionally, widening differences in GFR between young and older patients is a potential cause of concern in using HP diet in older people.

Branched-Chain Amino Acid Metabolism: Implications for Establishing Safe Intakes

There are several features of the metabolism of the indispensable BCAAs that set them apart from other indispensable amino acids. BCAA catabolism involves 2 initial enzymatic steps that are common to all 3 BCAAs; therefore, the dietary intake of an individual BCAA impacts on the catabolism of all 3. The first step is reversible transamination followed by irreversible oxidative decarboxylation of the branched-chain alpha-keto acid transamination products, the branched chain alpha-keto acids (BCKAs). The BCAA catabolic enzymes are distributed widely in body tissues and, with the exception of the nervous system, all reactions occur in the mitochondria of the cell. Transamination provides a mechanism for dispersing BCAA nitrogen according to the tissues requirements for glutamate and other dispensable amino acids. The intracellular compartmentalization of the branched-chain aminotransferase isozymes (mitochondrial branched-chain aminotransferase, cytosolic branched-chain aminotransferase) impacts on intra- and interorgan exchange of BCAA metabolites, nitrogen cycling, and net nitrogen transfer. BCAAs play an important role in brain neurotransmitter synthesis. Moreover, a dysregulation of the BCAA catabolic pathways that leads to excess BCAAs and their derivatives (e.g., BCKAs) results in neural dysfunction. The relatively low activity of catabolic enzymes in primates relative to the rat may make the human more susceptible to excess BCAA intake. It is hypothesized that the symptoms of excess intake would mimic the neurological symptoms of hereditary diseases of BCAA metabolism.

Branched-chain amino acids and neurotransmitter metabolism: Expression of cytosolic branched-chain aminotransferase (BCATc) in the cerebellum and hippocampus

In the brain, catabolism of the branched‐chain amino acids (BCAAs) provides nitrogen for the synthesis of glutamate and glutamine. Glutamate is formed through transfer of an amino group from BCAA to α‐ketoglutarate in reaction catalyzed by branched‐chain aminotransferases (BCAT). There are two isozymes of BCAT: cytosolic BCATc, which is found in the nervous system, ovary, and placenta, and mitochondrial BCATm, which is found in all organs except rat liver. In cell culture systems, BCATc is found only in neurons and developing oligodendrocytes, whereas BCATm is the isoform in astroglia. In this study, we used immunohistochemistry to examine the distribution of BCATc in the rat brain, focusing on the well‐known neural architecture of the cerebellum and hippocampus. We show that BCATc is expressed only in neurons in the adult rat brain. In glutamatergic neurons such as granule cells of the cerebellar cortex and of the dentate gyrus, BCATc is localized to axons and nerve terminals. In contrast, in GABAergic neurons such as cerebellar Purkinje cells and hippocampal pyramidal basket cells, BCATc is concentrated in cell bodies. A common function for BCATc in these neurotransmitter systems may be to modulate amounts of glutamate available either for release as neurotransmitter or for use as precursor for synthesis of GABA. Particularly striking in our findings is the strong expression of BCATc in the mossy fiber pathway of the hippocampal formation. This result is discussed in light of the effectiveness of the anticonvulsant drug gabapentin, which is a specific inhibitor of BCATc. J. Comp. Neurol. 477:360–370, 2004.

Widespread neuronal expression of branched-chain aminotransferase in the CNS: implications for leucine/glutamate metabolism and for signaling by amino acids.

Transamination of the branched‐chain amino acids produces glutamate and branched‐chain α‐ketoacids. The reaction is catalyzed by branched‐chain aminotransferase (BCAT), of which there are cytosolic and mitochondrial isoforms (BCATc and BCATm). BCATc accounts for 70% of brain BCAT activity, and contributes at least 30% of the nitrogen required for glutamate synthesis. In previous work, we showed that BCATc is present in the processes of glutamatergic neurons and in cell bodies of GABAergic neurons in hippocampus and cerebellum. Here we show that this metabolic enzyme is expressed throughout the brain and spinal cord, with distinct differences in regional and intracellular patterns of expression. In the cerebral cortex, BCATc is present in GABAergic interneurons and in pyramidal cell axons and proximal dendrites. Axonal labeling for BCATc continues into the corpus callosum and internal capsule. BCATc is expressed by GABAergic neurons in the basal ganglia and by glutamatergic neurons in the hypothalamus, midbrain, brainstem, and dorsal root ganglia. BCATc is also expressed in hypothalamic peptidergic neurons, brainstem serotoninergic neurons, and spinal cord motor neurons. The results indicate that BCATc accumulates in neuronal cell bodies in some regions, while elsewhere it is exported to axons and nerve terminals. The enzyme is in a position to influence pools of glutamate in a variety of neuronal types. BCATc may also provide neurons with sensitivity to nutrient‐derived BCAAs, which may be important in regions that control feeding behavior, such as the arcuate nucleus of the hypothalamus, where neurons express high levels of BCATc.

A Nonlinear Model for Hippocampal Cognitive Prosthesis: Memory Facilitation by Hippocampal Ensemble Stimulation

Collaborative investigations have characterized how multineuron hippocampal ensembles encode memory necessary for subsequent successful performance by rodents in a delayed nonmatch to sample (DNMS) task and utilized that information to provide the basis for a memory prosthesis to enhance performance. By employing a unique nonlinear dynamic multi-input/multi-output (MIMO) model, developed and adapted to hippocampal neural ensemble firing patterns derived from simultaneous recorded CA1 and CA3 activity, it was possible to extract information encoded in the sample phase necessary for successful performance in the nonmatch phase of the task. The extension of this MIMO model to online delivery of electrical stimulation delivered to the same recording loci that mimicked successful CA1 firing patterns, provided the means to increase levels of performance on a trial-by-trial basis. Inclusion of several control procedures provides evidence for the specificity of effective MIMO model generated patterns of electrical stimulation. Increased utility of the MIMO model as a prosthesis device was exhibited by the demonstration of cumulative increases in DNMS task performance with repeated MIMO stimulation over many sessions on both stimulation and nonstimulation trials, suggesting overall system modification with continued exposure. Results reported here are compatible with and extend prior demonstrations and further support the candidacy of the MIMO model as an effective cortical prosthesis.

Donor/recipient enhancement of memory in rat hippocampus.

The critical role of the mammalian hippocampus in the formation, translation and retrieval of memory has been documented over many decades. There are many theories of how the hippocampus operates to encode events and a precise mechanism was recently identified in rats performing a short-term memory task which demonstrated that successful information encoding was promoted via specific patterns of activity generated within ensembles of hippocampal neurons. In the study presented here, these “representations” were extracted via a customized non-linear multi-input multi-output (MIMO) mathematical model which allowed prediction of successful performance on specific trials within the testing session. A unique feature of this characterization was demonstrated when successful information encoding patterns were derived online from well-trained “donor” animals during difficult long-delay trials and delivered via online electrical stimulation to synchronously tested naïve “recipient” animals never before exposed to the delay feature of the task. By transferring such model-derived trained (donor) animal hippocampal firing patterns via stimulation to coupled naïve recipient animals, their task performance was facilitated in a direct “donor-recipient” manner. This provides the basis for utilizing extracted appropriate neural information from one brain to induce, recover, or enhance memory related processing in the brain of another subject.

Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism

In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron.