Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody.

BACKGROUND: The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. METHODS: A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. RESULTS: The most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30-60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2-16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. CONCLUSION: The pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

Real-time environmental surveillance of SARS-CoV-2 aerosols.

Real-time surveillance of airborne SARS-CoV-2 virus is a technological gap that has eluded the scientific community since the beginning of the COVID-19 pandemic. Offline air sampling techniques for SARS-CoV-2 detection suffer from longer turnaround times and require skilled labor. Here, we present a proof-of-concept pathogen Air Quality (pAQ) monitor for real-time (5 min time resolution) direct detection of SARS-CoV-2 aerosols. The system synergistically integrates a high flow (~1000 lpm) wet cyclone air sampler and a nanobody-based ultrasensitive micro-immunoelectrode biosensor. The wet cyclone showed comparable or better virus sampling performance than commercially available samplers. Laboratory experiments demonstrate a device sensitivity of 77-83% and a limit of detection of 7-35 viral RNA copies/m3 of air. Our pAQ monitor is suited for point-of-need surveillance of SARS-CoV-2 variants in indoor environments and can be adapted for multiplexed detection of other respiratory pathogens of interest. Widespread adoption of such technology could assist public health officials with implementing rapid disease control measures.

Rapid Direct Detection of SARS-CoV-2 Aerosols in Exhaled Breath at the Point of Care.

Airborne transmission via virus-laden aerosols is a dominant route for the transmission of respiratory diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Direct, non-invasive screening of respiratory virus aerosols in patients has been a long-standing technical challenge. Here, we introduce a point-of-care testing platform that directly detects SARS-CoV-2 aerosols in as little as two exhaled breaths of patients and provides results in under 60 s. It integrates a hand-held breath aerosol collector and a llama-derived, SARS-CoV-2 spike-protein specific nanobody bound to an ultrasensitive micro-immunoelectrode biosensor, which detects the oxidation of tyrosine amino acids present in SARS-CoV-2 viral particles. Laboratory and clinical trial results were within 20% of those obtained using standard testing methods. Importantly, the electrochemical biosensor directly detects the virus itself, as opposed to a surrogate or signature of the virus, and is sensitive to as little as 10 viral particles in a sample. Our platform holds the potential to be adapted for multiplexed detection of different respiratory viruses. It provides a rapid and non-invasive alternative to conventional viral diagnostics.

Enhanced in Vivo Blood Brain Barrier Transcytosis of Macromolecular Cargo Using an Engineered pH-sensitive Mouse Transferrin Receptor Binding Nanobody.

Background: The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. Methods: A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1 R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. Results: The most effective histidine mutant, M1 R56H, P96H, Y102H -neurotensin, caused >8°C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1 56,96,102His -13A7-13A7 in capillary depleted brain lysates peaked at 1 hour and were 60% retained at 8 hours. A control construct with no brain targets was only 15% retained at 8 hours. Addition of the albumin-binding Nb80 nanobody to make M1 R56H, P96H, Y102H -13A7-13A7-Nb80 extended blood half-life from 21 minutes to 2.6 hours. At 30-60 minutes, biotinylated M1 R56H, P96H, Y102H -13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2-16 hours it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1 R56H, P96H, Y102H -13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. Conclusion: The pH-sensitive mouse transferrin receptor binding nanobody M1 R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

Selection of single domain anti-transferrin receptor antibodies for blood-brain barrier transcytosis using a neurotensin based assay and histological assessment of target engagement in a mouse model of Alzheimer's related amyloid-beta pathology.

The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (Aß) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aß plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6°C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-Aß VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and Aß plaques at concentrations as low as 0.22 nM. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex, likely because the concentration required for NT-induced hypothermia was lower than the concentration required to produce in situ labeling. These findings reveal an important dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with Aß-plaques in cortex. Additional methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological, and pathological disease in a Syrian hamster model of COVID-19.

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor-binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.Abbreviations: ACE2 - angiotensin converting enzyme 2BSA - buried surface areaCDR - complementary determining regionRBD - receptor binding domainRBM - receptor-binding motifSARS-CoV-2 - severe acute respiratory syndrome coronavirus 2.

Unbiased high-content screening reveals Aß- and tau-independent synaptotoxic activities in human brain homogenates from Alzheimer's patients and high-pathology controls.

Alzheimer's disease (AD) is tightly correlated with synapse loss in vulnerable brain regions. It is assumed that specific molecular entities such as Aß and tau cause synapse loss in AD, yet unbiased screens for synaptotoxic activities have not been performed. Here, we performed size exclusion chromatography on soluble human brain homogenates from AD cases, high pathology non-demented controls, and low pathology age-matched controls using our novel high content primary cultured neuron-based screening assay. Both presynaptic and postsynaptic toxicities were elevated in homogenates from AD cases and high pathology non-demented controls to a similar extent, with more modest synaptotoxic activities in homogenates from low pathology normal controls. Surprisingly, synaptotoxic activities were found in size fractions peaking between the 17-44 kDa size standards that did not match well with Aß and tau immunoreactive species in these homogenates. The fractions containing previously identified high molecular weight soluble amyloid beta aggregates/"oligomers" were non-toxic in this assay. Furthermore, immunodepletion of Aß and tau did not reduce synaptotoxic activity. This result contrasts with previous findings involving the same methods applied to 3xTg-AD mouse brain extracts. The nature of the synaptotoxic species has not been identified. Overall, our data indicates one or more potential Aß and tau independent synaptotoxic activities in human AD brain homogenates. This result aligns well with the key role of synaptic loss in the early cognitive decline and may provide new insight into AD pathophysiology.

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological and pathological disease in a Syrian hamster model of COVID-19.

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses, including the currently dominant Delta variant. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.

Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under "basal" circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents.

Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein.

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.

Sensitive detection of extremely small iron oxide nanoparticles in living mice using MP2RAGE with advanced image co-registration.

Magnetic resonance imaging (MRI) is a widely used non-invasive methodology for both preclinical and clinical studies. However, MRI lacks molecular specificity. Molecular contrast agents for MRI would be highly beneficial for detecting specific pathological lesions and quantitatively evaluating therapeutic efficacy in vivo. In this study, an optimized Magnetization Prepared-RApid Gradient Echo (MP-RAGE) with 2 inversion times called MP2RAGE combined with advanced image co-registration is presented as an effective non-invasive methodology to quantitatively detect T1 MR contrast agents. The optimized MP2RAGE produced high quality in vivo mouse brain T1 (or R1 = 1/T1) map with high spatial resolution, 160 × 160 × 160 µm3 voxel at 9.4 T. Test-retest signal to noise was > 20 for most voxels. Extremely small iron oxide nanoparticles (ESIONPs) having 3 nm core size and 11 nm hydrodynamic radius after polyethylene glycol (PEG) coating were intracranially injected into mouse brain and detected as a proof-of-concept. Two independent MP2RAGE MR scans were performed pre- and post-injection of ESIONPs followed by advanced image co-registration. The comparison of two T1 (or R1) maps after image co-registration provided precise and quantitative assessment of the effects of the injected ESIONPs at each voxel. The proposed MR protocol has potential for future use in the detection of T1 molecular contrast agents.

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme.

There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12-15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1-5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates.

Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer's disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the MTT assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications.

Prospective natural history study of C9orf72 ALS clinical characteristics and biomarkers.

OBJECTIVE: To define the natural history of the C9orf72 amyotrophic lateral sclerosis (C9ALS) patient population, develop disease biomarkers, and characterize patient pathologies. METHODS: We prospectively collected clinical and demographic data from 116 symptomatic C9ALS and 12 non-amyotrophic lateral sclerosis (ALS) full expansion carriers across 7 institutions in the United States and the Netherlands. In addition, we collected blood samples for DNA repeat size assessment, CSF samples for biomarker identification, and autopsy samples for dipeptide repeat protein (DPR) size determination. Finally, we collected retrospective clinical data via chart review from 208 individuals with C9ALS and 450 individuals with singleton ALS. RESULTS: The mean age at onset in the symptomatic prospective cohort was 57.9 ± 8.3 years, and median duration of survival after onset was 36.9 months. The monthly change was -1.8 ± 1.7 for ALS Functional Rating Scale-Revised and -1.4% ± 3.24% of predicted for slow vital capacity. In blood DNA, we found that G4C2 repeat size correlates positively with age. In CSF, we observed that concentrations of poly(GP) negatively correlate with DNA expansion size but do not correlate with measures of disease progression. Finally, we found that size of poly(GP) dipeptides in the brain can reach large sizes similar to that of their DNA repeat derivatives. CONCLUSIONS: We present a thorough investigation of C9ALS natural history, providing the basis for C9ALS clinical trial design. We found that clinical features of this genetic subset are less variant than in singleton ALS. In addition, we identified important correlations of C9ALS patient pathologies with clinical and demographic data.

Repetitive Concussive and Subconcussive Injury in a Human Tau Mouse Model Results in Chronic Cognitive Dysfunction and Disruption of White Matter Tracts, But Not Tau Pathology.

Due to the unmet need for a means to study chronic traumatic encephalopathy (CTE) in vivo, there have been numerous efforts to develop an animal model of this progressive tauopathy. However, there is currently no consensus in the field on an injury model that consistently reproduces the neuropathological and behavioral features of CTE. We have implemented a repetitive Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA) injury paradigm in human transgenic (hTau) mice. Animals were subjected to daily subconcussive or concussive injuries for 20 days and tested acutely, 3 months, and 12 months post-injury for deficits in social behavior, anxiety, spatial learning and memory, and depressive behavior. Animals also were assessed for chronic tau pathology, astrogliosis, and white matter degeneration. Repetitive concussive injury caused acute deficits in Morris water maze performance, including reduced swimming speed and increased distance to the platform during visible and hidden platform phases that persisted during the subacute and chronic time-points following injury. We found evidence of white matter disruption in animals injured with subconcussive and concussive injuries, with the most severe disruption occurring in the repetitive concussive injury group. Severity of white matter disruption in the corpus callosum was moderately correlated with swimming speed, while white matter disruption in the fimbria showed weak but significant correlation with worse performance during probe trial. There was no evidence of tau pathology or astrogliosis in sham or injured animals. In summary, we show that repetitive brain injury produces persistent behavioral abnormalities as late as 1 year post-injury that may be related to chronic white matter disruption, although the relationship with CTE remains to be determined.

Soluble amyloid-beta buffering by plaques in Alzheimer disease dementia versus high-pathology controls.

An unanswered question regarding Alzheimer disease dementia (ADD) is whether amyloid-beta (Aß) plaques sequester toxic soluble Aß species early during pathological progression. We previously reported that the concentration of soluble Aß aggregates from patients with mild dementia was higher than soluble Aß aggregates from patients with modest Aß plaque burden but no dementia. The ratio of soluble Aß aggregate concentration to Aß plaque area fully distinguished these groups of patients. We hypothesized that initially plaques may serve as a reservoir or sink for toxic soluble Aß aggregates, sequestering them from other targets in the extracellular space and thereby preventing their toxicity. To initially test a generalized version of this hypothesis, we have performed binding assessments using biotinylated synthetic Aß1-42 peptide. Aß1-42-biotin peptide was incubated on unfixed frozen sections from non-demented high plaque pathology controls and patients with ADD. The bound peptide was measured using ELISA and confocal microscopy. We observed no quantitative difference in Aß binding between the groups using either method. Further testing of the buffering hypothesis using various forms of synthetic and human derived soluble Aß aggregates will be required to definitively address the role of plaque buffering as it relates to ADD.

Age-Dependent Effects of apoE Reduction Using Antisense Oligonucleotides in a Model of ß-amyloidosis.

The apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer disease. Previous studies suggest that reduction of apoE levels through genetic manipulation can reduce Aß pathology. However, it is not clear how reduction of apoE levels after birth would affect amyloid deposition. We utilize an antisense oligonucleotide (ASO) to reduce apoE expression in the brains of APP/PS1-21 mice homozygous for the APOE-ε4 or APOE-ε3 allele. ASO treatment starting after birth led to a significant decrease in Aß pathology when assessed at 4 months. Interestingly, ASO treatment starting at the onset of amyloid deposition led to an increase in Aß plaque size and a reduction in plaque-associated neuritic dystrophy with no change in overall plaque load. These results suggest that lowering apoE levels prior to plaque deposition can strongly affect the initiation of Aß pathology while lowering apoE after Aß seeding modulates plaque size and toxicity.

Diversity of Amyloid-beta Proteoforms in the Alzheimer's Disease Brain.

Amyloid-beta (Aß) plays a key role in the pathogenesis of Alzheimer's disease (AD), but little is known about the proteoforms present in AD brain. We used high-resolution mass spectrometry to analyze intact Aß from soluble aggregates and insoluble material in brains of six cases with severe dementia and pathologically confirmed AD. The soluble aggregates are especially relevant because they are believed to be the most toxic form of Aß. We found a diversity of Aß peptides, with 26 unique proteoforms including various N- and C-terminal truncations. N- and C-terminal truncations comprised 73% and 30%, respectively, of the total Aß proteoforms detected. The Aß proteoforms segregated between the soluble and more insoluble aggregates with N-terminal truncations predominating in the insoluble material and C- terminal truncations segregating into the soluble aggregates. In contrast, canonical Aß comprised the minority of the identified proteoforms (15.3%) and did not distinguish between the soluble and more insoluble aggregates. The relative abundance of many truncated Aß proteoforms did not correlate with post-mortem interval, suggesting they are not artefacts. This heterogeneity of Aß proteoforms deepens our understanding of AD and offers many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.

Non-canonical soluble amyloid-beta aggregates and plaque buffering: controversies and future directions for target discovery in Alzheimer's disease.

The specific amyloid-beta (Aß) species or other amyloid-precursor protein cleavage products that are most directly related to human neurodegeneration and clinical dementia of the Alzheimer's type have not yet been directly identified. Without a clear understanding of the most relevant species, it is difficult to determine whether therapeutic candidates successfully engaged the correct target(s). Here, we review some of the controversies regarding soluble Aß aggregates (also termed oligomers, dimers, trimers, Aß*56, amylospheroids, etc.) and propose experiments designed to move forward towards new therapeutic approaches. Specifically, we review the increasing evidence for the relevance of non-canonical forms of Aß, the much more potent toxicity attributable to native species than to synthetic Aß, and the evidence implicating the ratio of soluble Aß aggregates to plaques in differentiating demented patients from non-demented high Aß plaque pathology controls. To move forward, we propose four related directions. 1) Narrowing the focus to species derived from human Alzheimer's disease (AD) brain tissue, as opposed to synthetic Aß, cell culture-derived species, or species primarily present in animal models. 2) Careful study of differences between patients with dementia of the Alzheimer's type vs. non-demented controls with high Aß plaque pathology. This will involve testing the hypothesis that, under some circumstances, plaques may buffer soluble toxic species, but later release them into the surrounding milieu. 3) Investigations of other protein constituents of soluble Aß aggregates in addition to Aß itself. Our initial data based on chemical cleavage experiments indicate that other proteins do appear to be part of the human brain soluble Aß aggregates. 4) Multimodal experimental assessments of toxicity, including longer term effects on synapse loss, related deleterious cellular responses, and degeneration in human-derived neuron-like cells. Overall, the goal is to identify specific Aß species, other amyloid precursor protein cleavage products, or other key proteins in aggregates present in human AD brains, less abundant in non-demented high pathology control brains, and robustly toxic in a wide variety of relevant assays. These species themselves, the enzymatic or cellular processes involved in their production, and their routes of clearance would be highly relevant therapeutic targets for dementia of the Alzheimer's type.

Polarity of varicosity initiation in central neuron mechanosensation.

Little is known about mechanical regulation of morphological and functional polarity of central neurons. In this study, we report that mechanical stress specifically induces varicosities in the axons but not the dendrites of central neurons by activating TRPV4, a Ca2+/Na+-permeable mechanosensitive channel. This process is unexpectedly rapid and reversible, consistent with the formation of axonal varicosities in vivo induced by mechanical impact in a mouse model of mild traumatic brain injury. In contrast, prolonged stimulation of glutamate receptors induces varicosities in dendrites but not in axons. We further show that axonal varicosities are induced by persistent Ca2+ increase, disassembled microtubules (MTs), and subsequently reversible disruption of axonal transport, and are regulated by stable tubulin-only polypeptide, an MT-associated protein. Finally, axonal varicosity initiation can trigger action potentials to antidromically propagate to the soma in retrograde signaling. Therefore, our study demonstrates a new feature of neuronal polarity: axons and dendrites preferentially respond to physical and chemical stresses, respectively.