Cerebrospinal Fluid Proteomics For Identification Of α2-Macroglobulin As A Potential Biomarker To Monitor Pharmacological Therapeutic Efficacy In Dopamine Dictated Disease States Of Parkinson’s Disease And Schizophrenia

Figures & data

Table 1 Clinical Profile Of Patients Recruited For iTRAQ Experiments

Code	Sex	Age (Years)	Phenotype	Treatment Status
S2	Male	23	Schizophrenia	Drug-naïve
S3	Male	28	Schizophrenia	Drug-naive
P3	Male	42	Parkinson’s disease	Drug-naive
P4	Male	50	Parkinson’s disease	Drug-naive
N2	Male	53	Benign prostatic hypertrophy	Posted for surgery
N4	Male	60	Carcinoma of urinary blader	Posted for surgery

Table 2 Demographic Profile Of Patients Recruited For ELISA

Clinical Phenotype	Mean ± SD Of Age In Years	Median (IQR) of Age In Years	Naive	Treated	Gender		Total
					Female	Male
Schizophrenia^‡	25.0±5.1	23.0 (21.0–28.0)	3	4	1	6	7
Neurological Control	61.4±11.8	61.0 (50.0–70.0)	15	6	9	15
Parkinson^†	51.5±13.4	51.0 (42.5–58.5)	8	28	4	32	36

Notes: ^‡minimum BPRS: 19; ^†minimum UPDRS: 14; H&Y: Stage I.

Abbreviations: SD, standard deviation; IQR, interquartile range.

Table 3 Identification Of Proteins That Show Differential Expression Across The Clinical Dopaminergic Spectrum

Protein	Minimum Coverage (%)	Peptides Identified (95% Confidence)	Number Of Peptides Showing A Linear Correlation Along Dopamine Spectrum	Extent Of Relation (m)	Accuracy Of Linearity (R^2)
MCAM	5.6	3	2	+0.1	0.95
α2-Macroglobulin	63.1	160	144	+0.6	0.88
Somatomedin A	26.7	3	3	−0.1	0.98
Apolipoprotein D	33.5	35	21	−0.0.2	0.78
IgG Fc-binding protein	5.4	10	8	+0.1	0.99
Apolipoprotein C-III	37.6	3	3	+0.6	0.90
Complement C3	68.3	217	176	+0.3	0.89
Complement factor I	20.7	10	10	+0.1	0.88
IGL	53.2	72	57	+0.4	0.99
Ectonucleotide pyrophosphatase	38.6	33	26	−0.2	0.98

Table 4 Identified Proteins, Their Interactions And Implications In Dopaminergic Pathways

Protein	Interacting Proteins	Pathways	References
α2-Macroglobulin	Rab3A-α synuclein	Decreased dopamine release in PD Affects binding and synaptic activity in PD Disrupts cellular homeostasis in PD	^Citation75 ^Citation76 ^Citation77
Apolipoprotein C-III	ADP-ribosylation factorGTPase activating protein 1-Leucine-rich repeat kinase 2	GTP-mediated cell death in PD	^Citation78 ^Citation79
IGL	Ubiquilin1-amyloid precursor protein	Development of dementia in PD	^Citation50 ^Citation53 ^Citation80 ^Citation54
Complement C3	Complement factor I	Complement-mediated inflammation in PD	^Citation48
Complement factor I (C3b/C4b inactivator)	Glucagon-like peptide 1 receptor- G-protein-coupled receptor 37	Oxidative protection of dopaminergic neurons in PD Modulate dopamine uptake and behavioral response to dopaminergic drugs in PD	^Citation81 ^Citation82 ^Citation83
MCAM	Laminin-galectin 8-dardarin	Cytokine-mediated dopaminergic cell death in PD	^Citation84 ^Citation85 ^Citation86 ^Citation36
IgG Fc-binding protein	DNA mismatch repair protein MLH1-Caspase3	Apoptotic death of dopaminergic neurons in PD	^Citation87 ^Citation88 ^Citation89
Apolipoprotein D	Arachidonic acid and HDL	Lipid metabolism is dopamine-related	^Citation90 ^Citation91

Figure 1 Proteins identified in the CSF of Parkinson’s disease, neurological controls and schizophrenia patients. The total numbers and their respective percentages are shown.

Figure 2 Graphical representation of the biological annotation and molecular function of identified proteins. (A) Pie chart representations in Parkinson’s disease, neurological control and schizophrenia patients. (B) Pie chart representations in Parkinson’s disease, neurological control and schizophrenia patients.

Abbreviations: Im, immune process; Mp, metabolic process; Rs, response to stimulus; Lo, localization; In, interaction with cells and organisms; Dp, developmental process; Ot, others; Re, regulation; Cp, cellular processes; Ao, antioxidant activity; St, structural activity; Mt, molecular transducer activity; Ca, ctalytic activity; Bi, binding).

Figure 3 Graphical depiction of protein expression with respect to dopamine concentration in the brain. (A) Protein expressions that are inversely related to the dopamine spectrum comprising Parkinson’s disease, neurological control and schizophrenia patients. (a) Alpha-2-macroglobulin, (b) apolipoprotein C-III, (c) IGL@ protein, (d) complement C3, (e) melanoma cell adhesion molecule, (f) complement factor I light chain, and (g) IgG Fc-binding protein. (B) Protein expressions that are directly related to the dopamine spectrum comprising Parkinson’s disease, neurological control and schizophrenia patients. (h) Insulin-like growth factor 2, (i) apolipoprotein D, and (j) ectonucleotide pyrophosphatase/phosphodiesterase family member 2. Diagrammatic representation of the dopamine concentration in cerebrospinal fluid is shown along the x-axis.^73,74

Figure 4 Pathway analysis shows the identified proteins and their respective interactions. The identified proteins are shown in white nodes, interacting nodes in Parkinson’s disease pathway are highlighted in green, interacting nodes in the schizophrenia pathway are highlighted in red, and nodes that are common to both groups are highlighted in yellow. Direct interacting nodes are indicated by thick-bordered nodes. Interactions of the identified proteins are shown by grey lines.

Table 5 Pearson’s Correlation Between α2M Concentrations And Age, Gender And Disease Duration

Clinical Phenotype	Age (P-value)	Gender (P-value)	Disease Duration (P-value)
Parkinson	0.63	0.64	0.36
Neurological control	0.71	0.46	–
Schizophrenia	0.42	0.86	0.97

Table 6 Pharmaco-Therapeutic Monitoring Value Of CSF α-2-Macroglobulin In Parkinson’s Disease And Schizophrenia

Biomarker	Cut-off Values To Differentiate Healthy Controls From The Disease	Parameters		Likelihood Ratio
		Sensitivity (%)	Specificity (%)
α-2-Macroglobulin	>0.78 μg/mL (Parkinson’s disease)	100	93.3	15
α-2-Macroglobulin	<0.61 μg/mL (schizophrenia)	100	93.3	15

Figure 5 ELISA for the expression of alpha-2-macroglobulin in the CSF of schizophrenia patients, neurological controls and Parkinson’s disease patients. Mean ± standard error of mean of the values are shown by horizontal lines. The bars represent the concentrations as the average of duplicate readings of each patient sample. The trend line across the three phenotypes is shown as a blue dotted line (y = −0.43x+ 1.58; correlation coefficient: R² = 0.99). Diagrammatic representation of the dopamine concentration in cerebrospinal fluid is shown along the x-axis.^73,74 ***Indicates statistical significance with P<0.001.

Table 7 Protein Biomarkers In CSF With Potential Use In Neurological Diseases

No.	Protein	Tissue	Clinical Phenotype	Application	References
1	μ-Crystallin protein kinase C-gamma Glial fibrillary acidic protein	CSF	Schizophrenia	Diagnostic biomarker	^Citation92
2	Acute-phase markers Neuronal synaptic markers	CSF	Parkinsonism	Diagnostic biomarker	^Citation93
3	Chromogranin B Rho GTPase activating protein 10	CSF	Parkinson’s disease Alzheimer’s disease Lewy body dementia	Diagnostic biomarker	^Citation94
4	α-2-Macroglobulin	CSF	Parkinson’s disease schizophrenia	Therapeutic biomarker	This study

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