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Method for regulating neurite growth

Inactive Publication Date: 2008-12-25
LOVEJOY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009]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.

Problems solved by technology

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.
Although, the literature on brain acidosis is extensive, brain alkalosis, is not well understood (Robertson, 2002).

Method used

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  • Method for regulating neurite growth
  • Method for regulating neurite growth
  • Method for regulating neurite growth

Examples

Experimental program
Comparison scheme
Effect test

example 1

Peptide Synthesis

[0096]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.

[0097]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 hexfluorophosphate; 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-dimethylfor...

example 2

Cell Morphology Analysis

[0098]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).

[0099]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 8g 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 ...

example 3

Effect of TCAP on Cell Proliferation and Viability

[0101]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 50111 aliquots were vortexed and counted on a hemocytometer.

[0102]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 solut...

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Abstract

This invention relates to a method of inhibiting neuronal cell death, including protecting neronal 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 hypoxiaischemia and brain alkalosis or various brain or spinal cord injuries due to physical or physiological stresse. 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.

Description

RELATED APPLICATIONS[0001]This application 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. This application 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”. This application also claims priority from U.S. provisional patent application No., US 60 / 783,321, filed Mar. 21, 2005, entitled “Method for Regulating Neurite Growth”. All of these references are incorporated in their entirety by ref...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61P25/00
CPCC07K2/00C07K14/705A61P25/00
Inventor LOVEJOY, DAVIDAL CHAWAF, ARIJ
Owner LOVEJOY
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