Figure 1.
Low correlation between 14-3-3 protein levels and LDH activity in CSF of CJD patients.
CSF samples from CJD patients (N = 40) and non-CJD controls (N = 16), were obtained by lumbar puncture. Then, A. 14-3-3 levels in CSF were analyzed by Western blot. B. Relative levels of 14-3-3 protein band were quantified using densitometric analysis. C. LDH activity was measured in each sample as described in Material and Methods. D. A correlation between 14-3-3 protein and LDH activity from panels B and C is presented. E. Quantitative analysis of total protein levels in CSF samples of panel A was performed. In B, C and E the red line represents the mean of all samples analyzed. Statistical significance was calculated using Student's t-test.
Figure 2.
Altered PrP expression pattern in CSF of CJD patients.
CSF samples from CJD patients (N = 40) and non-CJD controls (N = 16), were obtained by lumbar puncture. A. PrP levels in CSF were examined by Western blot. B. and C. Quantification of relative levels of total PrP protein in CSF and the ratio of the PrP band 1 (upper) and PrP band 2 (lower) in CSF, respectively. D. Comparison between ratios of PrP band 1 and 2 in sCJD versus fCJD E. Correlation of total PrP protein levels with 14-3-3 protein in each CSF samples presented in Fig 1. In B, C and D the red line represents the mean of all samples analyzed. Statistical significance was calculated using Student's t-test.
Figure 3.
Alterations in PrP expression pattern in CSF are specific to CJD patients.
CSF samples from healthy control subjects (N = 3), CJD patients (N = 5), dementia patients (N = 3) and paraparesis patients (N = 3), were obtained by lumbar puncture. A. Assessment of PrP levels (two upper panels with different exposure), and 14-3-3 protein levels (third panel) by Western blot analysis, and total protein content by Ponceau Red staining (lower panel, a selected band is presented). B. and C. Quantification of relative total PrP protein levels in CSF and the ratio of the PrP band 1 (upper) and PrP band 2 (lower) in CSF, respectively. The asterisk in A indicates one sample with a technical problem in the blot detection that was excluded for quantification of panel B and C. In B and C the red line represents the mean of all samples analyzed. Statistical significance was calculated using Student's t-test.
Figure 4.
Changes in the glycosylation pattern of PrP in CSF of CJD patients.
A. The pattern of expression of PrP in two representative CSF samples (from one healthy control individual and one CJD patient) is compared to the PrP pattern observed in control brain tissue using Western blot analysis. B. CSF samples from one healthy control individual, one CJD patient and one healthy control brain tissue were treated with PNGase F and then analyzed by Western blot. Data represent the analysis of three control and five individual CJD samples. C. and D. CSF samples from one healthy control individual and one CJD patient were treated with various concentrations of PK or thermolysin (Ther), respectively, followed by Western blot analysis. Data represent the analysis of three control and five individual CJD samples.
Figure 5.
Changes in 14-3-3 protein and PrP levels in CSF from six CJD patients overtime.
PrP and 14-3-3 protein levels were analyzed in CSF samples obtained by lumbar puncture of 6 CJD patients (CJD-1 to CJD-6) at different time points during disease. Total protein content was assessed by Ponceau Red staining, the predominant band (albumin) is shown. The asterisk in samples from CJD-2, CJD-5 and CJD-6 indicates an additional PrP protein band detected between PrP band 1 and band 2.
Figure 6.
Postmortem examination of 14-3-3 in the brain of sCJD cases.
A. H&E staining was performed in the post mortem brain cortex obtained from of CJD patients 5 and 6 (CJD-5 and CJD-6) and one patient with cerebral infarction (control, non-affected hemisphere). B. 14-3-3 protein expression levels were monitored by immunohistochemistry in post-mortem brain sections of patient CJD-5 and CJD-6 compared to a control subject. Brain cortex, thalamus and cerebellum were analyzed. C. In addition, the expression pattern of PrP was also analyzed in cortex presented in A. Magnification 400×.
Table 1.
General information for CSF analysis.