Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.     

蛋白激酶A介导慢性压迫背根节神经元的肾上腺素能兴奋反应

本实验在奶节（ＤＲＧ）慢性压迫模型上,采用离体灌流ＤＲＧ和单纤维记录神经元自发放电的方法研究了初级感觉神经元的交感－感觉耦联作用及其细胞内机制。用外源性支甲肾上腺素（ＮＥ,１０μｍｏｌ／Ｌ）浸浴损伤的ＤＲＧ时,在９５个ＤＲＧ神经元中有８５个自发放电的神经元产生明显反应。其中,４４个呈现单纯兴奋效应,２１个表现先兴奋后抑制效应,６个出现兴奋－抑制交替振荡现象,１４个表现抑制效应。ＮＥ对损伤神经元的兴     

Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation

Bone marrow mesenchymal stem cells (MSC) are multipotent cells. To explain their plasticity, we postulated that undifferentiated MSC may express proteins from other tissues such as neuronal tissues. MSC are obtained by two different approaches: plastic adhesion or negative depletion (RosetteSep and magnetic beads CD45/glycophorin A). MSC are evaluated through FACS analysis using a panel of antibodies (SH2, SH3, CD14, CD33, CD34, CD45, etc.). To confirm the multipotentiality in vitro, we have differentiated MSC into adipocytes, chondrocytes, osteocytes, and neuronal/glial cells using specific induction media. We have evaluated neuronal and glial proteins (Nestin, Tuj-I, betaIII Tubulin, tyrosine hydroxylase [TH], MAP-2, and GFAP) by using flow cytometry, Western blots, and RT-PCR. We found that MSC constituently express native immature neuronal proteins such as Nestin and Tuj-1. After only five passages, MSC can already express more mature neuronal or glial proteins, such as TH, MAP-2, and GFAP, without any specific induction. We noticed an increase in the expression of more mature neuronal/glial proteins (TH, MAP-2, and GFAP) after exposure to neural induction medium, thus confirming the differentiation of MSC into neurons and astrocytes. The constitutive expression of Nestin or Tuj-1 by MSC suggests that these cells are "multidifferentiated" cells and thus can retain the ability for neuronal differentiation, enhancing their potentiality to be employed in the treatment of neurological diseases.     

Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus

Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3'-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development.     

GDNF augments survival and differentiation of TH-positive neurons in neural progenitor cells

GDNF plays an important role in the survival and differentiation of primary dopaminergic neurons, but it requires multiple factors for its entire range of activities. This study investigated the effects of GDNF and its cofactors on the development of bFGF-responsive neural progenitor cells (NPCs), mesencephalic and cortical progenitor cells (MP and CP). Various factors were found to have significant inductive effects on the survival and maintenance of these cells in late developmental stages. MP had greater potential than CP to differentiate into dopaminergic neurons. Treatment of NPCs with GDNF and its cofactors enhanced MAP-2 and TH expression, particularly the latter. These findings suggest that NPCs, particularly MP, could develop into more specific neurons if the appropriate factors were applied during the final cell fate specification. They might thus become beneficial sources of donor cells in the treatment of neurological disorders.     

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.