Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates.

Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer's disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer's disease patients.

[Cell-surface Initial Aggregation Process of Amyloid-ß Peptides Studied by Fluorescence Correlation Spectroscopy].

The formation of neurotoxic aggregates by amyloid-ß peptide (Aß) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous-phase aggregation of Aß. Membrane-mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Fluorescence correlation spectroscopy (FCS) is a sensitive method to monitor molecular aggregation processes by measuring diffusion of fluorophore-labeled molecules. Here, we monitored the initial aggregation process of Aß on cell membranes of SH-SY5Y neuroblastoma cells. For example, aggregation of Aß-(1-42) was evaluated by using Aß-(1-42) (5 µM) doped with fluorophore-Aß-(1-42) (10 nM). A membrane-bound Aß component was detected in FCS autocorrelation curve after 1-h incubation of the Aßs with the cells. Following incubation for ～10 h, Aß-(1-42) formed oligomers composed of ～10 Aß molecules. These Aß oligomers formed on membranes did not induce activation of caspase-3, an effector caspase for apoptosis, therefore were not neurotoxic, in contrast to reported Aß oligomers prepared in aqueous phase. Formation of larger Aß fibrils on membranes was found to be critical for Aß neurotoxicity. We also report that trace amounts of pyroglutaminated Aß-(3-42), a minor species of Aß, can enhance initial aggregation process of Aß-(1-42) on the cell membranes. These results indicate the usefulness of FCS detection of small Aß oligomers formed on cell surface, which can act as pathogenic seeds for amyloid fibrils responsible for neurotoxicity.

Live Cell FRET Imaging Reveals Amyloid ß-Peptide Oligomerization in Hippocampal Neurons.

Amyloid ß-peptide (Aß) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aß oligomerization in neurons still need to be revealed. Förster resonance energy transfer (FRET) is a simple but effective way to study molecular interactions. Here, we used a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detected Aß42 oligomerization in primary neurons. The neurons were incubated with fluorescently labeled Aß42 in the cell culture medium for 24 h. Aß42 were internalized and oligomerized in the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aß42 were significantly higher than for Aß40. These findings provide a better understanding of Aß42 oligomerization in neurons.

Detection of pathological alpha-synuclein aggregates in human iPSC-derived neurons and tissue.

The accumulation of proteins into insoluble aggregates is a common feature of several neurodegenerative diseases. Aggregated α-synuclein is a major component of Lewy bodies that pathologically define Parkinson's disease (PD). Here, we present methods for the detection of pathogenic conformations of α-synuclein in induced pluripotent stem cell (iPSC) patient-derived neuron models and brain tissue. These methods can be applied to studies of PD pathogenesis and the discovery of novel therapeutics that restore physiological α-synuclein. For complete details on the use and execution of this protocol, please refer to Cuddy et al. (2019) and Zunke et al. (2018).

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Three-dimensional real time imaging of amyloid ß aggregation on living cells.

Alzheimer's disease (AD) is a progressive disorder of the brain that gradually decreases thinking, memory, and language abilities. The aggregation process of amyloid ß (Aß) is a key step in the expression of its neurocytotoxicity and development of AD because Aß aggregation and accumulation around neuronal cells induces cell death. However, the molecular mechanism underlying the neurocytotoxicity and cell death by Aß aggregation has not been clearly elucidated. In this study, we successfully visualized real-time process of Aß42 aggregation around living cells by applying our established QD imaging method. 3D observations using confocal laser microscopy revealed that Aß42 preferentially started to aggregate at the region where membrane protrusions frequently formed. Furthermore, we found that inhibition of actin polymerization using cytochalasin D reduced aggregation of Aß42 on the cell surface. These results indicate that actin polymerization-dependent cell motility is responsible for the promotion of Aß42 aggregation at the cell periphery. 3D observation also revealed that the aggregates around the cell remained in that location even if cell death occurred, implying that amyloid plaques found in the AD brain grew from the debris of dead cells that accumulated Aß42 aggregates.

FAM222A encodes a protein which accumulates in plaques in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by amyloid plaques and progressive cerebral atrophy. Here, we report FAM222A as a putative brain atrophy susceptibility gene. Our cross-phenotype association analysis of imaging genetics indicates a potential link between FAM222A and AD-related regional brain atrophy. The protein encoded by FAM222A is predominantly expressed in the CNS and is increased in brains of patients with AD and in an AD mouse model. It accumulates within amyloid deposits, physically interacts with amyloid-ß (Aß) via its N-terminal Aß binding domain, and facilitates Aß aggregation. Intracerebroventricular infusion or forced expression of this protein exacerbates neuroinflammation and cognitive dysfunction in an AD mouse model whereas ablation of this protein suppresses the formation of amyloid deposits, neuroinflammation and cognitive deficits in the AD mouse model. Our data support the pathological relevance of protein encoded by FAM222A in AD.

Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients.

CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.

Dendrimers in the context of nanomedicine.

Dendrimers are globular structures, presenting an initiator core, repetitive layers starting radially from the core and terminal groups on the surface, resembling tree architecture. These structures have been studied in many biological applications, as drug, DNA, RNA and proteins delivery, as well as imaging and radiocontrast agents. With reference to that, this review focused in providing examples of dendrimers used in nanomedicine. Although most studies emphasize cancer, there are others which reveal action in the neurosystem, reducing either neuroinflammation or protein aggregation. Dendrimers can carry bioactive compounds by covalent bond (dendrimer prodrug), or by ionic interaction or adsortion in the internal space of the nanostructure. Additionally, dendrimers can be associated with other polymers, as PEG (polyethylene glycol), and with targeting structures as aptamers, antibodies, folic acid and carbohydrates. Their products in preclinical/clinical trial and those in the market are also discussed, with a total of six derivatives in clinical trials and seven products available in the market.

In vivo detection of tau fibrils and amyloid ß aggregates with luminescent conjugated oligothiophenes and multiphoton microscopy.

The detection of amyloid beta deposits and neurofibrillary tangles, both hallmarks of Alzheimer's disease (AD), is key to understanding the mechanisms underlying these pathologies. Luminescent conjugated oligothiophenes (LCOs) enable fluorescence imaging of these protein aggregates. Using LCOs and multiphoton microscopy, individual tangles and amyloid beta deposits were labeled in vivo and imaged longitudinally in a mouse model of tauopathy and cerebral amyloidosis, respectively. Importantly, LCO HS-84, whose emission falls in the green region of the spectrum, allowed for the first time longitudinal imaging of tangle dynamics following a single intravenous injection. In addition, LCO HS-169, whose emission falls in the red region of the spectrum, successfully labeled amyloid beta deposits, allowing multiplexing with other reporters whose emission falls in the green region of the spectrum. In conclusion, this method can provide a new approach for longitudinal in vivo imaging using multiphoton microscopy of AD pathologies as well as other neurodegenerative diseases associated with protein aggregation in mouse models.

Development of novel theranostic agents for in vivo amyloid imaging and protective effects on human neuroblastoma cells.

Brain amyloid deposits have been identified as the main neuropathological hallmarks of Alzheimer's diseases (AD) and intensive efforts have been devoted to develop aggregation inhibitors preventing the formation of toxic oligomeric Aß for therapeutic. In addition, evidence indicates that the formation and accumulation of ß-amyloid plaques probably precede clinical symptoms by around 20 years and imaging of such plaques would be beneficial for early-stage AD detection. In this study, we investigated phenothiazine-based compounds as novel promising theranostic agents for AD. These multifunctional agents exhibited BBB permeability, low neurotoxicity, good bio-stability as well as strong turn-on fluorescence with a Stokes shift upon binding to Aß aggregates. They had metal-chelating property which could delay Aß aggregation and displayed high binding affinity for ß-amyloid aggregates. Moreover, they have been simultaneously applied to perform in vivo near-infrared fluorescence imaging of ß-amyloid plaques in double transgenic AD mouse model, to prevent self-aggregation of Aß monomer from forming toxic oligomers and to protect human neuroblastoma SH-SY5Y cells against Aß-induced toxicity and oxidative stress.

Environment-Sensitive Near-Infrared Probe for Fluorescent Discrimination of Aß and Tau Fibrils in AD Brain.

The early noninvasive diagnosis of Alzheimer's disease targeted ß-amyloid (Aß) plaques or Tau tangles is a major challenge because of the coshared ß-sheet structure of the target. In contrast to tailoring probes to specific amyloids, here, we showed that near-infrared (NIR) environment-sensitive probe 18 could fluorescently discriminate Aß and Tau from artificial aggregates to pathological change in the brain tissue. The biological evaluation demonstrated that the substantial fluorescence enhancement, large blueshift in the emission upon interactions with the aggregates, and the high binding affinity significantly contributed to the fluorescent discrimination. A simplified Ooshika-Lippert-Mataga equation provided an effective means of correlating 18 with the static relative permittivity (ε0) of proteins, elucidating the origin of the distinction capabilities, and quantitatively estimating the dielectric properties of proteins. Moreover, 18 possessed high bioavailability, including sufficient blood-brain barrier penetration, in vivo NIR imaging, and ex vivo histology in living mice.

Excitation-Energy-Dependent Molecular Beacon Detects Early Stage Neurotoxic Aß Aggregates in the Presence of Cortical Neurons.

There is now crucial medical importance placed on understanding the role of early stage, subvisible protein aggregation, particularly in neurodegenerative disease. While there are strategies for detecting such aggregates in vitro, there is no approach at present that can detect these toxic species associated with cells and specific subcellular compartments. We have exploited excitation-energy-dependent fluorescence edge-shift of recombinant protein labeled with a molecular beacon, to provide a sensitive read out for the presence of subvisible protein aggregates. To demonstrate the potential utility of the approach, we examine the major peptide associated with the initiation of Alzheimer's disease, amyloid ß-protein (Aß) at a patho-physiologically relevant concentration in mouse cortical neurons. Using our approach, we find preliminary evidence that subvisible Aß aggregates are detected at specific subcellular regions and that neurons drive the formation of specific Aß aggregate conformations. These findings therefore demonstrate the potential of a novel fluorescence-based approach for detecting and imaging protein aggregates in a cellular context, which can be used to sensitively probe the association of early stage toxic protein aggregates within subcellular compartments.

Line excitation array detection fluorescence microscopy at 0.8 million frames per second.

Three-dimensional, fluorescence imaging methods with ~1 MHz frame rates are needed for high-speed, blur-free flow cytometry and capturing volumetric neuronal activity. The frame rates of current imaging methods are limited to kHz by the photon budget, slow camera readout, and/or slow laser beam scanners. Here, we present line excitation array detection (LEAD) fluorescence microscopy, a high-speed imaging method capable of providing 0.8 million frames per second. The method performs 0.8 MHz line-scanning of an excitation laser beam using a chirped signal-driven longitudinal acousto-optic deflector to create a virtual light-sheet, and images the field-of-view with a linear photomultiplier tube array to generate a 66 × 14 pixel frame each scan cycle. We implement LEAD microscopy as a blur-free flow cytometer for Caenorhabditis elegans moving at 1 m s-1 with 3.5-µm resolution and signal-to-background ratios >200. Signal-to-noise measurements indicate future LEAD fluorescence microscopes can reach higher resolutions and pixels per frame without compromising frame rates.

Neurogenetic contributions to amyloid beta and tau spreading in the human cortex.

Tau and amyloid beta (Aß) proteins accumulate along neuronal circuits in Alzheimer's disease. Unraveling the genetic background for the regional vulnerability of these proteinopathies can help in understanding the mechanisms of pathology progression. To that end, we developed a novel graph theory approach and used it to investigate the intersection of longitudinal Aß and tau positron emission tomography imaging of healthy adult individuals and the genetic transcriptome of the Allen Human Brain Atlas. We identified distinctive pathways for tau and Aß accumulation, of which the tau pathways correlated with cognitive levels. We found that tau propagation and Aß propagation patterns were associated with a common genetic profile related to lipid metabolism, in which APOE played a central role, whereas the tau-specific genetic profile was classified as 'axon related' and the Aß profile as 'dendrite related'. This study reveals distinct genetic profiles that may confer vulnerability to tau and Aß in vivo propagation in the human brain.

Structure-Property Relationships of Polyethylene Glycol Modified Fluorophore as Near-Infrared Aß Imaging Probes.

To optimize the lipophilicity and improve in vivo pharmacokinetics of near-infrared probes targeted Aß plaques, we designed, synthesized, and evaluated a series of polyethylene glycol modified probes with hydroxyl and methoxyl terminals. The relationships between chemical structure and optical, biological properties were systemically elucidated. The results indicated that a desired Aß probe should keep a balance among molecular rigidity, size, and lipophilicity. Probe 12d displayed improved properties including intense and selective response to Aß1-42 aggregates ( Kd = 7.3 nM, 22-fold fluorescence enhancement and emission maxima at 715 nm upon interaction with Aß1-42 aggregates), sufficient blood-brain barrier penetration (3.04% ID/g), and fast wash out from the brain (brain2 min/brain60 min = 10.1). Clear fluorescence signals retention in transgenic mice than control mice in in vivo near-infrared imaging. Hence, polyethylene glycol modified probes retained favorable optical properties but displayed great improvement of biological properties for Aß detection.

Does inflammation precede tau aggregation in early Alzheimer's disease? A PET study.

OBJECTIVE: Our aim was to assess with positron emission tomography (PET) the temporal and spatial inter-relationships between levels of cortical microglial activation and the aggregated amyloid-ß and tau load in mild cognitive impairment (MCI) and early Alzheimer's disease (AD). METHODS: Six clinically probable AD and 20 MCI subjects had inflammation (11C-(R)-PK11195), amyloid (11C-PiB) and tau (18F-flortaucipir) PET, magnetic resonance imaging (MRI) and a neuropsychological assessment. Parametric images of tracer binding were interrogated at a voxel level and by region of interest analyses. RESULTS: 55% of MCI and 83% of AD subjects had a high amyloid-ß load. We have previously reported that clusters of correlated amyloid and inflammation levels are present in cortex. Here we found no correlation between levels of inflammation (11C-(R)-PK11195 BPND) and tau (18F-flortaucipir SUVR) or MMSE scores in high amyloid-ß cases. INTERPRETATION: While correlated levels of amyloid-ß and inflammation can be seen in MCI, we did not detect an association between levels of cortical tau tangles and inflammation in our series of high amyloid-ß cases. High levels of inflammation could be seen in amyloid-ß positive MCI cases where 18F-flortaucipir signals were low suggesting microglial activation precedes tau tangle formation. Inflammation levels were higher in high amyloid-ß MCI than in early AD cases, compatible with it initially playing a protective role.

Super-resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin†T Molecules.

Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super-resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid-ß aggregation, as well as the structural remodeling of amyloid-ß fibrils by the compound epi-gallocatechin gallate.