Glial cells maintain the brain's lipid and energy balance, and their breakdown is increasingly recognized as a causal contributor to Alzheimer's disease (AD). While this concept is established, no approach has directly shown how glial homeostatic failure manifests across brain regions and microenvironments or how it links local pathology, such as plaques, to global metabolic imbalance. To address this gap, we developed iMIST, an integrated platform that combines MALDI-based metabolite imaging, histology, and spatial transcriptomics within a single tissue section to align molecular and anatomical information. Using a mouse model of late-onset AD that recapitulates both amyloid deposition and metabolic vulnerability, iMIST revealed that glial lipid dysregulation is widespread but spatially specialized. In gray matter, plaque-associated microglia were associated with upregulated glycerophospholipid-remodeling in cortico-thalamic areas indicating metabolic stress around local pathology. In contrast, white matter tracts rich in lipid-producing oligodendrocytes show plaque-independent deficits in galactosylceramide metabolism reflecting their high myelin demand. Both processes intensify with age, transforming adaptive glial responses into persistent metabolic dysfunction. Together, these findings demonstrate the spatial interplay between global glial metabolic imbalance and local microenvironmental stressors associated with AD pathology. By integrating transcriptomic and metabolomic information in situ, iMIST provides a framework for uncovering how regional glial vulnerability shapes the pathogenesis of neurodegenerative diseases.

Multiple system atrophy (MSA) and the Lewy body diseases (LBDs) are caused by α-synuclein misfolding into distinct conformations, or strains, with unique biological properties. MSA patient samples readily transmit disease following intracranial (i.c.) inoculation into humanized mice, whereas LBD samples typically do not. Unexpectedly, we identified one LBD patient sample that, following i.c. inoculation, transmitted neurological disease to four out of six mice over an extended incubation period. In light of these unexpected results, we sought to identify the α-synuclein strain responsible for disease onset. Using immunohistochemistry, we identified both Lewy bodies and oligodendroglial inclusions with a glial cytoplasmic inclusion-like appearance in the substantia nigra of the patient sample. To determine if these glial inclusions were due to the presence of low titer MSA α-synuclein in the sample, we performed a secondary passage of two terminal mouse brains from the primary passage and found that the humanized mice developed disease with a shortened incubation period. Unexpectedly, using our panel of mutant α-syn140–YFP cells to analyze the primary and secondary passage samples showed that the strain isolated in the in vivo studies has unique biological properties compared to the MSA and LBD strains. These data suggest that the oligodendroglial pathology in the LBD patient sample was not caused by MSA co-pathology, and provide evidence for the isolation of a novel distinct α-synuclein strain.

Aggregation of microtubule-associated protein tau into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes) such as thioflavin T have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here we leverage advances in protein-adaptive differential scanning fluorimetry to screen the Aurora collection of 300+ fluoroprobes against multiple synthetic fibril polymorphs, including those formed from tau, α-synuclein and islet amyloid polypeptide. This screen—coupled with excitation-multiplexed bright-emission recording (EMBER) imaging and orthogonal secondary assays—revealed pan-fibril-binding chemotypes, as well as fluoroprobes selective for fibril subsets. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer’s disease and rodent models. We propose that these scaffolds represent entry points for developing fibril-selective ligands. Amyloid fibrils can adopt a range of distinct conformations, yet it is challenging to rapidly discriminate between these polymorphs. Now methods have been developed to screen large, diverse libraries of turn-on fluorescent dyes to rapidly identify probes that recognize fibril subsets.

Prions are proteins that adopt alternative conformations that become self-propagating; the PrP(Sc) prion causes the rare human disorder Creutzfeldt-Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T-YFP) and TgM83(+/-) mice expressing α-synuclein (A53T). The TgM83(+/-) mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83(+/+) mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83(+/-) mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T-YFP in cultured cells, whereas none of six Parkinson's disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83(+/+) mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimer's disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to tau's accumulation or what mechanisms might be involved. To explore these questions, we focus on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to weaken its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 reveals the mechanism of this clash, allowing design of a mutation (CHIPD134A) that partially restores binding and turnover of pS416 tauC3. We confirm that, in our models, pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a molecular mechanism for how phosphorylation at a discrete site contributes to accumulation of a tau proteoform. Hyper-phosphorylated tau accumulates in Alzheimer's disease; but the roles of these phosphorylation sites are unclear. Here the authors show that phosphorylation of a single site, pSer416, limits its turnover and promotes accumulation.

Both wild-type and mutant tau proteins can misfold into prions and self-propagate in the central nervous system of animals and people. To extend the work of others, we investigated the molecular basis of tau prion-mediated neurodegeneration in transgenic (Tg) rats expressing mutant human tau (P301S); this line of Tg rats is denoted Tg12099. We used the rat Prnp promoter to drive the overexpression of mutant tau (P301S) in the human 0N4R isoform. In Tg12099(+/+) rats homozygous for the transgene, ubiquitous expression of mutant human tau resulted in the progressive accumulation of phosphorylated tau inclusions, including silver-positive tangles in the frontal cortices and limbic system. Signs of central nervous system dysfunction were found in terminal Tg12099(+/+) rats exhibiting severe neurodegeneration and profound atrophy of the amygdala and piriform cortex. The greatest increases in tau prion activity were found in the corticolimbic structures. In contrast to the homozygous Tg12099(+/+) rats, we found lower levels of mutant tau in the hemizygous rats, resulting in few neuropathologic changes up to 2 years of age. Notably, these hemizygous rats could be infected by intracerebral inoculation with recombinant tau fibrils or precipitated tau prions from the brain homogenates of sick, aged homozygous Tg12099(+/+) rats. Our studies argue that the regional propagation of tau prions and neurodegeneration in the Tg12099 rats resembles that found in human primary tauopathies. These findings seem likely to advance our understanding of human tauopathies and may lead to effective therapeutics for Alzheimer's disease and other tau prion disorders.

Aggregation of microtubule-associated protein tau (MAPT/tau) into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes), such as thioflavin T (ThT), have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here, we leverage advances in protein adaptive differential scanning fluorimetry (paDSF) to screen the Aurora collection of 300+ fluorescent dyes against multiple synthetic tau fibril polymorphs. This screen, coupled with orthogonal secondary assays, revealed pan-fibril binding chemotypes, as well as fluoroprobes selective for subsets of fibrils. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer's disease patients. We propose that these scaffolds represent entry points for development of selective fibril ligands and, more broadly, that high throughput, fluorescence-based dye screening is a platform for their discovery.

Synaptic dysfunction and loss are central to neurodegenerative diseases and correlate with cognitive decline. Synaptic Vesicle Protein 2A (SV2A) is a promising PET-imaging target for assessing synaptic density in vivo, but comprehensive mapping in the human brain is needed to validate its biomarker potential. This study used quantitative immunohistochemistry and Western blotting to map SV2A and synaptophysin (SYP) densities across six cortical regions in healthy controls and patients with early-onset Alzheimer's disease (EOAD), late-onset Alzheimer's disease (LOAD), progressive supranuclear palsy (PSP), and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-GRN). We identified region in SV2A density among controls and observed disease- and region-specific reductions, with the most severe in FTLD-GRN (up to 59.5%) and EOAD. EOAD showed a 49% reduction in the middle frontal gyrus (MFG), while LOAD had over 30% declines in the inferior frontal gyrus (IFG) and hippocampus (CA1). In PSP, smaller but significant reductions were noted in the hippocampal formation, with the inferior temporal gyrus (ITG) relatively unaffected. A strong positive correlation between SV2A and SYP densities confirmed SV2A's reliability as a synaptic integrity marker. This study supports the use of SV2A PET imaging for early diagnosis and monitoring of neurodegenerative diseases, providing essential data for interpreting in vivo PET results. Further research should explore SV2A as a therapeutic target and validate these findings in larger, longitudinal studies.

ABSTRACT Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimer’s disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to tau’s accumulation or what mechanisms might be involved. To explore these questions, we focused on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to block its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 revealed the mechanism of this clash and allowed design of a mutation (CHIP D134A ) that partially restores binding and turnover of pS416 tauC3. We find that pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a discrete molecular mechanism for how phosphorylation at a specific site contributes to accumulation of an important tau proteoform.

In multiple system atrophy (MSA), the protein α-synuclein misfolds into a prion conformation that self-templates and causes progressive neurodegeneration. While many point mutations in the α-synuclein gene, SNCA, have been identified as the cause of heritable Parkinson’s disease (PD), none have been identified as causing MSA. To examine whether MSA prions can transmit disease to mice expressing wild-type (WT) human α-synuclein, we inoculated transgenic (Tg) mice denoted TgM20+/− with brain homogenates prepared from six different deceased MSA patients. All six samples transmitted CNS disease to the mice, with an average incubation period of ~ 280 days. Interestingly, TgM20+/− female mice developed disease > 60 days earlier than their male counterparts. Brains from terminal mice contained phosphorylated α-synuclein throughout the hindbrain, consistent with the distribution of α-synuclein inclusions in MSA patients. In addition, using our α-syn–YFP cell lines, we detected α-synuclein prions in brain homogenates prepared from terminal mice that retained MSA strain properties. To our knowledge, the studies described here are the first to show that MSA prions transmit neurological disease to mice expressing WT SNCA and that the rate of transmission is sex dependent. By comparison, TgM20+/− mice inoculated with WT preformed fibrils (PFFs) developed severe neurological disease in ~ 210 days and exhibited robust α-synuclein neuropathology in both limbic regions and the hindbrain. Brain homogenates from these animals exhibited biological activities that are distinct from those found in MSA-inoculated mice when tested in the α-syn–YFP cell lines. Differences between brains from MSA-inoculated and WT PFF-inoculated mice potentially argue that α-synuclein prions from MSA patients are distinct from the PFF inocula and that PFFs do not replicate MSA strain biology.

In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson’s disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47 +/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47 +/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF–inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20 +/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47 +/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.

Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein α-synuclein. With no treatment currently available, we sought to characterize the spread of α-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83+/- mice, which express human α-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active α-synuclein prions using a cell-based assay and examined for α-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable α-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of α-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of α-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that α-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions.

Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83 +/− , which uses the prion protein promoter to express human α-synuclein harboring the A53T mutation found in familial Parkinson’s disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human α-synuclein on a mouse α-synuclein knockout background [Tg( SNCA +/+ )Nbm, Tg( SNCA *A30P +/+ )Nbm, and Tg( SNCA *A53T +/+ )Nbm]. In contrast to studies using TgM83 +/− mice, motor deficits were not observed by 330–400 days in any of the Tg( SNCA )Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure α-synuclein prions, we found brain homogenates from Tg( SNCA *A53T +/+ )Nbm mice inoculated with MSA patient samples contained α-synuclein prions, whereas control mice did not. Moreover, these α-synuclein aggregates retained the biological and biochemical characteristics of the α-synuclein prions in MSA patient samples. Intriguingly, Tg( SNCA *A53T +/+ )Nbm mice developed α-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83 +/− mice, which develop exclusively neuronal α-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83 +/− mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg( SNCA *A53T +/+ )Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg( SNCA *A53T +/+ )Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83 +/− animals. The TgM83 +/− mice inoculated with mouse-passaged MSA developed motor dysfunction and α-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced α-synuclein prion formation in Tg( SNCA *A53T +/+ )Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of α-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.

Significance An expanding body of evidence argues that the Aβ and tau proteins share important characteristics of prion propagation to cause pathogenesis in Alzheimer’s disease (AD). Aβ and tau form a number of amyloids (β-sheet–rich structures) with distinct conformations (“strains”), some of which give rise to different diseases and associated pathologies. We develop new probes of amyloid structure and use these to identify conformational strains of Aβ in heritable and sporadic forms of AD patient samples. We demonstrate that distinct strains of Aβ can be discerned in different disease types, or in different brain compartments within a given patient. Our findings may potentially explain the spectrum of clinical and pathologic features observed in AD.

Significance In Parkinson’s disease (PD) and multiple system atrophy (MSA), the accumulation and spread of α-synuclein prions leads to the progressive degeneration seen in patients. These diseases are thought to arise from unique conformations of α-synuclein prions—or strains. To investigate this hypothesis, we infected cell lines expressing PD-causing point mutations in α-synuclein with MSA patient samples. Unexpectedly, the E46K mutation inhibited MSA prion replication. This observation indicates that the α-synuclein prion conformation found in PD is different from the strain found in MSA, providing biological evidence that these diseases arise from distinct prion strains. The ability of α-synuclein recombinant fibrils to infect all cell lines tested argues that synthetic α-synuclein prions are not predictive of the disease-causing strains in human patients. In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson’s disease (PD), we developed nine α-synuclein−YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein–YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.

IMPORTANCE Accumulation of the protein tau is a defining characteristic of several neurodegenerative diseases. Thorough assessment of transgenic (Tg) mouse lines that replicate this process is critical for establishing the models used for testing anti-tau therapeutics in vivo.& para;& para;OBJECTIVE To define a consistent mouse model of disease for use in future compound efficacy studies.& para;& para;DESIGN, SETTING, AND PARTICIPANTS In this time course study, cohorts of Tg and control mice were euthanized at defined intervals. Collected brains were bisected down the midline. One half was frozen and used to measure the tau prion content, while the other half w as fixed for immunostaining with anti-tau antibodies. All mice were maintained at the Hunters Point Animal Facility at the University of California, San Francisco, and all experiments were performed at the Mission Bay Campus of the University of California, San Francisco. Study animals were PS19, homozygous and hemizygous Tg(MAPT*P3O1S), and B6/J mice. The study dates were August 9, 2010, to October3 , 2016 .& para;& para;MAIN OUTCOMES AND MEASURES Tau prions were measured using a cell-based assay. Neuropathology was measured by determining the percentage area positive for immunostaining in defined brain regions. A separate cohort of mice was aged until each mouse developed neurological signs as determined by trained animal technicians to assess mortality.& para;& para;RESULTS A total of 1035 mice were used in this time course study. These included PS19 mice (51.2% [126 of 246] male and 48.8% [120 of 246] female), Tg(MAPT*P3O1S(+/+)) mice (52.3% [216 of 413] male, 43.8% [181 of 413] female, and 3.9% [16 of 413] undetermined), Tg(MAPT*P3O1S(+/+)) mice (51.8% [101 of 195] male and 48.2% [94 of 195] female), and B6/J mice (49.7% [90 of 181] male and 50.3% [91 of 181] female). While considerable interanimal variability in neuropathology, disease onset, and tau prion formation in the PS19 mice was observed, all 3 measures of disease were more uniform in the Tg(MAPT*P3O1S(+/+)) mice. Comparing tau prion formation in Tg(MAPT*P3O1S(+/+)) mice with B6/J controls, the 95% CIs for the 2 mouse lines diverged before age 5 weeks, and significant (P < .05) neuropathology in the hindbrain of 24-week-old mice was quantifiable.& para;& para;CONCLUSIONS AND RELEVANCE The assessment of disease progression using 3 criteria showed that disease onset in PS19 mice is too variable to obtain reliable measurements for drug discovery research. However, the reproducibility of tau prion formation in young Tg(MAPT*P3O1S(+/+)) mice establishes a rapid assay for compound efficacy in vivo.

Misfolding of tau proteins into prions and their propagation along neural circuits are thought to result in neurodegeneration causing Alzheimer's disease, progressive supranuclear palsy, chronic traumatic encephalopathy, and other tauopathies. Little is known about the molecular processes mediating tau prion replication and spreading in different brain regions. Using transgenic (Tg) mice with a neuronal promoter driving expression of human mutant (P301S) tau, we found that tau prion formation and histopathologic deposition is largely restricted to the hind-brain. Unexpectedly, tau mRNA and protein levels did not differ between the forebrain and hindbrain, suggesting that other factors modulating the conversion of tau into a prion exist and are region specific. Using a cell-based prion propagation assay, we discovered that tau prion replication is suppressed by forebrain-derived inhibitors, one of which is sortilin, a lysosomal sorting receptor. We also show that sortilin expression is higher in the forebrain than the hindbrain across the life span of the Tg mice, suggesting that sortilin, at least in part, inhibits forebrain tau prion replication in vivo. Our findings provide evidence for selective vulnerability in mice resulting in highly regulated levels of tau prion propagation, thus affording a model for identification of additional molecules that could mitigate the levels of tau prions in human tauopathies.

The larger brain of the rat enables a much greater repertoire of complex behaviors than mice, likely making rats preferential for investigating neurodegeneration. Because molecular tools for specific expression of transgenes in the rat brain are sparse, we chose Prnp encoding the prion protein (PrP) to develop a novel vector to drive transgene expression in the rat brain. We compared the rat Prnp sequence with mouse and Syrian hamster Prnp sequences, identifying conserved genetic elements and hypothesizing that these elements would be able to drive neuronal transgene expression. We investigated this by generating a vector termed RaPrnp that encompasses portions of the rat Prnp gene. Importantly, we replaced the rat Prnp open reading frame (ORF) with a cloning site for rapid and seamless In-Fusion cloning. To validate the in vivo neuronal specificity of the RaPrnp vector in rats, we generated stable RaPrnp-LacZ/enhanced green fluorescent protein (EGFP) transgenic (Tg) rat lines, which led to robust LacZ activity and high EGFP fluorescence in the central nervous system of embryos and adult animals. Next, we restored the rat Prnp ORF and generated multiple Tg(RaPrnp-PrP) lines, demonstrating that overexpression of Prnp accelerates the onset of scrapie. While the incubation time in wild-type (WT) rats was 175 ± 3 days post inoculation (dpi), one line, Tg2919, overexpressed RaPrPC at 4.4-fold and exhibited a reduced incubation time of 149 ± 2 dpi. The second line, Tg2922, overexpressed RaPrPC at 9.7-fold compared with WT animals and had an incubation time of 112 ± 0 dpi. Tg2922 rats inoculated with rat RML showed extensive vacuolation of the brainstem in contrast to WT and Tg2919 animals in which vacuolation was most prominent in the hippocampus and striatum as well as the motor and sensory cortices. It is possible that construction of Tg rats with modified phenotypes will prove more advantageous than mice for neurodegeneration studies.