Comprehensive mapping of synaptic vesicle protein 2A (SV2A) in health and neurodegenerative diseases: a comparative analysis with synaptophysin and ground truth for PET-imaging interpretation.

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).

Methods for high throughput discovery of fluoroprobes that recognize tau fibril polymorphs.

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.

Phosphorylation of tau at a single residue inhibits binding to the E3 ubiquitin ligase, CHIP.

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.

Severe neurodegeneration in brains of transgenic rats producing human tau prions.

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.

Phosphorylation of a Cleaved Tau Proteoform at a Single Residue Inhibits Binding to the E3 Ubiquitin Ligase, CHIP.

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.

The E46K mutation modulates α-synuclein prion replication in transgenic mice.

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.

Multiple system atrophy prions transmit neurological disease to mice expressing wild-type human α-synuclein.

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.

Kinetics of α-synuclein prions preceding neuropathological inclusions in multiple system atrophy.

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.

Multiple system atrophy prions retain strain specificity after serial propagation in two different Tg(SNCA*A53T) mouse lines.

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.

Structural heterogeneity and intersubject variability of Aβ in familial and sporadic Alzheimer's disease.

Point mutations in the amyloid-β (Aβ) coding region produce a combination of mutant and WT Aβ isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aβ determines WT Aβ conformation through prion template-directed misfolding.

Familial Parkinson's point mutation abolishes multiple system atrophy prion replication.

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.

Evidence for sortilin modulating regional accumulation of human tau prions in transgenic mice.

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 hindbrain.

A novel vector for transgenesis in the rat CNS.

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.

Kinetics of Human Mutant Tau Prion Formation in the Brains of 2 Transgenic Mouse Lines.

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.

Objective: To define a consistent mouse model of disease for use in future compound efficacy studies.

MSA prions exhibit remarkable stability and resistance to inactivation.

In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrP) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrP transmission.

Oligodendroglial deletion of ESCRT-I component TSG101 causes spongiform encephalopathy.

Background Information: Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery.

Guinea Pig Prion Protein Supports Rapid Propagation of Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease Prions.

Unlabelled: The biochemical and neuropathological properties of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) prions are faithfully maintained upon transmission to guinea pigs. However, primary and secondary transmissions of BSE and vCJD in guinea pigs result in long incubation periods of ∼450 and ∼350 days, respectively. To determine if the incubation periods of BSE and vCJD prions could be shortened, we generated transgenic (Tg) mice expressing guinea pig prion protein (GPPrP).

Towards authentic transgenic mouse models of heritable PrP prion diseases.

Attempts to model inherited human prion disorders such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) disease, and fatal familial insomnia (FFI) using genetically modified mice have produced disappointing results. We recently demonstrated that transgenic (Tg) mice expressing wild-type bank vole prion protein (BVPrP) containing isoleucine at polymorphic codon 109 develop a spontaneous neurodegenerative disorder that exhibits many of the hallmarks of prion disease. To determine if mutations causing inherited human prion disease alter this phenotype, we generated Tg mice expressing BVPrP containing the D178N mutation, which causes FFI; the E200K mutation, which causes familial CJD; or an anchorless PrP mutation similar to mutations that cause GSS.

Optimization of Aryl Amides that Extend Survival in Prion-Infected Mice.

Developing therapeutics for neurodegenerative diseases (NDs) prevalent in the aging population remains a daunting challenge. With the growing understanding that many NDs progress by conformational self-templating of specific proteins, the prototypical prion diseases offer a platform for ND drug discovery. We evaluated high-throughput screening hits with the aryl amide scaffold and explored the structure-activity relationships around three series differing in their N-aryl core: benzoxazole, benzothiazole, and cyano.

Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases.

Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases.

Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism.

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.

Propagation of prions causing synucleinopathies in cultured cells.

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells.

Prion Disease Induces Alzheimer Disease-Like Neuropathologic Changes.

We examined the brains of 266 patients with prion disease (PrionD) and found that 46 patients (17%) had Alzheimer disease (AD)-like changes. To explore potential mechanistic links between PrionD and AD, we exposed human brain aggregates (BrnAggs) to a brain homogenate from a patient with sporadic Creutzfeldt-Jakob disease and found that neurons in human BrnAggs produced many β-amyloid (Aβ; Aβ42) inclusions, whereas uninfected control-exposed human BrnAggs did not. Western blot analysis of 20 pooled Creutzfeldt-Jakob disease-infected BrnAggs verified Aβ42 levels higher than those in controls.

Different 2-Aminothiazole Therapeutics Produce Distinct Patterns of Scrapie Prion Neuropathology in Mouse Brains.

Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days.

Use of a 2-aminothiazole to Treat Chronic Wasting Disease in Transgenic Mice.

Treatment with the 2-aminothiazole IND24 extended the survival of mice infected with mouse-adapted scrapie but also resulted in the emergence of a drug-resistant prion strain. Here, we determined whether IND24 extended the survival of transgenic mice infected with prions that caused scrapie in sheep or prions that caused chronic wasting disease (CWD; hereafter "CWD prions") in deer, using 2 isolates for each disease. IND24 doubled the incubation times for mice infected with CWD prions but had no effect on the survival of those infected with scrapie prions.