N-acetylaspartate in the vertebrate brain: metabolism and function

N-Acetyl-l-aspartate (NAA) is an amino acid that is present in the vertebrate brain. Its concentration is one of the highest of all free amino acids and, although NAA is synthesized and stored primarily in neurons, it cannot be hydrolyzed in these cells. Furthermore, neuronal NAA is dynamic and turns over more than once each day by virtue of its continuous efflux, in a regulated intercompartmental cycling via extracellular fluids, between neurons and a second compartment in oligodendrocytes. The metabolism of NAA, between its anabolic compartment in neurons and its catabolic compartment in oligodendrocytes, and its possible physiological role in the brain has been the subject of much speculation. There are two human inborn errors in metabolism of NAA. One is Canavan disease (CD), in which there is a buildup of NAA (hyperacetylaspartia) and associated spongiform leukodystrophy, caused by a lack of aspartoacylase activity. The other is a singular human case of lack of NAA (hypoacetylaspartia), where the enzyme that synthesizes NAA is apparently absent. There are two animal models currently available for studies of CD. One is a rat with a natural deletion of the catabolic enzyme, and the other a gene knockout mouse. In addition to the presence of NAA in neurons, its prominence in ¹H nuclear magnetic resonance spectroscopic studies has led to its wide use in diagnostic human medicine as both an indicator of brain pathology and of disease progression in a variety of CNS diseases. In this review, various hypotheses regarding the metabolism of NAA and its possible role in the CNS are evaluated. Based on this analysis, it is concluded that although NAA may have several functions in the CNS, an important role of the NAA intercompartmental system is osmoregulatory, and in this role it may be the primary mechanism for the removal of intracellular water, against a water gradient, from myelinated neurons.     

Caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells in mutant SOD1 transgenic mouse spinal cord: a study using laser microdissection and real-time RT-PCR

Amyotrophic lateral sclerosis is characterized by selective motor neuron degeneration. An apoptotic pathway is thought to be involved. It is difficult, however, to analyze the molecular pathogenic mechanism in single motor neurons because of complexity in the neural tissue, which consists of multiple lineages of cells neighboring motor neurons. We quantified the caspase-1 and -3 mRNA in single motor neurons and neighboring glial cells isolated from the spinal ventral horn of mutant SOD1 transgenic (Tg) mice and littermates. Motor neurons and neighboring glial cells were isolated from spinal sections by laser microdissection, and the mRNAs were quantified by RT-PCR. In the Tg mice, caspase-1 mRNA was first upregulated in motor neurons and second in glial cells. The caspase-3 mRNA was increased in motor neurons following the caspase-1 mRNA. These results indicated that caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells of the Tg mice, and that mRNAs in isolated cells can be accurately assessed using our procedures.     

The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons

In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.     

A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.     

Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Differential Response of Neural Cells to Trauma-Induced Free Radical Production In Vitro

CNS trauma has been associated with an increase in free radical production, but the cellular sources of this increase or the mechanism involved in the production of free radicals are not known. We, therefore, investigated the effects of trauma on free radical production in cultured neurons, astrocytes and BV-2 microglial cells. Free radicals were measured with the fluorescent dye DCFDA following in vitro trauma. At 30 and 60 min following trauma, there was a 132% and 64% increase, respectively, in free radical production in neurons when compared to controls. In astrocytes, there was a 94% and 133% increase at 30 and 60 min, respectively. Microglial cells, however, displayed no significant increase in free radicals at 30, 60 or 120 min following trauma. Since trauma can induce the mitochondrial permeability transition (MPT), a process associated with mitochondrial dysfunction, we further investigated whether cyclosporin A (CsA), an agent known to block the MPT, could prevent free radical formation following trauma. In neurons CsA did not block free radical production at 30 min but blocked it by 90% at 60 min. In contrast, in astrocytes CsA completely blocked free radical production at 30 min but did not block it at 60 min. Our results indicate that a differential sensitivity to trauma-induced free radical production exists in neural cells; that the MPT may be involved in the production of free radical post-trauma; and that the CsA-sensitive phase of free radical production is different in neurons and astrocytes.