Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration

Abstract

Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid-based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water (2H2O), distinct, newly synthesized 2H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct 2H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected 2H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After 2H2O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson's disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects.

Figures

Figure 1. Effects of nocodazole on CSF-based secretion kinetics of neuronal cargo proteins and on MT turnover in mice.

(A) Decline in plasma body water 2H enrichment (n = 10 per time point in duplicate; mean ± SD). Body water decay curves did not differ between nocodazole-infused mice and vehicle controls. Body water 2H2O content decayed to low levels by 3–5 days after bolus injection. (B) Delays in the time of appearance, Tmax, and disappearance of 2H-CHGB and 2H-NRG1 were observed in CSF from nocodazole-infused mice compared with vehicle controls (n = 10 per time point in duplicate; mean ± SD). CSF was collected 1, 2, 3, 5, and 10 days after labeling. 2H-CHGB and 2H-NRG1 were sequentially immunoprecipitated and purified to homogeneity from albumin/Ig-depleted CSF. *P < 0.001. (C) CSF-based secretion kinetics of 2H-CHGB and 2H-NRG1 in vehicle-infused, nocodazole-infused, nocodazole/noscapine-treated, and nocodazole/taxol-treated mice (mean ± SD). Treatment with noscapine and taxol normalized MT-mediated transport rates of 2H-CHGB and 2H-NRG1 to the levels observed in CSF of age-matched vehicle controls. *P < 0.001. (D) 2H-label incorporation of hippocampal tubulin in dimers and MTs (n = 4 per group; 10-week-old males, mean ± SD). In nocodazole-infused mice, an increase in 2H-free tubulin dimers correlated with a reduction in 2H-labeling of MTs associated with TAU and MAP2. The nocodazole/noscapine and nocodazole/taxol treatment groups showed 2H-labeling of TAU- and MAP2-associated MTs similar to vehicle controls. *P < 0.001.

Figure 2. Noscapine and taxol partially reverse nocodazole-induced MT disassembly in living cells.

(A) Measurement of FDAP to determine MT dynamics in neuronal processes. PC12 cells were transiently transfected to express PAGFP-tagged tubulin, neuronally differentiated and focally irradiated with an UV laser in the middle of a process. FDAP, as an indicator for the ratio of soluble to polymerized tubulin, was determined in the activation spot, as indicated in the color-coded filled contour plots of 2D intensity function. (B) FDAP plots of cells treated with vehicle (0.01% DMSO), nocodazole alone, or nocodazole in combination with taxol or noscapine. Total fluorescence demonstrates photostability of the activated protein. Taxol and noscapine partially reversed the nocodazole-induced increase in FDAP, indicative of MT stabilization by these drugs. FDAP plots show the mean of n = 15–27 measurements per experimental condition.

Figure 3. Differential delays in transport rates of neuronal cargo proteins in symptomatic SOD1G93A mice and MPTP-infused mice.

(A) Delays in the time of appearance, Tmax, and disappearance of 2H-CHGB, 2H-NRG1, and 2H-sAPPα were observed in CSF from symptomatic 13-week-old SOD1G93A mice compared with age-matched WT controls (n = 5 male and 5 female per time point in duplicate, mean ± SD). The appearance curve of 2H-sAPPα suggested less of an effect in delay of secretion rates than that measured for 2H-NRG1 and 2H-CHGB in CSF from symptomatic 13-week-old SOD1G93A animals. CSF secretion kinetics of 2H-SNCA did not change compared with age-matched WT mice. CSF was collected 1, 2, 3, 5, and 10 days after labeling. 2H-labeled cargo proteins were sequentially immunoprecipitated and purified to homogeneity from albumin/Ig-depleted CSF. *P < 0.001. (B) Delays in the time of appearance, Tmax, and disappearance of 2H-CHGB, 2H-SNCA and 2H-sAPPα were observed in CSF from symptomatic MPTP-injected 8-week-old male mice compared with age-matched vehicle controls (n = 10 per time point in duplicate, mean ± SD). CSF secretion kinetics of 2H-NRG1 did not change compared with age-matched vehicle control mice. Mice received a pulse 2H2O labeling at 10 days after the last MPTP injection, and CSF was collected from vehicle- and MPTP-injected mice 1, 2, 3, 5, and 10 days after labeling. 2H-labeled cargo proteins were sequentially immunoprecipitated and purified to homogeneity from albumin/Ig-depleted CSF. *P < 0.001.

Figure 4. Noscapine treatment restores transport rates of neu­ronal cargo proteins in symptomatic SOD1G93A and MPTP-injected mice.

(A) CSF-based secretion kinetics (appearance, Tmax, and disappearance) of 13-week-old 2H-CHGB, 2H-NRG1, 2H-sAPPα, and 2H-SNCA in SOD1G93A mice after 3 weeks of treatment with noscapine or riluzole (n = 5 males and 5 females per time point in duplicate, mean ± SD). Noscapine normalized MT-mediated transport rates of 2H-CHGB, 2H-NRG1, and 2H-sAPPα in symptomatic SOD1G93A mice to the levels observed in CSF of age-matched WT controls, whereas riluzole had much less of an effect. Normal kinetics of 2H-SNCA was not adversely affected by noscapine treatment. *P < 0.001. (B) CSF-based secretion kinetics in 8-week-old symptomatic MPTP-injected mice. Treatment with noscapine normalized the kinetics of secretion of 2H-CHGB, 2H-sAPPα, and 2H-SNCA (n = 10 males per time point in duplicate, mean ± SD). Normal kinetics of 2H-NRG1 were not adversely affected by noscapine treatment. Noscapine was administered 7 days after the last MPTP injection, treatment was for 10 days, and a pulse 2H2O labeling was administered on the last day of treatment (17 days after the fourth and final MPTP injection). CSF was collected from WT, vehicle control, SOD1G93A, and MPTP mice 1, 2, and 3 days after labeling. 2H-labeled cargo proteins were sequentially immunoprecipitated and purified to homogeneity from albumin/Ig-depleted CSF. *P < 0.001.

Figure 5. Transport rates of neuronal cargo proteins in CSF of non-PD volunteers.

(A) Decline in plasma body water 2H enrichment in control (Ctrl) volunteers. Body 2H2O enrichments peaked at day 7, then died away with a half-life of approximately 7 days, consistent with known turnover rate of body water in humans. (B) Enrichment curves in CSF cargo proteins from 6 control non-PD volunteers (mean ± SD, generated by technical replicates of repeated sample preparations and analyses). 4 LPs were conducted in 6 controls (days 2–15, 21–43) after beginning 2H2O administration (7-day labeling protocol). The line represents the curve fit of the data. 2H-labeled cargo proteins were sequentially immunoprecipitated and purified to homogeneity from albumin/Ig-depleted CSF.

Figure 6. Differential delays in transport rates of neuronal cargo proteins in CSF of PD subjects.

(A) Decline in plasma body water 2H enrichment in control non-PD volunteers and PD subjects. Body 2H2O enrichments peaked at day 7, then decayed with a half-life of approximately 7 days, consistent with known turnover rate of body water in humans. (B) 4 LPs were conducted in 1 PD patient (PD 6084; days 3, 9, 21, and 38), and a single LP was conducted in 11 PD subjects (days 15, 21, 22, or 23) after starting 2H2O administration (7-day labeling protocol). Delays in the time of appearance of neuronal 2H-CHGB, 2H-SNCA, and 2H-sAPPα, but not that of 2H-NRG1, were observed in CSF from PD subjects compared with 6 control volunteers (mean ± SD, generated by technical replicates of repeated sample preparations and analyses). Solid and dashed lines represent curve fits of the data for controls and PD volunteers, respectively. 2H-labeled cargo proteins were sequentially immunoprecipitated and purified to homogeneity from albumin/Ig-depleted CSF. *P < 0.001.

Figure 7. Model and kinetic interpretation of transport dynamics of released cargo proteins in CSF.

Newly synthesized cargo proteins destined for secretion travel from the cell body of neurons, where they are primarily produced, to the nerve terminal, where they are released into the CSF. MT-based transport kinetics are based on the timing of appearance and disappearance of newly synthesized 2H-labeled neuronal cargo proteins in CSF, after in vivo metabolic labeling. The differences between kinetics of appearance in and disappearance from CSF of 2H-CHGB and 2H-NRG1 in controls and PD subjects are summarized. Solid and dashed lines represent the curve fits of the data for controls and PD volunteers, respectively. CHGB and NRG1 concentrations were both markedly reduced in CSF of PD subjects (see Table 2). Delayed kinetics of appearance in and disappearance from CSF of 2H-labeled cargo proteins were independent of CSF protein concentrations.