Altered kinetics of neuronal transport in CSF of murine models of neurodegeneration.
To evaluate in CSF the time of appearance of secreted neuronal cargo proteins as biomarkers of axonal transport, we used a pulse-chase 2
O labeling protocol in mice. This labeling paradigm allows highly labeled proteins synthesized over the first approximately 48–72 hours of label exposure to be monitored as they appear in and disappear from CSF (Figure A). Cargo proteins were selected on the basis of the following published criteria: (a) high expression in neurons; (b) presence in CSF (Table ); and (c) association with neurodegeneration and neuronal survival (11
Effects of nocodazole on CSF-based secretion kinetics of neuronal cargo proteins and on MT turnover in mice.
Level of cargo molecules in murine CSF
To test whether changes in MT dynamics are causally involved in altered kinetics of axonal transport, we infused mice with nocodazole, a MT-depolymerizing agent, 1 day before CSF sampling. Measurement of newly synthesized 2
H-CHGB and 2
H-NRG1 in CSF from nocodazole-infused mice revealed major delays compared with vehicle-treated controls (Figure B). In vehicle-treated mice, the times of first appearance (day 1 or 2), peak (Tmax
; day 3), and disappearance (days 5–10) were markedly earlier than in nocodazole-treated mice. Similar changes in CSF secretion kinetics were observed for other axonally transported cargo, 2
H-SNCA and 2
H-sAPPα, in nocodazole-treated mice (Supplemental Figure 1A; supplemental material available online with this article; doi:
). Amyloid precursor protein is expressed in adult CNS, and its processing is known to generate the sAPPα fragment, which has been linked to neuroprotection (22
) and regulation of the choline transporter activity at the neuromuscular junction (34
). The SNCA protein has been linked to impairment of MT-dependent transport in PD models (11
). CHGB and NRG1 have been linked to motoneuron degeneration and survival (25
). CHGB, NRG1, sAPPα, and SNCA have been previously reported to be released extracellularly (22
To confirm that the effects of nocodazole are on transport kinetics, rather than protein metabolism, we next isolated cortical synaptic vesicles (Supplemental Figure 2). Anti-synaptophysin antibody staining was used to confirm the isolation of synaptic vesicles from nocodazole-treated and vehicle-treated control mice (Supplemental Figure 2A). The relative abundance of CHGB and NRG1 proteins in cortical synaptic vesicles was not different in nocodazole-treated compared with vehicle-treated control mice, as detected by Western blotting (Supplemental Figure 2B), consistent with unchanged pool sizes and production rates of these proteins. However, pulse 2H2O labeling revealed a significant delay in the time of disappearance of vesicular 2H-CHGB and 2H-NRG1 cargo within the cortical axons of nocodazole- compared with vehicle-treated mice (Supplemental Figure 2C). These results were consistent with concomitant delays in time of appearance we observed for these 2H-labeled cargo molecules in CSF in the absence of altered protein concentrations or synthesis rates (Figure B).
Noscapine and taxol have been identified as MT-targeting agents (14
). Thus, we next measured their protective effects against nocodazole-induced defects in neuronal transport in vivo. Noscapine and taxol reversed the nocodazole-induced reduction in kinetics of 2
H-CHGB and 2
H-NRG1 in CSF compared with the vehicle control group (Figure C). Similar results were obtained for secretion kinetics of 2
H-sAPPα and 2
H-SNCA in CSF of taxol- or noscapine-treated mice (Supplemental Figure 1A). Altered CSF-based kinetics of 2
H-cargo proteins were mirrored by changes in MT turnover in nocodazole-treated mice (Figure D). Specifically, 2
H-label incorporation into TAU- and MAP2-associated (axonal and dendritic, respectively) MTs from hippocampus (Figure D) and cortex (Supplemental Figure 1B) was reduced by nocodazole treatment compared with the basal levels found in vehicle-treated mice.
To confirm that taxol or noscapine directly reversed the nocodazole-induced effects on MT polymerization, we also used a live cell imaging assay to determine MT dynamics in living neuronal cells. The method is based on determination of fluorescence decay after photoactivation (FDAP) of photoactivatable GFP–tagged (PAGFP-tagged) tubulin in a spot of a neuronal process (Figure A). FDAP is an indicator of the ratio of soluble to polymerized tubulin; as expected, nocodazole treatment increased FDAP compared with vehicle-treated cells (Figure B). Both taxol and noscapine partially reversed the effect of nocodazole, suggestive of protective activity on MTs. These findings are consistent with the known MT-stabilizing activities of taxol and noscapine (14
) and provide a mechanistic explanation for the restoration of MT-based transport and appearance of 2
H-labeled cargo proteins in CSF that we observed.
Noscapine and taxol partially reverse nocodazole-induced MT disassembly in living cells.
We previously reported that hyperdynamic MTs underlie impairment of axonal transport in SOD1G93A
). Based on these findings, we examined secretion curves of 2
H-labeled neuronal cargo proteins into CSF of symptomatic SOD1G93A
). Pulse 2
O labeling revealed major delays in time of appearance and disappearance for 2
H-CHGB and 2
H-NRG1 in CSF from symptomatic SOD1G93A
compared with age-matched WT mice (Figure A). The secretion curve of 2
H-sAPPα in CSF from symptomatic SOD1G93A
mice was similar to — but somewhat less affected than — those of 2
H-CHGB and 2
H-NRG1, whereas the secretion curve for 2
H-SNCA was no different from WT controls (Figure A). These data suggest the existence of altered rates of cargo transport, over time and space, from a broad population of neurons affected in symptomatic SOD1G93A
Differential delays in transport rates of neuronal cargo proteins in symptomatic SOD1G93A mice and MPTP-infused mice.
The environmental toxin MPTP replicates the main biochemical and pathological hallmarks of PD (32
). Several studies indicate that this toxin exerts its effect by inhibiting mitochondrial complex 1 of substantia nigra dopaminergic neurons (32
). MPTP also causes loss of dopaminergic neurons in a complex1-independent mechanism by increasing phosphorylation of MT-associated TAU and MT dysfunction, which precede mitochondria injury (13
). Because MPTP-induced MT dysfunction and TAU phosphorylation may affect neuronal transport, we investigated whether MPTP-injected mice show altered CSF kinetics for 2
H-labeled neuronal cargo proteins. Delayed CSF secretion kinetics of 2
H-sAPPα, and 2
H-SNCA were observed in MPTP-injected mice compared with vehicle-treated controls (Figure B). In contrast, the CSF appearance kinetics of 2
H-NRG1 was identical to those of vehicle-treated controls, unlike those seen in SOD1G93A
mice (Figure , A and B). Moreover, in MPTP-injected mice, changes in CSF appearance of 2
H-sAPPα, and 2
H-SNCA were associated with altered MT dynamics in substantia nigra and striatum 3 and 7 days after the last MPTP injection (Supplemental Figure 3A). Hyperdynamic TAU-MTs correlated with an increase in TAU phosphorylation at Ser262 in MPTP-injected mice (Supplemental Table 1), which is uniquely located within one of the MT-binding regions of TAU (40
). A similar degree of hyperdynamicity was observed in these brain regions and MT populations isolated from transgenic mice expressing human A53T mutant SNCA on a Snca
-null background (Supplemental Figure 3B and ref. 41
We previously reported that reduction of hyperdynamic MTs by noscapine resulted in recovery of axonal transport, increased motoneuron survival, delayed symptoms, and life extension in SOD1G93A
). Based on these findings, we next examined the effects of noscapine and the drug riluzole, an agent approved clinically for the treatment of ALS, on alterations of MT-based transport in symptomatic SOD1G93A
mice, based on the appearance of 2
H-labeled neuronal cargo proteins in CSF. Treatment with noscapine normalized CSF kinetics of affected neuronal cargo (i.e., 2
H-NRG1, and 2
H-sAPPα) without adversely affecting the normal kinetics of 2
H-SNCA, whereas riluzole had no significant effects on kinetics of any cargo molecules (Figure A).
Noscapine treatment restores transport rates of neuronal cargo proteins in symptomatic SOD1G93A and MPTP-injected mice.
Similarly, in MPTP-injected mice, treatment with noscapine normalized CSF kinetics of affected neuronal cargo (i.e., 2H-CHGB, 2H-SNCA, and 2H-sAPPα) without adversely affecting the normal kinetics of 2H-NRG1 (Figure B); reduced hyperdynamic MTs (Supplemental Figure 3A); and resulted in symptom reversal (Supplemental Figure 4, A–C).
Alterations in CSF kinetics of neuronal 2H-cargo proteins were not caused by general defects in CSF protein clearance, as the replacement rate (half-life) of total CSF proteins was identical in symptomatic SOD1G93A, MPTP-injected, and control mice (Supplemental Figure 5, A and B).
It is also possible that generic suppression of neurotransmission may reduce the release of the cargo proteins in the CSF. However, the altered cargo kinetics observed were induced by treatment with a MT-depolymerizing agent and reversed by treatment with a MT-stabilizing agent (36
In summary, these results demonstrate, first, that measurement of appearance/disappearance kinetics of pulse-labeled neuronal cargo proteins into CSF represents a biomarker for monitoring axonal transport defects and, second, that treatments targeting MT dynamics can improve altered cargo transport kinetics in symptomatic murine models of neurodegeneration.
Altered kinetics of neuronal transport in CSF of PD patients.
Next, we asked whether this approach could reveal differences in patients with neurodegenerative disease compared with non-PD control subjects. The technique for measurement of neuronal transport kinetics in human subjects involves a simple outpatient procedure consisting of daily oral intake of 3 drinks of 2
O (3× 50 ml of 70% 2
O) for a week (the pulse labeling period), followed by 1 or more LPs to collect CSF and collection of blood samples to measure body 2
O enrichments. 2
O has been extensively administered to humans for over 60 years without evidence of toxicities (42
) and has no serious adverse effects in animal systems until it reaches levels greater than 20% of total body water — more than an order of magnitude greater than the levels achieved in these subjects. The labeling protocol of a 7-day pulse dose of 2
O was given to 6 non-PD controls and 12 PD subjects (see Table for clinical details and biochemical assessment in body fluids).
Clinical details of PD patients and biochemical assessment in body fluids
The 6 non-PD control subjects showed a minimal lag period between changes in 2H2O enrichment in body water (the precursor pool) and 2H-cargo appearance (the product) (Figure , A and B), indicative of rapid transit of newly synthesized cargo proteins from the cellular site of synthesis to the site of sampling. PD patients exhibited markedly slower transport kinetics of 2H-cargo, characterized by a pattern of strikingly prolonged release into the CSF (Figure , A and B). Specifically all 12 PD subjects showed persistence of 2H-CHGB, 2H-sAPPα, and 2H-SNCA in CSF at days 15, 21, 22, 23, and 38, instead of a return to baseline enrichments in parallel with the fall in body 2H2O enrichment, as observed in the non-PD control subjects. In 1 PD subject, PD6084, repeated LPs were performed at days 3, 9, 21, and 38, which clearly showed delayed appearance in addition to persistence of 2H-cargo in CSF (Figure B). Stated differently, the 2H-cargo present in CSF during the 2H2O label decay phase from days 15–38 in control subjects had enrichments that reflected almost exactly the Tmax possible at the low body 2H2O enrichments present at the date of CSF sampling, whereas the 2H-cargo in PD patients had enrichments much greater than the possible Tmax if the cargo molecules had been synthesized on that day. Therefore, the cargo must have been synthesized when the body 2H2O enrichments were higher (i.e., at least several days previously).
Transport rates of neuronal cargo proteins in CSF of non-PD volunteers.
Differential delays in transport rates of neuronal cargo proteins in CSF of PD subjects.
These results were consistent with a defect in axonal transport. Interestingly, the secretion curves for 2H-NRG1 in the PD subjects were similar to those observed in controls (Figure B), suggestive of altered rates of cargo transport, as reflected in CSF from a distinct degeneration of neuronal subpopulations affected in PD patients.
The persistence of 2H-cargo proteins in CSF of PD patients could not be explained by defective protein clearance from CSF, since no significant changes in the turnover rate (half-life) of total CSF proteins were observed in the PD patients (Supplemental Figure 5C).
A previous radiolabeling study of axonal transport (43
) showed slowing with aging, particularly for slow axonal transport of SNCA. It is therefore conceivable that the transport abnormalities in the PD patients were simply a result of their older age, not of pathology. Despite the difficulties in obtaining a large control cohort of elderly volunteers for routine LP, 4 controls that were close in age to the PD patients (Table ) did not show altered cargo transport kinetics compared with the younger controls (Figure B). Thus, aging per se appears unlikely to explain the changes in CSF secretion rates of cargo molecules observed in PD patients.
In summary, these results demonstrated that CSF kinetic biomarkers of axonal transport were translatable into human subjects and may be useful to assess the status of neurodegeneration and potentially to monitor therapeutic modulation.