ABNORMAL ALTERATIONS OF PKC ISOZYMES PROCESSING IN ALZHEIMER'S DISEASE PERIPHERAL CELLS
The present invention provides a method for the diagnosis of AD from non-AD conditions by using a PKC Isozyme Index obtained by determining ratios of ratios of different PKC Isozymes in peripheral cells of a test subject in the absence and presence of a beta-amyloid peptide, and optionally, in the presence of a PKC activator.
1 . A method for determining the presence or absence of Alzheimer's Disease in a candidate subject, which method comprises:
i) determining the protein levels of a first PKC isozyme in peripheral cells from a candidate subject in the absence of and in the presence of an Aβ peptide to generate a first ratio;
ii) determining the protein levels of a second PKC isozyme in peripheral cells from a candidate subject in the absence of and in the presence of the Aβ peptide, wherein the second PKC isozyme is not known to be differentially modulated by the Aβ peptide in AD cells compared to non-AD cells, to generate a second ratio;
iii) generating a PKC isozyme Index by dividing the first ratio by the second ratio, wherein a PKC isozyme Index of about 1.0 or lower indicates a diagnosis of Alzheimer's Disease and a PKC isozyme Index of greater than 1.0 indicates the absence of Alzheimer's Disease.
2 . The method of claim 1 , wherein the PKC isozyme Index is generated using steady state levels of the PKC isozymes as represented by the following Equation I:
I
[
PKC
-
x
]
/
[
PKC
-
x
]
A
β
[
PKC
-
z
]
/
[
PKC
-
z
]
A
β
=
PKC
-
x
Index
wherein “x” represents the first PKC isozyme, “z” represents the second PKC isozyme, and Aβ represents the cells contacted with the Aβ peptide.
3 . The method of claim 1 , wherein the PKC isozyme Index is generated using phosphorylated levels of the PKC isozymes as represented by the following Equation II:
II
[
p
-
PKC
-
x
]
/
[
p
-
PKC
-
x
]
A
β
[
p
-
PKC
-
z
]
/
[
p
-
PKC
-
z
]
A
β
=
p
-
PKC
-
x
Index
wherein “x” represents the first PKC isozyme, “z” represents the second PKC isozyme, and Aβ represents the cells contacted with the Aβ peptide, and p-PKC-x and p-PKC-z represent phosphorylated PKC isozymes.
4 . The method of claim 1 , wherein the method further comprises determining the protein levels of the first and second PKC isozymes in steps i and ii in the presence of a PKC activator.
5 . The method of claim 1 , wherein the first PKC isozyme is PKC-α and the second PKC isozyme is PKC-γ.
6 . The method of claim 1 , wherein the first PKC isozyme is PKC-ε and the second PKC isozyme is PKC-γ.
7 . The method of claim 2 , wherein the first PKC isozyme is PKC-α and the second PKC isozyme is PKC-γ.
8 . The method of claim 2 , wherein the first PKC isozyme is PKC-ε and the second PKC isozyme is PKC-γ.
9 . The method of claim 3 , wherein the first PKC isozyme is PKC-α and the second PKC isozyme is PKC-γ.
10 . The method of claim 3 , wherein the first PKC isozyme is PKC-ε and the second PKC isozyme is PKC-γ.
11 . The method of claim 1 , wherein the Aβ peptide is Aβ (1-40) or Aβ (1-42).
12 . The method of claim 1 , wherein the peripheral cells are skin cells, skin fibroblast cells, blood cells or buccal mucosa cells.
13 . The method of claim 12 , wherein the peripheral cells are skin fibroblast cells.
14 . The method of claim 1 , wherein the Aβ peptide is present at a concentration of from about 1.0 nM to 10 μM.
15 . The method of claim 14 , wherein the Aβ peptide is present at a concentration of about 1.0 μM
16 . A method for monitoring the progression of Alzheimer's Disease in a subject, which method comprises:
i) generating a PKC isozyme Index from peripheral cells of a test subject at a first time point, according to the method of claim 1 , wherein the test subject has been diagnosed with Alzheimer's Disease, to obtain a reference PKC isozyme Index for the subject;
ii) generating the same PKC isozyme Index from peripheral cells of the same subject at one or more time points after the first time point;
iii) determining whether there is a decrease in the PKC isozyme Index obtained from the from the one or more time points after the first time point when compared with the PKC isozyme Index from the first time point;
wherein a decrease in the PKC isozyme Index from the one or more time points after the first time point when compared with the PKC isozyme Index from the first time point indicates progression of Alzheimer's Disease.
17 . The method of claim 16 , wherein the test subject has been diagnosed with early Alzheimer's Disease at the first time point.
18 . The method of claim 16 , wherein the test subject has been diagnosed with mild Alzheimer's Disease at the first time point.
19 . A method for monitoring the progression from a non-Alzheimer's disease condition to Alzheimer's Disease in a subject, which method comprises:
i) generating a PKC isozyme Index from peripheral cells of a test subject at a first time point, according to the method of claim 1 , wherein the test subject does not have a diagnosis of Alzheimer's Disease, to obtain a reference PKC isozyme Index for the subject;
ii) generating the same PKC isozyme Index from peripheral cells of the same subject at one or more time points after the first time point;
iii) determining whether there is a decrease in the PKC isozyme Index obtained from the from the one or more time points after the first time point when compared with the PKC isozyme Index from the first time point;
wherein a decrease in the PKC isozyme Index from the one or more time points after the first time point when compared with the PKC isozyme Index from the first time point indicates progression to Alzheimer's Disease.
20 . The method of claim 19 , wherein the non-Alzheimer's disease condition is mild cognitive impairment.
21 . The method of claim 20 , wherein the mild cognitive impairment is amnestic cognitive impairment.
22 . A kit comprising one or more Aβ peptides, at least one antibody specific for a PKC isozyme known to be differentially modulated by the Aβ peptide in AD cells compared to non-AD cells; at least one antibody specific for a PKC isozyme that is not known to be differentially modulated by the Aβ peptide in AD cells compared to non-AD cells; and instructions for determining a PKC Isozyme Index.
23 . The kit of claim 22 , wherein the Aβ peptide is Aβ (1-40) or Aβ (1-42).
24 . The kit of claim 22 , wherein the PKC isozyme known to be differentially modulated by the Aβ peptide in AD cells is PKC-α.
25 . The kit of claim 22 , wherein the PKC isozyme known to be differentially modulated by the Aβ peptide in AD cells is PKC-ε.
26 . The kit of claim 22 , wherein the PKC isozyme known not to be differentially modulated by the Aβ peptide in AD cells is PKC-γ.