New data on an assortment of therapeutic possibilities appear this week, featuring the clearance of vascular amyloid by passive immunization, a calpain protease inhibitor that seems to block the synaptic effects of soluble Aβ, and a new approach to inhibiting tau phosphorylation by stimulating its glycosylation. Here follows a roundup of the studies, ranked from most clinically advanced candidate to the newest entrant.
The strategies of active and passive vaccination against Aβ have shown a lot of promise, but also present enormous complexities. Human clinical trials continue, as do persistent questions about how to design vaccines to have the best chance of enhancing cognitive function with the fewest side effects. One problem, observed in several mouse models of AD, is that mobilization of parenchymal plaques by Aβ antibodies results in an increase in vascular Aβ deposition and the appearance of microhemorrhages (see ARF related news story; Wilcock et al., 2004; Racke et al., 2005), although how these events are related remains unclear.
To try to sort out the relationship between parenchymal plaque clearance, vascular amyloid, and the risk of hemorrhages, Dora Games and colleagues from Elan Pharmaceuticals, South San Francisco, California, looked at the effect of Aβ antibodies on vascular amyloid and microhemorrhages in PDAPP mice. The work, published in the July 2 issue of the Journal of Neuroscience, shows that an antibody targeted to the N-terminal region of the peptide can clear vascular Aβ and does not cause microhemorrhage when used at moderate doses. The results raise the possibility that current active and passive immunization protocols, which involve N-terminal reactive antibodies, could also result in clearance of vascular Aβ, and that Aβ can be removed from the vasculature without triggering vessel leakage.
The research team, headed up by first author Sally Schroeter, compared the effects of chronic six-month treatment of year-old mice with two different Aβ antibodies. 3D6, which recognizes the amino-terminal 5 residues of Aβ, binds to plaques and clears parenchymal Aβ deposits, while 266, which recognizes central region residues 16-23, binds to soluble AAβ but does not clear plaques. The investigators found that 3D6 cleared vascular Aβ in a dose-dependent manner, while 266 did not. At the highest 3D6 dose (3 mg/kg), vascular Aβ was nearly completely prevented or cleared in the mice. At lower doses, the antibody caused a partial removal of vascular amyloid. Only the high dose of 3D6 was associated with a significantly elevated incidence of microhemorrhage as indicated by hemosiderin staining. The authors conclude that 3D6 can clear vascular amyloid, and that lowering antibody exposure lowers the risk of hemorrhage, presumably by slowing the clearance process.
One caveat to the study is that PDAPP mice deposit only small amounts of vascular amyloid compared to some other mouse models and to humans with cerebral amyloid angiopathy, which is associated with spontaneous hemorrhage. The study leaves open the question of what happens to vessels more heavily laden with amyloid, a situation that occurs in the majority of AD cases.
Taking Aim at Synaptic Changes
A second paper approaches treatment from the synaptic angle, providing evidence that inhibition of the calcium-activated protease calpain might be a way to prevent the impairment in synaptic function that occurs in AD. In addition to demonstrating the promising effects of two calpain inhibitors in vitro, the researchers, from the lab of Ottavio Arancio at Columbia University in New York, show that calpain inhibition improves cognitive performance on two different memory tests in the APP/PS1 mouse model of AD. The results appeared July 1 in the Journal of Clinical Investigation.
The calpain family of proteases has been implicated in AD in multiple ways. Calpain 1 is located at synapses, and its activity is increased in AD brain. Substrates for calpains include a host of proteins that play a role in APP production and tau phosphorylation, and calpain cleaves the Cdk5 regulator p35 to the constitutively active p25 form, which is also elevated in AD brain and appears to be involved in neurodegeneration.
In the new study, first author Fabrizio Trinchese and colleagues found that two different calpain inhibitors were able to restore normal synaptic function to cultured hippocampal neurons from APP/PS1 mice. The neurons show elevated spontaneous neurotransmitter release and fail to respond to glutamate. Treatment of the cells with E64, a general inhibitor of cysteine proteases, or the calpain-specific, orally administrable inhibitor BDA-410 (Li et al., 2007), returned synaptic activity to normal. The inhibitors had no effect on normal cells.
Studies confirmed the restoration of normal synaptic activity and long-term potentiation (LTP) in hippocampal slices from seven-month-old APP/PS1 mice that had been treated for the previous five months with either inhibitor, suggesting the strategy works in vivo. The effect of the calpain inhibitors depended on the production of Aβ, not PS1 overexpression, since the same effect on LTP was seen in APP mice, which do not overexpress PS1. There was no effect of inhibitors on LTP in normal mice. In addition, BDA-410 was able to block the inhibition of LTP seen when normal hippocampal slices were perfused with soluble Aβ oligomers.
The synaptic normalization observed by electrophysiological means was also reflected in improved behavioral measures. The animals’ performance in a radial arm water maze or in an associative learning test was both normalized by treatment with E64 or BDA-410. There was no effect on normal mice.
These results suggest that calpain activation plays a role in the synaptic toxicity of Aβ oligomers. The inhibitors appear to work downstream of Aβ production, since treatment did not change the levels of Aβ or plaque load in the mice. Treatment was associated with normalization of markers of synaptic remodeling, including phosphorylation of the transcription factor and calpain target CREB, and redistribution of the synaptic protein synapsin I. “Collectively, these data strongly support the possibility that calpain inhibitors act by reestablishing the increase in pCREB [phosphorylated CREB], thus rescuing the impairment of synaptic plasticity caused by overexpression of the APP and PS1 transgenes,” the authors write.
The data support the further development of calpain inhibitors to treat AD, Arancio told ARF. He sees the calpain approach as complementary to therapies aimed at reducing Aβ levels. “We feel it’s possible to improve disease by acting downstream of Aβ, in addition to decreasing Aβ. If you improve memory, that’s what counts,” he said. In March, Arancio received an NIH grant to work with chemist Gregory Thatcher of the University of Illinois at Chicago to synthesize and test new calpain inhibitors, and he said they are trying the first compounds now. Their efforts appear especially timely in light of recent developments highlighting the potential role of abnormally high calcium levels in brain to AD (see ARF related news story).
Hitting the Sweet Spot: Tau Glycosylation
Inhibiting the pathological phosphorylation of the microtubule-associated protein tau could provide a way to detoxify AD tangles and treat other tauopathies. In this regard, kinase inhibitors have gotten a lot of attention (see ARF related news story). In this week’s online edition of Nature Chemical Biology, David Vocadlo and colleagues at Simon Fraser University, Burnaby, British Columbia, Canada, offer up an alternative way to block tau phosphorylation. Taking advantage of the reciprocal relationship between tau phosphorylation and O-linked glycosylation (the two modifications both occur on serine/threonine residues and thus are mutually exclusive; see Liu et al., 2004), Vocadlo’s group shows that boosting glycosylation with an inhibitor of the sugar-removing enzyme O-GlcNAcase inhibits tau phosphorylation.
In the new work, first author Scott Yuzwa and colleagues describe the structure-based design of a specific O-GlcNAcase inhibitor and its effects on tau in vivo. The compound, thiamet-G, caused large increases in O-GlcNAc-modified proteins in PC12 cells with no sign of toxicity. Increased glycosylation was associated with a two- to threefold reduction in tau phosphorylation at the pathological residues Ser396 and Thr231. Thiamet-G was orally available and crossed the blood-brain barrier in rats, where it resulted in increased total brain O-GlcNAc-modified proteins and decreased tau phosphorylation at Ser396, Thr231, and Ser422. Immunohistochemistry showed that tau phosphorylation was decreased in the hippocampus, alongside a general increase in O-GlcNAc-specific staining.
Still to be determined is whether the changes in tau phosphorylation observed in healthy rats can be achieved in animals with established tau pathology. The authors write that these experiments are now underway. Another concern is the large number of substrates for the target enzyme, a problem that “needs to be addressed by further study,” Vocadlo told ARF by e-mail. “A number of proteins are modified with O-GlcNAc, making on-target toxicity a potential complication. Some work suggests that O-GlcNAc is involved in regulating glucohomeostasis and modulating insulin resistance (see, e.g., Yang et al., 2008),” he wrote. In the meantime, the specificity of thiamet-G will aid chemical biology efforts to understand the function of O-GlcNAc in brain.—Pat McCaffrey