Cerium oxide nanoparticles for treatment and prevention of Alzheimer's disease, Parkinson's disease, and disorders associated with free radical production and/or mitochondrial dysfunction

Abstract

Cerium oxide nanoparticles (CeONP) can be used to treat or prevent neurodegenerative diseases, including for example Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, AIDS-related dementia, ALS, progressive supranuclear palsy, and encephalitis, as well as mitochondrial diseases and diseases associated with mitochondrial damage. In particular, CeONP having an average size of about 2 nm to about 100 nm can be administered in an amount sufficient to block production of hydroxyl or superoxide radicals, block free radical production by A.sub.(1-42), block A.sub.(1-42)-induced neuronal death, block A.sub.(1-42)-induced [Ca.sup.2+].sub.i dysfunction in neurons, block A.sub.(1-42)-induced lipid peroxidation, decrease loss of dopaminergic neurotransmission, or reduce mitochondrial dysfunction in a cell. CeONP can also be effective in treating conditions involving toxic exposures to compounds that induce mitochondrial dysfunction, such as rotenone, cyanide, carbon monoxide, polychlorinated biphenyls (PCBs) and other mitochondrial toxins.

Claims

1. A method of treating a subject for Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, or amyotrophic lateral sclerosis (ALS), said method comprising: administering a non-agglomerating composition comprising cerium oxide nanoparticles having an average particle diameter size of about 10 nm to about 20 nm in an amount sufficient to provide a therapeutically effective dose of about 10 ng, 50 ng, 100 ng, 500 ng, 1 g, 5 g, 10 g, or 50 g per kg body mass to a subject having Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, or amyotrophic lateral sclerosis (ALS).

2. The method according to claim 1, wherein the non-agglomerating composition includes a carrier without any phosphate buffer.

3. A method of treating a subject for a mitochondrial disease or suffering from the effects of a mitochondrial toxin, comprising: administering a non-agglomerating composition comprising cerium oxide nanoparticles having an average particle diameter size of about 10 nm to about 20 nm in an amount sufficient to provide a therapeutically effective dose of about 10 ng, 50 ng, 100 ng, 500 ng, 1 g, 5 g, 10 g, or 50 g per kg body mass to a subject having mitochondrial disease or suffering from the effects of a mitochondrial toxin.

4. The method according to claim 3, wherein the effects of mitochondrial toxins result from exposure of a cell to rotenone, cyanide, carbon monoxide, or polychlorinated biphenyls (PCBs).

5. The method according to claim 4, wherein the effects of mitochondrial toxins are mitochondrial failure from inhibition of electron transport in the mitochondria.

6. The method of claim 3, wherein the subject has a mitochondrial disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an EPR tracing showing the effect of the presence of cerium oxide nanoparticles on quenching hydroxyl radical production.

(2) FIG. 2 shows an EPR tracing showing the effect of the presence of cerium oxide nanoparticles on quenching superoxide radical production.

(3) FIG. 3 shows an EPR tracing showing that cerium oxide nanoparticles block A.sub.(1-42)-induced free radical formation.

(4) FIG. 4 is a bar graph that shows cerium oxide nanoparticles decrease A.sub.(1-42)-induced neuronal death.

(5) FIGS. 5A, 5B, and 5C show light micrographs of cortical neuronal cultures, untreated, treated with 10 M A.sub.(1-42), and treated with 10 nM cerium oxide nanoparticles (average size 10-20 nm) and exposed to A.sub.(1-42).

(6) FIG. 6 is a bar graph that demonstrates that cerium oxide nanoparticles reduce A-induced elevation of basal intracellular free calcium in neurons.

(7) FIG. 7 is a bar graph that shows that cerium oxide nanoparticles protect neurons from A.sub.(1-42)-induced alterations in glutamate-mediated calcium signaling.

(8) FIG. 8 is a bar graph that shows that cerium oxide nanoparticles inhibit A.sub.(1-42)-induced lipid peroxidation.

(9) FIGS. 9A and 9B are bar graphs that show that cerium oxide nanoparticles extend the lifespan of male and female Drosophila after exposure to high dose (10 mM) paraquat.

(10) FIGS. 10A and 10B are line graphs that show that cerium oxide nanoparticles protect female and male Drosophila from low dose (1 mM) paraquat and extend post-paraquat lifespan.

(11) FIGS. 11A and 11B are bar graphs that show that cerium oxide nanoparticles preserve motor function in paraquat-challenged female and male Drosophila.

(12) FIGS. 12A and 12B are bar graphs that show that cerium oxide nanoparticles preserve the climbing motor function of paraquat-challenged female and male Drosophila.

(13) FIG. 13 is an electron micrograph that shows CeONP localized in mitochondria of mixed organotypic neuronal cultures.

(14) FIG. 14 is a bar graph that shows that cerium oxide nanoparticles protect mixed organotypic brain cell cultures from cell injury induced by rotenone.

(15) FIG. 15 is a bar graph that shows that cerium oxide nanoparticles preserve mitochondrial membrane potential after rotenone challenge.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

(16) Reference will now be made in detail to various exemplary embodiments of the invention. The following detailed description is presented for the purpose of describing certain embodiments in detail. Thus, the following detailed description is not to be considered as limiting the invention to the embodiments described. Rather, the true scope of the invention is defined by the claims.

(17) In the examples that follow, the inventors used cerium oxide nanoparticles available from Nanophase Technologies Corporation (Romeoville, Ill.). Synthesis of these particles has been described in the following patents: U.S. Pat. Nos. 6,669,823, 5,460,701, 5,514,349, 5,874,684; Japanese Patents JP2980987 and JP3383608; European Patent EP0711217B1; German Patent DE69426886; French Patent FR94922757; Great Britain Patent GB94922757; and Australian Patent AU068582882, the disclosures of which are hereby incorporated by reference in their entirety. Advantages of using these CeONP are further described in the inventors' prior work, e.g., WO 2007/002662, the disclosure of which is hereby incorporated by reference in its entirety.

(18) The inventors have found that CeONP having an average particle size ranging from about 2 nm to about 100 nm in diameter may be used for preventing or treating neurodegenerative diseases, mitochondrial diseases, and effects of mitochondrial toxins. The inventors have found, more particularly, that CeONP ranging from about 2 nm to about 50 nm are preferable. CeONP ranging from about 10 nm to about 20 nm may be even more preferable. Of course, depending on the application, any specific size or size range within these general sizes can be provided, the size being selected by the practitioner based on situation-specific parameters.

(19) The present invention provides methods of treating individuals suffering from, or suspected of suffering from neurodegenerative diseases, as well as methods of preventing such diseases. The invention also provides methods of treating or preventing injury resulting or caused by exposure to toxic substances. The methods include in vitro, in vivo, and/or ex vivo methods of treating or preventing neurodegenerative diseases. Administering CeONP according to the invention can comprise any act that provides cerium oxide nanoparticles to a subject (e.g., individual, animal, patient, etc.) in a way that the particles can function for their intended purpose. In general, a dosing of about 0.01 ng to about 1 g, such as about 0.05 ng, 0.1 ng, 0.5 ng, 1 ng, 10 ng, 50 ng, 100 ng, 500 ng, 1 ug, 5 ug, 10 ug, 50 ug, 100 ug, 500 ug, or 1 g per administration or per kg body mass per administration should be effective in providing the desired therapeutic or prophylactic result. Of course, depending on the application and according to the practitioner's specifications, any dose in this range may be used to prevent or treat neurodegenerative diseases.

(20) I. Alzheimer's Disease.

(21) 1.1. Cerium Oxide Nanoparticles block hydroxyl radical production in vitro. To demonstrate that CeONP directly inhibit free radical formation, we used electron paramagnetic resonance spectroscopy (EPR) to measure the ROS scavenging activity of CeONP. Hydroxyl radicals were generated by adding 250 l of 0.2 M H.sub.2O.sub.2 to a solution containing 0.025M DMPO (spin trap) and 1.7 mM FeSO.sub.4 in 0.17M potassium phosphate buffer, pH 7.4. Radical production was detected with a Bruker ER 200D ESR spectrometer, at 37 C.

(22) In the tracings shown in FIG. 1, the number of radicals produced is proportional to the intensity of the peaks shown in the EPR signal. Control experiments demonstrated that CeONP alone (in the presence of DMPO) did not induce hydroxyl radical formation (data not shown). Hydroxyl radicals generated via the Fenton reaction are shown in trace (a). In trace (b), 0.5 mg CeONP (average size about 10-20 nm) was added. As shown, hydroxyl radical production was almost completely quenched. CeONP with average sizes from about 2 nm to 50 nm produced similar quenching of hydroxyl radicals, with an average size of about 10-20 nm being the most effective in vitro size.

(23) 1.2. Cerium oxide nanoparticles block superoxide radical production in vitro. The ability of CeONP to block superoxide radical production in vitro was also examined, as is shown in FIG. 2. Superoxide radicals were generated by irradiating a solution of riboflavin (0.53 mM) and DMPO (0.1 M) in potassium phosphate buffer (0.17M) pH 7.4, with a UV lamp for 10 min. Trace (a) shows the superoxide generated by EPR measurement of the spectrum of the DMPO/O2-adduct. In trace (b), 0.5 mg of 7 nm ceria NP was added, which reduced superoxide production. In trace (c), 0.5 mg of 10-20 nm (average size) CeONP was added, showing almost complete quenching of superoxide production. Similar quenching of superoxide production was observed with 50 nm average size CeONP (data not shown). CeONP alone produced no superoxide radicals. Taken together, these EPR results demonstrate directly that CeONP, preferably having a size range of about 2-50 nm, reduce superoxide radical production in vitro.

(24) 1.3. Cerium oxide nanoparticles block free radical production by A.sub.(1-42). It has been shown that the toxic or disease producing fragment of A, A.sub.(1-42), produces free radicals (Butterfield (2007); Kanski, J, S Varadarajan, M Aksenova, & D A Butterfield, Role of glycine-33 and methionine-35 in Alzhemier's amyloid -peptide 1-42-associated oxidative stress and neurotoxicity, Biochim. Biophys. Acta., 1586, 190-198, 2001; and Varadarajan, S, J Kanski, M Aksenova, C. Lauderbeck, and D. A. Butterfield, Different mechanisms of oxidative stress and neurotoxicity for Alzheimers A (1-42) and Ab(25-35), J. Am. Chem. Soc., 123, 5625-5631, 2001), which are suspected to be an initiating event in the development of AD. We reproduced the generation of free radicals by A.sub.(1-42) as described by the Butterfield group (Butterfield (2007); Kanski (2001); and Varadarajan (2001), the disclosures of which are incorporated by reference herein in their entirety).

(25) A.sub.1-42 (1 mg/ml) was incubated at 37 C. in chelexed phosphate buffered saline containing PBN as a spin trap, in an EPR flat cell. Incubation periods were varied from 4-16 days. Cohen, C. A., et al. (2006), CeO2 Nanoparticles Extend Lifespan and Protect Drosophila Melanogaster from Paraquat-Induced Oxidative Stress, Free. Rad. Biol. & Med., 123, the disclosure of which is incorporated by reference herein in its entirety. Flat cells were examined by EPR at 12-24 hr intervals, for production of free radicals. As shown in FIG. 3 (trace A), A.sub.1-42 produced a characteristic fingerprint free radical spectra beginning on day 4 and persisting through day 16 (trace B). Incubation of A.sub.142 with CeONP (0.5 mg) completely blocked free radical production induced by A.sub.1-42 (FIG. 3, trace C). To ascertain whether cerium simply delayed free radical production, flat cells containing A.sub.1-42 or cerium+A.sub.1-42 were monitored via EPR for up to 16 days (data not shown). Free radicals continued to be produced by A.sub.1-42 solutions, but were completely blocked at all time points by addition of cerium oxide (data not shown). Cerium oxide alone (trace E) or cerium plus albumin (trace D) showed no free radical production, demonstrating that nonspecific protein incubation does not generate free radicals. Traces F and G represent controls of CeONP alone and CeONP with spin trap, demonstrating no radical production. Taken together, these results suggest that CeONP block free radical production associated with A.sub.1-42 in vitro.

(26) 1.4. Cerium oxide nanoparticles reduce A.sub.(1-42) toxicity in pure neuronal cultures. It is well known that A.sub.1-42 is toxic to neurons in culture. Pure neuronal cultures were prepared from the cortices of embryonic rats using established procedures (Weber, J. T., Rzigalinski, B. A., Willoughby, K. A., Moore, S. F., & Ellis, E. F. (1999), Alterations in calcium-mediated signal transduction and intracellular calcium stores after in vitro injury of pure embryonic neurons, Cell Calcium, 26, 289-299, the disclosure of which is incorporated herein by reference in its entirety) and treated with CeONP (10 nM, 10-20 nanometer average size) or vehicle (normal saline) on day 6 in vitro, for 48 hrs.

(27) Prior studies (see, e.g., WO 2007/002662; Rzigalinski I (2006); Rzigalinski II (2006); Rzigalinski 2005; and Singh 2007, the disclosures of which are incorporated by reference herein in their entirety) demonstrate that CeONP are taken up by the cells during this time period. Molar references (i.e., 10 nM CeONP) correlate to a CeONP suspension containing 10 nM of cerium, since the exact molecular weight of a single CeONP is unknown.

(28) CeONP solutions for delivery were prepared by 2 min probe sonication of concentrated stocks and all serial dilutions. On day 10 in vitro, neurons were exposed to vehicle (saline) or A.sub.1-42 (10 M) and cell death was assessed by propidium iodide staining 48 hrs later. Propidium iodide is normally excluded from uninjured cells, but enters cells with damaged or disrupted membranes, staining the nucleus a brilliant orange. As shown in FIG. 4, CeONP inhibited A.sub.1-42-induced cell death 42-53%, depending on the dose. FIGS. 5A, 5B, and 5C show light micrographs of pure neuronal cultures treated with saline or CeONP, followed by challenge with 10 M A.sub.(1-42). FIG. 5A shows healthy 12 day old neuronal cultures. Note distinct cells bodies and tightly cabled axonal processes. FIG. 5B shows neurons treated with A.sub.(1-42) on day 10 in vitro. Note the disintegrating cell bodies and fragmented axons. FIG. 5C shows neurons treated with CeONP, followed by A.sub.(1-42), demonstrating preservation of healthy, normal, neurons.

(29) 1.5. Cerium oxide nanoparticles block A.sub.(1-42)-induced calcium dysregulation in neurons. Intracellular free calcium ([Ca.sup.2+].sub.i) is an important signaling process in neurons and other cells. In unstimulated cells, [Ca.sup.2+].sub.i is generally maintained at a low level (50-100 nM). During a signaling event such as muscle contraction or neurotransmission in the brain, [Ca.sup.2+].sub.i is elevated as a signal is propagated. In order to effectively transmit and propagate signaling information, [Ca.sup.2+].sub.i must be maintained at a normal basal level. As neurons and other cells are injured and go on to die, failure of ionic gradients such as [Ca.sup.2+].sub.i occur, and basal [Ca.sup.2+].sub.i becomes elevated, often prior to cell death.

(30) We measured [Ca.sup.2+].sub.i in neurons exposed to 10 M A.sub.(1-42) with and without CeONP treatment. [Ca.sup.2+].sub.i was measured with Fura-2 microspectrophotometry, as previously described. (See, e.g., Weber (1999); Zhang, L., B. A. Rzigalinski, E. F. Ellis, & L. S. Satin (1997), Reduction of voltage-dependent Mg2+ blockade of NMDA currents in mechanically injured cortical neurons, Science 274, 1805-1976; Rzigalinski, B. A., Willoughby, K. A., Hoffman, S., Falck, J. R., & Ellis, E. F. (1999) Calcium influx factor: Further evidence it is 5,6-epoxyeicosatrienoic acid, J. Biol. Chem., 274, 175-182; and Ahmed, S. M., Weber, J. T., Rzigalinski, B. A., & Ellis, E. F. (2002), NMDA receptor contributes to a portion of the decreased mitochondrial membrane potential and elevated intracellular free calcium in strain-injured neurons, J. Neurotrauma, 19, 1619-29, the disclosures of which are incorporated by reference herein in their entirety.)

(31) As shown in FIG. 6, pure cultures of rat embryonic cortical neurons were treated with CeONP (avg size 10-20 nm) on day 6 in vitro. For the control, neurons were treated with saline. On day 10, cultures were treated with 10 M A.sub.(1-42). Results are derived from fields of 10-15 neurons in 3 separate experiments. Intracellular free calcium ([Ca.sup.2+].sub.i) was measured with Fura-2 microspectrophotometery on days 12-13. As shown, control and CeONP-treated neurons had similar basal [Ca.sup.2+].sub.i levels, while A.sub.(1-42) significantly increased basal (unstimulated) [Ca.sup.2+].sub.i in neurons, which was reduced to normal levels by pretreatment with 10 and 100 nM cerium oxide nanoparticles (i.e. pretreatment with CeONP protected against the A.sub.(1-42)-induced elevation in basal [Ca.sup.2+].sub.i).

(32) In addition to basal [Ca.sup.2+].sub.i levels, neurons also undergo an elevation in [Ca.sup.2+].sub.i during a neurotransmission event. A primary excitatory neurotransmitter in the brain is glutamate. In AD, both cholinergic and glutamatergic signaling decline as the disease progresses. Therefore, we examined the effect of CeONP on neuronal glutamate signaling after A.sub.(1-42) challenge. Neurons were treated with CeONP followed by A.sub.(1-42) challenge as described in 1.4 above.

(33) Glutamate-stimulated [Ca.sup.2+].sub.i signaling was assessed as previously described (see, e.g., Weber (1999); Zhang (1997); Rzigalinski (1999); and Ahmed (2002), the disclosures of which are incorporated by reference herein in their entirety). Results shown are derived from populations of 10-15 neurons from 3 separate experiments. FIG. 7 shows the average glutamate-stimulated change in [Ca.sup.2+].sub.i produced by 100 M glutamate, 42771 nM in controls. Pure cultures of rat embryonic cortical neurons were treated with cerium oxide nanoparticles (avg size 10-20 nm) on day 6 in vitro. On day 10, cultures were treated with 10 M A.sub.(1-42). Intracellular free calcium was measured with Fura-2 microspectrophotometery as described in FIG. 6. Glutamate (100 M) was used as a stimulus and the change in [Ca.sup.2+].sub.i was measured. As shown, A.sub.(1-42) decreased the glutamate-stimulated change in [Ca.sup.2+].sub.i, which was blocked by CeONP. In particular, CeONP treated neurons had similar levels of glutamate-stimulated [Ca.sup.2+].sub.i elevation (bars 3 and 4). Neurons treated with A.sub.(1-42) showed a significantly decreased response to glutamate, 23042 (2.sup.nd bar), demonstrating that A.sub.(1-42) decreases glutamate signaling in neurons. In CeONP-treated neurons, A.sub.(1-42) had no effect on glutamate signaling, which was maintained within the normal range (bars 5 and 6).

(34) Taken together, these data demonstrate that CeONP effectively prevent the A.sub.(1-42)-induced dysfunction in [Ca.sup.2+].sub.i signaling and protect neurons from the deleterious effects of A.sub.(1-42).

(35) 1.6. Cerium oxide nanoparticles decrease A.sub.(1-42)-induced lipid peroxidation in neurons. AD and A.sub.(1-42) are also associated with formation of free radical damage products to cellular macromolecules, particularly lipids. (Markesbery, W. R. & Lovell, M. A., Damage to lipids, proteins, DNA, and RNA in mild cognitive impairment, Arch. Neurol., 64, 954-966, 2007.)

(36) We assessed the effect of CeONP on A.sub.(1-42)-induced lipid damage by measuring lipid peroxidation products in A.sub.(1-42)-treated neuronal cultures, as shown in FIG. 8. Pure cultures of rat embryonic cortical neurons were treated with cerium oxide nanoparticles (avg size 10-20 nm) on day 6 in vitro. On day 10, cultures were treated with 10 M A.sub.(1-42). Two days after A.sub.(1-42) treatment, lipid peroxidation products (LPO) were measured with a spectrophotometric kit from Invitrogen, and represent the sum total of malondialdehyde (MDA) and hydroxynonenal (HNE), both of which are toxic lipid peroxidation products. Lipid peroxidation products were normalized to the amount of protein in each cell culture well, using a modified Lowry protein assay. As shown in FIG. 8, A.sub.(1-42) treatment increased lipid peroxidation, which was inhibited by CeONP treatment. In particular, the levels of LPO in controls treated with saline or CeONP alone (as described in section 1.4) are shown in the first three bars of FIG. 8. There was no significant difference in LPO in CeONP vs. saline treated neurons on day 12 in vitro. A.sub.(1-42) treatment dramatically increased LPO in controls as shown. The A.sub.(1-42)-induced increase in LPO was blocked by CeONP administration, as shown in the last 2 bars of FIG. 8. These results demonstrate that CeONP block A.sub.(1-42)-induced lipid peroxidation in neurons.

(37) II. Parkinson's Disease.

(38) Here, we demonstrate the utility of CeONP in treatment of Parkinson's Disease using a Drosophila model of Parkinson's disease, exposure to the herbicide toxin paraquat. See, e.g., Meulener M, Whitworth A J, Armstrong-Gold C E, Rizzu P, Heutink P, Wes P D, Pallanck L G, Bonini N M, Drosophila DJ-1 mutants are selectively sensitive to environmental toxins associated with Parkinson's disease, Curr. Biol., 15, 1572-1577, 2005; and Cicchetti F, Lapointe N, Roberge-Tremblay A, Saint-Pierre M, Jimenez L, Ficke B W, Gross R E, Systemic exposure to paraquat and maneb models early Parkinson;s disease in young adult rats, Neurobiol. Dis., 20, 360-371, 2005, the disclosures of which are incorporated by reference herein in their entirety. FIGS. 9A-B, 10A-B, 11A-B, and 12A-B show the results.

(39) In Drosophila, paraquat induces a dose dependent motor dysfunction and death, in part, by destruction of dopaminergic neurons through superoxide generation. For these studies, male and female Drosophila of the Oregon R strain were cultured as previously described. Rzigalinski I (2006); and Cohen (2006), the disclosures of which are incorporated by reference herein in their entirety.

(40) Experiments were conducted on cohorts of 100 male or female flies. One hundred newly enclosed male or female flies were placed in control or CeONP treated food (10 nM-100 M, 10 flies per vial) for days 1-30 of their lifespan. Fly food consisted of Jazz Mix with CeONP added in suspensions containing 1 M docusate sodium for even dispersion of nanoparticles in the food mix. On day 30, flies were placed in empty vials for 1 hr, followed by transfer to vials containing filter paper saturated with 5% sucrose containing 1 or 10 mM paraquat, for 1 hr. After paraquat exposure, flies were returned to their respective food groups. Death counts were performed at intervals thereafter. Results shown represent 2-3 separate experiments on cohorts of 100 flies.

(41) 2.1. Cerium oxide nanoparticles protect Drosophila from paraquat. CeONP protected against paraquat toxicity, decreasing mortality and increasing lifespan in treated flies. CeONP protected against paraquat toxicity in male flies, at a higher dose than that utilized in females.

(42) In particular, as shown in FIG. 9A, exposure to 10 mM paraquat resulted in rapid death of untreated female flies, with over 75% death by 22 hrs post-paraquat. However females treated with 10 nM-100 M CeONP showed enhanced survival and outlived their untreated age matched controls. Similar results were observed in male Drosophila (FIG. 9B). However the dose required for paraquat resistance in males was higher (1 M-100 M) than that required for females.

(43) A paraquat dose of 10 mM is a high, lethal dose for flies at age 30 (mid-life). Therefore, we also examined a lower dose of paraquat, 1 mM, as shown in FIGS. 10A and 10B. One hundred female and one hundred male Drosophila were treated with CeONP delivered in the fly food, as described above. The mortality curve for female flies exposed to 1 mM paraquat on day 30 is shown in FIG. 10A, black solid line. Flies died steadily after paraquat exposure (time zero on the graph) with all flies in the cohort of 100 dying by 44 days post-paraquat. CeONP treated flies showed increased survival post-paraquat, with CeONP treated groups surviving 52-56 days post-paraquat. Similar results were observed in male flies exposed to 1 mM paraquat on day 30, as shown in FIG. 10B. Taken together, these results demonstrate that CeONP increase survival and decrease mortality rate in male and female flies exposed to paraquat, a Drosophila model for Parkinson's disease.

(44) 2.2 Cerium oxide nanoparticles protect Drosophila from paraquat-induced motor dysfunction. One of the hallmarks of PD is motor dysfunction, induced by loss of dopaminergic neurotransmission. Paraquat exposure in Drosophila also induces dopaminergic neuronal loss. Meulener (2005); and Cicchetti (2005). Using the Drosophila model detailed above, we assessed the effects of CeONP on motor dysfunction in response to paraquat challenge. Motor function was assessed in drosophila by examining a) total vertical activity and b) ability to ascend the vial to an 8 cm height, using a Trikinetics activity monitor.

(45) For measurement of total vertical activity, an empty vial containing 10 flies was placed vertically in the monitor, which has 3 levels at which upward movement of flies can be measured, 3, 6 and 8 cm. Movement of a fly past a given level is registered as the fly crosses a beam of light. For a vial of 10 flies, the monitor assesses the number of times a fly crosses the light path at each respective height in the vial. The total number of beam crosses at all heights is then divided by the number of flies in the measurement group, giving a measure of activity/fly. Activity was monitored for 15 minutes per vial, for all flies within the group. All activity measurements were made at least 2 hrs after first exposure to light for the day, and 2 hrs prior to the lights turning off, to assure that measurement was not affected by diurnal activity changes.

(46) The effect of CeONP on total activity of paraquat-treated female flies is shown in FIG. 11A. Activity was first measured just prior to paraquat exposure (at 30 days of age) (first set of bars). As shown in FIG. 11A, middle aged CeONP fed flies had higher levels of activity than middle aged control flies, consistent with previous work demonstrating that CeONP preserve motor function in aging flies. Twenty-four hours (24 hrs) post paraquat (second group of bars), flies treated with paraquat alone exhibited a substantial decline in total activity. However the decline in activity post-paraquat was significantly less in CeONP fed flies. The effects persisted through 7 days post paraquat (last group of bars). Similar results were observed in male Drosophila, shown in FIG. 11B. These results demonstrate that CeONP preserves motor function in paraquat-treated flies.

(47) In addition to measuring total activity, we also measured the number of times flies ascended the vial to the 8 cm height, a measurement of the robustness of motor activity. FIG. 12A shows the ability of female flies to ascend to 8 cm at 30 days of age, prior to paraquat exposure. Once again, CeONP treated females showed enhanced ability to ascend to the 8 cm height, as compared to controls. Twenty-four hours (24 hrs) post paraquat, control flies were unable to ascend to 8 cm. This decline in function persisted through 7 days post-paraquat in all surviving flies. However surviving CeONP treated flies retained some ability to ascend to this level, which persisted in flies surviving 7 days post paraquat. Similar results were observed for male flies (FIG. 12B), where CeONP treated males were able to climb to 8 cm more often than controls. In particular, after treatment with 10 mM paraquat, controls were unable to climb the walls of the vial to this height, while CeONP preserved this motor function in surviving flies.

(48) Taken together, these activity measurements demonstrate that CeONP reduces motor dysfunction in a Drosophila model of PD.

(49) III. Mitochondrial Diseases and Mitochondrial Dysfunction.

(50) 3.1. Cerium oxide nanoparticles localize, in part, to mitochondria. Previous work has shown that CeONP readily enter cells and tissues and localize to cytoplasmic areas. See, e.g., WO 2007/002662; Rzigalinski I (2006); Rzigalinski II (2006); Rzigalinski 2005; and Singh 2007. However, CeONP also localize, in part, to mitochondria, as shown in FIG. 13. Their role within the mitochondria has not been identified. To assess the effects of CeONP on cell demise associated with mitochondrial dysfunction, we utilized rotenone. Rotenone inhibits mitochondrial energy production through inhibition of Complex 1, and disrupts oxidative phosphorylation and the flow of electrons through the electron transport chain. This decreases ATP production and disrupts mitochondrial membrane potential (MMP).

(51) 3.2. Cerium oxide nanoparticles protect cells from death associated with inhibition of mitochondrial Complex I with rotenone. We exposed mixed organotypic rat brain cell cultures to CeONP (10 nM) or saline on day 10 in vitro, for 48 hrs. On day 14 in vitro, cultures were exposed to the mitochondrial Complex I inhibitor, rotenone (1 M), for 24 hrs, and cell death was determined by propidium iodide uptake according to a published methodology. See, e.g., Ahmed (2002); and Ahmed, S. M., Rzigalinski, B. A., Willoughby, K. A., Sitterding, H. A., & Ellis, E. F (2000), Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons, J. Neurochem., 74, 1951-1960, the disclosures of which are hereby incorporated by reference in their entirety.

(52) CeONP treatment significantly reduced cell injury induced by rotenone. As shown in FIG. 14, CeONP significantly reduced propidium iodide uptake after rotenone exposure, suggesting that CeONP protects cells from damage associated with inhibition of mitochondrial Complex I. The organotypic cultures examined contained an astrocyte monolayer growing on the bottom of the culture well, with a layer of neurons attached to the top of the astrocytes. Although propidium iodide uptake does not differentiate between astrocytes and neurons, it appeared that the primary cell type taking up propidium iodide at 24 hrs post-rotenone were neurons, due to the increased propidium iodide staining observed in the cells above the confluent astrocyte monolayer. In CeONP-treated cultures, this neuronal layer appeared to be largely viable, without propidium iodide uptake.

(53) 3.3. Cerium oxide nanoparticles protect cells from mitochondrial failure associated with inhibition of mitochondrial complex I with rotenone. We also examined mitochondrial membrane potential (MMP) in rotenone-treated cells. MMP is a measurement of the ability of mitochondrial to undergo oxidative phosphorylation and produce ATP for cellular energy. MMP is decreased in dysfunctional mitochondria and in mitochondrial associated with several diseases including PD, AD, and mitochondrial disorders.

(54) For these experiments, cells were treated with CeONP or saline for 48 hrs as described above, followed by rotenone (1 M). MMP was measured 6 hrs post rotenone using Rhodamine 123 (Rh123) as previously described. Ahmed (2002); and Ahmed (2000), the disclosures of which are hereby incorporated by reference in their entirety.

(55) As shown in FIG. 15, MMP was slightly higher in CeONP treated controls as compared to untreated controls treated (first 2 bars), however this was not significant. At 6 hrs post-rotenone, MMP declined dramatically in rotenone challenged cells, and rotenone almost completely abolished MMP in untreated controls. However MMP was preserved in CeONP-treated cells challenged with rotenone. Taken together, these results suggest that CeONP protects cells from damage to Complex I of the electron transport chain and preserves mitochondrial function. Thus, CeONP can be used as an effective treatment of mitochondrial diseases and diseases associated with mitochondrial damage. CeONP can also be effective in treating conditions involving toxic exposures to compounds that induce mitochondrial dysfunction, such as rotenone, cyanide, carbon monoxide, polychlorinated biphenyls (PCBs) and other mitochondrial toxins.

(56) The present invention has been described with reference to particular embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that these features may be used singularly or in any combination based on the requirements and specifications of a given application or design. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. The description of the invention provided is merely exemplary in nature and, thus, variations that do not depart from the essence of the invention are intended to be within the scope of the invention.