Fetal striatal transplants restore electrophysiological sensitivity to dopamine in the lesioned striatum of rats with experimental Huntington's disease

Dopamine (DA), a major neurotransmitter used in the striatum, is involved in movement disorders such as Parkinson's disease and Huntington's chorea. With the loss of neurons in the striatum of patients with Huntington's disease (HD), there is an associated downregulation of DA receptors, which may alter DA-mediated responses. In the present study, DA-mediated electrophysiological depression was studied in animals with quinolinic acid (QA)-induced experimental HD. QA was directly applied to the right striatum of adult female Sprague-Dawley rats. Animals receiving QA developed ipsilateral rotation after the application of apomorphine. Fetal striatal tissue transplants grafted 1 month after lesioning attenuated apomorphine-induced rotation. Six months after lesioning, the animals were anesthetized with urethane for electrophysiological study. DA, applied directly to neurons by pressure microejection, inhibited spontaneous single-unit activity in the striatal neurons of nonlesioned, lesioned and lesioned/grafted rats. QA lesioning reduced responses to DA in the striatal neurons. The dose of DA required to inhibit striatal neuron activity in the lesioned rats was significantly increased compared to that in the nonlesioned rats. Transplantation of fetal striatal tissue restored the electrophysiological sensitivity to DA in the lesioned striatum. The dose of DA used to suppress striatal neuron activity was reduced after grafting. Immunohistostaining showed survival of gamma-aminobutyric acid neurons at the graft site. Tyrosine hydroxylase-positive terminals were found innervating the striatal grafts. In conclusion, our data demonstrate that fetal striatal transplants restore electrophysiological sensitivity to DA in the lesioned striatum of animals with experimental HD.     

Increase in Kynurenic Acid in Huntington''s Disease Motor Cortex

Huntington's disease is a neurological disorder characterised by a progressive chorea and dementia. Recent evidence has suggested that dysfunction involving endogenous excitatory amino acids may be important in the pathogenesis of this disease. Following the recent demonstration that kynurenic acid is present in the brain, we examined the levels in various areas of brain from patients who died with Huntington's disease and from age/sex-matched controls. Blocks (100-500 mg) of cortex (Brodmann's areas 4 and 10) and caudate nucleus and globus pallidus (lateral and medial parts) were obtained from the Cambridge Brain Bank. The tissue was then processed for the extraction and analysis of kynurenic acid. Whereas no differences in the content of kynurenic acid were observed in the caudate nucleus, lateral or medial globus pallidus, or prefrontal cortex (area 10) between controls' brains and those from patients who died with Huntington's disease, there was a 94% (p less than 0.01; n = 5) increase in the kynurenic acid content in the motor cortex (area 4) from Huntington's disease brains, relative to those of controls. Some time ago we suggested that a subtle change in the relative concentrations of quinolinic and kynurenic acids might be important in the pathogenesis of neurodegeneration. It is possible that the observation of raised kynurenic acid levels supports this supposition. Further work is now in progress to determine whether the change in kynurenic acid is a primary effect or a compensatory response to an increase in excitatory activity.     

Energetic Dysfunction in Quinolinic Acid-Lesioned Rat Striatum

Abstract: Impairment of mitochondrial energy metabolism may contribute to the selective neuronal degeneration observed in Huntington's disease and other neurodegenerative disorders. Intrastriatal injection of the excitotoxin, quinolinic acid, produces a pattern of neuronal death similar to that seen in Huntington's disease. However, little is known about the effects of quinolinic acid on striatal energetics. In the present work, time-dependent changes in energy metabolism caused by injection of quinolinic acid into rat striatum were examined. Oxygen consumption by free and synaptic mitochondria was quantified and correlated with the concentrations of nucleotides and amino acids at different times after injection. Compared with saline-treated controls, a decrease in ADP-stimulated (state 3) to basal (state 4) oxygen consumption (respiratory control ratio) by free mitochondria was apparent in quinolinic acid-injected striata as early as 6 h after treatment. No significant changes were seen in nucleotide concentrations at this time. By 12 h after injection, the decline in the respiratory control ratio was more pronounced (45%), and reductions in ATP, NAD, aspartate, and glutamate (30–60%) were also observed. These results show that injection of quinolinic acid in vivo produces progressive mitochondrial dysfunction, which may be a common and critical event in the cell death cascade initiated in Huntington's disease and in animal models of this neurodegenerative disorder. The indicators of mitochondrial function examined in this study, therefore, may be useful in evaluating the efficacy of neuroprotective agents.     

Aminooxyacetic Acid Results in Excitotoxin Lesions by a Novel Indirect Mechanism

Aminooxyacetic acid (AOAA) is an inhibitor of several pyridoxal phosphate-depedent enzymes in the brain. In the present experiments intrastriatal injections of AOAA produced dose-dependent excitotoxic lesions. The lesions were dependent on a pyridoxal phosphate mechanisms because pyridoxine blocked them. The lesions were blocked by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK-801 and by coinjection of kynurenate, a result indicating an NMDA receptor-mediated excitotoxic process. Electrophysiologic studies showed that AOAA does not directly activate ligand-gated ion channels in cultured cortical or striatal neurons. Pentobarbital anesthesia attenuated the lesions. AOAA injections resulted in significant increases in lactate content and depletions of ATP levels. AOAA striatal lesions closely resemble Huntington's disease both neurochemically and histologically because they show striking sparing of NADPH-diaphorase and large neurons within the lesioned area. AOAA produces excitotoxic lesions by a novel indirect mechanism, which appears to be due to impairment of intracellular energy metabolism, secondary to its ability to block the mitochondrial malate-aspartate shunt. These results raise the possibility that a regional impairment of intracellular energy metabolism may secondarily result in excitotoxic neuronal death in chronic neurodegenerative illnesses, such as Huntington's disease.     

Normal excretion of quinolinic acid in Huntington's disease

We measured the excretion of the endogenous neurotoxin quinolinic acid in 14 patients with Huntington's disease and in 11 age matched control subjects. Huntingtonian patients excreted less quinolinic acid, than controls. When normalised to urea or creatinine output quinolinic acid excretion was normal. We conclude that Huntington's disease is not associated with a generalised disturbance of quinolinic acid metabolism, however, a local hyperproduction of quinolinic acid cannot be excluded from our results.     

Changes in GAD67 mRNA expression evidenced by in situ hybridization in the brain of R6/2 transgenic mice

Huntington's disease is an autosomal dominant disorder with degeneration of medium size striatal neurones. As the disease evolves, other neuronal populations are also progressively affected. A transgenic mouse model of the disease (R6/2) that expresses exon 1 of the human Huntington gene with approximately 150 CAG repeats has been developed, but GABA concentrations are reported to be normal in the striatum of these animals. In the present study, we analysed the status of GABAergic systems by means of glutamic acid decarboxylase (GAD)67 mRNA in situ hybridization in the brain of R6/2 transgenic mice and wild-type littermates. We show that GAD67 expression is normal in the striatum, cerebellum and septum but decreased in the frontal cortex, parietal cortex, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata of R6/2 mice. These data, which may, in part, account for the behavioural changes seen in these animals, indicate that at 12.5 weeks of age the pathological features seen in the mice differ from those seen in humans with Huntington's disease.     

Cross-tolerance of dopamine metabolism to baclofen, γ-butyrolactone and HA-966 in the striatum and olfactory tubercle of the rat

Rats received 7 daily injections with baclofen (40 mg/kg), GBL (750 mg/kg) or HA-966 (100 mg/kg). Dopamine (DA) was measured in the striatum and olfactory tubercle (OT) of rats, sacrificed 0.5 h or 1 h after the last injection. Marked tolerance and cross-tolerance for the DA-elevating effect of these drugs was seen in the striatum, but not in OT. When on day 7 a unilateral lesion of the nigrostriatal pathway was made, also some tolerance to the DA increase in the striatum on the lesioned side was seen in HA-966-pretreated rats, but it was small compared to the tolerance after an additional drug administration in non-lesioned animals. A low dose of apomorphine (0.25 mg/kg, i.p.) had no effect on DA, dihydroxyphenylacetic acid DOPAC) or homovanillic acid (HVA) levels in the lesioned striata, whether the rats had been pretreated for 6 days with HA-966 or not. However, this dose of apomorphine had a significantly more lowering effect on striatal DOPAC and HVA levels on the unlesioned side of HA-966 pretreated rats. The results show that tolerance develops to the increase of DA synthesis, which is possibly receptor-mediated. This tolerance develops more readily in the striatum than in the olfactory tubercle.     

Treating the periphery to ameliorate neurodegenerative diseases

Abnormalities in the kynurenine pathway are associated with neurodegenerative disorders. Zwilling et?al. (2011) show that inhibition of kynurenine 3-monooxygenase in the body's periphery leads to an increase in kyneuric acid, a neuroprotective compound, in the brain. This intervention ameliorates neurodegeneration in mouse models of Alzheimer's disease and Huntington's disease.     

Enzyme Activities in Relation to pH and Lactate in Postmortem Brain in Alzheimer-Type and Other Dementias

Phosphate-activated glutaminase, glutamic acid decarboxylase, pyruvate dehydrogenase, succinic dehydrogenase, pH, and lactate were measured in frontal cortex and caudate nucleus of postmortem brains from cases of Alzheimer-type dementia (ATD), Down's syndrome, Huntington's disease, and one case of Pick's disease, as well as from sudden death and agonal controls. Lactate levels were higher and pH, phosphate-activated glutaminase, and glutamic acid decarboxylase levels were lower in the agonal controls than in the sudden death controls. Phosphate-activated glutaminase and glutamic acid decarboxylase were correlated with tissue pH and lactate, and also were reduced by in vitro acidification, suggesting that the low activities of these enzymes in agonal controls were related to decreased pH consequent upon lactate accumulation. Compared with control tissues at the same pH, phosphate-activated glutaminase and glutamic acid decarboxylase were unaltered in ATD and Down's frontal cortex and reduced in Huntington's caudate nucleus, and glutamic acid decarboxylase was reduced in Huntington's frontal cortex. These data suggest that GABAergic neurons are not affected in ATD and confirm the GABAergic defect in Huntington's disease. Pyruvate dehydrogenase and succinic dehydrogenase activities were the same in agonal controls and sudden death controls and were unaffected by acid pH and lactate in vitro, and pyruvate dehydrogenase was not correlated with pH or lactate. Reduced pyruvate dehydrogenase in frontal cortex of individual ATD, Down's, and Pick's cases, and in the caudate nucleus of Huntington's and Down's cases, was accompanied by gliosis/neuron loss. We conclude that decreased pyruvate dehydrogenase reflects neuronal loss.     

Phosphate-Activated Glutaminase in Relation to Huntington''s Disease and Agonal State

Involvement of phosphate-activated glutaminase in Huntington's disease and agonal state was investigated in caudate nucleus and frontal cortex from postmortem brains. In Huntington's disease the activities of phosphate-activated glutaminase, glutamic acid decarboxylase, succinic dehydrogenase, choline acetyltransferase, and acetylcholinesterase were significantly reduced in the caudate nucleus, but not in the frontal cortex. The activity of phosphate-activated glutaminase, and to a lesser extent of glutamic acid decarboxylase, was reduced in cases of terminal illness, as compared with cases of sudden death. Succinic dehydrogenase and choline acetyltransferase were reduced only in the few cases of prolonged and severe terminal illness. Enzyme activities of the caudate nucleus were more affected by agonal state than were those of frontal cortex. Results indicate that phosphate-activated glutaminase could be a useful marker of neuronal damage due to agonal state, and that phosphate-activated glutaminase and succinic dehydrogenase are reduced in Huntington's disease.     

Enzyme histochemical changes in retinal ganglion cells and the optic nerve after goldfish optic nerve lesion. A regenerative and hypertrophic phenomena study

After sectioning of the goldfish optic nerve a number of enzyme histochemical changes are observed in the hypertrophied retinal ganglion cells and in the optic nerve. Between one and eighteen days postoperatively an increase in the amount of acid phosphatase reaction product is noted. The enhanced activity decreased to normal first in the optic nerve, followed by the optic tract and tectum. Four days postoperatively higher levels of activity were noted in the hypertrophic retinal ganglion cells for the enzymes NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase. The same enzymes also showed an activity increase in the lesioned optic nerve after four to ten days postoperatively, beginning at the cut and gradually spreading towards the optic tectum. Between fifteen and eighteen days the activity dropped to normal in the hypertrophic retinal ganglion cells, while in the lesioned nerve raised levels of reaction products could be seen till days thirty-five and/or forty-five. It was concluded that the degeneration of the optic pathway is marked by the increase of acid phosphatase activity, whereas the process of regeneration is characterized by an increase of NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase activities. The possible functional implications of these enzymes in the regenerative phenomena are discussed.     

Sex and age alter plasma membranes of cultured fibroblasts

Human skin fibroblasts were taken from age-matched male and female subjects. The cells were then cultured under identical conditions and passage-number matched. Plasma membranes were isolated and membrane enzyme activities, lipid composition, and structure of isolated plasma membranes were measured in order to determine the presence of significant sex differences in human fibroblast membrane properties. The results indicated that plasma membranes from normal female subjects had a 1.6-fold and 3.6-fold higher cholesterol/phospholipid ratio and oleic acid (18:2) content than normal male subjects. The limiting anisotropy and the rotational relaxation time of fluorescence probe molecules such as trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in the plasma membranes was not significantly different from fibroblasts of male versus female normal subjects. The total activity of plasma membrane (Na+, K+)-ATPase was significantly higher in female than male normal subjects. A potential 'membrane structural disorder', Huntington's disease, was confirmed in fibroblast membranes from male but not from female Huntington's disease subjects. The possibility that Huntington's disease was a 'premature membrane aging' phenomenon was considered. A comparison of plasma membrane enzymes, lipids, and structure from old and young Huntington's disease subjects did not show differences consistent with accelerated membrane aging as explaining the molecular basis for the disease. The age-dependent differences noted in aged Huntington's disease subjects: increased phosphatidylcholine/phosphatidylethanolamine ratio and sphingomyelin + lysophosphatidylcholine content of fibroblast plasma membranes were not significantly altered when compared to normal age-matched controls. However, (Na+, K+)-ATPase activity was significantly enhanced in fibroblast plasma membranes of older Huntington's disease subjects unlike those of control subjects. In conclusion, sex and age differences in membrane properties of cultured cells represent important potential variables in the elucidation of human genetic disorders that may be membrane-related.     

Alterations in the mouse and human proteome caused by Huntington's disease

Huntington's disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that alpha1-antitrypsin and alphaB-crystalline both decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors alpha1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington's disease patients, alpha1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington's disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.     

Human CSF GABA Concentrations: Revised Dowd for Controls, but Not Decreased in Huntington''s Chorea

gamma-Aminobutyric acid (GABA) concentrations were measured in CSF specimens from two large groups of control subjects, one without neurological or psychiatric disease, and one with a variety of neurological disorders not known to involve altered GABAergic function in brain. CSF GABA was also measured in patients with Huntington's chorea and in patients with other choreiform disorders. GABA was measured in CSF by a modification of the ion exchange-fluorometric method that featured use of a relatively large cation exchange column, and a markedly decreased quantity of sulfosalicylic acid for deproteinization of CSF. Mean BABA concentrations in CSF were 87 and 77 nmol/liter for neurologically normal and abnormal control subjects, 82 nmol/liter for the Huntington's chorea patients, and 105 nmol/liter for patients with other forms of chorea. The mean concentration of homocarnosine was not reduced in CSF of Huntington's chorea patients as compared with controls. Mean CSF GABA concentrations found in control subjects were less than half the lowest control means previously reported. These low values are attributable in part to a reduction in on-column hydrolysis of conjugated forms of GABA in CSF, which can be produced by excessive sulfosalicylic acid, and in part to improved chromatographic resolution of GABA from other unknown o-phthalaldehyde-reactive compounds in CSF. Analysis of free GABA in CSF does not appear useful for diagnosis of suspected Huntington's chorea, nor as a possible predictive test for persons genetically at risk for Huntington's chorea.     

L-dihydroxyphenylalanine-induced asymmetric changes in striatal uric acid peak: determination by in vivo electrochemical detection in rats with unilateral nigrostriatal lesions

Rats were unilaterally lesioned with 6-hydroxydopamine to destroy the nigrostriatal pathway. Following injection with 25 mg/kg L-dopa, circling behavior away from the lesioned side was monitored. Concurrently, in vivo electrochemical detection was used to compare changes in striatal extracellular levels of ascorbic acid (Peak 1) and uric acid (Peak 2) on the lesioned and unlesioned sides. Peak 1 changes did not differ, but Peak 2 changed asymmetrically on the two sides. The difference in the changes in Peak 2 was highly correlated with the circling behavior, with the greater increase on the lesioned side.     

Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington''s Disease

Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50 = 6.1 microM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89-3.75 microM and Vmax values of 33.4-72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2-fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at -20 degrees C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.     

Does tissue transglutaminase play a role in Huntington's disease?

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.     

Huntington's disease: modeling the gait disorder and proposing novel treatments

Huntington's disease is a movement disorder originated from malfunctioning of Basal Ganglia (BG). There are some models for this disease, most of them being conceptual. So, it seems that considering all physiological information and structural specifications to develop a holistic model is needed. We introduce a computational model based on experimental and physiological findings. Parts of the brain known to be involved in Huntington's disease are all considered in our model and most features of the movement disorders have been appeared in the model. This mathematical model has considered the involved parts of the brain in a fairly accurate way, explaining the behavior and mechanism of the disease according to the physiological information. Our model has several advantages. It is able to simulate the normal and Huntington's disease stride time intervals. It shows how the present treatment, i.e. diazepam, is able to ameliorate the gait disorder. In this research we assessed the effects of changing some neurotransmitter levels in order to propose new treatments. Although we showed that gamma amino butyric acid (GABA) blockers reduce Huntington's disease movement disorder, but we discussed that it is unfair to use this route for treatment. We evaluated our model response to increment of GABA, alone and observed that the gait disorder was strengthened. Our novel idea in this regard is resuscitation of BG loop in order to maintain its major physiological functions, and at the same time raising the threshold in order to weaken the internal disturbances. Our last idea about BG treatment is to decrease glutamate. Our model was able to show the effectiveness of this treatment on Huntington's disease disturbances. We propose that experimental studies should be designed in which these two novel methods of treatment will be evaluated. This validation would implement a milestone in treatment of such a debilitating disease at Huntington.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

Huntington's disease: studies on brain free amino acids

We studied the levels of free amino acids in putamen and Brodmann's area 10 of 12 patients who died with Huntington's disease and 13 non-neurologic controls. GABA, glutamate and alpha-amino-n-butyric acid concentrations were found to be reduced in putamen of Huntington's disease patients. In Brodmann's area 10 the levels of glutamate, histidine and lysine were decreased, but the content of aspartate, GABA, glycine, serine and taurine was increased in the same group of patients.