Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability.

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt ß-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

Progressive tau aggregation does not alter functional brain network connectivity in seeded hTau.P301L mice.

Progressive accumulation of hyperphosphorylated tau is a hallmark of various neurodegenerative disorders including Alzheimer's disease. However, to date, the functional effects of tau pathology on brain network connectivity remain poorly understood. To directly interrogate the impact of tau pathology on functional brain connectivity, we conducted a longitudinal experiment in which we monitored a fibril-seeded hTau.P301L mouse model using correlative whole-brain microscopy and resting-state functional MRI. Despite a progressive aggravation of tau pathology across the brain, the major resting-state networks appeared unaffected up to 15 weeks after seeding. Targeted analyses also showed that the connectivity of regions with high levels of hyperphosphorylated tau was comparable to that observed in controls. In line with the ostensible retention of connectivity, no behavioural changes were detected between seeded and control hTau.P301L mice as determined by three different paradigms. Our data indicate that seeded tau pathology, with accumulation of tau aggregates throughout different regions of the brain, does not alter functional connectivity or behaviour in this mouse model. Additional correlative functional studies on different mouse models should help determine whether this is a generalizable trait of tauopathies.

A Novel SOD1 Intermediate Oligomer, Role of Free Thiols and Disulfide Exchange.

Wild-type human SOD1 forms a highly conserved intra-molecular disulfide bond between C57-C146, and in its native state is greatly stabilized by binding one copper and one zinc atom per monomer rendering the protein dimeric. Loss of copper extinguishes dismutase activity and destabilizes the protein, increasing accessibility of the disulfide with monomerization accompanying disulfide reduction. A further pair of free thiols exist at C6 and C111 distant from metal binding sites, raising the question of their function. Here we investigate their role in misfolding of SOD1 along a pathway that leads to formation of amyloid fibrils. We present the seeding reaction of a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) to exclude variables caused by these free cysteines. Completely reduced fibril seeds decreasing the kinetic barrier to cleave the highly conserved intramolecular disulfide bond, and accelerating SOD1 reduction and initiation of fibrillation. Presence or absence of the pair of free thiols affects kinetics of fibrillation. Previously, we showed full maturation with both Cu and Zn prevents this behavior while lack of Cu renders sensitivity to fibrillation, with presence of the native disulfide bond modulating this propensity much more strongly than presence of Zn or dimerization. Here we further investigate the role of reduction of the native C57-C146 disulfide bond in fibrillation of wild-type hSOD1, firstly through removal of free thiols by paired mutations C6A, C111S (AS-SOD1), and secondly in seeded fibrillation reactions modulated by reductant tris (2-carboxyethyl) phosphine (TCEP). Fibrillation of AS-SOD1 was dependent upon disulfide reduction and showed classic lag and exponential growth phases compared with wild-type hSOD1 whose fibrillation trajectories were typically somewhat perturbed. Electron microscopy showed that AS-SOD1 formed classic fibrils while wild-type fibrillation reactions showed the presence of smaller "sausage-like" oligomers in addition to fibrils, highlighting the potential for mixed disulfides involving C6/C111 to disrupt efficient fibrillation. Seeding by addition of sonicated fibrils lowered the TCEP concentration needed for fibrillation in both wild-type and AS-SOD1 providing evidence for template-driven structural disturbance that elevated susceptibility to reduction and thus propensity to fibrillate.