INHIBITORS OF ALPHA-TUBULIN ACETYLATION FOR THE TREATMENT OF PAIN

Abstract

The present invention pertains to novel analgesics useful for treating mechanical pain. The invention suggests the use of inhibitors of α-tubulin acetylation for inhibition of neurological sensations that are mediated by sensory neurons. The perception of mechanical pain is can be modulated by altering the α-tubulin acetylation, in context of the invention in particular by modulation of the expression and/or activity of the enzyme α-tubulin acetyltransferase (Atat). The invention provides the medical application of α-tubulin acetyltransferase inhibitors as analgesics and a screening method for the identification of compounds useful in the treatment of pain.

Claims

1-15. (cancelled)

16. A method for reducing expression of ATAT1 in a subject experiencing mechanical pain mediated by sensory neurons, the method comprising: administering to the subject an effective amount of a nucleic acid that is capable of reducing expression of ATAT1; wherein the mechanical pain is selected from the group consisting of: inflammatory pain, acute mechanical pain, chronic mechanical pain, mechanical hyperalgesia, mechanical allodynia, visceral pain, and labor pain.

17. The method of claim 16, wherein the acute mechanical pain is due to physical trauma selected from soft tissue damage, infection and inflammation.

18. The method of claim 16, wherein the acute mechanical pain is selected from pain due to surgery, cuts, bruises, fractured or broken bones, hemorrhoids, intestinal gas, dyspepsia and dental pain such as toothache, denture pain and nerve root pain.

19. The method of claim 16, wherein the chronic mechanical pain is a pain that lasts longer than 1 month or beyond the resolution of an acute tissue injury or is recurring or is associated with tissue injury and/or chronic diseases that are expected to continue or progress.

20. The method of claim 16, wherein the chronic mechanical pain is selected from pain due to inflammatory disease, cancer, arthritis, chronic wounds, cardiovascular incidents, spinal cord disorders, central nervous system disorder, recovery from surgery, and neuropathy.

21. The method of claim 16, wherein the chronic mechanical pain is selected from pain due to osteoarthritis, rheumatoid arthritis, fibromyalgia, meralgia paresthetica, back pain, angina, carpel tunnel syndrome, menstruation, and hemorrhoids.

22. The method of claim 16, wherein the visceral pain is selected from pain due to menstruation, inflammation, cancer, dyspepsia, and intestinal gas.

23. The method of claim 16, wherein the nucleic acid is capable of reducing expression of ATAT1 through RNA interference.

24. The method of claim 23, wherein the nucleic acid encodes a shRNA.

25. The method of claim 23, wherein the nucleic acid encodes a siRNA.

26. The method of claim 16, wherein mechanical pain is reduced.

27. A method for reducing pain in a subject experiencing mechanical pain mediated by sensory neurons, the method comprising: administering to the subject an effective amount of a nucleic acid that is capable of reducing expression of ATAT1; wherein the mechanical pain is selected from the group consisting of: inflammatory pain, acute mechanical pain, chronic mechanical pain, mechanical hyperalgesia, mechanical allodynia, visceral pain, and labor pain.

28. The method of claim 27, wherein the acute mechanical pain is due to physical trauma selected from soft tissue damage, infection, and inflammation.

29. The method of claim 27, wherein the acute mechanical pain is selected from pain due to surgery, cuts, bruises, fractured or broken bones, hemorrhoids, intestinal gas, dyspepsia and dental pain such as toothache, denture pain, and nerve root pain.

30. The method of claim 27, wherein the chronic mechanical pain is a pain that lasts longer than 1 month or beyond the resolution of an acute tissue injury or is recurring or is associated with tissue injury and/or chronic diseases that are expected to continue or progress.

31. The method of claim 27, wherein the chronic mechanical pain is selected from pain due to inflammatory disease, cancer, arthritis, chronic wounds, cardiovascular incidents, spinal cord disorders, central nervous system disorder, recovery from surgery, and neuropathy.

32. The method of claim 27, wherein the chronic mechanical pain is selected from pain due to osteoarthritis, rheumatoid arthritis, fibromyalgia, meralgia paresthetica, back pain, angina, carpel tunnel syndrome, menstruation, and hemorrhoids.

33. The method of claim 27, wherein the visceral pain is selected from pain due to menstruation, inflammation, cancer, dyspepsia, and intestinal gas.

34. The method of claim 27, wherein the nucleic acid is capable of reducing expression of ATAT1 through RNA interference.

35. The method of claim 34, wherein the nucleic acid encodes a shRNA.

36. The method of claim 34, wherein the nucleic acid encodes a siRNA.

37. The method of claim 27, wherein mechanical pain is reduced.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0068] The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

[0069] FIG. 1A is a bar chart summarising the results of a tape test to assay low threshold mechanosensation. Atat1.sup.cKO mice demonstrated significantly less response events over the 5 minute counting period (t-Test, P<0.05). FIG. 1B is a bar chart showing the cotton swab analysis assaying low threshold mechanosensation. Atat1.sup.cKO mice demonstrated significantly less response events then Atat1.sup.Control counterparts (t-Test, P<0.01). FIG. 1C is a graph of von Frey thresholds showing the significantly lower response frequency in Atat1.sup.cKO animals (RM ANOVA, Holm-Sidak method, P<0.001). FIG. 1D is a bar chart showing latency to awareness of a clip attached to the base of the tail. Atat1.sup.cKO animals take significantly longer to respond to the stimulus (t-Test, P<0.01). FIG. 1E is a bar chart showing that there are no significant differences in the responses recorded to noxious heat between Atat1.sup.cKO and Atat1.sup.Control animals (t-Test, P>0.05). FIG. IF is a bar chart showing that there are no significant differences in motor performance as assayed using the Rotorod test (RM ANOVA, Holm-Sidak method, P>0.05). Error bars indicate s.e.m.

[0070] FIG. 2A shows responses (top) and stimulus-response function (bottom) of slowly adapting mechanoreceptor fibers (SAM), FIG. 2B shows rapidly adapting mechanoreceptor fibers (RAM), FIG. 2C shows D-hair afferents, FIG. 2D shows Aδ-mechanonociceptors (AM) and FIG. 2E show C-fibre nociceptors from αTAT1control and αTAT1cko mice (two-way ANOVA with post-hoc Bonferroni's test, SAM: P<0.001; RAM: P<0.0001; D-hair: P<0.0001; AM: P<0.0001; C-fibre: P<0.0001). FIG. 2F shows Mean von Frey thresholds for C fibre discharge (Mann-Whitney test, P<0.01). The number of fibres recorded is indicated in parentheses in each panel. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Error bars indicate s.e.m.

[0071] FIG. 3A shows stacked histograms showing the proportion of different mechanogated currents activated by neurite indentation in sensory neurons from control Atat1.sup.Control and Atat1.sup.cKO mice (χ.sup.2 test, P<0.05). NR, non-responsive to given displacement 512 nm. FIG. 3B shows representative traces of RA currents elicited by increasing probe displacement on soma of Atat1.sup.Control and Atat1.sup.cKO sensory neurons. FIG. 3C shows threshold of activation of RA currents was determined as mechanical stimulus that elicited a current ≥20 pA. Closed circles indicate individual recorded cells. Note the marked increase in the displacement threshold in Atat1.sup.cKO sensory neurons (Mann-Whitney test, P<0.01). e FIG. 3D shows stimulus response curve of RA currents evoked by increasing probe displacement. Genetic depletion of αTAT1 in sensory neuron significantly reduced RA-currents amplitude (two-way ANOVA with post-hoc Bonferroni's test, P<0.0001). FIG. 3E shows stacked histograms showing the proportions of different mechano-gated currents observed in Atat1.sup.cKO sensory neurons transfected with EGFP, αTAT1-YFP or αTAT1-GGL-YFP cDNA. Transfection of wild-type αTAT1 rescued the loss of mechanosensitivity, while transfection of catalytically inactive αTAT1 (αTAT1-GGL-YFP) failed to restore it in Atat1.sup.cKO sensory neurons (χ.sup.2 test, EGFP versus αTAT1-YFP, P<0.05; EGFP versus αTAT1-GGL-YFP, P>0.05). FIG. 3F shows stacked histograms showing the proportions of different mechano-gated currents observed in Atat1.sup.cKO sensory neurons transfected with EGFP, α-tubulin.sup.K40R-IBES-YFP (K40R) or α-tubulin.sup.K40Q-IRES-YFP (K40Q) cDNA. Transfection of acetylated α-tubulin mimics (K40Q) but not non-acetylatable α-tubulin mutant (K40OR) restored mechanosensitivity in Atat1.sup.cKO sensory neurons (χ.sup.2 test, EGFP versus K40Q, P<0.05; EGFP versus K40R, P>0.05). The number of neurons recorded is indicated in parentheses in each panel. ** P<0.01; **** P<0.0001; Error bars indicate s.e.m.

[0072] FIG. 4A shows anti-acetylated-α-tubulin staining of Atat1.sup.Control cultured DRG cells (corresponding surface plot below). Note the prominent sub-membrane localisation of acetylated tubulin (Scale bar 5 μm). FIG. 4B shows anti α-tubulin staining of Atat1.sup.Control cultured DRG cells (Scale bar 5 μm). FIG. 4C shows anti-acetylated-α- tubulin staining of Atat1.sup.Control MEFs (Scale bar 20 μm). Note the even distribution of acetylated tubulin in this cell type. FIG. 4D shows anti α-tubulin staining of Atat1.sup.Control MEFs (Scale bar 20 μm). FIG. 4E shows immunohistochemical staining of nerve fibres within the saphenous nerve taken from Atat1.sup.Control mice. Anti-acetylated-α-tubulin staining is in green) and myelin basic protein (MBP) is in red (Scale bar 10 μm). Note the sub-membrane localisation of the anti-acetylated tubulin stain. FIG. 4F shows a fluorescent image of free nerve endings in a whole mount cornea preparation from a Avil-Cre::SNAP.sup.CaaX mouse. Acetylated tubulin is in green) and membrane bound SNAP staining is in red (Scale bar 30 μm). Note the strong co-localisation of signals. FIG. 4G shows a super-resolution image of an anti α-tubulin staining of Atat1.sup.Control cultured DRG colour coded by depth (red close to objective, Scale bar 5 μm). FIG. 4H shows a super-resolution image of an Anti-α tubulin stain in Atat1.sup.cKO cultured DRG (Scale bar 5 μm). FIG. 41 is a graphical summary of AFM analysis showing the pressure required to indent the membrane to 200, 400 and 600 nm respectively, using a blunt ended cantilever in cultured DRG taken from Atat1.sup.Control and Atat1.sup.cKO mice. A significantly higher pressure is required to indent the membranes of Atat1.sup.cKO neurons over Atat1.sup.Control cells (Mann-Whitney test, P<0.01). FIG. 4J is a graph showing the relative shrinkage of axonal outgrowths from Atat1.sup.Control and Atat1.sup.cKO DRG loaded with calcein (2 μM) in response to a hyperosmotic shock over time. Deletion of Atat1 leads to a significant decrease in the percentage shrinking of axons relative to control samples (ANOVA on ranks, multiple comparison Dunn's Method, P<0.05). FIG. 4K is an image from a cultured DRG cell showing an overlay of C8 SIR tubulin labelled microtubules before (purple) and after (green) hyperosmotic shock. Note the clear compression of the microtubule cytoskeleton after shrinking (Scale bar 10 μm). FIG. 4L is a bar chart summarising osmotically induced microtubule compression in DRG neurons from Atat1.sup.Control, Atat1.sup.cKO, and Atat1.sup.cKO neurons transfected with tubulin-K40Q. There is significantly less compression in Atat1.sup.cKO than Atat1.sup.Control neurons, which is rescued by transfection of tubulin-K40Q (ANOVA on ranks, multiple comparison Dunn's Method, P<0.05). Error bars indicate s.e.m.

EXAMPLES

Materials and Methods

Animals and Behavioural Experiments

[0073] To study the effect on touch sensitivity of deleting the ATAT1 gene we crossed ATAT1.sup.fl/+ mice.sup.17 with a peripheral nervous system specific Cre line Avil.sup.cre/+ mouse line.sup.18 to obtain Avil-Cre::ATAT1.sup.fl/+ (control) and Avil-Cre::Atat1.sup.fl/+ (cKO) animals. Mice were genotyped as described previously.sup.18,19 and maintained at the EMBL Mouse Biology Unit, Monterotondo, Italy, in accordance with Italian legislation (Art. 9, 27. January 1992, no 116) under license from the Italian Ministry of Health.

[0074] For the tape response assay mice were left to acclimate in plexiglass containers for 15 min. A 3 cm long by 1 cm wide piece of tape (Identi tape) was then gently applied along the spinal column on the back of the animal. The mice were then monitored for 5 min and the number of behavioural responses recorded. A response was recorded whenever a mouse attempted to remove the tape by scratching, biting or shaking.

[0075] For the cotton swab test, mice were placed in plexiglass boxes atop an elevated mesh base, and allowed to habituate for 30 min. A cotton swab was then ‘puffed out’ by pulling with forceps to increase its size. This enlarged swab was then applied to the hind paw of the animal using a gentle brushing manner, firstly to the right and subsequently the left hind paw.

[0076] For von Frey testing, mice were placed inside an open topped plexiglass container on an elevated mesh platform to acclimate for 1 h. A series of von Frey filaments (North Coast Medical, NC12775-99) with final force of 0.02 g to 1 g were applied to the animal's hind paw alternating left and right paw and a yes/no paw withdrawal response was recorded.

[0077] For the Tail clip assay, an alligator clip, covered with rubber tubing (to reduce tissue damage) and calibrated to exert 400 g force was attached to the base of the tail of Atat1.sup.Control and Atat1.sup.cKO mice Animals were placed in plexiglass containers and the latency to awareness of the clip as indicated by biting, vocalization or grasping was measured.

[0078] For the hot plate assay mice were placed on a hot plate (Ugo Basile, 35150) pre-heated to 55° C. and latency time was measured until a jump, hind paw flick or hind paw lick were observed. In case of the lack of any response the mice were removed from the hot plate after 30 s.

[0079] A modified version of the rotarod test was performed on naive Atat1.sup.Control and Atat1.sup.cKO mice. Briefly, mice were habituated for 5 mM to the stationary dowels of the rotarod (Rotarod 3375-5 TSE systems). Each step, either habituation or test was followed by a 5 minute rest period with food and water ad libitum. Mice were then habituated for 5 mM to the moving dowels at 5 RPM. Following this the mice were tested at 5, 10, 15, 20 and 25 RPM respectively for 2 mM a trial. The time spent on the dowels was then calculated. A fall or two full spins while gripping the dowel was considered a fail during the test.

Ex Vivo Electrophysiology

[0080] The skin nerve preparation was used essentially as previously described.sup.27. Briefly, mice were sacrificed using CO2 inhalation, and the saphenous nerve together with the skin of the hind limb was dissected free and placed in an organ bath. The chamber was perfused with a synthetic interstitial fluid (SIF buffer) consisting of (in mM): NaCl, 123; KCl, 3.5; MgSO4, 0.7; NaH2PO4, 1.7; CaCl2, 2.0; sodium gluconate, 9.5; glucose, 5.5; sucrose, 7.5; and HEPES, 10 at a pH of 7.4.) The skin was placed with the corium side up, and the nerve was placed in an adjacent chamber for fiber teasing and single-unit recording. Single units were isolated with a mechanical search stimulus applied with a glass rod and classified by conduction velocity, von Frey hair thresholds and adaptation properties to suprathreshold stimuli. A computer-controlled nanomotor (Kleindiek Nanotechnik) was used to apply mechanical ramp-and-hold stimuli of known amplitude and velocity. Standardized displacement stimuli of 2 s or 10 s duration were applied to the receptive field at regular intervals (interstimulus period, 30 s). The probe was a stainless steel metal rod with a flat circular contact area of 0.8 mm. The signal driving the movement of the linear motor and raw electrophysiological data were collected with a Powerlab 4.0 system and Labchart 7.1 software (AD instruments), Spikes were discriminated off-line with the spike histogram extension of the software.

Patch Clamping

[0081] DRG neurons were collected from mice and dissociated as described.sup.27. In some cases they were transfected using the Nucleofector system (Lonza AG) in 20 μl of Mouse Neuron Nucleofector solution from the SCN nucleofector kit (Lonza AG) and a total 4-5 μg of plasmid DNA at room temperature using the preinstalled program SCN Basic Neuro program 6. After electroporation, the cell suspension was transferred to 500 μl of RPMI 1640 medium (Gibco) for 10 min at 37° C. This suspension, supplemented with 10% horse serum, was used to plate the cells onto glass coverslips for recording. The RPMI medium supplemented with 100 ng/ml nerve growth factor (NGF), 50 ng/ml BDNF was replaced with the standard DRG medium 3-4 h later. Electrophysiology experiments began 12 h after plating.

[0082] Whole-cell recordings from isolated DRG neurons were made as previously described.sup.23. Recordings were made from DRG neurons using fire-polished glass electrodes with a resistance of 3-7 Mg. Extracellular solution contained (mM): NaCl 140, MgCl2 1, CaCl2 2, KCl 4, glucose 4 and HEPES 10 (pH 7.4), and electrodes were filled with a solution containing (mM): KCl 130, NaCl 10, MgCl2 1, EGTA 1 and HEPES 10 (pH 7.3). Cells were perfused with drug-containing solutions by moving an array of outlets in front of the patched cells (WAS02; Ditel, Prague). Observations were made with Observer A1 inverted microscope (Zeiss) equipped with a CCD camera and the imaging software AxioVision. Membrane current and voltage were amplified and acquired using EPC-10 amplifier sampled at 40 kHz; acquired traces were analyzed using Patchmaster and Fitmaster software (HEKA). Pipette and membrane capacitance were compensated using the auto function of Pulse. For most of the experiments, to minimize the voltage error, 70% of the series resistance was compensated and the membrane voltage was held at −60 mV with the voltage-clamp circuit. After establishing whole-cell configuration, voltage-gated currents were measured using a standard series of voltage commands Briefly, the neurons were pre-pulsed to −120 mV for 150 ms and depolarized from −65 to +55 mV in increments of 5 mV (40 ms test pulse duration). Next the amplifier was switched to current-clamp mode and current injection was used to evoke action potentials. If the membrane capacitance and resistance changed more than 20% after the mechanical stimulus, the cell was regarded as membrane damaged and the data discarded. Mechanical stimuli were applied using a heat-polished glass pipette (tip diameter 3-5 μm), driven by a MM3A Micromanipulator system (Kleindiek), and positioned at an angle of 45 degrees to the surface of the dish. The probe was positioned near the neurite, moved forward in steps of 200-600 nm for 500 ms and then withdrawn. For analysis of the kinetic properties of mechanically activated current, traces were fit with single exponential functions using the Fitmaster software (HEKA). Data are presented as mean±s.e.m.

Immunofluorescence and Staining

[0083] For microtubule staining in DRG cultures, cells were washed once with PBS, and then fixed for 15 mM in cytoskeleton buffer (CB) pH 6.3 containing 3% paraformaldehyde, 0.25% triton and 0.2% glutaraldehyde at room temperature. Cells were then washed 3 times with PBST (0.3% triton). Samples were then subsequently blocked with 5% normal goat serum (NGS) in PBS for 1 h at room temperature. Cells were then placed overnight at 4° C. with primary anti α-tubulin (1:1000) (Sigma-Aldrich, T9026) or anti-acetylated-α-tubulin (1:1000) (Sigma-Aldrich, T7451) in PBS. Cells were then washed with PBS and incubated for 1 h with fluorescently labelled secondary antibodies (1:1000) (Alexa Fluor 546 Lifetechnologies) for 1 h at room temperature. All images were acquired using a 40× objective on a Leica SP5 confocal microscope. Processing of images and generation of surface plots were performed using ImageJ Images were deconvoluted using Huygens software.

[0084] Actin filaments in DRG primary cultures were stained with Alexa488-phalloidin at 0.5 μg/ml (Lifetechnologies). Briefly cells were fixed with fresh 4% PFA (EM grade, TAAB) in cytoskeleton buffer (10 mM MES, 138 mM KCl, 3 mM MgCl, 2 mM EGTA) freshly added supplemented of 0.3 M sucrose, permeabilized in 0.25% Triton-X-100 (Sigma-Aldrich), and blocked in 2% BSA (Sigma-Aldrich).

[0085] Immunostaining of saphenous nerves was performed on paraffin sections after fixation with PFA. Following rehydration, antigen retrieval was performed with 10 mM sodium citrate (pH 6) at boiling temperature for 10 mM. Subsequently, sections were permeabilized (0.3% Triton X-100), blocked (5% goat serum) and stained with anti-acetylated-α-tubulin (Sigma-Aldrich, T7451) and anti-myelin basic protein (Chemicon, MAB386).

[0086] For cornea staining, the eyes were removed and fixed for 1 h in 4% PFA at room temperature. The cornea was then dissected and permeabilized with PBS-Triton 0.03% for 30 mM. Following this, the cornea was immersed in PBS-Triton 0.03% containing 1 μM SNAP surface 546 (New England Biolabs) for 30 min. The samples were then washed with PBS-Triton 0.03% for 20 min and subsequently blocked with 5% normal goat serum in PBS-Triton 0.03% for 30 mM. The tissue was then stained with anti-acetylated-α-tubulin (1:500) overnight. Samples were then washed with PBS and a secondary antibody (Alexa Fluor 488 Life-technologies) was added for 5 h. The samples were again washed with PBS and stained with DAPI 10 mM. The cornea was then mounted on glass with 100% glycerol and imaged.

[0087] For whole mount axon outgrowth assays, individual DRG were extracted from mice and grown in Matrigel (Corning) for 7 days. Preparations were fixed with 4% PFA for 5 minutes and labelled with the primary antibody PGP9.5 (1:200) overnight at 4° C. The samples were then labeled with secondary antibodies (1:1000) Alexa Fluor 546 Lifetechnologies) for 1 h at room temperature. All images were acquired using a Leica LMD 7000.

[0088] SNAP-tag labelling was carried out by intradermal injection of the finger in anaesthetized mice of 2 ∞M BG TMRstar substrate as described previously.sup.28. After five hours the animals were sacrificed and the samples were mounted in 80% glycerol for imaging.

Electron Microscopy of Saphenous Nerve

[0089] Saphenous nerves were dissected and postfixed for 24 h with fresh 4% (w/v) PFA, 2.5% (w/v) Glutaraldehyde (TAAB) in 0.1 M Phosphate buffer at 4 C. Following postfixation, the samples were incubated for 2 h with 1% (w/v) OsO4 supplemented with 1.5% (w/v) Potassium Ferrocyanide, samples were dehydrated in Ethanol and infiltrated with propylene oxide/Epon (Agar) (1:1) followed by resin embedding. Ultrathin sections were cut (Ultracut S, Leica), counter-stained with Uranyl Acetate and Lead Citrate and observed with a Transmission Electron Microscope (TEM) Jeol 1010 equipped with a MSC 791 CCD camera (Gatan).

Microfluidics

[0090] DRG neurons were suspended in 1:1 Matrigel in 10% FBS DMEM and seeded onto a two-chamber microfluidic chip (Xona Microfluidics, SD150). Axons were allowed to grow across the microchannels connecting the two chambers for 3-5 days. On the day of the experiment, media in both the cell body and axon chambers was replaced with media with no serum for 3 h. 1 μM mono-biotinylated NGF purified in house from eukaryotic cells was coupled with 1 μM streptavidin conjugated quantum dots 655 (Life Technologies) for 30 min on ice, then diluted to 5 nM in imaging buffer (as above) and then used to replace the media in the axon chamber. A 25% volume difference was kept between the cell body and the axon chamber to avoid backflow from the axon to the cell body chamber. After 1 h incubation at 37° C. in 5% CO2, retrograde transport of NGF-Qdot655 containing endosomes was imaged using a confocal Ultraview Vox (Perkin Elmer) equipped with a 5% CO2 humidified chamber at 37° C. 100 s time lapses were recorded using 300 ms exposure time Images were analyzed with Imaris software using the particle tracking function and autoregressive motion track generation setting.

Superresolution Microscopy

[0091] The cells were washed once with 3 ml of warm PBS. Subsequently, the cells were fixed and permeabilized for 2 min in cytoskeleton buffer containing 0.3% Glutaraldehyde and 0.25% Triton X-100. Following this, the cells were fixed for 10 min in cytoskeleton buffer containing 2% Glutaraldehyde and treated for 7 min with 2 ml of 0.1% Sodium Borohydride (NaBH4) in PBS. Cells were then washed 3 times for 10 min in PBS. The cells were incubated with primary antibody for 30 min (mouse anti α-tubulin, Neomarker, 1:500) in PBS+2% BSA After washing 3 times for 10 min with PBS, the cells were transferred to the secondary antibody (goat anti mouse Alexa 647, 1:500, Molecular Probes A21236) at room temperature for 30 min. The cells were then washed 3 times with PBS for 10 min and then mounted for PALM imaging At the time of imaging cells were overlaid with PALM blinking buffer: 50 mM Tris pH 8.0, 10 mM NaCl, 10% Glucose, 100 U/ml Glucose Oxidase (Sigma-Aldrich), 40 ug/ml Catalase (Sigma-Aldrich).

[0092] The analysis of microtubule (MT) network morphology was done using the open source software CellProfiler.sup.29. The MT signal was enhanced by a top-hat filter and then binarised with the same manual threshold for all images. Binary images were skeletonized using CellProfiler's “skelPE” algorithm and the resulting skeleton was subjected to branchpoint detection. As an approximation for MT network complexity we divided the number of branchpoints by the number of pixels in the skeleton. Moreover, we measured the local angular distribution of the MTs in order to assess whether they run in parallel, or in a crossing manner (angular variance). To this end, we subjected each pixel to a rotating morphological filter using a linear structural element with a length of 11 pixels, and recorded at which angle we obtained a maximum response. The inventors computed the response for angles from 0 to 170 degrees at steps of 10 degrees since there is no information on MT polarity. Next we measured the local circular variance.sup.3 of the MT orientations in a sliding window with a diameter of 51 pixels, using angle doubling as it is commonly done for axial data.sup.3. The circular variance has a value 1 if the MTs in a given region are completely parallel and has smaller values (down to 0) if the MTs are oriented in various directions. Finally, we computed the average circular variance of all MT pixels in a given cell. If this value were close to 1 it would mean that locally, on a length scale of 51 pixels, the MTs are parallel in most of the cell.

Atomic Force Microscopy (AFM)

[0093] Force spectroscopy measurements were performed by using a NanoWizard AFM (JPK Instruments, Berlin, Germany) equipped with a fluid chamber (Biocell; JPK) for live cell analysis and an inverted optical microscope (Axiovert 200; Zeiss) for sample observation.

[0094] DRG cells were seeded on glass coverslip previously coated with a first layer of polylysine (500 μg/ml for about 1 h room temperature) and a second layer of laminin (20 μg/ml for about 1 h at 37° C.). The cells were then cultured for at least 15 h before measurements. Then, the sample was inserted into the fluid chamber (Biocell; JPK) immersed in culture medium and measurements were carried out at room temperature. The status of cells was constantly monitored by optical microscope.

[0095] Indenters for probing cell elasticity were prepared by mounting silica microspheres of 4.5 μm nominal diameter (Bangs Laboratories Inc.) to tipless V-shaped silicon nitride cantilevers having nominal spring constants of 0.32 N/m or 0.08 N/m (NanoWorld, Innovative Technologies) by using UV sensitive glue (Loxeal UV Glue). Silica beads were picked under microscopy control. Before measurements the spring constant of the cantilevers was calibrated by using the thermal noise method.

[0096] By using the optical microscope the bead-mounted cantilever was brought over the soma of single DRG and pressed down to indent the cell. The motion of the z-piezo and the force were recorded. On each cell eight-about ten force-distance curves were acquired with a force load of 500 pN and at a rate of 5 μm sec-1 in closed loop feed-back mode.

[0097] Cell elastic properties were assessed by evaluating the Young's modulus (E) of the cell. This value was obtained by analyzing the approaching part of the recorded F-D curves using the JPK DP software. The software converts the approaching curve into force-indentation curves by subtracting the cantilever bending from the signal height to calculate indentation. Afterwards force-indentation curves were fitted by Hertz-Sneddon model for a spherical indenter according to this equation:

[00001]$F=\frac{E}{1-v^2}\left[\frac{a^2+R_s^2}{2}\ln \frac{R_s+a}{R_s-a}-{\mathrm{aR}}_s\right]$$\delta =\frac{a}{2}\ln \frac{R_s+a}{R_s-a}$

[0098] Here, δ is the indentation depth, a is the contact radius of the indenter, R is the silica bead radius, v is the sample's Poisson ratio (set to 0.5 for cell).sup.30 and E is Young's modulus. Fitting was performed at different indentations 200, 400 and 600 nm (see SI for examples of fitting curves obtained).

[0099] For Young's modulus values, the statistical difference between two groups of data was evaluated by using the non-parametric statistical analysis of the Mann-Whitney test (two-tailed distribution) by GraphPad Prism 5.0. A p value <0.05 was considered statistically significant.

Osmotic Shrinking Assays

[0100] Cultured DRG were loaded with 500 nM C8 SIR-Tubulin for 1 h at 37° C. and/or 2 μM calcein dye (Invitrogen C3100MP) for 30 mM in DRG at 37° C. The cells were then transferred to imaging buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose) at 320 mOsm. Following a 5 mM acclimatization period the cells were subjected to a 440 mOsm (osmolarity adjusted with mannitol) hyperosmotic shock for 3 min. All imaging was carried out using a Leica SP5 resonant scanner.

Example 1

Conditional Atat1 Knock-Out Mice Have Impaired Sensation of Mechanical Innoxious and Noxious Stimuli

[0101] To investigate cell autonomous effects of Atat1 disruption in sensory neurons the inventors took a conditional gene deletion strategy. Atat1.sup.fl mice.sup.18 were crossed with a sensory neuron specific Cre driver line Avil-Cre.sup.19 to generate Avil-Cre::Atat1.sup.fl/fl (referred to as Atat1.sup.cKO) and control Avil-Cre::Atat1.sup.fl/+ mice (referred to as Atat1.sup.Control). Mice were then subjected to a series of behavioural assays. The inventors first tested their ability to detect an innocuous mechanical stimulus applied to the hairy skin. Adhesive tape was fixed gently to the backs of animals and the number of responses counted over a 5 minute observation period. While control mice made regular attempts to remove the tape, Atat1.sup.cKO mice effectively ignored it for much of the time, and the total number of responses was significantly lower (FIG. 1a).

[0102] The inventors next investigated the sensitivity of mice to innocuous mechanical stimuli applied to the glabrous skin by lightly stroking the underside of the paw with a diffuse cotton swab.sup.20. Again, Atat1.sup.cKO mice responded significantly less to this stimulus than Atat1.sup.Control mice (FIG. 1b). The inventors also examined whether mechanical sensitivity to punctate stimuli was altered in Atat1.sup.cKO mice by applying von Frey filaments of calibrated forces to the hindpaw of mice. Control animals responded to forces as low as 0.07 g with a linear increase in detection into the noxious range. However, Atat1.sup.cKO mice required significantly higher forces to evoke a response throughout the range of von-Frey filaments (FIG. 1c).

[0103] To investigate noxious mechanical sensitivity in more detail, the inventors analysed responses to a clip applied to the base of the tail. Atat1.sup.cKO mice displayed substantially longer latencies to awareness of the clip compared to Atat1.sup.Control mice and again, essentially ignored it for much of the time (FIG. 1d).

[0104] The inventors further tested whether noxious thermal detection was effected by Atat1 deletion by measuring the time to response on a hotplate. The inventors observed no difference in withdrawal latencies to noxious temperatures between Atat1.sup.cKO and Atat1.sup.Control mice (FIG. 1e). Finally the inventors assessed the motor coordination of Atat1.sup.cKO mice by evaluating their performance on a rotorod device. Atat1.sup.cKO and Atat1.sup.Control mice displayed statistically similar latencies to fall from the rotating drum across all speeds tested. Thus, Atat1 is required for the detection of innocuous and noxious mechanical touch but not for noxious heat or proprioceptive coordination.

Example 2

Sensory Neuron Electrophysiological Responses are Impaired in Atat1 Conditional Knockout Mice

[0105] Sensory neuron axons terminate in the skin and form a diverse range of functionally distinct mechanoreceptors that underlie the sense of touch.sup.1. They can be classified by their conduction velocity (into Aβ, Aδ and C fibres), their adaptation properties (into rapidly adapting or slowly adapting) and by their mechanical thresholds. To determine how Atat1 deletion affects each of these populations and regulates touch sensitivity, the inventors utilized an ex vivo skin-nerve preparation to record from single cutaneous sensory neurons in the saphenous nerve. The inventors first considered fast conducting Aβ fibres, separating them into slowly adapting (SAM) and rapidly adapting (RAM) mechanoreceptors. The inventors observed a striking reduction in the mechanical sensitivity of SAM fibres that was apparent as a reduced number of action potentials per stimulus indentation (FIG. 2a) and a ˜10-fold increase in the latency of the response in Atat1.sup.cKO mice. Reductions in firing frequencies were evident during both the ramp phase of the mechanical stimulus and during the static phase. RAM fibres displayed a similar reduction in their stimulus response function (FIG. 2b) and an increased latency to the highest displacement stimulus. A characteristic of these fibres is that they display higher firing frequencies with increasing stimulus speed.sup.21, a feature which was also reduced in Atat1.sup.cKO mice. Mechanical, electrical thresholds and conduction velocities were unchanged in Aβ fibres in the absence of Atat1. The inventors next examined Aδ fibres which can be classified as D-hair and Aδ-mechanonociceptors (AM) units by their mechanical threshold and adaptation properties. Both populations of mechanoreceptor displayed significant reductions in their stimulus response function (FIGS. 2c and d), longer latencies for mechanical activation, and decreased sensitivity to dynamic stimuli. Electrical thresholds and conduction velocities were unchanged in the absence of Atat1. Finally the inventors considered C-fibres, the largest population of sensory afferents. Similar to all other fibre types, C-fibres exhibited a reduced number of action potentials evoked by indentation stimuli (FIG. 2e), and no change in electrical properties or conduction velocity. Strikingly, mechanical thresholds of C-fibres were also significantly elevated in Atat1.sup.cKO mice (FIG. 2f). Thus Atat1 is required for mechanical sensitivity across all major fibre types innervating the skin.

Example 3

Modulation of Atat1 Enzymatic Activity Regulates Mechanosensitivity

[0106] To determine how deletion of Atat1 influences mechanotransduction in sensory neurons the inventors recorded mechanosensitive currents from cultured DRG neurons indented with a blunt glass probe. Such a stimulus can evoke mechanically gated currents in ˜90% of DRG neurons that are further classified as rapidly adapting (RA), intermediate-adapting (IA) and slowly adapting (SA) responses.sup.23. In the absence of Atat1, the inventors observed a marked loss in the number of mechanically sensitive neurons in the DRG that was evident across each subtype of current (FIG. 3a). Furthermore, the small proportion of neurons which still displayed mechanosensitive currents in Atat1.sup.cKO mice exhibited significantly reduced current amplitudes and higher thresholds (FIG. 3b-d), but no difference in their activation kinetics. Other functional parameters such as voltage gated channel activity, resting membrane potential, action potential threshold, and pH sensitivity were indistinguishable between Atat1.sup.Control and Atat1.sup.cKO mice indicating that the reduced mechanical sensitivity of DRG neurons does not arise from compromised membrane properties.

[0107] The inventors next asked whether the reduction in mechanosensitivity observed in Atat1.sup.cKO mice is dependent upon the α-tubulin acetyltransferase activity of αTAT1 by testing if mechanically activated currents could be re-established by expression of exogenous cDNAs. As a positive control the inventors determined that transfection of an Atat1-YFP construct rescued mechanosensitivity in Atat1.sup.cKO cultures and that the proportion of RA, IA and SA responses across the DRG returned to control levels (FIG. 3e). The inventors subsequently transfected a catalytically inactive form of αTAT1 that has no acetyltransferase activity but remains functional.sup.24 (termed αTAT1-GGL). Expression of αTAT1-GGL did not restore mechanosensitivity in Atat1.sup.cKO neurons, and the inventors observed no difference in the proportion of mechanically activated current types compared to mock eGFP transfection (FIG. 3e). Atat1 has also been demonstrated to acetylate other substrates in addition to α-tubulin.sup.25. Therefore, to determine whether α-tubulin acetylation underlies the mechanosensory phenotype in Atat1.sup.cKO mice, the inventors transfected a K40Q point mutant of α-tubulin that genetically mimics α-tubulin lysine 40 acetylation. Expression of K40Q α-tubulin rescued mechanosensitivity of Atat1.sup.cKO DRG neurons to Atat1.sup.Control levels, while a charge conserving control mutation (K40R) had no significant effect (FIG. 3f). Collectively these data indicated that the acetyltransferase activity of αTAT1 regulates mechanosensitivity and that acetylated α-tubulin is the likely effector.

Example 4

Microtubule Organization in Peripheral Sensory Neurons

[0108] The inventors investigated a potential structural contribution of acetylated tubulin to mechanosensitivity by examining the distribution of acetylated microtubules in sensory neurons. Strikingly, the inventors observed that acetylated α-tubulin was concentrated in a prominent band directly under the plasma membrane in cultured DRG neurons (FIG. 4a), while total α-tubulin was distributed evenly across the cytoplasm of all cells (FIG. 4b). Importantly, this band was not present in non-mechanosensory cells such as fibroblasts where acetylated α-tubulin was present throughout the microtubule network (FIGS. 4c and d). The inventors further examined the distribution of acetylated α-tubulin in intact preparations of the peripheral nervous system. Again, acetylation was highly enriched under the membrane of axons in the saphenous nerve (FIG. 4e) and also at sensory neuron terminal endings in the cornea (FIG. 4f).

[0109] The loss of the acetylated ct-tubulin sub-membrane band in Atat1.sup.cKO mice could potentially impact upon the organization of microtubules in DRG neurons and thereby influence mechanosensitivity. Indeed, it has been recently shown that the arrangement of microtubules is important for mechanosensitivity of hypothalamic osmosensory neurons.sup.26. Utilizing super-resolution microscopy and automated analysis of ct-tubulin distribution, the inventors were unable however to detect any difference in the spatial arrangement of microtubules in sensory neurons from Atat1.sup.Control and Atat1.sup.cKO mice (FIGS. 4g and h). Furthermore, the organization of the actin cytoskeleton also appeared unaltered in Atat1.sup.cKO mice.

[0110] What then is the function of the acetylated α-tubulin band, and how does it impact upon mechanosensitivity across the range of mechanoreceptors in the skin? One possibility is that it sets the rigidity of cells thereby influencing the amount of force required to displace the plasma membrane and activate mechanosensitive channels. The inventors explored this by directly measuring membrane elasticity using atomic force microscopy. In DRG neurons from Atat1.sup.cKO mice the inventors observed that cellular stiffness was significantly higher across a range of indentations extending from displacements that perturbed mainly the membrane (200 nm) to those that deformed the underlying cytoskeleton (600 nm) (FIG. 4i). Thus higher forces are required to indent sensory neurons from Atat1.sup.cKO mice than Atat1.sup.Control mice. The inventors investigated this further by assaying the sensitivity of neurons to hyperosmotic induced shrinkage. In the absence of Atat1, sensory neuron axons displayed less shrinkage than their control counterparts, an effect that could be rescued by expression of the acetylation mimicking mutation ct-tubulin K40Q (FIG. 4j).

[0111] Finally the inventors examined how the microtubule cytoskeleton responds to compression induced by osmotic pressure. Using a novel tubulin labelling fluorescent dye the inventors were able to resolve individual microtubule bundles in live imaging experiments. Strikingly, in DRG neurons from Atat1.sup.cKO mice the inventors observed significantly reduced microtubule displacement upon application of hyperosmotic solutions (FIG. 4k and 4l), again supporting the premise that in the absence of α-tubulin acetylation sensory neurons are more resistant to mechanical deformation.

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