Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma SH-SY5Y cells

Tissue transglutaminase is a normal constituent of the central and peripheral nervous systems and in rats transglutaminase activity in brain and spinal cord is highest during fetal stages when axonal outgrowth is occurring. Further, treatment of human neuroblastoma SH-SY5Y cells with retinoic acid results in the cells withdrawing from the cell cycle and extending neurites, in the same time frame that tissue transglutaminase expression significantly increases. Considering these and other previous findings, this study was carried out to determine whether tissue transglutaminase is involved in neuronal differentiation of SH-SY5Y cells. For these studies SH-SY5Y cells stably overexpressing wild-type tissue transglutaminase, an inactive tissue transglutaminase mutant (C277S) or an antisense tissue transglutaminase construct (which decreased endogenous tissue transglutaminase below detectable levels) were used. SH-SY5Y cells overexpressing wild-type tissue transglutaminase spontaneously differentiated into a neuronal phenotype when grown in low-serum media. In contrast, cells overexpressing inactive tissue transglutaminase or the antisense tissue transglutaminase continued to proliferate and exhibit a flat polygenic morphology even when maintained in low-serum conditions. In addition, increased tissue transglutaminase expression in response to retinoic acid was abolished in the antisense tissue transglutaminase cells, and antisense and mutant tissue transglutaminase expressing cells did not extend neurites in response to retinoic acid. Moreover, wild-type and inactive tissue transglutaminase exhibited differential intracellular localization.These data indicate that tissue transglutaminase is necessary and sufficient for neuronal differentiation of human neuroblastoma SH-SY5Y cells.     

Guanine nucleotide exchange factors for the Rho GTPases: a role in human disease?

 The Rho family of low molecular weight GTP-binding proteins control important aspects of cell shape, adhesion, movement and growth. The DBL-homology (DH) protein family of upstream regulators of Rho GTPases has recently been identified, and deregulated expression of these proteins can have dramatic cellular consequences. This review examines the possible role of DH proteins and Rho GTPases in oncogenesis, metastasis and development. Received: 29 March 1996 / Accepted: 3 June 1996     

Stimulation of neurite outgrowth in a human nerve scaffold designed for peripheral nerve reconstruction

The clinical outcome of microsurgical reconstruction of large peripheral nerve lesions depends on the availability of suitable graft material. Allogenic nerve grafts are rejected by the immune system. Extracellular-matrix proteins, in contrast to the resident cells, are of low immunogenicity in allografts. Here, human tibial nerve segments were extracted with lysophosphatidyl choline. The obtained cell-free and myelin-free scaffold consisted of empty endoneural tubes with maintained extracellular matrix architecture. The nerve scaffold had mechanical properties comparable to intact nerve, making it suitable for microsurgical reconstruction. Sections of the nerve scaffold were tested as a substrate for the adhesion and neuronal differentiation of human neuroblastoma-derived LAN-5 cells. Nerve extraction removed laminin-2, an isoform of laminin important for peripheral nerve regeneration. Laminin-2 reloading of the nerve scaffold did not improve cell adhesion and axon growth. Chemical crosslinking of heparan sulfate, on the other hand, increased the percentage of adherent cells with outgrowing neurites from 48 to 85%. Combined laminin-2 reloading and heparan sulfate crosslinking reduced the percentage of neurite-forming cells to 22% of the number of adherent cells. Implantation of the nerve scaffold into the peritoneal cavity of mice was not cytotoxic, and neovascularization of the graft material was observed within weeks. In conclusion, extraction of human nerve with detergents revealed a biocompatible nerve scaffold supporting neuronal cell adhesion. Heparan sulfate crosslinking to the scaffold surface improved neurite outgrowth, presumably mediated by midkine, a member of the neurokine family of growth factors, which is secreted by neuroblastoma-derived cells and binds to heparan sulfate.     

Aberrant cortical synaptic plasticity and dopaminergic dysfunction in a mouse model of Huntington's disease

Predictive genetic testing for Huntington's disease (HD) has revealed early cognitive deficits in asymptomatic gene carriers, such as altered working memory, executive function and impaired recognition memory. The perirhinal cortex processes aspects of recognition memory and the underlying mechanism is believed to be long-term depression (LTD) of excitatory neurotransmission, the converse of long-term potentiation (LTP). We have used the R6/1 mouse model of HD to assess synaptic plasticity in the perirhinal cortex. We report here a progressive derailment of both LTD and short-term plasticity at perirhinal synapses. Layer II/III neurones gradually lose their ability to support LTD, show early nuclear localization of mutant huntingtin and display a progressive loss of membrane integrity (depolarization and loss of cell capacitance) accompanied by a reduction in the expression of D1 and D2 dopamine receptors visualized in layer I of the perirhinal cortex. Importantly, abnormalities in both short-term and long-term plasticity can be reversed by the introduction of a D2 dopamine receptor agonist (Quinpirole), suggesting that alterations in dopaminergic signalling may underlie early cognitive dysfunction in HD.     

NTera2: a model system to study dopaminergic differentiation of human embryonic stem cells

NTera2, a human embryonal carcinoma (EC) stem cell line, shares many characteristics with human embryonic stem cells (hESCs). To determine whether NTera2 can serve as a useful surrogate for hESCs, we compared global gene expression between undifferentiated NTera2, multiple undifferentiated hESC cell lines, and their differentiated derivatives, and we showed that NTera2 cells share multiple markers with hESCs. Similar to hESCs, NTera2 cells differentiated into TH-positive cells that express dopaminergic markers including AADC, DAT, Nurr1, TrkB, TrkC, and GFRA1 when co-cultured with PA6 cells. Flow cytometry analysis showed that tyrosine hydroxylase (TH) and neural cell adhesion molecule (NCAM) expression increased, whereas SSEA4 expression decreased as cells differentiated. Medium conditioned by PA6 cells stimulated differentiation of NTera2 cells to generate TH-positive cells that expressed dopaminergic markers. Flow cytometry selected polysialylated (PSA-NCAM) cells responded to medium conditioned by PA6 cells by differentiating into TH-positive cells and expressed dopaminergic markers. Sorted cells differentiated for 4 weeks in PA6 cell conditioned media included functional neurons that responded to neurotransmitters and exhibited electronic excitability. Therefore, NTera2 cell dopaminergic neuronal differentiation and PSA-NCAM enrichment provides a useful system for the future study of hESCs.     

Basal ganglia efferents to the brainstem centers controlling postural muscle tone and locomotion: a new concept for understanding motor disorders in basal ganglia dysfunction

The present study is designed to elucidate how basal ganglia afferents from the substantia nigra pars reticulata (SNr) to the mesopontine tegmental area of the brainstem contribute to gait control and muscle-tone regulation. We used unanesthetized and acutely decerebrated cats (n=27) in which the striatum, thalamus and cerebral cortex were removed but the SNr was preserved. Repetitive stimulation (50 Hz, 10-60 microA, for 5-20 s) applied to a mesencephalic locomotor region (MLR), which corresponded to the cuneiform nucleus, and adjacent areas, evoked locomotor movements. On the other hand, stimulation of a muscle-tone inhibitory region in the pedunculopontine tegmental nucleus (PPN) suppressed postural muscle tone. An injection of either glutamatergic agonists (N-methyl-D-aspartic acid and kainic acid) or GABA antagonists (bicuculline and picrotoxin) into the MLR and PPN also induced locomotion and muscle-tone suppression, respectively. Repetitive electrical stimuli (50-100 Hz, 20-60 microA for 5-20 s) delivered to the SNr alone did not alter muscular activity. However stimulating the lateral part of the SNr attenuated and blocked PPN-induced muscle-tone suppression. Moreover, weaker stimulation of the medial part of the SNr reduced the number of step cycles and disturbed the rhythmic alternation of limb movements of MLR-induced locomotion. The onset of locomotion was delayed as the stimulus intensity was increased. At a higher strength SNr stimulation abolished the locomotion. An injection of bicuculline into either the PPN or the MLR diminished the SNr effects noted above. These results suggest that locomotion and postural muscle tone are subject to modulation by GABAergic nigrotegmental projections which have a partial functional topography: a lateral and medial SNr, for regulation of postural muscle tone and locomotion, respectively. We conclude that disorders of the basal ganglia may include dysfunction of the nigrotegmental (basal ganglia-brainstem) systems, which consequently leads to the production of abnormal muscle tone and gait disturbance.     

Adenosine A2 receptors: selective localization in the human basal ganglia and alterations with disease.

Adenosine A2 receptors were labeled and visualized by autoradiography in tissue sections of the human brain using the A2-selective agonist ligand [3H](2-p-(2-carboxyethyl)phenylamino)-5'-N-carboxamidoadenosine (CGS 21680). The binding of this ligand was of high affinity, reversible, and was blocked by adenosine A2 agents. Autoradiographic mapping of adenosine A2 sites revealed them to be exclusively restricted to the caudate nucleus, putamen, nucleus accumbens, olfactory tubercle and the lateral segment of the globus pallidus. The densities of adenosine A2 receptors in other brain areas did not differ from background levels. This selective localization prompted us to study the consequences of neurodegenerative diseases such as Parkinson's disease and Huntington's chorea on the densities and localization of these sites in the basal ganglia. In Parkinson's disease the density of adenosine A2 binding sites was comparable to that seen in control cases. In contrast, density values of A2 sites were dramatically decreased, compared to control values, in the basal ganglia of patients with Huntington's chorea. Similar losses of A2 receptors were observed in the guinea-pig striatum after local application of quinolinic acid while lesioning of the dopaminergic neurons was without effect. All these results taken together suggest that adenosine A2 receptors are localized on striatal output neurons which degenerate in Huntington's chorea.     

Multiple benzodiazepine receptors in the human basal ganglia: a detailed pharmacological and anatomical study

The pharmacological characteristics and anatomical distribution of benzodiazepine receptors in the striatum (dorsal striatum, comprising the caudate nucleus and putamen, and ventral striatum) and globus pallidus (dorsal pallidum, comprising the external and internal segments, and ventral pallidum) of the human basal ganglia were examined in twelve cases aged 4-71 years. The pharmacology of the receptors was studied using computerized, non-linear least-squares regression analysis of [3H]flunitrazepam displacement by flunitrazepam, CL218,872 and ethyl beta-carboline-3-carboxylate binding to membranes. The results showed that the dorsal striatum (caudate nucleus and putamen) contained higher concentrations of receptors than the dorsal pallidum (external and internal segments). The dorsal striatum contained equal numbers of sites with high affinity (Type I) and low affinity (Type II) for CL218,872 and ethyl beta-carboline-3-carboxylate whereas the globus pallidus contained sites with only high affinity (Type I) for these ligands. The anatomical localization of the benzodiazepine receptor subtypes (Type I and II) was studied using quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam in the absence or presence of the discriminating ligand CL218,872. The autoradiograms showed that benzodiazepine receptors were distributed throughout all regions of the human striatum in a heterogeneous fashion, i.e. high-density patches of receptors were set against a background matrix of lower receptor densities. The highest densities of receptors were seen in the ventral striatum where the patches were particularly extensive and showed densities 56% higher than the receptor densities in the dorsal striatal patches. Quantitative analysis showed that the patches in all striatal regions contained mainly Type II receptors (83%-86%) whereas the matrix regions in the ventral and dorsal striatum contained different proportions of the receptor subtypes; Type I receptors predominated (60%) in the matrix of the ventral striatum and Type II receptors predominated (67%-71%) in the matrix of the dorsal striatum. By contrast, the autoradiograms showed that the globus pallidus contained considerably lower concentrations of receptors than the striatum. The highest density of receptors in the globus pallidus was present in the ventral pallidum with successively lower concentrations in the external (26% less) and internal (66% less) segments of the dorsal pallidum. In agreement with the membrane binding studies the receptors in the globus pallidus were mainly of the Type I variety.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue-specific effects of wild-type and mutant connexin 31: a role in neurite outgrowth

Channels formed by connexins (Cx), the major protein subunits of gap junctions, allow passage of ions and molecular messengers between cells to provide a mechanism of synchronized cellular response. Twenty human Cx isoforms have been identified and mutations in the gene GJB3 encoding the 31 kDa isoform, Cx31, can cause dominant or recessive skin disease, dominant or recessive deafness or dominant neuropathy with deafness. Cx31 is expressed in differentiating keratinocytes in skin. Here, we also demonstrate endogenous Cx31 expression in human neuronal cell lines, particularly in differentiated neurones. Exogenous Cx31 expression induced neurite outgrowth in human neuronal cell lines, but not differentiation in primary human keratinocytes. Though neither the neuropathy and hearing loss mutation (66delD)Cx31 nor the skin disease associated mutation (R42P)Cx31 is able to traffic to the plasma membrane, the R42P mutant induced neurite outgrowth to a level equal to wild-type Cx31. In contrast, there was significantly reduced neurite outgrowth after (66delD)Cx31 expression. In addition to indicating a potential disease mechanism for the neuropathy/deafness mutation, this work demonstrates a tissue-specific function for Cx31.     

Implantation of c-mycER TAM immortalized human mesencephalic-derived clonal cell lines ameliorates behavior dysfunction in a rat model of Parkinson's disease

Human neural stem cells offer the hope that a cell therapy treatment for Parkinson's disease (PD) could be made widely available. In this study, we describe two clonal human neural cell lines, derived from two different 10-week-old fetal mesencephalic tissues and immortalized with the c-mycER(TAM) transgene. Under the growth control of 4-hydroxytamoxifen, both cell lines display stable long-term growth in culture with a normal karyotype. In vitro, these nestin-positive cells are able to differentiate into tyrosine hydroxylase (TH)-positive neurons and are multipotential. Implantation of the undifferentiated cells into the 6-OHDA substantia nigral lesioned rat model displayed sustained improvements in a number of behavioral tests compared with noncell-implanted, vehicle-injected controls over the course of 6 months. Histological analysis of the brains showed survival of the implanted cells but no evidence of differentiation into TH-positive neurons. An average increase of approximately 26% in host TH immunoreactivity in the lesioned dorsal striatum was observed in the cell-treated groups compared to controls, with no difference in loss of TH cell bodies in the lesioned substantia nigra. Further analysis of the cell lines identified a number of expressed trophic factors, providing a plausible explanation for the effects observed in vivo. The exact mechanisms by which the implanted human neural cell lines provide behavioral improvements in the PD model are not completely understood; however, these findings provide evidence that cell therapy can be a potent treatment for PD acting through a mechanism independent of dopaminergic neuronal cell replacement.     

Survival motor neuron (SMN) protein: role in neurite outgrowth and neuromuscular maturation during neuronal differentiation and development

Childhood spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by absent or deficient full-length survival motor neuron (SMN) protein. Clinical studies and animal models suggest that SMA is a developmental defect in neuromuscular interaction; however, the role of SMN in this process remains unclear. In the present study, we have determined the subcellular localization of SMN during retinoic-acid-induced neuronal differentiation of mouse embryonal teratocarcinoma P19 cells as well as in skeletal muscle during the critical period of neuromuscular maturation. We demonstrate, for the first time, SMN accumulation in growth-cone- and filopodia-like structures in both neuronal- and glial-like cells, identifying SMN as a new growth cone marker. Indeed, SMN was present at the leading edge of neurite outgrowths, suggesting that SMN may play a role in this process. In addition, SMN was detected as small dot-like particles within the cytoplasm of skeletal muscle during the first 2 weeks after birth, but their number peaked by P6. Intense SMN staining in neuromuscular junctions was observed throughout the entire postnatal period examined. Taken together, these results suggest that SMN may indeed fulfill neuronal- and muscle-specific functions, providing a more plausible mechanism explaining motor neuron degeneration and associated denervation atrophy of skeletal muscles in SMA. The primary SMA pathology most likely initiates in the peripheral axon--the result of deficient neurite outgrowth and/or neuromuscular maturation.     

Advantages and limitations of the equine disease, pituitary pars intermedia dysfunction as a model of spontaneous dopaminergic neurodegenerative disease

Parkinson's disease (PD) is a leading cause of neurologic disability in the aged population. Remarkable progress has been made in the past decade to understand the cellular and molecular events that occur in PD. However attempts to unravel the early, initiating factors in the pathogenesis of dopaminergic neurodegeneration and PD have been limited by the lack of a suitable animal model. Models in which there has been genetic or environmental manipulation are not of use in determining the natural cause of a disease. While a large scale prospective human study would be ideal, the relatively low prevalence of PD makes this approach economically and logistically infeasible. Equine pituitary pars intermedia dysfunction (PPID) is a spontaneous, progressive neuroendocrine disease that commonly affects aged horses and ponies. PPID results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. PPID is 10-20 times more prevalent than PD and may be readily diagnosed without a need for advanced imaging technology. Although the diseases are anatomically distinct, recent evidence suggests the pathogenesis of dopaminergic neuronal damage in PPID may have significant similarities to that of PD. In this review, the similarities and differences in the pathology of neurodegeneration in PPID and PD are compared. The potential utility of the horse as a model of spontaneous dopaminergic neurodegeneration is discussed.     

Primate basal ganglia activity in a precued reaching task: preparation for movement

Single cell activity was recorded from the primate putamen, caudate nucleus, and globus pallidus during a precued reaching movement task. Two monkeys were trained to touch one of several target knobs mounted in front of them after an LED was lighted on the correct target. A precue was presented prior to this target go cue by a randomly varied delay interval, giving the animals partial or complete advance information about the target for the movement task. The purpose of this design was to examine neuronal activity in the major structures of the basal ganglia during the preparation phase of limb movements when varying amounts of advance information were provided to the animals. The reaction times were shortest with complete precues, intermediate with partial precues, and longest with precues containing no information, demonstrating that the animals used precue information to prepare partly or completely for the reaching movement before the target go cue was given. Changes in activity were seen in the basal ganglia during the preparatory period in 30% of neurons in putamen, 31% in caudate nucleus, and 27% in globus pallidus. Preparatory changes were stronger and more closely linked to the time of movement initiation in putamen than in caudate nucleus. Although the amount of information contained in the precues had no significant effect on preparatory activity preceding the target go cue, a directional selectivity during this period was observed for a subset of neurons with preparatory changes (15% in putamen, 11% in caudate nucleus, 14% in globus pallidus) when the precue contained information about the upcoming direction of movement. A smaller subset of neurons showed selectivity for the preparation of movement amplitude. A larger number of preparatory changes showed selectivity for the direction or amplitude of movement following the target go cue than in the delay period before the cue. The intensity of preparatory changes in activity in many cases depended on the length of the delay interval preceding the target go cue. Even following the target go cue, the intensity of the preparatory changes in activity continued to be significantly influenced by the length of the preceding delay interval for 11% of changes in putamen, 8% in caudate nucleus, and 18% in globus pallidus. This finding suggests that preparatory activity in the basal ganglia takes part in a process termed motor readiness. Behaviorally, this process was seen as a shortening of reaction time regardless of precue information for trials in which the delay interval was long and the animals showed an increased readiness to move. Preparatory activity in putamen following the target go cue was most intense in trials with a short delay interval, in which motor readiness had not achieved its maximum level prior to the go cue. The results of this study indicate that the basal ganglia are involved in multiple aspects of preparatory processing for limb movement. Preparatory processing is therefore unlikely to be divided anatomically along the functional lines examined in this study. In the basal ganglia, preparatory processing reflects both preparation for target selection and control of timing the onset of movement (motor readiness). These characteristics can be integrated in a functional scheme in which the basal ganglia are predominantly responsible for the automated execution of well-trained behavior.     

Synaptic plasticity in the basal ganglia: a similar code for physiological and pathological conditions

It is widely accepted that the complexity and adaptability of neuronal communication, which is necessary for integrative and higher functions of the brain, is amply represented by plastic changes occurring at synaptic level. Therefore, long-term modifications of synaptic efficacy between neurons have been considered the cellular basis of learning and memory. Accordingly, there is a plethora of experimental evidence supporting this contention. Indeed, synaptic modifications in the hippocampus, the cerebral and cerebellar cortices regulate composite neuronal functions such those related to cognition, awareness, memory storage, and motion.

In recent years, the concept that enduring changes of excitatory glutamatergic synaptic potentials [long-term potentiation (LTP) and long-term depression (LTD)] are not limited to the hippocampus and cortices but occur also in other brain areas has emerged. For instance, plasticity at different excitatory pathways has been clearly demonstrated in the basal ganglia.

Here we present an overview of the experimental data regarding synaptic plasticity in the basal ganglia and highlight how results reported in the literature are often contradictory, especially when compared to those obtained in the hippocampal area. In trying to propose possible explanations to some of these contradictions, we present a holistic approach that re-interprets the basal ganglia synaptic plasticity in terms of expression of physiological and pathological phenomena and therapeutic effects of drugs.     

Neurotensin receptors in Parkinson's disease and progressive supranuclear palsy: an autoradiographic study in basal ganglia

The technique of receptor autoradiography was used to study the distribution of neurotensin receptors in post mortem brain tissues from patients affected by Parkinson's disease, progressive supranuclear palsy and from age-matched controls. [125I]Neurotensin was used as ligand. Significant receptor decreases were found in the substantia nigra, both pars compacta and reticulata, and in the putamen in Parkinson's disease and progressive supranuclear palsy. In addition, significant decreases of neurotensin receptors were found in the ventral tegmental area, nucleus accumbens and dorsal part of caudate head in patients with Parkinson's disease but not in patients with progressive supranuclear palsy, indicating differential involvement of neurotensin receptors in these two neurological disorders. In addition, both in Parkinson's disease and progressive supranuclear palsy the decrement of striatal neurotensin binding sites was less than expected from the reported decrease of dopamine content in this nucleus, suggesting only partial localization of neurotensin receptors on mesostriatal dopaminergic projections.     

Integrating glutathione metabolism and mitochondrial dysfunction with implications for Parkinson's disease: a dynamic model

Oxidative/nitrosative stress and mitochondrial dysfunction have been implicated in the degeneration of dopaminergic neurons in the substantia nigra during Parkinson's disease (PD). During early stages of PD, there is a significant depletion of the thiol antioxidant glutathione (GSH), which may lead to oxidative stress, mitochondrial dysfunction, and ultimately neuronal cell death. Mitochondrial complex I (CI) is believed to be the central player to the mitochondrial dysfunction occurring in PD. We have generated a dynamic, mechanistic model for mitochondrial dysfunction associated with PD progression that is activated by rotenone, GSH depletion, increased nitric oxide and peroxynitrite. The potential insults independently inhibit CI and other complexes of the electron transport chain, drop the proton motive force, and reduce ATP production, ultimately affecting the overall mitochondrial performance. We show that mitochondrial dysfunction significantly affects glutathione synthesis thereby increasing the oxidative damage and further exacerbating the toxicities of these mitochondrial agents resulting in neurodegeneration. Rat dopaminergic neuronal cell culture and in vitro experiments using mouse brain mitochondria were employed to validate important features of the model. MAJOR CONCLUSIONS: Using a combination of experimental and in silico modeling approaches, we have demonstrated the interdependence of mitochondrial function with GSH metabolism in relation to neurodegeneration in PD.     

The pattern of neurodegeneration in Huntington's disease: a comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington's disease

In order to investigate the sequence and pattern of neurodegeneration in Huntington's disease, the distribution and density of cannabinoid CB(1), dopamine D(1) and D(2), adenosine A(2a) and GABA(A) receptor changes were studied in the basal ganglia in early (grade 0), intermediate (grades 1, 2) and advanced (grade 3) neuropathological grades of Huntington's disease. The results showed a sequential pattern of receptor changes in the basal ganglia with increasing neuropathological grades of Huntington's disease. First, the very early stages of the disease (grade 0) were characterized by a major loss of cannabinoid CB(1), dopamine D(2) and adenosine A(2a) receptor binding in the caudate nucleus, putamen and globus pallidus externus and an increase in GABA(A) receptor binding in the globus pallidus externus. Second, intermediate neuropathological grades (grades 1, 2) showed a further marked decrease of CB(1) receptor binding in the caudate nucleus and putamen; this was associated with a loss of D(1) receptors in the caudate nucleus and putamen and a loss of both CB(1) and D(1) receptors in the substantia nigra. Finally, advanced grades of Huntington's disease showed an almost total loss of CB(1) receptors and the further depletion of D(1) receptors in the caudate nucleus, putamen and globus pallidus internus, and an increase in GABA(A) receptor binding in the globus pallidus internus.These findings suggest that there is a sequential but overlapping pattern of neurodegeneration of GABAergic striatal efferent projection neurons in increasing neuropathological grades of Huntington's disease. First, GABA/enkephalin striatopallidal neurons projecting to the globus pallidus externus are affected in the very early grades of the disease. Second, GABA/substance P striatonigral neurons projecting to the substantia nigra are involved at intermediate neuropathological grades. Finally, GABA/substance P striatopallidal neurons projecting to the globus pallidus internus are affected in the late grades of the disease. In addition, the finding that cannabinoid receptors are dramatically reduced in all regions of the basal ganglia in advance of other receptor changes in Huntington's disease suggests a possible role for cannabinoids in the progression of neurodegeneration in Huntington's disease.     

Serotonin-1 receptor binding sites in the human basal ganglia are decreased in Huntington's chorea but not in Parkinson's disease: a quantitative in vitro autoradiography study

Serotonin-1 receptors were examined in post-mortem human brains, using quantitative in vitro autoradiography. [3H]Serotonin was used as a ligand. Serotonin-1 receptor subtypes were defined with 8-hydroxy-2-(di-n-propylamino)-tetralin and mesulergine. In the control human basal ganglia, the highest density of serotonin-1 binding sites was observed in both lateral and medial globus pallidus and substantia nigra reticulata. Lower densities were seen in the substantia nigra pars compacta, the nucleus accumbens, caudate and putamen. The majority of these serotonin-1 sites belonged to the serotonin-1D class. No significant alteration of the density and distribution of these sites was observed in Parkinson's disease brains. In contrast, a marked decrease in the density of the receptor binding was seen in the basal ganglia and the substantia nigra from patients dying with Huntington's disease. These results suggest that serotonin-1D receptors are expressed by cells intrinsic to the striatum which degenerate in Huntington's disease and project to the substantia nigra reticulata where these receptors are probably presynaptically localized. These observations in pathological human brains agree with the results of lesion studies in animal models and further support a role for serotoninergic mechanisms in movement control.     

Glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex I subunits: implications for Parkinson's disease

Oxidative stress and mitochondrial dysfunction signify two important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models and in affected tissues from the disease itself have demonstrated a selective inhibition of mitochondrial complex I activity that appears to affect normal mitochondrial physiology leading to neuronal cell death. Earlier experiments from our laboratory have demonstrated that induced depletion of glutathione (GSH + GSSG) in cultured dopaminergic cells resulted in increased oxidative stress and a decrease in mitochondrial function. Furthermore, this dysfunction was linked to a selective decrease in mitochondrial complex I activity that appears to be due to oxidation of this complex. Glutathione depletion is the earliest detectable biochemical event during PD progression and occurs prior to complex I inhibition. Recent observations have also indicated that oxidative damage to complex I via naturally occurring free radicals such as peroxynitrite leads to modification of tyrosine and/or cysteine residues resulting in complex I inhibition. Using the sucrose gradient method, we detected in complex I-enriched fractions from a glutathione-depleted dopaminergic cell line two bands corresponding to approximately 25-kDa and approximately 30-kDa polypeptides that demonstrate anti-nitrotyrosine immunoreactivity, suggesting the possible involvement of protein nitration by peroxynitrite in glutathione depletion-mediated complex I inhibition.