Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and alpha-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of alpha-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of alpha-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or alpha-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from alpha-actinin by two distinct but converging calcium-dependent processes: competitive displacement of alpha-actinin by calmodulin and reduction in the affinity of alpha-actinin for C0 after binding of calcium to alpha-actinin.

Recent human trials of treatments for Alzheimer’s disease (AD) have been largely unsuccessful, raising the idea that treatment may need to be started earlier in the disease, well before cognitive symptoms appear. An early marker of AD pathology is therefore needed and it is debated as to whether amyloid-βAβ? plaque load may serve this purpose. We investigated this in the hAPP-J20 AD mouse model by studying disease pathology at 6, 12, 24 and 36 weeks. Using robust stereological methods, we found there is no neuron loss in the hippocampal CA3 region at any age. However loss of neurons from the hippocampal CA1 region begins as early as 12 weeks of age. The extent of neuron loss increases with age, correlating with the number of activated microglia. Gliosis was also present, but plateaued during aging. Increased hyperactivity and spatial memory deficits occurred at 16 and 24 weeks. Meanwhile, the appearance of plaques and oligomeric Aβ were essentially the last pathological changes, with significant changes only observed at 36 weeks of age. This is surprising given that the hAPP-J20 AD mouse model is engineered to over-expresses Aβ. Our data raises the possibility that plaque load may not be the best marker for early AD and suggests that activated microglia could be a valuable marker to track disease progression.

The amyloid hypothesis has driven drug development strategies for Alzheimer's disease for over 20 years. We review why accumulation of amyloid-beta (Aβ) oligomers is generally considered causal for synaptic loss and neurodegeneration in AD. We elaborate on and update arguments for and against the amyloid hypothesis with new data and interpretations, and consider why the amyloid hypothesis may be failing therapeutically. We note several unresolved issues in the field including the presence of Aβ deposition in cognitively normal individuals, the weak correlation between plaque load and cognition, questions regarding the biochemical nature, presence and role of Aβ oligomeric assemblies in vivo, the bias of pre-clinical AD models toward the amyloid hypothesis and the poorly explained pathological heterogeneity and comorbidities associated with AD. We also illustrate how extensive data cited in support of the amyloid hypothesis, including genetic links to disease, can be interpreted independently of a role for Aβ in AD. We conclude it is essential to expand our view of pathogenesis beyond Aβ and tau pathology and suggest several future directions for AD research, which we argue will be critical to understanding AD pathogenesis.