Factors promoting survival of mesencephalic dopaminergic neurons

Growth factors promoting survival of mesencephalic dopaminergic neurons are discussed in the context of their requirement during development and adulthood. The expression of growth factors should be detectable in the nigrostriatal system during critical periods of development, i.e., during the period of ontogenetic cell death and synaptogenesis and during neurite extension and neurotransmitter synthesis. Growth factors discussed include members of the family of glial-cell-line-derived neurotrophic factors (GDNF), neurotrophins, transforming growth factors beta, and low molecular compounds mimicking growth factor activities. To date, the available data support the notion that GDNF is a highly promising candidate, although GDNF-null mice lack a dopaminergic phenotype. There remains a possibility that endogenous dopaminotrophic factors remain to be discovered.The authors work is supported by grants from the Deutsche Forschungsgemeinschaft through the DFG-Research Center for Molecular Physiology of the Brain     

Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.     

Contrasting and brain region-specific roles of neurogenin2 and mash1 in GABAergic neuron differentiation in vitro

We have cultivated highly uniform populations of neural precursor cells, which retain their region-specific identities, from various rat embryonic brain regions. The roles of the proneural basic-helix-loop-helix (bHLH) factors neurogenin2 (Ngn2) and Mash1 in gamma-aminobutyric acid (GABA) neuron differentiation were explored in the region-specific cultures. Consistent with previous in vivo studies, forced expression of Mash1 promoted GABA neuron formation from the precursors derived from the developing forebrains, whereas Ngn2 displayed an inhibitory role in forebrain GABA neuron differentiation. Functional analyses of mutant bHLH proteins indicated that the helix-loop-helix domains of Mash1 and Ngn2, known as the structures for protein-protein interactions, impart the distinct activities. Intriguingly, the regulatory activities of Mash1 and Ngn2 in GABA neuron differentiation from the hindbrain- and spinal cord-derived precursor cells were completely opposite of those observed in the forebrain-derived cultures: increased GABA neuron yield by Ngn2 and decreased yield by Mash1 were shown in the precursors of those posterior brain regions. No clear difference that depended on dorsal-ventral brain regions was observed in the bHLH-mediated activities. Finally, we demonstrated that Otx2, the expression of which is developmentally confined to the regions anterior to the isthmus, is a factor responsible for the anterior-posterior region-dependent opposite effects of the bHLH proteins.     

Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures

Mutations in alpha-synuclein, parkin and ubiquitin C-terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinson's disease (PD). This suggests that failure of the ubiquitin-proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin-mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)-induced impairment of ubiquitin C-terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures. We demonstrate that both lactacystin and UbA caused concentration-dependent and preferential degeneration of dopaminergic neurons. Inhibition of 26/20S proteasomal function was accompanied by the accumulation of alpha-synuclein and ubiquitin, and the formation of inclusions that were immunoreactive for these proteins, in the cytoplasm of VM neurons. Inhibition of UCH was associated with a loss of ubiquitin immunoreactivity in the cytoplasm of VM neurons, but there was a marked and localized increase in alpha-synuclein staining which may represent the formation of inclusions bodies in VM neurons. These findings provide direct evidence that impaired protein clearance can induce dopaminergic cell death and the formation of proteinaceous inclusion bodies in VM neurons. This study supports the concept that defects in the UPS may underlie nigral pathology in familial and sporadic forms of PD.     

Frequency switching in a two-compartmental model of the dopaminergic neuron

Mid-brain dopaminergic (DA) neurons display two functionally distinct modes of electrical activity: low- and high-frequency firing. The high-frequency firing is linked to important behavioral events in vivo. However, it cannot be elicited by standard manipulations in vitro. We had suggested a two-compartmental model of the DA cell that united data on firing frequencies under different experimental conditions. We now analyze dynamics of this model. The analysis was possible due to introduction of timescale separation among variables. We formulate the requirements for low and high frequencies. We found that the modulation of the SK current gating controls the frequency rise under applied depolarization. This provides a new mechanism that limits the frequency in the control conditions and allows high-frequency responses to depolarization if the SK current gating is downregulated. The mechanism is based on changing Ca^2 + balance and can also be achieved by direct modulation of the balance. Interestingly, such changes do not affect the high-frequency oscillations under NMDA. Therefore, altering Ca^2 + balance allows combining the high-frequency response to NMDA activation with the inability of other treatments to effectively elevate the frequency. We conclude that manipulations affecting Ca^2 + balance are most effective in controlling the frequency range. This modeling prediction gives a clue to the mechanism of the high-frequency firing in the DA neuron in vivo and in vitro.     

Spatiotemporal expression of sonic hedgehog signalling molecules in the embryonic mesencephalic dopaminergic neurons

Midbrain dopaminergic neurons (mDA) play an important role in controlling the voluntary motor movement, reward, and emotion-based behaviour. Differentiation of mDA neurons from progenitors depends on several secreted proteins, such as sonic hedgehog (SHH). The present study attempted to elucidate the possible role(s) of some SHH signaling components (Ptch1, Gli1, Gli2 and Gli3) in the spatiotemporal development of mDA neurons along the rostrocaudal axis of the midbrain and their possible roles in differentiation and survival of mDA neurons and the significance of using in vitro models for studying the development of mDA neurons. At E12 and E14, only Ptch1 and Gli1 were expressed in ventrolateral midbrain domains. All examined SHH signalling molecules were not detected in mDA area. Whereas, in MN9D cells, many SHH signalling molecules were expressed and co-localized with the dopaminergic marker; tyrosine hydroxylase (TH), and their expression were upregulated with SHH treatment of the MN9D cells. These results suggest that mDA neurons differentiation and survival might be independent of SHH in the late developmental stages (E12-18). Besides, MN9D cell line is not the ideal in vitro model for investigating the differentiation of mDA and hence, the ventral midbrain primary culture might be favored over MN9D line.     

Catalpol protects dopaminergic neurons from LPS-induced neurotoxicity in mesencephalic neuron-glia cultures

Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Microglia, the resident immune cells in the central nervous system, are pivotal in the inflammatory reaction. Activated microglia can induce expression of inducible nitric-oxide synthase (iNOS) and release significant amounts of nitric oxide (NO) and TNF-alpha, which can damage the dopaminergic neurons. Catalpol, an iridoid glycoside, contained richly in the roots of Rehmannia glutinosa, was found to be neuroprotective in gerbils subjected to transient global cerebral ischemia. But the effect of catalpol on inflammation-mediated neurodegeneration has not been examined. In this study, microglia in mesencephalic neuron-glia cultures were activated with lipopolysaccharide (LPS) and the aim of the study was to examine whether catalpol could protect dopaminergic neurons from LPS-induced neurotoxicity. The results showed that catalpol significantly reduced the release of reactive oxygen species (ROS), TNF-alpha and NO after LPS-induced microglial activation. Further, catalpol attenuated LPS-induced the expression of iNOS. As determined by immunocytochemical analysis, pretreatment by catalpol dose-dependently protected dopaminergic neurons against LPS-induced neurotoxicity. These results suggest that catalpol exerts its protective effect on dopaminergic neurons by inhibiting microglial activation and reducing the production of proinflammatory factors. Thus, catalpol may possess therapeutic potential against inflammation-related neurodegenerative diseases.     

Role of activity-dependent mechanisms in the control of dopaminergic neuron survival

Dopaminergic neurons that constitute the nigrostriatal pathway are characterized by singular electrical properties that allow them to discharge in vivo spontaneously in a spectrum of patterns ranging from pacemaker to random and bursting modes. These electrophysiological features allow dopaminergic neurons to optimize the release of dopamine in their terminal fields. However, there is emerging evidence indicating that electrical activity might also participate in the control of dopaminergic neuron survival, not only during development, but also in the adult brain, thus raising the possibility that alterations in ionic currents could contribute actively to the demise of these neurons in Parkinson disease. This review focuses on the mechanisms by which activity-dependent mechanisms might modulate dopaminergic cell survival.     

Method of evaluating the synaptic input to a single mesencephalic neuron]

The mathematical model of the spike activity of a neuron with synaptic input from many other neurons [1], describes adequately the firing of 5 from 7 neurons in the tegmentum of mesencephalic cat and changes of their activity evoked by glutamate iontophoresis. For these 5 neurons the estimates of the PSPs' average frequency of the threshold depolarization and of the constant decay of the EPSP were received. For different neurons the values of these parameters are 4--100 KHz, 100--800 average unitary EPSPs and 4--30 msec correspondingly. The stationary value of the average membrane potential (SVAMP) in all 5 neurons was removed significantly from the resting potential toward the threshold potential. SWAMP could be changed by the glutamate iontophoresis in such a degree to overlap the threshold potential.     

A two-compartment phenomenological model of a dopaminergic neuron

A two-compartment model of a dopaminergic neuron based on modified FitzHugh-Nagumo oscillators for each compartment has been built. The compartments correspond to the soma and dendrites and differ in the values of small parameters. The influence of stimuli (imposed current for the soma compartment and synaptic activation for the dendrite compartment) on the model has been studied. Activation of AMPA and NMDA synaptic currents is shown to cause generation of high-frequency bursts by the neuron. The mechanisms underlying burst generation are considered.