Cx3cr1, the receptor for the chemokine Cx3cl1 (fractalkine), has been implicated in the progression and severity of Alzheimer's disease-like pathology in mice, but the underlying mechanisms remain unclear. A complicating factor is that Cx3cr1 has been demonstrated in both neurons and microglia. Here, we have dissected the differences between neuronal and microglial Cx3cr1, specifically by comparing direct amyloid-β-induced toxicity in cultured, mature, microglia-depleted hippocampal neurons from wild-type and Cx3cr1-/- mice. Wild-type neurons expressed both Cx3cl1 and Cx3cr1 and released Cx3cl1 in response to amyloid-β. Knockout of neuronal Cx3cr1 abated amyloid-β-induced lactate dehydrogenase release. Furthermore, amyloid-β differentially induced depression of pre- and postsynaptic components of miniature excitatory postsynaptic currents, in a peptide conformation-dependent manner. Knockout of neuronal Cx3cr1 abated effects of both amyloid-β conformational states, which were differentiable by aggregation kinetics and peptide morphology. We obtained similar results after both acute and chronic treatment of cultured neurons with the Cx3cr1 antagonist F1. Thus, neuronal Cx3cr1 may impact Alzheimer's disease-like pathology by modulating conformational state-dependent amyloid-β-induced synaptotoxicity.

Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson's disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.

The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer's disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon--prostate tissue remodelling in middle-aged and elderly men.

Although the aggregation of the amyloid-β peptide (Aβ) into amyloid fibrils is a well-established hallmark of Alzheimer's disease, the complex mechanisms linking this process to neurodegeneration are still incompletely understood. The nematode worm C. elegans is a valuable model organism through which to study these mechanisms because of its simple nervous system and its relatively short lifespan. Standard Aβ-based C. elegans models of Alzheimer's disease are designed to study the toxic effects of the overexpression of Aβ in the muscle or nervous systems. However, the wide variety of effects associated with the tissue-level overexpression of Aβ makes it difficult to single out and study specific cellular mechanisms related to the onset of Alzheimer's disease. Here, to better understand how to investigate the early events affecting neuronal signalling, we created a C. elegans model expressing Aβ42, the 42-residue form of Aβ, from a single-copy gene insertion in just one pair of glutamatergic sensory neurons, the BAG neurons. In behavioural assays, we found that the Aβ42-expressing animals displayed a subtle modulation of the response to CO2, compared to controls. Ca2+ imaging revealed that the BAG neurons in young Aβ42-expressing nematodes were activated more strongly than in control animals, and that neuronal activation remained intact until old age. Taken together, our results suggest that Aβ42-expression in this very subtle model of AD is sufficient to modulate the behavioural response but not strong enough to generate significant neurotoxicity, suggesting that slightly more aggressive perturbations will enable effectively studies of the links between the modulation of a physiological response and its associated neurotoxicity.

Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions.