Cystamine is one of the very few candidate drugs being considered for the treatment of HD. Here we have investigated the molecular mechanisms of action of cystamine, and our study has revealed that an FDA-approved reduced form of cystamine produces very similar biological effects in vitro and in vivo. We demonstrate that part of the neuroprotective effect of cystamine is due to its promotion of secretion of the neuronal survival factor BDNF. Cystamine has 2 quite distinct actions in this regard. First, it increases the steady-state levels of the Hsp HSJ1b mRNA, which stimulates the secretory pathway through its action on CCV formation, and, second, it inhibits TGase, which has a negative effect on BDNF sorting.
HSJ1b belongs to the large family of DnaJ-like proteins that contain the typical Hsp40 chaperones HDJ1/Hsp40 and HDJ2/HSDJ. In most cases, these chaperones have been reported to reduce polyQ-induced aggregation and toxicity in various models (for reviews, see refs. 20
). We found that HSJ1b functions in a qualitatively different way inasmuch as it appears not to prevent aggregation or the formation of NIIs. However, we found that HSJ1b is relevant to HD, as it strongly inhibits polyQ-huntingtin–induced neuronal death in vitro and rescues neuronal dysfunction in a nematode model of HD. Although we found that in mammalian cells, HSJ1b stimulates BDNF secretion, the exact mechanism by which HSJ1b operates in nematodes remains to be established. Indeed, BDNF is absent in nematodes. HSJ1b could, however, enhance the secretion of other specific C. elegan
s factors. In C. elegans
, trophic factors other than BDNF exist. In particular, the mesencephalic, astrocyte-derived neurotrophic factor, MANF, is well conserved from C. elegans
to humans and could be the target of HSJ1b (50
). Furthermore, BDNF second messengers are conserved. Therefore, it is possible that HSJ1b acts through a pathway involving these molecules.
In mammalian cells the neuroprotective properties of HSJ1b are linked to its ability to enhance neurotrophic support. HSJ1b positively regulates the sorting of BDNF-containing vesicles from the Golgi/TGN, leading to an increase in BDNF release. These findings are in agreement with the function of HSJ1b in the inhibition of the uncoating of CCVs (35
). Most of the vesicles budding from the Golgi/TGN region are CCVs, and assembly of the clathrin coat on the forming bud is an important step (37
). HSJ1b could therefore promote the budding of BDNF-containing vesicles by stabilizing this assembly step. Consistent with this idea, we observed an increased percentage of BDNF-containing vesicles that were clathrin positive when HSJ1b expression was increased, whereas reducing HSJ1b levels decreased it. Interestingly, we found significantly less HSJ1b in postmortem brain extracts from HD patients than from control brains, suggesting a potential alteration in the processing of BDNF at the Golgi/TGN during HD pathogenesis. A defect in clathrin coating processes in HD is supported by the fact that huntingtin interacts with HIP1 and HIP12/HIP1R, 2 proteins that are components of clathrin coats and that regulate clathrin assembly by directly interacting with clathrin (51
). HSJ1b is thought to inhibit uncoating by interfering with the interactions of Hsc70 with specialized uncoating DnaJ-like proteins such as auxilin (35
). Whether it also regulates the activity of huntingtin interactors such HIP1 remains to be determined.
The second consequence of cystamine treatment is promotion of the secretion of BDNF vesicles through a mechanism involving TGase. Our findings that TGase 2 colocalizes with BDNF at the Golgi and the observation that TGases regulate the secretion of BDNF is consistent with previous observations from the 1980s indicating a role of TGases in inhibiting secretion and/or release of various hormones and neurotransmitters such as insulin, serotonin, and dopamine (41
). For instance, monodansylcadaverine (MDC), a potent TGase inhibitor, enhances dopamine release from rat brain synaptosomes in basal and in potassium-stimulated conditions (41
). Moreover, reports that MDC blocks clathrin-mediated endocytosis (55
) accord with our finding that cystamine and TGases regulate the clathrin pathway.
We and others have reported a lack of BDNF support in HD that involves defects in BDNF synthesis (25
) and transport (27
). Our findings suggest that, in addition, a defect in BDNF sorting from the Golgi/TGN occurs in HD and that such BDNF processing is regulated by HSJ1b and TGases.
Cystamine was first described as a TGase inhibitor in vitro, and several studies are consistent with the possibility that cystamine is beneficial in HD by this mechanism (10
). However, whether cystamine is directly inhibiting TGase 2 in vivo remains to be clearly established. First, the reduced form of cystamine, cysteamine, could act as a competitive inhibitor of TGase 2 in vivo (57
). Second, other metabolites of cystamine and cysteamine could mediate the neuroprotective effect, as they are rapidly metabolized and low to undetectable levels of cystamine and cysteamine are found in the brain of cystamine-treated mice (17
). In addition, cystamine or its metabolites could act through a TGase-independent mechanism (16
). Cystamine inhibits caspase 3 activity and increases glutathione levels in cells (18
). In vivo, the beneficial effect of cystamine could involve the increase in l-cysteine, which has antioxidant properties (17
We now show that cystamine and cysteamine target HSJ1b and TGase to increase the release of BDNF, a trophic factor that is depleted in HD and that is crucial for the survival of striatal neurons in HD. Our data further emphasize that cystamine or its metabolites acts at multiple levels to protect against polyQ-huntingtin–induced toxicity and therefore add to the motivation for optimizing a therapy with cystamine or related compounds.
We demonstrate that cysteamine is as efficient as cystamine in increasing levels of BDNF in the brain. We also report that cysteamine is neuroprotective in HD mice by increasing levels of BDNF in brain. BDNF levels can also be measured in blood as a biomarker for pathological stages. We found that in HD knock-in mice and in a primate model of HD, the levels of BDNF in serum were reduced compared with those in controls, whereas such decreases in brain BDNF could not be detected in HD knock-in mice and in R6/1 mice at 15 and 16 weeks of age. Interestingly, at these early stages, these mice do not show overt phenotypes (45
). This suggests that blood BDNF could be used to follow disease progression and validate the neuroprotective effects of drugs acting on BDNF levels.
We found cysteamine-induced release of BDNF in brain to be transient. This is consistent with the rapid clearance of cysteamine from the plasma of healthy individuals (58
) and patients with nephropathic cystinosis (59
) and suggests that, as for the treatment of nephropathic cystinosis, repeated doses of cysteamine at short intervals would be appropriate for the treatment of HD. Such limited and controlled release of BDNF is of particular interest for therapy, as an excessive stimulation of the BDNF/TrkB pathway leads to tumorigenesis in mice (60
). Moreover, the efficacy of a repeated treatment is unlikely to diminish with time, as we found that the cysteamine-induced increase in brain and serum BDNF levels was still detected after 12 weeks continuous treatment (Supplemental Figure 3).
We propose the use of cysteamine as a therapeutic approach to treat HD. Indeed, the safety of cysteamine in humans is well documented, as cysteamine is used to treat cystinosis (42
). Moreover, the tolerated cysteamine dose has been evaluated in HD patients (43
). Finally, our findings indicate that the efficacy of cysteamine treatment in HD patients could be monitored by measuring serum levels of BDNF as a convenient biomarker.