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.

TPL2 kinase activity regulates microglial inflammatory responses and promotes neurodegeneration in tauopathy mice.

Tumor progression locus 2 (TPL2) (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates microglia activation states and brain cytokine levels. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNA sequencing analysis indicates that protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity can promote multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS (inducible nitric oxide synthase) induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions.

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection.

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

Nav1.7 is essential for nociceptor action potentials in the mouse in a manner independent of endogenous opioids.

Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.

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.

Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease.

Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.

Complement C1q-dependent excitatory and inhibitory synapse elimination by astrocytes and microglia in Alzheimer's disease mouse models.

Microglia and complement can mediate neurodegeneration in Alzheimer's disease (AD). By integrative multi-omics analysis, here we show that astrocytic and microglial proteins are increased in TauP301S synapse fractions with age and in a C1q-dependent manner. In addition to microglia, we identified that astrocytes contribute substantially to synapse elimination in TauP301S hippocampi. Notably, we found relatively more excitatory synapse marker proteins in astrocytic lysosomes, whereas microglial lysosomes contained more inhibitory synapse material. C1q deletion reduced astrocyte-synapse association and decreased astrocytic and microglial synapses engulfment in TauP301S mice and rescued synapse density. Finally, in an AD mouse model that combines ß-amyloid and Tau pathologies, deletion of the AD risk gene Trem2 impaired microglial phagocytosis of synapses, whereas astrocytes engulfed more inhibitory synapses around plaques. Together, our data reveal that astrocytes contact and eliminate synapses in a C1q-dependent manner and thereby contribute to pathological synapse loss and that astrocytic phagocytosis can compensate for microglial dysfunction.

Genetic ablation of Gpnmb does not alter synuclein-related pathology.

The gene GPNMB is known to play roles in phagocytosis and tissue repair, and is upregulated in microglia in many mouse models of neurodegenerative disease as well as in human patients. Nearby genomic variants are associated with both elevated Parkinson's disease (PD) risk and higher expression of this gene, suggesting that inhibiting GPNMB activity might be protective in Parkinson's disease. We tested this hypothesis in three different mouse models of neurological diseases: a remyelination model and two models of alpha-synuclein pathology. We found that Gpnmb deletion had no effect on histological, cellular, behavioral, neurochemical or gene expression phenotypes in any of these models. These data suggest that Gpnmb does not play a major role in the development of pathology or functional defects in these models and that further work is necessary to study its role in the development or progression of Parkinson's disease.

Antibody semorinemab reduces tau pathology in a transgenic mouse model and engages tau in patients with Alzheimer's disease.

Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.

Genetic inactivation of RIP1 kinase activity in rats protects against ischemic brain injury.

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.

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.

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.

Genetic inactivation of RIP1 kinase does not ameliorate disease in a mouse model of ALS.

RIP1 kinase is proposed to play a critical role in driving necroptosis and inflammation in neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). Preclinical studies indicated that while pharmacological inhibition of RIP1 kinase can ameliorate axonal pathology and delay disease onset in the mutant SOD1 transgenic (SOD1-Tg) mice, genetic blockade of necroptosis does not provide benefit in this mouse model. To clarify the role of RIP1 kinase activity in driving pathology in SOD1-Tg mice, we crossed SOD1-Tgs to RIP1 kinase-dead knock-in mice, and measured disease progression using functional and histopathological endpoints. Genetic inactivation of the RIP1 kinase activity in the SOD1-Tgs did not benefit the declining muscle strength or nerve function, motor neuron degeneration or neuroinflammation. In addition, we did not find evidence of phosphorylated RIP1 accumulation in the spinal cords of ALS patients. On the other hand, genetic inactivation of RIP1 kinase activity ameliorated the depletion of the neurotransmitter dopamine in a toxin model of dopaminergic neurodegeneration. These findings indicate that RIP1 kinase activity is dispensable for disease pathogenesis in the SOD1-Tg mice while inhibition of kinase activity may provide benefit in acute injury models.

Global proteomics of Ubqln2-based murine models of ALS.

Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.

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.

Ubiquitin Ligase COP1 Suppresses Neuroinflammation by Degrading c/EBPß in Microglia.

Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPß) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPß in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPß accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPß-dependent gene expression programs in microglia.

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.

Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

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.