Conformational plasticity of the Gerstmann-Sträussler-Scheinker disease peptide as indicated by its multiple aggregation pathways

The existence of several prion strains and their capacity of overcoming species barriers seem to point to a high conformational adaptability of the prion protein. To investigate this structural plasticity, we studied here the aggregation pathways of the human prion peptide PrP82-146, a major component of the Gerstmann-Sträussler-Scheinker amyloid disease.By Fourier transform infrared (FT-IR) spectroscopy, electron microscopy, and atomic force microscopy (AFM), we monitored the time course of PrP82-146 fibril formation. After incubation at 37 °C, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands. At a critical oligomer concentration, the emergence of a new FT-IR band allowed to detect fibril formation. A different intermolecular β-sheet interaction of the peptides in oligomers and in fibrils is, therefore, detected by FT-IR spectroscopy, which, in addition, suggests a parallel orientation of the cross β-sheet structures of PrP82-146 fibrils. By AFM, a wide distribution of PrP82-146 oligomer volumes—the smallest ones containing from 5 to 30 peptides—was observed. Interestingly, the statistical analysis of AFM data enabled us to detect a quantization in the oligomer height values differing by steps of ∼ 0.5 nm that could reflect an orientation of oligomer β-strands parallel with the sample surface. Different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly.Thermal aggregation of PrP82-146 was also investigated by FT-IR spectroscopy, which indicated for these aggregates an intermolecular β-sheet interaction different from that observed for oligomers and fibrils. Unexpectedly, random aggregates, induced by solvent evaporation, were found to display a significant α-helical structure as well as several β-sheet components.All these results clearly point to a high plasticity of the PrP82-146 peptide, which was found to be capable of undergoing several aggregation pathways, with end products displaying different secondary structures and intermolecular interactions.     

A Chinese patient of P102L Gerstmann-Sträussler-Scheinker disease contains three other disease-associated mutations in SYNE1

Gerstmann-Sträussler-Scheinker disease (GSS) with the P102L mutation in PRNP gene is characterized with progressive cerebellar dysfunction clinically and PrP^Sc plaques neurologically. Due to the cerebellar ataxia in the early stage, GSS P102L is often misdiagnosed as other neurodegenerative disorders. We presented here a 49-year-old female patient with proven P102L PRNP mutation, and three heterologous mutations in hereditary ataxias associated gene SYNE1, including p.V3643L, p.M3376V and p.T2860A. The patient appeared progressive unsteady gait in early stage and developed the Creutzfeldt-Jacob disease (CJD) – associated clinical manifestations, including progressive dementia, myoclonus, pyramidal and extrapyramidal signs. She is still alive but with akinetic mutism 21 months after onset. Observation of intense signal changes in cortical regions (cortical ribboning) in diffusion weighted imaging (DWI) MRI scanning and positive protein 14-3-3 in cerebrospinal fluid (CSF) proposed the diagnosis of sporadic CJD. The final diagnosis of P102L GSS was made after PRNP sequencing.     

Gerstmann-Sträussler-Scheinker disease amyloid protein polymerizes according to the "dock-and-lock" model

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired prion diseases such as Gerstmann-Str?ussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues approximately 82-146, which when synthesized as a peptide, readily forms fibrils featuring GSS amyloid. The present study employed surface plasmon resonance (SPR) to characterize the binding events underlying PrP82-146 oligomerization at the first stages of fibrillization, according to evidence suggesting a pathogenic role of prefibrillar oligomers rather than mature amyloid fibrils. We followed in real time the binding reactions occurring during short term (seconds) addition of PrP82-146 small oligomers (1-5-mers, flowing species) onto soluble prefibrillar PrP82-146 aggregates immobilized on the sensor surface. SPR data confirmed very efficient aggregation/elongation, consistent with the hypothesis of nucleation-dependent polymerization process. Much lower binding was observed when PrP82-146 flowed onto the scrambled sequence of PrP82-146 or onto prefibrillar Abeta42 aggregates. As previously found with Abeta40, SPR data could be adequately fitted by equations modeling the "dock-and-lock" mechanism, in which the "locking" step is due to sequential conformational changes, each increasing the affinity of the monomer for the fibril until a condition of irreversible binding is reached. However, these conformational changes (i.e. the locking steps) appear to be faster and easier with PrP82-146 than with Abeta40. Such differences suggest that PrP82-146 has a greater propensity to polymerize and greater stability of the aggregates.     

Molecular origin of Gerstmann-Sträussler-Scheinker syndrome: insight from computer simulation of an amyloidogenic prion peptide

Prion proteins become pathogenic through misfolding. Here, we characterize the folding of a peptide consisting of residues 109–122 of the Syrian hamster prion protein (the H1 peptide) and of a more amyloidogenic A117V point mutant that leads in humans to an inheritable form of the Gerstmann-Sträussler-Scheinker syndrome. Atomistic molecular dynamics simulations are performed for 2.5 μs. Both peptides lose their α-helical starting conformations and assume a β-hairpin that is structurally similar in both systems. In each simulation several unfolding/refolding events occur, leading to convergence of the thermodynamics of the conformational states to within 1 kJ/mol. The similar stability of the β-hairpin relative to the unfolded state is observed in the two peptides. However, substantial differences are found between the two unfolded states. A local minimum is found within the free energy unfolded basin of the A117V mutant populated by misfolded collapsed conformations of comparable stability to the β-hairpin state, consistent with increased amyloidogenicity. This population, in which V117 stabilizes a hydrophobic core, is absent in the wild-type peptide. These results are supported by simulations of oligomers showing a slightly higher stability of the associated structures and a lower barrier to association for the mutated peptide. Hence, a single point mutation carrying only two additional methyl groups is here shown to be responsible for rather dramatic differences of structuring within the unfolded (misfolded) state.     

Aggregation/fibrillogenesis of recombinant human prion protein and Gerstmann-Sträussler-Scheinker disease peptides in the presence of metal ions

In this study we investigated the role of Cu(2+), Mn(2+), Zn(2+), and Al(3+) in inducing defective conformational rearrangements of the recombinant human prion protein (hPrP), which trigger aggregation and fibrillogenesis. The research was extended to the fragment of hPrP spanning residues 82-146, which was identified as a major component of the amyloid deposits in the brain of patients affected by Gerstmann-Str?ussler-Scheinker (GSS) disease. Variants of the 82-146 wild-type subunit [PrP-(82-146)(wt)] were also examined, including entirely, [PrP-(82-146)(scr)], and partially scrambled, [PrP-(82-146)(106)(-)(126scr)] and [PrP-(82-146)(127)(-)(146scr)], peptides. Al(3+) strongly stimulated the conversion of native hPrP into the altered conformation, and its potency in inducing aggregation was very high. Despite a lower rate and extent of prion protein conversion into altered isoforms, however, Zn(2+) was more efficient than Al(3+) in promoting organization of hPrP aggregates into well-structured, amyloid-like fibrillar filaments, whereas Mn(2+) delayed and Cu(2+) prevented the process. GSS peptides underwent the fibrillogenesis process much faster than the full-length protein. The intrinsic ability of PrP-(82-146)(wt) to form fibrillar aggregates was exalted in the presence of Zn(2+) and, to a lesser extent, of Al(3+), whereas Cu(2+) and Mn(2+) inhibited the conversion of the peptide into amyloid fibrils. Amino acid substitution in the neurotoxic core (sequence 106-126) of the 82-146 fragment reduced its amyloidogenic potential. In this case, the stimulatory effect of Zn(2+) was lower as compared to the wild-type peptide; on the contrary Al(3+) and Mn(2+) induced a higher propensity to fibrillation, which was ascribed to different binding modalities to GSS peptides. In all cases, alteration of the 127-146 sequence strongly inhibited the fibrillogenesis process, thus suggesting that integrity of the C-terminal region was essential both to confer amyloidogenic properties on GSS peptides and to activate the stimulatory potential of the metal ions.     

Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation

Gerstmann-Str?ussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP(Sc) isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP(Sc) has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP(Sc) allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP(Sc) quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.     

A 7-kDa prion protein (PrP) fragment, an integral component of the PrP region required for infectivity, is the major amyloid protein in Gerstmann-Sträussler-Scheinker disease A117V

Gerstmann-Str?ussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Str?ussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.     

Gerstmann-Sträussler-Scheinker disease with P102L-V129 mutation: a case with psychiatric manifestations at onset

Gerstmann-Str?ussler-Scheinker disease (GSS) is an adult onset, rare, genetically determined autosomal dominant prion disease. Clinically, it is characterized predominantly by slowly progressive spino-cerebellar dysfunction with ataxia, absent reflexes in the legs and cognitive impairment. Onset is usually in the fifth decade and in the early phase, ataxia is predominant. Mutations in the prion protein gene (PRNP) had been identified and the most important of these is at codon 129. A genotype-phenotype relationship with genetic polymorphism at residue 129 between methionine and valine has been supposed. We describe a patient with GSS and P102L-V129 mutation in which the onset with prominent psychiatric features characterized by apathy and depression and not with cerebellar sign and the clinical course with seizures, nor observed in P102L-V129 cases, allow us to confirm observations that the GSS caused by the 102 mutation is influenced by the codon 129 polymorphism with a specific genotype-phenotype influence, but probably other additional factors might be considered as background for phenotypic variability.     

Specific amyloid-β42 deposition in the brain of a Gerstmann-Sträussler-Scheinker disease patient with a P105L mutation on the prion protein gene

ABSTRACT

Although colocalization of amyloid β (Aβ) with prion protein (PrP) in the kuru plaque has previously been observed in the brain of prion diseases patients, the participating Aβ species has not been identified. Here, we present an immunohistochemical assessment of the brain and spinal cord of a 69-year-old Japanese female patient with Gerstmann-Sträussler-Scheinker disease with a P105L mutation on the PRNP gene (GSS-P105L). Immunohistochemical assessment of serial brain sections was performed using anti-PrP and -Aβ antibodies in the hippocampus, frontal and occipital lobes. She died 69 years after a 21-year clinical course. Immunohistochemistorical examination revealed that ~50% of the kuru plaques in the cerebrum were colocalized with Aβ, and Aβ42 was predominantly observed to be colocalized with PrP-plaques. The Aβ deposition patterns were unique, and distinct from diffuse plaques observed in the normal aging brain or Alzheimer’s disease brain. The spinal cord exhibited degeneration in the lateral corticospinal tract, posterior horn, and fasciculus gracilis. We have demonstrated for the first time that Aβ42, rather than Aβ40, is the main Aβ component associated with PrP-plaques, and also the degeneration of the fasciculus gracilis in the spinal cord in GSS-P105L, which could be associated with specific clinical features of GSS-P105L.     

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter Ⅰ (GAT1)

It is well documented that γ-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABA_A and GABA_B receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter Ⅰ (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wi     

Pyroglutamate amyloid β (Aβ) aggravates behavioral deficits in transgenic amyloid mouse model for Alzheimer disease

Pyroglutamate-modified Aβ peptides at amino acid position three (Aβ(pE3-42)) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). Aβ(pE3-42) is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated Aβ(pE3-42) levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces Aβ(pE3-42). TBA42 mice showed age-dependent behavioral deficits and Aβ(pE3-42) accumulation. The Aβ profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-terminal truncated, and modified Aβ peptides: Aβ(1-42), Aβ(1-40), Aβ(pE3-40), Aβ(pE3-42), Aβ(3-42), Aβ(4-42), and Aβ(5-42). 5XFAD and TBA42 mice were then crossed to generate transgenic FAD42 mice. At 6 months of age, FAD42 mice showed an aggravated behavioral phenotype compared with single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that Aβ(pE3) levels were elevated in FAD42 mice. No change in Aβ(x)(-42) or other Aβ isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of Aβ(pE-42) in FAD42 mice.     

Genetic and pharmacological evidence of intraneuronal Aβ accumulation in APP transgenic mice

Intraneuronal punctate immunostaining in Alzheimer’s disease brain and amyloid-β precursor protein (APP) transgenic mice has been suggested to represent Aβ, but this is somewhat controversial. Here we show that both biochemical Aβ levels and intraneuronal immunostaining are reduced in APP transgenic mice when γ-secretase is inhibited. Moreover, BACE-1 deficient APP transgenic mice show neither Aβ production nor intraneuronal immunostaining. Our findings suggest that the punctate immunostaining with APP antibodies is due to Aβ that has accumulated inside neurons. Similar type of intraneuronal Aβ accumulation, which precedes senile plaque formation, may link Aβ to tauopathy and neurodegeneration in Alzheimer’s disease pathogenesis.     

A synthetic amino acid substitution of Tyr10 in Aβ peptide sequence yields a dominant negative variant in amyloidogenesis

Alzheimer's disease (AD) is the most common cause of dementia in elderly people, and age is the major nongenetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly of the amyloid beta-peptide (Aβ) in the form of oligomers and fibrils. However, how aging induces Aβ aggregation is not yet fully determined. Some residues in the Aβ sequence seem to promote Aβ-induced toxicity in association with age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Aβ sequence in promoting aggregation following interaction with the plasma membrane is not yet demonstrated. As Tyr10 is implicated in the induction of oxidative stress and stabilization of Aβ aggregation, we substituted Tyr 10 with a synthetic amino acid that abolishes Aβ-induced oxidative stress and shows an accelerated interaction with GM1. This variant peptide shows impaired aggregation properties and increased affinity for GM1. It has a dominant negative effect on amyloidogenesis in vitro, in cellulo, and in isolated synaptosomes. The present study shed new light in the understanding of Aβ-membrane interactions in Aβ-induced neurotoxicity. It demonstrates the relevance of Aβ sequence in (i) Aβ-membrane interaction, underlining the role of age-dependent enhanced GM1 content in promoting Aβ aggregation, (ii) Aβ aggregation, and (iii) Aβ-induced oxidative stress. Our results open the way for the design of peptides aimed to inhibit Aβ aggregation and neurotoxicity.     

Effect of tranexamic acid and δ-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or δ-aminovaleric acid (δ-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or δ-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1.5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1.4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.     

Astrocytic gap junctional communication is reduced in amyloid-β-treated cultured astrocytes,but not in Alzheimer's disease transgenic mice

Alzheimer''s disease is characterized by accumulation of amyloid deposits in brain, progressive cognitive deficits and reduced glucose utilization. Many consequences of the disease are attributed to neuronal dysfunction, but roles of astrocytes in its pathogenesis are not well understood. Astrocytes are extensively coupled via gap junctions, and abnormal trafficking of metabolites and signalling molecules within astrocytic syncytia could alter functional interactions among cells comprising the neurovascular unit. To evaluate the influence of amyloid-β on astrocyte gap junctional communication, cultured astrocytes were treated with monomerized amyloid-β_1–40 (1 μmol/l) for intervals ranging from 2 h to 5 days, and the areas labelled by test compounds were determined by impaling a single astrocyte with a micropipette and diffusion of material into coupled cells. Amyloid-β-treated astrocytes had rapid, sustained 50–70% reductions in the area labelled by Lucifer Yellow, anionic Alexa Fluor® dyes and energy-related compounds, 6-NBDG (a fluorescent glucose analogue), NADH and NADPH. Amyloid-β treatment also caused a transient increase in oxidative stress. In striking contrast with these results, spreading of Lucifer Yellow within astrocytic networks in brain slices from three regions of 8.5–14-month-old control and transgenic Alzheimer''s model mice was variable, labelling 10–2000 cells; there were no statistically significant differences in the number of dye-labelled cells among the groups or with age. Thus amyloid-induced dysfunction of gap junctional communication in cultured astrocytes does not reflect the maintenance of dye transfer through astrocytic syncytial networks in transgenic mice; the pathophysiology of Alzheimer''s disease is not appropriately represented by the cell culture system.     

A deuterohemin peptide protects a transgenic Caenorhabditis elegans model of Alzheimer’s disease by inhibiting Aβ1–42 aggregation

Alzheimer’s disease (AD) is a progressive neurodegenerative brain disease and is the most common cause of dementia in the elderly. The main hallmark of AD is the deposition of insoluble amyloid (Aβ) outside the neuron, leading to amyloid plaques and neurofibrillary tangles in the brain. Deuterohemin-Ala-His-Thr-Val-Glu-Lys (DhHP-6), a novel porphyrin-peptide, has both microperoxidase activity and cell permeability. In the present study, DhHP-6 efficiently inhibited the aggregation of Aβ and reduced the β-sheet percentage of Aβ from 89.1% to 78.3%. DhHP-6 has a stronger affinity (K_D = 100 ± 12 μM) for binding with Aβ at Phe⁴, Arg⁵, Val¹⁸, Glu¹¹ and Glu²². In addition, DhHP-6 (100 μM) significantly prolonged lifespan, alleviated paralysis and reduced Aβ plaque formation in the Aβ_1–42 transgenic Caenorhabditis elegans CL4176 model of AD. Our results demonstrate that DhHP-6 is a potential drug candidate that efficiently protects a transgenic C. elegans model of Alzheimer’s disease by inhibiting Aβ aggregation.     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.