Natsuki Matsushita

Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons

To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitored in vitro with brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP+ cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP+ cells were transplanted into a rat model of Parkinsons disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinsons disease.

Survival of Developing Motor Neurons Mediated by Rho GTPase Signaling Pathway through Rho-Kinase

A variety of neurons generated during embryonic development survive or undergo programmed cell death (PCD) at later developmental stages. Survival or death of developing neurons is generally considered to depend on trophic support from various target tissues. The small GTPase Rho regulates diverse cellular processes such as cell morphology, cell adhesion, cell motility, and apoptosis. Rho-dependent serine–threonine protein kinase (Rho-kinase–ROK–ROCK), one of the effector proteins, transmits signals for some Rho-mediated processes. Here, we report the in vivo role of the Rho signaling pathway through Rho-kinase during development of motor neurons (MNs) in the spinal cord. We performed conditional expression of a dominant-negative form for RhoA (RhoA DN) or for Rho-kinase (Rho-K DN) in transgenic mice by using the Cre-loxP system to suppress the activity of these signaling molecules in developing MNs. Expression of RhoA DN reduced the number of MNs in the spinal cord because of increased apoptosis while preserving the gross patterning of motor axons. Expression of Rho-K DN produced developmental defects similar to those observed in RhoA DN expression. In addition, analysis of transgenic mice expressing Rho-K DN showed that the increased apoptosis of MNs was induced at the early embryonic stages before the initiation of PCD, and that MN death at the late embryonic stages corresponding to the period of PCD was moderately enhanced in the transgenic mice. These findings indicate that the Rho signaling pathway, primarily through Rho-kinase, plays a crucial role in survival of spinal MNs during embryogenesis, particularly at the early developmental stages.

Age-dependent and tissue-specific CAG repeat instability occurs in mouse knock-in for a mutant Huntington's disease gene

Huntingtons disease (HD) is a neurodegenerative disorder characterized by the expansion of CAG repeats in exon 1 of the HD gene. To clarify the instability of expanded CAG repeats in HD patients, an HD model mouse has been generated by gene replacement with human exon 1 of the HD gene with expansion to 77 CAG repeats. Chimeric proteins composed of human mutated exon 1 and mouse huntingtin are expressed ubiquitously in brain and peripheral tissues. One or two CAG repeat expansion was found in litters from paternal transmission, whereas contraction of CAG repeat in litters was observed through maternal transmission. Elderly mice show greater CAG repeat instability than younger mice, and a unique case was observed of an expanded 97 CAG repeat mouse. Somatic CAG repeat instability is particularly pronounced in the liver, kidney, stomach, and brain but not in the cerebellum of 100‐week‐old mice. The same results of expanded CAG repeat instability as observed in this HD model mouse were confirmed in the human brain of HD patients. Glial fibrillary acidic protein (GFAP)‐positive cells have been found to be increased in the substantia nigra (SN), globus pallidus (GP), and striatum (St) in the brains of 40‐week‐old affected mice, although without neuronal cell death. The CAG repeat instability and increase in GFAP‐positive cells in this mouse model appear to mirror the abnormalities in HD patients. The HD model mouse may therefore have advantages for investigations of molecular mechanisms underlying instability of CAG repeats. J. Neurosci. Res. 65:289–297, 2001.

Motor and learning dysfunction during postnatal development in mice defective in dopamine neuronal transmission

Mice lacking expression of tyrosine hydroxylase (TH), the first and rate‐limiting enzyme of the catecholamine biosynthetic pathway, in dopaminergic neuronal cell types were generated by a transgenic rescue approach to clarify the role of dopamine signaling during postnatal development. Introduction of the TH transgene directed by the dopamine β‐hydroxylase gene promoter into TH knockout mice restored noradrenaline and adrenaline synthesis, preventing perinatal lethality and cardiac dysfunction in the knockout mice. Lack of TH expression in the cells that normally express the dopaminergic phenotype resulted in a marked reduction of dopamine accumulation in the tissues, which led to multiple behavioral abnormalities at the juvenile stage. These abnormalities were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine‐induced hyperactivity, cataleptic behavior, and defects in active avoidance learning. In contrast, development of the pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained, despite defective dopamine synthesis. These results demonstrate that dopamine neurotransmission is essential for controlling spontaneous and voluntary movement and associative learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. J. Neurosci. Res. 54: 450–464, 1998.

Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro.

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P < 0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinsons disease.

Enhanced flexibility of place discrimination learning by targeting striatal cholinergic interneurons

Behavioural flexibility is mediated through the neural circuitry linking the prefrontal cortex and basal ganglia. Here we conduct selective elimination of striatal cholinergic interneurons in transgenic rats by immunotoxin-mediated cell targeting. Elimination of cholinergic interneurons from the dorsomedial striatum (DMS), but not from the dorsolateral striatum, results in enhanced reversal and extinction learning, sparing the acquisition of place discrimination. This enhancement is prevented by infusion of a non-selective muscarinic acetylcholine receptor agonist into the DMS either in the acquisition, reversal or extinction phase. In addition, gene-specific silencing of M4 muscarinic receptor by lentiviral expression of short hairpin RNA (shRNA) mimics the place reversal learning promoted by cholinergic elimination, whereas shRNA-mediated gene silencing of M1 muscarinic receptor shows the normal performance of reversal learning. Our data indicate that DMS cholinergic interneurons inhibit behavioural flexibility, mainly through the M4 muscarinic receptor, suggesting that this role is engaged to the stabilization of acquired reward contingency and the suppression of response switch to changed contingency.

Fate of transient catecholaminergic cell types revealed by site-specific recombination in transgenic mice.

Catecholamine‐producing cell types are generated from specified neuronal lineages during vertebrate development. The catecholaminergic phenotype is also expressed transiently in some cell types in non‐catecholaminergic tissues, including the sensory ganglia, enteric ganglia, and ventral portions of the neural tube during embryonic development. The fate of the transient catecholaminergic cell types at later developmental stages, however, has not been elucidated. We developed a Cre‐loxP‐mediated recombination system under the control of the dopamine β‐hydroxylase (DBH) promoter, which drives gene expression in typical noradrenergic and adrenergic cell groups as well as in transient catecholaminergic cell types. Expression of Cre recombinase in transgenic mice resulted in an efficient recombination in noradrenergic and adrenergic cell groups at the adult stage. The recombination was also induced in the cranial nerve/spinal cord motor neurons and sensory/enteric ganglion neurons. Analysis of recombination patterns in transgenic mouse embryos showed the occurrence of recombination during prenatal development in both cell types exhibiting the typical and transient catecholaminergic phenotypes. Because the DBH gene promoter is expressed transiently in the ventral neural tube and sensory ganglion during embryonic development, our results provide evidence that the cell types showing a transient catecholaminergic phenotype in these tissues are destined to become mature motor neurons or sensory ganglion neurons during subsequent differentiation.