Neutral or Detrimental Effects of TREM2 Agonist Antibodies in Preclinical Models of Alzheimer's Disease and Multiple Sclerosis.

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.

TREM2-dependent microglial function is essential for remyelination and subsequent neuroprotection.

Disability in multiple sclerosis (MS) is driven in part by the failure of remyelination and progressive neurodegeneration. Microglia, and specifically triggering receptor expressed on myeloid cells 2 (TREM2), a factor highly expressed in microglia, have been shown to play an important role in remyelination. Here, using a focal demyelination model in the brain, we demonstrate that demyelination is persistent in TREM2 knockout mice, lasting more than 6 weeks after lysolecithin injection and resulting in substantial neurodegeneration. We also find that TREM2 knockout mice exhibit an altered glial response following demyelination. TREM2 knockout microglia demonstrate defects in migration and phagocytosis of myelin debris. In addition, human monocyte-derived macrophages from subjects with a TREM2 mutation prevalent in human disease also show a defect in myelin debris phagocytosis. Together, we highlight the central role of TREM2 signaling in remyelination and neuroprotection. These findings provide insights into how chronic demyelination might lead to axonal damage and could help identify novel neuroprotective therapeutic targets for MS.

Disease-associated oligodendrocyte responses across neurodegenerative diseases.

Oligodendrocyte dysfunction has been implicated in the pathogenesis of neurodegenerative diseases, so understanding oligodendrocyte activation states would shed light on disease processes. We identify three distinct activation states of oligodendrocytes from single-cell RNA sequencing (RNA-seq) of mouse models of Alzheimer's disease (AD) and multiple sclerosis (MS): DA1 (disease-associated1, associated with immunogenic genes), DA2 (disease-associated2, associated with genes influencing survival), and IFN (associated with interferon response genes). Spatial analysis of disease-associated oligodendrocytes (DAOs) in the cuprizone model reveals that DA1 and DA2 are established outside of the lesion area during demyelination and that DA1 repopulates the lesion during remyelination. Independent meta-analysis of human single-nucleus RNA-seq datasets reveals that the transcriptional responses of MS oligodendrocytes share features with mouse models. In contrast, the oligodendrocyte activation signature observed in human AD is largely distinct from those observed in mice. This catalog of oligodendrocyte activation states (http://research-pub.gene.com/OligoLandscape/) will be important to understand disease progression and develop therapeutic interventions.

Multiple sclerosis risk gene Mertk is required for microglial activation and subsequent remyelination.

In multiple sclerosis (MS) and other neurological diseases, the failure to repair demyelinated lesions contributes to axonal damage and clinical disability. Here, we provide evidence that Mertk, a gene highly expressed by microglia that alters MS risk, is required for efficient remyelination. Compared to wild-type (WT) mice, Mertk-knockout (KO) mice show impaired clearance of myelin debris and remyelination following demyelination. Using single-cell RNA sequencing, we characterize Mertk-influenced responses to cuprizone-mediated demyelination and remyelination across different cell types. Mertk-KO brains show an attenuated microglial response to demyelination but an elevated proportion of interferon (IFN)-responsive microglia. In addition, we identify a transcriptionally distinct subtype of surviving oligodendrocytes specific to demyelinated lesions. The inhibitory effect of myelin debris on remyelination is mediated in part by IFNγ, which further impedes microglial clearance of myelin debris and inhibits oligodendrocyte differentiation. Together, our work establishes a role for Mertk in microglia activation, phagocytosis, and migration during remyelination.

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations.

Studying myelination in vitro and in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs) in vitro, although scalable, does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe an ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0-2 mice, after which they myelinate ex vivo for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment.

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Activin receptors regulate the oligodendrocyte lineage in health and disease.

The most prevalent neurological disorders of myelin include perinatal brain injury leading to cerebral palsy in infants and multiple sclerosis in adults. Although these disorders have distinct etiologies, they share a common neuropathological feature of failed progenitor differentiation into myelin-producing oligodendrocytes and lack of myelin, for which there is an unmet clinical need. Here, we reveal that a molecular pathology common to both disorders is dysregulation of activin receptors and that activin receptor signaling is required for the majority of myelin generation in development and following injury. Using a constitutive conditional knockout of all activin receptor signaling in oligodendrocyte lineage cells, we discovered this signaling to be required for myelination via regulation of oligodendrocyte differentiation and myelin compaction. These processes were found to be dependent on the activin receptor subtype Acvr2a, which is expressed during oligodendrocyte differentiation and axonal ensheathment in development and following myelin injury. During efficient myelin regeneration, Acvr2a upregulation was seen to coincide with downregulation of Acvr2b, a receptor subtype with relatively higher ligand affinity; Acvr2b was shown to be dispensable for activin receptor-driven oligodendrocyte differentiation and its overexpression was sufficient to impair the abovementioned ligand-driven responses. In actively myelinating or remyelinating areas of human perinatal brain injury and multiple sclerosis tissue, respectively, oligodendrocyte lineage cells expressing Acvr2a outnumbered those expressing Acvr2b, whereas in non-repairing lesions Acvr2b+ cells were increased. Thus, we propose that following human white matter injury, this increase in Acvr2b expression would sequester ligand and consequently impair Acvr2a-driven oligodendrocyte differentiation and myelin formation. Our results demonstrate dysregulated activin receptor signaling in common myelin disorders and reveal Acvr2a as a novel therapeutic target for myelin generation following injury across the lifespan.

Oligodendrocyte-encoded HIF function couples postnatal myelination and white matter angiogenesis.

Myelin sheaths provide critical functional and trophic support for axons in white matter tracts of the brain. Oligodendrocyte precursor cells (OPCs) have extraordinary metabolic requirements during development as they differentiate to produce multiple myelin segments, implying that they must first secure adequate access to blood supply. However, mechanisms that coordinate myelination and angiogenesis are unclear. Here, we show that oxygen tension, mediated by OPC-encoded hypoxia-inducible factor (HIF) function, is an essential regulator of postnatal myelination. Constitutive HIF1/2α stabilization resulted in OPC maturation arrest through autocrine activation of canonical Wnt7a/7b. Surprisingly, such OPCs also show paracrine activity that induces excessive postnatal white matter angiogenesis in vivo and directly stimulates endothelial cell proliferation in vitro. Conversely, OPC-specific HIF1/2α loss of function leads to insufficient angiogenesis in corpus callosum and catastrophic axon loss. These findings indicate that OPC-intrinsic HIF signaling couples postnatal white matter angiogenesis, axon integrity, and the onset of myelination in mammalian forebrain.

Parallel states of pathological Wnt signaling in neonatal brain injury and colon cancer.

In colon cancer, mutation of the Wnt repressor APC (encoding adenomatous polyposis coli) leads to a state of aberrant and unrestricted high-activity signaling. However, the relevance of high Wnt tone in non-genetic human disease is unknown. Here we demonstrate that distinct functional states of Wnt activity determine oligodendrocyte precursor cell (OPC) differentiation and myelination. Mouse OPCs with genetic Wnt dysregulation (high tone) express multiple genes in common with colon cancer, including Lef1, Sp5, Ets2, Rnf43 and Dusp4. Surprisingly, we found that OPCs in lesions of hypoxic human neonatal white matter injury upregulated markers of high Wnt activity and lacked expression of APC. We also found that lack of Wnt repressor tone promoted permanent white matter injury after mild hypoxic insult. These findings suggest a state of pathological high-activity Wnt signaling in human disease tissues that lack predisposing genetic mutation.

M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination.

The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia and macrophages, which can be polarized to distinct functional phenotypes: pro-inflammatory (M1) and anti-inflammatory or immunoregulatory (M2). We found that a switch from an M1- to an M2-dominant response occurred in microglia and peripherally derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 cell conditioned media and impaired in vivo following intra-lesional M2 cell depletion. M2 cell densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger mouse and in multiple sclerosis lesions that normally show remyelination. Blocking M2 cell-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Thus, our results indicate that M2 cell polarization is essential for efficient remyelination and identify activin-A as a therapeutic target for CNS regeneration.

Expression profiling of Aldh1l1-precursors in the developing spinal cord reveals glial lineage-specific genes and direct Sox9-Nfe2l1 interactions.

Developmental regulation of gliogenesis in the mammalian CNS is incompletely understood, in part due to a limited repertoire of lineage-specific genes. We used Aldh1l1-GFP as a marker for gliogenic radial glia and later-stage precursors of developing astrocytes and performed gene expression profiling of these cells. We then used this dataset to identify candidate transcription factors that may serve as glial markers or regulators of glial fate. Our analysis generated a database of developmental stage-related markers of Aldh1l1+ cells between murine embryonic day 13.5-18.5. Using these data we identify the bZIP transcription factor Nfe2l1 and demonstrate that it promotes glial fate under direct Sox9 regulatory control. Thus, this dataset represents a resource for identifying novel regulators of glial development.

Identification of endothelin 2 as an inflammatory factor that promotes central nervous system remyelination.

The development of new regenerative therapies for multiple sclerosis is hindered by the lack of potential targets for enhancing remyelination. The study of naturally regenerative processes such as the innate immune response represents a powerful approach for target discovery to solve this problem. By 'mining' these processes using transcriptional profiling we can identify candidate factors that can then be tested individually in clinically-relevant models of demyelination and remyelination. Here, therefore, we have examined a previously described in vivo model of the innate immune response in which zymosan-induced macrophage activation in the retina promotes myelin sheath formation by oligodendrocytes generated from transplanted precursor cells. While this model is not itself clinically relevant, it does provide a logical starting point for this study as factors that promote myelination must be present. Microarray analysis of zymosan-treated retinae identified several cytokines (CXCL13, endothelin 2, CCL20 and CXCL2) to be significantly upregulated. When tested in a cerebellar slice culture model, CXCL13 and endothelin 2 promoted myelination and endothelin 2 also promoted remyelination. In studies to identify the receptor responsible for this regenerative effect of endothelin 2, analysis of both remyelination following experimental demyelination and of different stages of multiple sclerosis lesions in human post-mortem tissue revealed high levels of endothelin receptor type B in oligodendrocyte lineage cells. Confirming a role for this receptor in remyelination, small molecule agonists and antagonists of endothelin receptor type B administered in slice cultures promoted and inhibited remyelination, respectively. Antagonists of endothelin receptor type B also inhibited remyelination of experimentally-generated demyelination in vivo. Our work therefore identifies endothelin 2 and the endothelin receptor type B as a regenerative pathway and suggests that endothelin receptor type B agonists represent a promising therapeutic approach to promote myelin regeneration.

Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination.

Permanent damage to white matter tracts, comprising axons and myelinating oligodendrocytes, is an important component of brain injuries of the newborn that cause cerebral palsy and cognitive disabilities, as well as multiple sclerosis in adults. However, regulatory factors relevant in human developmental myelin disorders and in myelin regeneration are unclear. We found that AXIN2 was expressed in immature oligodendrocyte progenitor cells (OLPs) in white matter lesions of human newborns with neonatal hypoxic-ischemic and gliotic brain damage, as well as in active multiple sclerosis lesions in adults. Axin2 is a target of Wnt transcriptional activation that negatively feeds back on the pathway, promoting ß-catenin degradation. We found that Axin2 function was essential for normal kinetics of remyelination. The small molecule inhibitor XAV939, which targets the enzymatic activity of tankyrase, acted to stabilize Axin2 levels in OLPs from brain and spinal cord and accelerated their differentiation and myelination after hypoxic and demyelinating injury. Together, these findings indicate that Axin2 is an essential regulator of remyelination and that it might serve as a pharmacological checkpoint in this process.

Analysis of A47, an immunoprevalent protein of vaccinia virus, leads to a reevaluation of the total antiviral CD8+ T cell response.

Vaccinia virus (VACV) is the prototypic orthopoxvirus and was the live vaccine used to eradicate smallpox. In addition, VACV is a possible vector for recombinant vaccines. Despite these reasons for study, the roles of many VACV genes are unknown, and some fundamental aspects, such as the total size of immune responses, remain poorly characterized. VACV gene A47L is of interest because it is highly transcribed, has no sequence similarity to any nonpoxvirus gene, and contains a larger-than-expected number of CD8(+) T cell epitopes. Here it is shown that A47L is not required for growth in vitro and does not contribute to virulence in mice. However, we confirmed that this one protein primes CD8(+) T cells to three different epitopes in C57BL/6 mice. In the process, one of these epitopes was redefined and shown to be the most dominant in A47 and one of the more highly ranked in VACV as a whole. The relatively high immunogenicity of this epitope led to a reevaluation of the total CD8(+) T cell response to VACV. By the use of two methods, the true size of the response was found to be around double previous estimates and at its peak is on the order of 60% of all CD8(+) T cells. We speculate that more CD8(+) T cell epitopes remain to be mapped for VACV and that underestimation of responses is unlikely to be unique to VACV, so there would be merit in revisiting this issue for other viruses.

Sodium channelopathy induced by mild axonal trauma worsens outcome after a repeat injury.

There is great concern that one mild traumatic brain injury (mTBI) predisposes individuals to an exacerbated response with a subsequent mTBI. Although no mechanism has been identified, mounting evidence suggests traumatic axonal injury (TAI) plays a role in this process. By using a cell culture system, a threshold of mild TAI was found where dynamic stretch of cortical axons at strains lower than 5% induced no overt pathological changes. However, the axons were found to display an increased expression of sodium channels (NaChs) by 24 hr. After a second, identical mild injury, pathologic increases in [Ca(2+)](i) were observed, leading to axon degeneration. The central role of NaChs in this response was demonstrated by blocking NaChs with tetrodotoxin prior to the second injury, which completely abolished postinjury increases in [Ca(2+)](i). These data suggest that mild TAI induces a form of sodium channelopathy on axons that greatly exaggerates the pathophysiologic response to subsequent mild injuries.

Correction of clawed hallux deformity: comparison of the Jones procedure and FHL transfer in a cadaver model.

BACKGROUND: A clawed hallux is defined as extension of the first metatarsophalangeal (MTP) joint combined with flexion of the interphalangeal (IP) joint. Two operative procedures, the modified Jones procedure and flexor hallucis longus (FHL) transfer, are indicated for correction. The purpose of this study were to evaluate the overall effectiveness of these two procedures in correcting both the clawed hallux deformity and its mechanical consequences and to compare their effect on postoperative plantar pressures. METHODS: The modified Jones procedure and FHL transfer were done on cadaver specimens that were tested before and after surgery in a specialized foot-loading frame. We quantified the angular correction of the MTP and the IP joints, as well as the plantar pressures under the head of the first metatarsal and the hallux. RESULTS: Both surgeries were equally effective in correcting the angular deformity at the MTP and IP joints (p = 0.037 and 0.0020, respectively). A significant reduction in the plantar pressure (p = 0.015) beneath the first metatarsal was observed with both the modified Jones procedure and the FHL transfer. Overall, there was no significant difference between preoperative and postoperative pressures beneath the hallux (p = 0.5); however, for the FHL overpull group there was significantly less pressure beneath the hallux after surgery (p = 0.014). CONCLUSIONS: The two surgeries produced similar results, but the FHL transfer does not require fusion of the hallux, which is considered an undesirable co-morbidity of the modified Jones procedure.

A comparison of gastrocnemius muscle-tendon unit length during gait using anatomic, cadaveric and MRI models.

Computing the length of a specific muscle is often of interest both in the study of human movement and the optimization of surgical techniques. The objective of this study was to compare three widely used methods of calculating gastrocnemius muscle-tendon unit (MTU) length. Gastrocnemius MTU length was calculated from the sagittal knee and ankle kinematics of 20 normal adults using three different methods: one based on cadaveric dissection and joint manipulation, one based on anatomic computer software modeling with insertion points obtained from cadavers and the third based on MRI measurements. All methods were normalized to percent anatomic neutral length (%ANL) with 100% at full knee extension and neutral ankle plantar-dorsiflexion. At the longest length (terminal stance), mean values (% ANL+/-standard deviation) were found to be 99.3+/-0.8, 101.8+/-0.5 and 102.4+/-0.7 for the MRI, cadaveric and anatomic methods, respectively. During the shortest length (early swing), the MRI, cadaveric and anatomic methods had mean values of 93.0+/-0.6, 92.2+/-2.0 and 91.9+/-2.1% ANL, respectively. All methods showed statistically significant differences (p<0.01) from each other in terminal stance and early swing, while loading and terminal swing lengths were similar among all methods. The gastrocnemius MTU calculated using the MRI-based method was found to be the shortest. Additional research is needed in order to validate a model for calculating gastrocnemius MTU length using in vivo methods of estimating the gastrocnemius moment arm at the knee and ankle.

The midtarsal joint locking mechanism.

BACKGROUND: The midtarsal joint, consisting of the talo-navicular and the calcaneocuboid joints, is presumed to be responsible for the foot being both flexible and rigid during different parts of the stance phase of gait. However, this mechanism has never been well quantified. This study explores the midtarsal joint locking mechanism by comparing the effect of hindfoot inversion and eversion on midfoot and forefoot mobility. METHODS: Motion of the tibia, talus, calcaneus, navicular, cuboid and the first, second, and fifth metatarsals were measured in nine cadaver feet using Polhemus Fastrak electromagnetic sensors (EST GmbH and Co. KG, Kaiserslautern, Germany). The talus was fixed to the tibia, and then the forefoot was maximally dorsiflexed, plantarflexed, inverted, and everted, with the hindfoot in maximal eversion and inversion, for a total of eight test positions. The range of motion of the individual bones between maximal forefoot dorsiflexion and plantarflexion and between maximal forefoot inversion and eversion was calculated for the hindfoot in maximal eversion and inversion. RESULTS: For the range of motion from maximal dorsiflexion to maximal plantarflexion there was significantly increased movement of the first, second, and fifth metatarsals in the sagittal plane (p-value = 0.003, 0.007, and 0.002, respectively) when the calcaneus was maximally everted compared to when the calcaneus was maximally inverted. No significant differences were detected for the range of motion from forefoot inversion to eversion for the two hindfoot positions. CONCLUSIONS: This study demonstrated that motion in the forefoot is influenced by hindfoot position through the midtarsal joint. Specifically, the sagittal plane range of motion of the metatarsals is increased when the hindfoot is in valgus.