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.

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.

TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia.

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3-IFT-A interaction suggests how these proteins cooperate during neural tube patterning.

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.

Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia.

Advancement in human induced pluripotent stem cell (iPSC) neuron and microglial differentiation protocols allow for disease modeling using physiologically relevant cells. However, iPSC differentiation and culturing protocols have posed challenges to maintaining consistency. Here, we generated an automated, consistent, and long-term culturing platform of human iPSC neurons, astrocytes, and microglia. Using this platform we generated a iPSC AD model using human derived cells, which showed signs of Aß plaques, dystrophic neurites around plaques, synapse loss, dendrite retraction, axon fragmentation, phospho-Tau induction, and neuronal cell death in one model. We showed that the human iPSC microglia internalized and compacted Aß to generate and surround the plaques, thereby conferring some neuroprotection. We investigated the mechanism of action of anti-Aß antibodies protection and found that they protected neurons from these pathologies and were most effective before pTau induction. Taken together, these results suggest that this model can facilitate target discovery and drug development efforts.

Alternative splicing controls selective trans-synaptic interactions of the neuroligin-neurexin complex.

Formation of synapses requires specific cellular interactions that organize pre- and postsynaptic compartments. The neuroligin-neurexin complex mediates heterophilic adhesion and can trigger assembly of glutamatergic and GABAergic synapses in cultured hippocampal neurons. Both neuroligins and neurexins are encoded by multiple genes. Alternative splicing generates large numbers of isoforms, which may engage in selective axo-dendritic interactions. We explored whether alternative splicing of the postsynaptic neuroligins modifies their activity toward glutamatergic and GABAergic axons. We find that small extracellular splice insertions restrict the function of neuroligin-1 and -2 to glutamatergic and GABAergic contacts and alter interaction with presynaptic neurexins. The neuroligin isoforms associated with GABAergic contacts bind to neurexin-1alpha and a subset of neurexin-1betas. In turn, these neurexin isoforms induce GABAergic but not glutamatergic postsynaptic differentiation. Our findings suggest that alternative splicing plays a central role in regulating selective extracellular interactions through the neuroligin-neurexin complex at glutamatergic and GABAergic synapses.

CRISPR whole-genome screening identifies new necroptosis regulators and RIPK1 alternative splicing.

The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.

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.

Discovery of Novel Blood-Brain Barrier Targets to Enhance Brain Uptake of Therapeutic Antibodies.

The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies for neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aß reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.

Is reelin the answer to synapse elimination at the neuromuscular junction?

The formation of mature neuronal circuits during development involves elimination of a large number of synapses by activity-dependent processes. A recent study suggests that synapse elimination at the neuromuscular junction is impaired in reeler mutant mice, which are lacking the extracellular matrix protein Reelin. In this process, Reelin acts through an unexpected, proteolytic mechanism that is independent of Disabled 1, a cytoplasmic factor that mediates Reelin signaling in the central nervous system. This Perspective discusses possible models for Reelin function in the framework of activity-dependent synapse elimination at the neuromuscular junction.

A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain.

Using RNAi screening, proteomics, cell biological and mouse genetics approaches, we have identified a complex of nine proteins, seven of which are disrupted in human ciliopathies. A transmembrane component, TMEM231, localizes to the basal body before and independently of intraflagellar transport in a Septin 2 (Sept2)-regulated fashion. The localizations of TMEM231, B9D1 (B9 domain-containing protein 1) and CC2D2A (coiled-coil and C2 domain-containing protein 2A) at the transition zone are dependent on one another and on Sept2. Disruption of the complex in vitro causes a reduction in cilia formation and a loss of signalling receptors from the remaining cilia. Mouse knockouts of B9D1 and TMEM231 have identical defects in Sonic hedgehog (Shh) signalling and ciliogenesis. Strikingly, disruption of the complex increases the rate of diffusion into the ciliary membrane and the amount of plasma-membrane protein in the cilia. The complex that we have described is essential for normal cilia function and acts as a diffusion barrier to maintain the cilia membrane as a compartmentalized signalling organelle.

Functional characterization of putative cilia genes by high-content analysis.

Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates.

The diacylglycerol-binding protein alpha1-chimaerin regulates dendritic morphology.

The morphological and functional differentiation of neuronal dendrites is controlled through transcriptional programs and cell-cell signaling. Synaptic activity is thought to play an important role in the maturation of dendritic arbors, but the signaling pathways that couple neuronal activity and morphological changes in dendrites are not well understood. We explored the function of alpha1-chimaerin, a neuronal diacylglycerol-binding protein with a Rho GTPase-activating protein domain that inactivates Rac1. We find that stimulation of phospholipase Cbeta-coupled cell surface receptors recruits alpha1-chimaerin to the plasma membrane of cultured hippocampal neurons. We further show that alpha1-chimaerin protein levels are controlled by synaptic activity and that increased alpha1-chimaerin expression results in the pruning of dendritic spines and branches. This pruning activity requires both the diacylglycerol-binding and Rac GTPase-activating protein activity of alpha1-chimaerin. Suppression of alpha1-chimaerin expression resulted in increased process growth from the dendritic shaft and from spine heads. Our data suggest that alpha1-chimaerin is an activity-regulated Rho GTPase regulator that is activated by phospholipase Cbeta-coupled cell surface receptors and contributes to pruning of dendritic arbors.

Control of excitatory and inhibitory synapse formation by neuroligins.

The normal function of neural networks depends on a delicate balance between excitatory and inhibitory synaptic inputs. Synapse formation is thought to be regulated by bidirectional signaling between pre- and postsynaptic cells. We demonstrate that members of the Neuroligin family promote postsynaptic differentiation in cultured rat hippocampal neurons. Down-regulation of neuroligin isoform expression by RNA interference results in a loss of excitatory and inhibitory synapses. Electrophysiological analysis revealed a predominant reduction of inhibitory synaptic function. Thus, neuroligins control the formation and functional balance of excitatory and inhibitory synapses in hippocampal neurons.

Disorder-associated mutations lead to functional inactivation of neuroligins.

Autism is a neuro-developmental syndrome that affects 0.1-0.5% of the population. It has been proposed that alterations in neuronal circuitry and/or neuronal signaling are responsible for the behavioral and cognitive aberrations in autism patients. However, the cellular basis of such alterations is unknown. Recently, point mutations in a family of neuronal cell adhesion molecules called neuroligins have been linked to autism-spectrum disorders and mental retardation. We investigated the consequences of these disease-associated mutations on neuroligin function. We demonstrate that the point mutation at arginine 451 and a nonsense mutation at aspartate 396 of neuroligin-3 and -4 (NL3 and NL4), respectively, result in intracellular retention of the mutant proteins. Over-expression of wild-type NL3 and NL4 proteins in hippocampal neurons stimulates the formation of presynaptic terminals, whereas the disease-associated mutations result in a loss of this synaptic function. Our findings suggest that the previously identified mutations in neuroligin genes are likely to be relevant for the neuro-developmental defects in autism-spectrum disorders and mental retardation since they impair the function of a synaptic cell adhesion molecule.