NEUROPROTECTION BY PSAMMAPLYSENE A

By

Robert KaIb

This application claims priority under 35 U.S. C. §119(e) to U.S. Provisional Patent Application 60/918,653, filed on March 16, 2007. The foregoing application is incorporated by reference herein.

Pursuant to 35 USC §202(c), it is acknowledged that the United States Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health Grant No. ROl NS052325.

FIELD OF THE INVENTION

The present invention relates to the fields of neurology, molecular biology and pharmacology. More specifically, the invention provides methods for inhibiting neurodegeneration, particularly in patients with ALS.

BACKGROUND OF THE INVENTION

Several publications are cited throughout the specification in order to describe the state of the art to which this invention pertains. Full citations for these publications are found within the text and at the end of the specification. Each of these citations is incorporated by reference herein as though set forth in full.

In most cases of sporadic amyotrophic lateral sclerosis (ALS), motor neuron death is triggered by the interaction of a genetic predisposition and environmental factors (1). Correlative evidence suggests that aging is also a risk factor for the development of ALS, as well as other adult-onset neurodegenerative disorders (2). Studies from a variety of experimental systems have provided insight into the genetic factors controlling aging, in particular, the insulin/insulin-like growth factor signaling pathway (3, 4). In Caenorhabditis elegans, hypomorphic alleles of the daf-2 gene (mammalian homolog, insulin/insulin-like growth factor receptor) and the downstream signaling molecule age-1 (mammalian homolog, phosphotidylinositol-3' -kinase, PI3-K) promote longevity and this requires the activity the daf-16 transcription factor (mammalian homolog, FOXO3a) (5).

Daf-16/FOXO3a shuttles between the cytoplasm (where it is inactive) and the nucleus in a process that is controlled by its phosphorylation state. Phosphorylation of Daf-16/FOXO3a by the PD-K substrate kinases Akt and SGK leads to the 14-3-3 protein-dependent export of nuclear Daf-16/FOXO3a and re-entry into the nucleus requires dephosphorylation and release of 14-3-3 (6, 7). Within the nucleus, Dafl6/FOXO3a leads to the expression of a number of genes that help cope with stress and play an essential role in its longevity promoting activity (8). Reducing the activity of the insulin/insulin-like growth factor signaling pathway leads to increased Daf-16 transcription and enhanced stress resistance.

Some of the factors that contribute to motor neuron death in ALS include excitotoxicity, reactive oxygen species, accumulation of insoluble aggregates of neurofilaments, and defects in axonal transport (9, 10). Expression of mutant forms of proteins implicated in familial ALS (such as superoxide dysmutase or pl50 ^glued ) will also cause motor neuron death in vivo and in vitro (11). This collection of observations raises the possibility that pharmacological manipulations that activate FOXO3a signaling might protect motor neurons. In light of the role motor neuron death plays in ALS, a clear need exists for methods of treatment that preserve motor neuron viability.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method for preventing motor neuron death upon insult is provided. An exemplary method comprises administration of an effective amount of an activator of the FOXO3a signaling pathway to a patient in need thereof, thereby inhibiting or reducing neuronal death. Preferably, the activator is Psammaplysene A (PA). The person of skill in the art is aware that other nuclear export inhibitors and PI3K/AKT specific inhibitory compounds can be used in the invention, for example, see figure 4 of Kau et al. (Cancer Cell (2003) 4(6): 463-476) (15). Neurodegenerative disorders that may be treated using the methods of the invention include, without limitation, ALS, stroke, traumatic brain injury, epilepsy.

In another embodiment of the invention, screening assays for identifying agents which protect neurons from toxic insult are provided. An exemplary method entails providing a population of neuronal cells which are exposed to excitotoxic conditions.

The cells are then incubated in the presence and absence of the agent and the amount of cell death is determined. Agents which inhibit cell death should have efficacy in the prevention or treatment of conditions associated with neurodegeneration. The cells can also be assessed for other phenotypic and biochemical alterations, including, without limitation, activation of the FOXO3a signaling pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Triple-mutant FOXO3a is retained in the nucleus where it protects against excitotoxic insult and induces target gene expression in stressed neurons.

Panel A. Mixed spinal cord neurons were transfected using Lipofectamine 2000 (Invitrogen) with HA-tagged wild type FOXO3a (WT-F0X03a) or triple mutant FOXO3a (TM-FOXO3a) and 24 hours later fixed and immunostained with anti-HA antibody and Alexa 488 conjugated secondary antibody. In the left frame, WT-FOXO3a is present in neurites and the soma cytoplasm while the nucleus appears devoid of immunoreactivity. In the right frame, TM-FOXO3a is seen largely concentrated in the nucleus while soma and neurite staining is weak. Calibration bar = 24 microns. Panel B. Mixed spinal cord cultures were maintained for 14 DIV before infection with HSV-LacZ, HSV, WT-FOXO3a, HSV-TM-FOXO3a, HSV-KD-AKT or HSV-KD-SGK. 24 hrs later, the cultures underwent a 1 hr excitotoxic insult and 24 hrs after that, the cultures fixed and stained for SMI-32. Number of SMI32(+) motor neurons is noted on the ordinate axis. A statistically significant reduction in motor neuron number is seen in all groups except the cultured treated with HSV-TM-FOXO3a. Panel C. 14 DIV mixed spinal cord cultures were infected with HSV-LacZ, HSV-KD- Akt or HSV-SGK and 24 hrs. later lysates were prepared for western blotting for phosphoFOXO3a. Equal amounts of protein were loaded in each lane (concentration determined using BCA reagents, Pierce). There is a modest reduction in the phosphorylated species in the KD-AKT and KD-SGK lanes in comparison with actin control. Panel D. Western blots from 14 DIV mixed spinal cord cultures infected with either HSV-LacZ, HSV-WT-FOXO3a or HSV-TM- FOXO3a. Lysates were prepared either 24-28 hours after viral infection ("baseline") or 24-28 hours after viral infection and post 4 hour excitotoxic challenge ("p excitotoxic insult"). Histograms display the densitometric analysis of the autoradiograms; the values

on the ordinate axis represent fold-difference in comparison with LacZ controls (noted with horizontal dashed line). Above each histogram are representative images from blots. Only small differences are seen in the abundance of each of the proteins under the three conditions, pre-insult and none were statistically significant. Four hours after an excitotoxic insult, cultures infected with HSV-TM-FOXO3a displayed a prominent and statistically significant increase in the expression of p27 ^KIP1 , MnSOD and PLK.

Figure 2. Psammaplysene A (PA) drives FOXO3a into the neuronal nucleus and protects against the proteotoxicity of mutant SOD and mutant pl50 ^glued . Panel A. The chemical structure of PA. Panel B. 14 DIV spinal cord cultures were treated with PA or vehicle for 24 hrs prior to fixation and immunostaining for FOXO3a or the neuronal nuclear antigen recognized by anti-NeuN. Confocal images show that PA treatment leads to nuclear localization of FOXO3a and localization with NeuN immunoreactivity as seen in the merge image. In vehicle treated cells, FOXO3a is cytoplasmic (excluded from nucleus) and clearly distinct from the nuclear staining using anti-NeuN (merge image). Calibration bar = 7 microns. Panel C. Western blotting for various proteins after 24 hr treatment of 14 DIV mixed spinal cord cultures with PA. Drug treatment leads to an increase in the abundance of p27 ^KIP1 and a small increase in cyclin B abundance. No changes were noted in the abundance of the other proteins. Panel D. One set of 14 DIV mixed spinal cord cultures were infected with HSV-LacZ, HSV-WT-SOD or HSV-mutant SOD and a second set of cultures were infected with HSV-LacZ, HSV-WT-p 150 ^glued or HSV-mutant pl50 ^glued . Survival of SMI-32 stained motor neurons was determined 4 days later. Infected cultures were either treated with Vehicle ("- PA") or PA ("+ PA"). Mutant SOD led to a statistically significant reduction in motor neuron number (by ANOVA, see text for details) and this was prevented in PA treated cultures. Similarly mutant p 15O ^glued led to a statistically significant reduction in motor neuron number and this was prevented in PA treated cultures. * p < 0.05.

Figure 3. Mutant forms of SOD and pl50 ^glued inhibit the expression of some FOXO3a target genes and PA reverses these events. One set of 14 DIV mixed spinal cord cultures were infected with HSV-LacZ, HSV-WT-SOD or G87R SOD and a second

set of 14 DIV mixed spinal cord cultures were infected with HSV-LacZ, HSV-WT- pl50 ^glued or G59S pl50 ^glued . Two days later lysates were prepared for western blotting. Histograms display the densitometric analysis of the autoradiograms; the values on the ordinate axis represent fold-difference in comparison with LacZ controls (noted with horizontal dashed line). Above each histogram are representative images from blots. Mutant pl50 ^{g ue} led to an increase in the abundance of phosphoFOXO3a and an inhibition in the expression of p27 ^KIP1 (panel B) and cyclin B (panel D). Mutant SOD led to an increase in the abundance of phosphoFOXO3a and an inhibition in the expression of MnSOD and cyclin B. Results in the right column show spinal cord cultures were treated as above except that PA was administered at 14 DIV. No differences in the abundance of phosphoFOXO3a were seen in cultures expressing WT versus mutant pl50 ^glued or WT versus mutant SOD. In the mutant pl50 ^gIued expressing cultures, the drug treatment normalized cyclin B expression but did not reverse the depression of p27 ^KIP1 expression. In the mutant SOD expressing cultures, the drug treatment normalized MnSOD expression and led to an increase in the expression of cyclin B.

Figure 4. Effects of PA on the distribution of WT and mutant SOD in vitro; inhibition of FOXO3a signaling in presymptomatic mutant SOD expressing mice.

Panel A. Mixed spinal cord cultures infected with HSV-WT-SOD or HSV-G87R-SOD were treated with PA or vehicle for 3 days prior to fixation and immunostaining for human SOD. WT-SOD is homogenously distributed throughout the cytoplasm in the neuronal soma and processes in the absence of PA ("- PA") and in the presence of PA ("+ PA"). In contrast, G87R SOD assumes a punctate pattern of expression indicative of aggregates of the protein, both in the absence of PA ("- PA") and in the presence of PA ("+ PA"). Calibration bar = 20 microns. Below, using cultures infected with HSV-G87R- SOD, motor neurons are stained with SMI-32 (panel a-1) and anti-human SOD (panel b- 1). Aggregated mutant SOD is localized within motor neurons (panel c-1). Calibration bar = 15 microns. Panel B. Inhibition of F0X03a signaling in presymptomatic G93A SOD mice. Western blots of lumbar spinal cord lysates from 2 wild type (labeled 1 and 2) and G93A mutant SOD mice (labeled 1 and 2). Representative western blots show an

increase in the abundance of phosphoFOXO3a in the lysates from the mutant mice as well as a decrease in the abundance of MnSOD. No changes were noted in the abundance of p27 ^κπ>1 or actin. Quantification of these results is shown in the histograms below.

Figure 5. Psammaplysene A (PA) prevents necrotic death of C. elegans head neurons. glt-3;nuls5 worms were maintained under standard conditions and PA was added to the media. L = larval stage (e.g., Ll, L2, L3, and L4).

Figure 6. Biochemical evidence that Psammaplysene A promotes nuclear localization of FOXO3a. Subcellular fractionation of spinal cord neuron cultures treated with PA or vehicle was performed followed by western blot.

DETAILED DESCRIPTION OF THE INVENTION

Aging is hypothesized to be a risk factor for the development of adult-onset neurodegenerative diseases. The central pathologic event in ALS is the selective degeneration of motor neurons. The death of motor neurons results from excitotoxic insult or mutant gene expression. One problem that compounds the lack of understanding behind the signals that cause neurodegeneration in mammals is suppression of the molecular pathways which promote stress resistance and longevity.

The susceptibility of motor neurons to excitotoxic insults can be limited by enhancing the FOXO3a signaling pathway. This can be achieved by pharmacological manipulation using Psammaplysene A (PA). In addition to protecting motor neurons from excitotoxic insult, this agent also prevents toxicity that follows expression of mutant proteins in familial cases of ALS.

Thus, in accordance with the present invention, compositions and methods are provided which effectively enhance FOXO3a signaling, thereby inhibiting or preventing motor neuron death known to be relevant to ALS. The protective effects of PA have been demonstrated herein using tissue culture and animal models. PA may be used therapeutically in patients who suffer from neuronal damage including ALS.

For therapeutic use, the compounds of the invention may be administered in any

conventional dosage form in any conventional manner. Routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intrasynovial, infusion, sublingual, transdermal, oral, topical or inhalation via a nebulizer. The preferred mode of administration is intraventricular injection.

The compounds of this invention may be administered alone or in combination with adjuvants that enhance the stability of the therapeutic agent, facilitate its administration, enhance its activity, and the like. This includes the use of other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies. Compounds of the invention may be physically combined with the conventional therapeutics for the treatment of neurodegeneration (e.g., minocyclin, riluzole) or other adjuvants into a single pharmaceutical composition. Medications to help with the symptoms of ALS can also be used in combination therapy with PA-derivatives and compositions, for example, Baclofen, branched-chain amino acids, phenytoin, tricyclic antidepressants and antidepressants are commonly prescribed as ALS progresses. Advantageously, the compounds may then be administered together in a single dosage form. In some embodiments, the pharmaceutical compositions comprising such combinations of compounds contain at least about 15%, but more preferably at least about 20%, of a compound of the invention or a combination thereof. Alternatively, the compounds may be administered separately (either serially or in parallel). Separate dosing allows for greater flexibility in the dosing regime.

As mentioned above, dosage forms of the compounds of this invention include pharmaceutically acceptable carriers and adjuvants known to those of ordinary skill in the art. These carriers and adjuvants include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, buffer substances, water, salts or electrolytes and cellulose-based substances. Preferred dosage forms include, tablet, capsule, caplet, liquid, solution, suspension, emulsion, lozenges, syrup, reconstitutable powder, granule, suppository and transdermal patch. Methods for preparing such dosage forms are known (see, for example, H. C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th ed., Lea and Febiger (1990)). Dosage levels and

requirements are well-recognized in the art and may be selected by those of ordinary skill in the art from available methods and techniques suitable for a particular patient. In some embodiments, dosage levels range from about 10-1000 mg/dose for a 70 kg patient. Although one dose per day may be sufficient, up to 5 doses per day may be given. For oral doses, up to 2000 mg/day may be required. As the skilled artisan will appreciate, lower or higher doses may be required depending on particular factors. For instance, specific dosage and treatment regimens will depend on factors such as the patient's general health profile, the severity and course of the patient's disorder or disposition thereto, the patient's age, and the judgment of the treating physician.

Alternatively, a time release or slow release preparation may be utilized which allows for periodic or constant release of the therapeutic agent over a given time period. This method would allow for a single dose of the therapeutic agent in a given day. Methods for preparing such capsules are well known to those of skill in the art of drug delivery.

The following definitions are provided to facilitate and understanding of the present invention.

I. Definitions:

The following definitions are provided to facilitate an understanding of the present invention:

The phrase "excitotoxicity" as used herein refers to excessive activation of glutamate receptors in neurodegeneration which can be brought about, for example, with treatment of kainic acid.

The phrase "proteotoxicity" as used herein refers to neuronal death resulting from mutant form of genes, for example, mutated SOD or pl50 ^glued .

The phrase "FOXO3a signaling" as used herein refers to the mammalian signaling cascade which activates the gene expression of p27 ^KIP1 , MnSOD, cyclin B or PLK.

"Psammaplysene A" or "PA" as used herein refers to a marine sponge derived compound which prevents FOXO nuclear export by specifically inhibiting PI3-K/AKT signal transduction. This definition of PA also includes synthetic Psammaplysene A, also referred to as B6-7-1 (17).

A "derivative" or "analog" of a compound or a fragment thereof means a compound modified by varying the structure of the molecule, e.g. by manipulation of the side-chain residues or the compound itself. Such derivatives of the compound may involve insertion, addition, deletion or substitution at one or more positions of the molecule shown in Figure 2A.

As used herein, the term "administration" refers to the methods of delivery of the compounds of the invention (e.g., routes of administration such as, without limitation, intravenous, intramuscular, subcutaneous, intrasynovial, infusion, sublingual, transdermal, oral, topical or inhalation via a nebulizer).

As used herein, a "conventional ALS agent" refers to riluzole, minocyclin, Baclofen, branched-chain amino acids, phenytoin, tricyclic and general antidepressants.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the invention for performing a method of the invention. The instructional material of a kit of the invention can, for example, be affixed to a container which contains a kit of the invention to be shipped together with a container which contains the kit. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and kit be used cooperatively by the recipient.

II. Kits

Kits are provided for practicing the methods of the instant invention. The kits may comprise materials and reagents to facilitate the delivery of the therapeutic agent of the invention to a patient and instructional materials.

The following materials and methods are provided to facilitate practice of the present invention:

Source of Reagents: Trophic factors (ciliary neuronotrophic factor (CNTF), brain- derived neurotrophic factor (BDNF), neurotrophin 4 (NT 4), cardiotrophin 1 (CT 1) and glial-derived neurotrophic factor (GDNF)) were obtained from Alomone Labs

(Jerusalem, Israel). Psammaplysene A was synthesized as described (17). All other reagents were obtained from Sigma (St. Louis, MO) and were of the highest grade available.

Tissue Culture: Embryonic Sprague-dawley rat spinal cord neurons were grown on confluent monolayers of cortical astrocytes, as previously described (35). The substratum was acid washed glass coverslips when imaging was performed and Primaria tissue culture plasticware (Falcon, Becton-Dickinson) when biochemistry was performed. Astrocyte-conditioned culture media was supplemented with 10 ng/ml CNTF, BDNF, NT 4, CT 1 and GDNF and 50% of media was replaced with fresh media every 3 days.

Recombinant HSV: cDNAs were cloned into the PrpUC amplicon plasmid to generate recombinant HSV as previously described (36). The titer of virus used in these studies was routinely 3-5 x 10 ⁷ plaque-forming units/ml. The sources of constructs were: Michael Greenberg, Harvard University (HA-tagged wild type and triple mutant human FOXO3a), Philip Cohen, University of Dundee (DN-SGK), Thomas Francke, Columbia University (DN- Akt), David Borchelt, University of Florida (WT and mutant SOD), Erika Holzbaur, University of Pennsylvania (WT and mutant pl50 ^glued ).

Excitotoxicity Assay: After 14 days in vitro (DIV), culture media was removed (and saved) and cells were exposed to 100 μM kainic acid (KA) for 1 hour. Subsequently they were washed three times in Locke's buffer not containing KA, the original media was replaced and incubated for another 24 hours at 37 ⁰ C. in 5% CO ₂ before fixation in 4% paraformaldehyde. Motor neurons were identified in mixed culture by immunostaining for unphosphorylated neurofilaments and counting only labeled cells with a cell body that were 25 μm or greater. This method has previously been validated as a means of specifically recognizing motor neurons (Figure 1 in (H)). In experiments involving recombinant HSV, 1 μL of viral stock was added to ImL of culture media 24 hours or more prior to the next manipulation. Tubes containing viruses were color coded so that the operator was blinded to the specific virus used.

Quantification of motor neurons: The number of immunostained cells was counted in 3 randomly selected fields/coverslip and the mean value obtained. In each experiment 3+ independent coverslips were used per condition and the results presented were obtained for 4+ independent cultures and experiments.

Western Blots and quantification: Cultures were lysed in NP-40 lysis buffer (1% NP- 40, 40 mM Tris pH = 7.4, 0.15 M NaCl, 10% glycerol, 0,1% SDS, 0.1% deoxycholate + protease inhibitors and phosphatase inhibitors), sonicated, centrifuged to remove particulate matter, and subjected to PAGE-SDS prior to transfer to nitrocellose. Equal amounts of protein (determined using BCA reagents from Pierce) were loaded in each lane. After blocking in 5% milk in phosphate buffer saline, membranes were incubated in primary antibody overnight, washed, incubated with secondary antibody, washed and visualized to GE Healthcare (Buckinghamshire, U.K.) chemiluminescent substrate according to the manufacturer's directions. Densitometric analysis of films was obtained from 4+ independent experiments, the results averaged and mean values ± S.E. formed the basis of the statistical comparisons. Quantitative data on band intensity was expressed as the fold change in comparison with values of HSV-LacZ infected cultures. The displayed western blot data are representative of the results obtained from at least 4 independent cultures. The source of primary antibodies was: mouse anti-human cyclin B 1 and mouse anti-p27 ^KIP1 (BD Pharmingen, San Diego, CA), rabbit anti-human Plkl (Calbiochem, Oncogene Research Products, rabbit anti-MnSOD (Stressgen Bioreagents, Victoria, British Columbia, Canada), rabbit anti- FKHRLl/FOXO3a and anti-phospho FKHRLl/FOXO3a (Thr 32) (Upstate, Lake Placid, NY), rabbit anti-Bim (Affinity BioReagents, Golden, Colo), and rabbit anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA). Species-specific HRP-conjugated secondary antibodies were from Amersham (GE Healthcare, Buckinghamshire, U.K.) and Alexa 488-conjugated anti rabbit secondary antibody from Molecular Probes, Invitrogen (Eugene, OR).

Study of mice: B 6SJL-Tg(SOD 1-G93 A) lGur/J mice were obtained from Jackson labs and were bred to the Fl generation of C57B1/6 x SJL mice. The offspring of this cross was used for all of the biochemical studies (n=4 in each experimental group). Lumbar

spinal cord was obtained at P85, frozen on dry ice until use. The spinal cord segments were homogenized with a dounce in NP-40 buffer as above (10:1 volume to weight). All other manipulation of the lysates was as described tissue culture cells.

Immunocytochemistry: Tissue culture cells were fixed in freshly prepared 4% paraformaldehyde in 0.1 M pH 7.4 phosphate buffer for 30 minutes prior to extensive washing in phosphate buffered saline. Overnight incubation with primary antibody was performed at room temperature and after washing, coverslips were incubated with Alexafluor conjugated secondary antibody (2 - 4 hrs.). When double labeling experiments were performed, species-specific secondary antibodies with distinct emission spectra were employed. Coverslips were washed prior to mounting in PermaFluor (Thermo Electron Corporation) and viewing on an Olympus FV300 Fluoview laser confocal microscope.

Nematode studies: A C. elegans strain was generated by crosses ofnulsδ with glt-3 mutants. The nuls5 strain contains GFP expressed under the control of the GIuRl gene promoter and also expresses a hyperactive mutation). The glt-3 mutants is a null for the glt-3 glutamate transporter. The nuls5;glt3 worms show a reliable loss of GFP labeled head neurons through larval development. Worms were grown under standard conditions with or without PA added. Counts of head neurons in worms at various larval stages were performed.

Neuronal nuclei isolation and western blot: Nuclear and cytoplasmic fractions were prepared using CelLytic™ NuCLEAR™ Extraction Kit (Sigma, NXTRACT). Briefly, cultured cells were washed three times with PBS. Cells were then collected and incubated in 150μl (for 60 mm dish) hypotonic lysis buffer (1OmM HEPES, pH 7.9, 1.5mM MgCl ₂ and 1OmM KCl) containing protease inhibitors cocktail (Sigma P8340) on ice for 15 min. 10% NP-40 solution was added into the lysate to the final concentration of 0.6%. The mixture was vortexed vigorously for 10 sec. 50 μl lysate was transferred to a fresh tube. The remainder was subject to centrifuge for 30 sec. at 10,000-1 l,000g. The supernatant was transferred to a fresh tube as cytoplasmic portion and nuclear protein lysate was prepared by resuspending the pellet in lOOμl RIPA (Tris 5OmM pH7.4, ImM EDTA, 15OmM NaCl, 1% TritonX-100, 1% Sodium Deoxycholate, 0.1% SDS). The antibodies

and dilution used for Western blot are as followed: FOXO3a, BioVision 3673-100, 1:1000; Lamin, abeam, abl6048, 1:1000; Actin, Sigma, A 2066, 1:1000.

Statistics: Pairwise comparisons employed two-tailed Student's t-test and when three or more groups were compared Analysis of Variance (ANOVA) and post hoc analysis with significance set at p< 0.05 was utilized.

The following example is provided to illustrate an embodiment of the invention. It is not intended to limit the scope of the invention in any way.

EXAMPLE 1

In fibroblasts, expression of FOXO3a with mutations in three phosphorylation sites (Thr-32, Ser-253 and Ser-315) leads to nuclear retention of the transcriptionally active protein (6). Cultures of spinal cord neurons were infected with recombinant HSV engineered to express the triple mutant (TM) or wild type (WT) FOXO3a. Immuno- staining for the transgene demonstrated that the WT-FOXO3a is restricted to the neuronal cytoplasm and the TM-FOXO3a is largely nuclear (Figure IA). The transgenes were detectable in all neurons for >5days without any apparent toxicity. To determine if these transgenes influenced the susceptibility of motor neurons to excitotoxic insult, 14 days in vitro (DIV) mixed spinal cord cultures were infected with HSV- WT-FOXO3a, HSV- TM-FOXO3a, HSV-LacZ or no virus, and the following day exposed to an excitotoxic challenge (100 μM kainic acid (KA) or vehicle for Ih). The number of surviving motor neurons determined 24 hours later. KA led to statistically significant motor neuron death in all groups except neurons expressing TM-FOXO3a (F(s _, i ₂ ) = 15.68; p< 0.001, ANOVA, post-hoc analysis using Scheffe's F test, significance set atp < 0.05). Thus expression of the TM-FO XO3a protects motor neurons from excitotoxic insult.

Since Akt and SGK can phosphorylate FOXO3a and sequester the protein in the cytoplasm, kinase dead (KD- ) versions of these kinases (which can act in a dominant negative manner (12-14)) were tested to determine if they could block FOXO3a phosphorylation and be neuroprotective. Approximately 45 % of motor neurons were

killed by KA in cultures infected with HSV-LacZ, HSV-KD-Akt or HSV-KD-SGK (Figure IB) {F _(5J8) = 85.44, p < 0.0001, ANOVA and in post-hoc comparisons). To determine if KD-Akt and KD-SGK have redundant biochemical activities, their effect on FOXO3a phosphorylation was examined. Both KD-Akt and KD-SGK led to small reductions in the abundance of the phosphorylated species in comparison with LacZ but the effect was incomplete (Figure 1C). These results suggest that multiple FOXO3a kinases exist in neurons and that inhibiting a single FOXO3a kinase will not completely suppress FOXO3a phosphorylation. The limited capacity to block FOXO3a phosphorylation and thus permit its transcriptional activation may underlie the inability of KD-Akt or KD-SGK to protect motor neurons from insult.

The effect of WT-, TM-FOXO3a and LacZ on the expression of several FOXO3a target genes was determined. Surprisingly, the abundance of p27 ^KIP1 , MnSOD, cyclin B or PLK were indistinguishable in lysates from cultures expressing any of the 3 transgenes (Figure ID). To examine the possibility that the effects of the transgenes might become manifest under stressful circumstances, cultures were treated with recombinant HSV and 24 hours later subjected to an excitotoxic insult for 1 hour. Four hours later (before any neuron death occurs), lysates were prepared for western blotting. The combination of

KTP1

TM-FOXO3a + excitotoxic stress led to an increase in the abundance of p27 , MnSOD and PLK in comparison with LacZ + excitotoxic stress (figure ID). WT- FOXO3a + excitotoxic stress led to a modest increase in the abundance of p27 ^KIP1 and no change in the expression of the other proteins. Thus the effects of expressing TM- F0X03a on target genes depend upon the state of stress within neurons.

A chemical-genetic screen recently reported the identification of a series of compounds that can inhibit FOXOIa nuclear export. Compounds fell into two classes: 1) inhibitors of general nuclear export machinery and 2) inhibitors specific to the PI3- K/Akt/FOXOla pathway (15). Investigation into whether compounds in the second class would also block the nuclear export of FOXO3a was undertaken since they would be predicted, based on the results above, to display neuro-protective activity. Psammaplysene A (PA), isolated from marine sponge, was the focus of the study since it is the most potent of the class 2 inhibitors (Figure 2A) (16).

The effect of a synthetic sample of PA (i.e., B6-7-1) on the distribution of

FOXO3a endogenously expressed by neurons was examined (17). In vehicle treated cultures, FOXO3a was cytoplasmic and clearly excluded from the nucleus. In contrast, 24 hours treatment of cultures with 10 nM PA led to strong nuclear localization of FOXO3a (Figure 2B). Next, the effect of PA on the expression of several genes whose transcription is controlled by FOXO3a was explored. The abundance of several FOXO3a targets was increased by PA (such as cyclin B and p27 ^KIP1 ) but many others were unchanged (such as PLK, MnSOD) (Figure 2C). Bim (another FOXO3a target) was not detected in our cultures, and the abundance of phosphorylated FOXO3a itself was not altered by PA. These results indicate that PA leads to the nuclear retention of two members of the FOXO transcription factor family and promotes the expression, under basal conditions, of some of the FOXO transcriptome.

To determine if PA had neuroprotective activity, spinal cord cultures were treated with the drug (10 nM) for two days and then subjected to an excitotoxic challenge. The percent of KA induced cell death was 55 ± 4 % in vehicle treated cultures and 3 ± 1 % in PA treated cultures (p < 0.01, Student's t-test) indicating that PA protected motor neurons from excitotoxic challenge. Recently it was found that expressing mutant forms of SOD or pl50 ^glued in neurons (but not their wild type counterparts) leads to a time-dependent motor neuron death (18). In light of this finding, examination was conducted to determine whether PA blocked the proteotoxicity of these mutant proteins. Spinal cord cultures were infected with HSV engineered to express the WT or mutant forms of SOD or the WT or mutant forms of pl50 ^glued and received PA (or vehicle) every other day for 4 days. The drug treatment had no effect on transgene expression. After 4 days, the cultures were fixed and motor neuron number was determined. ANOVA revealed statistically significant difference between groups in LacZ versus WT SOD versus mutant SOD (± PA) comparisons (F ₍₅ j ₂₎ = 18.41, p < 0.001) as well as LacZ versus WT pl50 ^glued versus mutant 150 ^glued (± PA) comparisons (F _(5J2 ) = 19.26, p < 0.001) (Figure 2D). The post hoc analysis revealed that statistically significant protection against the toxicity of mutant SOD or pl50 ^glued was conferred by drug treatment on motor neurons survival. PA had no adverse effect on survival on motor neurons expressing LacZ or wild type versions of SOD or pl50 ^glued . Thus, PA protected against three different insults that are directly relevant to motor neuron death in ALS.

If the proteotoxicity of mutant SOD or pl50 ^glued is linked to alterations in aging pathway signaling, one might anticipate that these toxic proteins lead to suppression of FOXO3a-dependent gene expression. To the extent that the neuroprotective activity of PA is mediated by influencing FOXO3a activity, this agent might de-repress these biochemical events. To explore these issues, the expression of some FOXO3a target genes from cultures expressing WT or mutant SOD or pl50 ^glued in the absence of PA (when motor neurons die) and in its presence were monitored. Spinal cord cultures were infected with either with HSV-LacZ, HSV-WT-SOD or HSV-G87R-SOD or HSV-LacZ, HSV-WT-pl50 ^glued or HSV-G59S-pl50 ^glued and received PA (or vehicle) for two days. At this point, no motor neuron death occurs (18). Western blots for phosphorylated FOXO3a revealed a marked induction of this species in cultures expressing G87R-SOD or G59S-pl50 ^glued (Figure 3). A more modest level of ρhosphoFOXO3a was induced by the wild type protein in comparison with the LacZ control. These findings suggest that ALS-causing mutant proteins might stimulate kinases that phosphorylate FOXO3a and inactive it, and are consistent with the observed increased PI3-K activity in spinal cords of ALS patients (19) and reduced PTEN expression in mouse ALS tissue (20). PA treatment blocked the mutant protein-induced rise in phosphoFOXO3a, suggesting that the drug alleviates proteotoxic cellular stress and results in less stimulation of FOXO3a kinases.

The expression of some FOXO3a target genes was studied (Figure 3), and expression of G87R-SOD led to a marked reduction in the abundance of MnSOD (panel C) and a less prominent reduction the abundance of cyclin B (panel D). No significant changes in the abundance of p27 ^κπ>1 (panel B) or PLK (panel E) were noted. Figure 3 shows expression of G59S-pl50 ^glued led to a marked reduction in the abundance of p27 ^KIP1 (panel B) and a less prominent reduction the abundance of cyclin B (panel D). No significant changes in the abundance of MnSOD (panel C) or PLK (panel E) were noted. Thus, alterations in the expression of FOXO3a target genes are evoked by the expression of ALS-causing mutant proteins, but the specific alterations in protein expression is context-dependent in that they depend on the particular mutant protein expressed.

The effects of PA treatment on these changes in protein expression were

subsequently examined. The prominent reduction in MnSOD (panel C) expression in G87R-SOD expressing cultures was completely prevented by PA treatment, as was the suppression of cyclin B (panel D) expression (Figure 3). In addition, PA evoked a small increase in PLK expression (panel E) over the level seen in cultures expressing Lac Z or WT-SOD. The effects of PA on MnSOD expression are particularly interesting in light of the recent report of impaired mitochondrial anti-oxidant defense in SODl -related motor neuron death and its amelioration by the antioxidant ebselen (21).

Unexpectedly, the prominent reduction in p27 ^KU>1 in cultures expressing G59S pl5O ^glued was not reversed by PA treatment (Figure 3, panel B). Instead the major drug- treatment evoked change was a normalization of the abundance of cyclin B (panel D). No alterations in MnSOD (panel C) or PLK (panel E) were noted. Thus, PA treatment does not reverse all the alterations in gene expression that follow from mutant pl5O ^glued expression. In addition, the effects of PA on gene expression differ as a function of the mutant protein expressed, indicating that the nature of the stressful stimulus dictates its activity on F0X03a-regulated gene expression.

Insoluble aggregates of mutant SOD are detectable within cells from transgenic mice engineered to express mutant SOD (22, 23). To determine if WT or mutant SOD aggregated in neurons in vitro and if treating cultures with PA influenced the cellular distribution of transgene human SOD, immunocytological experiments were performed. Detection of human SOD in cultures infected with HSV-WT-SOD revealed that the protein is homogeneously distributed throughout the cytoplasm and extends centrifugally for >100 microns into axons and dendrites (Figure 4A). In contrast, in cultures infected with HSV- G87R, human SOD immunoreactivity is concentrated into puncta (the cytological signature of insoluble aggregated proteins) in the soma cytoplasm and neurites. Double labeling studies reveal that mutant SOD punta are present in motor neurons as well as non-motor neurons in our cultures (Figure 4A, panel c-1). Treatment of cultures with PA had no effect on the subcellular distribution of human SOD in cultures infected with either of the recombinant HSVs. Although the pathophysiological significance of aggregated protein is controversial, these results indicate that the neuroprotective action PA is dissociable from the accumulation of aggregated mutant SOD. A similar observation has been made in C. elegans wherein Daf-16 protects

against Aβ].4 ₂ toxicity but does not influence the accumulation of protein aggregates (24).

Next, lysates from spinal cords of mice expressing the G93A mutant form of human SOD or wild type controls were examined for the expression of phosphoFOXO3a and target genes. The mice were 87 days old, a time when they are asymptomatic in terms of weakness, but do manifest other subtle abnormalities. A consistent increase in phosphoFOXO3a was seen in the G93A mice in comparison with the wild type animals (n=4 in each experimental group) (Figure 4B). This was associated with a reduction in MnSOD in the G93A mice in comparison with the wild type animals. No differences were noted in the abundance of p27 ^KIP1 in the mutant versus wild type animals. Thus a reduction in the abundance of a FOXO3a transcriptional target was detected in the spinal cord of pre-symptomatic mutant SOD mice, further supporting the notion that the disease process is intimately associated with inhibition of the FOXO3a stress resistance/longevity program.

The effects of PA on neuron protection in a worm model system of necrotic death was also performed (Figure 5). Counts of head neurons at various larval stages (Ll, L2, L3, and L4) reveal a substantial neuroprotective effect of PA particularly at the L2 and L3 stages. In another set of experiments, nuclei were isolated from neurons in vitro which had been treated with PA or vehicle control (Figure 6). Western blot of the nuclear fraction for FOXO3a shows that PA treatment leads in an increase in nuclear FOXO3a. There is no drug effect on total FOXO3a abundance. The nuclear protein lamin is enriched in the nuclear fraction (as expected) and absent from the cytoplasmic fractions. The abundance of total lamin and actin is unaffected by PA treatment.

Discussion

The conserved biochemical pathway that regulates longevity in yeast, C. elegans, Drosophila melanogaster and mice also plays a fundamental role in resistance to stresses such as UV radiation, oxidative conditions, heat shock and misfolded and aggregation- prone proteins (25). Two transcription factors, Heat-shock factor 1 and Daf-16/FOXO3a, are essential mediators of this longevity/stress resistance program in worms (26, 27). Here, it was demonstrated that genetic and pharmacological maneuvers that enhance FOXO3a signaling protect mammalian motor neurons from 3 insults directly relevant to ALS. Furthermore, it was determined that prior to inducing neuronal death, mutant

versions of SOD and pl50 ^glued lead to alterations in FOXO3a phosphorylation (and thus activity) and target gene expression. These observations suggest that the pathophysiological events in ALS involve inhibition of FOXO3a-dependent gene expression and reversal of these alterations prevents the demise of motor neurons.

Mitochondrial dysfunction occurs in sporadic and familial ALS, and is accompanied by evidence for oxidative modification of macromolecules (28). The pathogenic role of disturbances in mitochondrial bioenergetics is supported by the observation that mutant SOD-suppression of several mitochondrial proteins including peroxiredoxin 3 (Prx3), a thioredoxin-dependent hydroperoxidase that reduces H ₂ O ₂ to H ₂ O in mitochondria (21). Here, it was shown that the expression of FOXO3a target MnSOD is suppressed in neurons expressing G87R SOD and the neuroprotective activity of PA treatment is associated with a de-repression of MnSOD expression. Therapies directed towards limiting oxidative stress such as free radical traps or other types of antioxidants are neuro-protective reinforcing the idea that mitochondrial dysfunction and oxidative stress are fundamental problems in ALS (2, 29).

Inactivation of forkhead transcription factors in Gl allows for cell cycle entry and aberrant entry into the cell cycle has been seen in pathological tissue samples from Alzheimer's Disease and ALS patients (30-32). Alterations in the expression of several cyclins (e.g., E, C and Al) have been found in postmortem spinal cords from ALS victims (33). p27 ^KIP1 expression, which is suppressed in G59S-pl50 ^glued -expressing neurons, is a cyclin-dependent kinase inhibitor that can bind to and inhibit the activity Cyclin D-Cdk4, Cyclin E-Cdk2 and Cyclin A-Cdk2 (34). Deregulation of Cdk4 in G37R SOD mice has been linked to aberrant signaling at the Gl-S checkpoint and apoptosis (30), a process that could be facilitated by the finding that the mutant protein-evoked reduction in p27 ^KIP1 expression. While PA did not reverse the suppression of p27 ^KIP1 in G59S-pl50 ^glued -expressing neurons, p27 ^KIP1 was upregulated in TM-FOXO3a expressing neurons after excitotoxic stress. A comprehensive analysis of the expression of cyclin- dependent kinases and cyclin regulatory subunits (many of which are known to be FOXO3a substrates) under conditions of stress and stress-resistance may provide insight into the pathogenic role of abortive attempts of neurons to re-enter the cell cycle. FOXO transcription factors have remarkable variety of activities (some antipodal) and their

unique capacity to regulate processes that are aberrant in ALS (i.e., oxidative stress and abortive entry into the cell cycle) make them attractive therapeutic targets.

EXAMPLE II

To further validate the activity of the synthetic Psammaplysene A (i.e., B6-7-1) composition of the invention in vivo, non-human animal models, for example, murine, rat, zebrafish and primate models, can be used. In one aspect of in vivo testing, the compositions of the invention can be injected into animal, and assessed for the presence of the composition in the brain. As another model of in vivo assessment, the synthetic Psammaplysene A compositions of the invention could be conjugated with other moieties to facilitate entry into the central nervous system. For example, the compositions of the invention could be conjugated to a herpesvirus protein which confers neurotropic targeting and facilitates passage of the compositions across the BBB. The compositions of the invention can also be delivered to the brain via nanoparticles.

Surgical method for delivering pharmaceutical agents (e.g., B6-7-1) to the brain is also possible; for example, intra-cranial injection or delivery utilizing a cannula device are suitable methods. Briefly, for cannula-delivery, a burr hole will be placed in the cranium. Next, a cannula device is secured within the burr hole; the cannula device provides multiple points of access for the insertion of surgical catheters. In the next step, a surgical catheter could be passed through the cannula device in the vicinity of the neurons at a variety of angles. Lastly, pharmaceutical agents will be delivered to the brain through the surgical catheters. Suitable methods are described in U.S. Patent 5,792,110 and U.S. Patent Application 20080004630, both of which are incorporated herein by reference.

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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. It will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope of the

present invention, as set forth in the following claims.