Normal hypocretin-1 (orexin-A) levels in the cerebrospinal fluid of patients with Huntington's disease

A significant atrophy and loss of hypocretin neurons in the brains of human patients with Huntington's disease (HD) and in R6/2 mice have been reported. We included 10 patients with HD and 12 patients with chorea-like hyperkinetic movement disorders (non-HD). All patients of the HD group and eleven patients of the non-HD group showed normal hypocretin-1 levels. Thus, hypocretin-1 may not serve as an additional diagnostic marker for HD.     

Late onset neurodegeneration in the Cln3-/- mouse model of juvenile neuronal ceroid lipofuscinosis is preceded by low level glial activation

Mouse models of neuronal ceroid lipofuscinosis (NCL) exhibit many features of the human disorder, with widespread regional atrophy and significant loss of GABAergic interneurons in the hippocampus and neocortex. Reactive gliosis is a characteristic of all forms of NCL, but it is unclear whether glial activation precedes or is triggered by neuronal loss. To explore this issue we undertook detailed morphological characterization of the Cln3 null mutant (Cln3(-/-)) mouse model of juvenile NCL (JNCL) that revealed a delayed onset neurodegenerative phenotype with no significant regional atrophy, but with widespread loss of hippocampal interneurons that was first evident at 14 months of age. Quantitative image analysis demonstrated upregulation of markers of astrocytic and microglial activation in presymptomatic Cln3(-/-) mice at 5 months of age, many months before significant neuronal loss occurs. These data provide evidence for subtle glial responses early in JNCL pathogenesis.     

Effects of the Ca2+ antagonist nimodipine on functional deficits in the peripheral and central nervous system of streptozotocin-diabetic rats

Diabetes mellitus can lead to functional and structural deficits in both the peripheral and central nervous system. The pathogenesis of these deficits is multifactorial, probably involving, among others, microvascular dysfunction and alterations in intracellular calcium homeostasis. The present study examined the effects of treatment with the Ca2+ antagonist nimodipine (20 mg/kg, intraperitoneal injection, every 48 h) on functional deficits in the peripheral and central nervous system in streptozotocin-diabetic rats. In a prevention experiment, treatment was initiated immediately after diabetes induction and continued for 10 weeks. In a reversal experiment, treatment was initiated 16 weeks after diabetes induction and continued for 12 weeks. Sciatic nerve motor and sensory conduction velocity, brainstem auditory-evoked potentials, and visual-evoked potentials were measured in control, untreated, and nimodipine-treated diabetic rats. In addition, long-term potentiation, a form of synaptic plasticity used as a model for learning and memory at the cellular level, was examined in hippocampal slices. Nimodipine treatment partially prevented deficits in nerve conduction velocity and hippocampal long-term potentiation in diabetic rats. However, nimodipine intervention treatment was unable to reverse established deficits in nerve conduction velocity, evoked potential latencies, or long-term potentiation. It is concluded that nimodipine can partially prevent early functional deficits in the peripheral and central nervous system of streptozotocin-diabetic rats but is unable to reverse late deficits.     

Aspartoacylase deficiency does not affect N-acetylaspartylglutamate level or glutamate carboxypeptidase II activity in the knockout mouse brain

Aspartoacylase (ASPA)-deficient patients [Canavan disease (CD)] reportedly have increased urinary excretion of N-acetylaspartylglutamate (NAAG), a neuropeptide abundant in the brain. Whether elevated excretion of urinary NAAG is due to ASPA deficiency, resulting in an abnormal level of brain NAAG, is examined using ASPA-deficient mouse brain. The level of NAAG in the knockout mouse brain was similar to that in the wild type. The NAAG hydrolyzing enzyme, glutamate carboxypeptidase II (GCP II), activity was normal in the knockout mouse brain. These data suggest that ASPA deficiency does not affect the NAAG or GCP II level in the knockout mouse brain, if documented also in patients with CD.     

Alterations in neurofilament light in optic nerve in rat kainate and monkey ocular hypertension models

The purpose of this study is to evaluate the changes in neurofilament light (NF-L) protein in the optic nerve in rat kainate and monkey ocular hypertension models. In the rat model, optic nerve damage was induced by kainate injection into the vitreous body. In the monkey model, photocoagulation of the trabecular meshwork led to laser-induced ocular hypertension. NF-L in optic nerve extract was quantified by quantitative immunoblot using an imaging analyzer. The amount of NF-L in optic nerve was compared between normal and kainate-treated groups at 7 days after kainate injection. Specimens from rat optic nerves and retinas were evaluated histologically to examine the correlations between damage and amount of NF-L in the optic nerve. In monkeys, the amount of NF-L in the optic nerve was compared between control fellow and ocular hypertensive eyes. Injection of kainate induced morphological optic nerve and retinal damage in rats. The amount of NF-L in the optic nerve was significantly reduced in kainate-treated eyes (vs. normal eyes). The amount of NF-L correlated with the cell count in the ganglion cell layer and the axon number in the optic nerve at 7 days after kainate injection. Further, 6-cyano-7-nitro-quinoxaline-2,3-dione, a non N-methyl-D-aspartate receptor antagonist, suppressed the kainate-induced reduction in NF-L and retinal damage. In the monkey model, ocular hypertension and morphological optic nerve damage were shown by laser-treated eyes. The amount of NF-L in the optic nerve was reduced in laser-treated eyes. In conclusion, in rat kainate and monkey ocular hypertension models, immunoblot evaluation of NF-L shows the reduction of the amount of NF-L in the optic nerves of treated-eyes. The amount of NF-L correlated with the morphological retinal and optic nerve damage in rats. These findings indicate that immunoblot evaluation of NF-L in the optic nerve may provide a quantitative index of optic nerve damage.     

Experimental allergic encephalomyelitis is exacerbated in mice deficient for 12/15-lipoxygenase or 5-lipoxygenase

12/15-Lipoxygenase (12/15-LO) produces 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) which are agonists for peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma agonists reduce clinical severity of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. In contrast, 5-lipoxygenase (5-LO) produces the generally proinflammatory leukotrienes (LTs) which would be expected to worsen EAE. We tested the hypotheses that EAE severity would be exacerbated in 12/15-LO-deficient mice and attenuated in 5-LO-deficient mice. 12/15-LO deficiency conferred a significantly worse disease course, and surprisingly, 5-LO deficiency also caused significantly more severe EAE compared to control mice. These data suggest that PPARgamma-regulated gene expression and that 5-LO production of certain LTs have the ability to diminish EAE. Continued analysis will provide insight into the endogenous LO-generated effectors that assist in tempering EAE.     

Expression of PrP(C) in the rat brain and characterization of a subset of cortical neurons

The cellular prion protein (PrP(C)) is a membrane-bound glycoprotein mainly present in the CNS. The scrapie prion protein (PrP(Sc)) is an isoform of PrP(C), and it is responsible for transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases affecting both humans and animals. The presence of the cellular form is necessary for the establishment and further evolution of prion diseases. Here, we map the regional distribution of PrP(C) in the rat brain and study the chemical nature of these immunopositive neurons. Our observations are congruent with retrograde transport of prions, as shown by the ubiquitous distribution of PrP(C) throughout the rat brain, but especially in the damaged areas that send projections to primarily affected nuclei in fatal familial insomnia. On the other hand, the presence of the cellular isoform in a subset of GABAergic neurons containing calcium-binding proteins suggests that PrP(C) plays a role in the metabolism of calcium. The lack of immunostaining in neurons ensheathed by perineuronal nets indicates that prions do not directly interact with components of these nets. The destruction of these nets is more likely to be the consequence of a factor needed for prions during the early stages of TSEs. This would cause destruction of these nets and death of the surrounded neurons. Our results support the view that destruction of this extracellular matrix is caused by the pathogenic effect of prions and not a primary event in TSEs.     

Proteomic identification of proteins oxidized by Abeta(1-42) in synaptosomes: implications for Alzheimer's disease

Protein oxidation has been implicated in Alzheimer's disease (AD) and can lead to loss of protein function, abnormal protein turnover, interference with cell cycle, imbalance of cellular redox potential, and eventually cell death. Recent proteomics work in our laboratory has identified specifically oxidized proteins in AD brain such as: creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, alpha-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, excitotoxicity and/or synaptic plasticity, protein turnover, and neuronal communication. Synapse loss is known to be an early pathological event in AD, and incubation of synaptosomes with amyloid beta peptide 1-42 (Abeta 1-42) leads to the formation of protein carbonyls. In order to test the involvement of Abeta(1-42) in the oxidation of proteins in AD brain, we utilized two-dimensional gel electrophoresis, immunochemical detection of protein carbonyls, and mass spectrometry to identify proteins from synaptosomes isolated from Mongolian gerbils. Abeta(1-42) treatment leads to oxidatively modified proteins, consistent with the notion that Abeta(1-42)-induced oxidative stress plays an important role in neurodegeneration in AD brain. In this study, we identified beta-actin, glial fibrillary acidic protein, and dihydropyrimidinase-related protein-2 as significantly oxidized in synaptosomes treated with Abeta(1-42). Additionally, H+-transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, gamma-actin, and elongation factor Tu were identified as increasingly carbonylated. These results are discussed with respect to their potential involvement in the pathogenesis of AD.     

Protective effect of donepezil against Abeta(1-40) neurotoxicity in rat septal neurons

Donepezil, a potent acetylcholinesterase (AChE) inhibitor used for the treatment of Alzheimer's disease (AD), is thought to have a neuroprotective effect in AD patients. Because a deficit in cholinergic neurotransmission is a major feature in AD, and amyloid-beta (Abeta) accumulation has been proposed as a possible causative phenomenon, we were interested to examine the effect of donepezil on Abeta(1-40) induced neurotoxicity in primary cultures of rat septal neurons. Using immunohistochemical staining, almost all the neurons were found to be positive for vesicular acetylcholine transporter (VAChT) in these septal cultures. Septal neuronal cells were cultured for 7 days and then 15 micromol/L of Abeta(1-40) was added to the cell medium for 48 h. The cultured septal neurons were highly susceptible to Abeta toxicity, as shown by morphological examination and lactate dehydrogenase (LDH) assay. Donepezil concentration-dependently reduced the LDH efflux induced by Abeta(1-40), and the effect was significant at 100 nmol/L and above. Donepezil decreased both the negative peak at around 215 nm in the circular dichroism (CD) spectrum and the fluorescence intensity of thioflavin T in the presence of Abeta(1-40). These results suggest that donepezil exerts a neuroprotective effect by reducing the amount of the toxic form of Abeta fibrils in septal neuron cultures. These findings support the idea that the clinical efficacy of donepezil in AD is due to not only activation of cholinergic transmission, but also attenuation of neuronal damage.     

Altered distribution of striatal activity-dependent synaptic plasticity in the 3-nitropropionic acid model of Huntington's disease

Huntington's disease (HD) is a neurodegenerative disorder characterized by involuntary choreiform movements, neuropsychiatric disturbances and cognitive decline. The hyperkinetic phenomenology has commonly been attributed to a disturbance of the basal ganglia function, mainly the neostriatum, but its pathophysiology mechanisms remain unclear. Activity-dependent long-term changes in synaptic efficacy, such as long-term potentiation (LTP) and long-term depression (LTD), are widely considered to be the cellular models for acquisition and storage of information in neuronal networks. Both LTP and LTD have been described at the corticostriatal pathway and they might be probably involved not only in physiological motor behavior processing but also in disease states affecting that pathway. Systemic injection of 3-nitropropionic acid (3-NP) induces excitotoxic striatal lesions and abnormal movements in rodents, resembling those seen in HD. We examined synaptic plasticity in dorsolateral striatum slices prepared from both control and 3-NP-treated rats by recording extracellular field potentials. Our results reinforce the idea that both forms of activity-dependent synaptic plasticity can be recorded at the dorsolateral region of striatum by the same stimulating protocol in control rats and suggest that 3-NP-induced striatal lesions may be associated with suppression of LTD expression in the sensorimotor striatum.     

Failure of FK506 (tacrolimus) to alleviate apomorphine-induced circling in rat Parkinson model in spite of some cytoprotective effects in SH-SY5Y dopaminergic cells

The mechanism of action of the neurotoxin 6-hydroxydopamine (6-OHDA) is thought to involve the generation of free radicals and subsequent apoptotic processes. We have demonstrated in vitro that the neuroimmunophilin, FK506 (10-100 nM), dose dependently and significantly restored the ROS production to the control level, increased the Bcl-2 protein level, partly inhibited the cytochrome C release from mitochondria and reduced the caspase-3 activation in SH-SY5Y cells. On the other hand, there was no significant restoration of the ATP level by FK506 and the toxin activated proteins, p53 and Bax, were not normalized by FK506. In support of these latter results, daily administration of FK506 for 7 days to rats (0.5, 1 and 3 mg/kg i.p.) did not significantly prevent the apomorphine-induced contralateral circling, measured 2 weeks after unilateral nigral lesioning. Moreover, FK506 pretreatment did not significantly lower the toxin elevated lipid peroxidation levels, indicating that oxidative stress was present even after the FK506 treatment in the lesioned striatum. Taken together, our results with FK506 are inconsistent. We confirm the antioxidant nature of FK506, that is, it blocks ROS production in SH-SY5Y cells. However, there were no significant protective effects in any apoptotic analyses in SH-SY5Y cells and in animal studies, a 7-day FK506 pre-treatment was not able to reverse the toxic effect of 6-OHDA in a rat model of Parkinson's disease.     

Immunofluorescent labeling of increased calpain expression and neuronal death in the spinal cord of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice

Parkinson's disease (PD) is a movement disorder characterized by rigidity, tremor, and bradykinesia, originating from degeneration of dopaminergic neurons in the substantia nigra (SN), retrorubral area, and locus ceoruleus (LC). Calpain has been implicated in the pathophysiology of neurodegenerative diseases. Since the spinal cord (SC) and brain are integrally connected and calpain is involved in cell death and mitochondrial dysfunction, we hypothesized that SC neurons are also affected in PD. In order to examine this hypothesis, we examined both brain and SC from mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To identify cells expressing calpain, double immunofluorescent labeling was performed with antibodies specific for calpain and a cell type (OX-42, GFAP, or NeuN). Combined terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and double immunofluorescent labeling were used to identify death of specific cells in the central nervous system (CNS). There was an increase in calpain expression in microglia, astrocytes, and neurons in the SC of MPTP-treated mice at 1 and 7 days, as compared to controls. TUNEL-positive neurons in the SC and SN showed apoptotic characteristics. These results demonstrated that neuronal death occurred not only in SN but also in the SC of MPTP-treated mice and has provided evidence for a possible calpain-mediated SC neuronal death in MPTP-induced parkinsonism in mice.     

Protection but maintained dysfunction of nigral dopaminergic nerve cell bodies and striatal dopaminergic terminals in MPTP-lesioned mice after acute treatment with the mGluR5 antagonist MPEP

The mGluR5 antagonist MPEP was used to study the role of mGluR5 in MPTP-induced injury of the nigrostriatal DA neurons. The findings indicate that acute blockade of mGluR5 may result in neuroprotective actions against MPTP neurotoxicity on nigral DA cell bodies and striatal DA terminals using stereological analysis of TH immunoreactivity and microdensitometry. Biochemical analysis showed no restoration of DA levels and metabolism indicating a maintained reduction of DA transmission.     

Accelerated alpha-synuclein aggregation after differentiation of SH-SY5Y neuroblastoma cells

Alpha-synuclein (alpha-syn) is a major component of inclusion bodies in Parkinson's disease (PD) and other synucleinopathies. To clarify the possible roles of alpha-syn in the molecular pathogenesis of neurodegenerative diseases, we have established a novel cellular model based on the differentiation of SH-SY5Y cells that overexpress alpha-syn. In the presence of ferrous iron, differentiation of the cells led to the formation of large perinuclear inclusion bodies, which developed from scattered small aggregates seen in undifferentiated cells. The iron-induced alpha-syn-positive inclusions co-localized largely with ubiquitin, and some of them were positive for nitrotyrosine, lipid, gamma-tubulin and dynein. Notably, treatment with nocodazole, a microtubule depolymerizing agent, interrupted the aggregate formation but led to a concomitant increase of apoptotic cells. Therefore, it appears that an intracellular retrograde transport system via microtubules plays a crucial role in the aggregate formation and also that the aggregates may represent a cytoprotective response against noxious stimuli. This cellular model will enable better understanding of the molecular pathomechanisms of synucleinopathy.     

3-Nitrotyrosine-dependent dopaminergic neurotoxicity following direct nigral administration of a peroxynitrite but not a nitric oxide donor

The presence of 3-nitrotyrosine (3-NT) adducts in Lewy bodies in Parkinson's disease suggests a role for nitrative stress in dopaminergic cell death. Whether this is a direct effect of increased nitric oxide (NO) formation or requires its reaction with superoxide to form peroxynitrite is not clear. In the present study, we show that direct nigral administration of a NO donor, SNOG, in the rat produced only local toxicity to dopaminergic neurones pre-labeled with fluorogold with no 3-NT formation. However, administration of a peroxynitrite donor, SIN-1, caused widespread damage to dopaminergic neurones and marked expression of 3-NT immunoreactivity. Importantly, dopaminergic cell loss and the expression of 3-NT were completely prevented when SIN-1 was co-administered with the NO/peroxynitrite scavenger, carboxy-PTIO. The results suggest that increased NO formation is not inherently toxic to dopaminergic neurons, but when both oxidative and nitrative stress combine to cause peroxynitrite formation, neurotoxicity occurs.     

Overexpression of the human beta-amyloid precursor protein downregulates cortistatin mRNA in PDAPP mice

We measured preprocortistatin mRNA expression in young and aged transgenic (Tg) mice overexpressing the human beta-amyloid precursor protein (hbetaAPP) under the platelet-derived growth factor-beta promoter. Our findings suggest that the significant increase in hippocampal cortistatin mRNA expression during normal aging is significantly attenuated in Tg mice at an age known to exhibit beta-amyloid protein (Abeta) deposition. These deficits in cortistatin expression may play a role in the deficits in hippocampal-dependent spatial learning and sleep/wake states previously demonstrated in aged Tg mice.     

GABA(A) and mu-opioid receptor binding in the globus pallidus and endopeduncular nucleus of animals symptomatic for and recovered from experimental Parkinsonism

The expression of parkinsonian motor symptoms may be partly attributed to an increase in GABAergic neurotransmission from hyperactive GABA/enkephalinergic striatopallidal efferents. The present study measured pallidal GABA_A and μ-opioid receptor binding in normal cats and cats symptomatic for and recovered from MPTP-induced parkinsonism. GABA_A receptor binding was significantly decreased in the globus pallidus (GP) in symptomatic cats and returned to normal levels in spontaneously recovered cats. Mu-opioid receptor binding in the GP was significantly decreased in symptomatic cats and remained significantly decreased in recovered cats. These results suggest that GABA_A but not μ-opioid receptor binding may correlate with the expression of parkinsonian motor deficits and may reflect increased pallidal GABA and ENK release in parkinsonian animals. Upon recovery from experimental parkinsonism, however, pallidal GABA_A receptor binding returns to normal levels while μ-opioid receptor binding reflecting enkephalin release remains elevated.     

Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity

Oxidative stress caused by various stimuli lead to oxidation of glutathione (GSH), the major redox power of the cell. Amyloid beta [Abeta(1-42)] is one of the key components of senile plaques and is involved in the progress initiation and triggers of Alzheimer's disease (AD). Lower GSH levels correlated with the activation of mitogen-activated proteins kinases (MAPK) have been demonstrated in AD, Parkinson's disease (PD) and other neurodegenerative disorders and have been proposed to play a central role in the deterioration of the aging and neurodegenerative brain. In this study, we evaluated the ability of low molecular weight thiol amides, N-acetyl cysteine amide (AD4) that replenishes GSH levels, N-acetyl glycine cysteine amide (AD7) and N-acetyl-Cys-Gly-Pro-Cys-amide (CB4) to protect primary neuronal culture against the oxidative and neurotoxic effects of Abeta(1-42) and to inhibit cisplatin- and hydrogen-peroxide-induced phosphorylation of two MAP kinases (MAPK), p38 and ERK1/2, in NIH3T3 cells. Cell death induced by Abeta(1-42) in primary neuronal cells was reversed by the thiol amides. Likewise, protein oxidation, loss of mitochondrial function and DNA fragmentation all returned to control levels by pretreatment with the three thiol amides. Elevated phosphorylation of ERK1/2 and p38 induced by cisplatin or H2O2 in NIH3T3 cells was lowered by AD4, AD7 and CB4 in a dose-dependent manner. Taken together, these results suggest that the thiol amides AD4, AD7 and CB4 protect neuronal cells against Abeta(1-42) toxicity by attenuating oxidative stress in correlation with inhibiting the MAPK phosphorylation cascade. These results are consistent with the notion that these small molecular thiol amides may play a viable protective role in the oxidative and neurotoxicity induced by Abeta(1-42) in AD brain.