Impaired brain clearance and abnormal A? degradation are considered key events for the formation and progressive accumulation of soluble neurotoxic oligomers and the development synaptic pathology, one of the strongest correlates to cognitive impairment. Clearance studies mostly centered in monomeric A?40 have provided a basic assessment of the participating mechanisms but the complex molecular and structural heterogeneity of the brain A? peptidome has been largely overlooked. The most obvious heterogeneity resides in the multiple N- and C-terminal truncated fragments that populate the A? peptidome; surprisingly, little is known about their homeostasis or their potential contribution to disease pathogenesis. Our studies indicate that while C-terminal truncations increase solubility and abrogate oligomerization/fibrillogenesis, properties associated with clearance mechanisms, N-terminal truncations, particularly A?4-x, exhibit enhanced pro-amyloidogenic and neurotoxic characteristics in vitro and in vivo, being particularly abundant in parenchymal deposits in AD and multiple APP Tg models and requiring detergents/formic acid for tissue extraction. Proteomic studies of tissue deposits and novel antibodies specific for the free N-terminus of A?4-x showed their topographic association with fibrillar plaque cores. Notably, these antibodies prevented A?4-x oligomerization, abrogated toxicity, and rescued behavioral deficits in a passive immunization pilot study. We hypothesize that N-terminal truncation of A? at position 4 generates pro-amyloidogenic fragments that progressively accumulate in brain deposits as a result of their high oligomerization propensity, poor brain removal, and resistance to proteolytic degradation, perpetuating the amyloidogenic loop, and postulate that passive immunization constitute a feasible therapeutic approach to reduce formation of A?4-x toxic oligomers and fibrillar deposits, restoring synaptic function and improving cognitive parameters. We propose to compare A?4-x clearance mechanisms with those of full-length A?40/A?42 in WT mice, assessing involvement of A? efflux transporters LRP-1 and P-gp and how brain efflux is influenced by oligomerization, local proteolytic degradation, and aging (Aim 1). The temporal accruement of A?4-x species as the disease establishes and progresses will be evaluated by proteomic analysis of the brain A? peptidome in APPswe/PS1?E9 and 3xTg of different age as well as in human specimens from MCI, mild, and severe AD. Intracerebral inoculation of AD-isolated A?4-x species will be used to evaluate seeding capabilities in young Tgs (Aim 2). Preventive and therapeutic benefits of MAb anti-A?4-x will be tested in passive immunization protocols in the same Tg models, assessing changes in behavioral parameters, biochemical composition of the brain A? peptidome, type and distribution of parenchymal A? lesions and ratio A?4-x to A?40/42. Cognitive changes will be correlated with the preservation/rescue of synaptic markers, electrophysiological parameters, and metabolic / bioenergetic features in isolated synaptosomes, all elements directly linked to synaptic function (Aim 3)
Understanding the molecular basis that regulate the delicate balance among A? production, its dynamics of aggregation and rate of clearance is a crucial step towards the identification of brain homeostatic mechanisms that modulate the undesirable formation of neurotoxic oligomers and an alternative path in the search for new targets of therapeutic interventions. Our proposal centers in the often-neglected heterogeneity of the brain A? peptidome ? going well beyond the classic A?40/A?42 dichotomy ? by focusing specifically in the brain clearance mechanisms and seeding potential of the pro-amyloidogenic and neurotoxic A?4-x species and their contribution to the synaptic dysfunction and cognitive impairment characteristic of Alzheimer's disease. A translational approach using antibodies highly specific for the A?4-x species in passive immunization protocols provides additional proof-of-concept to the contribution of these fragments to the disease pathogenesis while unveiling a not previously recognized novel therapeutic target.
