Lysosomal dysfunction and microglial hyperactivation in models of progranulin deficiency

In my thesis I focused on the pivotal role of microglia in neurodegenerative disease, their different activation stages upon progranulin (PGRN) or triggering receptor expressed on myeloid cells 2 (TREM2) deficiency and the connection between lysosomal deficiency and microglial hyperactivation. Microglia majorly contribute to the progression and pathology of neurodegenerative disorders like Alzheimer`s disease (AD) and frontotemporal lobal degeneration (FTLD) and additionally recent advances of genome wide association studies (GWAS) have identified genetic association, as rare variants of genes that are predominantly expressed by microglia increase the risk of developing neurodegenerative disease. Among these risk genes are progranulin (GRN) and the triggering receptor expressed on myeloid cells 2 (TREM2). While the heterozygous loss of PGRN leads to FTLD, the complete loss of PGRN results in the lysosomal storage disease neuronal ceroid lipofuscinosis, indicating a major role of PGRN in lysosomal protein degradation in the brain. Our laboratory has previously shown, that progranulin knockout mice (Grn-/-) and GRN-associated FTLD patients exhibit increased levels of the lysosomal protein cathepsin (Cat) D, however the exact role of PGRN in lysosomal protein degradation remained unclear. In a collaborative effort with Julia K. Götzl, Alessio-Vittorio Colombo and Kathrin Fellerer, I therefore analyzed microglia and other brain cells regarding changes in expression, maturation and enzymatic activity of lysosomal proteins like Cat D, B and L. We found a striking age-depended increase of lysosomal proteases associated with increased enzymatic activity. Interestingly, we demonstrated that microglia show early lysosomal deficits, even before enhanced Cat transcription levels were observed. Our laboratory has previously shown, that PGRN loss of function (LOF) leads to hyperactivated microglia that exhibit increased phagocytosis, proliferation and migration. The opposite microglial phenotype is found in TREM2 LOF models, where microglia appear to be locked in a homeostatic state, unable to react to pathological insults. In addition to the lysosomal dysfunction discussed above, PGRN LOF microglia also increase TREM2 expression. To test the hypothesis that hyperactivation of microglia in PGRN LOF is TREM2-dependent and that microglia can reversibly switch between activation stages, I used genetic and pharmacological TREM2 antagonistic approaches to prevent the transition of homeostatic microglia to a disease-associated microglia (DAM) state. To further investigate the microglial contribution to disease pathology in PGRN LOF models, I generated Grn x Trem2 double knockout mice to analyze the expression of DAM genes, lysosomal dysfunction, glucose uptake, lipid metabolism and microglia morphology and activation status. Here, I found that ablating TREM2 in PGRN LOF mice reduces the expression of DAM genes, suggesting that suppression of TREM2 can lower microglia hyperactivation and is likely to be upstream PGRN-mediated microglial transcriptional changes. To further explore whether pharmacological modulation of TREM2 has beneficial functions on microglia states, I used antibodies antagonistic for TREM2, developed at Denali Therapeutics, to treat macrophages isolated from GRN-FTLD patients. Treatment of the cells with these antibodies resulted in reduced TREM2 signaling, due to its enhanced shedding. To confirm these findings, I collaborated with Sophie Robinson, who generated PGRN-deficient microglia derived from human-induced pluripotent stem cells (iPSC). Treatment of these cells with antagonistic TREM2 antibodies resulted in reduced microglia hyperactivation, TREM2 signaling and phagocytic activity. However, we did not observe any effects on lysosomal dysfunction in PGRN deficient iPSC after antibody treatment. In line with this, Grn x Trem2 double knockout mice not only failed to rescue effects on lysosomal dysfunction, lipid metabolism and microglia morphology, but also further increased synaptic loss and neurofilament light-chain (Nfl) levels, a marker of neuronal damage in the brain. My results suggest that with PGRN deficiency, lysosomal dysfunction is upstream to the microglia hyperactivation. In addition, these findings imply a protective role of TREM2-dependent chronic activation of microglia and show the dynamic nature of microglia kinetics and their ability to reversibly switch between activation stages

Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

Background: Heterozygous loss-of-function mutations in the progranulin gene (GRN) lead to frontotemporal lobar degeneration (FTLD) while the complete loss of progranulin (PGRN) function results in neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Thus the growth factor-like protein PGRN may play an important role in lysosomal degradation. In line with a potential lysosomal function, PGRN is partially localized and processed in lysosomes. In the central nervous system (CNS), PGRN is like other lysosomal proteins highly expressed in microglia, further supporting an important role in protein degradation. We have previously reported that cathepsin (Cat) D is elevated in GRN-associated FTLD patients and Grn knockout mice. However, the primary mechanism that causes impaired protein degradation and elevated CatD levels upon PGRN deficiency in NCL and FTLD remains unclear. Methods: mRNA expression analysis of selected lysosomal hydrolases, lysosomal membrane proteins and autophagy-related genes was performed by NanoString nCounter panel. Protein expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts (MEF) and brains of Grn knockout mice were investigated. To selectively characterize microglial and non-microglial brain cells, an acutely isolated microglia fraction using MACS microbeads (Miltenyi Biotec) conjugated with CD11b antibody and a microglia-depleted fraction were analyzed for protein expression and maturation of selected cathepsins. . Results: We demonstrate that loss of PGRN results in enhanced expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts and brain extracts of aged Grn knockout mice. Consistent with an overall enhanced expression and activity of lysosomal proteases in brain of Grn knockout mice, we observed an age-dependent transcriptional upregulation of certain lysosomal proteases. Thus, lysosomal dysfunction is not reflected by transcriptional downregulation of lysosomal proteases but rather by the upregulation of certain lysosomal proteases in an age-dependent manner. Surprisingly, cell specific analyses identified early lysosomal deficits in microglia before enhanced cathepsin levels could be detected in other brain cells, suggesting different functional consequences on lysosomal homeostasis in microglia and other brain cells upon lack of PGRN. Conclusions: The present study uncovers early and selective lysosomal dysfunctions in Grn knockout microglia/macrophages. Dysregulated lysosomal homeostasis in microglia might trigger compensatory lysosomal changes in other brain cells

Depletion and activation of microglia impact metabolic connectivity of the mouse brain

AimWe aimed to investigate the impact of microglial activity and microglial FDG uptake on metabolic connectivity, since microglial activation states determine FDG-PET alterations. Metabolic connectivity refers to a concept of interacting metabolic brain regions and receives growing interest in approaching complex cerebral metabolic networks in neurodegenerative diseases. However, underlying sources of metabolic connectivity remain to be elucidated.Materials and methodsWe analyzed metabolic networks measured by interregional correlation coefficients (ICCs) of FDG-PET scans in WT mice and in mice with mutations in progranulin (Grn) or triggering receptor expressed on myeloid cells 2 (Trem2) knockouts ((-/-)) as well as in double mutant Grn(-/-)/Trem2(-/-) mice. We selected those rodent models as they represent opposite microglial signatures with disease associated microglia in Grn(-/-) mice and microglia locked in a homeostatic state in Trem2(-/-) mice;however, both resulting in lower glucose uptake of the brain. The direct influence of microglia on metabolic networks was further determined by microglia depletion using a CSF1R inhibitor in WT mice at two different ages. Within maps of global mean scaled regional FDG uptake, 24 pre-established volumes of interest were applied and assigned to either cortical or subcortical networks. ICCs of all region pairs were calculated and z-transformed prior to group comparisons. FDG uptake of neurons, microglia, and astrocytes was determined in Grn(-/-) and WT mice via assessment of single cell tracer uptake (scRadiotracing).ResultsMicroglia depletion by CSF1R inhibition resulted in a strong decrease of metabolic connectivity defined by decrease of mean cortical ICCs in WT mice at both ages studied (6-7 m;p = 0.0148, 9-10 m;p = 0.0191), when compared to vehicle-treated age-matched WT mice. Grn(-/-), Trem2(-/-) and Grn(-/-)/Trem2(-/-) mice all displayed reduced FDG-PET signals when compared to WT mice. However, when analyzing metabolic networks, a distinct increase of ICCs was observed in Grn(-/-) mice when compared to WT mice in cortical (p < 0.0001) and hippocampal (p < 0.0001) networks. In contrast, Trem2(-/-) mice did not show significant alterations in metabolic connectivity when compared to WT. Furthermore, the increased metabolic connectivity in Grn(-/-) mice was completely suppressed in Grn(-/-)/Trem2(-/-) mice. Grn(-/-) mice exhibited a severe loss of neuronal FDG uptake (- 61%, p < 0.0001) which shifted allocation of cellular brain FDG uptake to microglia (42% in Grn(-/-) vs. 22% in WT).ConclusionsPresence, absence, and activation of microglia have a strong impact on metabolic connectivity of the mouse brain. Enhanced metabolic connectivity is associated with increased microglial FDG allocation

Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency

Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection

The wide genetic landscape of clinical frontotemporal dementia: systematic combined sequencing of 121 consecutive subjects

PurposeTo define the genetic spectrum and relative gene frequencies underlying clinical frontotemporal dementia (FTD).MethodsWe investigated the frequencies and mutations in neurodegenerative disease genes in 121 consecutive FTD subjects using an unbiased, combined sequencing approach, complemented by cerebrospinal fluid Aβ1-42 and serum progranulin measurements. Subjects were screened for C9orf72 repeat expansions, GRN and MAPT mutations, and, if negative, mutations in other neurodegenerative disease genes, by whole-exome sequencing (WES) (n = 108), including WES-based copy-number variant (CNV) analysis.ResultsPathogenic and likely pathogenic mutations were identified in 19% of the subjects, including mutations in C9orf72 (n = 8), GRN (n = 7, one 11-exon macro-deletion) and, more rarely, CHCHD10, TARDBP, SQSTM1 and UBQLN2 (each n = 1), but not in MAPT or TBK1. WES also unraveled pathogenic mutations in genes not commonly linked to FTD, including mutations in Alzheimer (PSEN1, PSEN2), lysosomal (CTSF, 7-exon macro-deletion) and cholesterol homeostasis pathways (CYP27A1).ConclusionOur unbiased approach reveals a wide genetic spectrum underlying clinical FTD, including 11% of seemingly sporadic FTD. It unravels several mutations and CNVs in genes and pathways hitherto not linked to FTD. This suggests that clinical FTD might be the converging downstream result of a delicate susceptibility of frontotemporal brain networks to insults in various pathways