TMEM106B reduction does not rescue GRN deficiency in iPSC-derived human microglia and mouse models.

Heterozygous mutations in the granulin (GRN) gene are a leading cause of frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP). Polymorphisms in TMEM106B have been associated with disease risk in GRN mutation carriers and protective TMEM106B variants associated with reduced levels of TMEM106B, suggesting that lowering TMEM106B might be therapeutic in the context of FTLD. Here, we tested the impact of full deletion and partial reduction of TMEM106B in mouse and iPSC-derived human cell models of GRN deficiency. TMEM106B deletion did not reverse transcriptomic or proteomic profiles in GRN-deficient microglia, with a few exceptions in immune signaling markers. Neither homozygous nor heterozygous Tmem106b deletion normalized disease-associated phenotypes in Grn -/-mice. Furthermore, Tmem106b reduction by antisense oligonucleotide (ASO) was poorly tolerated in Grn -/-mice. These data provide novel insight into TMEM106B and GRN function in microglia cells but do not support lowering TMEM106B levels as a viable therapeutic strategy for treating FTD-GRN.

Polarized microtubule remodeling transforms the morphology of reactive microglia and drives cytokine release.

Microglial reactivity is a pathological hallmark in many neurodegenerative diseases. During stimulation, microglia undergo complex morphological changes, including loss of their characteristic ramified morphology, which is routinely used to detect and quantify inflammation in the brain. However, the underlying molecular mechanisms and the relation between microglial morphology and their pathophysiological function are unknown. Here, proteomic profiling of lipopolysaccharide (LPS)-reactive microglia identifies microtubule remodeling pathways as an early factor that drives the morphological change and subsequently controls cytokine responses. We find that LPS-reactive microglia reorganize their microtubules to form a stable and centrosomally-anchored array to facilitate efficient cytokine trafficking and release. We identify cyclin-dependent kinase 1 (Cdk-1) as a critical upstream regulator of microtubule remodeling and morphological change in-vitro and in-situ. Cdk-1 inhibition also rescues tau and amyloid fibril-induced morphology changes. These results demonstrate a critical role for microtubule dynamics and reorganization in microglial reactivity and modulating cytokine-mediated inflammatory responses.

Integrative in situ mapping of single-cell transcriptional states and tissue histopathology in a mouse model of Alzheimer's disease.

Complex diseases are characterized by spatiotemporal cellular and molecular changes that may be difficult to comprehensively capture. However, understanding the spatiotemporal dynamics underlying pathology can shed light on disease mechanisms and progression. Here we introduce STARmap PLUS, a method that combines high-resolution spatial transcriptomics with protein detection in the same tissue section. As proof of principle, we analyze brain tissues of a mouse model of Alzheimer's disease at 8 and 13 months of age. Our approach provides a comprehensive cellular map of disease progression. It reveals a core-shell structure where disease-associated microglia (DAM) closely contact amyloid-ß plaques, whereas disease-associated astrocyte-like (DAA-like) cells and oligodendrocyte precursor cells (OPCs) are enriched in the outer shells surrounding the plaque-DAM complex. Hyperphosphorylated tau emerges mainly in excitatory neurons in the CA1 region and correlates with the local enrichment of oligodendrocyte subtypes. The STARmap PLUS method bridges single-cell gene expression profiles with tissue histopathology at subcellular resolution, providing a tool to pinpoint the molecular and cellular changes underlying pathology.

Translational Approaches for Brain Delivery of Biologics via Cerebrospinal Fluid.

Delivery of biologics via cerebrospinal fluid (CSF) has demonstrated potential to access the tissues of the central nervous system (CNS) by circumventing the blood-brain barrier and blood-CSF barrier. Developing an effective CSF drug delivery strategy requires optimization of multiple parameters, including choice of CSF access point, delivery device technology, and delivery kinetics to achieve effective therapeutic concentrations in the target brain region, whereas also considering the biologic modality, mechanism of action, disease indication, and patient population. This review discusses key preclinical and clinical examples of CSF delivery for different biologic modalities (antibodies, nucleic acid-based therapeutics, and gene therapy) to the brain via CSF or CNS access routes (intracerebroventricular, intrathecal-cisterna magna, intrathecal-lumbar, intraparenchymal, and intranasal), including the use of novel device technologies. This review also discusses quantitative models of CSF flow that provide insight into the effect of fluid dynamics in CSF on drug delivery and CNS distribution. Such models can facilitate delivery device design and pharmacokinetic/pharmacodynamic translation from preclinical species to humans in order to optimize CSF drug delivery to brain regions of interest.

TREM2-independent oligodendrocyte, astrocyte, and T cell responses to tau and amyloid pathology in mouse models of Alzheimer disease.

Non-neuronal responses in neurodegenerative disease have received increasing attention as important contributors to disease pathogenesis and progression. Here we utilize single-cell RNA sequencing to broadly profile 13 cell types in three different mouse models of Alzheimer disease (AD), capturing the effects of tau-only, amyloid-only, or combined tau-amyloid pathology. We highlight microglia, oligodendrocyte, astrocyte, and T cell responses and compare them across these models. Notably, we identify two distinct transcriptional states for oligodendrocytes emerging differentially across disease models, and we determine their spatial distribution. Furthermore, we explore the impact of Trem2 deletion in the context of combined pathology. Trem2 knockout mice exhibit severely blunted microglial responses to combined tau and amyloid pathology, but responses from non-microglial cell types (oligodendrocytes, astrocytes, and T cells) are relatively unchanged. These results delineate core transcriptional states that are engaged in response to AD pathology, and how they are influenced by a key AD risk gene, Trem2.

Trem2 restrains the enhancement of tau accumulation and neurodegeneration by ß-amyloid pathology.

Loss-of-function TREM2 mutations strongly increase Alzheimer's disease (AD) risk. Trem2 deletion has revealed protective Trem2 functions in preclinical models of ß-amyloidosis, a prominent feature of pre-diagnosis AD stages. How TREM2 influences later AD stages characterized by tau-mediated neurodegeneration is unclear. To understand Trem2 function in the context of both ß-amyloid and tau pathologies, we examined Trem2 deficiency in the pR5-183 mouse model expressing mutant tau alone or in TauPS2APP mice, in which ß-amyloid pathology exacerbates tau pathology and neurodegeneration. Single-cell RNA sequencing in these models revealed robust disease-associated microglia (DAM) activation in TauPS2APP mice that was amyloid-dependent and Trem2-dependent. In the presence of ß-amyloid pathology, Trem2 deletion further exacerbated tau accumulation and spreading and promoted brain atrophy. Without ß-amyloid pathology, Trem2 deletion did not affect these processes. Therefore, TREM2 may slow AD progression and reduce tau-driven neurodegeneration by restricting the degree to which ß-amyloid facilitates the spreading of pathogenic tau.

Inducible EphA4 knockout causes motor deficits in young mice and is not protective in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss that ultimately leads to fatal paralysis. Reducing levels or function of the tyrosine kinase, ephrin type-A receptor 4 (EphA4), has been suggested as a potential approach for slowing disease progression in ALS. Because EphA4 plays roles in embryonic nervous system development, study of constitutive knockout (KO) of EphA4 in mice is limited due to confounding phenotypes with homozygous knockout. We used a tamoxifen-inducible EphA4 conditional KO mouse to achieve strong reduction of EphA4 levels in postnatal mice to test for protective effects in the SOD1G93A model of ALS. We found that EphA4 KO in young mice, but not older adult mice, causes defects in muscle function, consistent with a prolonged postnatal role for EphA4 in adolescent muscle growth. When testing the effects of inducible EphA4 KO at different timepoints in SOD1G93A mice, we found no benefits on motor function or disease pathology, including muscle denervation and motor neuron loss. Our results demonstrate deleterious effects of reducing EphA4 levels in juvenile mice and do not provide support for the hypothesis that widespread EphA4 reduction is beneficial in the SOD1G93A mouse model of ALS.

Trem2 Deletion Reduces Late-Stage Amyloid Plaque Accumulation, Elevates the Aß42:Aß40 Ratio, and Exacerbates Axonal Dystrophy and Dendritic Spine Loss in the PS2APP Alzheimer's Mouse Model.

TREM2 is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of Trem2 in a mouse model of ß-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking Trem2 (PS2APP;Trem2ko) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2ko mice, with Trem2-deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE. Plaque abundance was elevated in PS2APP;Trem2ko females at 6-7 months; but by 12 or 19-22 months of age, it was notably diminished in female and male PS2APP;Trem2ko mice, respectively. Across all ages, plaque morphology was more diffuse in PS2APP;Trem2ko brains, and the Aß42:Aß40 ratio was elevated. The amount of soluble, fibrillar Aß oligomers also increased in PS2APP;Trem2ko hippocampi. Associated with these changes, axonal dystrophy was exacerbated from 6 to 7 months onward in PS2APP;Trem2ko mice, notwithstanding the reduced plaque load at later ages. PS2APP;Trem2ko mice also exhibited more dendritic spine loss around plaque and more neurofilament light chain in CSF. Thus, aggravated neuritic dystrophy is a more consistent outcome of Trem2 deficiency than amyloid plaque load, suggesting that the microglial packing of Aß into dense plaque is an important neuroprotective activity.SIGNIFICANCE STATEMENT Genetic studies indicate that TREM2 gene mutations confer increased Alzheimer's disease (AD) risk. We studied the effects of Trem2 deletion in the PS2APP mouse AD model, in which overproduction of Aß peptide leads to amyloid plaque formation and associated neuritic dystrophy. Interestingly, neuritic dystrophies were intensified in the brains of Trem2-deficient mice, despite these mice displaying reduced plaque accumulation at later ages (12-22 months). Microglial clustering around plaques was impaired, plaques were more diffuse, and the Aß42:Aß40 ratio and amount of soluble, fibrillar Aß oligomers were elevated in Trem2-deficient brains. These results suggest that the Trem2-dependent compaction of Aß into dense plaques is a protective microglial activity, limiting the exposure of neurons to toxic Aß species.

Characterization of the selective in vitro and in vivo binding properties of crenezumab to oligomeric Aß.

BACKGROUND: Accumulation of amyloid ß (Aß) in the brain is proposed as a cause of Alzheimer's disease (AD), with Aß oligomers hypothesized to be the primary mediators of neurotoxicity. Crenezumab is a humanized immunoglobulin G4 monoclonal antibody that has been shown to bind to synthetic monomeric and aggregated Aß in vitro; however, less is known about the binding characteristic in vivo. In this study, we evaluated the binding patterns of crenezumab to synthetic and native forms of Aß both in vitro and in vivo. METHODS: Crenezumab was used to immunoprecipitate Aß from synthetic Aß preparations or brain homogenates from a PS2APP mouse model of AD to determine the forms of Aß that crenezumab interacts with. Following systemic dosing in PS2APP or nontransgenic control mice, immunohistochemistry was used to localize crenezumab and assess its relative distribution in the brain, compared with amyloid plaques and markers of neuritic dystrophies (BACE1; LAMP1). Pharmacodynamic correlations were performed to investigate the relationship between peripheral and central target engagement. RESULTS: In vitro, crenezumab immunoprecipitated Aß oligomers from both synthetic Aß preparations and endogenous brain homogenates from PS2APP mice. In vivo studies in the PS2APP mouse showed that crenezumab localizes to regions surrounding the periphery of amyloid plaques in addition to the hippocampal mossy fibers. These regions around the plaques are reported to be enriched in oligomeric Aß, actively incorporate soluble Aß, and contribute to Aß-induced neurotoxicity and axonal dystrophy. In addition, crenezumab did not appear to bind to the dense core region of plaques or vascular amyloid. CONCLUSIONS: Crenezumab binds to multiple forms of amyloid ß (Aß), particularly oligomeric forms, and localizes to brain areas rich in Aß oligomers, including the halo around plaques and hippocampal mossy fibers, but not to vascular Aß. These insights highlight a unique mechanism of action for crenezumab of engaging Aß oligomers.

Complement C3 Is Activated in Human AD Brain and Is Required for Neurodegeneration in Mouse Models of Amyloidosis and Tauopathy.

Complement pathway overactivation can lead to neuronal damage in various neurological diseases. Although Alzheimer's disease (AD) is characterized by ß-amyloid plaques and tau tangles, previous work examining complement has largely focused on amyloidosis models. We find that glial cells show increased expression of classical complement components and the central component C3 in mouse models of amyloidosis (PS2APP) and more extensively tauopathy (TauP301S). Blocking complement function by deleting C3 rescues plaque-associated synapse loss in PS2APP mice and ameliorates neuron loss and brain atrophy in TauP301S mice, improving neurophysiological and behavioral measurements. In addition, C3 protein is elevated in AD patient brains, including at synapses, and levels and processing of C3 are increased in AD patient CSF and correlate with tau. These results demonstrate that complement activation contributes to neurodegeneration caused by tau pathology and suggest that blocking C3 function might be protective in AD and other tauopathies.

Changes in the Synaptic Proteome in Tauopathy and Rescue of Tau-Induced Synapse Loss by C1q Antibodies.

Synapse loss and Tau pathology are hallmarks of Alzheimer's disease (AD) and other tauopathies, but how Tau pathology causes synapse loss is unclear. We used unbiased proteomic analysis of postsynaptic densities (PSDs) in Tau-P301S transgenic mice to identify Tau-dependent alterations in synapses prior to overt neurodegeneration. Multiple proteins and pathways were altered in Tau-P301S PSDs, including depletion of a set of GTPase-regulatory proteins that leads to actin cytoskeletal defects and loss of dendritic spines. Furthermore, we found striking accumulation of complement C1q in the PSDs of Tau-P301S mice and AD patients. At synapses, C1q decorated perisynaptic membranes, accumulated in correlation with phospho-Tau, and was associated with augmented microglial engulfment of synapses and decline of synapse density. A C1q-blocking antibody inhibited microglial synapse removal in cultured neurons and in Tau-P301S mice, rescuing synapse density. Thus, inhibiting complement-mediated synapse removal by microglia could be a potential therapeutic target for Tau-associated neurodegeneration.

Diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse Models.

Microglia, the CNS-resident immune cells, play important roles in disease, but the spectrum of their possible activation states is not well understood. We derived co-regulated gene modules from transcriptional profiles of CNS myeloid cells of diverse mouse models, including new tauopathy model datasets. Using these modules to interpret single-cell data from an Alzheimer's disease (AD) model, we identified microglial subsets-distinct from previously reported "disease-associated microglia"-expressing interferon-related or proliferation modules. We then analyzed whole-tissue RNA profiles from human neurodegenerative diseases, including a new AD dataset. Correcting for altered cellular composition of AD tissue, we observed elevated expression of the neurodegeneration-related modules, but also modules not implicated using expression profiles from mouse models alone. We provide a searchable, interactive database for exploring gene expression in all these datasets (http://research-pub.gene.com/BrainMyeloidLandscape). Understanding the dimensions of CNS myeloid cell activation in human disease may reveal opportunities for therapeutic intervention.

Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12) with Activity in a Model of Alzheimer's Disease.

Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.

Widespread brain distribution and activity following i.c.v. infusion of anti-ß-secretase (BACE1) in nonhuman primates.

BACKGROUND AND PURPOSE: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. EXPERIMENTAL APPROACH: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against ß-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. KEY RESULTS: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-ß (Aß) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aß in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aß levels and had a low (~0.6 nM) antibody concentration in the brain. CONCLUSION AND IMPLICATIONS: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aß concentrations, due to enhanced brain exposure to antibody.

Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative disease.

Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.

BACE1 across species: a comparison of the in vivo consequences of BACE1 deletion in mice and rats.

Assessing BACE1 (ß-site APP cleaving enzyme 1) knockout mice for general health and neurological function may be useful in predicting risks associated with prolonged pharmacological BACE1 inhibition, a treatment approach currently being developed for Alzheimer's disease. To determine whether BACE1 deletion-associated effects in mice generalize to another species, we developed a novel Bace1-/- rat line using zinc-finger nuclease technology and compared Bace1-/- mice and rats with their Bace1+/+ counterparts. Lack of BACE1 was confirmed in Bace1-/- animals from both species. Removal of BACE1 affected startle magnitude, balance beam performance, pain response, and nerve myelination in both species. While both mice and rats lacking BACE1 have shown increased mortality, the increase was smaller and restricted to early developmental stages for rats. Bace1-/- mice and rats further differed in body weight, spontaneous locomotor activity, and prepulse inhibition of startle. While the effects of species and genetic background on these phenotypes remain difficult to distinguish, our findings suggest that BACE1's role in myelination and some sensorimotor functions is consistent between mice and rats and may be conserved in other species. Other phenotypes differ between these models, suggesting that some effects of BACE1 inhibition vary with the biological context (e.g. species or background strain).

Characterization of a sensitive mouse Aß40 PD biomarker assay for Alzheimer's disease drug development in wild-type mice.

AIM: Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. RESULTS: We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aß40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aß reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. CONCLUSION: This sensitive and well-characterized mouse Aß40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.

Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ß-secretase (BACE1) can cross the BBB and reduce brain amyloid-ß (Aß) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aß in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aß both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.

Chronic GluN2B antagonism disrupts behavior in wild-type mice without protecting against synapse loss or memory impairment in Alzheimer's disease mouse models.

Extensive evidence implicates GluN2B-containing NMDA receptors (GluN2B-NMDARs) in excitotoxic-insult-induced neurodegeneration and amyloid ß (Aß)-induced synaptic dysfunction. Therefore, inhibiting GluN2B-NMDARs would appear to be a potential therapeutic strategy to provide neuroprotection and improve cognitive function in Alzheimer's disease (AD). However, there are no reports of long-term in vivo treatment of AD mouse models with GluN2B antagonists. We used piperidine18 (Pip18), a potent and selective GluN2B-NMDAR antagonist with favorable pharmacokinetic properties, for long-term dosing in AD mouse models. Reduced freezing behavior in Tg2576 mice during fear conditioning was partially reversed after subchronic (17 d) Pip18 treatment. However, analysis of freezing behavior in different contexts indicated that this increased freezing likely involves elevated anxiety or excessive memory generalization in both nontransgenic (NTG) and Tg2576 mice. In PS2APP mice chronically fed with medicated food containing Pip18 for 4 months, spatial learning and memory deficits were not rescued, plaque-associated spine loss was not affected, and synaptic function was not altered. At the same time, altered open field activity consistent with increased anxiety and degraded performance in an active avoidance task were observed in NTG after chronic treatment. These results indicate that long-term treatment with a GluN2B-NMDAR antagonist does not provide a disease-modifying benefit and could cause cognitive liabilities rather than symptomatic benefit in AD mouse models. Therefore, these results challenge the expectation of the therapeutic potential for GluN2B-NMDAR antagonists in AD.

A death receptor 6-amyloid precursor protein pathway regulates synapse density in the mature CNS but does not contribute to Alzheimer's disease-related pathophysiology in murine models.

Recent studies implicate death receptor 6 (DR6) in an amyloid precursor protein (APP)-dependent pathway regulating developmental axon pruning, and in a pruning pathway operating during plastic rearrangements in adult brain. DR6 has also been suggested to mediate toxicity in vitro of Aß peptides derived from APP. Given the link between APP, Aß, and Alzheimer's disease (AD), these findings have raised the possibility that DR6 contributes to aspects of neurodegeneration in AD. To test this possibility, we have used mouse models to characterize potential function(s) of DR6 in the adult CNS and in AD-related pathophysiology. We show that DR6 is broadly expressed within the adult CNS and regulates the density of excitatory synaptic connections onto pyramidal neurons in a genetic pathway with APP. DR6 knock-out also gives rise to behavioral abnormalities, some of which are similar to those previously documented in APP knock-out animals. However, in two distinct APP transgenic models of AD, we did not observe any alteration in the formation of amyloid plaques, gliosis, synaptic loss, or cognitive behavioral deficits with genetic deletion of DR6, though we did observe a transient reduction in the degree of microglial activation in one model. Our results support the view that DR6 functions with APP to modulate synaptic density in the adult CNS, but do not provide evidence for a role of DR6 in the pathophysiology of AD.