Detecting the effect of genetic diversity on brain composition in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is broadly characterized by neurodegeneration, pathology accumulation, and cognitive decline. There is considerable variation in the progression of clinical symptoms and pathology in humans, highlighting the importance of genetic diversity in the study of AD. To address this, we analyze cell composition and amyloid-beta deposition of 6- and 14-month-old AD-BXD mouse brains. We utilize the analytical QUINT workflow- a suite of software designed to support atlas-based quantification, which we expand to deliver a highly effective method for registering and quantifying cell and pathology changes in diverse disease models. In applying the expanded QUINT workflow, we quantify near-global age-related increases in microglia, astrocytes, and amyloid-beta, and we identify strain-specific regional variation in neuron load. To understand how individual differences in cell composition affect the interpretation of bulk gene expression in AD, we combine hippocampal immunohistochemistry analyses with bulk RNA-sequencing data. This approach allows us to categorize genes whose expression changes in response to AD in a cell and/or pathology load-dependent manner. Ultimately, our study demonstrates the use of the QUINT workflow to standardize the quantification of immunohistochemistry data in diverse mice, - providing valuable insights into regional variation in cellular load and amyloid deposition in the AD-BXD model.

Variation in Mesenchymal KITL/SCF and IGF1 Expression at Middle Age Underlies Steady-State Hematopoietic Stem Cell Aging.

Intrinsic molecular programs and extrinsic factors including pro-inflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single cell transcriptomic profiles of hematopoietic and non-hematopoietic cell types in five young adult and nine middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated which extrinsic regulatory cell types and factors would predict variance in these signatures between individual middle-aged mice. Decline in signaling mediated by ADIPOQ, KITL and IGF1 from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated pro-inflammatory cytokines including IL1, IL6, TNF and RANTES. In sum, we find that Kitl and Igf1 expression are co-regulated and variable between individual mice at middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

New directions for Alzheimer's disease research from the Jackson Laboratory Center for Alzheimer's and Dementia Research 2022 workshop.

INTRODUCTION: In September 2022, The Jackson Laboratory Center for Alzheimer's and Dementia Research (JAX CADR) hosted a workshop with leading researchers in the Alzheimer's disease and related dementias (ADRD) field. METHODS: During the workshop, the participants brainstormed new directions to overcome current barriers to providing patients with effective ADRD therapeutics. The participants outlined specific areas of focus. Following the workshop, each group used standard literature search methods to provide background for each topic. RESULTS: The team of invited experts identified four key areas that can be collectively addressed to make a significant impact in the field: (1) Prioritize the diversification of disease targets, (2) enhance factors promoting resilience, (3) de-risk clinical pipeline, and (4) centralize data management. DISCUSSION: In this report, we review these four objectives and propose innovations to expedite ADRD therapeutic pipelines.

Detecting the effect of genetic diversity on brain composition in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is characterized by neurodegeneration, pathology accumulation, and progressive cognitive decline. There is significant variation in age at onset and severity of symptoms highlighting the importance of genetic diversity in the study of AD. To address this, we analyzed cell and pathology composition of 6- and 14-month-old AD-BXD mouse brains using the semi-automated workflow (QUINT); which we expanded to allow for nonlinear refinement of brain atlas-registration, and quality control assessment of atlas-registration and brain section integrity. Near global age-related increases in microglia, astrocyte, and amyloid-beta accumulation were measured, while regional variation in neuron load existed among strains. Furthermore, hippocampal immunohistochemistry analyses were combined with bulk RNA-sequencing results to demonstrate the relationship between cell composition and gene expression. Overall, the additional functionality of the QUINT workflow delivers a highly effective method for registering and quantifying cell and pathology changes in diverse disease models.

Translational approaches to understanding resilience to Alzheimer's disease.

Individuals who maintain cognitive function despite high levels of Alzheimer's disease (AD)-associated pathology are said to be 'resilient' to AD. Identifying mechanisms underlying resilience represents an exciting therapeutic opportunity. Human studies have identified a number of molecular and genetic factors associated with resilience, but the complexity of these cohorts prohibits a complete understanding of which factors are causal or simply correlated with resilience. Genetically and phenotypically diverse mouse models of AD provide new and translationally relevant opportunities to identify and prioritize new resilience mechanisms for further cross-species investigation. This review will discuss insights into resilience gained from both human and animal studies and highlight future approaches that may help translate these insights into therapeutics designed to prevent or delay AD-related dementia.

Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration.

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.

Leveraging genetic diversity in mice to inform individual differences in brain microstructure and memory.

In human Alzheimer's disease (AD) patients and AD mouse models, both differential pre-disease brain features and differential disease-associated memory decline are observed, suggesting that certain neurological features may protect against AD-related cognitive decline. The combination of these features is known as brain reserve, and understanding the genetic underpinnings of brain reserve may advance AD treatment in genetically diverse human populations. One potential source of brain reserve is brain microstructure, which is genetically influenced and can be measured with diffusion MRI (dMRI). To investigate variation of dMRI metrics in pre-disease-onset, genetically diverse AD mouse models, we utilized a population of genetically distinct AD mice produced by crossing the 5XFAD transgenic mouse model of AD to 3 inbred strains (C57BL/6J, DBA/2J, FVB/NJ) and two wild-derived strains (CAST/EiJ, WSB/EiJ). At 3 months of age, these mice underwent diffusion magnetic resonance imaging (dMRI) to probe neural microanatomy in 83 regions of interest (ROIs). At 5 months of age, these mice underwent contextual fear conditioning (CFC). Strain had a significant effect on dMRI measures in most ROIs tested, while far fewer effects of sex, sex*strain interactions, or strain*sex*5XFAD genotype interactions were observed. A main effect of 5XFAD genotype was observed in only 1 ROI, suggesting that the 5XFAD transgene does not strongly disrupt neural development or microstructure of mice in early adulthood. Strain also explained the most variance in mouse baseline motor activity and long-term fear memory. Additionally, significant effects of sex and strain*sex interaction were observed on baseline motor activity, and significant strain*sex and sex*5XFAD genotype interactions were observed on long-term memory. We are the first to study the genetic influences of brain microanatomy in genetically diverse AD mice. Thus, we demonstrated that strain is the primary factor influencing brain microstructure in young adult AD mice and that neural development and early adult microstructure are not strongly altered by the 5XFAD transgene. We also demonstrated that strain, sex, and 5XFAD genotype interact to influence memory in genetically diverse adult mice. Our results support the usefulness of the 5XFAD mouse model and convey strong relationships between natural genetic variation, brain microstructure, and memory.

AD-linked R47H-TREM2 mutation induces disease-enhancing microglial states via AKT hyperactivation.

The hemizygous R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), a microglia-specific gene in the brain, increases risk for late-onset Alzheimer's disease (AD). Using transcriptomic analysis of single nuclei from brain tissues of patients with AD carrying the R47H mutation or the common variant (CV)­TREM2, we found that R47H-associated microglial subpopulations had enhanced inflammatory signatures reminiscent of previously identified disease-associated microglia (DAM) and hyperactivation of AKT, one of the signaling pathways downstream of TREM2. We established a tauopathy mouse model with heterozygous knock-in of the human TREM2 with the R47H mutation or CV and found that R47H induced and exacerbated TAU-mediated spatial memory deficits in female mice. Single-cell transcriptomic analysis of microglia from these mice also revealed transcriptomic changes induced by R47H that had substantial overlaps with R47H microglia in human AD brains, including robust increases in proinflammatory cytokines, activation of AKT signaling, and elevation of a subset of DAM signatures. Pharmacological AKT inhibition with MK-2206 largely reversed the enhanced inflammatory signatures in primary R47H microglia treated with TAU fibrils. In R47H heterozygous tauopathy mice, MK-2206 treatment abolished a tauopathy-dependent microglial subcluster and rescued tauopathy-induced synapse loss. By uncovering disease-enhancing mechanisms of the R47H mutation conserved in human and mouse, our study supports inhibitors of AKT signaling as a microglial modulating strategy to treat AD.

Discovery of small molecules that normalize the transcriptome and enhance cysteine cathepsin activity in progranulin-deficient microglia.

Patients with frontotemporal dementia (FTD) resulting from granulin (GRN) haploinsufficiency have reduced levels of progranulin and exhibit dysregulation in inflammatory and lysosomal networks. Microglia produce high levels of progranulin, and reduction of progranulin in microglia alone is sufficient to recapitulate inflammation, lysosomal dysfunction, and hyperproliferation in a cell-autonomous manner. Therefore, targeting microglial dysfunction caused by progranulin insufficiency represents a potential therapeutic strategy to manage neurodegeneration in FTD. Limitations of current progranulin-enhancing strategies necessitate the discovery of new targets. To identify compounds that can reverse microglial defects in Grn-deficient mouse microglia, we performed a compound screen coupled with high throughput sequencing to assess key transcriptional changes in inflammatory and lysosomal pathways. Positive hits from this initial screen were then further narrowed down based on their ability to rescue cathepsin activity, a critical biochemical readout of lysosomal capacity. The screen identified nor-binaltorphimine dihydrochloride (nor-BNI) and dibutyryl-cAMP, sodium salt (DB-cAMP) as two phenotypic modulators of progranulin deficiency. In addition, nor-BNI and DB-cAMP also rescued cell cycle abnormalities in progranulin-deficient cells. These data highlight the potential of a transcription-based platform for drug screening, and advance two novel lead compounds for FTD.

Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain.

Microglia are resident immune cells that play critical roles in maintaining the normal physiology of the central nervous system (CNS). Remarkably, microglia have an intrinsic capacity to repopulate themselves after acute ablation. However, the underlying mechanisms that drive such restoration remain elusive. Here, we characterized microglial repopulation both spatially and temporally following removal via treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. We show that microglia were replenished via self-renewal, with no contribution from nonmicroglial lineages, including Nestin+ progenitors and the circulating myeloid population. Interestingly, spatial analyses with dual-color labeling revealed that newborn microglia recolonized the parenchyma by forming distinctive clusters that maintained stable territorial boundaries over time, indicating the proximal expansive nature of adult microgliogenesis and the stability of microglia tiling. Temporal transcriptome profiling at different repopulation stages revealed that adult newborn microglia gradually regain steady-state maturity from an immature state that is reminiscent of the neonatal stage and follow a series of maturation programs, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, interferon immune activation, and apoptosis. Importantly, we show that the restoration of microglial homeostatic density requires NF-κB signaling as well as apoptotic egress of excessive cells. In summary, our study reports key events that take place from microgliogenesis to homeostasis reestablishment.

Differential effects of partial and complete loss of TREM2 on microglial injury response and tauopathy.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the abnormal accumulation of amyloid plaques and hyperphosphorylated tau aggregates, as well as microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) are associated with elevated risk for developing late-onset AD. These variants are hypothesized to result in loss of function, mimicking TREM2 haploinsufficiency. However, the consequences of TREM2 haploinsufficiency on tau pathology and microglial function remain unknown. We report the effects of partial and complete loss of TREM2 on microglial function and tau-associated deficits. In vivo imaging revealed that microglia from aged TREM2-haploinsufficient mice show a greater impairment in their injury response compared with microglia from aged TREM2-KO mice. In transgenic mice expressing mutant human tau, TREM2 haploinsufficiency, but not complete loss of TREM2, increased tau pathology. In addition, whereas complete TREM2 deficiency protected against tau-mediated microglial activation and atrophy, TREM2 haploinsufficiency elevated expression of proinflammatory markers and exacerbated atrophy at a late stage of disease. The differential effects of partial and complete loss of TREM2 on microglial function and tau pathology provide important insights into the critical role of TREM2 in AD pathogenesis.

Endo-lysosomal dysfunction: a converging mechanism in neurodegenerative diseases.

Endo-lysosomal pathways are essential in maintaining protein homeostasis in the cell. Numerous genes in the endo-lysosomal pathways have been found to associate with neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia (FTD). Mutations of these genes lead to dysfunction in multiple steps of the endo-lysosomal network: autophagy, endocytic trafficking and lysosomal degradation, resulting in accumulation of pathogenic proteins. Although the exact pathogenic mechanism varies for different disease-associated genes, dysfunction of the endo-lysosomal pathways represents a converging mechanism shared by these diseases. Therefore, strategies that correct or compensate for endo-lysosomal dysfunction may be promising therapeutic approaches to treat neurodegenerative diseases.

Individuals with progranulin haploinsufficiency exhibit features of neuronal ceroid lipofuscinosis.

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.

In silico to in vitro screening of hydroxypyridinones as acetylcholinesterase inhibitors.

We have previously shown the improved acetylcholinesterase inhibitory activity of a model hydroxypyridinone compound transforming the hydroxyl group on the main ring into an N,N-dimethylcarbamate group; in the course of that study we developed a computational model to screen compounds for enzymatic activity. Herein we report development of second generation libraries. Candidates that adhere to drug-like criteria from a virtual library of compounds were tested using computational docking studies. Synthesis and characterization of chosen test compounds and their acetylcholinesterase inhibitory activity are presented.

3-Hydroxy-4-pyridinone derivatives designed for fluorescence studies to determine interaction with amyloid protein as well as cell permeability.

Finding a cure for Alzheimer's disease is an urgent goal. Multifunctional metal binders are used to elucidate its pathological features and investigated as potential therapeutics. The use of physicochemical and TD-DFT calculations constituted successful strategy in the design of 1-(4-(benzo[d]oxazol-2-yl)phenyl)-3-hydroxy-2-methylpyridin-4(1H)-one (HL21) and 1-(4-(benzo[d]thiazol-2-yl)phenyl)-3-hydroxy-2-methylpyridin-4(1H)-one (HL22). We report the synthesis and full characterization of these compounds, including X-ray crystallography. Using fluorescent signal as the readout, it was determined that HL22 interacts with amyloid-beta protein fibrils, and permeates into bEnd.3 cells used as a mimic of the blood-brain barrier. This provides the first example of direct investigation of our hydroxypyridinone compounds within a biological setting.

Synthesis, characterization, and cytotoxicity studies of Cu(II), Zn(II), and Fe(III) complexes of N-derivatized 3-hydroxy-4-pyridiones.

The deleterious role of metal ions in Alzheimer's disease has inspired the study of various metal chelators. We previously showed the synthesis and in vitro activity of several bidentate hydroxypyridinone compounds, including 3-hydroxy-2-methyl-1-phenyl-4(1H)-pyridinone (1), 1-(4-aminophenyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (2), and 1-(2-benzothiazolyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (3). While the focus has been on the Cu(II) ion, the other biorelevant metals, Zn(II) and Fe(III) have been largely neglected. Herein, we report the synthesis of Zn(II) and Fe(III) complexes of ligands 1, 2, and 3, and their characterization by infrared (IR) spectroscopy, high resolution mass spectrometry (HR-MS), elemental analysis, and NMR, where applicable. Solid state structures of Zn(1)2, Fe(1)3, and Cu(3)2 are analyzed with X-ray crystallography. The cytotoxicity of pro-ligands 1, 2, and 3, and the three metal complexes of 2 are examined in a neuronal cell line to determine the effect of metal chelation on toxicity of the compounds.

3-Hydroxy-4-pyridinone derivatives as metal ion and amyloid binding agents.

Metal ions have been implicated in several neurodegenerative diseases, including Alzheimer's disease, as their dyshomeostasis may lead to production of reactive oxygen species as well as increased toxicity of amyloid protein aggregates. In this work, we present design and synthesis of three novel multifunctional hydroxypyridinone ligands, HL11, HL12, and HL13, bearing benzothiazole and benzoxazole functionalities. We study the ability of these compounds to bind metal ions Cu(II), Zn(II), and Fe(III), as well as their antioxidant activity and cytotoxicity. Additionally, we determine the pro-ligands' (compounds prior to chelation) propensity to target amyloid protein. Through these studies we determine the effect of combining amyloid- and metal-binding functionalities within the HPO scaffold on different aspects of AD pathology.

Exploring the multifunctionality of thioflavin- and deferiprone-based molecules as acetylcholinesterase inhibitors for potential application in Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disease that affects millions of people worldwide. With no prevention or cure available, this progressive disease has a significant impact on society - dementia patients and their caretakers, healthcare systems, and the economy. Previously, we have reported initial developments of multifunctional drug candidates for AD based on two scaffolds - thioflavin-T and deferiprone. Individually, these molecules have shown several favorable functionalities, including dissociation of toxic amyloid-ß aggregates, antioxidant and/or metal chelating ability that can pacify reactive oxygen species, plaque targeting, and blood-brain barrier penetration. In this work, the two scaffolds are augmented with a new functionality - acetylcholinesterase inhibition. This functionality is incorporated by derivatization with a carbamate group, which is the active group in some AD drugs currently in the market. We present the rationale for designing three novel compounds, their synthesis and characterization, including X-ray crystallographic data, and encouraging results from in vitro and computational acetylcholinesterase inhibition studies. Also, we evaluate the compounds as potential drug candidates by Lipinski's rules and cytotoxicity studies in a neuronal cell line. Overall, we demonstrate the feasibility of improving on two well established scaffolds, as well as show in vitro efficacy plus initial mode of action and biological compatibility data.

Werner coordination chemistry and neurodegeneration.

Neurodegenerative diseases are capturing the world's attention as being the next set of diseases we must tackle collectively. Not only are the patients experiencing gradual cognitive and physical decline in most cases, but these diseases are fatal with no prevention currently available. As these diseases are progressive, providing care and symptom treatment for the ageing population is becoming both a medical and a financial challenge. This review discusses how Werner coordination chemistry plays a role in three diseases - those of Alzheimer's, Parkinson's, and prions. Metal ions are considered to be involved in these diseases in part via their propensity to cause toxic aggregation of proteins. First, the coordination of metal ions, with emphasis on copper(II), to metalloproteins that are hallmarks of these diseases - amyloid ß, α-synuclein, and prion, respectively - will be discussed. We will present the current understanding of the metal coordination environments created by the amino acids of these proteins, as well as metal binding affinity. Second, a diverse set of examples of rationally designed metal chelators to outcompete this deleterious binding will be examined based on coordination mode and affinity toward bio-relevant metal ions. Overall, this review will give a general overview of protein and metal chelator coordination environments in neurodegenerative diseases.

Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a tetrabutoxy-calix[4]arene scaffold.

A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide public health challenge. A potent compound based on a tetrabutoxy-calix[4]arene scaffold that possesses dual inhibition for both HIV and HCV is described. Structural activity relationship studies demonstrate the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interacting head groups on the calix[4]arene play key roles for its potent dual antiviral activities.