Alexander Storch

Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells

Clonogenic neural stem cells (NSCs) are self-renewing cells that maintain the capacity to differentiate into brain-specific cell types, and may also replace or repair diseased brain tissue. NSCs can be directly isolated from fetal or adult nervous tissue, or derived from embryonic stem cells. Here, we describe the efficient conversion of human adult bone marrow stromal cells (hMSC) into a neural stem cell-like population (hmNSC, for human marrow-derived NSC-like cells). These cells grow in neurosphere-like structures, express high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, MSl1 as well as otx1 and nestin, but lose the characteristics of mesodermal stromal cells. In the presence of selected growth factors, hmNSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Clonal analysis demonstrates that individual hmNSCs are multipotent and retain the capacity to generate both glia and neurons. Our cell culture system provides a powerful tool for investigating the molecular mechanisms of neural differentiation in adult human NSCs. hmNSCs may therefore ultimately help to treat acute and chronic neurodegenerative diseases.

Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells.

We report on generation of dopamine neurons from long-term cultures of human fetal mesencephalic precursor cells. These CNS precursor cells were successfully expanded in vitro using the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Incubation of these cultures in 3% atmospheric oxygen resulted in higher cellular yields than room air. Following incubation in differentiation media containing interleukin (IL)-1b (IL-1b), IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF), up to 1% of the precursor cells converted into cells immunoreactive for tyrosine hydroxylase (TH), a marker for dopamine neurons. The TH immunoreactive cells exhibited morphological and functional properties characteristic of dopamine neurons in culture. These precursor cells might serve as a useful source of human dopamine neurons for studying the development and degeneration of human dopamine neurons and may further serve as a continuous, on-demand source of cells for therapeutic transplantation in patients with Parkinsons disease.

Increased susceptibility to sporadic Parkinson's disease by a certain combined α‐synuclein/apolipoprotein E genotype

Parkinsons disease (PD) is one of the most common neurodegenerative disorders affecting about 1% of Western populations older than age 50. The pathological hallmark of PD are Lewy bodies, that is, intracytoplasmic inclusion bodies in affected neurons of the substantia nigra. Recently, α‐synuclein (α‐SYN) has been identified as the main component of Lewy bodies in sporadic PD, suggesting involvement in neurodegeneration via protein accumulation. The partially overlapping pathology of PD and Alzheimers disease, as well as striking structural similarities of α‐SYN and apolipoprotein E, which is a major risk factor for late‐onset Alzheimers disease, prompted us to investigate the influence of different α‐SYN and apolipoprotein E alleles for developing sporadic PD. We performed association studies in 193 German PD patients and 200 healthy control subjects matched for age, sex, and origin. A polymorphism in the promoter region of the α‐SYN gene (NACP‐Rep1) as well as of the closely linked DNA markers D4S1647 and D4S1628 revealed significant differences in the allelic distributions between PD patients and the control group. Furthermore, the Apoε4 allele but not the Th1/E47 promoter polymorphism of the apolipoprotein E gene was significantly more frequent among early‐onset PD patients (age at onset, <50 years) than in late‐onset PD. Regarding the combination of the Apoε4 allele and allele 1 of the α‐SYN promoter polymorphism, a highly significant difference between the group of PD patients and control individuals has been found, suggesting interactions or combined actions of these proteins in the pathogenesis of sporadic PD. PD patients harboring this genotype have a 12.8‐fold increased relative risk for developing PD during their lives. Ann Neurol 1999;45:611–617

A clonal line of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines: a source of cells for transplantation in Parkinson's disease.

Neural progenitor cells potentially provide a limitless, on-demand source of cells for grafting into patients with Parkinsons disease (PD) if the signals needed to control their conversion into dopamine (DA) neurons could be identified. We have recently shown that cytokines which instruct cell division and differentiation within the hematopoeitic system may provide similar functions in the central nervous system. We have shown that mitotic progenitor cells can be isolated from embryonic rat mesencephalon and that these cells respond to a combination of interleukin-1, interleukin-11, leukemia inhibitory factor, and glial cell line-derived neurotrophic factor yielding a tyrosine hydroxylase-immunoreactive (THir) phenotype in 20-25% of total cells. In the present study, 24 clonal cell lines derived from single cells of mesencephalic proliferation spheres were examined for their response to the cytokine mixture. The clone yielding the highest percentage of THir neurons (98%) was selected for further study. This clone expressed several phenotypic characteristics of DA neurons and expression of Nurr1. The response to cytokines was stable for several passages and after cryopreservation for several months. When grafted into the striatum of DA-depleted rats, these cells attenuated rotational asymmetry to the same extent as freshly harvested embryonic DA neurons. These data demonstrate that mesencephalic progenitor cells can be clonally expanded in culture and differentiated in the presence of hematopoietic cytokines to yield enriched populations of DA neurons. When transplanted, these cells provide significant functional benefit in the rat model of PD.

FTDP-17: An early-onset phenotype with parkinsonism and epileptic seizures caused by a novel mutation

Recently, mutations in the tau gene on chromosome 17 were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism (FTDP‐17). We describe a family carrying a missense mutation at nucleotide 1137 C → T, resulting in the amino acid substitution P301S. Methods of investigations include clinical, electrophysiological, and imaging techniques. This kindred presents with a novel phenotype characterized by an early onset of rapidly progressive frontotemporal dementia and parkinsonism in combination with epileptic seizures. We define the dopaminergic deficits as being predominantly presynaptic by the use of single‐photon emission computed tomography with a dopamine transporter ligand. The association of this early‐onset phenotype with P301S mutation is not entirely consistent with current criteria for the diagnosis of frontotemporal dementias and may encourage the search for tau mutations in diseases similar but not identical to FTDP‐17. Also, the change from proline to serine suggests that this mutation might contribute to tau hyperphosphorylation.

Dopamine transporter: involvement in selective dopaminergic neurotoxicity and degeneration.

Summary.The carrier molecule that transports dopamine (DA) into dopamine neurons by an electrogenic, Na+- and Cl−-transport-coupled mechanism is known as the dopamine transporter (DAT). This uptake system is exclusively expressed in DA neurons with significantly higher levels of DAT expression in cells of the substantia nigra pars compacta than those of the ventral tegmental area and arcuate hypothalamic neurons. The expression density of DAT strongly correlates with the extent of DA cell loss in Parkinson’s disease (PD). There are also DAT gene polymorphisms associated with PD. These data suggest a role of the DAT in the pathogenesis of PD. Though selective for its respective neurotransmitter, the DAT can also transport synthetic/natural analogues of the transmitter. Should such compounds interact with vital intracellular structures, their penetration into the neuron might have significant consequences. This sequence of toxic events could indeed demonstrated for the synthetic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces selective degeneration of DA neurons characteristic of PD. Dopaminergic toxicity of its active metabolite 1-methyl-4-pyridinium (MPP+) is mediated by the DAT through accumulation into DA neurons, where it inhibits mitochondrial complex I activity. Various endogenous and exogenous heterocyclic molecules, which are structurally related to MPTP/MPP+, such as isoquinolines and β-carbolines, have been reported to exhibit similar toxic properties on DA cells, which are conferred by their uptake by the DAT. Taken together, there is large body of evidence from morphological, molecular biological and toxicological studies indicating that the DAT might be responsible for the selectivity of DA cell death in PD.

Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis

Chorea-acanthocytosis (ChAc) is an autosomal recessive neurological disorder whose characteristic features include hyperkinetic movements and abnormal red blood cell morphology. Mutations in the CHAC gene on 9q21 were recently found to cause chorea-acanthocytosis. CHAC encodes a large, novel protein with a yeast homologue implicated in protein sorting. In this study, all 73 exons plus flanking intronic sequence in CHAC were screened for mutations by denaturing high-performance liquid chromatography in 43 probands with ChAc. We identified 57 different mutations, 54 of which have not previously been reported, in 39 probands. The novel mutations comprise 15 nonsense, 22 insertion/deletion, 15 splice-site and two missense mutations and are distributed throughout the CHAC gene. Three mutations were found in multiple families within this or our previous study. The preponderance of mutations that are predicted to cause absence of gene product is consistent with the recessive inheritance of this disease. The high proportion of splice-site mutations found is probably a reflection of the large number of exons that comprise the CHAC gene. The CHAC protein product, chorein, appears to have a certain tolerance to amino-acid substitutions since only two out of nine substitutions described here appear to be pathogenic.

1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) is toxic to dopaminergic neuroblastoma SH-SY5Y cells via impairment of cellular energy metabolism.

The endogenous neurotoxin 1-methyl-6,7-dihydroxy-1,2,3, 4-tetrahydroisoquinoline (salsolinol), which is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been reported to inhibit mitochondrial complex I (NADH-Q reductase) activity as does the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP(+)). However, the mechanism of salsolinol leading to neuronal cell death is still unknown. Thus, we correlated indices of cellular energy production and cell viability in human dopaminergic neuroblastoma SH-SY5Y cells after exposure to salsolinol and compared these results with data obtained with MPP(+). Both toxins induce time and dose-dependent decrease in cell survival with IC(50) values of 34 microM and 94 microM after 72 h for salsolinol and MPP(+), respectively. Furthermore, salsolinol and MPP(+) produce a decrease of intracellular net ATP content with IC(50) values of 62 microM and 66 microM after 48 h, respectively. In contrast to MPP(+), salsolinol does not induce an increase of intracellular net NADH content. In addition, enhancing glycolysis by adding D-glucose to the culture medium protects the cells against MPP(+) but not salsolinol induced cellular ATP depletion and cytotoxicity. These results suggest that cell death induced by salsolinol is due to impairment of cellular energy supply, caused in particular by inhibition of mitochondrial complex II (succinate-Q reductase), but not complex I.

Protective effects of riluzole on dopamine neurons: involvement of oxidative stress and cellular energy metabolism.

Abstract: Riluzole is neuroprotective in patients with amyotrophiclateral sclerosis and may also protect dopamine (DA) neurons in Parkinsonsdisease. We examined the neuroprotective potential of riluzole on DA neuronsusing primary rat mesencephalic cultures and human dopaminergic neuroblastomaSH‐SY5Y cells. Riluzole (up to 10 μM) alone affected neither thesurvival of DA neurons in primary cultures nor the growth of SH‐SY5Y cellsafter up to 72 h. Riluzole (1‐10 μM) dose‐dependently reduced DAcell loss caused by exposure to MPP+ in both types of cultures.These protective effects were accompanied by a dose‐dependent decrease ofintracellular ATP depletion caused by MPP+ (30‐300 μM)in SH‐SY5Y cells without affecting intracellular net NADH content, suggestinga reduction of cellular ATP consumption rather than normalization ofmitochondrial ATP production. Riluzole (1‐10 μM) also attenuatedoxidative injury in both cell types induced by exposure to L‐DOPA and6‐hydroxydopamine, respectively. Consistent with its antioxidative effects,riluzole reduced lipid peroxidation induced by Fe3+ and L‐DOPA inprimary mesencephalic cultures. Riluzole (10 μM) did not alterhigh‐affinity uptake of either DA or MPP+. However, in the samecell systems, riluzole induced neuronal and glial cell death withconcentrations higher than those needed for maximal protective effects(≥100 μM). These data demonstrate that riluzole has protectiveeffects on DA neurons in vitro against neuronal injuries induced by (a)impairment of cellular energy metabolism and/or (b) oxidative stress. Theseresults provide further impetus to explore the neuroprotective potential ofriluzole in Parkinsons disease.

Selective dopaminergic neurotoxicity of isoquinoline derivatives related to Parkinson’s disease: studies using heterologous expression systems of the dopamine transporter

Endogenous isoquinoline (IQ) derivatives structurally related to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridine (MPP(+)) may contribute to dopaminergic neurodegeneration in Parkinsons disease. We addressed the importance of the DAT molecule for selective dopaminergic toxicity by testing the differential cytotoxicity of 22 neutral and quaternary compounds from three classes of isoquinoline derivatives (3, IQs; 4,3,4-dihydroisoquinolines and 15, 1,2,3,4-tetrahydroisoquinolines) as well as MPP(+) in non-neuronal and neuronal heterologous expression systems of the DAT gene (human embryonic kidney HEK-293 and mouse neuroblastoma Neuro-2A cells, respectively). Cell death was estimated using the MTT assay and the Trypan blue exclusion method. Nine isoquinolines and MPP(+) showed general cytotoxicity in both parental cell lines after 72hr with half-maximal toxic concentrations (TC(50) values) in the micromolar range. The rank order of toxic potency was: papaverine>salsolinol=tetrahydropapaveroline=1-benzyl-TIQ=norsalsolinol>tetrahydropapaverine>2[N]-methyl-salsolinol>2[N]-methyl-norsalsolinol>2[N]-Me-IQ(+)=MPP(+). Besides MPP(+), only the 2[N]-methylated compounds 2[N]-methyl-IQ(+), 2[N]-methyl-norsalsolinol and 2[N]-methyl-salsolinol showed enhanced cytotoxicity in both DAT expressing cell lines with 2- to 14-fold reduction of TC(50) values compared to parental cell lines. The rank order of selectivity in both cell systems was: MPP(+)>>2[N]-Me-IQ(+)>2[N]-methyl-norsalsolinol=2[N]-methyl-salsolinol. Our results suggest that 2[N]-methylated isoquinoline derivatives structurally related to MPTP/MPP(+) are selectively toxic to dopaminergic cells via uptake by the DAT, and therefore may play a role in the pathogenesis of Parkinsons disease.