# Patent

## Title
Method for regulating neurite growth

## Abstract

This invention relates to a method of inhibiting neuronal cell death, including protecting neuronal cells from cell death and the effects of stress, such as high or low pH, comprising administering to the cells an effective amount of Teneurin C-terminal Associated Peptide (TCAP). The invention provides the use of TCAP to prevent and/or treat a number of brain conditions, such as hypoxia-ischemia and brain alkalosis or various brain or spinal cord injuries due to physical or physiological stresses. In one aspect the invention provides a use of TCAP to increase β-tubulin, β-actin levels in neuronal cells and/or to increase fasciculation among neuronal cells, in culture or in tissue. In another aspect, the invention provides a method of treating various pH induced neuronal conditions.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/706,375, filed on Feb. 15, 2007, which is a continuation-in-part of U.S. application Ser. No. 10/510,959, filed Aug. 10, 2005, entitled “Teneurin C-Terminal Associated Peptides (TCAP) and Uses Thereof” which was a national phase entry of PCT/CA2003/00622 filed May 2, 2003, which was a non-provisional of U.S. provisional patent application No. U.S. 60/376,879, filed May 2, 2002, and a non-provisional of U.S. provisional patent application No. U.S. 60/377,231, filed May 3, 2002, and a non-provisional of U.S. provisional patent application No. U.S. 60/424,016, filed Nov. 6, 2002. U.S. application Ser. No. 11/706,375 also claims priority from U.S. provisional patent application No., U.S. 60/73,309, filed Feb. 15, 2006, entitled “A Method for Inhibiting Neuronal Cell Death”. U.S. application Ser. No. 11/706,375 also claims priority from U.S. provisional patent application No. U.S. 60/783,321, filed Mar. 21, 2005, entitled “Method for Regulating Neurite Growth”. All of these references are incorporated in their entirety by reference. Further, all sequence listings enclosed herewith and associated with this file in computer readable form are incorporated herein by reference.


FIELD OF THE INVENTION
This invention relates to a method for regulating neurite growth. In another aspect, it relates to a method for inhibiting neuronal cell death. In another aspect, it further relates to the neuroprotective effects of teneurin C-terminal associate peptides (TCAP) and to methods and uses of TCAP as a neuroprotective agent and/or to inhibit neuronal cell death and to regulate neurite growth. It further relates to the use of TCAP to induce neuronal growth, increase β-tubulin and β-actin levels in neuronal cells and induce fasciculation of neuronal cells, cultures or tissue, such as primary embryonic hippocampal cultures.
BACKGROUND OF THE INVENTION
The teneurins are a family of four vertebrate type II transmembrane proteins preferentially expressed in the central nervous system (Baumgartner et al., 1994). The teneurins are about 2800 amino acids long and possess a short membrane spanning region. The extracellular face consists of a number of structurally distinct domains suggesting that the protein may possess a number of distinct functions (Minet and Chiquet-Ehrismann, 2000; Minet et al., 1999; Oohashi et al., 1999). The gene was originally discovered in Drosophila as a pair rule gene and was named tenascin-major (Ten-M) or Odz (Baumgartner et al., 1994; Levine et al., 1994). It is expressed in the Drosophila nervous system and targeted disruption of the genes leads to embryonic lethality (Baumgartner et al., 1994). In immortalized mouse cells, expression of the teneurin protein led to increased neurite outgrowth (Rubin et al., 1999).
The extracellular C-terminal region of each teneurin is characterized by a 40 or 41 amino acid sequence flanked by enzymatic cleavage sites, which predicts the presence of an amidated cleaved peptide (Qian et al., 2004; Wang et al., 2005). A synthetic version of this peptide was named teneurin C-terminus associated peptide (TCAP) and is active in vivo and in vitro. The mouse TCAP from teneurin-1 (TCAP-1) can modulate cAMP concentrations and proliferation in mouse hypothalamic cell lines as well as regulate the teneurin protein in a dose dependent manner (Wang et al, 2004). Intracerebroventricular injection of TCAP-1 into rats can induce changes in the acoustic startle response three weeks after administration (Wang et al., 2005). [Also see, PCT/CA2003/000622. filed May 2, 2003, published Nov. 13, 2003, herein incorporated by reference.]
Currently, it is thought that following initial trauma, neurons die by necrosis, apoptosis or a combination of the two (Thompson, 1995; Columbano., 1995; Rosser and Gores, 1995; Watson, 1995). Necrosis has been defined as unprogrammed cell death induced by physiological trauma, such as hypoxia, injury, infection and cancer. The role of pH in the brain during these times of stress depends upon the trauma inflicted as both phenomenon can occur simultaneously depending upon pathological conditions, physiological activators, physical trauma, environmental toxins and carcinogenic chemicals (Wyllie et al., 1980; Arends and Willie, 1991; Buja et al., 1993; Majno and Jorris, 1995). Various neurodegenerative diseases, such as brain ischemia and Huntington's Disease, exist contingent upon various forms of cell death that in turn are mediated by their environments' surrounding pH. Although extracellular pH changes under normal metabolic circumstances, a number of pathological conditions affect pH and lead to cell death.
One of the logistical problems in understanding cell death and its corroborating factors is the ambiguity surrounding cell death. The current research indicates that many characteristics that were once thought to pertain only to apoptosis, now apply to necrosis as well. The current consensus is that following the initial insult such as during brain ischemia, brain cells die by necrosis, apoptosis or a combination of the two and pH plays a pivotal role during these times, specifically alkaline pH (Levine et al., 1992; Robertson, 2002).
Although, the literature on brain acidosis is extensive, brain alkalosis, is not well understood (Robertson, 2002). Intracellular alkalinization has been observed in cells undergoing cytokine deprivation (Khaled, 1999) as well as hypoxia-ischemia (HI) (Robertson, 2002). For example, during brain ischemia, brain pH levels indicated a progression from early acidosis to subacute alkalosis (Levine et al., 1992).
There is a need to counteract the effects of stress, such as pH induced cellular stress on the brain and to develop methods and compounds to protect cells against said effects, accordingly. Further there is a need to regulate neurite growth which may be beneficial in the diagnosis and treatment of various neuronal conditions.
SUMMARY OF THE INVENTION
In one aspect the invention provides a method for inhibiting neuronal cells against cell death. The inventors have surprisingly found that TCAP treated cells survive better in stress conditions, for instance in pH induced stress conditions, and in one aspect in alkaline pH conditions compared to vehicle treated cells.
As such, in one aspect the invention provides a method for inhibiting neuronal cells against cell death by administering an effective amount of TCAP, pharmaceutically acceptable salt or ester thereof or obvious chemical equivalent thereof to the cells. In another embodiment, administration of TCAP to the cells is administration of TCAP to a patient in need thereof comprising said cells. In one aspect the patient in need thereof is a patient who sustained or is suspected to have sustained a physiological trauma. In one aspect, a pharmaceutical composition comprising TCAP, pharmaceutically acceptable salt or ester or obvious chemical equivalent thereof and a pharmaceutically acceptable carrier is administered.
In one aspect, the invention provides a method of inhibiting and/or preventing neuronal cell death comprising administering to the cell an effective amount of TCAP, a pharmaceutical acceptable salt or ester thereof or obvious chemical equivalent thereof.
In one embodiment, inhibiting neuronal cell death comprises inhibiting and/or protecting and/or preventing neuronal cells from cell death under conditions where cell death may occur, such as a result of physiological trauma.
In one embodiment, conditions wherein cell death may occur are conditions conducive to necrosis. As such, in one aspect the invention provides a method of inhibiting, preventing or protecting neuronal cells from cell death by necrosis by administering an effective amount of TCAP, pharmaceutically acceptable salt or ester thereof or obvious chemical equivalent thereof.
In one embodiment, conditions where cell death may occur is stress-induced neuronal cell death, such as pH-induced neuronal cell death. In one aspect, pH-induced neuronal cell death is alkalosis-stress induced neuronal cell death or cell death as a result of high pH conditions. In one aspect, high pH conditions are conditions wherein pH is greater than 7.4. In another aspect, the pH is 8.0 or greater. In another aspect, the pH is from 8.0 to 9.0, 8.0 to 8.5, or 8.0 to 8.4. In another aspect, one condition of pH induced stress is from 6.0 to 7.4 or at pH 6.8.
In another aspect, the physiological trauma is selected from the group consisting of: hypoxia, injury, infection, cytokine deprivation, carcinogenic agents and cancer and/or is related to or the result of a neurodegenerative disease.
In one aspect, the neurodegenerative disease is selected from the group consisting of: Alzheimer's, Parkinson's, Huntington's, Multiple Sclerosis and brain ischemia.
In yet another embodiment, the physiological trauma is selected from the group consisting of: hypothermia, hypoxia, acute ischemia, hypoxia-ischemia, respiratory alkalosis, metabolic alkalosis and brain alkalosis. In another embodiment, it is traumatic injury to the brain or spinal cord or a result of secondary energy failure post the physiological trauma.
In one embodiment, the invention provides a method for using an effective amount of TCAP, pharmaceutical acceptable salt or ester thereof or obvious chemical equivalent thereof in the treatment of a neuronal condition associated with alkaline neuronal cell pH, by administering said TCAP to the patient in need thereof. In one aspect said condition is related to pH conditions greater than 7.4, 8.0 or greater, from 8.0 to 9.0, or from 8.0 to 8.4.
In one embodiment of the aforementioned methods of the invention, the neuronal cell is a immortalized mouse hypothalamic cell.
In one embodiment, the invention provides a method of screening of modulators of the neuronal cell death inhibitory effects of TCAP, comprising administering TCAP to neuronal cells under conditions that would normally induce neuronal cell death if TCAP were not present (e.g. pH induced cell death, alkalosis induced cell death); administering a suspected modulator of said TCAP function and determining the effects of said suspected modulator on TCAP inhibition of neuronal cell death. If said suspected modulator enhances TCAP inhibition of neuronal cell death or decreases TCAP inhibition of neuronal cell death, then it is a modulator of TCAP inhibition of neuronal cell death. In one embodiment, said suspected modulator is administered to the cells prior to, simultaneously with and/or after administration of TCAP. In another embodiment, determining the effects of said modulator comprises comparing the levels of neuronal cell death and/or survival with a control, such as cell death absent the presence of TCAP or modulator; in the presence of TCAP alone or modulator alone, or compared to established baseline effects of neuronal cell death under various conditions.
In another aspect of the invention, the invention provides a method for increasing neuronal cell proliferation under conditions of neutral pH or acidosis pH conditions. In one embodiment, the pH conditions are pH of 7.4 or less. In another embodiment, the pH conditions are 6.8 or less. In yet another embodiment the pH conditions are between 6.8 and 7.4.
In another embodiment, the invention provides a method to regulate neurite growth by administering TCAP to neuronal cells. In another embodiment, the invention provides of a method of inducing neuronal growth by administering an effective amount of TCAP to neuronal cells. In another aspect, the invention provides a use of TCAP to increase β-tubulin and/or β-actin levels in neuronal cells. In one aspect, the invention provides a method for treating conditions related to β-tubulin and/or β-actin levels, such as memory loss, learning disorders, neurodegenerative diseases and necrosis or inflammation resulting from trauma to the central nervous system.
In another aspect, the invention provides a method or use of TCAP to induce fasciculation of neuronal cells, cultures or tissue, such as primary embryonic hippocampal cultures. In yet another embodiment, the invention provides a method for treating a condition that can be treated by increasing fasciculation among neuronal cells, such as in the treatment of physiological or physical trauma to neuronal cells, such brain d injuries.
In another embodiment, the invention provides a method or use of TCAP as a guidance molecule. Axonal guidance and pathfinding is anormal and necessary aspect of neuroregeneration and restoration of function following a trauma. As such, in one aspect, the invention includes a method for axonal guidance or neurogeneration comprising administering an effective amount of TCAP to a neuron or patient in need thereof.
Additional aspects and advantages of the present invention will be apparent in view of the description which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE FIGURES
The invention will now be described in relation to the drawings, in which:
FIG. 1A. Cell Morphology of N38 cells at 48 hrs as a function of pH treatment.
FIG. 1B. Cell Morphology of N38 cells at 72 hrs as a function of pH treatment.
FIG. 1C. Example of necrotic cell.
FIG. 1D. Quantification of small crenated (necrotic) cells. The levels of significance were determined by two-way ANOVA using Bonferroni's Post Test.
FIG. 2A. Proliferation of N38 cells a function of pH. TCAP-1(10−7 M) increased the number of cells post 48 hrs after treatment at pH extremes 6.8, 8.0, 8.4. The level of significance was determined using a two-way analysis of variance (ANOVA).
FIG. 2B. Changes in cell viability, over 48 hours as determined by trypan blue. TCAP increased the number of viable cells at pH 6.8 (p<0.10) pH 8.0 (p<0.001) and pH 8.4 (p<0.05). The level of significance was determined using a two-way analysis of variance (ANOVA).
FIG. 3. Changes in mitochondrial metabolism of N38 cells as determined by the MTT assay. TCAP-1 (10−7M) increased the number of viable cells post 48 hrs after treatment at pH extremes 8.0 and 8.4. The level of significance was determined using a two-way analysis of variance (ANOVA).
FIG. 4A. Apoptotic, necrotic and healthy cells fluorescent microscopy quantification analyses post 48 hrs. Cell types are characterized by colour: apoptosis (green) necrosis (red) healthy (blue).
FIG. 4B. Example of apoptotic cell.
FIG. 4C. Apoptotic, necrotic and healthy cells fluorescent microscopy quantification analyses. TCAP significantly decreased the amount of necrotic cells post 48 hrs at pH extremes 6.8 (P<0.0001), 8.0 (P<0.0001), 8.4 (P<0.0001). A two way ANOVA was used to determine levels of significance.
FIG. 5A. Caspase 8 colorimetric assay at pH extremes.
FIG. 5B. Caspase 3 colorimetric assay at pH extremes.
FIG. 5C. Caspase 3 western blot.
FIG. 6A. PARP quantification using transformed data.
FIG. 6B. PARP western blot detection at pH extremes. Post 48 hrs TCAP-1 (10−7M).
FIG. 6C PARP optical density quantification.
FIG. 7A. Akt quantification using transformed data.
FIG. 7B. Akt western blot detection at pH extremes. Post 48 hrs TCAP-1 (10−7M).
FIG. 7C. Phospho-Akt western blot detection at pH extremes. There was no indication of AKT phosphorylation in any sample except for the control, thus TCAP is not rescuing cells through the AKT cell survival pathway.
FIG. 8. BrdU colorimetric assay at pH extremes.
FIG. 9 illustrates immortalized mouse hypothalamic N38 cells treated with 1 nM and 100 nM mouse TCAP-1 and measurements of neurite lengths. FIG. 9A illustrates untreated cells at 8 hours. FIG. 9B illustrates cells treated with 100 nM of TCAP-1 at 8 hours. FIG. 9C illustrates percent change in neurite length in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4 and 8 hours post TCAP administration. FIG. 9D illustrates percent change of number of neurites in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4, and 8 hours post TCAP administration. FIGS. 9E and 9F illustrate the frequency distribution in neurite length of the cell population in untreated (9E) and 100 nM TCAP-1 treated (9F) samples.
FIG. 10: Analysis of gene expression following TCAP stimulation. N-38 immortalized neurons were treated with 1 or 100 nM TCAP or vehicle over a 8 h timecourse. Total RNA was isolated at the indicated timepoints. Real-time RT-PCR was performed for β-actin; α-actinin 4; and β-tubulin. All genes were normalized to 18S rRNA levels as a loading control. Statistical significance was determined using a two way analysis of variance (n=5-8).
FIG. 11: β-tubulin protein expression is increased after 1 hour of 100 nM TCAP-1 treatment. A. Protein levels in N38 cells were assayed using western blotting. 100 nM TCAP-1 induced a significant increase at 1 hour (two-way ANOVA with Bonferroni's post-hoc test p<0.05) B. Representative blots for the different time-points C. Mean and SE of the optical density of the blots at 1 hour.
FIG. 12; Cytoskeletal β-actin protein expression is upregulated in N38 cells after 1 hour of TCAP-1 treatment. A. 1 and 100 nM TCAP-1 induces a significant increase in β-actin levels in cells treated for one hour (two way ANOVA with Bonferroni's post-hoc test p<0.001) B. Representative blots for the different time points C. Mean and SE of the optical density of the blots at 1 hour.
FIG. 13 Effect of TCAP-1 treatment on α-actinin-4 protein levels. A. TCAP-1 treatment did not cause any real change over 8 hours. B. Representative western blots.
FIG. 14: Confocal immunofluorescence of beta tubulin in N38 cells Confocal analysis of 100 nM TCAP-1 effects on localization of β-tubulin in N38 cells. Immunofluorescence analysis of cells treated with 1 hour TCAP-1 show an increase in β-tubulin expression both in the perinuclear and the whole cell region. A. Ten central cells from each image was analyzed for the number of pixels at maximal intensity (149) and expressed as a ratio of total pixels in B. the perinuclear region and C. the whole cell (Student's t-test with Welch's correction for unequal variances p=0.05, minimum 30 cells per group). Perinculear region and cell size not different in control and treated cells. Bar=20 μm.
FIG. 15 illustrates that 100 nM TCAP administered to a developing axon of an N38 cell in the direction of the arrow (A) causes expansion of the growth cone area followed by repulsion away from the source of TCAP (B). Bar=1 μm.
FIG. 16. Effect of TCAP-1 on primary cultures of hippocampal cells. A. Reverse image of hippocampal cultures. Top panel, vehicle treated cells, bottom panel TCAP treated cells. B. There were significantly (p<0.01) greater number of cells and cell clusters in the TCAP-1 treated cultures (n=4, two way students t test). C. Histograms of mean pixel intensity (n=4). Standard error of the mean is indicated.
FIG. 17: TCAP-1 in culture medium caused an increase in dendritic density and fasciculation in primary E18 hippocampal cultures. Anti-β-tubulin III immunocytochemistry cultured in the presence of vehicle or 100 nM TCAP-1 for seven days. Boxes indicate regions shown in the subsequent image. A. ×40 magnification, bar=0.25 mm B. ×100 magnification, bar=100 μm C. ×4000 magnification, arrows point to areas of fasciculation, bar=25 μm.
FIG. 18 illustrates the results as described in Example 8, wherein FIG. 18A illustrates the presence of the superoxide radical measured indirectly by the conversion of a soluble tetrazolium salt in cells after 48 hours. Absorbance of the substrate is proportional to superoxide radical activity. FIGS. 18B and 18C illustrate the presence of superoxide dismutase directly by western blot (FIG. 18C) and change relative to vehicle treated cells (percent) versus pH (FIG. 18B). FIG. 18D illustrates superoxide dismustase gene expression as measured by real-time PCR, while FIG. 18E illustrate superoxide copper chaperone expression as measured by real-time PCR.
FIG. 19 illustrates the results as described in Example 8. FIG. 19A illustrates that TCAP-1 showed a significant increase in MIT activity relative to the vehicle-treated at 6-48 hours in cells treated with 50 uM H2O2. FIG. 19B illustrates the results of a catalase assay on pH treated cells. FIG. 19C illustrates catalase gene expression as determined by real-tie PCR.



DETAILED DESCRIPTION OF THE INVENTION
As described herein, teneurin C-terminus associated peptide (TCAP) inhibits neuronal cell death, such as during timed of pH induced cellular stress in the brain. In another aspect, TCAP has a neuroprotective effect, protecting neuronal cells from cell death, such as, during times of pH induced cellular stress in the brain. In one aspect of the invention, such pH induced cellular stress in the brain is related to hypoxia-ischemia and/or brain alkalosis. In the examples described herein, an immortalized hypothalamic mouse cell line (N38) was treated with medium buffered at pHs 6.8, 7.4, 8.0 and 8.4 treated with 100 nM TCAP and examined at 24 and 48 hours. TCAP significantly increased cell proliferation at pH 6.8 and inhibited declines in cell proliferation at pHs 8.0 and 8.4 as determined by direct cell viability assays. TCAP did not significantly alter caspase 8 and 3 activity, nor induce PARP cleavage. TCAPs effects on the S phase of cell cycling were investigated through a bromodeoxyuridine (BrdU) uptake assay, the results showing that TCAP does not have a major effect during the S phase of cell proliferation. The incidence of necrosis was tested via cell viability (Trypan Blue) assay and fluorescence microscopy utilizing fluorophores to Annexin V and Ethidium Homodimer III as well as morphological analyses. The results indicate that TCAP can protect cells from necrosis. In one aspect, TCAP has a neuroprotective role during times of cellular stress, such as induced pH stress. As such, TCAP can be used in the treatment of physiological effects of pH in the brain during trauma, such as hypoxia-ischemia.
In another aspect of the invention, TCAP was shown to enhance neurite length, β-tubulin and β-actin levels in neuronal cells and to enhance fasciculation of neuronal cells in cell culture or tissue. All these can contribute to TCAP's neuronal protective effects against death and to inhibit neuronal cell death. In another aspect, it illustrates the use of TCAP in the treatment neuronal conditions resulting from traumatic or epigenetically associated necrosis. In one aspect, TCAP can regulate neurite and axonal growth. In another aspect, it was shown that TCAP can alter interneuron communication via changes in neurite and axon outgrowth.
Definitions
“Administering to the cell(s)” as used herein means both in vitro and in vivo administration to the cells and can be direct or indirect administration, as long as the cells are at some point exposed to the substance being administered. In the case of a peptide, it can also include methods to increase expression of the peptide or peptides to enhance exposure of the desired target to said peptide.
“Apoptosis” as used herein means “programmed cell death” and is a necessary event of normal development. It is a normal process for eliminating unwanted cells.
“Effective Amount” and “Therapeutically Effective Amount” as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results. For example, an effective amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
“Homeostasis” as used herein means the inherent tendency in an organism or cell toward maintenance of physiological stability and making automatic adjustments in relation to its environment. Other wise known as normal stability of the internal environment (Sapolsky, 1992).
“Inhibiting Neuronal Cell Death” as used herein include inhibiting, preventing, and protecting neuronal cells (including rescuing neuronal cells) from, cell death.
“Necrosis” as used herein means unprogrammed cell death induced by physiological trauma, such as hypoxia, injury, infection and cancer/carcinogenic agents.
“Neuronal Cells” as used herein includes immortalized mouse hypothalamic neurons.
“Obvious Chemical Equivalents” as used herein means, in the case of TCAP, any variant that does not have a material effect upon the way the invention works and would be known to a person skilled in the art. For instance, this could include but not necessarily be limited to any salts, esters, conjugated molecules comprising TCAP, truncations or additions to TCAP.
“Pharmaceutically Acceptable Carrier” as used herein means any medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered. It includes any carrier, excipient, or vehicle, which further includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials such as absorbants that may be needed in order to prepare a particular composition. Examples of carriers, excipient or vehicles include but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The use of such media and agents for an active substance is well known in the art (e.g., “Remington: The Sciences and Practice of Pharmacy, 21st Edition”, (University of the Sciences in Philadelphia, 2005)
“Neuronal condition associated with alkaline neuronal cell pH” as used herein means any neuronal condition that is caused by or causes or results in or is associated with alkaline neuronal cell pH. Such conditions include, but are not limited to brain ischemia, neurodegenerative diseases such as, Alzheimer's, Parkinson's, Huntington's, brain ischemia and multiple sclerosis, and brain injury associated with physiological trauma.
“Stressor” is defined as anything that disrupts physiological balance, be it physical or psychological (Sapolsky, 1992)
“Stress-related brain or neuronal condition” as used herein means any brain neuronal condition associated with neuronal cells not being in a state of homeostasis.
“TCAP” as used herein means a 38-41 amino acid sequence, preferably a 40-41 amino acid sequence from the C-terminal end of a teneurin peptide and all analogs, homologs, fragments, derivatives, salts, esters of the TCAP peptide which have the desired activity, and obvious chemical equivalents thereto, as described in PCT/CA2003/000622. filed May 2, 2003, published Nov. 13, 2003, and which is herein incorporated by reference. For instance, in one embodiment, TCAP includes human or mouse TCAP, such as TCAP 1, such as SEQ. ID. NOs. 37-44 (mouse) or 69-76 (human) of PCT/CA2003/000622 and analogs, homologs, fragments, derivatives, salts, esters and obvious chemical equivalents thereof. In one embodiment the TCAP is mouse TCAP-1 having the amino acid sequence:
QQLLGTGRVQGYDGYFVLSVEQYLELSDSANNIHFMRQSEI-NH2 (accession number nm 011855) (SEQ. ID. NO. 38).
In one embodiment TCAP is prepared by solid phase synthesis and stored as a lyophilized powder at −80° C. reconstituted by alkalinizing with ammonium hydroxide and dissolved into physiological saline at 10−4 M stock solution.
“A nucleotide encoding TCAP” as used herein means a nucleotide sequence that encodes TCAP, including DNA and RNA. Such suitable sequences are described in PCT/CA2003/000622, which is herein incorporated by reference.
Applications: The Use of TCAP to Inhibit Neuronal Cell Death
The invention broadly contemplates the use of TCAP, including an isolated TCAP, or a nucleotide encoding TCAP to inhibit neuronal cell death. In another aspect, the invention broadly contemplates the use of TCAP to increase fasciculation of neuronal cells in culture or in tissue, and in another aspect to increase β-tubulin and/or β-actin levels.
(a) Necrosis in Neurodegenerative Diseases
Necrotic cell death in the central nervous system follows acute ischemia or traumatic injury to the brain or spinal cord (Linnik, 1993; Emery, 1998). It occurs in areas that are most severely affected by abrupt biochemical collapse, which leads to the generation of free radicals and excitotoxins (e.g., glutamate, cytotoxic cytokines, and calcium). The histologic features of necrotic cell death are mitochondrial and nuclear swelling, dissolution of organelles, and condensation of chromatin around the nucleus. These events are followed by the rupture of nuclear and cytoplasmic membranes and the degradation of DNA by random enzymatic cuts in the molecule (Martin, 2001). Given these mechanisms and the rapidity with which the process occurs, necrotic cell death is extremely difficult to treat or prevent. The present inventors herein describe a method of treating and/or preventing necrotic cell death using TCAP.
(b) pH in Necrosis
According to Potapenko et al., brain alkalinization induces an increase of Ca2+ in neurons due to Ca2+ sequestering structures, such as the mitochondria and endoplasmic reticulum, and elevated cytoplasmic Ca2+ is implicated in neuronal cell death, more specifically, necrosis during brain ischemia (Yuan et al., 2003). As mentioned previously such excessive rises in Ca2+ may be induced by excitoxicity caused by brain ischemia, subsequently over stimulating postsynaptic glutamate receptors; of these glutamate-gated channels, NMDA receptor channels play a key role in excitotoxicity as they conduct both Na+ and Ca2+ (Bonfoco et al. 1995).
(c) Brain Injuries Related to Alkalosis
Insults to the brain can quite often lead to shifts in pH and based on the data presented it appears that TCAP is rescuing neurons from necrosis consistently at high pH extremes, specifically pH 8.0 and 8.4. Dying neurons are a clear indication of many neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's, brain ischemia and multiple sclerosis (Siao, 2002). These neuro degenerative conditions are characterized by their deleterious effects on brain function resulting from deterioration of neurons. The destruction of neurons in these conditions may be regulated by various forms of cell death and can be caused by damaged mitochondrion, increased levels of excitotoxins such as glutamate, which increases calcium influx into the neurons and activates calcium dependent enzymes such as calpain and caspases (Randall & Thayer, 1992; Brorson et al., 1995) and pH. Brain pH during times of neurodegenerative stress is not well understood, however, calcium and pH are not mutually exclusive, during both respiratory and metabolic alkalosis, increases in calcium occur in rat neurons due to intracellular calcium accumulating structures such as the mitochondrion (Potapenko, 2004), this is also substantiated by the fact that glutamate induced neuron death requires mitochondrial calcium uptake (Stout et al., 1998).
Recent studies on brain energy metabolism using phosphorous and proton magnetic resonance (MR) spectroscopy have allowed an understanding of energy changes within the brain following (HI) (Thornton, 1998; Moon, 1973). A phenomenon named the “secondary energy failure” that occurs some 8-24 hours after the initial insult has been recently discovered, and have correlated the magnitude of this disruption with the eventual neurodevelopmental outcome (Thornton, 1998). A similar relationship between intracellular alkalosis and the severity of brain injury in infants has also found that babies with the most alkaline brain cells had more severe changes on MR imaging within the first 2 weeks of life and the worst neurodevelopmental outcome at one year (Robertson, 2002). Thus, a means of identifying neuropeptides with pH protective properties would be a pivotal finding as it would provide novel therapeutic treatments. The inventors have shown herein that TCAP is a neuroprotective peptide and can inhibit neuronal cell death. As such, it can be used to treat a number of neuronal conditions, such as a neuronal condition associated with alkaline neuronal cell pH.
(d) Neuronal Cell Death Inhibition/Neuroprotective Role of TCAP During Times of Stress
The potential for neuropeptides to regulate brain processes during times of stress (e.g. as a result of a stress-related brain or neuronal condition) is an important paradigm in the search for novel ways of coping with neurodegenerative diseases and physiological stress and examples of neuropeptides being connected with therapeutic uses are plentiful. (Gozes et al., 1994; Glazer et al. 1994; Zhang et al., 2001) The teneurin C-terminus associated peptides (TCAP) have a neuroprotective effect from cell death, during times of pH induced cellular stress in the brain such as during hypoxia-ischemia. The present inventors herein describe a method of treatment or use of TCAP in the treatment of such stress-related brain or neuronal conditions and the use of TCAP in the preparation of a medicament for the treatment of such conditions.
(e) Screening for Potential Modulators of TCAP Inhibition of Neuronal Cell Death.
In on embodiment, the invention provides a method for screening compounds that modulate TCAP inhibition of neuronal cell death, comprising, administering TCAP to neuronal cells under conditions that promote inhibition of neuronal cell death in the presence of a potential TCAP modulator and monitoring the affects of said potential modulator on the viability of the neuronal cells. In one embodiment, this can be done in comparison to a control, such as the potential modulator with or without TCAP and/or with TCAP but no potential modulator. In one aspect of the invention the administration of TCAP can occur in a number of ways including, but not necessarily limited to: administering the TCAP in a suitable form of peptide to the cells, administering a substance that will enhance TCAP expression and availability of TCAP to the cell; administration of a nucleic acid encoding TCAP that will result in enhanced TCAP expression to the cell.
(f) The Use of TCAP to Regulate Neurite Growth—TCAP as a Neuroplastic Agent
In one embodiment of the invention, TCAP alters interneuron communication via changes in neurite and axon outgrowth. Synthetic mouse/rat TCAP-1 was used to treat cultured immortalized mouse hypothalamic cells to determine if TCAP-1 could directly regulate neurite and axon growth. TCAP-1 treated cells showed a significant increase in the length of neurites, accompanied by a marked increase in β-tubulin transcription and translation as determined by real-time PCR and western blot analysis, respectively, although changes in α-actinin 4 transcription and β-actin translation were also noted. Immunofluorescence confocal microscopy using β-tubulin antisera showed enhanced resolution of β-tubulin cytoskeletal elements throughout the cell. In order to determine if the effects of TCAP-1 could be reproduced in primary neuronal cultures, primary cultures of day E18 rat hippocampal cells were treated with 100 nM TCAP-1. The TCAP-1 treated hippocampal cultures showed a significant increase in both the number of cells and the presence of large and fasciculated β-tubulin immunoreactive axons. The data indicates the TCAP acts as a functional region of the teneurins to regulate neurite and axonal growth of neurons.
It is also herein shown that TCAP-1 increases neurite length and alters the levels and distribution of key cytoskeletal proteins and genes associated with axon outgrowth in immortalized neuronal cell lines. Moreover, because both TCAP-1 expression (Wang et al., 2005) and teneurin-1 (Zhou et al., 2003) expression is high in hippocampal cells the effects of TCAP-1 was studied on primary cultures of hippocampal cells. In these cultures TCAP-1 dramatically increased the incidence of axon formation, e.g. in primary cultures of hippocampal cells. The TCAP/teneurin system represents a new mechanism in neuroplasticity.
This has implications in the treatment of certain conditions and inducing changes in the brain, such as changes in acoustic startle response, learning, memory, anxiety or other brain or neuronal conditions. TCAP can be used to treat such conditions.
One can screen for modulators of TCAP, neurite growth or neuroplasticity, by administering the suspected modulator to a neuron or neurons or tissue comprising neurons in the presence of TCAP under conditions that promote neurite growth or neuroplasticity and monitoring the effects of the suspected modulator on said activities. The effect can be compared to a control, such as known baseline levels of activity, or a control such as in the presence or absence of TCAP and/or the suspected modulator. In one embodiment, a modulator can enhance the effects of TCAP. In another embodiment, the modulator can diminish the effects of TCAP.
Pharmaceutical Compositions and Modes of Administration
TCAP, pharmaceutically acceptable salts or esters thereof or obvious chemical equivalents thereof can be administered by any means that produce contact of said active agent with the agent's sites of action in the body of a subject or patient to produce a therapeutic effect, in particular a beneficial effect, in particular a sustained beneficial effect. The active ingredients can be administered simultaneously or sequentially and in any order at different points in time to provide the desired beneficial effects. A compound and composition, of the invention can be formulated for sustained release, for delivery locally or systemically. It lies with the capability of a skilled physician or veterinarian to select a form and route of administration that optimizes the effects of the compositions and treatments of the present invention to provide therapeutic effects, in particular beneficial effects, more particularly sustained beneficial effects.
In one embodiment, administration of TCAP includes any mode that produce contact of said active agent with the agent's sites of action in vitro or in the body of a subject or patient to produce the desired or therapeutic effect, as the case may be. As such it includes administration of the peptide to the site of action—directly or through a mode of delivery (e.g. sustained release formulations, delivery vehicles that result in site directed delivery of the peptide to a particular cell or site in the body. It also includes administration of a substance that enhances TCAP expression and leads to delivery of TCAP to a desired cell or site in the body. This would include but is not limited to the use of an oligonucleotide encoding TCAP, e.g. via gene therapy or through a TCAP expression system in vitro or in vivo, as the case may be that results in enhanced expression of TCAP. It can also include administration of a substance to the cell or body that enhances TCAP levels at the desired site.
The above described substances including TCAP and nucleic acids encoding TCAP or other substances that enhance TCAP expression may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. By “biologically compatible form suitable for administration in vivo” is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans, and animals.
Thus in one embodiment, the invention provides the use of TCAP or modulator thereof in the preparation of a medicament for the inhibition of neuronal cell death and/or the treatment of related conditions. In one embodiment, a therapeutically effective amount of TCAP or a pharmaceutical composition as described herein is administered to a patient in need thereof. A patient in need thereof is any animal, in one embodiment a human, that may benefit from TCAP and its effect on inhibition of neuronal cell death or increase neuronal growth, β-tubulin or β-actin levels, increase in fasciculation or as a guidance molecule.
An active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the compound. In one embodiment, TCAP is administered directly to or proximate to the desired site of action, by injection or by intravenous. If the active substance is a nucleic acid encoding, for example, a TCAP peptide it may be delivered using techniques known in the art.
The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutical acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutical acceptable vehicle or carrier. Suitable vehicles or carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985 or Remington's The Sciences and Practice of Pharmacy, 21st Edition”, (University of the Sciences in Philadelphia, 2005) or Handbook of Pharmaceutical Additives (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995)). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutical acceptable vehicles, carriers or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids. In this regard, reference can be made to U.S. Pat. No. 5,843,456.
As will also be appreciated by those skilled, administration of substances described herein may be by an inactive viral carrier. In one embodiment TCAP can be administered in a vehicle comprising saline and acetic acid.
Further, in one embodiment, TCAP may be administered in a form that is conjugated to another peptide to facilitate delivery to a desired site, or in a vehicle, eg. a liposome or other vehicle or carrier for delivery. For instance, in one embodiment TCAP can be conjugated to a brain targeting vector, which is a peptide or peptidomimetic monoclonal antibody (MAb), that is transported into brain from blood via an endogenous blood brain barrier (bBB) transport system, which has shown to significantly reduce stroke volume (e.g. see Zhang et al. (2001)). Thus, in one embodiment, brain ischemia can be treated by neuropeptides, such as TCAP, with noninvasive intravenous administration. In one embodiment, the peptide is conjugated to a BBB drug targeting system such as transferrin, for example as described in Vuisser et al. (2004) or Kang et al. (1994). In another embodiment, TCAP does not require a transport mechanism to cross the blood brain barrier.
The present invention is described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
EXAMPLES
Example 1—Peptide Synthesis
Mouse TCAP-1 (i.e., SEQ. ID. NO. 38) was prepared by solid phase synthesis as previously described (Qian et al., 2004). The peptide was solubilized in phosphate buffered saline (PBS) at a concentration of 2×10−7 M before being diluted in the appropriate medium.
More particularly, a mouse paralogue of the putative peptide sequence from teneurin-1 was synthesized on an automated peptide synthesizer, Model Novayn Crystal (NovaBiochem, UK Ltd., Nottingham, UK) on PEG-PS resin using continuous flow Fmoc chemistry (Calbiochem-Novabiochem Group, San Diego, Calif.). Eight times excess diisopropyl ethylamine (Sigma-Aldrich Canada Ltd.) and four times excess Fmoc-amino acid activated with HATU (O-(7-azabenzotriazol)-1-3,3-tetramethyluronium hexyluorophosphate; Applied Biosystems, Foster City, Calif.) at a 1:1 (mol/mol) ratio were used during the coupling reaction. The reaction time was 1 h. A solution of 20% piperidine (Sigma-Aldrich Canada Ltd.) in N,N-dimethylformide (DMF; Caledon Laboratories Ltd., Canada) was used for the deprotection step in the synthesis cycle. The DMF was purified in-house and used fresh each time as a solvent for the synthesis. The cleavage/deprotection of the final peptide was carried out with trifluoroacetic acid (TFA), thioanisole, 1,2 ethandithiol, m-cresole, triisopropylsilane, and bromotrimethyl silane (Sigma-Aldrich Canada Ltd.) at a ratio of 40:10:5:1:1:5. Finally, it was desalted on a Sephadex G-10 column using aqueous 0.1% TFA solution and lyophilized. The peptide was solubilized by exposure to ammonium hydroxide vapors for 2 minutes before dilution in phosphate-buffered saline (PBS) pH 7.4 with 10 nM sodium phosphate.
Example 2—Cell Morphology Analysis
The effect of TCAP-1 on cell morphology was conducted using the N38 cells immortalized mouse hypothalamic cell line (Belsham et al, 2004). Cells were grown in six-well culture plate with 2 ml of Dulbeco's Modified Eagle Medium (DMEM) with high glucose, L-glutamate, 25 mM HEPES buffer, pyridoxine hydrochloride in the absence of sodium pyruvate, 5 ml penicillin with 10% fetal bovine serum (FBS) at pH 7.4 (all from Gibco-Invitrogen, Burlington, Canada).
At 24 and 48 hrs, the medium was replaced with medium buffered at pH 6.8, 7.4, 8.0 or 8.4. Half of the cell groups received (10−7M) TCAP-1, whereas the other half received phosphate buffered saline (PBS) pH 7.4 containing 8 g NaCl, 0.2 g KCl, 1.4 g Na2HPO4, 0.2 g KH2PO4 in 800 mL ddH2O. For all groups, 4 replicates were run. Digital pictures were taken at 24, 48 and 72 hrs using an Olympus IX&1 inverted microscope at magnification and analyzed using Lab Works 4.0 Image Acquisition and Analysis Software (Ultraviolet Products Ltd., CA)
Results
TCAP did not induce any observable morphological changes in the cells cultured at pH 7.4. However, there was significant increase in the number of small round cell types (necrotic cells) in the vehicle-treated cultures at pH 6.8 (p<0.05), 8.0 (p<0.001) and 8.4 (p<0.001) as compared to the TCAP-treated samples at 48 hrs (F=96.16). At 72 hrs, TCAP significantly decreased the number of rounded cells in pH 8.0 (p<0.001) and pH 8.4 (p<0.001) (F=51.13) relative to the vehicle-treated cells. (FIGS. 1A-1D).
Example 3—Effect of TCAP on Cell Proliferation and Viability
The effect of TCAP-1 on cell proliferation at each pH was examined by direct counts using a hemocytometer and indirectly by assessing mitochondrial activity using a colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay on cultured N38 cells. For hemocytometer counts, the cultures were incubated for 24 and 48 hrs. The cells were suspended using 1 ml of 0.25% Trypsin with EDTA (Gibco-Invitrogen, Burlington, Canada), centrifuged at 1600 RPM for 4 min, and resuspended with PBS. The cells in 50 μl aliquots were vortexed and counted on a hemocytometer.
The proportion of viable cells in the samples was determined by measuring Trypan Blue uptake. At 48 hrs, the cells from the four pH treatments were suspended using 1 ml of Trypsin EDTA, centrifuged at 1600 RPM for 4 min and resuspended in 1 ml of BSS (Hank's Balanced Salt Solution) (Sigma, St. Louis). An aliquot of 0.5 ml of 0.04% Trypan Blue solution was transferred to a 1.5 ml tube, 0.03 ml of BSS was added to 0.2 ml of the cell suspension; the samples were mixed thoroughly and the cell suspension-Trypan Blue mixture was allowed to stand for 10 minutes and then counted on a hemocytometer. Separate counts were kept for both viable and non viable cells.
A (3-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) MTT assay was conducted using the In Vitro Toxicology Assay Kit: MIT based (Sigma, St. Louis). The cells were cultured for the MTT assay at 24 and 48 hours and were incubated at 37° C. in 5% CO2 for 3 hrs in the presence of MTT 200 μl/2 ml medium. The samples were mixed by shaking the plate horizontally for 30 min. The background absorbance of the multi-well plates was determined at 690 nm and subtracted from the 570 nm measurement.
Results
There were no significant differences in the total number of cells, as determined by direct hemocytometer counts, between the vehicle- and TCAP-treated cells at 24 hrs under any pH condition (FIG. 2A). There was a marked reduction in the number of total cells at pH 8.0 and 8.4 in the vehicle-treated cells at both 24 and 48 hrs. However, TCAP inhibited the decrease in total cell numbers relative to the vehicle-treated cells at pH 6.8 (P<0.001) 8.0 (P<0.001) and 8.4 (P<0.01) (F=38.10) after 48 hrs of incubation.
A Trypan Blue stain was conducted in order to estimate the proportion of viable cells in a population (FIG. 2B). TCAP-1 treatment caused a significant decrease in the number of cells that took up the trypan blue stain at 48 hrs in cells cultured at pH 6.8 (p<0.05), pH 8.0 (p<0.0001) and at pH 8.4 (p<0.001) (F=58.27) but not pH 7.4. Although TCAP did not induce a significant effect on MTT activity at pH 7.4 or pH 6.8 there was a significant increase in optical density at 48 hrs in TCAP-1-treated samples cultured in pH 8.0 (p<0.01) and pH 8.4 (p<0.001) (F=21.19) (FIG. 3).
Example 4—Fluorescent Microscopy of Necrosis and Apoptosis Markers
N38 cells were cultured on poly-D-Lysine treated coverslips (VWR, Mississauga) in each of the four pH condition, and cells were washed twice with PBS, each fluorochrome was added to each well: 5 μl Fluorescein (FITC)-Annexin V in Tris EDTA buffer containing 0.1% BSA (Bovine serum albumin) and 0.1% NaN3, pH 7.5, 5 μl rhodamine EtD-III 200 μM in PBS and 5 μl 4′,6-Diamidino-2-phenylindole (DAPI) Hoechst 33342 5 μg/mL in PBS (Biotium, Inc. Hayward). The samples were incubated in the dark for 15 min, then washed before being placed on slides. The cells were viewed under a LEICA DM 4500 inverted fluorescent microscope and digitally analyzed using OpenLab software.
Results
Annexin V labelled with fluorescein (FITC) was used to identify apoptotic cells in green. Ethidium homodimer III (EtD-III) is a positively charged nucleic acid probe, which is impermeable to live or apoptotic cells but stains necrotic cells with red fluorescence (rhodamine) and Hoechst 3342 (4′,6-Diamidino-2-phenylindole (DAPI) emits bright blue fluorescence upon binding to DNA in living cells.
TCAP-1 decreased the number of rhodamine-fluorescing cells at pH 6.8 (p<0.001), 8.0 (p<0.001) and 8.4 (p<0.001) (F=348.2) but not in the pH 7.4 samples (FIGS. 4A and 4C). There were nominal amounts of FITC-labelled cells located intermittently throughout samples where only a total of 3 green cells were counted (see inset, FIG. 4B and inset).
Summary of Examples 3 and 4—Necrosis
Necrosis occurs when cells are exposed to extreme variance from physiological conditions such as hypothermia and hypoxia, which may result in damage to the plasma membrane (Majno and Jorris, 1995). Necrosis begins with an impairment of the cell's ability to maintain homeostasis, leading to an influx of water and extracellular ions. Intracellular organelles, most notably the mitochondria, and the entire cell swell and rupture (cell lysis) (Linnik et al, 1993). Due to the ultimate degeneration of the plasma membrane, the cytoplasmic contents including lysosomal enzymes are released into the extracellular fluid. Therefore, in vivo, necrotic cell death is often associated with extensive tissue damage resulting in an intense inflammatory response (Emery et al, 1993). Necrosis was determined as the form of cell death occurring based on expected morphological alterations affecting the plasma membrane including massive production of small surface evaginations (bubbles) caused by the cells inability to control water influx through the plasma membrane (Rello et al., 2005). The Trypan Blue Stain (Example 3) is based on an acid dye that contains two azo chromophores. The reactivity of this dye is dependent on the negatively charged chromophore binding to cytoplasmic material when the membrane is damaged. Staining facilitates the visualization of cell morphology since it is only the dead cells that take up the dye, thus identifying cells that are necrotic or are in the very late stages of apoptosis. The fluorescent microscopy study (Example 4) also solidifies this assumption as TCAP decreases the number of necrotic cells and not apoptotic cells. These findings are significant as necrosis plays an integral role in neurodegenerative diseases.
Example 5—Apoptosis (Caspase and PARP) Markers
Apoptosis, otherwise known as “programmed cell death” is a necessary event of normal development. The apoptotic pathway is mediated by a family of death proteins, caspases, These signaling proteins are proteolytic enzymes that when inactive, lay dormant as zymogens until they are activated by various triggers (Hengartner, 2000). Upon activation of caspase 3 certain nuclear proteins are cleaved such as Poly ADP-ribose polymerase (PARP). PARP, a 116 kDa nuclear polymerase, is involved in DNA repair usually in response to environmental stress (Hengartner, 2000; Willie, 1980; Kerr, 1972). The protein can be cleaved by many interleukin-converting enzyme-like (ICE-like) proteases (Willie, 1980; Liu, 1997). (PARP) was one of the first proteins reported to be cleaved during apoptosis, and is a target of the Yama/CPP32 protease, caspase-3 (Kaufmann, 1989; Kaufman et al, 1993). Cleavage products occurring due to apoptosis result in western blot bands at 89 KDa. The following experiments were conducted to determine whether TCAP works through the apoptotic pathway.
(a) Colorimetric Caspase Assays
Caspase 8 and 3 colorimetric assays were performed on the N38 cells at all pH conditions. The assay was based on the detection of the chromophore pNA after cleavage from the labeled substrate IETD-pNA and DEVD-pNA for caspase 8 and 3, respectively. Comparison of the pNA absorbance from the suspected apoptotic sample was compared to the uninduced neutral pH sample. Caspase 8 and 3 were analysed using the Caspase-3 Colorimetric Activity Assay (Chemicon, Temecula USA) and Caspase-8 Colorimetric Activity Assay (Chemicon, Temecula USA). The cells from each pH treatment described previously at 24 and 48 hrs were removed using a cell scraper and centrifuged at 1500 rpm for 10 minutes. The cells were resuspended in 350 μl of chilled cell lysis buffer containing 500 μl PBS, 5 μl 1% Triton ×100 (Sigma, St. Louis), 25 μl proteinase inhibitor cocktail set III (VWR, Mississauga), 0.5 μl 1M dithiothreitol (DTT) (Sigma, St. Louis) and 2.5 μl phenylmethylsulphonylfluoride (PMSF) diluted in 1 mL of methanol (EM Science, Gibbstown), then incubated on ice for 10 min and centrifuged for 5 minutes at 10,000 rpm. The supernatant, consisting of cytosolic extracts, was transferred to a new tube and a bicinchoninic acid (BCA) protein assay (Pierce, Rockford) was conducted to determine total protein concentration. The absorbance of each sample was measured on a SPECTRAmax Microplate spectrophotometer at 405 nm after an incubation period of 2 hours at 37° C. Changes in caspase 3 activity were determined by comparing the absorbance reading from the induced sample with the level of the uninduced control. Background readings from the buffer were subtracted from the reading of both the induced (pH 6.8, 8.0, 8.4) and uninduced (pH 7.4) samples before calculating changes in caspase 3 activity. The same was done for the detection of caspase 8. As a control, N38 cells were cultured with pH 7.4 DMEM and incubated for 4 days, apoptosis was then induced using 10 μM/ml etoposide and lysed according to the above protocol and used a control for all subsequent caspase 3 detection. All assays were performed with 4 replications.
(b) Caspase 3 and Poly(ADP-Ribose)Polymerase (PARP) Cleavage by Immunoblot
Detection of caspase 3 cleavage was determined at 48 hrs. The samples at each pH and control (see above) were lysed using total protein isolation lysis buffer (described above). An aliquot of 25 μl of each sample was combined with 25 μl of 2×20% sodium dodecyl sulphate (SDS) sample buffer and loaded onto a 4-10% HCL-Tris pre cast polyacrylamide gel (BioRad, Mississauga). The gel was run at 200 v for 35 min and proteins were electrotransfered to a Hybond-C nitrocellulose membrane (Amersham, Baie d'Urfé) for 75 min at 100 v. After transfer, the membrane was washed with 10 ml of PBS with 0.05% Tween 20 (PBST) for 5 min at room temperature (RT) and the membrane was incubated in 10 ml of PBST-milk for one hour at RT followed by 3 times for 5 min washes with 10 ml of PBST. The membrane was then incubated with cleaved caspase 3 primary antiserum (Cell Signaling Technology, Beverly) at a titre of 1:500 in 6 ml of PBST-milk with gentle agitation overnight at 4° C. The membranes were washed 3 times for 5 min with 10 ml of PBST followed by membrane incubation with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham, Baie d'Urfé) at 1:3000 in 6 ml of PBST-milk with gentle agitation for 1 hr at RT. The membranes were then washed 3 times for 5 min with 10 ml of PBST then exposed to Kodak X-OMAT Blue scientific imaging film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) for 30 min.
Using the same protocol, changes in PARP expression were determined at 48 hrs. The membrane was incubated with PARP primary antibody (Cell Signaling Technology, Beverly) at a titre of 1:100. The membranes were washed 3 times for 5 min with 10 ml of PB ST followed by membrane incubation with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham, Baie d'Urfé) at 1:3000 in 6 ml of PBST-milk with gentle agitation for 1 hr at RT. The membranes were then washed 3 times for 5 min with 10 ml of PBST then exposed to Kodak X-OMAT Blue scientific imaging film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) for 30 min. Total optical density of the blots, were quantified using LabWorks 4.0 Image Acquisition and Analysis Software from Ultra-Violet Products Ltd. (UVP).
Results
Etoposide was used to determine the amount of caspase 8 (FIG. 5A) and 3 (FIG. 5B) activation under apoptotic conditions. Etoposide induced a greater than 3-fold increase in caspase 8 and 3.5-fold increase in caspase-3 relative to the vehicle-treated cells at pH 7.4. Although TCAP-1 increased caspase 8 activity in pH 7.4 samples (P<0.001) (F=20.80) and increased caspase 3 activity in pH 6.8 samples (P<0.05) (F=2.117), the relative level of caspase activity was about 70% and 40% of the etoposide-induced increase for caspase 8 and 3 respectively. There were no significant differences in caspase 8 and 3 activity between the TCAP-1- and vehicle-treated cells at pH 8.0 and 8.4. As a further determination of caspase 3 activity, four replicates of western blots were conducted on pH treated N38 cells at the 48 hr mark in order to detect the cleaved and activated caspase 3 (17/19 kDa) (FIG. 5C). The caspase 3 cleavage product was clearly visible in the protein extracts of the etoposide-treated cell but could not be observed in any of the TCAP-1 or vehicle-treated cells at any of the pH conditions.
Four replicates of western blots were conducted on pH treated N38 cells at the 48 hour mark in order to detect endogenous levels of full length PARP, as well as the large fragment (89 kDa) and small fragment (24 kDa) of PARP resulting from caspase cleavage. The western blot (FIG. 6B) revealed endogenous PARP at all pH treatments as well as vehicles samples and based on a two way ANOVA using Bonferroni's Post Test, there were no significant differences between vehicle and TCAP treated samples (FIGS. 6A and 6C).
Based on the studies conducted and described in Example 5, TCAP is not protecting neuronal cells by inhibiting the apoptotic pathway.
Example 6—Kinase B/Akt Cell Survival Pathway
Protein kinase B or Akt (PKB/Akt) is a serine/threonine kinase, which functions to promote cell survival by inhibiting apoptosis by means of its ability to phosphorylate and inactivate several targets including BAD and forkhead transcription factors (Crowder, 1998). AKT, also referred to as PKB or Rac, plays a critical role in controlling the balance between cell survival and cell death in neurons (Dudek, 1997). The present example was conducted to determine whether TCP acts through this particular survival pathway.
Western blots using Akt and phosphorylated Akt (P-Akt) primary antibodies were conducted on all conditions of the cultured N38 cells to determine whether TCAP was preventing cell death by phosphorylation. The same western blot procedure outlined above was repeated with an Akt primary antibody (Cell Signalling, Beverly) at a titer of 1:500, followed by membrane incubation with anti-rabbit HRP-conjugated secondary antibody (Amersham, Baie d'Urfé) at 1:3000, followed by exposure on Kodak X-OMAT Blue film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) for 30 min. Phospho Akt expression at 48 hrs was determined using the method described above with a PAkt primary antibody (Cell Signalling 9271) at 1:1000 followed by membrane incubation with anti-rabbit HRP-conjugated secondary antibody (Amersham, Baie d'Urfé) at 1:2000 followed by exposure on Kodak X-OMAT Blue film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) overnight. Cultured N38 cells were serum-starved for 48 hours in order to induce phosphorylation and following the same protocol above were loaded as a control. Total optical density of the blots, were quantified using LabWorks 4.0 Image Acquisition and Analysis Software from Ultra-Violet Products Ltd. (UVP).
Results
Western blots were conducted using an Akt antibody, which detected total levels of endogenous Akt (FIG. 7B). The blot revealed endogenous Akt in all treatments as well as the vehicle, however according to a two way ANOVA using Bonferroni's Post Test, there appears to be no difference in endogenous Akt between vehicle and TCAP treated samples (FIG. 7A). Total optical density of the blots were quantified using LabWorks 4.0 Image Acquisition and Analysis Software from Ultra-Violet Products Ltd. (UVP).
Western blots were conducted using a Phospho-Akt antibody, which detected total levels of endogenous Akt1 only when phosphorylated at serine 473. The blot revealed no bands in any samples, thus phosphylation of Akt is not occurring. Phsophorylation of cells was induced by serum starvation and loaded as a control, the blot revealed a band, however no other bands were detected (FIG. 7C).
Example 7—The Effect of TCAP on Cell Cycling: Bromodeoxyuridine (BrdU) Incorporation Assay
The evaluation of cell cycle progression is important when assessing the viability of a cell population. The cell cycle is a sequence of stages that a cell passes through between one division and the next. The cell cycle oscillates between mitosis and the interphase, which is divided into G, S, and G 2. In the G phase there is a high rate of biosynthesis and growth; in the S phase there is the doubling of the DNA content as a consequence of chromosome replication; in the G 2 phase the final preparations for cell division (cytokinesis) are made (Raza, 1985). In order to determine whether TCAP was increasing cell cycle efficiency, a bromodeoxyuridine (BrdU) non-isotopic enzyme immunoassay was conducted (Calbiochem, Canada). BrdU incorporation into newly synthesized DNA of actively proliferating cells enables one to quantify cell cycle progression and the population of cells entering the S phase (Gratzner, 1982; Raza, 1985).
N38 cells were grown in a 96-well culture plate using 100 μl at an initial density of 2×105 cells/ml. Controls consisted of a blank, one well containing only DMEM with no cells and background, and one well with cells but with no BrdU label added. A working stock of BrdU was prepared by diluting the BrdU label 1:2000 into fresh DMEM, 20 μl of the working stock was added to each well to be labelled, the BrdU was allowed to incubate with the cells for 2 hrs at 37° C. The contents of the wells were then removed and 200 μl of the enclosed Fixative/Dentauring solution was added to each well and incubated for 30 min at Room Temperature (RT). The contents of the wells were removed and Anti-BrdU Antibody (1:100) was added to each well and incubated for 1 hr at RT. Wells were washed 3 times with wash buffer, the plate was then gently blotted on paper towel. The conjugate was prepared by diluting the reconstituted in (1×PBS) peroxidase goat anti-Mouse IgG HRP conjugate in the enclosed conjugate diluent and loaded onto a syringe filter through 0.2 μm filter and a 100 μl aliquot of this solution was transferred to each well and incubated for 30 min at RT. The wells were washed with wash buffer, the entire plate was then flooded with double deonized water and the contents of the wells were removed. An aliquot of 100 μl of BrdU substrate solution was added to each well, the plate was then incubated in the dark at RT for 15 min. 100 μl of stop solution containing 2.5N sulphuric acid was added to each well in the same order as the previously added substrate solution. Absorbance was measured on a SPECTRAmax Microplate spectrophotometer at dual wavelengths at 450-540 nm.
Results
Based on a two way ANOVA using Bonferroni's Post Test there were no significant results at 24 or 48 hrs (FIG. 8).
This investigation indicates that synthetic TCAP-1 has a neuroprotective effect on immortalized hypothalamic mouse cells. The data described in this study suggest a significant neuroprotective role for TCAP during times of pH induced cellular stress. Several lines of evidence point to this. Based on haemocytometer counts and an MTT assay conducted on pH stressed N38 cell samples, TCAP has a positive affect on cell viability during pH induced cellular stress, suggesting that TCAP could be inhibiting cells from undergoing apoptosis, acting through a cell survival pathway or rescuing cells from necrosis. The Examples herein indicate that this neuroprotective effect occurs by the inhibition of mechanisms regulating necrosis and to a lesser extent by regulating apoptotic, survival, or cell cycle pathways.
Example 8—TCAP Modulates Neurite Length in Immortalized Hypothalamic N38 Cells
Immortalized mouse hypothalamic N38 cells were treated with 1 nM and 100 nM mouse TCAP-1 and measurements of neurite lengths were taken over 8 hours post TCAP administration. FIG. 9A illustrates untreated cells at 8 hours. FIG. 9B illustrates cells treated with 100 nM of TCAP-1 at 8 hours. FIG. 9C illustrates percent change in neurite length in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4 and 8 hours post TCAP administration. FIG. 9D illustrates the percent change of number of neuritis in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4, and 8 hours post TCAP administration. FIGS. 9E and 9F illustrate the frequency distribution in neurite length of the cell population in untreated (9E) and 100 nM TCAP-1 treated (9F) samples.
Results
The results of these experiments illustrate that TCAP is useful in enhancing neurite length. TCAP-1 induced 25% and 31% increase (p<0.001, one way ANOVA with Bonferroni's post-test, n=4) in neurite length at 100 nM, at 4 hr and 8 hr, respectively, relative to the length at the beginning of treatment. After 8 hrs, 100 nM TCAP-1 induced about a 45% (p<0.001) reduction in the number of neurites per cell. A frequency distribution of the neurite length indicated that 100 nM TCAP-1 promoted longer but fewer neurites per cell.
Example 9—TCAP Upregulates β-Tubulin and β-Actin Levels In Immortalized N38 Cells
β-tubulin and β-actin expression levels in immortalized mouse N38 cells were studied.
Materials and Methods
Primary Antisera
All antisera used in this study are rabbit polyclonal antisera. β-Actin and GAPDH were purchased from Abcam (Cambridge, Mass.). α-actinin 4 antisera were purchased from Alexis Biochemicals (Lausen, Switzerland). The β-tubulin antisera was purchased from Neomarkers, Lab Vision (Fremont, Calif.) and β-tubulin III was purchased from Sigma-Aldrich Canada (Oakville, ON).
Morphological Analyses of Immortalized Neurons
N-38 immortalized mouse hypothalamic cells were cultured in quadruplet in 6 well tissue culture plates until 70% confluent at which time fresh DMEM with 10% fetal bovine serum (Invitrogen Canada, Burlington, ON) containing 1 nM TCAP-1, 100 nM TCAP-1 or vehicle (PBS) was added. Each well was digitally imaged twice at 0, 4 and 8 hours using an inverted Zeiss Axiovert 200M. A minimum of 90 cells were analyzed per condition using Labworks V4.0.0.8 (UVP, Upland, Calif.) and scored for number of neurites per cell, neurite length and cell size.
Quantitative Real Time-PCR
Total RNA from N-38 cells was isolated by the guanidinium thiocyanate phenol chloroform extraction method (Chomczynski and Sacchi, 1987). First strand cDNA was synthesized from 1 μg deoxyribonuclease I-treated RNA, using SuperScript reverse transcriptase (RT) and random primers (Invitrogen, Carlsbad, Calif.), as described in the Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, Calif.). The specificity of each amplification reaction was monitored in control reactions, where amplification was carried out on samples in which the RT was omitted (RT−). Quantitative “real time” RTPCR was performed as described in the SYBR Green PCR Master Mix and PCR Protocol (Applied Biosystems, Foster City, Calif.). Briefly, cDNA was synthesized from 1 μg total RNA in a total volume of 20 ul. 50-100 ng cDNA as template was amplified with SYBR Green Master Mix (Applied Biosystems) and 300 nM primers in a 10 μl reaction for 40 cycles (15 sec at 95 C, 1 min 60 C). The primers used for RT-PCR are: 18s rRNA; gtaacccgttgaaccccatt, ccatccaatcggtagtagcg: α-actinin 4; gagaagcagcagcgcaaga, ccgaagatgagagttgcacca: β-actin; ggccaaccgtgaaaagatga, cacagcctggatggctacgt: β-catenin; agcagtttgtggagggcgt, cgagcaaggatgtggagagc: α-tubulin 1; acaggattcgcaagctggc, ccaagaagccctggagacc: and β-tubulin 4; tgaggccacaggtggaaactatgt, aagttgtctggccgaaagatctgg. All primers were designed using Primer Express software (Applied Biosystems) and synthesized by ACGT Corp. (Toronto, ON) or Integrated DNA Technologies, Inc. (Coralville, Iowa). Data was represented as mean quantity, defined as the average of the replicate group (n>3), analyzed using ABI Prism 7000 SDS software package (Applied Biosystems). Copy number of amplified gene was standardized to 18S rRNA levels. The final fold differences in expression were relative to the vehicle treatment at each individual timepoint.
Western Blot Analysis of Cytoskeletal Proteins
N38 immortalized hypothalamic cells were cultured as described previously (Belsham et al, 2004) in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal bovine serum (Invitrogen Canada, Burlington, ON). At 70% confluency, cells were treated with medium containing 1 nM TCAP-1, 100 nM TCAP-1 or vehicle (phosphate buffered saline (PBS) pH 7.4) for 0.5, 1, 4 or 8 hours after which total cell proteins were extracted. Briefly, cells were removed in the presence of cold PBS and centrifuged. The cells were resuspended in PBS containing 1% Triton X-100 (Sigma), 1 mM dithiothreitol (DTT) and protease inhibitors ((5% Protease inhibitor cocktail set III (Calbiochem, EMD Biosciences, San Diego, Calif.) and 1 mM phenylmethyl sulfonyl fluoride (PMSF, EM Science)). Following vortex mixing, the cells were spun for 15 minutes at 15,300 g at 4° C. The supernatant containing total proteins was stored at −20 C.° until further analysis. The protein concentration was determined using a BCA protein assay kit (Pierce Chemical Co, Rockford, Ill.). For SDS PAGE, the appropriate μg loading volumes were determined for each antiserum. Samples were mixed with sample buffer containing SDS and boiled for 5 minutes at 90 C.° and were run in duplicate to test for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The proteins were resolved on 4-20% Tris-HCl Ready gels (Bio-Rad, Hercules, Calif.) using a Mini-PROTEAN 3 Cell (Bio-Rad) electrophoresis unit for 35 minutes at 200V. Transfers were performed using the Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad) with Hybond C Nitrocellulose membranes (GE Healthcare, Piscataway, N.J.) at 100V for 2 hours. Membranes were blocked in 0.2% PBS-Tween 20(v/v) containing 5% nonfat milk (w/v) and probed with primary antiserum overnight at 4 C.° at the appropriate dilution. The dilutions are as follows: β-actin, 1:4000; β-3-tubulin, 1:500; α-actinin-4, 1:5000. The secondary antibody conjugated to horse radish peroxidase was used at a concentration of 1:5000. For all analyses the GAPDH antiseraum was used at a dilution of 1:2000. A ECL Western Blotting Analysis System (GE Healthcare, Piscataway, N.J.) was used to detect the proteins using X OMAT Blue XB1 film. Blots were scanned and optical density was determined using an Epi Chemi II Darkroom and Lab works V4.0.0.8 (UVP, Upland, Calif.).
Gene and Protein Expression and Confocal Studies
Gene Expression
Significant changes in mRNA expression, as determined by real-time PCR were not observed in any of the 1 nM TCAP treatments (FIG. 10). However there were indications of expression increase in α-actinin-4 and β-tubulin mRNA after 4 hours, although these changes were not statistically significant. In contrast, at a concentration of 100 nM TCAP, there was a significant increase in synthesis as determined by a two-way analysis of variance (ANOVA) for β-catenin (p=0.0158; F=6.192), α-actinin-4 (p=0.0265; F=5.329) and β-tubulin (p=0.0042; F=9.320). Expression of mRNA for β-catenin and α-actinin-4 showed increases between 30 and 40% within 1 hr of treatment and remained high for 8 h. β-tubulin expression was more modest with a maximal increase of 25 to 30%, although inter-experimental variability as assessed by standard errors were low.
Protein Expression
Treatment of cells with 1 nM TCAP-1 did not result in any significant changes in β-tubulin protein levels over 8 hours (FIG. 11A-C). In contrast, cells treated with 100 nM showed a significant increase of 60% at 1 hour in β-tubulin relative to the vehicle treated cells at the same time point (two-way ANOVA with Bonferroni post-test, p<0.05, F=1.48). TCAP-1 treated cells also experienced a significant change with regards to β-actin expression (FIG. 12A-C). A concentration of 1 nM TCAP-1 showed an expression level of 184±4.1% of vehicle at 0.5 hrs. Similarly 100 nM TCAP-1 induced an expression level of 192±10.5%. No significant effects were noted at any other time points or on α-actinin-4 (FIG. 13A and B). Due to the consistent high levels of both β-tubulin mRNA and protein levels, βtubulin immunoreactivity was used as a marker to examine subsequent TCAP induced effects on cellular morphology.
Immunofluorescence Confocal Microscopy
In one study, a confocal analysis of 100 nM TCAP-1 effects on localization of B-tubulin in N38 cells was conducted.
FIG. 14A show B-tubulin immunoreactivity with Alexa fluor 488 in vehicle and TCAP-1 treated cells after 1 hour post-vehicle or mouse TCAP-1 treatment, respectively. Ten central cells from each image were then analyzed for number of pixels at maximal intensity (149) and expressed as a ratio of total pixels in the perinuclear region (FIG. 14B) and the whole cell (FIG. 14C) (Student's t-test with Welch's correction for unequal variances P=0.05; bar=20 μm). Perinuclear region and cell size were not different in 30 cells per group.
Results
Overall, the TCAP-treated cells were characterized by greater clarity and number of observable β-tubulin strands in the cells and the neuritis (FIG. 14A). The 100 nM TCAP-1 treatment resulted in a significant increase in whole cell immunofluorescence (FIG. 14B).
The results indicate that cells treated with TCAP show an increased expression of B-tubulin in the cell and perinuclear region of neuronal cells and increase in B-tubulin protein levels. The results further illustrate that cytoskeletal B-actin is upregulated in TCAP-treated cells. Actin synthesis and expression is a normal and required component of neuron function, migration and axon elongation. Regulation of actin synthesis and expression is required for restoration of function following necrotic or inflammatory degenerative conditions.
Example 10—TCAP Induces Repulsion in Growing Axons
100 μM TCAP-1 was puffed on the neurite of an N38 cell in the direction of the arrow in FIG. 15A. The neurite was imaged over one hour. TCAP caused expansion of the growth cone area followed by repulsion away from the source of TCAP (FIG. 15B). Bar—1 um.
Results
The images clearly show that TCAP induce repulsion in growing axons and can be used as a guidance molecule for neuronal growth and potentially fasciculation.
Example 11—Increases Growth and Fasciculation of Primary Embryonic Hippocampal Cultures
This example illustrates the immunohistochemistry of B-tubulin III in primary hippocampal E18 cultures treated with vehicle or 100 nM TCAP-1 for seven days.
Timed-pregnant Sprague-Dawley rats (Charles River, Boston, Mass.) on day 18 (E18) of gestation were euthanized in a CO2 chamber. The uteri were surgically removed and embryos were collected in Hank's balanced salts solution (HBSS) with 15 mM HEPES and 10 mM sodium bicarbonate (Sigma-Aldrich Canada, Oakville, ON). The embryos were decapitated the hippocampi dissected. The hippocampi were trypsinized for 15 minutes at 37 C.°, centrifuged for 5 minutes at 1600 rpm and the pellets washed two times in HESS. The cell pellets were suspended in Neurobasal medium supplemented with B27, 0.5 mM Glutamax, and penicillin/streptomycin and this medium was subsequently used for culturing. Following trituration with a fire polished glass pipette, 300 000 cells were plated into 6-well plates containing 12 mm glass coverslips coated with poly-D-lysine (VWR, Mississauga, ON). After 24 hours, fresh medium containing 100 nM TCAP or vehicle was used. The medium was replaced twice a week. On the eighth day of culture, coverslips were processed.
The coverslips with cells were rinsed with PBS (pH 7.4) twice before fixing with 1 ml 4% paraformaldehyde for 15 minutes. Following two washes for 5 minutes, cells were permeabilized by addition with 0.2% Triton X100 solution in PBS for 90 seconds. After washing twice for 2 minutes, the cells were incubated with 0.5% normal goat serum (NGS) in PBS. A 1:100 dilution of β-tubulin III antiserum in 0.5% NGS was applied to the coverslips and incubated at room temperature for 1 hour. The detection and staining was done according to instructions provided by the Vectastain ABC kit (Vector Laboratories, Burlington, ON, Canada). The biotinylated goat anti-rabbit serum was applied at 1:200 dilution in serum for 1 hour as well. The Vectastain reagents, avidin DH and biotinylated horse radish peroxidase H were mixed and incubated with the cells for 30 minutes before washing for 5 minutes with PBS. The DAB substrate (Vector Laboratories) was then added for 8 minutes and cells washed with distilled water for 5 minutes. The cells were dehydrated with ethanol, cleared with Xylene and the coverslip mounted on a slide using Vectamount mounting medium (Vector Laboratories). The stained cells were visualized using an Olympus (BX60) microscope and imaged with a CCD CoolSNAP camera (Photometrics, Tuscon, Ariz.).
Results
The results shown in FIGS. 16 and 17 clearly show that TCAP caused an increase in dendritic density and fasciculation compared to control.
The β-tubulin-III immunoreactivity in primary hippocampal cultures treated with 100 nM TCAP-1 was enhanced (FIG. 16A-C). A frequency distribution of pixel intensity indicated a significant (p<0.05) effect of TCAP-1 using a Chi square test for trends (FIG. 16C. The increase in immunoreactivity was due to both an increase in total number of cells and cell processes as indicated by the increase in the number of pixels in the dark gray to black regions (FIG. 16A). TCAP-1 treated cultures show a significantly greater (p=0.0142) mean number of cell clusters (270±22) over the vehicle treated cells (175±17) as determined by a two-tailed Students t-test (FIG. 16B). A much denser mesh of cell processes were observed in TCAP treated cells.
TCAP treated hippocampal cells showed a much greater incidence in the number of large axons and axon bundles relative to the vehicle treated cells. Higher magnification of both groups of cultures revealed that the TCAP treated cells showed a much greater tendency for fasciculation along with a greater incidence of neural processes outgrowth (FIG. 17).
The results further illustrate that TCAP can be used to increase fasciculation among neurons and in addition to supporting the effects of TCAP on inhibiting neuronal cell death, it illustrates that TCAP may be used in the treatment of a number of conditions, such as brain injury, especially if administered with 8 or 24 hours of said injury to minimize any secondary injury effects.
TCAP 1 is a novel putative neuropeptide that bears the structural hallmarks of a bioactive peptide. TCAP-1 can modulate cell growth and anxiety-related behaviors. The present study shows that TCAP-1 has the ability to stimulate neurite outgrowth in part by increasing the synthesis of components of the cytoskeleton. The TCAP-1 mediated neurite outgrowth is coupled with an increase in the synthesis and translation off β-tubulin and possibly the enhanced translation of β-actin. In primary hippocampal cultures, the increase in β-tubulin expression is associated with an increase in the number of immunoreactive β-tubulin cells and large axonal processes. Because many long term behavioural effects are associated with changes in neuronal circuitry, the effects observed with TCAP can be explained by changes in the morphological properties of neurons.
The morphological characteristics of cells treated with TCAP were examined. An immortalized hypothalamic cell line (N38) previously known to be responsive to TCAP-1 (Belsham et al, 2004; Wang et al, 2005) was used in the Examples. Cell cultures were held at 70-80% confluency as a maximal as beyond that, the cells went into a stress response. TCAP-1 treated cells showed a dose-dependent increase in the number of longer neurites and a decrease in the number of shorter neurites.
Together, the present studies with the N38 cell line indicate that TCAP 1 stimulates neurite outgrowth and increases the synthesis and translation of β-tubulin while enhancing β-actin translation only. TCAP induced an increase in the incidence of axon formation and fasciculation. In one embodiment, TCAP and the teneurins can be used to regulate neuronal process outgrowth in the hippocampus and in the potentiation of learning and memory.
In one embodiment, the Examples indicate that TCAP may exert its effects at least in part by inducing changes in axonal and dendritic outgrowth. Changes in dendritic morphology are important since they are the mechanism behind many diseases and disorders. Specifically, the hippocampus is a neuroplastic part of the brain whose cells when exposed to effectors can undergo morphological changes associated with disorders such as stress and depression (McEwen, 1999). The present Examples also indicate that the TCAP and teneurin system is associated with neuroplasticity, learning and anxiety.
Example 12—Superoxide Dismutase-Catalase Data
Superoxide Dismutase Detection and Measurement
Examination of the superoxide dismutase-associated system was investigated as a possible mechanism for necrosis after the apoptotic, survival and cell cycle experiments did not show a robust effect. The presence of the superoxide radical was measured indirectly by the conversion of a soluble tetrazolium salt in cells after 48 hours (FIG. 18A). The TCAP-1 treated cells showed a 40% (p<0.05) and 60% (p<0.01) decrease in the absorbance of the substrate, which is proportional to superoxide radical activity, at pHs 8.0 and 8.4, respectively. However, because this method shows only the indirect presence of the superoxide radical, and by inference, the presence of superoxide dismutase, we also examined the presence of this enzyme protein directly by western blot (FIG. 18B,C). Relative to the vehicle-treated cells at pH 7.4, superoxide dismutase levels in the vehicle-treated cells showed a significant (p<0.05) decrease as a function of pH, as determined by a one-way ANOVA. There were no significant differences in the expression of the superoxide dismutase protein at pHs 6.8 and 7.4. In contrast, at pH 8.0 and 8.4, TCAP-1 significantly (p<0.05 and p<0.01, respectively) reduced the pH-induced decline in superoxide dismutase levels. The superoxide dismutase expression levels at pH 8.0 and 8.4 were not significantly different than that of the vehicle-treated cells at pH 7.4.
Superoxide dismustase gene expression as measured by real-time PCR indicated a significant (p<0.01) increase over the vehicle treated cells at pH 7.4 and 8.4 (FIG. 18D). A greater effect on gene expression was noted in superoxide copper chaperone (CCSD) expression where CCSD expression levels in the TCAP-1 treated cells at pH 8.4 was increased almost 4.5 fold over the vehicle treated cells (FIG. 18E).
H2O2 Toxicity and Catalase Activity
TCAP-1 showed a significant increase in MTT activity relative to the vehicle-treated at 6 to 48 hours in cells treated with 50 μM H2O 2 (FIG. 19A). The results indicate that TCAP-1 significantly increased mitochondrial activity at 6, 12 and 48 hours (p<0.001) (F=168.2) as compared with the vehicle-treated cells. There was also a less significant effect at 24 hours (p<0.05) and no effect at 0 hours.
A catalase assay was performed on the pH treated cells in order to determine whether TCAP-1 was conferring survivability to the cells via upregulation of catalase and thus increasing H2O 2 breakdown into H2O and O2 (FIG. 19B). The results indicate that TCAP-1 significantly increased catalase levels at pH 8.4 (p<0.001) (F=24.42) as compared to the vehicle treated cells according to a two-way ANOVA with a Bonferroni's post hoc test. There was also a significant TCAP-1 effect at pH 8.0 (p<0.01) but no significant effects at either pH 6.8 or pH 7.4 compared to the vehicle treated cells. Bovine liver was also assayed as a positive control. Catalase gene expression, as determined by real-time PCR indicated that TCAP induced mRNA levels by 3 fold (p<0.001) and 5 fold (p<0.001) at pHs 8.0 and 8.4, respectively (FIG. 19C).
Superoxide dismutase is an enzyme that is responsible for catalyzing the highly reactive oxygen radical, superoxide (O2-) into hydrogen peroxide (H2O2). Hydrogen peroxide is in turn, catalysed to water by the enzyme catalase. Superoxide dismutase is bound to copper atoms for full activity. The protein superoxide dismutase copper chaperone acts to effect the transfer of copper to superoxide dismutase. Together, these three proteins act to protect the cells from the toxic effects of reactive oxygen species (ROS). High concentrations of ROS have been implicated in the destruction of cellular membranes and proteins and play a significant role in the onset of neurodegenerative disorders. The findings that TCAP enhances the activity and expression of the superoxide dismutase-catalase system is indicative that TCAP inhibits cellular necrosis.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.
All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
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## Claims

1. A method of inhibiting neuronal necrosis and inducing neurite growth in vitro comprising administering to neuronal cells an effective amount of a teneurin c-terminal associated peptide (TCAP peptide), or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of said TCAP peptide consists essentially of:
(i) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 69, 70, and 101; or
optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

2. The method of claim 1, wherein the neuronal cells sustained a physiological trauma.

3. A method of claim 1 for increasing fasciculation in neuronal cells.

4. The method of claim 1 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 69 and 70, optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

5. The method of claim 4 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence Identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

6. The method of claim 1 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence Identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 38 and 101 optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it Is in the form of pyroglutamic acid.

7. The method of claim 1 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence identity to an amino acid sequence of SEQ ID NO: 38, optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

8. A method for increasing β-tubulin and/or β-actin levels in neuronal cells in vitro comprising administering to the cells an effective amount of a teneurin c-terminal associated peptide (TCAP peptide), or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of said TCAP peptide consists essentially of:
(i) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 69, 70, and 101; or
optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

9. The method of claim 8 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, and 70, optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

10. The method of claim 9 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, optionally wherein:
(a) the carboxy terminal end of said TCAP peptide Is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

11. The method of claim 9 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 38 and 101 optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

12. The method of claim 9 wherein the amino acid sequence of said TCAP peptide or pharmaceutically acceptable salt thereof consists essentially of an amino acid sequence having at least 95% sequence identity to an amino acid sequence of SEQ ID NO: 38, optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.

13. A method of inhibiting neuronal necrosis and inducing neurite growth comprising administering to neuronal cells in vitro an effective amount of a teneurin c-terminal associated peptide-1 (TCAP-1 peptide), or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of said TCAP-1 peptide consists essentially of: SEQ ID NO: 70 or 38 or peptides having substitutions thereof at the first and fifth amino acid positions of the peptide, optionally wherein:
(a) the carboxy terminal end of said TCAP peptide is amidated or comprises an amidation signal sequence; or
(b) when the amino terminal amino acid of said TCAP peptide is glutamine, it is in the form of pyroglutamic acid.