Abstract Body

The neuropathological diagnosis of Lewy body disease (LBD) may be stated according to several staging systems, which include the Braak Lewy body stages (Braak), the consensus criteria by McKeith and colleagues (McKeith), the modified McKeith system by Leverenz and colleagues (Leverenz), the Unified Staging System by Beach and colleagues (Beach), and the recently established Lewy pathology consensus criteria (LPC). Braak, McKeith, Leverenz and Beach use semi-quantitative scoring of Lewy pathology (LP) in defined cortical and subcortical areas. While these systems are widely used, some suffer from low inter-rater reliability and/ or an inability to unequivocally classify all cases with LP. The LPC on the other hand are based on a dichotomous approach for the scoring of LP and includes amygdala predominant and olfactory only stages.

In a multicentre study we found that McKeith, Leverenz, and LPC systems reached good, while both Braak and Beach systems had lower inter-rater reliability, respectively. Using the LPC system all cases could be unequivocally classified by the majority of raters, which was also seen for 97.1% when the Beach system was used. However, a considerable proportion of cases could not be classified when using Leverenz (11.8%), McKeith (26.5%), or Braak (29.4%) systems.

We established that the LPC system has good reproducibility and allows classification of all cases into distinct categories. We expect that it will be reliable and useful in routine diagnostic practice and therefore suggest that it should be the standard future approach for the basic post-mortem evaluation of LP.

Abstract Body

Recent advances in the development of novel Alzheimer’s disease (AD) measures of amyloid, tau, and neurodegeneration have enabled a better understanding of the links between amyloid-beta (Aβ), tau species and neurodegeneration in AD and related dementias. The discoveries of novel tau species in CSF and blood, including specific phospho-tau (p-tau) and truncated species containing the microtubule binding region of tau (MTBR-tau) which aggregates in tau tangles, have greatly expanded our understanding of tau biology and tau target development. The longitudinal Aβ, tau, and neurofilament light chain (NfL) changes previously measured in CSF are now being measured accurately in blood, enabling interpretation and understanding of AD progression in much larger and more robust populations.

The longitudinal results indicate that a pathophysiological cascade of events begin with altered Aβ42/Aβ40 ratios, followed by increases in amyloid plaques as measured by amyloid PET, associated with increased phosphorylation of specific CSF tau species (e.g., p-tau217, p-tau181), before increases in p-tau205, NfL, total tau concentrations, hypometabolism, and atrophy. Finally, some species of MTBR-tau increase before or with tau aggregation by tau PET and onset of clinical symptoms and correlate with longitudinal tau aggregation and clinical progression.

These findings indicate that CSF and blood plasma biomarkers of Aβ, p-tau, MTBR-tau and NfL measures can accurately identify stages of preclinical and clinical AD and inform about the sequential progression of AD.

Abstract Body

Several papers show that the core AD pathophysiologies amyloid deposition (A), tau pathology (T), neurodegeneration (N), as well as glial activation (G) can accurately be identified and monitored using CSF and blood tests. For amyloid deposition, the Ab42/40 ratio, shows high concordance (AUCs of 80-90%) with brain amyloidosis evaluated by PET, but despite high AUCs, the fold change in PET positive cases is lower in plasma than in CSF. Several plasma P-tau species (181, 217, and 231), show a specific increase in plasma in AD, high concordance with tau PET, and increase also in elderly people with PET evidence of brain amyloidosis but with negative tau PET scans. Recent studies show high correlations between these P-tau species in both CSF and plasma, suggesting that differences are minor.

To implement the blood biomarkers in clinical routine diagnostics, full analytical validation of the analytical methods is needed, and optimally also Reference Measurement Procedures and Certified Reference Materials to assure batch-to-batch stability and comparable results across laboratories. In addition, data on the robustness, defined as the difference between the biomarker variability [combined effects of patient-related, biological, pre-analytical, analytical, and longitudinal bias] and the fold change [percent difference between patients and controls]. For a biomarker to be robust and clinically useful, the variability needs to be substantially lower than the fold change. In summary, although more data on assay performance and the certainty of classification into normal/abnormal is needed, blood tests show great promise as easily accessible screening tools for AD in the clinic.

Abstract Body

It has been over 25 years since the first symptomatic treatment for Alzheimer’s disease was approved by the US Food and Drug Administration (FDA) in 1993. Since then, the Alzheimer’s disease research community has made tremendous strides, learning much about biomarkers underlying disease pathophysiology, improving the design of clinical trials, and increasing the diversity of potential therapeutic targets. Emerging positive clinical trial data from several new anti-amyloid beta (Aβ) monoclonal antibodies (mAbs) has given new hope to physicians and patients. Most recently, the FDA’s approval of aducanumab, a human IgG1 mAb that targets Aβ aggregates, via the accelerated approval pathway has ushered in a new era and, for the first time, patients will be treated with a therapy that targets the underlying pathophysiology of the disease. Clinical trial data and real-world evidence generated from ongoing trials of aducanumab will inform key clinical questions such as the optimal duration of treatment and the ideal biomarker profile for benefit, at initiation and as the disease progresses. In addition to these developments, innovations in biomarkers and diagnostics have evolved our collective understanding of how Alzheimer’s disease may be detected, monitored and managed. Together, these advances will help to answer questions about the long-term outcomes of treatment and the impact of delayed progression on the lives of patients, caregivers, and society. Most importantly, these data will also contribute to the generation of new hypotheses for future research that will continue to move the field forward.

Abstract Body

Tau proteins aggregate into filaments in brain cells in Alzheimer’s disease and tauopathies. Tau immunotherapy is a promising approach. However, tau is also found within brain cells and alternative solutions must be explored. Here, we used fragments of camelid heavy chain-only antibodies (VHHs or single domain antibody fragments) targeting Tau as immuno-modulators of seeding.

Different anti-tau VHHs were screened, and one was selected for its properties to bind microtubule-binding domains, composing the core of Tau fibrils.

This lead VHH was optimized to improve its biochemical and biological properties (intracellular solubility, affinity), resulting in VHH Z70. VHH Z70 was more efficient than the lead to inhibit in vitro Tau aggregation in heparin-induced assays. Expression of VHH Z70 in the Marc Diamond’s HEK cell model of Tau seeding also decreased the FRET signal. Finally, viral vectors encoding VHH Z70 were stereotactically injected in the hippocampus of an established tauopathy mouse seeding model. VHH Z70 demonstrated its capacity to mitigate accumulation of pathological Tau in neuronal cells.

VHH Z70, by targeting Tau inside brain neurons, may be considered as a new immunological tool to target the intra-cellular compartment in Alzheimer’s disease and tauopathies.

Abstract Body

One of the main components of senile plaques in Alzheimer’s disease (AD)-affected brain is the Aβ peptide species harboring a pyroglutamate at position three (pE3-Aβ). Several studies indicated that pE3-Aβ is toxic, prone to aggregation and serves as a seed of Aβ aggregation. The cyclisation of the glutamate in position 3 requires prior removal of the Aβ N-terminal aspartyl residue to allow subsequent biotransformation. We have identified aminopeptidase A (APA)and dipeptidyl peptidase 4 (DDD4) as the main exopeptidases involved in an additional manner. First, we show by mass-spectrometry that human recombinant APA and DPP4 truncate synthetic Aβ1-40 to yield Aβ2-40 and Aβ3-40, respectively. We demonstrate that the pharmacological blockade of APA and DPP4 with theirs selective inhibitors RB150 and sitagliptin restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aβ-precursor protein (βAPP). Pharmacological reduction of enzymes activities and lowering of their expression by shRNA affect Aβ pE3-42- and Aβ1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA and DPP4 inhibitors and specific shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of Aβ pE3-42-positive plaques, APA and DPP4 activity are augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA and DPP4 are two key exopeptidases involved in Aβ N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with Aβ-related AD pathology.

Abstract Body

Methods to enhance adult neurogenesis are needed to enable production of replacement neurons for those lost to disease in the adult nervous system. Here we describe a therapeutically viable approach that may bring this concept to clinical application, building on our discovery that injection of antisense oligonucleotides (ASOs) into cerebral spinal fluid can produce transient target gene silencing throughout the nervous system. ASOs have slowed disease progression or produced disease reversal in models of inherited ALS and Huntington’s disease, with clinical trials in progress in inherited ALS, including C9orf72-mediated ALS/FTD, and to suppress tau in Alzheimer’s, or LRRK2 or alpha synuclein in Parkinson’s.

Remarkably, “identity conversion” of glia into new functional hippocampal or cortical neurons within the aged adult mouse brain can be achieved by transient depletion of the RNA binding protein Polypyrimidine-Tract-Binding-Protein-1 (PTB) with an ASO delivered by a single injection into cerebral spinal fluid. Radial glial-like cells and other GFAP-expressing cells convert into new neurons that over a two-month period acquire mature neuronal character in a process mimicking normal neuronal maturation. The new neurons functionally integrate into endogenous circuits, receive excitatory and inhibitory inputs, and send axons into CA3, modifying mouse hippocampal-dependent behavior. ASO suppression of PTB after chemically-induced Parkinson’s disease in mice converts glia into nigral neurons, restoring striatal dopamine.

Thus, generation of new neurons within the aging brain can be achieved with ASO-mediated suppression of PTB delivered in a therapeutically feasible manner and may be a generalizable approach for treating neurodegenerative disorders, including Parkinson’s and Alzheimer’s.

Abstract Body

AIM: To study NGF trophic support in Health and Disease.

METHODS: Neurochemical and pharmacological procedures.

RESULTS: We have shown that this brain NGF Metabolic pathway

-Sustains the cholinergic phenotype of basal forebrain (BF) neurons.

-In Alzheimer’s disease (AD) pathology there is diminished proNGF to mNGF conversion and increased mNGF degradation

-In AD, the normal NGF synthesis and the failed pro-to-mature NGF conversion provokes a brain build-up of proNGF

-The above NGF metabolic deregulation is found in non-cognitively impaired (NCI) individuals with preclinical Aβ-amyloid pathology and remains physiologically normal in NCI brain samples lacking significant Aβ amyloidosis, proNGF levels correlating with cognitive scores.

-The NGF pathway is deregulated in Down Syndrome (DS) brains at clinical and preclinical AD stages.

-In DS body fluids, proNGF levels are increasingly elevated in their transition from DS without clinical AD to DS with clinical AD and predict subsequent cognitive decline.

CONCLUSIONS The failure of the NGF metabolic pathway, even at preclinical AD stages, would explain the early loss of trophic support to the NGF-dependent cholinergic neurons of the basal forebrain. The correction of this brain metabolic dysfunction at stages preclinical AD should prevent their neuronal atrophy and synaptic loses. The investigation of levels of key molecules of NGF metabolic pathway in blood and cerebrospinal fluids offers an opportunity to reveal the ongoing, silent, preclinical AD pathology.

Abstract Body

There are 126 agents in clinical trials for Alzheimer’s disease (AD) as reported on clinicaltrials.gov (index date 1/4/2021). Of these 104 are putative disease modifying therapies (DMT), and 22 are directed at cognitive enhancement (13) or neuropsychiatric symptoms (9). Thirty-one of the agents are biologicals (e.g., monoclonal antibodies, vaccines) and 73 are small molecules. There at 28 agents in Phase 3, 74 in Phase 2, and 24 in Phase 1. Therapeutic targets represented by multiple agents in the pipeline include amyloid, tau, inflammation, synaptic plasticity, and metabolism/bioenergetics. Agents targeting neuropsychiatric symptoms include a repertoire of treatments for agitation; development programs also address apathy sleep, and psychosis.

The landscape of AD therapies is rapidly evolving. AD drug development has produced its first approved treatment in 17 years. Aducanumab (Aduhelmä) is an anti-amyloid monoclonal antibody directed at amyloid plaque and high molecular weight amyloid species. The FDA accepted amyloid lowering as indicative of an effect on the underlying pathophysiology of AD (e.g., a DMT). The approval of aducanumab using an accelerated approval mechanism based on amyloid lowering as demonstrated by amyloid PET provides both a new drug and a new pathway for drug approval. This will have profound consequences for AD drug development going forward. There are three monoclonal antibodies shown to reduce plaque amyloid that could be candidates for accelerated approval: lecanemab, donanemab, and gantenerumab. Previous drugs targeting amyloid and not shown to have a drug-placebo difference have not shown amyloid lowering.

Abstract Body

Classification of tauopathies is based on immunohistochemical analyses of brain tissue. Conformations of tau assemblies, or seeds, in tauopathy brain lysate could provide a complementary metric for standard neuropathology. Biosensor cells that express tau repeat domain fused to fluorescent proteins will propagate self-replicating tau assemblies (strains) derived from patient material. Certain strains propagate indefinitely, and produce faithfully transmissible neuropathology upon inoculation into mice. Thus tau can be reasonably considered a mammalian prion. We hypothesize that, as for PrP prions, a “ground truth” of diagnosis in tauopathy could derive from underlying tau assembly conformation, rather than patterns of neuropathology. To build a relatively unbiased classification system, we have created a library of mutants with an alanine scan through the tau repeat domain. Using biosensor cells we have probed assembly of each alanine mutant onto existing stable strains, and onto transient intracellular assemblies induced by exposure to brain lysates. The relative incorporation of alanine mutants into intracellular aggregates is dictated by artificial “species barriers” they create. We thus derive a “fingerprint” of amino acids critical to build a given tau assembly. This unbiased, functional genetic dendrogram of tau prions predicts patterns of neuropathology following inoculation into a tauopathy mouse model. For human brain extracts, this method readily distinguishes different tauopathies. It also implicates amino acids outside the amyloid core defined by cryoEM as critical for stability. In ongoing work we are testing the validity of this structure-based classification scheme.