Christian Arriagada

Behavioral effects of manganese injected in the rat substantia nigra are potentiated by dicumarol, a DT-diaphorase inhibitor

The purpose of this study was to evaluate the contribution of DT-diaphorase inhibition to in vivo neurodegenerative effects of dopamine (DA) oxidation to the corresponding o-quinones. The neurotoxicity to nigrostriatal DA neurons was induced by injection of manganese pyrophosphate (Mn(3+)) complex as a prooxidizing agent alone or together with the DT-diaphorase inhibitor dicumarol into the right rat substantia nigra. The behavioral effects were compared with those induced after selective lesions of dopaminergic neurons with 6-hydroxydopamine (6-OHDA). Intranigral injection of Mn(3+) and Mn(3+) plus dicumarol produced significant impairment in motor behavior compared with control animals. However, the effect seen in the Mn(3+) plus dicumarol injected group was significantly more severe than that observed in the Mn(3+) alone injected group. In motor activity and rearing behavior, the simultaneous injection of Mn(3+) plus dicumarol produced a 6-OHDA-like impairment. Similar effects were observed in the acquisition of a conditioned avoidance response (CAR). Dicumarol significantly impaired avoidance conditioning although without affecting the motor behavior. The behavioral effects were correlated to the extent of striatal tyrosine hydroxylase (TH)-positive fiber loss. Rats receiving unilateral intranigral Mn(3+) and Mn(3+) plus dicumarol injections exhibited a significant reduction in nigrostriatal TH-positive fiber density in medial forebrain bundle compared with the contralateral noninjected side. In conclusion, this study provides evidence that the neurotoxicity of Mn(3+) in vivo is potentiated by DT-diaphorase inhibition, suggesting that this enzyme could play a neuroprotective role in the nigrostriatal DA systems.

Impaired cholinergic function in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

Murine trisomy 16 is an animal model of human Downs syndrome. We have successfully established permanently growing cell lines from the cerebral cortex of normal and trisomy 16 foetal mice using an original procedure. These lines, named CNh (derived from a normal animal) and CTb (derived from a trisomic foetus), express neuronal markers. Considering that Downs syndrome exhibits cholinergic deficits, we examined cholinergic function in these lines, using incorporation of [3H]‐choline and fractional release studies. After 1, 3 and 5 min of [3H]‐choline incubation, CTb cell uptake was lower by ∼ 50% compared to controls. Hemicholinium‐3 significantly reduced the incorporation of [3H]‐choline in both CNh and CTb cells at high concentration (10 μm), suggesting high‐affinity choline transport. However, CTb cells exhibited greater sensitivity to the blocker. For fractional release experiments, the cells were stimulated by K+ depolarization, glutamate or nicotine. When depolarized, CTb cells showed a 68% reduction in fractional release of [3H]‐acetylcholine compared to CNh cell line, and a 45% reduction when stimulated by nicotine. Interestingly, glutamate induced similar levels of release in both cell types. The results indicate the existence of cholinergic dysfunction in CTb cells when compared to CNh, similar to that reported for primary cultures of trisomy 16 brain tissue ( Fiedler et al. 1994 , Brain Res., 658, 27–32). Thus, the CTb cell line may serve as a model for the study of Downs syndrome pathophysiology.

Establishment and characterization of immortalized neuronal cell lines derived from the spinal cord of normal and trisomy 16 fetal mice, an animal model of Down syndrome.

We report the establishment of continuously growing cell lines from spinal cords of normal and trisomy 16 fetal mice. We show that both cell lines, named M4b (derived from a normal animal) and MTh (trisomic) possess neurological markers by immunohistochemistry (neuron specific enolase, synaptophysin, microtubule associated protein‐2 [MAP‐2], and choline acetyltransferase) and lack glial traits (glial fibrillary acidic protein and S100). MTh cells were shown to overexpress mRNA of Cu/Zn superoxide dismutase, whose gene is present in autosome 16. We also studied intracellular Ca2+ signals ([Ca2+]i) induced by different agonists in Indo‐1 loaded cells. Basal [Ca2+]i was significantly higher in MTh cells compared to M4b cells. Glutamate (200 μM) and (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACDP) (100 μM) induced rapid, transient increases in [Ca2+]i in M4b and MTh cells, indicating the presence of glutamatergic metabotropic receptors. N‐methyl‐D‐aspartate (NMDA) and kainate, but not alpha‐amino‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA), produced [Ca2+]i rises in both cell types. MTh cells exhibited faster time‐dependent decay phase kinetics in glutamate‐induced responses compared to M4b cells. Nicotine induced a transient increase in [Ca2+]i in M4b and MTh cells, with significantly greater amplitudes in the latter compared to the former. Further, both cell types responded to noradrenaline. Finally, we examined cholinergic function in both cell lines and found no significant differences in the [3H]‐choline uptake, but fractional acetylcholine release induced by either K+, glutamate or nicotine was significantly higher in MTh cells. These results show that M4b and MTh cells have neuronal characteristics and the MTh line shows differences which could be related to neuronal pathophysiology in Downs syndrome.

Neuronal dysfunction in Down syndrome: Contribution of neuronal models in cell culture

Down syndrome (DS) in humans, or trisomy of autosome 21, represents the hyperdiploidy that most frequently survives gestation, reaching an incidence of 1 in 700 live births. The condition is associated with multisystemic anomalies, including those affecting the central nervous system (CNS), determining a characteristic mental retardation. At a neuronal level, our group and others have shown that the condition determines marked alterations of action potential and ionic current kinetics, which may underlie abnormal processing of information by the CNS. Since the use of human tissue presents both practical and ethical problems, animal models of the human condition have been sought. Murine trisomy 16 (Ts16) is a model of the human condition, due to the great homology between human autosome 21 and murine 16. Both conditions share the same alterations of electrical membrane properties. However, the murine Ts16 condition is unviable (animals die in utero), thus limiting the quantity of tissue procurable. To overcome this obstacle, we have established immortal cell lines from normal and Ts16 mice with a method developed by our group that allows the stable in vitro immortalization of mammalian tissue, yielding cell lines which retain the characteristics of the originating cells. Cell lines derived from cerebral cortex, hippocampus, spinal cord and dorsal root ganglion of Ts16 animals show alterations of intracellular Ca2+ signals in response to several neurotransmitters (glutamate, acetylcholine, and GABA). Gene overdose most likely underlies these alterations in cell function, and the identification of the relative contribution of DS associated genes on such specific neuronal dysfunction should be investigated. This could enlighten our understanding on the contribution of these genes in DS, and identify new therapeutic targets.

Behavioral effects of aminochrome and dopachrome injected in the rat substantia nigra.

The exact mechanism of cell death in neurodegenerative diseases remains obscure, although there is evidence that their pathogenesis may involve the formation of free radicals originating from the oxidative metabolism of catecholamines. The purpose of this study was to evaluate the degree of neurodegenerative changes and behavioral impairments induced by unilateral injection into the rat substantia nigra of cyclized o-quinones, aminochrome and dopachrome, derived from oxidizing dopamine and L-DOPA, respectively, with Mn(3+)-pyrophosphate complex. The behavioral changes were compared with those induced after selective lesions of dopaminergic neurons with 6-hydroxydopamine (6-OHDA). Intranigral injection of aminochrome and dopachrome produced impairment in motor and cognitive behaviors. The behavioral impairment was also revealed by apomorphine-induced rotational asymmetry. Apomorphine (0.5 mg/kg sc) significantly increased rotational behavior in rats injected with aminochrome and dopachrome. These rats presented a clear motor bias showing a significant contralateral rotation activity, similar but less vigorous that in rats injected with 6-OHDA. The avoidance conditioning was seriously impaired in rats injected with aminochrome and dopachrome although only dopachrome-injected rats showed a similar hypomotility to 6-OHDA-injected rats. The behavioral effects were correlated to the extent of striatal tyrosine hydroxylase (TH)-positive fiber loss. Rats receiving unilateral intranigral aminochrome and dopachrome injections exhibited a 47.9+/-5.1% and a 39.7+/-4.4% reduction in nigrostriatal TH-positive fiber density. In conclusion, this study provided evidence that oxidizing DA and L-DOPA to cytotoxic quinones, aminochrome and dopachrome appears to be an important mediator of oxidative damage in vivo.

Cell lines derived from hippocampal neurons of the normal and trisomy 16 mouse fetus (a model for Down syndrome) exhibit neuronal markers, cholinergic function, and functional neurotransmitter receptors.

We have established hippocampal cell lines from normal and trisomy 16 fetal mice, a model of human trisomy 21. Both cell lines, named H1b (derived from a normal animal) and HTk (trisomic) possess neuronal markers by immunohistochemistry (enolase, synaptophysin, microtubule associated protein-2, and choline acetyltransferase) and lack glial markers (glial fibrillary acidic protein and S-100). Also, we evaluated intracellular Ca(2+) levels ([Ca(2+)](i)) in response to neurotransmitter agonists, in cells loaded with the fluorescent Ca(2+) indicators Indo-1 and Fluo-3. Both cell lines responded to glutamatergic stimuli induced by glutamate, N-methyl-D-aspartate, I-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate. Glutamate responses were only partially prevented by addition of 5 mM EGTA and the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), increased [Ca(2+)](i) in both cell types. These results confirm the presence of glutamatergic metabotropic receptors. In glutamate-induced responses, HTk cells exhibited slower time-dependent decay kinetics than H1b cells. Cholinergic agonists (nicotine and muscarine) induced a rapid, transient increase in [Ca(2+)](i) in both cell types. Furthermore, some cells were sensitive to histamine and norepinephrine. All responses to the aforementioned agonists were prevented by addition of specific antagonists. We also studied incorporation and release of [(3)H]choline in the cells, and observed no differences in uptake parameters. However, release induced by K(+) and nicotine depolarization was greatly reduced in HTk cells. The results show that H1b and HTk cells retain neuronal characteristics and respond to specific neurotransmitter stimuli. The HTk differences could be related to neuronal pathophysiology in Down syndrome.

Angiotensin receptor II is present in dopaminergic cell line of rat substantia nigra and it is down regulated by aminochrome

Angiotensin receptor II mRNA was found to be expressed in dopaminergic neuronal cell line RCSN3 of rat substantia nigra using RT-PCR reaction. Aminochrome (150 μM), a metabolite of the dopamine oxidative pathway, was found to down regulate the expression of angiotensin receptor mRNA in RCSN3 cells by 83% (p < 0.05).

A dorsal root ganglia cell line derived from trisomy 16 fetal mice, a model for Down syndrome

We have established two immortalized cell lines from dorsal root ganglia of normal (G4b) and trisomy 16 mice (GT1), a model for Down syndrome. By immunohistochemistry, both cell lines exhibit neuronal traits and lack glial markers. GTl cells exhibited greater [3H]choline uptake than G4b cells. K+ and nicotine-mediated acetylcholine release was greater in GT1 cells. Basal intracellular Ca2+ concentration ([Ca2+]i) was significantly lower in GTl cells. More GTl cells responded to neurotransmitters with a transient [Ca2+]i increase compared to G4b cells, but both cell types showed similar amplitudes of [Ca2+]i responses. The results show that both cell lines retain neuronal characteristics and respond to specific neurotransmitter stimuli. Altered GT1 cell responses could be related to neuronal pathophysiology in Downs syndrome.

Endosomal abnormalities related to amyloid precursor protein in cholesterol treated cerebral cortex neuronal cells derived from trisomy 16 mice, an animal model of Down syndrome

The CNh and CTb cell lines are derived from the cerebral cortex of normal and trisomy 16 mice, an animal model of human trisomy 21, Down syndrome (DS), and represent in vitro models to study cellular events associated with the human condition. Amyloid precursor protein (APP) plays an important role in the development of neuropathology associated with DS and cholesterol in the amyloidogenic processing of APP. There is also increasing evidence of alterations in the recycling pathway of the early endosome compartment in nervous tissue from DS. In the present study, we report endosomal abnormalities related to amyloid precursor protein in cholesterol-treated CTb cells. Colocalization studies revealed the presence of APP-derived products in early endosomal compartments in both cell lines. Using internalization and immunoprecipitation techniques, differential effects were observed between the normal and trisomic cell lines when treated with cholesterol. Internalization experiments showed that the CTb cell line accumulates internalized APP in intracellular compartments for longer periods of time when compared to the CNh cell line. Immunoprecipitation revealed a differential interaction between the trafficking-related protein Rab4 and APP in the neuronal cell lines CNh and CTb. The present study suggests a putative mechanism by which overexpressed APP accumulates in intracellular compartments related to the endosomal trafficking pathway in individuals with DS, and highlights the usefulness of the CTb cell line as a model to study altered APP metabolism related to this genetic condition.

Apoptosis is directly related to intracellular amyloid accumulation in a cell line derived from the cerebral cortex of a trisomy 16 mouse, an animal model of Down syndrome

Human Down syndrome (DS) represents the most frequent cause of mental retardation associated to a genetic condition. DS also exhibits a characteristic early onset of neuropathology indistinguishable from that observed in Alzheimers disease (AD), namely the deposition of the beta-amyloid peptide. Early endosomal dysfunction has been described in individuals with DS and AD, suggesting an important role of this subcellular compartment in the onset and progression of the pathology. On the other hand, cholesterol activates the amyloidogenic processing pathway for the amyloid precursor protein, and the lipoprotein receptor-related peptide interacts with the beta-amyloid peptide. In the present work, using cell lines derived from the cortex of both normal and trisomy 16 mice (Ts16), an animal model of DS, we showed that the application of exogenous beta-amyloid has cytotoxic effects, expressed in decreased viability and increased apoptosis. Supplementation of the culture media with cholesterol associated to lipoprotein increased cell viability in both cell lines, but apoptosis decreased only in the normal cell line. Further, intracellular beta-amyloid content was elevated in trisomic cells following cholesterol treatment, with higher values in the trisomic cell line. Immunocytochemical detection showed intracellular accumulation of exogenous beta-amyloid in Rab4-positive compartments, which are known to be associated to endosomal recycling. The results suggest that the intracellular beta-amyloid pool plays a central role in apoptosis-mediated cell death in the trisomic condition.