Microglial-derived C1q integrates into neuronal ribonucleoprotein complexes and impacts protein homeostasis in the aging brain.

Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.

CSMD1 regulates brain complement activity and circuit development.

Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.

Convergent mechanisms of microglia-mediated synaptic dysfunction contribute to diverse neuropathological conditions.

Changes in synaptic function are an early hallmark of neuropathological conditions that often precede symptom onset, with mounting genetic, transcriptional, and epidemiological evidence implicating microglia in this process. The correlation between infection and neurocognitive sequelae further suggests that environmental exposures modulate neuroimmune interactions and contribute to synaptic alterations. Recent studies investigating functional roles of microglia across broad neuropathological contexts including neurodegeneration, aging, neuropsychiatric and neurodevelopmental disorders, and neurotropic infections reveal convergent mechanisms underlying microglial-mediated synaptic dysfunction. We propose that early microglial changes, driven by genetic alterations coupled with environmental neuroimmune modulation, may be a common denominator that contributes to early synaptic pathologies. Here we review the evidence and discuss how microglia respond, and contribute, to synaptopathies across diverse neurological conditions, spotlighting their importance as broadly relevant therapeutic targets within neurological diseases.

Engulfed by Glia: Glial Pruning in Development, Function, and Injury across Species.

Phagocytic activity of glial cells is essential for proper nervous system sculpting, maintenance of circuitry, and long-term brain health. Glial engulfment of apoptotic cells and superfluous connections ensures that neuronal connections are appropriately refined, while clearance of damaged projections and neurotoxic proteins in the mature brain protects against inflammatory insults. Comparative work across species and cell types in recent years highlights the striking conservation of pathways that govern glial engulfment. Many signaling cascades used during developmental pruning are re-employed in the mature brain to "fine tune" synaptic architecture and even clear neuronal debris following traumatic events. Moreover, the neuron-glia signaling events required to trigger and perform phagocytic responses are impressively conserved between invertebrates and vertebrates. This review offers a compare-and-contrast portrayal of recent findings that underscore the value of investigating glial engulfment mechanisms in a wide range of species and contexts.

Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia.

Neuronal circuit assembly requires the fine balance between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized as key players, the neuronal molecular components that specify synapses to be eliminated are still undefined. Here, we show that exposed phosphatidylserine (PS) represents a neuronal "eat-me" signal involved in microglial-mediated pruning. In hippocampal neuron and microglia co-cultures, synapse elimination can be partially prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, PS exposure at both hippocampal and retinogeniculate synapses and engulfment of PS-labeled material by microglia occurs during established developmental periods of microglial-mediated synapse elimination. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, have elevated presynaptic PS exposure and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial-mediated synapse pruning and identify a novel role of developmentally regulated neuronal PS exposure that is common among developing brain structures.

Heterozygous carriers of galactocerebrosidase mutations that cause Krabbe disease have impaired microglial function and defective repair of myelin damage.

This review addresses two puzzling findings related to mutations in galactocerebrosidase (GALC) that cause Krabbe disease (KD), a severe lysosomal storage disorder characterized by extensive myelin damage in children with mutations in both GALC alleles. First, heterozygous carriers of KD-causing mutations, which include the biological parents of children with KD, exhibit increased risk for developing other diseases. Second, variants in the GALC locus increase the risk of developing multiple sclerosis (MS), another disease characterized by extensive myelin damage. What explains these correlations? In studies on cuprizone-induced myelin damage in heterozygous (GALC+/-) mice carrying one copy of a mutation that causes KD-like disease, the extent of damage was similar in GALC+/- and wild-type (WT) mice. In contrast, GALC+/- mice had striking defects in repair of cuprizone-induced damage. We further found unexpected microglial defects in myelin debris clearance and in the ability to up-regulate the Trem2 microglial protein critical for debris uptake. These defects were rescued by exposure to a lysosomal re-acidifying drug discovered in our studies on KD, and which provides multiple clinically relevant benefits in the twitcher (GALC+/-) mouse model of KD. Thus, heterozygous GALC mutations cause effects on biological function that may help to understand the increased disease risk in heterozygous carriers of such mutations and to understand why GALC variations increase the risk of MS. Our findings indicate that while some genetic risk factors may contribute to complex diseases by increasing the risk of tissue damage, others may do so by compromising tissue repair.

Heterozygote galactocerebrosidase (GALC) mutants have reduced remyelination and impaired myelin debris clearance following demyelinating injury.

Genome-wide association studies are identifying multiple genetic risk factors for several diseases, but the functional role of these changes remains mostly unknown. Variants in the galactocerebrosidase (GALC) gene, for example, were identified as a risk factor for Multiple Sclerosis (MS); however, the potential biological relevance of GALC variants to MS remains elusive. We found that heterozygote GALC mutant mice have reduced myelin debris clearance and diminished remyelination after a demyelinating insult. We found no histological or behavioral differences between adult wild-type and GALC +/- animals under normal conditions. Following exposure to the demyelinating agent cuprizone, however, GALC +/- animals had significantly reduced remyelination during recovery. In addition, the microglial phagocytic response and elevation of Trem2, both necessary for clearing damaged myelin, were markedly reduced in GALC +/- animals. These altered responses could be corrected in vitro by treatment with NKH-477, a compound discovered as protective in our previous studies on Krabbe disease, which is caused by mutations in both GALC alleles. Our data are the first to show remyelination defects in individuals with a single mutant GALC allele, suggesting such carriers may have increased vulnerability to myelin damage following injury or disease due to inefficient myelin debris clearance. We thus provide a potential functional link between GALC variants and increased MS susceptibility, particularly due to the failure of remyelination associated with progressive MS. Finally, this work demonstrates that genetic variants identified through genome-wide association studies may contribute significantly to complex diseases, not by driving initial symptoms, but by altering repair mechanisms.

Lysosomal Re-acidification Prevents Lysosphingolipid-Induced Lysosomal Impairment and Cellular Toxicity.

Neurodegenerative lysosomal storage disorders (LSDs) are severe and untreatable, and mechanisms underlying cellular dysfunction are poorly understood. We found that toxic lipids relevant to three different LSDs disrupt multiple lysosomal and other cellular functions. Unbiased drug discovery revealed several structurally distinct protective compounds, approved for other uses, that prevent lysosomal and cellular toxicities of these lipids. Toxic lipids and protective agents show unexpected convergence on control of lysosomal pH and re-acidification as a critical component of toxicity and protection. In twitcher mice (a model of Krabbe disease [KD]), a central nervous system (CNS)-penetrant protective agent rescued myelin and oligodendrocyte (OL) progenitors, improved motor behavior, and extended lifespan. Our studies reveal shared principles relevant to several LSDs, in which diverse cellular and biochemical disruptions appear to be secondary to disruption of lysosomal pH regulation by specific lipids. These studies also provide novel protective strategies that confer therapeutic benefits in a mouse model of a severe LSD.