Russell T. Matthews

NEUROPROTECTIVE EFFECTS OF CREATINE IN A TRANSGENIC ANIMAL MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

Mitochondria are particularly vulnerable to oxidative stress, and mitochondrial swelling and vacuolization are among the earliest pathologic features found in two strains of transgenic amyotrophic lateral sclerosis (ALS) mice with SOD1 mutations. Mice with the G93A human SOD1 mutation have altered electron transport enzymes, and expression of the mutant enzyme in vitro results in a loss of mitochondrial membrane potential and elevated cytosolic calcium concentration. Mitochondrial dysfunction may lead to ATP depletion, which may contribute to cell death. If this is true, then buffering intracellular energy levels could exert neuroprotective effects. Creatine kinase and its substrates creatine and phosphocreatine constitute an intricate cellular energy buffering and transport system connecting sites of energy production (mitochondria) with sites of energy consumption, and creatine administration stabilizes the mitochondrial creatine kinase and inhibits opening of the mitochondrial transition pore. We found that oral administration of creatine produced a dose-dependent improvement in motor performance and extended survival in G93A transgenic mice, and it protected mice from loss of both motor neurons and substantia nigra neurons at 120 days of age. Creatine administration protected G93A transgenic mice from increases in biochemical indices of oxidative damage. Therefore, creatine administration may be a new therapeutic strategy for ALS.

Electrophysiological evidence for excitation of rat ventral tegmental area dopamine neurons by morphine.

A considerable body of evidence indicates that opiates have an important influence on midbrain dopaminergic neurons. However, little data exist concerning the effects of opiates on the activity of single dopaminergic neurons, particularly the dopaminergic neurons of the ventral tegmental area. Firing rates of mesencephalic dopaminergic neurons were recorded extracellularly, and the effects of morphine, administered systemically or applied locally onto dopaminergic cells, were tested in paralyzed, unanesthetized or chloral hydrate anesthetized rats. In general, dopaminergic neurons were excited by both systemically and locally applied morphine. When mesencephalic dopaminergic neurons were subdivided into substantia nigra zona compacta (A9) and ventral tegmental area (A10) neurons, A10 neurons were excited 2-3 times more than A9 neurons by systemic morphine. Systemic administration of the specific opiate antagonist, naloxone, in large part reversed the effects of morphine. Microiontophoretic or micropressure ejection of morphine caused an apparent depolarization-induced excitation of both A10 and A9 dopaminergic neurons. These results provide direct evidence that morphine increases impulse flow of A10 dopaminergic neurons, which are known to be involved in locomotor stimulant and positive reinforcement effects of opiates.

Inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects against MPTP-induced neurotoxicity in mice.

Abstract: Several studies suggest that nitric oxide (NO•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7‐nitroindazole (7‐NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7‐NI dose‐dependently protected against MPTP‐induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7‐NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP‐induced depletions of both homovanillic acid and 3,4‐dihydroxyphenylacetic acid. 7‐NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO−), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3‐nitrotyrosine, which was attenuated by treatment with 7‐NI. Our results suggest that NO• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinsons disease.

Creatine and cyclocreatine attenuate MPTP neurotoxicity

Systemic administration of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) produces parkinsonism in experimental animals by a mechanism involving impaired energy production. MPTP is converted by monoamine oxidase B to 1-methyl-4-phenylpyridinium (MPP+), which blocks complex I of the electron transport chain. Oral supplementation with creatine or cyclocreatine, which are substrates for creatine kinase, may increase phosphocreatine (PCr) or cyclophosphocreatine (PCCr) and buffer against ATP depletion and thereby exert neuroprotective effects. In the present study we found that oral supplementation with either creatine or cyclocreatine produced significant protection against MPTP-induced dopamine depletions in mice. Creatine protected against MPTP-induced loss of Nissl and tyrosine hydroxylase immunostained neurons in the substantia nigra. Creatine and cyclocreatine had no effects on the conversion of MPTP to MPP+ in vivo. These results further implicate metabolic dysfunction in MPTP neurotoxicity and suggest a novel therapeutic approach, which may have applicability for Parkinsons disease.

Coenzyme Q10 attenuates the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced loss of striatal dopamine and dopaminergic axons in aged mice

We investigated whether oral administration of coenzyme Q10 (CoQ10) could attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in one-year-old mice. Four groups of one-year-old, male C57BL/6 mice received a either standard diet or a diet supplemented with CoQ10 (200 mg/kg/day) for five weeks. After four weeks, one group that had received the standard diet and one group that had received the CoQ10 supplemented diet were treated with MPTP. The four groups continued on their assigned diets for an additional week prior to sacrifice. Striatal dopamine concentrations were reduced in both groups treated with MPTP, but they were significantly higher (37%) in the group treated with CoQ10 and MPTP than in the group treated with MPTP alone. The density of tyrosine hydroxylase immunoreactive (TH-IR) fibers in the caudal striatum was reduced in both MPTP-treated groups, but the density of TH-IR fibers was significantly (62%) greater in the group treated with CoQ10 and MPTP than in the group treated with MPTP alone. Our results indicate that CoQ10 can attenuate the MPTP-induced loss of striatal dopamine and dopaminergic axons in aged mice and suggest that CoQ10 may be useful in the treatment of Parkinsons disease.

Brain-enriched Hyaluronan Binding (BEHAB)/Brevican Cleavage in a Glioma Cell Line Is Mediated by a Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) Family Member

Brain-enriched hyaluronan binding (BEHAB)/brevican is a brain-specific extracellular matrix protein containing a cleavage site between Glu395-Ser396, which bears remarkable homology to the “aggrecanase” site in the cartilage proteoglycan aggrecan. Expression of BEHAB/brevican is dramatically increased in human gliomas, notoriously invasive tumors. Recently, we showed that the rat 9L gliosarcoma cell line, which does not express BEHAB/brevican and forms non-invasive tumors when grown as intracranial grafts, can form invasive tumors when transfected with a 5′ cDNA fragment of BEHAB/brevican, but not when transfected with the full-length cDNA. In marked contrast, the highly invasive CNS-1 glioma cell line expresses and cleaves BEHAB/brevican protein when grown as an intracranial graft. These results suggest that both synthesis and cleavage of BEHAB/brevican protein may play a role in the invasiveness of gliomas. We report here, using an antibody developed to the neoepitope created by BEHAB/brevican cleavage at the Glu395-Ser396 site, that the CNS-1 cells are able to cleave the protein in vitro. We characterized the CNS-1-derived cleavage activity by assaying its ability to cleave BEHAB/brevican proteoglycan, and determined that the enzyme is a constitutively expressed, secreted activity. Using a variety of protease inhibitors, reverse transcriptase-polymerase chain reaction, and specific antibodies, we determined that this activity is likely to be a member of the ADAMTS family of metalloproteinases, specifically ADAMTS4. These results suggest a novel function for ADAMTS family members in BEHAB/brevican cleavage and glioma and indicate that inhibition of ADAMTS in glioma may provide a novel therapeutic strategy.

3‐Nitropropionic Acid Neurotoxicity Is Attenuated in Copper/Zinc Superoxide Dismutase Transgenic Mice

Abstract: The mitochondrial toxin 3‐nitropropionic acid (3‐NP) produces selective striatal lesions in both experimental animals and humans. The pathogenesis of the lesions involves secondary excitotoxicity that may then lead to free radical generation. To test this further we examined the effects of 3‐NP in both transgenic (Tg) mice that carry the complete sequence for the human copper/zinc superoxide dismutase (SOD) gene as well as non‐Tg littermate controls. The Tg‐SOD mice showed a pronounced attenuation of Nissl‐stained striatal lesions compared with non‐Tg mice. Systemic administration of 3‐NP resulted in production of hydroxyl free radicals as assessed by the conversion of salicylate to 2,3‐ and 2,5‐dihydroxybenzoic acid. This production was attenuated significantly in Tg‐SOD mice. In a similar way, 3‐NP produced significant increases in 3‐nitrotyrosine/tyrosine, a marker for peroxynitrite‐mediated damage, which were significantly attenuated in Tg‐SOD mice. These results support that oxygen free radicals and peroxynitrite play an important role in the pathogenesis of 3‐NP neurotoxicity.

The role of mitochondrial dysfunction and neuronal nitric oxide in animal models of neurodegenerative diseases

Excitotoxicity, mitochondrial dysfunction and free radical induced oxidative damage have been implicated in the pathogenesis of several different neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease. Much of the interest in the association of neurodegeneration with mitochondrial dysfunction and oxidative damage emerged from animal studies using mitochondrial toxins. Within mitochondria l-methyl-4-phenylpyridinium (MPP+), the active metabolite of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), acts to inhibit NADH-coenzyme Q reductase (complex I) of the electron transport chain. MPTP produces Parkinsonism in humans, primates, and mice. Similarly, lesions produced by the reversible inhibitor of succinate dehydrogenase (complex II), malonate, and the irreversible inhibitor, 3-nitropropionic acid (3-NP), closely resemble the histologic, neurochemical and clinical features of HD in both rats and non-human primates. The interruption of oxidative phosphorylation results in decreased levels of ATP. A consequence is partial neuronal depolarization and secondary activation of voltage-dependent NMD A receptors, which may result in excitotoxic neuronal cell death (secondary excitotoxicity). The increase in intracellular Ca2+ concentration leads to an actiation of Ca2+ dependent enzymes, including the constitutive neuronal nitric oxide synthase (cnNOS) which produces NO-. NO- may react with the Superoxide anion to form peroxynitrite. We show that systemic administration of 7-nitroindazole (7-NI), a relatively specific inhibitor of cnNOS in vivo, attenuates lesions produced by striatal malonate injections or systemic treatment with 3-NP or MPTP. Furthermore 7-NI attenuated increases in lactate production and hydroxyl radical and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. Our results suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role. (Mol Cell Biochem 174: 193–197, 1997)

Uncoupling Protein-2 Is Critical for Nigral Dopamine Cell Survival in a Mouse Model of Parkinson's Disease

Mitochondrial uncoupling proteins dissociate ATP synthesis from oxygen consumption in mitochondria and suppress free-radical production. We show that genetic manipulation of uncoupling protein-2 (UCP2) directly affects substantia nigra dopamine cell function. Overexpression of UCP2 increases mitochondrial uncoupling, whereas deletion of UCP2 reduces uncoupling in the substantia nigra-ventral tegmental area. Overexpression of UCP2 decreased reactive oxygen species (ROS) production, which was measured using dihydroethidium because it is specifically oxidized to fluorescent ethidium by the superoxide anion, whereas mice lacking UCP2 exhibited increased ROS relative to wild-type controls. Unbiased electron microscopic analysis revealed that the elevation of in situ mitochondrial ROS production in UCP2 knock-out mice was inversely correlated with mitochondria number in dopamine neurons. Lack of UCP2 increased the sensitivity of dopamine neurons to 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP), whereas UCP2 overexpression decreased MPTP-induced nigral dopamine cell loss. The present results expose the critical importance of UCP2 in normal nigral dopamine cell metabolism and offer a novel therapeutic target, UCP2, for the prevention/treatment of Parkinsons disease.

Sensory Deprivation Alters Aggrecan and Perineuronal Net Expression in the Mouse Barrel Cortex

An important role for the neural extracellular matrix in modulating cortical activity-dependent synaptic plasticity has been established by a number of recent studies. However, identification of the critical molecular components of the neural matrix that mediate these processes is far from complete. Of particular interest is the perineuronal net (PN), an extracellular matrix component found surrounding the cell body and proximal neurites of a subset of neurons. Because of the apposition of the PN to synapses and expression of this structure coincident with the close of the critical period, it has been hypothesized that nets could play uniquely important roles in synapse stabilization and maturation. Interestingly, previous work has also shown that expression of PNs is dependent on appropriate sensory stimulation in the visual system. Here, we investigated whether PNs in the mouse barrel cortex are expressed in an activity-dependent manner by manipulating sensory input through whisker trimming. Importantly, this manipulation did not lead to a global loss of PNs but instead led to a specific decrease in PNs, detected with the antibody Cat-315, in layer IV of the barrel cortex. In addition, we identified a key activity-regulated component of PNs is the proteoglycan aggrecan. We also demonstrate that these Cat-315-positive neurons virtually all also express parvalbumin. Together, these data are in support of an important role for aggrecan in the activity-dependent formation of PNs on parvalbumin-expressing cells and suggest a role for expression of these nets in regulating the close of the critical period.