THERAPY This application claims priority of U.S. Provisional Application No. 61/412,385, filed November 10, 2010, the content of which is hereby incorporated by reference in its entirety .

Throughout this application, certain patents and publications are referenced, the latter by authors, journal citation and publication date. The disclosures of these patents and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention relates.

FIELD OF INVENTION

The present invention provides methods relating to use of glycine uptake inhibitors for treating Parkinson' s disease (PD) .

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) is the second most common neurodegenerative disorder, with a life-time incidence of 1- 2%. As it is a disease of older people, it will become increasingly common as life expectancy increases. Parkinson's disease is named after James Parkinson, who was first to describe the disease in his 1817 assay "An Essay on the Shaking Palsy". PD is a chronic progressive neurodegenerative disorder. The symptoms of Parkinson's disease result from the loss of dopamine-producing neurons in the substantia nigra pars compacta (SNpc) region of the brain. The four primary symptoms of PD are 1) tremor, or trembling in hands, arms, legs, jaw, and face; 2) rigidity, or stiffness of the limbs and trunk; 3) bradykinesia, or slowness of movement; and 4) postural instability, or impaired balance and coordination. For a more thorough review, see Jankovic, 2008, J Neurol Neurosurg Psychiatry, 79: 368-376.

PD usually affects people over the age of 50. Early symptoms are subtle and occur gradually. In some people the disease progresses more quickly than in others. As these symptoms become more pronounced, patients may have difficulty walking, talking, or completing other simple tasks. As the disease progresses, the shaking, or tremor, which affects the majority of PD patients may begin to interfere with daily activities.

Other symptoms of PD may include depression and other emotional changes; difficulty in swallowing, chewing, and speaking; urinary problems or constipation; skin problems; and sleep disruptions.

PD is diagnosed based on clinical criteria; currently no blood or laboratory tests have been proven to help in diagnosing sporadic PD. Diagnoistic criteria have been developed by the National Institute of Neurological Disorders and Stroke (NINDS) . Gelb et al . , 1999, Arch Neurol, 56: 33-39. Doctors may sometimes request brain scans or laboratory tests in order to rule out other diseases.

There is no effective cure for PD, or any means of slowing or stopping disease progression. Present therapies for PD focus on symptomatic relief rather than treating the underlying cause for the disease. Usually, patients are given levodopa combined with carbidopa, which delays the conversion of levodopa into dopamine until it reaches the brain where the nerve cells can then use levodopa to synthesize dopamine. Although levodopa helps at least three-quarters of PD cases, not all symptoms respond equally to the drug. Bradykinesia and rigidity respond best, while tremor may be only marginally reduced. Problems with balance and other symptoms may not be alleviated at all.

Anticholinergics may help control tremor and rigidity. Other drugs, such as bromocriptine, pramipexole, and ropinirole, mimic the role of dopamine in the brain, causing the neurons to react as they would to dopamine. An antiviral drug, amantadine, also appears to reduce symptoms. In May 2006, the FDA approved rasagiline to be used along with levodopa for patients with advanced PD or as a single-drug treatment for early PD. Other treatments for PD, including gene therapy, are currently under investigation.

SUMMARY OF THE INVENTION

The present invention provides a method for promoting functional regeneration and sprouting of striatal dopaminergic fibers which comprises contacting cells with an agent which increases extracellular glycine level so as to enhance NMDA receptor-dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers.

This invention also provides a method for treating a disease comprises administering to a subject a compound which increases extracellular glycine level so as to enhance NMDA receptor-dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers.

This invention further provides a use of glycine uptake inhibitors in the preparation of a medicament for promoting functional regeneration and sprouting of striatal dopaminergic fibers wherein the medicament increases extracellular glycine level so as to enhance NMDA receptor-dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers. This invention even further provides a use of glycine uptake inhibitors in the preparation of a medicament for treating a disease wherein the medicament increases extracellular glycine level so as to enhance NMDA receptor-dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers. BRIEF DESCRIPTION OF THE FIGURES

Figure 1.

Figure 1A Overlay of axonal growth cone images taken at 0 and 60' time points (left) and following superfusion (right) with glutamate/0Mg ²⁺ for 3 min (70') and one hour later (130')· The graph below shows the shift to faster growth rates in response to 200uM glutamate/0Mg ²⁺ as well as to the NMDA receptor agonist tetrazol-glycine (20uM) .

Figure IB Images of a growth cone before and following a 3 min AMPA (50uM) stimulus. The graph shows the shift to slower rates in response to AMPA, as well as to glutamate/0Mg ²⁺ in the presence of AP-5 (50uM) , an NMDA receptor antagonist. (All treatments are significantly different from controls with p<0.01, Kolgomorow-Smirnov test, scale bar: 20um) . Figure 2.

Figure 2A Distribution of growth rate change in response to glutamate/0Mg ²⁺ (light green circles) and to glutamate/0Mg ²⁺ in the presence of the CaMKII inhibitor KN93 (light blue triangles) or its inactive form KN92 (dark green diamonds) . Figure 2B Distribution of growth rate change in response to AMPA (light red circles) and to glutamate/0Mg ²⁺ in the presence of the NMDA receptor AP-5 (dark red diamonds) , and for both stimuli in the presence of the calcineurin inhibitor cyclosporine A (light blue circles for AMPA, dark blue diamonds for glutamate/0Mg ²⁺ plus AP-5) . (Green and red traces are significantly different from controls with p<0.01, blue traces are not . ) Figure 3. RWI-D-132-1 Promotes Dopaminergic Sprouting In the Dorsal Striatum

Figure 3A Coronal striatal sections immune-stained for the dopamine transporter (DAT) . Three weeks after the 6-OHDA lesion the dorsal striatum was almost completely void of DAT- stained fibers, while the ventral striatum was spared. Four weeks later DAT-stained fibers reappeared in the dorsal striatum in controls (3+4 weeks) . Markedly, in RWI-D-132-1- treated mice the density of DAT stained fibers was substantially increased.

Figure 3B The density of DAT-stained fibers was expressed as region coverage in ~6 , normalized to control hemispheres in 4 regions in dorso-ventral direction. The region examined in each set of bars is indicated directly below as the red oval. As seen in the fourth bar group, three weeks after the lesion (red bars, n=4), approximately 40% of fibers were still preserved in that region. The largest difference between untreated (blue, n=5) and treated (green, n=5) mice was seen in the second to ventral-most region, but all regions showed significantly increased innervations with glycine uptake inhibitors .

Figure 4. RWI-D-132-1 Treatment Enhances Dopamine Release and Uptake Seven weeks following the 6-OHDA injection living brain slices of untreated and treated mice were prepared for cyclic voltammetry recordings of electrically evoked dopamine release .

Figure 4A Dopamine release was recorded at three sites in control and lesioned hemispheres as indicated. Example of signals evoked by a single electrical pulse are shown for the control and lesioned side (untreated) . The peak amplitude of the signal reflects release and uptake parameters (peak amplitude is higher with more release and less uptake) whereas the falling phase (half-life) mostly reflects uptake kinetics (wider with less uptake) .

Figure 4B Quantification of peak amplitude and half-life for signals recorded at the middle site: there was a trend to higher signal amplitudes and shorter half-life in the treated group (n=4 in each group) .

Figure 5. Enhanced Improvement In Mice Treated With Glycine Transport-1 Inhibitor

Figure 5A Illustration of mice behavior test.

Figure 5B A scatter plot of data of untreated and treated mice at 3 and 7 weeks. For all mice there was improvement, i.e. less lateralization, between the 3- and 7-week time points. There was a clear difference between untreated (open triangles) and treated (filled circles) mice. The average improvement in lateralization between 3 and 7 weeks was 18.5% in controlled mice, and 33.8% in treated mice.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides for a method for promoting functional regeneration and sprouting of striatal dopaminergic fibers which comprises contacting cells with an agent which increases extracellular glycine level so as to enhance NMDA receptor- dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers.

In one embodiment, the cells are substantia nigra neurons.

In one embodiment, the agent is a glycine uptake inhibitor. In one embodiment, the glycine uptake inhibitor is (R)-(N[3- ( 4 ' -fluorophenyl ) -3- ( 4 ' -phenylphenoxy) propyl] ) sarcosine.

In one embodiment, the glycine uptake inhibitor is 4-Phenyl piperdine sulfonyl.

In one embodiment, functional dopaminergic innervation of striatum is restored.

This invention also provides for a method for treating a disease comprises administering to a subject a compound which increases extracellular glycine level so as to enhance NMDA receptor-dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers.

In one embodiment, the disease is Parkinson's disease.

In one embodiment, the compound comprises a glycine uptake inhibitor and a carrier.

In one embodiment, the compound comprises a therapeutically effective amount of the glycine uptake inhibitor and a pharmaceutically acceptable carrier.

In one embodiment, the glycine uptake inhibitor is (R)-(N[3- ( 4 ' -fluorophenyl ) -3- ( 4 ' -phenylphenoxy) propyl] ) sarcosine. In one embodiment, the glycine uptake inhibitor is 4-Phenyl piperdine sulfonyl.

In one embodiment, the compound comprises lOmg of the glycine uptake inhibitor per kg of the body weight of the subject. In one embodiment, the compound is administered every other day .

In one embodiment, the glycine uptake inhibitor increases glycine level in prefrontal cortex of the subject by at least 40% at 60-90 minutes after administration. This invention further provides a use of glycine uptake inhibitors in the preparation of a medicament for promoting functional regeneration and sprouting of striatal dopaminergic fibers wherein the medicament increases extracellular glycine level so as to enhance NMDA receptor-dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers.

This invention even further provides a use of glycine uptake inhibitors in the preparation of a medicament for treating a disease wherein the medicament increases extracellular glycine level so as to enhance NMDA receptor-dependent transmission, thereby promoting functional regeneration and sprouting of striatal dopaminergic fibers.

The above embodiments are generally applicable to methods and uses provided by this invention. This invention is illustrated in the Experimental Details Section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to limit in any way the invention as set forth in the claims which follow thereafter. EXPERIMENTAL DETAILS

Example 1. Ca Imaging in Dopamine Neuron Culture System

We have found that in postnatal dopaminergic neuron cultures axonal growth rates increased up to 4 fold in response to a 3 min stimulus that activated NMDA receptors (both: 200uM glutamate in Mg ²⁺ -free Tyrodes's (glutamate/0Mg ²⁺ ) to relieve the Mg ²⁺ block of NMDA receptors, and an NMDA receptor agonist, see Fig. 1A) . In contrast, stimulation of AMPA receptors alone led to a halt of axonal growth (Fig. IB) . The growth acceleration in response to NMDA agonists was blocked by CaMKII inhibitor (Fig. 2A) , while the growth- halting action of AMPA receptor agonists was blocked by calcineurin inhibitors (Fig. 2B) . Calcium imaging revealed that the calcium signals in growth cones in response to NMDA receptor activation were of high amplitude and short lasting, while in response to AMPA receptor activation the signals were of lower amplitude but longer lasting (data not shown) . To complete our in vitro findings, we test whether the calcium signals in dopaminergic growth cones in response to NMDA or AMPA stimulation, are entirely due to calcium influx or whether calcium-induced calcium release from intracellular stores is also involved.

Ventral midbrain cultures are prepared from TH-GFP mice, cultured for 5-9 days, incubated with the ratiometric calcium- indicator fura-2 for 30 minutes, and transferred into imaging chambers. Cultures are stimulated for 3 minutes with either glutamate in Mg ²⁺ -free Tyrodes's (glutamate/0Mg ²⁺ ) or with AMPA with or without prior depletion of the Ca ²⁺ stores using caffeine, the SERCA inhibitor thapsigargin, or ryanodine. Calcium signals are acquired at 0.5 Hz for 2 minutes prior, 3 minutes during, and 5 minutes following the stimulus. The results of this experiment reveal the origin of the calcium signals observed in response to NMDA and AMPA receptor activation that determine whether axonal growth is accelerated or inhibited.

Example 2: Effects of Dopaminergic Cells' NMDA Receptors on Developmental Innervation of the Striatum In Vivo

To investigate whether NMDA receptors on dopaminergic cells have an influence on the developmental innervations of the striatum in vivo, we obtained NR1 receptor flox/flox mice from Dr. Susumu Tonegawa, which we crossed with DAT-Cre mice to obtain animals that lack the NMDA receptor only in dopaminergic cells. We have obtained animals with the desired genotype: NRlf/f DAT-Cre +/-. Striatal sections from brains of adult mice are immunostained for TH and assessed stereoligically for changes in the dopaminergic innervations density .

To examine the nigrostriatal projection using a functional approach, we also measure evoked dopamine release and uptake in striatal brain slices from these animals using cyclic voltammetry recordings (Schmitz et al . 2001. J Neurosci, 21, 5916) .

For stereology, at least 3 animals from each genotype (NRlf/f DAT-Cre +/- and DAT-Cre +/-) are used; for cyclic voltammetry recordings about 6 animals are used.

We find a sparser dopaminergic innervation of the striatum in the NR1 knockout mice with reduced dopamine release. This is in agreement with our in vitro data. Example 3: Enhancing NMDA Receptor-Dependent Transmission via Glycine Uptake Inhibitor To Promote Regeneration of Dopaminergic Nigrostriatal Fibers

Our in vitro data demonstrate that NMDA receptor activation stimulates axonal growth in dopaminergic neurons. NMDA agonists, however, can cause neurotoxicity and so a different approach has been pursued to enhance NMDA signaling in vivo, which is to raise the extracellular level of glycine, a co- agonist of the NMDA receptor, by blocking glycine uptake transporters (Lechner, 2006, Curr Opin Pharmacol , 6, 75; Lindsley et al . , 2006, Curr Top Med Chem, 6, 1883).

The glycine transporter-1 (GlyT-1) inhibitor, (R) - (N [ 3- (4 ' - fluorophenyl ) -3- ( 4 ' -phenylphenoxy) propyl ] ) sarcosine (NFPS) has been tested in small-scale trials, and has been found to improve positive, negative, cognitive and general psychiatric symptoms in schizophrenic patients with no significant side effects (Tsai et al . , 2004, Biol Psychiatry, 55, 452; Lane et al., 2005, Arch Gen Psychiatry, 62, 1196; Lane et al., 2008, Biol Psychiatry, 63, 9) . In mice, NFPS has been used in numerous studies (by oral, intraperitoneal and subcutaneous applications) and dosing, timing and duration of its effects have already been fully characterized (Atkinson et al . , 2001, Mol Pharmacol, 60, 1414; Hashimoto et al . , 2007, Eur Neuropsychopharmacol . in press; Kinney et al . , 2003, J Neurosci , 23, 7586) . Other examples of glycine uptake inhibitors include, but are not limited to, those compounds disclosed in U.S. Patent Application Publication No. 2007/0105902, PCT International Publication No. WO/2005/014563, and PCT International Publication No. WO/1997/020553, all of which are herein incorporated by reference . We use the 6-OHDA mouse model of Parkinson' s disease to validate whether NMDA receptor activity has an effect on regenerative sprouting of dopaminergic neuritis in vivo. Our collaborator, Dr. Robert Burke, is a prominent expert in the used of the 6-OHDA model, and assists with the injections and the analysis of the sections (Burke et al . , 1990, J Neurosci Methods, 35, 63) . Work on this model in rats has shown that striatal axon terminal loss reaches its peak at 4 weeks after lesioning. For lesion sizes that comprise more than 75% of substantia nigra (SN) cell bodies, no regenerative sprouting has been detected. For smaller lesions, however, dopaminergic reinnervation of the striatum occurred within 12 weeks after the peak of terminal loss, reaching an innervations density close to normal (Stanic et al . , 2003, Eur J Neurosci, 18, 1175) .

We study the effect of NFPS treatment on regeneration for different lesions sizes.

We determine the optimal 6-OHDA doses to achieve lesion sizes ranging from 0-30%, 30-70%, and >75%, and the optimal time point post-lesioning to assess recovery of the striatal innervations. Wild-type mice (WT) are injected with three different doses of 6-OHDA into the SNpc (between 5-20ug (Jeon et al . , 1995, Neurodegeneration, 4, 131)) and analyzed for cell body loss in the SNpc (using both neutral red stain for all cell bodies and TH stain for cell bodies expressing TH) and terminal loss in the striatum at 4, 12 and 16 weeks after lesioning (3 animals per dose and time point, total n=27) . If the lesions size or recovery times are inadequate, doses and/or time points are adjusted. Mice of both genotypes are injected with 3 previously determined doses of 6-OHDA (n=6 per dose) to achieve lesions of 0-30%, 30-70%, and >70%. The control group receives i.p. injections of saline, the treatment groups receive i.p. injections of NFPS (lOmg/kg) every other day starting at the peak of axonal loss in the striatum until the time at which about 50% of recovery is achieved (about 4 weeks in rats) . This dose of NFPS increases glycine levels in the prefrontal cortex by 40% at 60-90 min after oral administration (Atkinson et al., 2001, Mol Pharmacol, 60, 1414). The half life of NFPS exceeds 24 hours. By that time NFPS levels has only dropped to 80% of the peak concentration that has been found 5 hours following subcutaneous injection (Liu et al . , 2005, J Pharmacol Exp Ther, 313, 1254) .

There are 4 groups: 1. WT untreated; 2. WT treated; 3. NR1 KO untreated; and 4. NR1 KO treated. (Each group 3 doses: 3x6 animals, total of 36 WT and 36 KO mice) . We assess cell body numbers in the SNpc by neutral red and TH-stain to estimate the lesion size, and axon terminal density in the striatum by TH stain (previous work has shown that the outcome of TH versus DAT immunostaining produced identical results.)

We find that recovery of striatal dopaminergic innervations is accelerated and enhanced in the WT NFPS-treated group compared to WT saline-injected group. We also find that recovery is delayed and less complete in the NRl-untreated mice compared to WT-untreated mice. We further find that NFPS treatment in NR1 KO mice has no effect comparing to saline-injected KO mice.

When effects of NFPS treatment on the regeneration of the dopaminergic striatal innervations are established morphologically, we assess the functional regeneration by using CV recordings of dopamine release and uptake from striatal slices of NFPS-treated versus untreated mice. DISCUSSION

We are developing a new approach for Parkinson' s disease therapy: rather than focusing on cell survival, we are developing an approach to promote reinnervation of the brain areas that are depleted of dopamine from adjacent areas that survive in the disease. We found that glycine uptake inhibitors promote the dopaminergic reinnervation of dopamine depleted brain areas in a toxin-based mouse model of PD.

Glycine uptake inhibitors have been developed to date for treatment of schizophrenia, as there is evidence that inhibition of NMDA may be involved in the negative symptoms of that disease. Many companies have developed or are developing different classes of glycine uptake inhibitors intended as treatments for schizophrenia. However, to our knowledge none are testing these drugs for treatment of PD. The rationale for using glycine uptake inhibitors for treatment of Parkinson's disease comes from our recent work in the area.

While relatively close correlation between cell body loss in the substantia nigra (SN) and axon terminal loss in the putamen suggests a lack of compensatory sprouting (Porritt et al . , 2005 Mov Disord, 20, 810), compensatory sprouting and regeneration has been observed in toxin-based animal models using mice and macaques. Moreover, anecdotal evidence obtained from the GDNF Amgen trial indicates that dopaminergic sprouting can be induced in PD patients by growth promoting factors (Love et al . , 2005, Nat Med, 11, 703) . To achieve a functional restoration of the dopaminergic innervations of the striatum, sprouting needs to be directed to the right target, as undirected sprouting may cause adverse effects and not restore normal function. During late development, synaptic connections are shaped by activity and neurotransmitter release (Spitzer, 2006, Nature, 444, 707). Dopaminergic axon terminals express glutamate receptors and their major striatal target is the glutamatergic cortico-striatal synapse (Gracy, 1996, Brain Res, 739, 169) .

We tested initially whether axonal growth of dopaminergic neurons was affected by glutamate input in an in vitro cell culture system of postnatal ventral midbrain neurons. These experiments revealed that axonal growth in dopaminergic neurons responded to glutamate input in two ways. When NMDA receptors were activated together with AMPA receptors axonal growth accelerated. If only AMPA receptors were activated axonal growth was inhibited. Calcium imaging demonstrated that stimuli that activated AMPA and NMDA receptors produced a rise in calcium levels that was high and relatively short- lasting, whereas stimulation of AMPA receptors alone evoked a low amplitude, long-lasting calcium signal.

Additional experiments showed that growth acceleration induced by NMDA receptor activation involved a CaMKII-dependent pathway, whereas AMPA receptor-induced growth inhibition involved a calcineurin-dependent pathway. Thus our data show that enhancement of NMDA receptor-dependent transmission promotes sprouting of dopaminergic axons via a CaMKII- dependent pathway.

We investigate whether NMDA receptor activity has an influence on dopaminergic projection in vivo by using a dopamine neuron- specific NR1 receptor KO mouse, and test whether NMDA receptor activity influences the regeneration of dopaminergic fibers. A way of enhancing NMDA receptor activity in vivo without causing neurotoxicity is to increase extracellular levels of glycine (an NMDA receptor co-agonist) via inhibitors of glycine uptake, an approach that has already been used in animal models and in small-scale trials on schizophrenia in humans. Using the 6-OHDA-mouse model of PD in which regeneration of the nigrastriatal projection can be observed for lesions smaller than 75% of dopaminergic SN neurons, we determine that the glycine transporter-1 (GlyT-1) inhibitor, (R) - (N[3- ( 4 ' -fluorophenyl ) -3- ( 4 ' -phenylphenoxy) propyl ] ) sarcosine (NFPS) promotes functional regeneration and sprouting of striatal dopaminergic fibers. Therefore, glycine uptake inhibitors would be beneficial in therapeutic treatment of Parkinson's disease patients.

Example 4: Origin Of The Glutamate Input That Affects Dopamine Axon Growth

An important new goal related to the location of the glycine transport inhibitor effect is to identify from where the glutamate input that affects dopamine axon growth originates. An obvious source is cortico- and thalamo-striatal terminals, but the glutamate could also be released as an auto-feedback signal from dopaminergic axons themselves.

Dopamine neurons express the vesicular glutamate transporter 2 (Vglut2) during development and following 6-OHDA lesions (Dal Bo et al . , 2008, Neuroscience, 156 ( 1 ) : 59-70 ; Berube-Carriere et al., 2009, J Comp Neurol., 517 ( 6) : 873- 91 ) . The role of Vglut2 in dopamine neurons is not well understood. Glutamate transmission by dopamine neurons may play a role in the reward pathway in the N. accumben (Chuhma et al . , 2004, J Neurosci., 24 (4) : 972-81; Birgner et al . , 2010, PNAS, 107 (1) : 389-94) , and Vglut2 expression has been shown to modulate vesicular storage of dopamine (Hnasko et al . , 2010, Neuron, 65 (5) : 643-56) . We propose that glutamate release from dopamine neurons may serve a regulatory function in dopaminergic axonal arbor development. We examine this question with conditional Vglut2 KO mice, in which this transporter is selectively deleted in dopamine neurons (made available to use by Dr. Robert Edwards, UCSF) . If the effects of glycine transport inhibitors on dopaminergic re-innervation following 6-OHDA lesions are absent in this mouse line, this indicate that glutamate release by dopaminergic axons provides an auto-regulatory mechanism of axonal arborization. Thus, a mechanism to enhance glutamate co-release in dopamine neurons in adult brains, could potentially "rejuvenate" dopaminergic axons and provide an additional target for development of new therapies.

Example 5: Effects of Glycine Uptake Inhibitors In Genetically Based Mouse Models of Parkinson' s Disease We examine whether the beneficial effects of glycine uptake inhibitors are restricted to the 6-OHDA model, or whether axonal sprouting is also promoted in genetically based mouse models of Parkinson's disease. Mice that overexpress LRRK2 containing the R1441G mutation have been reported to show age- dependent deficits in motor behavior, dopamine release, and dopamine neurite degeneration (Li et al . , 2009, Nat Neurosci., 12 (7) : 826-8) .

While effects on the morphology may not be detectable in these mice, the functional read out, i.e. motor behavior and evoked dopamine release and uptake, can be straightforwardly determined. These transgenic mice may provide a useful model to show glycine uptake inhibitors promote functional compensatory sprouting of dopamine neurites while a subset of neurons develops neurite degeneration. Example 6: Enhancing NMDA Receptor-Dependent Transmission via Glycine Uptake Inhibitor To Promote Regeneration of Dopaminergic Fibers In 6-OHDA Mouse Model Of Parkinson' s Disease We assess effects of glycine uptake inhibitors on the regeneration of striatal dopaminergic innervations following 6-OHDA lesions using 1. histology, 2. cyclic voltammetry, which measure evoked dopamine release and uptake, 3. motor behavior tests, and 4. test glycine uptake inhibitors in conditional NMDA receptor KO mice, to reveal whether the effects are directly attributable to NMDA receptor activity in dopamine neurons .

Histological Assessment of Striatal Reinnervation Following Intrastriatal 6-OHDA Lesions We developed a protocol for intrastriatal 6-OHDA injections that consistently produced lesions of the dorsal striatum along the entire rostro-caudal axis while sparing dopaminergic fibers in the ventral striatum. We assess lesion extent and recovery at 3 and 7 weeks following the 6-OHDA injections. The results are shown in Figure 3.

At 3 weeks, when most of the 6-OHDA-induced cell death had occurred, almost no dopamine fibers (as stained for the dopamine transporter, DAT) remained in the dorsal striatum. At 7 weeks, re-innervation of the dorsal striatum was visible, and apparently originated from the spared ventral striatum: in coronal sections, the density of fibers declined gradually from the more ventral towards the more dorsal regions. The density of fibers was assessed in four regions (120,000 urn ² ) extending from directly under the corpus callosum (most dorsal) toward the ventral striatum. The average fiber density expressed as a percentage of density in the non- lesioned control side was 6% (most dorsal) , 23%, 37%, 66% (most ventral) , respectively (n=5) .

To test the effect of glycine uptake transporter 1 inhibitors on dopaminergic striatal re-innervation, mice were treated with compound RWI-D-132-1, provided by Dr. Craig Lindsley at Vanderbilt University (Wolkenberg et al . , 2009, Bioorg Med Chem Lett. 19(5) : 1492) . RWI-D-132-1 was injected (30mg/kg) i.p. 3 times a week for 4 weeks, beginning 3 weeks after the lesion . The treatment elicited substantial effects on dopaminergic re- innervation, resulting in a significantly increased fiber density that was 11%, 39%, 68%, and 96% in the four regions, respectively (n=5) . These data show efficacy in all regions, with the largest difference in the two ventral-most regions where fiber density was 30% higher in treated mice compared to controls .

Recordings of Evoked Dopamine Release and Uptake At 7 Weeks of Recovery From Intrastriatal 6-QHDA Lesions

To assess the effects of RWI-D-132-1 on evoked striatal dopamine release and uptake, coronal striatal brain slices were prepared from control and treated mice at 7 weeks. Dopamine release was evoked by bipolar electrical stimulation at 3 different sites: directly under the corpus callosum, in the middle of the dorsal striatum and in the ventral part of the dorsal striatum in control and lesioned hemispheres. In cyclic voltammetry recordings of evoked dopamine overflow, the peak of the signals reflects dopamine release and uptake and the half-life of the signal reflects uptake activity (Schmitz et al., 2001, J Neurosci, 21: 5916-24) As seen in Figure 4, there was an increase in peak amplitude and a marked decrease in the half-life of the dopamine signals of treated (n=4) as compared to untreated mice (n=4) recorded in the middle of the dorsal striatum. This indicates that both dopamine release and uptake were enhanced in treated mice. In the dorsal-most area, signals would either not be detected or were extremely small in both groups. In the ventral-most area there was no difference between the groups.

Thus, our preliminary data on evoked dopamine release confirm the histology data and indicate that the re-innervation of the striatum following the 6-OHDA lesion is functional and enhanced in mice treated with glycine uptake inhibitors.

Behavioral Test Following Intrastriatal 6-OHDA Lesions

As a behavioral test for the extent of motor behavior recovery, we used the "cylinder test", which unlike amphetamine-induced rotation, does not require exposure to drugs and correlates well with the lesion size (Iancu et al . , 2005, Behav Brain Res., 162 (1) : 1-10) . This test measures forelimb use during explorative activity when mice are placed in a glass cylinder and explore the novel environment by rearing and leaning their forepaws against the wall. Weight- bearing wall contacts made by each forelimb are counted.

To date, two control mice and four treated mice were tested and analyzed at 3 weeks and 7 weeks following the lesion. Two control mice do not show any improvement from 3 to 4 weeks after the lesion, whereas 2 of the 4 treated mice do. Assessment of Compound' s Effects in Conditional NR1 KO Mice

We bred conditional NR1 KO mice that lack the NMDA receptor only in neurons that express DAT to test whether the glycine transport inhibitor effects are directly attributable to NMDA receptor activity in dopamine neurons. 10 NR1 KO mice and 3 DAT-Cre+/- control mice have been injected with 6-OHDA. Of the 10 mice, 5 serve as controls, 5 treated with RWI-D-132-1 and their behavior and histology analyzed.

Evaluation of the re-innervation following 6-OHDA lesions in treated and untreated NR1 KO mice indicates the location of the glycine uptake inhibitor effects, i.e. the effects are directly attributable to enhanced NMDA receptor signaling in dopamine neurons .

DISCUSSION

In summary, the data demonstrate that glycine uptake inhibitors promote sprouting of dopaminergic fibers following intrastriatal 6-OHDA lesions. We have found that glycine uptake inhibitors that enhance NMDA glutamate receptor activity promote striatal dopaminergic re-innervation in a toxin-based mouse model of Parkinson's disease. Adult mice receive unilateral intrastriatal 6-OHDA injections. Three weeks after the lesion the dorsal striatum was devoid of dopamine neurites. After an additional four weeks, a gradual and partial dopaminergic reinnervation of the dorsal striatum occurred in untreated mice. This reinnervation was enhanced by 30% when mice were treated with a glycine uptake inhibitor, RWI-D-132-1, beginning three weeks after the lesion. Our data on evoked dopamine release and motor-behavior recovery further support a beneficial effect of glycine uptake inhibitors on dopamine release and uptake and paw usage. Thus, glycine uptake inhibitors, a class of drugs that is already being considered for treatment of symptoms in schizophrenia patients, promote functional dopaminergic sprouting and could reverse the dopaminergic fiber loss seen in Parkinson's disease patients. Example 7 : Glycine Uptake Inhibitor Treatment Enhances Behavioral Improvement In Mice

Enhanced improvement were observed in mice treated with glycine transporter-1 inhibitor in a behavioral test, the so called "corridor test", which is very sensitive to lesion size (Dowd et al . , 2005, Brain Research Bulletin, 68:24-30).

The test indicates lateralized sensory-motor neglect. As shown in Figure 5A, a hungry mouse is placed in a narrow corridor containing pairs of containers with sugar pellets placed adjacently along the left and right wall. Unilaterally lesioned mice tend to collect sugar pellets only from the ipsilateral side.

Mice were tested in the corridor test at 3 and 7 weeks post lesion. Figure 5B shows a scatter plot of the data of untreated and treated mice at 3 and 7 weeks. For all mice there was improvement, i.e. less lateralization, between the 3 and 7 week time points. There was a clear difference between untreated (open triangles) and treated (filled circles) mice. The average improvement in lateralization between 3 and 7 weeks was 18.5 % in control mice, and 33.8 % in treated mice.