Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Diacylglycerol Lipase α Knockout Mice Demonstrate Metabolic and Behavioral Phenotypes Similar to Those of Cannabinoid Receptor 1 Knockout Mice.

After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or ß (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (2) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side-effects.

Ligation of mouse L4 and L5 spinal nerves produces robust allodynia without major motor function deficit.

Spinal nerve L5/L6 ligation (SNL) in rats has become the standard for mechanistic studies of peripheral neuropathy and screening for novel analgesics. Conventional SNL in our hybrid mice resulted in a wide range of allodynia. Anatomical evaluation indicated that a variable number of lumbar vertebrae existed, resulting in L4/L5 or L5/L6 being ligated. Surprisingly, L4/L5 ligation did not result in ipsilateral hind limb paralysis and produced robust allodynia. Following a recent report that the mouse L4 neural segment is homologous with rat L5 we generated L4, L5 or both L4 and L5 (L4/L5) ligations in C57 mice after establishing a modified set of surgical landmarks. In contrast to rats, L4 ligation in these mice did not result in hind limb paralysis. Robust allodynia was observed in all three ligation groups. Nerve degeneration confirmed that L4 and L5, respectively, are primary contributors to the tibial and sural branches of the sciatic nerve in mice. A larger von Frey sensitive area reflected the wider distribution of Wallerian degeneration in the hindlimb of L4- compared to L5-ligated mice. Ligation of mouse L4 and L5 spinal nerves produces consistent, robust neuropathic pain behaviors and is suitable as a model for investigating mechanisms of neuropathic pain and for testing of novel analgesics. Gabapentin, used as a validation drug in neuropathic pain models and as a reference compound for novel analgesics, significantly reduced allodynia in the mice tested (L4/L5 ligations). Given the ease of surgery, robust allodynia, and larger von Frey sensitive area, we conclude that combined ligation of spinal nerves L4 and L5 optimizes the SNL model in mice.

Novel inhibitors of the high-affinity L-proline transporter as potential therapeutic agents for the treatment of cognitive disorders.

The incidence of cognitive disorders such as Alzheimer's disease continues to increase unabated. While cures for such diseases have eluded investigators, progress is being made on alleviating certain symptoms of these diseases. Mouse knockouts of the proline transporter (PROT), a high affinity Na(+)/Cl(-)-dependent transporter, indicated its potential as a novel therapeutic target for cognition improvement. Herein we report our investigation into a novel class of PROT inhibitors.

Characterization of PTPRG in knockdown and phosphatase-inactive mutant mice and substrate trapping analysis of PTPRG in mammalian cells.

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98-99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, "substrate trapping" mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.

A high-throughput screen for receptor protein tyrosine phosphatase-gamma selective inhibitors.

Protein tyrosine phosphatase-γ (PTP-γ) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP-γ genetic deletion model associated the loss of PTP-γ gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP-γ (RPTP-γ) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z'-LYTE technology to screen for inhibitors of RPTP-γ. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP-γ inhibitors in the assay. After chemical triage and clustering, compounds were assessed for potency and selectivity by IC(50) determination with RPTP-γ and two other phosphatases, PTP-1B and CD45. One hundred twenty-nine RPTP-γ selective (defined as IC(50) value greater than 5- to 10-fold over PTP-1B and CD45) inhibitors were identified and prioritized for evaluation. One of these hits, 3-(3, 4-dichlorobenzylthio) thiophene-2-carboxylic acid, was the primary chemotype for the initiation of a medicinal chemistry program.

Viable mouse gene ablations that robustly alter brain Aß levels are rare.

BACKGROUND: Accumulation of amyloid-ß (Aß) peptide in the brain is thought to play a key pathological role in Alzheimer's disease. Many pharmacological targets have therefore been proposed based upon the biochemistry of Aß, but not all are equally tractable for drug discovery. RESULTS: To search for novel targets that affect brain Aß without causing toxicity, we screened mouse brain samples from 1930 novel gene knock-out (KO) strains, representing 1926 genes, using Aß ELISA assays. Although robust Aß lowering was readily apparent in brains from a BACE1 KO strain, none of the novel strains exhibited robust decreases in brain Aß, including a GPR3 KO strain, which had previously been proposed as an Aß target. However, significantly increased Aß was observed in brain samples from two KO strains, corresponding to genes encoding the glycosylphosphatidylinositol mannosyl transferase PIGZ and quinolinate phosphoribosyltransferase (QPRT). CONCLUSIONS: Thus, gene ablations that are permissive for mouse survival and that also have a robust effect on Aß levels in the brain are rare.

Transmembrane and ubiquitin-like domain containing 1 (Tmub1) regulates locomotor activity and wakefulness in mice and interacts with CAMLG.

Tmub1 (C7orf21/HOPS) encodes a protein containing a ubiquitin-like domain. Tmub1 is highly expressed in the nervous system. To study its physiological function, we generated mice with Tmub1 deleted by homologous recombination. The knockout mice were grossly normal and viable. In a comprehensive behavioral testing battery, the only knockout phenotype displayed was a strong increase in home cage locomotor activity during the dark phase (subjective day) of the light:dark (L:D) cycle. There were no changes in activity during the light period. There were no changes in locomotor activity observed in other assays, e.g. novel open-field. The increase in dark phase locomotor activity persisted during a seven day D:D (complete darkness) challenge, and remained largely confined to the normally dark period. Telemetric recording in freely moving subjects for one 24 hr L:D cycle, revealed the same increase in locomotor activity in the dark phase. In addition, EEG analysis showed that the knockout mice exhibited increased waking and decreased NREM & REM times during the dark phase, but the EEG was otherwise normal. Using lacZ as a reporter we found Tmub1 expression prominent in a few brain structures including the thalamus, a region known to drive wakefulness and arousal via its projections to the cortex. We identified calcium modulating cyclophilin ligand CAMLG/CAML as a binding partner by a yeast two-hybrid screen of a brain library. The interaction of Tmub1 and CAMLG was confirmed by co-immunoprecipitation assays in HEK cells. The two proteins were also found to be co-localized to the cytoplasm when expressed in HEK cells. Both Tmub1 and CAMLG have been recently described in the regulation of membrane trafficking of specific receptors. Taken together our results implicate Tmub1 in the regulation of locomotor activity and wakefulness and suggest that Tmub1 binds to and functions together with CAMLG.

Loss of the putative catalytic domain of HDAC4 leads to reduced thermal nociception and seizures while allowing normal bone development.

Histone deacetylase 4 (HDAC4) has been associated with muscle & bone development [1]-[6]. N-terminal MEF2 and RUNX2 binding domains of HDAC4 have been shown to mediate these effects in vitro. A complete gene knockout has been reported to result in premature ossification and associated defects resulting in postnatal lethality [6]. We report a viral insertion mutation that deletes the putative deacetylase domain, while preserving the N-terminal portion of the protein. Western blot and immuno-precipitation analysis confirm expression of truncated HDAC4 containing N-terminal amino acids 1-747. These mutant mice are viable, living to at least one year of age with no gross defects in muscle or bone. At 2-4 months of age no behavioral or physiological abnormalities were detected except for an increased latency to respond to a thermal nociceptive stimulus. As the mutant mice aged past 5 months, convulsions appeared, often elicited by handling. Our findings confirm the sufficiency of the N-terminal domain for muscle and bone development, while revealing other roles of HDAC4.

Discovery and characterization of potent small molecule inhibitors of the high affinity proline transporter.

The mammalian proline transporter (PROT) is a high affinity Na(+)/Cl(-)-dependent transporter expressed in specific regions of the brain. It is homologous to other neurotransmitter transporters such as glycine, norepinephrine, serotonin, and dopamine transporters. PROT is enriched in glutamatergic synaptic terminals and may play an important role in the regulation of excitatory neurotransmission. No non-peptide small molecule inhibitors have been described for this transporter. To study its physiological role in the central nervous system and evaluate its potential as a therapeutic target, we established cell lines that stably express recombinant hPROT and characterized its kinetic properties for proline uptake. We then screened for inhibitors and identified a series of compounds that inhibit hPROT-mediated proline uptake. A known compound, benztropine, was found to inhibit hPROT with an IC(50) of 0.75microM. A series of novel compounds were also found, one of which, LP-403812, showed an IC(50) of approximately 0.1microM on both recombinant human and mouse PROT without significant inhibition of glycine and dopamine transporters at concentrations up to 10microM. This compound also inhibited proline transporter activity of mouse brain synaptosomes with the same potency. These inhibitors provide important tools for the understanding of PROT functions in the brain and may lead to the development of therapeutic agents for certain neurological disorders.

Differential sensitivity to SSRI and tricyclic antidepressants in juvenile and adult mice of three strains.

Clinical studies have shown differential efficacy of several antidepressants in children and adolescents compared to adults, yet few animal studies have sought to characterize this phenomenon. We compared effects of fluoxetine and imipramine in two common behavioral assays that hold high predictive validity for antidepressant activity, tail suspension and forced swim test, using juvenile (5 weeks) and adult (12 weeks) mice from 3 strains. C57BL/6J-Tyr(c-Brd) (C57), hybrid C57BL/6J-Tyr(c-Brd)x129S5/SvEvBrd (F2), and Balb/cAnNTac (Balb/C) mice were tested in forced swim test and tail suspension after i.p. dosing with either fluoxetine or imipramine. Brain tissues were analyzed to evaluate levels of VMAT2, a possible modulator of age-dependent sensitivity to antidepressants. Imipramine had more consistent antidepressant effect across age groups and strains. Imipramine increased struggle in mice of both ages. Fluoxetine did not have an effect on immobility in Balb/C of both ages in tail suspension. Fluoxetine also did not increase forced swim struggle behavior in juvenile mice of all strains, but was effective in increasing struggle in adults. Juvenile mice had higher immobility and lower struggle than adults in forced swim, and juveniles also had higher immobility in tail suspension test for Balb/C and C57. In addition, VMAT2 levels were increased in juveniles. These results confirm that standard antidepressants produce effects in both juveniles and adults but age-related differences were evident in both tests. Further examination of these effects is needed to determine whether it may be related to age-dependent difference in the clinical response to antidepressants of these classes.

Genetic disruption of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in models sensitive to antidepressants.

The neurotransmitter serotonin (5-HT) plays an important role in both the peripheral and central nervous systems. The biosynthesis of serotonin is regulated by two rate-limiting enzymes, tryptophan hydroxylase-1 and -2 (TPH1 and TPH2). We used a gene-targeting approach to generate mice with selective and complete elimination of the two known TPH isoforms. This resulted in dramatically reduced central 5-HT levels in Tph2 knockout (TPH2KO) and Tph1/Tph2 double knockout (DKO) mice; and substantially reduced peripheral 5-HT levels in DKO, but not TPH2KO mice. Therefore, differential expression of the two isoforms of TPH was reflected in corresponding depletion of 5-HT content in the brain and periphery. Surprisingly, despite the prominent and evolutionarily ancient role that 5-HT plays in both vertebrate and invertebrate physiology, none of these mutations resulted in an overt phenotype. TPH2KO and DKO mice were viable and normal in appearance. Behavioral alterations in assays with predictive validity for antidepressants were among the very few phenotypes uncovered. These behavioral changes were subtle in the TPH2KO mice; they were enhanced in the DKO mice. Herein, we confirm findings from prior descriptions of TPH1 knockout mice and present the first reported phenotypic evaluations of Tph2 and Tph1/Tph2 knockout mice. The behavioral effects observed in the TPH2 KO and DKO mice strongly confirm the role of 5-HT and its synthetic enzymes in the etiology and treatment of affective disorders.

Learning and memory impairment in Eph receptor A6 knockout mice.

Genetic inhibition of the ephrin receptor (EphA6) in mice produced behavioral deficits specifically in tests of learning and memory. Using a fear conditioning training paradigm, mice deficient in EphA6 did not acquire the task as strongly as did wild type (WT) mice. When tested in the same context 24h later, knockout (KO) mice did not freeze as much as WT mice indicating reduced memory of the consequences of the training context. The KO mice also displayed less freezing when presented with the conditioning stimulus (CS) in a separate context. In the hidden platform phase of the Morris water maze (MWM) task, KO mice did not reach the same level of proficiency as did WT mice. KO mice also exhibited less preference for the target quadrant during a probe trial and were significantly impaired on an initial reversal of the platform. These findings suggest that EphA6, in line with a number of other Eph receptors and their ephrin ligands, is involved in neural circuits underlying aspects of learning and memory.

Use of a platform in an automated open-field to enhance assessment of anxiety-like behaviors in mice.

The present report describes a setup for simultaneously measuring anxiety-like behaviors and locomotor activity in mice. Animals are placed in a brightly lit, standard automated open-field (OF) in which a rectangular ceramic platform 8 cm high covers one quadrant of the floor. Mice preferred to stay under the platform, avoiding the area with bright illumination. Activities under and outside the platform were measured for 5 min. Chlordiazepoxide and buspirone dose-dependently increased time spent outside the platform (L-Time) and the light distance to total OF distance ratio (L:T-TD) in both genders without changing total OF distance. By contrast, amphetamine decreased L-Time and L:T-TD in males, thus displaying an anxiogenic effect. Imipramine was without selective effect on L-Time or L:T-TD, but decreased total OF distance at the highest dose indicative of a sedative effect. Drug effects were also evaluated in the OF without platform using conventional anxiety measures. Introduction of the platform into the OF apparatus strongly enhanced the sensitivity to anxiolytics. Comparison of strains differing in activity or anxiety levels showed that L-Time and L:T-TD can be used as measures of anxiety-like behavior independent of locomotor activity. Changes in motor activity are reflected in the total distance traveled under and outside the platform. Therefore, the platform test is fully automated, sensitive to both anxiolytic and anxiogenic effects of drugs and genetic phenotypes with little evidence of gender-specific responses, and can be easily utilized by most laboratories measuring behavior.