Browsing by Author "Cracco, Laura"

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    Characterization of Prion Disease Associated with a Two-Octapeptide Repeat Insertion
    (MDPI, 2021-09-08) Brennecke, Nicholas; Cali, Ignazio; Mok, Tze How; Speedy, Helen; Hosszu, Laszlo L.P.; Stehmann, Christiane; Cracco, Laura; Puoti, Gianfranco; Prior, Thomas W.; Cohen, Mark L.; Collins, Steven J.; Mead, Simon; Appleby, Brian S.; Pathology and Laboratory Medicine, School of Medicine
    Genetic prion disease accounts for 10-15% of prion disease. While insertion of four or more octapeptide repeats are clearly pathogenic, smaller repeat insertions have an unclear pathogenicity. The goal of this case series was to provide an insight into the characteristics of the 2-octapeptide repeat genetic variant and to provide insight into the risk for Creutzfeldt-Jakob disease in asymptomatic carriers. 2-octapeptide repeat insertion prion disease cases were collected from the National Prion Disease Pathology Surveillance Center (US), the National Prion Clinic (UK), and the National Creutzfeldt-Jakob Disease Registry (Australia). Three largescale population genetic databases were queried for the 2-octapeptide repeat insertion allele. Eight cases of 2-octapeptide repeat insertion were identified. The cases were indistinguishable from the sporadic Creutzfeldt-Jakob cases of the same molecular subtype. Western blot characterization of the prion protein in the absence of enzymatic digestion with proteinase K revealed that 2-octapeptide repeat insertion and sporadic Creutzfeldt-Jakob disease have distinct prion protein profiles. Interrogation of large-scale population datasets suggested the variant is of very low penetrance. The 2-octapeptide repeat insertion is at most a low-risk genetic variant. Predictive genetic testing for asymptomatic blood relatives is not likely to be justified given the low risk.
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    Cryo-EM structures of prion protein filaments from Gerstmann-Sträussler-Scheinker disease
    (Springer, 2022) Hallinan, Grace I.; Ozcan, Kadir A.; Hoq, Md Rejaul; Cracco, Laura; Vago, Frank S.; Bharath, Sakshibeedu R.; Li, Daoyi; Jacobsen, Max; Doud, Emma H.; Mosley, Amber L.; Fernandez, Anllely; Garringer, Holly J.; Jiang, Wen; Ghetti, Bernardino; Vidal, Ruben; Pathology and Laboratory Medicine, School of Medicine
    Prion protein (PrP) aggregation and formation of PrP amyloid (APrP) are central events in the pathogenesis of prion diseases. In the dominantly inherited prion protein amyloidosis known as Gerstmann-Sträussler-Scheinker (GSS) disease, plaques made of PrP amyloid are present throughout the brain. The c.593t > c mutation in the prion protein gene (PRNP) results in a phenylalanine to serine amino acid substitution at PrP residue 198 (F198S) and causes the most severe amyloidosis among GSS variants. It has been shown that neurodegeneration in this disease is associated with the presence of extracellular APrP plaques and neuronal intracytoplasmic Tau inclusions, that have been shown to contain paired helical filaments identical to those found in Alzheimer disease. Using cryogenic electron microscopy (cryo-EM), we determined for the first time the structures of filaments of human APrP, isolated post-mortem from the brain of two symptomatic PRNP F198S mutation carriers. We report that in GSS (F198S) APrP filaments are composed of dimeric, trimeric and tetrameric left-handed protofilaments with their protomers sharing a common protein fold. The protomers in the cross-β spines consist of 62 amino acids and span from glycine 80 to phenylalanine 141, adopting a previously unseen spiral fold with a thicker outer layer and a thinner inner layer. Each protomer comprises nine short β-strands, with the β1 and β8 strands, as well as the β4 and β9 strands, forming a steric zipper. The data obtained by cryo-EM provide insights into the structural complexity of the PrP filament in a dominantly inherited human PrP amyloidosis. The novel findings highlight the urgency of extending our knowledge of the filaments' structures that may underlie distinct clinical and pathologic phenotypes of human neurodegenerative diseases.
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    Distinguishing post-translational modifications in dominantly inherited frontotemporal dementias: FTLD-TDP Type A (GRN) vs Type B (C9orf72)
    (Wiley, 2022) Cracco, Laura; Doud, Emma H.; Hallinan, Grace I.; Garringer, Holly J.; Jacobsen, Max H.; Richardson, Rose M.; Buratti, Emanuele; Vidal, Ruben; Ghetti, Bernardino; Newell, Kathy L.; Pathology and Laboratory Medicine, School of Medicine
    Aims: Frontotemporal dementias are neuropathologically characterised by frontotemporal lobar degeneration (FTLD). Intraneuronal inclusions of transactive response DNA-binding protein 43 kDa (TDP-43) are the defining pathological hallmark of approximately half of the FTLD cases, being referred to as FTLD-TDP. The classification of FTLD-TDP into five subtypes (Type A to Type E) is based on pathologic phenotypes; however, the molecular determinants underpinning the phenotypic heterogeneity of FTLD-TDP are not well known. It is currently undetermined whether TDP-43 post-translational modifications (PTMs) may be related to the phenotypic diversity of the FTLDs. Thus, the investigation of FTLD-TDP Type A and Type B, associated with GRN and C9orf72 mutations, becomes essential. Methods: Immunohistochemistry was used to identify and map the intraneuronal inclusions. Sarkosyl-insoluble TDP-43 was extracted from brains of GRN and C9orf72 mutation carriers post-mortem and studied by Western blot analysis, immuno-electron microscopy and mass spectrometry. Results: Filaments of TDP-43 were present in all FTLD-TDP preparations. PTM profiling identified multiple phosphorylated, N-terminal acetylated or otherwise modified residues, several of which have been identified for the first time as related to sarkosyl-insoluble TDP-43. Several PTMs were specific for either Type A or Type B, while others were identified in both types. Conclusions: The current results provide evidence that the intraneuronal inclusions in the two genetic diseases contain TDP-43 filaments. The discovery of novel, potentially type-specific TDP-43 PTMs emphasises the need to determine the mechanisms leading to filament formation and PTMs, and the necessity of exploring the validity and occupancy of PTMs in a prognostic/diagnostic setting.
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    Efficient transmission of human prion diseases to a glycan-free prion protein-expressing host
    (Oxford University Press, 2024) Cracco, Laura; Cali, Ignazio; Cohen, Mark L.; Aslam, Rabail; Notari, Silvio; Kong, Qingzhong; Newell, Kathy L.; Ghetti, Bernardino; Appleby, Brian S.; Gambetti, Pierluigi; Pathology and Laboratory Medicine, School of Medicine
    It is increasingly evident that the association of glycans with the prion protein (PrP), a major post-translational modification, significantly impacts the pathogenesis of prion diseases. A recent bioassay study has provided evidence that the presence of PrP glycans decreases spongiform degeneration and disease-related PrP (PrPD) deposition in a murine model. We challenged (PRNPN181Q/197Q) transgenic (Tg) mice expressing glycan-free human PrP (TgGlyc-), with isolates from sporadic Creutzfeldt-Jakob disease subtype MM2 (sCJDMM2), sporadic fatal insomnia and familial fatal insomnia, three human prion diseases that are distinct but share histotypic and PrPD features. TgGlyc- mice accurately replicated the basic histotypic features associated with the three diseases but the transmission was characterized by high attack rates, shortened incubation periods and a greatly increased severity of the histopathology, including the presence of up to 40 times higher quantities of PrPD that formed prominent deposits. Although the engineered protease-resistant PrPD shared at least some features of the secondary structure and the presence of the anchorless PrPD variant with the wild-type PrPD, it exhibited different density gradient profiles of the PrPD aggregates and a higher stability index. The severity of the histopathological features including PrP deposition appeared to be related to the incubation period duration. These findings are clearly consistent with the protective role of the PrP glycans but also emphasize the complexity of the conformational changes that impact PrPD following glycan knockout. Future studies will determine whether these features apply broadly to other human prion diseases or are PrPD-type dependent.
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    Gerstmann-Sträussler-Scheinker disease revisited: accumulation of covalently-linked multimers of internal prion protein fragments
    (Biomed Central, 2019-05-29) Cracco, Laura; Xiao, Xiangzhu; Nemani, Satish K.; Lavrich, Jody; Cali, Ignazio; Ghetti, Bernardino; Notari, Silvio; Surewicz, Witold K.; Gambetti, Pierluigi; Pathology and Laboratory Medicine, School of Medicine
    Despite their phenotypic heterogeneity, most human prion diseases belong to two broadly defined groups: Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). While the structural characteristics of the disease-related proteinase K-resistant prion protein (resPrPD) associated with the CJD group are fairly well established, many features of GSS-associated resPrPD are unclear. Electrophoretic profiles of resPrPD associated with GSS variants typically show 6-8 kDa bands corresponding to the internal PrP fragments as well as a variable number of higher molecular weight bands, the molecular nature of which has not been investigated. Here we have performed systematic studies of purified resPrPD species extracted from GSS cases with the A117V (GSSA117V) and F198S (GSSF198S) PrP gene mutations. The combined analysis based on epitope mapping, deglycosylation treatment and direct amino acid sequencing by mass spectrometry provided a conclusive evidence that high molecular weight resPrPD species seen in electrophoretic profiles represent covalently-linked multimers of the internal ~ 7 and ~ 8 kDa fragments. This finding reveals a mechanism of resPrPD aggregate formation that has not been previously established in prion diseases.
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    Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP type C
    (Springer Nature, 2024) Arseni, Diana; Nonaka, Takashi; Jacobsen, Max H.; Murzin, Alexey G.; Cracco, Laura; Peak-Chew, Sew Y.; Garringer, Holly J.; Kawakami, Ito; Suzuki, Hisaomi; Onaya, Misumoto; Saito, Yuko; Murayama, Shigeo; Geula, Changiz; Vidal, Ruben; Newell, Kathy L.; Mesulam, Marsel; Ghetti, Bernardino; Hasegawa, Masato; Ryskeldi-Falcon, Benjamin; Pathology and Laboratory Medicine, School of Medicine
    Neurodegenerative diseases are characterized by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies have established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in cryo-electron microscopy have enabled the structures of the protein filaments to be determined from the brains of patients1. All neurodegenerative diseases studied to date have been characterized by the self-assembly of proteins in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) types A and B3,4. Here we used cryo-electron microscopy to determine filament structures from the brains of individuals with FTLD-TDP type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/G284-N345 and ANXA11 residues L39-Y74 from their respective low-complexity domains. Regions of TDP-43 and ANXA11 that were previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as an approximately 22 kDa N-terminal fragment lacking the annexin core domain. Immunohistochemistry of brain sections showed the colocalization of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP type C. This work establishes a central role for ANXA11 in FTLD-TDP type C. The unprecedented formation of heteromeric amyloid filaments in the human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.
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    Novel histotypes of sporadic Creutzfeldt–Jakob disease linked to 129MV genotype
    (BMC, 2023-08-31) Cracco, Laura; Puoti, Gianfranco; Cornacchia, Antonio; Glisic, Katie; Lee, Seong‑Ki; Wang, Zerui; Cohen, Mark L.; Appleby, Brian S.; Cali, Ignazio; Pathology and Laboratory Medicine, School of Medicine
    The MV1 and MV2 subtypes of sporadic Creutzfeldt-Jakob disease (sCJD) are linked to the heterozygous methionine (M)/valine (V) polymorphism at codon 129 of the prion protein (PrP) gene. MV2 is phenotypically heterogeneous, whereas MV1, due to its low prevalence, is one of the least well characterized subtypes. In this study, we investigated the biochemical properties of PrPSc and phenotypic expression of cases diagnosed as sCJD MV1 and MV2. We describe four MV2 histotypes: 2C, with cortical (C) coarse pathology; 2K, with kuru (K) plaque deposits; 2C-K, with co-existing C and K histotypic features; and the novel histotype 2C-PL that mimics 2C in the cerebral cortex and cerebellum, but exhibits plaque-like (PL) PrP deposits in subcortical regions (e.g., basal nuclei, thalamus and midbrain). Histotype prevalence is highest for 2C-K (55%), intermediate for 2C (31%), and lowest for 2C-PL and 2K (7%). Nearly every MV2 case expressed both PrPSc types, with T2 being the predominant type ("MV2-1"). MV1 cases typically show a rapid disease course (≤ 4 months), and feature the 1C histotype, phenotypically identical to sCJDMM1. Co-existing PrPSc types, with T1 significantly exceeding T2 ("MV1-2"), are detected in patients diagnosed as MV1 with longer disease courses. We observed four histotypes among MV1-2 cases, including two novel histotypes: 1V, reminiscent of sCJDVV1; 1C-2C, resembling sCJDMM1-2 with predominant MM1 histotypic component; and novel histotypes 1C-2PL and 1C-2K, overall mimicking 1C in the cerebral cortex, but harboring T2 and plaque-like PrP deposits in subcortical regions (1C-2PL), and T2 and kuru plaques in the cerebellum (1C-2K). Lesion profiles of 1C, 1V, and 1C-2C are similar, but differ from 1C-2PL and 1C-2K, as the latter two groups show prominent hippocampal and nigral degeneration. We believe that the novel "C-PL" histotypes are distinct entities rather than intermediate forms between "C" and "C-K" groups, and that 1C-2PL and 1C-2K histotypes may be characterized by different T1 variants of the same size.