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Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation

Graphical Abstract

Highlights d Progranulin regulates lysosomal function and complements

production in microglia

d Grn!/! microglia preferentially eliminates inhibitory synapse

in ventral thalamus

d Grn!/! mice exhibit hyperexcitability in ventral thalamus and

OCD-like behaviors

d Loss of C1qa mitigates neurodegeneration and improves

survival in Grn!/! mice

Authors Hansen Lui, Jiasheng Zhang,

Stefanie R. Makinson, ..., Jeanne T. Paz,

Ben A. Barres, Eric J. Huang

Correspondence eric.huang2@ucsf.edu

In Brief Loss of progranulin, which occurs in

patients with frontotemporal dementia,

causes lysosomal defects and excessive

complement production, triggering

selective synaptic pruning by microglia

and behavioral deficits that can be

rescued by blocking complement

activation.

Accession Numbers GSE75083

Lui et al., 2016, Cell 165, 921–935 May 5, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2016.04.001

mailto:eric.huang2@ucsf.edu

http://dx.doi.org/10.1016/j.cell.2016.04.001

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Article

Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation Hansen Lui,1,18 Jiasheng Zhang,1 Stefanie R. Makinson,2 Michelle K. Cahill,1 Kevin W. Kelley,3 Hsin-Yi Huang,1,19

Yulei Shang,1 Michael C. Oldham,4 Lauren Herl Martens,5 Fuying Gao,6 Giovanni Coppola,6 Steven A. Sloan,7

Christine L. Hsieh,8 Charles C. Kim,9,20 Eileen H. Bigio,10 Sandra Weintraub,10 Marek-Marsel Mesulam,10

Rosa Rademakers,11 Ian R. Mackenzie,12 William W. Seeley,13 Anna Karydas,13 Bruce L. Miller,13 Barbara Borroni,14

Roberta Ghidoni,15 Robert V. Farese, Jr.,16 Jeanne T. Paz,2 Ben A. Barres,7 and Eric J. Huang1,17,* 1Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA 2Gladstone Institute of Neurological Disease and Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA 3Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA 4Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA 94158, USA 5FORUM Pharmaceuticals, Waltham, MA 02451, USA 6Semel Institute for Neuroscience and Human Behaviors, University of California, Los Angeles, Los Angeles, CA 90095, USA 7Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA 8Immunology Section, San Francisco VA Medical Center and Department of Medicine, University of California, San Francisco, San Francisco, CA 94121, USA 9Department of Medicine, University of California, San Francisco, San Francisco, CA 94110, USA 10Northwestern Alzheimer Disease Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA 11Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA 12Department of Pathology, University of British Columbia and Vancouver General Hospital, Vancouver, BC V5Z 1M9, Canada 13Department of Neurology and Memory and Aging Center, University of California, San Francisco, San Francisco, CA 94158, USA 14Centre for Ageing Brain and Neurodegenerative Disorders, Neurology Unit, University of Brescia, Brescia 25100, Italy 15Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio, Fatebenefratelli, Brescia 25100, Italy 16Department of Genetics and Complex Diseases, School of Public Health, Harvard University, Boston, MA 02115, USA 17Pathology Service (113B), San Francisco VA Medical Center, San Francisco, CA 94121, USA 18Present address: UC Berkeley-UCSF Joint Medical Program (JMP), School of Public Health, Berkeley, CA 94720, USA 19Present address: Department of Pathology, National Taiwan University Hospital, Taipei 10002, Taiwan 20Present address: Verily, Mountain View, CA 94043, USA *Correspondence: eric.huang2@ucsf.edu http://dx.doi.org/10.1016/j.cell.2016.04.001

SUMMARY

Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, pro- gressive upregulation of lysosomal and innate immu- nity genes, increased complement production, and enhanced synaptic pruning in microglia. During ag- ing, Grn!/! mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-com- pulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly re- duces synaptic pruning by Grn!/! microglia and mit- igates neurodegeneration, behavioral phenotypes, and premature mortality in Grn!/! mice. Together, our results uncover a previously unrecognized role

of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neu- rodegeneration caused by progranulin deficiency.

INTRODUCTION

Microglia are innate immune cells that repair injury and maintain homeostasis in the CNS (Ransohoff and Perry, 2009). Previous studies indicate that microglia employ a diverse repertoire of proteins in the innate immune system to regulate synapse forma- tion and maintenance. For example, in early postnatal life, micro- glia use the classical complement pathway to regulate synapse development in the lateral geniculate nucleus (Schafer et al., 2012; Stevens et al., 2007). During the aging process, however, progressive accumulation of complement C1qa in the dentate gyrus of hippocampus promotes cognitive decline and memory impairments (Stephan et al., 2013). In contrast, loss of comple- ment C3 protein protects against age-dependent declines in synaptic and dendritic spine density in the CA3 region of the

Cell 165, 921–935, May 5, 2016 ª2016 Elsevier Inc. 921

mailto:eric.huang2@ucsf.edu

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hippocampus and rescues attenuation of long-term potentiation (LTP) (Shi et al., 2015). In addition, microglia also use fractalkine receptor CX3CR1 to regulate the growth and maintenance of dendritic spines on hippocampal neurons, which in turn serve as the structural basis of synapse formation (Paolicelli et al., 2011). Finally, genetic ablation of microglia in the adult brain further reveals the essential role of microglia in the maintenance of synaptic functions and motor learning (Parkhurst et al., 2013). Given these results, it has been proposed that excessive mi- croglial activation may contribute to the pathogenesis of neuro- degenerative diseases (Aguzzi et al., 2013). However, whether microglial activation directly contributes to neurodegeneration remains unclear.

In this study, we investigate the role of aberrant microglial activation as the primary pathogenic factor for frontotemporal dementia (FTD), the second most common dementia affecting patients younger than 65 years of age (Rascovsky et al., 2011; Ratnavalli et al., 2002). We focus on the autosomal dominant mu- tations in the human progranulin (GRN) gene, which cause a drastic reduction in progranulin (PGRN) levels, contributing to the pathogenesis of one the most common forms of familial fron- totemporal lobar degeneration (FTLD) (Baker et al., 2006; Cruts et al., 2006; Finch et al., 2009; Ghidoni et al., 2008; Sleegers et al., 2009). Several studies indicate that PGRN is a key regu- lator of inflammation and that PGRN deficiency causes an aber- rant increase in phagocytosis and pro-inflammatory cytokine production in microglia and macrophages (Kao et al., 2011; Mar- tens et al., 2012; Yin et al., 2010). Furthermore, when exposed to the neurotoxin MPTP, both global Grn knockout (Grn!/!) and mi- croglia-specific Grn knockout (Cd11b-Cre;Grnfl/fl) mutant mice show a much more robust increase in microglial activation and neuronal loss (Martens et al., 2012), supporting the idea that PGRN is required to suppress excessive microglial activation. Finally, rare homozygous GRN mutations in humans have been shown to cause neuronal ceroid lipofuscinosis (NCL), which shares similar neuropathological features with FTLD patients with GRN mutations (Götzl et al., 2014; Smith et al., 2012). The connection between FTLD caused by GRN mutations and NCL is intriguing because neuroinflammation is a prominent and consistent feature in animal models of NCL and lysosomal stor- age diseases (Castaneda et al., 2008; Cotman et al., 2013). Together, these data suggest that PGRN deficiency may lead to a spectrum of neurodegenerative conditions in a dose-depen- dent manner.

Despite evidence supporting the role of PGRN in microglia function, it remains unclear how PGRN deficiency causes microglia activation and how PGRN-deficient microglia con- tribute to neurodegeneration in the aging brain. It is equally unclear whether blocking microglia activation in chronic PGRN deficiency could mitigate neurodegeneration. Here, we show that loss of PGRN causes an age-dependent upre- gulation of lysosomal and innate immunity genes in microglia, which increases complement production and synaptic pruning activity by microglia to preferentially eliminate inhibitory synapses in the ventral thalamus. These defects lead to hyperexcitability in the thalamocortical circuits and obses- sive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, blocking complement activation significantly re-

duces synaptic pruning by Grn!/! microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn!/! mice. These results uncover a previously unrecognized role of PGRN in suppressing microglia activa- tion and support an important conceptual advance that com- plement activation and microglia activation are major drivers, rather than consequences, of neurodegeneration caused by PGRN deficiency.

RESULTS

Transcriptional Profiling Reveals Age-Dependent Lysosomal and Innate Immunity Defects in Grn!/!

Microglia To investigate how PGRN deficiency contributes to neurodegen- eration during aging, we analyzed the transcriptomes of the ce- rebral cortex, hippocampus, and cerebellum in 2-, 6-, 9-, 12-, and 18-month-old Grn+/+, Grn+/!, and Grn!/! mice. We used weighted correlation network analysis (WGCNA) to identify highly correlated gene modules that were either up- or downre- gulated in an age-dependent and genotype-specific manner (Figure 1A) (Langfelder and Horvath, 2008; Zhang and Horvath, 2005). Principal component analyses showed no difference in the transcriptomes between Grn+/+ and Grn+/! brain regions, but revealed an age-dependent upregulation of one particular gene module in the cerebral cortex (magenta module) (Fig- ure 1B), hippocampus (purple module), and cerebellum (pink module) of Grn!/! mice (Figure S1A). DAVID gene ontology (GO) analyses of these modules revealed two major categories, the lytic vacuole genes in the lysosomal pathway and genes related to innate immune responses (Table S1). Compared to an independent cell-type enrichment dataset (Zhang et al., 2014), genes in the magenta module (cerebral cortex) showed exclusive association with the microglial lineage (p = 6.52 3 10!14) (Figure 1C, inset). System-level analyses of the top 40 genes from magenta, purple, and pink modules revealed exten- sive topographical overlap (Figures 1C, S1B, and S1C), support- ing functional interactions among these genes. At the center of this interconnected network were complement genes (C1qa, C1qb, C1qc, and C3), CD68, and Trem2, which have been implicated in innate immunity, lysosomal function, and microglial activation (Wang et al., 2015), respectively. These results are consistent with the cell-type enrichment transcriptome data, which shows that Grn mRNA is >50-fold enriched in microglia (>881.4 fragments per kilobase of exon per million fragments mapped [FPKM]) (Figure S1D) (Zhang et al., 2014) and support that microglia contribute to the age-dependent transcriptional upregulation of lysosomal and innate immunity genes in the ag- ing Grn!/! brain. To validate the brain region-specific transcriptome profiling re-

sults, we isolated microglia from 4- and 16-month-old Grn+/+ and Grn!/! mice using Percoll gradients and fluorescence-activated cell sorting (FACS) with microglia/macrophage/neutrophil markers, CD11b PE, CD45 APC, and anti-Ly6G PE-Cy7 (Fig- ure 1D). This approach revealed a 4-fold increase in the relative abundance of microglia (defined as CD45lo;CD11b+, pink gate in Figures 1E and 1F) in 16-month-old Grn!/! mice but no detectable increase in macrophage number (defined as

922 Cell 165, 921–935, May 5, 2016

CD45hi;CD11b+;Ly6G!, green gate in Figure 1E). We then analyzed the transcriptional profiles of FACS-sorted 4- and 16- month-old Grn+/+ and Grn!/! microglia using principle compo- nent analysis of the top 500 most variable transcripts and showed that the 4-month-old Grn+/+ and Grn!/! microglia replicates clus- tered closely, suggesting that loss of PGRN had only modest ef- fects on the transcriptional profiles of microglia at this age (Fig- ure 1G). In contrast, 16-month-old Grn!/! microglia showed much more profound alterations in transcriptional profiles than 16-month-old Grn+/+ microglia (Figure 1G). Hierarchical clus- tering analyses of the top 200 transcripts confirmed that the tran- scriptional profiles of Grn+/+ and Grn!/! microglia were similar at 4 months old, but became drastically different at 16 months old (Figure 1H). Together, these results support that PGRN is required to suppress aberrant microglial activation during aging.

Characterizations of PGRN in the Endolysosomal Pathway in Microglia To characterize how microglia contribute to the upregulation of lysosomal and innate immune response genes in Grn!/! brain, we analyzed lysosomal morphology in the microglia of 4- and 18-month-old Grn+/+ and Grn!/! brains. Using CD68 as a marker for lysosomes and Iba-1 for microglia, we found that Grn!/! mi- croglia showed a marked increase in lysosomal size at 18 months old (Figures 2A–2E). Although microglia in 4-month-old Grn+/+

and Grn!/! brains showed no difference in the lysosomal size, treatment with neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahy- dropyridine (MPTP) induced a significant increase in the size of CD68+ lysosomes in Grn!/! microglia (Figure 2E). Similar MPTP-induced increase in lysosomal size was also detected in the microglia of Cd11b-Cre;Grnf/f mutants, suggesting that the

Figure 1. Transcriptome Profiling in Grn+/+, Grn+/!, and Grn!/! Mice Reveal Age-Dependent Upregulation of Lysosomal and Innate Immunity Genes in Microglia (A) Diagram showing the procedures to characterize the transcriptomes of specific brain regions in Grn+/+, Grn+/!, and Grn!/! mice during aging.

(B) Weighted correlation network analysis (WGCNA) identifies highly correlated gene modules that are age-dependently upregulated in the cerebral cortex

of Grn!/! mice.

(C) The top 40 genes from the magenta (cerebral cortex) module are highly enriched with microglial genes (inset), and there is extensive topographical overlap

among their expression patterns, especially for complements C1qa, C1qb, C1qc and C3, Cd68, and Trem2.

(D) Diagram showing the procedures to isolate microglia from 4- and 16-month-old Grn+/+ and Grn!/! mice using discontinuous isotonic Percoll gradient, FACS,

preparation of amplified antisense RNA (aaRNA), microarray, and bioinformatics analyses.

(E) Dissociated cells from 16-month-old Grn+/+ and Grn!/! mice are incubated with CD11b PE (clone M1/70) (Invitrogen), CD45 APC (clone Ly5) (eBioscience),

and anti-Ly6G PE-Cy7 (clone 1A8) (BD Biosciences), and sorted by Beckman-Coulter MoFlo XDPs flow cytometer.

(F) Microglia are defined as CD45lo;CD11b+, whereas macrophages are defined as CD45hi;CD11b+ population in FACS. Data are presented as % of total events.

Student’s t test, n = 3 for Grn+/+ and Grn!/! mice. **p < 0.01; ns, not significant.

(G) Principle component analysis of the top 500 most variable transcripts from the FACS-sorted microglia from 4- and 16-month-old Grn+/+ and Grn!/! mice.

(H) Hierarchical clustering analyses of the top 200 transcripts from FACS-sorted microglia from 4- and 16-month-old Grn+/+ and Grn!/! mice.

See also Figure S3.

926 Cell 165, 921–935, May 5, 2016

mouse brains are more severely affected by the increases incom- plements and microglia and how this might contribute to neuro- degeneration. To test this, we performed qRT-PCR using mRNA from cerebral cortex, hippocampus, caudate/putamen, cerebellum, and thalamus from 12-month-old Grn+/+ and Grn!/!

brains and showed that C1qa and C3 mRNA were most abundant in the thalamus in Grn!/! brains, with an age-dependent increase (Figures 4A–4D). In addition, western blots showed that both C1qa and C3 cleavage product iC3b were age-dependently up- regulated in the thalamus of Grn!/! mice (Figures 4E and 4F). Given the role of complements in synaptic pruning, we asked

whether C1qa and C3 were deposited near or at the synapses in vivo. To address this, we performed immunostains and showed very low C1qa signal in the ventral thalamus of Grn+/+

mice at 4 and 18 months old (Figures 4G and 4H). In contrast, C1qa staining intensity showed a modest increase around the ventral thalamic nuclei of Grn!/! mice at 4 months old and a much more prominent increase at 18 months old (Figures 4I and 4J). Whereas no detectable C1qa was found in the cell body of Grn+/+ or Grn!/! thalamic neurons, abundant C1qa was found in the neuropil surrounding the cell bodies and pro- cesses of Grn!/! neurons (Figures 4K–4P). To test if C1qa was deposited near or at synapses in the

ventral thalamus of Grn!/! brain, we performed double labeling using antibodies for C1qa and the presynaptic marker synapto- physin (SPH). Consistent with our prediction, most C1qa signals were in close proximity to SPH+ puncta in the thalamus of Grn!/!

mice. No detectable C1qa signals were found near the synapses in Grn+/+ brain (Figures 4Q–4V). In addition, immunogold electron microscopy (IEM) showed that C1qa was immediately adjacent to synapses in Grn!/! thalamus, but not in Grn+/+ thalamus (Fig- ures 4W and 4X). Finally, to provide biochemical evidence for complement deposition at the synapses of Grn!/! mice, we used discontinuous sucrose gradient to isolate synaptosomes from 4-, 9-, and 16-month-old Grn+/+ and Grn!/! mouse brains (Carlin et al., 1980) and showed an age-dependent increase in C1qa and multiple C3 cleavage products in the synaptosomes from Grn!/! brain (Figure 4Y).

Removing C1qa in Grn!/!;C1qa!/! Mice Protects Synapse Loss, Restores Thalamic Microcircuit Function, Mitigates OCD-like Behaviors, and Improves Survival To determine whether synapse loss is a major phenotype in Grn!/! mouse brain and whether C1qa removal might protect synapse loss, we established an aging cohort of Grn+/+, Grn!/!, C1qa!/!, and Grn!/!;C1qa!/! mice and examined microgliosis and synaptic density in the ventral thalamus from 2 to 19 months old. During aging, the number of microglia in the ventral thalamus of Grn+/+ and C1qa!/! mice only showed a very modest in- crease, with inconspicuous cytoplasm and thin, delicate pro- cesses, consistent with resting, quiescent microglia morphology (Figures 5A, 5B, 5E, 5F, and 5I). In contrast, Grn!/! mice showed an age-dependent increase in microglia in the ventral thalamus, and the majority of Grn!/! microglia exhibited abundant cyto- plasm with short, prominent processes consistent with reactive microglia (Figures 5C, 5D, and 5I). Interestingly, compared to Grn!/! mice, Grn!/!;C1qa!/! mice showed a consistent and

significant reduction in the number of microglia in the ventral thalamus at 7, 12, and 19 months old (p = 0.0006, two-way ANOVA) (Figures 5G–5I). Furthermore, Grn!/!;C1qa!/! micro- glia showed mixed morphology similar to Grn+/+ or Grn!/! micro- glia (Figure 5H). Using SPH as a marker for synaptic density, we found no detectable loss of synapse in the ventral thalamus of Grn+/+ and C1qa!/! mice during aging (Figures 5J, 5L, and 5N). In contrast, Grn!/! mice showed significant reductions in SPH density in the ventral thalamus at 4, 7, 12, and 19 months old, whereas SPH density in Grn!/!;C1qa!/! mice was almost completely preserved (Figures 5K, 5M, and 5N). These results are consistent with data from microglia-neuron co-cultures and support the essential role of C1qa in mediating synaptic pruning by Grn!/! microglia in vivo. Given the progressive loss of SPH in the ventral thalamus of

Grn!/! mice, we asked whether synaptic pruning preferentially affects excitatory or inhibitory synapses. Within the ventral thal- amus, neurons in the ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei project to layer IV of somatosensory cortex and receive excitatory inputs from neurons in layer VI (Fig- ures S4A and S4B). The sole source of inhibition to this recipro- cally excitatory circuit comes from parvalbumin-positive (Parv+) inhibitory neurons in the thalamic reticular nuclei (TRN), which co-express vesicular GABA transporter VGAT (Figures 6A, 6A0, and S4C–S4J). To investigate whether C1qa protein deposits in the ventral thalamus of Grn!/! mice can perturb the balance of excitatory and inhibitory synaptic inputs, we used confocal im- ages to show that C1qa proteins extensively surrounded both VGLUT2+ excitatory and VGAT+ inhibitory synapses in the ventral thalamus of Grn!/! mice (Figures S5A–S5D). Although C1qa deposits showed no preference for excitatory or inhibitory synapses, the number of Parv+ synapses in the VPM and VPL of 12-month-old Grn!/! mice was significantly reduced (Figures 6B and 6B0). Many Parv+ synapses were in close proximity to Iba1+

microglial processes, suggesting that Grn!/! microglia were actively pruning these synapses. Similar to these data, the num- ber of VGAT+ synapses also showed significant reduction in the ventral thalamus of Grn!/! mice from 8 to 19 months old (Figures 6E, 6F, and 6I). In contrast, the number of VGAT+ synapses in Grn!/!;C1qa!/! mice showed complete preservation (Figures 6G–6I). Remarkably, unlike the VGAT+ synaptic phenotype, the number of VGLUT2+ excitatory synapses showed no reduction in Grn!/!, C1qa!/!, or Grn!/!;C1qa!/! mice (Figures S5E–S5I). To characterize how loss of inhibitory synapses in the ventral

thalamus of Grn!/! mice might alter circuit function, we per- formed multiunit array extracellular recording in VPM and VPL in freshly prepared brain slices from 12-month-old Grn+/+ and Grn!/! mice (Figure 6J). This approach preserves an intact intra-thalamic circuit and has been instrumental in determining the thalamic function in rodents (Paz et al., 2011, 2013). In the ventral thalamus of Grn+/+ mice, electrical stimulation of the in- ternal capsule generated occasional bursts of action potentials (AP) (Figure 6K). In contrast, the same stimulation in the ventral thalamus of Grn!/! brain slices evoked a long-lasting and sus- tained tonic firing, with significant increase in AP firing frequency and the relative probability of eliciting AP (p < 0.0001) (Figure 6L). Remarkably, loss of C1qa in Grn!/!;C1qa!/! mice completely reversed the hyperexcitability phenotype observed in the Grn!/!

Cell 165, 921–935, May 5, 2016 927

Figure 4. Age-Dependent C1qa Accumulation at the Synapses of the Ventral Thalamus in Grn!/! Mice (A and B) qRT-PCR detects the relative abundance of C1qa (A) and C3 (B) mRNA in cerebral cortex (CTX), hippocampus (HIP), caudate-putamen (CP), cerebellum

(CRB), and thalamus (THAL) of 12-month-old Grn+/+ and Grn!/! mice.

(C and D) qRT-PCR shows the progressive increase of C1qa (C) and C3 (D) mRNA in the thalamus of 4-, 9-, 12-, and 18-month-old Grn!/! mice.

(legend continued on next page)

928 Cell 165, 921–935, May 5, 2016

mice and restored the evoked firing pattern in the thalamus to a pattern similar to Grn+/+ mice (Figures 6K and 6L). Dysfunction in the thalamus and striatum has been implicated

in obsessive compulsive disorder (OCD), a key clinical feature in FTD (Fitzgerald et al., 2011; Rascovsky et al., 2011). Consistent with hyperexcitability in the ventral thalamus, Grn!/! mice ex- hibited increased grooming activity, consisting of bouts of high-frequency repetitive movement that covered the snout, face, ear, and back (Movie S1). These excessive grooming be- haviors began at 8 months old and persisted at 12 months old (Figures 6M and S6A). Due to excessive grooming, >60% of Grn!/! mice developed severe skin ulcerations. In addition, "10% of Grn!/! mice also developed motor dysfunction, including unsteady gait and imbalance (Movie S1). These two

phenotypes contributed to the early mortality in Grn!/! mice (median survival for Grn!/! mice was 529 days, compared to 735 days in Grn+/+ mice, p < 0.0001, log rank [Mantel-Cox] test) (Figures 6N and 6O). In contrast, Grn!/!;C1qa!/! mice showed significantly reduced grooming activity, more modest skin lesions, and improved survival (Figures 6M–6O and S6B– S6D). These results support that blocking complement activa- tion via C1qa gene deletion can mitigate the neurodegenerative phenotypes in Grn!/! mice.

Complement Activation as Biomarkers for FTLD with GRN Mutations Finally, we asked whether microglial and complement activa- tion also contribute to neurodegeneration in FTD patients with

(E and F) Western blots (E) and quantification (F) showing the relative abundance of C1qa and C3 proteins in the thalamus of 4-, 9-, and 18-month-old Grn+/+ and

Grn!/! mice. *p < 0.05, **p < 0.01, ***p < 0.005, Student’s t test. Error bars indicate SEM. n = 3 per age for Grn+/+ and Grn!/! mice.

(G–J) Immunostains in 4- and 18-month-old Grn+/+ (G and H) and Grn!/! (I and J) mouse brain detect C1qa signals in the VPM and VPL thalamic nuclei (dotted

areas). Scale bar, 500 mm (G).

(K–P) Confocal images using neuronal marker TuJ1 (K and N) and C1qa (L and O) antibodies in the ventral thalamus of 12-month-old Grn+/+ and Grn!/! mice.

(M) and (P) are merged images. Scale bar, 10 mm (P).

(Q–V) Colocalization of synaptophysin (Q and T) and C1qa (R and U) in the ventral thalamus of 12-month-old Grn+/+ and Grn!/! mice. (S) and (V) are merged

images. Scale bar, 10 mm (V).

(W and X) Immunogold EM detects C1qa deposits in synapses in the ventral thalamus of 12-month-old Grn+/+ (W) and Grn!/! (X) mice. Scale bars, 0.5 mm.

(Y) Western blots using synaptosomes from 4-, 9-, and 16-month-old Grn+/+ and Grn!/! brains detect C1qa and cleaved C3 proteins.

Figure 5. Reduced Microglia Number and Preservation of Synaptic Density in the Ventral Thalamus of Grn!/!;C1qa!/! Mutant Mice (A–H) Immunostains for Iba-1 in coronal sections of 12-month-old Grn+/+ (A and B), Grn!/! (C and D), C1qa!/! (E and F), and Grn!/!;C1qa!/! (G and H) mouse

brains at the level of anterior hippocampus. The square dotted boxes in (A), (C), (E), and (G) highlight the ventral thalamus, where the higher magnification images

are obtained. Scale bars, 500 mm (A); 50 mm (B).

(I) Iba-1+ microglial density in the ventral thalamus of Grn+/+, Grn!/!, C1qa!/!, and Grn!/!;C1qa!/! mouse brains at 2, 4, 7, 12, and 19 months old. ***p < 0.005,

****p < 0.001, two-way ANOVA, n = 4 per genotype per age.

(J–M) Confocal images of synaptophysin in 12-month-old Grn+/+ (J), Grn!/! (K, C1qa!/! (L), and Grn!/!;C1qa!/! (M) brains. Scale bar, 10 mm (J).

(N) Synaptophysin density in Grn+/+, Grn!/!, C1qa!/!, and Grn!/!;C1qa!/! brains at 2, 4, 7, 12, and 19 months old. *p < 0.05, **p < 0.01, ***p < 0.005, ns, not

significant. Student’s t test, n = 4 per genotype per age.

Cell 165, 921–935, May 5, 2016 929

Figure 6. Removing C1qa in Grn!/!;C1qa!/! Mice Protects Synaptic Pruning, Restores Thalamic Microcircuit Function, Mitigates OCD-like Behaviors, and Improves Survival (A–D0) Confocal images of parvalbumin (Parv) show the projection of Parv+ neurons in the reticular nucleus (TRN) to the ventroposterior medial (VPM) and

ventroposterior lateral (VPL) nuclei in the ventral thalamus of 12-month-old Grn+/+ (A and A0), Grn!/! (B and B0), C1qa!/! (C and C0), and Grn!/!;C1qa!/! (D and D0)

mice. Dashed lines highlight VPM and VPL nuclei, and squares highlight regions of higher magnification in (A0)–(D0). Scale bars, 500 mm (D); 20 mm (D0).

(E–H) Confocal images of VGAT+ synapses in the ventral thalamus in 19-month-old Grn+/+ (E), Grn!/! (F), C1qa!/! (G), and Grn!/!;C1qa!/! (H) mice. Scale bar,

25 mm (H).

(I) Quantification of VGAT+ synaptic density in the ventral thalamus of 8-, 12-, and 19-month-old Grn+/+, Grn!/!, C1qa!/!, and Grn!/!;C1qa!/! mice. Student’s

t test, *p < 0.05, **p < 0.01, ***p < 0.005; ns, not significant.

(J) Image of a thalamic slice showing the stimulating electrode in the internal capsule and the 16-channel linear array silicon probe that records multiunit firing in

VPM and VPL.

(K) Representative multiunit recordings (orange box in J) from the ventral thalamus of Grn+/+, Grn!/!, and Grn!/!;C1qa!/! mice. Black circle indicates stimulation

artifact. Bottom: rate meters showing consistent evoked spike rate across sweeps of stimulations (x axis) relative to time (y axis).

(L) Peri-stimulus time histogram of the population data from six Grn+/+, seven Grn!/!, and four Grn!/!;C1qa!/! mice. Inset bottom: enlargement of the black

dashed box in (L) showing the slope of the response is significantly different among genotypes (***p < 0.0001, F = 10.5554). Inset top: plot of the relative probability

of eliciting AP firing frequencies among Grn+/+, Grn!/! and Grn!/!;C1qa!/! mice analyzed by the Kolmogorov-Smirnov test (Grn+/+ versus Grn!/!, ***p < 0.0001,

D = 0.5783; Grn+/+ versus Grn!/!;C1qa!/!, ***p < 0.0001, D = 0.4980; Grn!/! versus Grn!/!;C1qa!/!, ***p < 0.0001, D = 0.7751). Error bars, SEM.

(M) Grooming activities in Grn+/+, Grn!/!, C1qa!/!, and Grn!/!;C1qa!/! mice is expressed as percentage of total time. *p < 0.05, **p < 0.01; ns, not significant,

Student’s t test.

(N and O) Kaplan-Meier curve for skin lesion onset (N) and survival (O) in Grn+/+, Grn!/!, C1qa!/!, and Grn!/!;C1qa!/! mice. *p < 0.05, ****p < 0.001; ns, not

significant, Long-rank (Mantel-Cox) test.

See also Figures S4, S5 and S6, and Movie S1.

930 Cell 165, 921–935, May 5, 2016

GRN mutations, and if so, whether complement activation could be a potential disease-specific biomarker. To this end, we examined microglial density in the frontal cortex of FTLD patients with GRN mutations (see the Supplemental Information and Table S2 for details). In addition, we included patients with sporadic Alzheimer’s disease (AD) and age-matched control cases with no evidence of neurodegeneration (Table S2). In control frontal cortex, only few microglia with quiescent morphology were identified (Figure 7A). Most microglia in con- trol individuals expressed very low levels of C1qa by immuno- histochemistry, and no C1qa was identified near synapses by IEM (Figures 7B and 7C). In contrast, FTLD GRN carriers showed a marked increase in microglial density, most promi- nently affecting layers I–III of the frontal cortex (Figure 7D).

Similar to the microglia Grn!/! mouse brain, microglia in FTLD GRN carriers exhibited prominent reactive features and abundant C1qa deposits (Figure 7E). Furthermore, immunogold EM identified prominent C1qa signals near or at synapses in the frontal cortex of GRN mutation carriers (Figure 7F). To determine whether microglial infiltration in the frontal cortex of FTLD GRN carriers is disease-specific, we examined frontal cortex of AD patients and found that the majority of microglia in these cases appeared to surround the amyloid plaques, where abundant C1qa deposits were found (Figures 7G and 7H). On average, the microglial density in the frontal cortex of FTLD GRN carriers showed a 3- to 4-fold increase, whereas the microglial density in AD patients was similar to controls (Figure 7I).

Figure 7. Microglial Pathology and CSF Complement Levels in FTLD Grn Mutation Carriers (A–H) Immunostains of frontal cortex from control, FTLD Grn carriers and AD patients detect the presence of microglia and C1qa (A, B, D, E, G, and H). In addition,

immunogold EM detects the presence of C1qa deposits at the synapses in FTLD Grn mutation carriers (C and F). Arrows in (A), (B), (D), (E), (G), and (H) indicate

microglia. Arrowhead in (G) indicates an amyloid plaque and in (H) indicates a blood vessel (BV). Arrowheads in (C) and (F) indicate presynaptic terminals, and

arrows in (F) indicate C1qa-positive immunogold particles in synapses.

(I) Quantification of Iba-1+ microglia in the frontal cortex of controls (n = 7), FTLD Grn carriers (n = 16), and AD patients (n = 8). ***p < 0.005, ****p < 0.001; ns, not

significant, Student’s t test.

(J) Quantification of CSF PGRN levels in controls and FTLD GRN carriers. Student’s t test, ****p < 0.0001.

(K and L) ELISA assays for C1qa (K) and C3b (L) protein levels in the CSF of controls (n = 23) and FTLD Grn carriers (n = 19). Chi-square goodness-of-fit test to

calculate R2 and p values.

See also Figure S7 and Tables S2 and S3.

Cell 165, 921–935, May 5, 2016 931

The results from the frontal cortex of FTD GRN carriers raise the possibility that complement proteins might be released into the cerebrospinal fluid (CSF) and could serve as biomarkers to predict disease onset and/or progression. Indeed, the utility of CSF was underscored by the "70% drop in PGRN levels in FTLD patients with GRN mutations (Figure 7J). Interestingly, the levels of C1qa and C3b showed extensive overlapping in controls and FTLD GRN carriers, indicating that complement protein levels in CSF can be quite variable. However, as dis- ease progressed in FTLD GRN carriers, the levels of C1qa and C3b progressively increased as cognitive functions declined (as determined by mini-mental status exam [MMSE] score) (Figures 7K and 7L). In contrast, analyses of previously published CSF data from AD patients (Smyth et al., 1994) showed that C1qa levels in AD patients were significantly lower than those in control and FTLD GRN carriers (controls: 479.5 ± 35.6, FTLD GRN carriers: 442.3 ± 33.0, AD: 268.0 ± 14.2, p < 0.0001, unpaired t test) (Figure S7A). Interestingly, as the MMSE declined in AD patients, the CSF C1qa levels showed progressive decrease (Figure S7B). Together, these results highlight the distinct differences in the microglia pathology and CSF complement protein levels between FTLD GRN car- riers and AD patients.

DISCUSSION

PGRN Deficiency and Excessive Microglia Activation in the Aging Brain Although PGRN has been implicated in lysosomal functions (Belcastro et al., 2011; Zhou et al., 2015), the exact mechanism remains poorly understood. Several lines of evidence from our study provide key mechanistic insights that PGRN regulates the formation and functions of lysosomes in the microglia. First, transcriptome profiling in Grn+/+, Grn+/!, and Grn!/! aging brains shows that loss of PGRN leads to progressive upregula- tion of genes that control lysosomal functions and the innate immunity response (Figures 1 and S1). Second, confocal micro- scopic analyses of cultured microglia show that abundant PGRN protein is expressed in the microglia and localized pri- marily in the Golgi apparatus, Sortilin+ vesicles, and lysosomes, suggesting that PGRN might regulate intracellular trafficking and the formation of lysosomes. In support of this idea, Grn!/!

microglia exhibit profound lysosomal defects that facilitate more efficient processing via the endolysosomal pathway. Such lysosomal phenotypes, working in conjunction with the upregulation of complement protein C3, promote proteolytic cleavage of C3 by C3 convertase in the lysosomes and lead to a marked increase in the release of biologically active C3 products, including C3b and iC3d (Figures 2, S2, and S3) (Lis- zewski et al., 2013; Naito et al., 2012). Finally, western blot an- alyses show age-dependent accumulation of C1qa and cleaved C3 products in the synaptosomes of Grn!/! brain. Consistent with these results, Grn!/! microglia show a marked increase in synaptic pruning activity in microglia-neuron co-cul- tures and in the ventral thalamus of Grn!/! brain (Figures 3, 4, 5, and 6). Together, these results support the idea that PGRN serves as an important ‘‘brake’’ to suppress excessive micro- glia activation in the aging brain by facilitating phagocytosis

and endolysosomal trafficking in microglia. Given the robust microglial activation in young mice exposed to the neurotoxin MPTP (Figure 2) (Martens et al., 2012), it is very likely that PGRN deficiency may have a broader role in suppressing mi- croglia activation in other injury paradigms. The identification of aberrant complement activation in Grn!/!

microglia provides key mechanistic insights into the pathogen- esis of neurodegeneration due to PGRN deficiency. The classical complement pathway serves as an important and well-recog- nized arm of the innate immunity surveillance system (Walport, 2001). Several previous studies indicate that complement-medi- ated synaptic pruning by microglia plays a critical role in the refinement of neural circuits during early postnatal development and in the normal aging process (Schafer et al., 2012; Shi et al., 2015; Stephan et al., 2013; Stevens et al., 2007). Although it has been postulated that excessive microglial activation may pro- mote neurodegeneration (Aguzzi et al., 2013), the exact mecha- nisms remain unclear. Remarkably, our results show that loss of C1qa reduces synaptic pruning activity in Grn!/! microglia, reduces synapse loss, protects against hyperexcitability in the thalamocortical circuit, reduces behavioral abnormalities, and improves survival in Grn!/!;C1qa!/! mice. These results support that lysosomal dysfunctions and complement activation in Grn!/! microglia are the main drivers that directly promotes neu- rodegeneration in a mouse model of FTLD.

Circuit-Specific Synaptic Pruning by Grn!/! Microglia One surprising neurodegenerative feature in Grn!/! brains is the selective loss of inhibitory synapses in the ventral thalamus, despite the fact that the accumulation of C1qa can be detected in both excitatory and inhibitory synapses (Figures 6 and S5). Such preferential elimination of inhibitory synapses by Grn!/!

microglia is unprecedented in other neurodegenerative models. It is possible that differential expression of complement recep- tors or other recognition molecules in different synaptic subtypes may contribute to this selective phenotype. Alternatively, intrinsic properties of excitatory and inhibitory synapses may determine the efficacy of pruning by Grn!/! microglia. Regard- less of the mechanism, the selective loss of inhibitory synapses in Grn!/! brain leads to increased excitability in the thalamocort- ical circuits. Another remarkable finding in Grn!/! mice is the much more

robust increase in microglia density in ventral thalamus, compared to other brain regions (Yin et al., 2010). This finding provides a neuroanatomical basis for the observed excessive grooming and OCD-like behaviors and is further supported by the well-established role of the ventral thalamus in inte- grating sensory inputs, frontostriatal circuits, and motor learning (Burguière et al., 2015). While it remains unclear why Grn!/! microglia show a preferential effect on the thalamocort- ical circuit, given the highly evolutionarily conserved function of this circuit in sensorimotor integration in mammals (Pe- tersen, 2007), it is possible that similar pathology may contribute to the clinical manifestations in FTLD patients. Consistent with this idea, perseverative and compulsive be- haviors are among the most important diagnostic criteria for FTLD (Rascovsky et al., 2011). Together, the results reveal pre- viously unrecognized mechanisms of PGRN in microglia

932 Cell 165, 921–935, May 5, 2016

function and how loss of PGRN may affect behaviors in a cir- cuit-specific manner. Despite the robust effects of deleting C1qa in mitigating

several key neurodegenerative phenotypes in the aging Grn!/! mice, it is important to note that loss of C1qa does not completely rescue all the Grn!/! phenotypes. Although the microglial infiltration is significantly reduced in the ventral thalamus of Grn!/!;C1qa!/! mice, the microglial den- sity is still significantly higher than age-matched Grn+/+ mice (Figure 5). Many microglia in Grn!/!;C1qa!/! mice continue to exhibit morphological features similar to those seen in Grn!/! mice. Furthermore, despite the significant improve- ment in survival, Grn!/!;C1qa!/! mice still show a modest in- crease in mortality compared to control Grn+/+ littermates. These results suggest that additional pathogenic factors, such as over-production of pro-inflammatory cytokines, other intrinsic microglia and/or neuronal defects, or intricate micro- glia-neuron interactions, may contribute to disease progres- sion in Grn!/!;C1qa!/! mice in the absence of complement activation. For instance, the interacting network of upregu- lated genes in Grn!/! brain also identifies Trem2 as a node that connects many other genes in the lysosomal and innate immunity pathways (Figure 1). Since the recent study indi- cates that TREM2 loss-of-function and TREM2-R47H variant attenuate lipid-sensing ability in microglia to mount a sus- tained response to neuronal injury in mouse AD models (Wang et al., 2015), these results raise the possibility that the elevated level of TREM2 in Grn!/! microglia may promote their synaptic pruning activity.

Microglia and Complement Activation as Critical Biomarkers for FTLD One important neuropathologic feature in the frontal cortex of FTLD patients with GRN mutations is microglial activation, char- acterized by reactive morphology and excessive complement production (Figure 7). These findings are strikingly similar to those observed in Grn!/! mouse brain and raise the possibility that GRN mutations in FTLD patients may lead to chronic PGRN deficiency in brain tissues, resulting in a de facto PGRN loss-of-function phenotype similar to Grn!/! mice (Baker et al., 2006; Cruts et al., 2006). Another interesting finding is that CSF samples from FTLD patients with Grn mutations show progres- sive increases in C1qa and C3b that correlate with cognitive decline (Figure 7). In contrast, the histopathologic characteristics of C1qa distribution in the frontal cortex and the positive correla- tion of complement elevation in the CSF with the decline of MMSE in FTLD GRN carriers are distinctly different from those in AD patients. These results support the disease specificity of complement activation in FTLD (Figures 7 and S7) (Smyth et al., 1994) and further indicate that the underlying mechanisms that regulate the complement activation pathway in these two neurodegenerative diseases may be fundamentally different. These results underscore the importance of microglia and com- plement activation in human FTLD caused by GRN mutations and support the feasibility of developing strategies that target complement proteins as biomarkers to track disease progres- sion and as valid therapeutic targets to mitigate neurodegenera- tion in FTLD.

EXPERIMENTAL PROCEDURES

Brain-Region-Specific Microarray and Bioinformatics Analyses An aging cohort of Grn+/+, Grn+/!, and Grn!/! mice, ranging from 2, 6, 9, 12,

and 18 months old, was used to characterize age-dependent changes in tran-

scriptomes caused by PGRN deficiency. Mice were perfused with ice-cold

PBS, and brains were removed and further dissected to isolate cerebral

cortex, hippocampus, and cerebellum. Tissues were homogenized using the

Bullet Blender (Next Advance) in Trizol (Invitrogen). RNA integrity was

measured by running samples on a Bioanalyzer (Agilent). RNA samples were

hybridized to Illumina Mouse8 version 2 microarray chips as previously

described (Rosen et al., 2011). Network analyses were performed as previ-

ously described, and gene module merging was accomplished using the

WGCNA package in R (Langfelder and Horvath, 2008). Differentially expressed

genes within a given module were compared against the murine background

for enrichment within gene ontology (GO) analysis (Table S1). These modules

were further subjected to system-level functional analyses by determining their

topographical overlap.

Human Brain Tissues and Cerebrospinal Fluid Samples Frozen frontal lobe tissues were procured from controls with no known neuro-

degenerative diseases, FTLD patients with GRN mutations, and Alzheimer’s

disease (AD) patients. All cases were clinically and neuropathologically evalu-

ated at the University of California San Francisco (UCSF), Northwestern Uni-

versity, and University of British Columbia. In addition, cerebrospinal fluid

(CSF) samples were collected from controls and patients with GRN mutations

and clinical diagnosis of FTD at UCSF and Italy. All human tissues and CSF

samples were collected with informed consents and institutional review board

(IRB) approvals. The demographic information, GRN mutations and pertinent

clinical data of these cases are provided in Tables S2 and S3.

ACCESSION NUMBERS

The accession number for the microarray data reported in this paper is GEO:

GSE75083.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

seven figures, three tables, and one movie and can be found with this article

online at http://dx.doi.org/10.1016/j.cell.2016.04.001.

AUTHOR CONTRIBUTIONS

H.L., J.Z., H.-Y.H., M.K.C., and Y.S. designed and performed the experiments

and analyzed data with E.J.H. J.T.P. and S.R.M. designed the electrophysi-

ology experiments, S.R.M. performed recordings, and S.R.M. and J.T.P.

analyzed data. L.M.H. and G.C. performed and analyzed microarray data.

J.Z. and C.L.H. performed FACS sorting of microglia. F.G., G.C., K.W.K.,

S.A.S., M.C.O., and C.C.K. provided bioinformatics analyses. E.H.B., S.W.,

M.-M.M., R.R., I.R.M., W.W.S., A.K., B.L.M., B.B., R.G., R.V.F., and B.A.B.

provided human samples and reagents. E.J.H. supervised the project, and

H.L., J.Z., M.K.C., H.Y.H., Y.S., and E.J.H. wrote the manuscript.

ACKNOWLEDGMENTS

We thank Ivy Hsieh for immunogold EM, Eric Bennett, Jordan Sorokin, and

John Huguenard for their feedback on the Matlab code for analyzing the

multi-unit recording in the thalamus, and Dr. Jennifer Cotter for critical com-

ments on the manuscript. This work has been supported by NIH AG013854

(E.H.B.), P50 AG023501 and P01 AG019724 (B.L.M. and W.W.S.), P30

AI027763 and P30 DK063720 (C.C.K.), Italian Ministry of Health (Ricerca Cor-

rente, R.G.), JPB Foundation (B.A.B.), Department of Veterans Affairs Career

Development Award-2 (C.L.H.), Merit Awards BX002690 (C.L.H.) BX001108

(E.J.H.), and RX002133 (E.J.H.), and Consortium for Frontotemporal Dementia

Research (CFR) and the Bluefield Project (B.L.M, W.W.S., and E.J.H.). Special

Cell 165, 921–935, May 5, 2016 933

http://dx.doi.org/10.1016/j.cell.2016.04.001

thanks to Dr. Laura Mitic and Dr. Rodney Pearlman for their unwavering

support.

Received: November 18, 2015

Revised: March 10, 2016

Accepted: March 31, 2016

Published: April 21, 2016

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