Direct three-dimensional visualization of membrane disruption by amyloid fibrils

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer’s, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from β₂-microglobulin (β₂m). Using cryoelectron tomography, we demonstrate that fragmented β₂m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15–25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid–protein interactions in membrane remodelling.The failure of molecular chaperones to prevent the accumulation of misfolded proteins results in protein aggregation and amyloid formation, processes associated with severe human degenerative diseases (1, 2). Despite the attention focused on these problems during the century since these disorders were first identified (3–5) and advances in understanding the structure of the cross-β conformation of amyloid fibrils in atomic detail (6, 7), the basic pathological mechanisms of amyloidosis remain poorly understood and therapeutic intervention is lacking. The identity of the toxic species and the mechanisms of cytotoxicity remain major unsolved problems. In some systems, there is evidence suggesting that prefibrillar oligomers, rather than the fully formed fibrils, are the source of toxicity (8, 9). In these cases, cytotoxicity is thought to result from the formation of specific membrane pores (10, 11) although alternative models including membrane destabilization or membrane thinning have also been proposed (12–15). In other cases, toxicity may reside with the amyloid fibrils themselves. Evidence that toxicity correlates with fibrillar assemblies has been reported for yeast and mammalian prion proteins (16, 17), human lysozyme (18), Huntingtin exon 1, α-synuclein (19), and Amyloid-β (Aβ) (20, 21). Furthermore, Aβ plaques have been shown to form rapidly in vivo and to precede neuropathological changes in a mouse model (22). The end surfaces of fibrils (herein termed “fibril ends”) are unusually reactive entities: they play a key role in catalyzing recruitment and conformational conversion of amyloid-forming proteins (23, 24) and provide the sites for templated elongation of amyloid fibril growth (25, 26). Recently, Xue et al. (27) showed that short fibrils of β₂-microglobulin (β₂m), α-synuclein, and hen lysozyme, each prepared by fragmentation of longer fibrils, cause increased damage to membranes and disruption to cellular function compared with the initial long fibrils. Short and long fibril preparations differ in the number of fibril ends at a given protein concentration. Because these are known to be reactive sites, the above observations suggest a role for fibril ends in amyloid–lipid interaction and possibly in amyloid pathogenesis (23, 24, 27). Fragmented fibrils of all three proteins were also found to induce dye leakage from negatively charged liposomes, the most susceptible of which contain a mixture of the cellular lipids phosphatidylcholine (PC) and phosphatidylglycerol (PG), but liposomes with a variety of compositions were damaged by the fibrils in all cases (27).Here, we use β₂m amyloid fibrils formed in vitro as a model system to investigate the structural basis of membrane damage by amyloid fibrils (27, 28). Previous studies have shown that these fibrils possess a parallel in register cross-β structure (29, 30) assembled into multidomain filaments coiled together, described by cryo-EM (28). These fibrils bind amyloid-specific ligands such as serum amyloid P component with a similar affinity to their ex vivo counterparts (31). Using the conditions under which β₂m amyloid fibrils induce dye release from liposomes (pH 7.4), we examined the effects of both long (∼1,400 nm) and fragmented (∼400 nm) β₂m fibrils (27), as well as various control preparations, on the 3D structures of the liposomes by confocal microscopy, cryo-EM, and tomography. We found pronounced distortions in the liposomes, interruptions to the bilayer structure, and extraction of lipids that were induced by the presence of amyloid fibrils. The most severe distortions were seen in proximity to the fibril ends, which are enriched in the fragmented fibril samples. This type of membrane remodelling appears distinct from the actions of other previously described proteins that induce membrane breakage, as in the action of membrane pore-forming proteins (32). The results suggest a role of fibrils in membrane damage that could contribute to the cellular dysfunction associated with amyloid disease.     

Measurement of energy landscape roughness of folded and unfolded proteins

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4–5 k_BT). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k_BT) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues.     

A case of splanchnic rhabdomyosarcoma

Rhabdomyosarcoma is a typical tumor of the skeletal musculature. A case personally observed, with possible splenic origin is presented.     

Experimental repair of spinal cord lesions by grafting from CNS to PNS

Based on the previous work of other researchers, we first removed 1 cm of the spinal cord of rats and grafted the gap with peripheral nerve. We obtained progression of regenerating axons inside the grafts, but regrowing axons halted at the distal cord level. We then tried directly to connect the cephalad stump of the cord with the sciatic nerve, several surgical models of which have been previously studied. The latest model uses a homologous sciatic nerve of an inbred Wistar rat, to bridge the gap between the lateral bundle of the cord, proximal to the origin of the sciatic roots, and the sciatic trunk distal to the fusion of its roots. Good reinnervation of the sciatic nerve and distal muscles was found. It was demonstrated by: a) the remyelination of regenerated nerve fibers, not only in the grafts but also in the sciatic nerve; b) re-innervation of motor endplates; and c) clinical and electromyographic muscle responses. Central nervous system (CNS) neurons are able to regenerate not only their axons through the CNS but also into peripheral nerves. The capacity of CNS neurons to neurotize peripheral nerves reaching terminal organs, is a basic finding. Many questions arise from the study, including: Can these results be extrapolated to humans? What would be the function of these regenerated fibers that might lack some of the control mechanisms peculiar to peripheral nerves?     

Photokinetic and ultrastructural studies on porphyrin photosensitization of HeLa cells

Liposome-bound haematoporphyrin or haematoporphyrin dimethylester, as well as haematoporphyrin dissolved in phosphate-buffered saline, were added to HeLa cell monolayers at a dose of 1 microgram of porphyrin per 10(5) cells. After 2 min or 20 min incubation liposome-bound porphyrins were accumulated by cells in an about two-fold larger amount than the water-dissolved haematoporphyrin. This caused a more efficient photosensitization of HeLa cells by liposome-delivered porphyrins upon illumination with 366 nm light. Ultrastructural studies of HeLa cells, which had been incubated in a physiological medium for 24h after the end of irradiation, showed that liposomal porphyrins induce an early and extensive endocytoplasmic damage, leading to swelling of the mitochondria and vesiculation; changes of the permeability of the cytoplasmic membrane are also evident, especially in the case of haematoporphyrin dimethylester. On the other hand, water-dissolved haematoporphyrin predominantly photosensitizes damage of the plasma membrane. The different pattern of cell photodamage probably reflects a different subcellular distribution of the photosensitizing drugs.     

Cardiovascular reflex responses in cluster headache patients: basal autonomic alterations

To investigate the involvement of the autonomic nervous system in Cluster Headache (CH) patients, we compared the cardiovascular reflex responses in a group of CH subjects and a group of controls. We considered five tests: 1) deep breathing test (DB); 2) lying to standing test; 3) heart rate response to Valsalva manoeuvre; 4) blood pressure response to standing; 5) blood pressure response to sustained handgrip. Our data confirm an autonomic dysfunction in CH, particularly regarding the parasympathetic system. Alternative interpretations of these results are discussed.     

Lack of association between BK virus and ependymomas, malignant tumors of pancreatic islets, osteosarcomas and other human tumors

BK virus (BKV) DNA sequences were not detected in 142 human tumors analyzed by DNA-DNA reassociation kinetics and blot hybridization. The investigation was focused mainly on those rare types of human tumors (ependymomas, choroid plexus papillomas, tumors of pancreatic islets and osteosarcomas) that are induced with highest frequency by BKV in experimental animals. In addition, other tumors of the urinary apparatus and of the central nervous system were analyzed. BKV tumor (T) antigen was not detected in neoplastic tissues, and BKV T antibodies were not found in sera and cerebrospinal fluids from patients with neoplasms. Sequences homologous to BKV DNA were found in normal tissue from a kidney carrying a carcinoma. The neoplastic tissue from the same organ, however, had no sequences homologous to BKV DNA. Such DNA does not belong to BKV but probably to another papovavirus related to BKV.