Tau aggregates are present in a large number of neurodegenerative diseases known as “tauopathies”... more Tau aggregates are present in a large number of neurodegenerative diseases known as “tauopathies”, including Alzheimer’s disease (AD). As there are six human tau isoforms in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions, we tested whether tau isoforms behave differently in aggregation. We discovered that all six tau isoforms are capable of forming PHF-tau like filaments and the 3R tau isoforms aggregate significantly faster than their 4R counterparts. We further mapped key segments of tau isoforms that contribute to their aggregation kinetics, where it was determined that microtubule binding domains R2 and R3 were the major contributors to tau aggregation. To evaluate the feasibility of using the six recombinant tau isoforms as substrates to amplify misfolded tau, we demonstrated that full-length human tau isoforms can seed and detect misfolded tau from the post-mortem AD brain tissues with high specificity by an ultrasensitive technology te...
The neuro-physiological properties of individuals with genetic pre-disposition to neurological di... more The neuro-physiological properties of individuals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of individuals with confirmed genetic mutations including PRNPE200K, trisomy 21 (T21), and LRRK2G2019S, which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson’s disease. We utilized no known disease/healthy COs (HC) as normal function controls. At 3–4 and 6–10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6–10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with the HC, including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the am...
ObjectiveTo determine whether (1) immunofluorescence is a reproducible technique in detecting mis... more ObjectiveTo determine whether (1) immunofluorescence is a reproducible technique in detecting misfolded α-synuclein in skin nerves and subsequently whether (2) immunofluorescence and real-time quaking-induced conversion (RT-QuIC) (both in skin and CSF) show a comparable in vivo diagnostic accuracy in distinguishing synucleinopathies from non-synucleinopathies in a large cohort of patients.MethodsWe prospectively recruited 90 patients fulfilling clinical and instrumental diagnostic criteria for all synucleinopathies variants and non-synucleinopathies (mainly including Alzheimer disease, tauopathies, and vascular parkinsonism or dementia). Twenty-four patients with mainly peripheral neuropathies were used as controls. Patients underwent skin biopsy for immunofluorescence and RT-QuIC; CSF was examined in patients who underwent lumbar puncture for diagnostic purposes. Immunofluorescence and RT-QuIC analysis were made blinded to the clinical diagnosis.ResultsImmunofluorescence showed rep...
Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human... more Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human cerebral cortex. COs are a promising new system for modelling pathological features of neurological disorders, including prion diseases. COs expressing normal prion protein (PrPC) are susceptible to prion infection when exposed to the disease isoforms of PrP (PrPD). This causes the COs to develop aspects of prion disease pathology considered hallmarks of disease, including the production of detergent-insoluble, protease-resistant misfolded PrPD species capable of seeding the production of more misfolded species. To determine whether COs can model aspects of familial prion diseases, we produced COs from donor fibroblasts carrying the E200K mutation, the most common cause of human familial prion disease. The mature E200K COs were assessed for the hallmarks of prion disease. We found that up to 12 months post-differentiation, E200K COs harbored no PrPD as confirmed by the absence of deterg...
Cellular prion protein (PrPC) is a GPI-anchored cell surface glycoprotein that is expressed in th... more Cellular prion protein (PrPC) is a GPI-anchored cell surface glycoprotein that is expressed in the brain, blood, bone marrow (BM), and lymphoid tissue. PrPC can be converted post-translationally into scrapie-PrP (PrPSc), which is involved in the pathogenesis of neurodegenerative diseases including Creutzfeldt-Jakob disease, Kuru disease in humans, and scrapie and bovine spongiform encephalopathy in animals. However, the biological function of PrPSc has yet to be conclusively elucidated. In order to understand the role of PrPC in the hematopoietic system, we compared bone marrow, lymphoid organs and peripheral blood of PrPC knockout mice (KO) to age and sex-matched transgenic mice used as background controls (WT) expressing human PrPC under the control of a mouse PrPC promoter with a slightly augmented expression (2-fold) of PrPC. Complete blood count (CBC) showed a significant increase of WBC in KO mice (KO 9.03 ± 5.16 x109/L vs. WT 4.13 ± 1.87 x109/L, p = 0.0405; Table 1 and Figure...
Diabetic retinopathy (DR), a major complication of diabetes caused by vascular damage and patholo... more Diabetic retinopathy (DR), a major complication of diabetes caused by vascular damage and pathological proliferation of retinal vessels, often progresses to vision loss. Vascular endothelial growth factor (VEGF) signaling plays a pivotal role in the development of DR, but the exact underlying molecular mechanisms remain ill-defined. Cellular prion protein (PrP) is a surface protein expressed by vascular endothelial cells, and the increased expression of PrP is associated with physiological and pathological vascularization. Nevertheless, a role for PrP in the development of DR has not been appreciated. Here, we addressed this question. We found that the development of streptozocin (STZ)-induced DR, but not the STZ-induced hyperglycemia/diabetes itself, was significantly attenuated in PrP-KO mice, compared to control wildtype (WT) mice, evident by measurement of retinal vascular leakage, retinal neovascularization, a retinopathy score and visual acuity assessment. Moreover, the attenu...
International journal of clinical and experimental medicine, 2015
Investigation of the physiological function of cellular prion protein (PrP(C)) has been developed... more Investigation of the physiological function of cellular prion protein (PrP(C)) has been developed by the generation of transgenic mice, however, the pathological mechanisms related to PrP(C) in prion diseases such as transmissible spongiform encephalopathies (TSEs) are still abstruse. Regardless of some differences, most studies describe the neuroprotective role of PrP(C) in environmental stresses. In this review, we will update the current knowledge on the responses of PrP(C) to various stresses, especially those correlated with cell signaling and neural degeneration, including ischemia, oxidative stress, inflammation and autophagy.
Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all... more Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far - a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimin...
The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded... more The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP(C) into its pathogenic isoform PrP(Sc 1). PrP(C) is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP(Sc) forms detergent-insoluble aggregates and is partially resistant to PK(2-6). The conversion of PrP(C) to PrP(Sc) is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP(Sc), intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain(7). Using a combination of biophysical and biochemical approaches, we identified insoluble PrP(C) aggregates (designated iPrP(C)) from uninfected mammalian brains and cultured neuronal cells(8, 9). Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, si...
Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and ... more Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and 91 of prion protein (PrP) gene is associated with inherited prion diseases. Most cases linked to this insertion examined by Western blotting showed detectable proteinase K-resistant PrPSc (rPrPSc) resembling PrPSc type 1 and type 2 in sporadic Creutzfeldt-Jakob disease (sCJD), or PrP7-8 in Gerstmann-Sträussler-Scheinker disease. However, cases lacking detectable rPrPSc also have been reported. Which PrP conformer is associated with neuropathological changes in the cases without detectable rPrPSc remains to be determined. Here we report that while all six but one subjects with the 144-bp insertion mutations examined display the pathognomonic PrP patches in the cerebellum, one of them exhibits no detectable typical rPrPSc even in PrPSc-enriched preparations. Instead, a large amount of abnormal PrP is captured from this case by gene 5 protein and sodium phosphotungstate, reagents that have ...
In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aβ plaq... more In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aβ plaques is proposed to be initiated by seeding the inoculated amyloid β (Aβ) peptides in the brain. Like the misfolded scrapie prion protein (PrPSc) in prion diseases, Aβ in AD shows a certain degree of resistance to protease digestion while the biochemical basis for protease resistance of Aβ remains poorly understood. Using in vitro assays, histoblotting, and electron microscopy, we characterize the biochemical and morphological features of synthetic Aβ peptides and Aβ isolated from AD brain tissues. Consistent with previous observations, monomeric and oligomeric Aβ species extracted from AD brains are insoluble in detergent buffers and resistant to digestions with proteinase K (PK). Histoblotting of AD brain tissue sections exhibits an increased Aβ immunoreactivity after digestion with PK. In contrast, synthetic Aβ40 and Aβ42 are soluble in detergent buffers and fully digested by PK. Elect...
The heterogeneity of the clinicopathological phenotype in human prion diseases is associated with... more The heterogeneity of the clinicopathological phenotype in human prion diseases is associated with the presence of the different forms of the abnormal prion protein, PrP(Sc). We have previously shown that PrP(Sc) in FFI and a subtype of familial CJD linked to the D178N mutation can be distinguished by their difference in gel mobility following proteinase K (PK) treatment. To further characterize the structural difference of PrP(Sc) in familial prion diseases, N-terminal sequencing and mass spectrometry were used to identify the protease cleavage sites in PrP(Sc) extracted from affected brains. We found that the main PK cleavage sites of PrP(Sc) are located at residue 97 in FFI, and residue 82 in both CJD178 and a GSS subtype linked to the P102L mutation. The differential accessibility to protease in the native PrP(Sc) suggests that PrP(Sc) exist as distinct conformers in different disease states.
A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its... more A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its β-sheet-rich pathological isoform (PrPSc) is the hallmark of prion diseases, a group of fatal transmissible encephalopathies that includes spontaneous and acquired forms. Recently, a PrPSc-like intermediate form characterized by the formation of insoluble aggregates and protease-resistant PrP species termed insoluble PrPC (iPrPC) has been identified in uninfected mammalian brains and cultured neuronal cells, providing new insights into the molecular mechanism(s) of these diseases. Here, we explore the molecular characteristics of the spontaneously formed iPrPC in cultured neuroblastoma cells expressing wild-type or mutant human PrP linked to two familial prion diseases. We observed that although PrP mutation at either residue 183 from Thr to Ala (PrPT183A) or at residue 198 from Phe to Ser (PrPF198S) affects glycosylation at both N-linked glycosylation sites, the T183A mutation that res...
Background: Functional connectivity in the default mode network (DMN) has been proposed as an ear... more Background: Functional connectivity in the default mode network (DMN) has been proposed as an early biomarker in amnestic lateonset Alzheimer’s disease (AD). Connectivity in the DMN and other networks has been less explored in early-onset AD patients, who present with greater clinical and anatomic heterogeneity, including focal non-amnestic presentations. Methods: The study included resting-state functional MRI scans of 64 non-familial AD patients (Table), including 23 with early-onset AD (EO-AD, age at onset <65 years, predominant amnestic/dysexecutive deficits), 25 with logopenic variant primary progressive aphasia (lvPPA, predominant language deficits) and 16 patients with posterior cortical atrophy (PCA, predominant visual deficits). 70 healthy controls were also included for comparison. Seed-based connectivity analyses were conducted using the peak intensity voxels of network templates (http://findlab.stanford.edu/functional_ROIs.html) for 3 DMN components (anterior, posterior and ventral), as well as 4 non-DMN networks clinically involved in AD variants: left and right executive-control, language and higher visual networks. Z scores were extracted from the resulting connectivity maps in each subject. Group comparisons were performed for all AD patients compared with controls as well as for individual AD variant groups, adjusting for age and gender. Results: Comparing all AD patients with controls, we found significantly reduced connectivity in the executive-control, language and higher visual networks (p<0.0001), whereas only the posterior DMN component showed reduced connectivity (p1⁄40.047, Figure). Connectivity was significantly lower in bilateral executive-control, language and higher visual networks compared to the DMN components in the AD patients (p<0.05). Looking at specific AD variants, connectivity was reduced in the bilateral executive-control, language and higher visual networks in EO-AD, lvPPA and PCA compared with controls (Figure). The posterior DMN showed a trend for reduced connectivity in EO-AD only (p1⁄40.07). No significant differences were found for the anterior and ventral DMN components. Comparing connectivity between AD variants revealed a trend towards lower connectivity in the higher visual network in PCA compared with lvPPA (p1⁄40.06). Conclusions: Functional networks outside the DMN were strongly affected in early-onset and non-amnestic AD variants, whereas the DMN was less affected, with only the posterior component showing reduced connectivity in the amnestic/dysexecutive variant. Connectivity in networks outside the DMN may represent a better biomarker in early-onset AD variants.
Dickson/Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders, 2011
ABSTRACT Protease-sensitive prionopathy is a novel disease which was first reported in 2008. It i... more ABSTRACT Protease-sensitive prionopathy is a novel disease which was first reported in 2008. It is associated with an abnormal prion protein that is mostly sensitive to proteases and cannot be diagnosed with ordinary Western blots. However, when detected with special antibodies or following enrichment, the protease-sensitive prionopathy-associated protease-resistant prion protein forms a ladder-like electrophoretic profile with prion protein fragments variously truncated at the N-terminus. Currently, there are 13 published cases of protease-sensitive prionopathy. They all are valine homozygous at codon 129 and carry no mutation on the prion protein gene coding region. Average age at onset and disease duration are 61.5 years and 24 months, respectively. Protease-sensitive prionopathy generally presents with mood changes, speech deficit or cognitive impairment. Spongiform degeneration is widespread but moderate to minimal; microplaques may be seen in the cerebellar cortex. Prion protein immunostains in a distinctive target-like granular pattern. Protease-sensitive prionopathy might be the sporadic form of Gerstmann–Sträussler–Scheinker disease.
HISTORICAL OUTLINE The first report of an inherited prion disease can be traced to an affected me... more HISTORICAL OUTLINE The first report of an inherited prion disease can be traced to an affected member of the “H” family carrying a neurologic disorder through multiple generations. It was presented at a meeting of the Viennese Neurological and Psychiatric Association in 1912 (Dimitz 1913). Two decades passed before Gerstmann in 1928 and Gerstmann, Straussler, and Scheinker in 1936 reported clinical presentations and neuropathologic findings for several affected members of the “H” family. These reports established the disease that is currently referred to as Gerstmann-Straussler-Scheinker disease or GSS (Gerstmann 1928; Gerstmann et al. 1936). The subject reported by Creutzfeldt in 1920 and 1921, who had a positive family history (Creutzfeldt 1920Creutzfeldt 1921), is unlikely to have been affected by the condition now called Creutzfeldt-Jakob disease (CJD). The first authentic familial case of CJD was recorded in 1924 by Kirschbaum (1924). However, it was Meggendorfer in 1930 who showed that the subject described by Kirschbaum was a member of a large kindred that became known as the “Backer” family, proven in subsequent publications to be affected by an inherited form of CJD (Meggendorfer 1930; Stender 1930; Jakob et al. 1950). In 1973, Gajdusek, Gibbs, and their colleagues first demonstrated the transmissibility of inherited prion diseases with a CJD-like phenotype to nonhuman primates (Roos et al. 1973). This finding followed earlier studies that reported the transmissibility of the sporadic form of CJD and kuru, a prion disease of the Fore tribe of New Guinea propagated through endocannibalism (Gajdusek et al. 1966; Gibbs...
Tau aggregates are present in a large number of neurodegenerative diseases known as “tauopathies”... more Tau aggregates are present in a large number of neurodegenerative diseases known as “tauopathies”, including Alzheimer’s disease (AD). As there are six human tau isoforms in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions, we tested whether tau isoforms behave differently in aggregation. We discovered that all six tau isoforms are capable of forming PHF-tau like filaments and the 3R tau isoforms aggregate significantly faster than their 4R counterparts. We further mapped key segments of tau isoforms that contribute to their aggregation kinetics, where it was determined that microtubule binding domains R2 and R3 were the major contributors to tau aggregation. To evaluate the feasibility of using the six recombinant tau isoforms as substrates to amplify misfolded tau, we demonstrated that full-length human tau isoforms can seed and detect misfolded tau from the post-mortem AD brain tissues with high specificity by an ultrasensitive technology te...
The neuro-physiological properties of individuals with genetic pre-disposition to neurological di... more The neuro-physiological properties of individuals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of individuals with confirmed genetic mutations including PRNPE200K, trisomy 21 (T21), and LRRK2G2019S, which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson’s disease. We utilized no known disease/healthy COs (HC) as normal function controls. At 3–4 and 6–10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6–10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with the HC, including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the am...
ObjectiveTo determine whether (1) immunofluorescence is a reproducible technique in detecting mis... more ObjectiveTo determine whether (1) immunofluorescence is a reproducible technique in detecting misfolded α-synuclein in skin nerves and subsequently whether (2) immunofluorescence and real-time quaking-induced conversion (RT-QuIC) (both in skin and CSF) show a comparable in vivo diagnostic accuracy in distinguishing synucleinopathies from non-synucleinopathies in a large cohort of patients.MethodsWe prospectively recruited 90 patients fulfilling clinical and instrumental diagnostic criteria for all synucleinopathies variants and non-synucleinopathies (mainly including Alzheimer disease, tauopathies, and vascular parkinsonism or dementia). Twenty-four patients with mainly peripheral neuropathies were used as controls. Patients underwent skin biopsy for immunofluorescence and RT-QuIC; CSF was examined in patients who underwent lumbar puncture for diagnostic purposes. Immunofluorescence and RT-QuIC analysis were made blinded to the clinical diagnosis.ResultsImmunofluorescence showed rep...
Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human... more Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human cerebral cortex. COs are a promising new system for modelling pathological features of neurological disorders, including prion diseases. COs expressing normal prion protein (PrPC) are susceptible to prion infection when exposed to the disease isoforms of PrP (PrPD). This causes the COs to develop aspects of prion disease pathology considered hallmarks of disease, including the production of detergent-insoluble, protease-resistant misfolded PrPD species capable of seeding the production of more misfolded species. To determine whether COs can model aspects of familial prion diseases, we produced COs from donor fibroblasts carrying the E200K mutation, the most common cause of human familial prion disease. The mature E200K COs were assessed for the hallmarks of prion disease. We found that up to 12 months post-differentiation, E200K COs harbored no PrPD as confirmed by the absence of deterg...
Cellular prion protein (PrPC) is a GPI-anchored cell surface glycoprotein that is expressed in th... more Cellular prion protein (PrPC) is a GPI-anchored cell surface glycoprotein that is expressed in the brain, blood, bone marrow (BM), and lymphoid tissue. PrPC can be converted post-translationally into scrapie-PrP (PrPSc), which is involved in the pathogenesis of neurodegenerative diseases including Creutzfeldt-Jakob disease, Kuru disease in humans, and scrapie and bovine spongiform encephalopathy in animals. However, the biological function of PrPSc has yet to be conclusively elucidated. In order to understand the role of PrPC in the hematopoietic system, we compared bone marrow, lymphoid organs and peripheral blood of PrPC knockout mice (KO) to age and sex-matched transgenic mice used as background controls (WT) expressing human PrPC under the control of a mouse PrPC promoter with a slightly augmented expression (2-fold) of PrPC. Complete blood count (CBC) showed a significant increase of WBC in KO mice (KO 9.03 ± 5.16 x109/L vs. WT 4.13 ± 1.87 x109/L, p = 0.0405; Table 1 and Figure...
Diabetic retinopathy (DR), a major complication of diabetes caused by vascular damage and patholo... more Diabetic retinopathy (DR), a major complication of diabetes caused by vascular damage and pathological proliferation of retinal vessels, often progresses to vision loss. Vascular endothelial growth factor (VEGF) signaling plays a pivotal role in the development of DR, but the exact underlying molecular mechanisms remain ill-defined. Cellular prion protein (PrP) is a surface protein expressed by vascular endothelial cells, and the increased expression of PrP is associated with physiological and pathological vascularization. Nevertheless, a role for PrP in the development of DR has not been appreciated. Here, we addressed this question. We found that the development of streptozocin (STZ)-induced DR, but not the STZ-induced hyperglycemia/diabetes itself, was significantly attenuated in PrP-KO mice, compared to control wildtype (WT) mice, evident by measurement of retinal vascular leakage, retinal neovascularization, a retinopathy score and visual acuity assessment. Moreover, the attenu...
International journal of clinical and experimental medicine, 2015
Investigation of the physiological function of cellular prion protein (PrP(C)) has been developed... more Investigation of the physiological function of cellular prion protein (PrP(C)) has been developed by the generation of transgenic mice, however, the pathological mechanisms related to PrP(C) in prion diseases such as transmissible spongiform encephalopathies (TSEs) are still abstruse. Regardless of some differences, most studies describe the neuroprotective role of PrP(C) in environmental stresses. In this review, we will update the current knowledge on the responses of PrP(C) to various stresses, especially those correlated with cell signaling and neural degeneration, including ischemia, oxidative stress, inflammation and autophagy.
Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all... more Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far - a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimin...
The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded... more The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP(C) into its pathogenic isoform PrP(Sc 1). PrP(C) is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP(Sc) forms detergent-insoluble aggregates and is partially resistant to PK(2-6). The conversion of PrP(C) to PrP(Sc) is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP(Sc), intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain(7). Using a combination of biophysical and biochemical approaches, we identified insoluble PrP(C) aggregates (designated iPrP(C)) from uninfected mammalian brains and cultured neuronal cells(8, 9). Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, si...
Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and ... more Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and 91 of prion protein (PrP) gene is associated with inherited prion diseases. Most cases linked to this insertion examined by Western blotting showed detectable proteinase K-resistant PrPSc (rPrPSc) resembling PrPSc type 1 and type 2 in sporadic Creutzfeldt-Jakob disease (sCJD), or PrP7-8 in Gerstmann-Sträussler-Scheinker disease. However, cases lacking detectable rPrPSc also have been reported. Which PrP conformer is associated with neuropathological changes in the cases without detectable rPrPSc remains to be determined. Here we report that while all six but one subjects with the 144-bp insertion mutations examined display the pathognomonic PrP patches in the cerebellum, one of them exhibits no detectable typical rPrPSc even in PrPSc-enriched preparations. Instead, a large amount of abnormal PrP is captured from this case by gene 5 protein and sodium phosphotungstate, reagents that have ...
In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aβ plaq... more In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aβ plaques is proposed to be initiated by seeding the inoculated amyloid β (Aβ) peptides in the brain. Like the misfolded scrapie prion protein (PrPSc) in prion diseases, Aβ in AD shows a certain degree of resistance to protease digestion while the biochemical basis for protease resistance of Aβ remains poorly understood. Using in vitro assays, histoblotting, and electron microscopy, we characterize the biochemical and morphological features of synthetic Aβ peptides and Aβ isolated from AD brain tissues. Consistent with previous observations, monomeric and oligomeric Aβ species extracted from AD brains are insoluble in detergent buffers and resistant to digestions with proteinase K (PK). Histoblotting of AD brain tissue sections exhibits an increased Aβ immunoreactivity after digestion with PK. In contrast, synthetic Aβ40 and Aβ42 are soluble in detergent buffers and fully digested by PK. Elect...
The heterogeneity of the clinicopathological phenotype in human prion diseases is associated with... more The heterogeneity of the clinicopathological phenotype in human prion diseases is associated with the presence of the different forms of the abnormal prion protein, PrP(Sc). We have previously shown that PrP(Sc) in FFI and a subtype of familial CJD linked to the D178N mutation can be distinguished by their difference in gel mobility following proteinase K (PK) treatment. To further characterize the structural difference of PrP(Sc) in familial prion diseases, N-terminal sequencing and mass spectrometry were used to identify the protease cleavage sites in PrP(Sc) extracted from affected brains. We found that the main PK cleavage sites of PrP(Sc) are located at residue 97 in FFI, and residue 82 in both CJD178 and a GSS subtype linked to the P102L mutation. The differential accessibility to protease in the native PrP(Sc) suggests that PrP(Sc) exist as distinct conformers in different disease states.
A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its... more A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its β-sheet-rich pathological isoform (PrPSc) is the hallmark of prion diseases, a group of fatal transmissible encephalopathies that includes spontaneous and acquired forms. Recently, a PrPSc-like intermediate form characterized by the formation of insoluble aggregates and protease-resistant PrP species termed insoluble PrPC (iPrPC) has been identified in uninfected mammalian brains and cultured neuronal cells, providing new insights into the molecular mechanism(s) of these diseases. Here, we explore the molecular characteristics of the spontaneously formed iPrPC in cultured neuroblastoma cells expressing wild-type or mutant human PrP linked to two familial prion diseases. We observed that although PrP mutation at either residue 183 from Thr to Ala (PrPT183A) or at residue 198 from Phe to Ser (PrPF198S) affects glycosylation at both N-linked glycosylation sites, the T183A mutation that res...
Background: Functional connectivity in the default mode network (DMN) has been proposed as an ear... more Background: Functional connectivity in the default mode network (DMN) has been proposed as an early biomarker in amnestic lateonset Alzheimer’s disease (AD). Connectivity in the DMN and other networks has been less explored in early-onset AD patients, who present with greater clinical and anatomic heterogeneity, including focal non-amnestic presentations. Methods: The study included resting-state functional MRI scans of 64 non-familial AD patients (Table), including 23 with early-onset AD (EO-AD, age at onset <65 years, predominant amnestic/dysexecutive deficits), 25 with logopenic variant primary progressive aphasia (lvPPA, predominant language deficits) and 16 patients with posterior cortical atrophy (PCA, predominant visual deficits). 70 healthy controls were also included for comparison. Seed-based connectivity analyses were conducted using the peak intensity voxels of network templates (http://findlab.stanford.edu/functional_ROIs.html) for 3 DMN components (anterior, posterior and ventral), as well as 4 non-DMN networks clinically involved in AD variants: left and right executive-control, language and higher visual networks. Z scores were extracted from the resulting connectivity maps in each subject. Group comparisons were performed for all AD patients compared with controls as well as for individual AD variant groups, adjusting for age and gender. Results: Comparing all AD patients with controls, we found significantly reduced connectivity in the executive-control, language and higher visual networks (p<0.0001), whereas only the posterior DMN component showed reduced connectivity (p1⁄40.047, Figure). Connectivity was significantly lower in bilateral executive-control, language and higher visual networks compared to the DMN components in the AD patients (p<0.05). Looking at specific AD variants, connectivity was reduced in the bilateral executive-control, language and higher visual networks in EO-AD, lvPPA and PCA compared with controls (Figure). The posterior DMN showed a trend for reduced connectivity in EO-AD only (p1⁄40.07). No significant differences were found for the anterior and ventral DMN components. Comparing connectivity between AD variants revealed a trend towards lower connectivity in the higher visual network in PCA compared with lvPPA (p1⁄40.06). Conclusions: Functional networks outside the DMN were strongly affected in early-onset and non-amnestic AD variants, whereas the DMN was less affected, with only the posterior component showing reduced connectivity in the amnestic/dysexecutive variant. Connectivity in networks outside the DMN may represent a better biomarker in early-onset AD variants.
Dickson/Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders, 2011
ABSTRACT Protease-sensitive prionopathy is a novel disease which was first reported in 2008. It i... more ABSTRACT Protease-sensitive prionopathy is a novel disease which was first reported in 2008. It is associated with an abnormal prion protein that is mostly sensitive to proteases and cannot be diagnosed with ordinary Western blots. However, when detected with special antibodies or following enrichment, the protease-sensitive prionopathy-associated protease-resistant prion protein forms a ladder-like electrophoretic profile with prion protein fragments variously truncated at the N-terminus. Currently, there are 13 published cases of protease-sensitive prionopathy. They all are valine homozygous at codon 129 and carry no mutation on the prion protein gene coding region. Average age at onset and disease duration are 61.5 years and 24 months, respectively. Protease-sensitive prionopathy generally presents with mood changes, speech deficit or cognitive impairment. Spongiform degeneration is widespread but moderate to minimal; microplaques may be seen in the cerebellar cortex. Prion protein immunostains in a distinctive target-like granular pattern. Protease-sensitive prionopathy might be the sporadic form of Gerstmann–Sträussler–Scheinker disease.
HISTORICAL OUTLINE The first report of an inherited prion disease can be traced to an affected me... more HISTORICAL OUTLINE The first report of an inherited prion disease can be traced to an affected member of the “H” family carrying a neurologic disorder through multiple generations. It was presented at a meeting of the Viennese Neurological and Psychiatric Association in 1912 (Dimitz 1913). Two decades passed before Gerstmann in 1928 and Gerstmann, Straussler, and Scheinker in 1936 reported clinical presentations and neuropathologic findings for several affected members of the “H” family. These reports established the disease that is currently referred to as Gerstmann-Straussler-Scheinker disease or GSS (Gerstmann 1928; Gerstmann et al. 1936). The subject reported by Creutzfeldt in 1920 and 1921, who had a positive family history (Creutzfeldt 1920Creutzfeldt 1921), is unlikely to have been affected by the condition now called Creutzfeldt-Jakob disease (CJD). The first authentic familial case of CJD was recorded in 1924 by Kirschbaum (1924). However, it was Meggendorfer in 1930 who showed that the subject described by Kirschbaum was a member of a large kindred that became known as the “Backer” family, proven in subsequent publications to be affected by an inherited form of CJD (Meggendorfer 1930; Stender 1930; Jakob et al. 1950). In 1973, Gajdusek, Gibbs, and their colleagues first demonstrated the transmissibility of inherited prion diseases with a CJD-like phenotype to nonhuman primates (Roos et al. 1973). This finding followed earlier studies that reported the transmissibility of the sporadic form of CJD and kuru, a prion disease of the Fore tribe of New Guinea propagated through endocannibalism (Gajdusek et al. 1966; Gibbs...
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