Progranulin Haploinsufficiency Research Articles

Peripheral expression of brain-penetrant progranulin rescues pathologies in mouse models of frontotemporal lobar degeneration.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD-GRN). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD-GRN, namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD-GRN-associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)-derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD-GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD-GRN and potentially other CNS disorders.

Biochemical, Biomarker, and Behavioral Characterization of the GrnR493X Mouse Model of Frontotemporal Dementia.

Heterozygous loss-of-function mutations in the progranulin gene (GRN) are a major cause of frontotemporal dementia due to progranulin haploinsufficiency; complete deficiency of progranulin causes neuronal ceroid lipofuscinosis. Several progranulin-deficient mouse models have been generated, including both knockout mice and knockin mice harboring a common patient mutation (R493X). However, the GrnR493X mouse model has not been characterized completely. Additionally, while homozygous GrnR493X and Grn knockout mice have been extensively studied, data from heterozygous mice is still limited. Here, we performed more in-depth characterization of heterozygous and homozygous GrnR493X knockin mice, which includes biochemical assessments, behavioral studies, and analysis of fluid biomarkers. In the brains of homozygous GrnR493X mice, we found increased phosphorylated TDP-43 along with increased expression of lysosomal genes, markers of microgliosis and astrogliosis, pro-inflammatory cytokines, and complement factors. Heterozygous GrnR493X mice did not have increased TDP-43 phosphorylation but did exhibit limited increases in lysosomal and inflammatory gene expression. Behavioral studies found social and emotional deficits in GrnR493X mice that mirror those observed in Grn knockout mouse models, as well as impairment in memory and executive function. Overall, the GrnR493X knockin mouse model closely phenocopies Grn knockout models. Lastly, in contrast to homozygous knockin mice, heterozygous GrnR493X mice do not have elevated levels of fluid biomarkers previously identified in humans, including neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in both plasma and CSF. These results may help to inform pre-clinical studies that use this Grn knockin mouse model and other Grn knockout models.

Progranulin AAV gene therapy for frontotemporal dementia: translational studies and phase 1/2 trial interim results

GRN mutations cause progranulin haploinsufficiency, which eventually leads to frontotemporal dementia (FTD-GRN). PR006 is an investigational gene therapy delivering the granulin gene (GRN) using an adeno-associated virus serotype 9 (AAV9) vector. In non-clinical studies, PR006 transduced neurons derived from induced pluripotent stem cells of patients with FTD-GRN, resulted in progranulin expression and improvement of lipofuscin, lysosomal and neuroinflammation pathologies in Grn-knockout mice, and was well tolerated except for minimal, asymptomatic dorsal root ganglionopathy in non-human primates. We initiated a first-in-human phase 1/2 open-label trial. Here we report results of a pre-specified interim analysis triggered with the last treated patient of the low-dose cohort (n = 6) reaching the 12-month follow-up timepoint. We also include preliminary data from the mid-dose cohort (n = 7). Primary endpoints were safety, immunogenicity and change in progranulin levels in cerebrospinal fluid (CSF) and blood. Secondary endpoints were Clinical Dementia Rating (CDR) plus National Alzheimer’s Disease Coordinating Center (NACC) Frontotemporal Lobar Degeneration (FTLD) rating scale and levels of neurofilament light chain (NfL). One-time administration of PR006 into the cisterna magna was generally safe and well tolerated. All patients developed treatment-emergent anti-AAV9 antibodies in the CSF, but none developed anti-progranulin antibodies. CSF pleocytosis was the most common PR006-related adverse event. Twelve serious adverse events occurred, mostly unrelated to PR006. Deep vein thrombosis developed in three patients. There was one death (unrelated) occurring 18 months after treatment. CSF progranulin increased after PR006 treatment in all patients; blood progranulin increased in most patients but only transiently. NfL levels transiently increased after PR006 treatment, likely reflecting dorsal root ganglia toxicity. Progression rates, based on the CDR scale, were within the broad ranges reported for patients with FTD. These data provide preliminary insights into the safety and bioactivity of PR006. Longer follow-up and additional studies are needed to confirm the safety and potential efficacy of PR006. ClinicalTrials.gov identifier: NCT04408625.

Epigenetic modulation through BET bromodomain inhibitors as a novel therapeutic strategy for progranulin-deficient frontotemporal dementia

Frontotemporal dementia (FTD) is a debilitating neurodegenerative disorder with currently no disease-modifying treatment options available. Mutations in GRN are one of the most common genetic causes of FTD, near ubiquitously resulting in progranulin (PGRN) haploinsufficiency. Small molecules that can restore PGRN protein to healthy levels in individuals bearing a heterozygous GRN mutation may thus have therapeutic value. Here, we show that epigenetic modulation through bromodomain and extra-terminal domain (BET) inhibitors (BETi) potently enhance PGRN protein levels, both intracellularly and secreted forms, in human central nervous system (CNS)-relevant cell types, including in microglia-like cells. In terms of potential for disease modification, we show BETi treatment effectively restores PGRN levels in neural cells with a GRN mutation known to cause PGRN haploinsufficiency and FTD. We demonstrate that BETi can rapidly and durably enhance PGRN in neural progenitor cells (NPCs) in a manner dependent upon BET protein expression, suggesting a gain-of-function mechanism. We further describe a CNS-optimized BETi chemotype that potently engages endogenous BRD4 and enhances PGRN expression in neuronal cells. Our results reveal a new epigenetic target for treating PGRN-deficient forms of FTD and provide mechanistic insight to aid in translating this discovery into therapeutics.

Progranulin haploinsufficiency mediates cytoplasmic TDP-43 aggregation with lysosomal abnormalities in human microglia

BackgroundProgranulin (PGRN) haploinsufficiency due to progranulin gene (GRN) variants can cause frontotemporal dementia (FTD) with aberrant TAR DNA-binding protein 43 (TDP-43) accumulation. Despite microglial burden with TDP-43-related pathophysiology, direct microglial TDP-43 pathology has not been clarified yet, only emphasized in neuronal pathology. Thus, the objective of this study was to investigate TDP-43 pathology in microglia of patients with PGRN haploinsufficiency.MethodsTo design a human microglial cell model with PGRN haploinsufficiency, monocyte-derived microglia (iMGs) were generated from FTD–GRN patients carrying pathogenic or likely pathogenic variants (p.M1? and p.W147*) and three healthy controls.ResultsiMGs from FTD–GRN patients with PGRN deficiency exhibited severe neuroinflammation phenotype and failure to maintain their homeostatic molecular signatures, along with impaired phagocytosis. In FTD–GRN patients-derived iMGs, significant cytoplasmic TDP-43 aggregation and accumulation of lipid droplets with profound lysosomal abnormalities were observed. These pathomechanisms were mediated by complement C1q activation and upregulation of pro-inflammatory cytokines.ConclusionsOur study provides considerable cellular and molecular evidence that loss-of-function variants of GRN in human microglia can cause microglial dysfunction with abnormal TDP-43 aggregation induced by inflammatory milieu as well as the impaired lysosome. Elucidating the role of microglial TDP-43 pathology in intensifying neuroinflammation in individuals with FTD due to PGRN deficiency and examining consequential effects on microglial dysfunction might yield novel insights into the mechanisms underlying FTD and neurodegenerative disorders.

Regulation of extracellular progranulin in medial prefrontal cortex

Progranulin is a secreted pro-protein that has anti-inflammatory and neurotrophic effects and is necessary for maintaining lysosomal function. Mutations in progranulin (GRN) are a major cause of frontotemporal dementia. Most pathogenic GRN mutations cause progranulin haploinsufficiency, so boosting progranulin levels is a promising therapeutic strategy. Progranulin is constitutively secreted, then taken up and trafficked to lysosomes. Before being taken up from the extracellular space, progranulin interacts with receptors that may mediate anti-inflammatory and growth factor-like effects. Modifying progranulin trafficking is a viable approach to boosting progranulin, but progranulin secretion and uptake by cells in the brain is poorly understood and may involve distinct mechanisms from other parts of the body. Understanding the cell types and processes that regulate extracellular progranulin in the brain could provide insight into progranulin's mechanism of action and inform design of progranulin-boosting therapies. To address this question we used microdialysis to measure progranulin in interstitial fluid (ISF) of mouse medial prefrontal cortex (mPFC). Grn+/− mice had approximately 50% lower ISF progranulin than wild-type mice, matching the reduction of progranulin in cortical tissue. Fluorescent in situ hybridization and immunofluorescence confirmed that microglia and neurons are the major progranulin-expressing cell types in the mPFC. Studies of conditional microglial (Mg-KO) and neuronal (N-KO) Grn knockout mice revealed that loss of progranulin from either cell type results in approximately 50% reduction in ISF progranulin. LPS injection (i.p.) produced an acute increase in ISF progranulin in mPFC. Depolarizing cells with KCl increased ISF progranulin, but this response was not altered in N-KO mice, indicating progranulin secretion by non-neuronal cells. Increasing neuronal activity with picrotoxin did not increase ISF progranulin. These data indicate that microglia and neurons are the source of most ISF progranulin in mPFC, with microglia likely secreting more progranulin per cell than neurons. The acute increase in ISF progranulin after LPS treatment is consistent with a role for extracellular progranulin in regulating inflammation, and may have been driven by microglia or peripheral immune cells. Finally, these data indicate that mPFC neurons engage in constitutive progranulin secretion that is not acutely changed by neuronal activity.

Analytical and clinical validation of a blood progranulin ELISA in frontotemporal dementias.

Heterozygous mutations in the granulin (GRN) gene may result in haploinsufficiency of progranulin (PGRN), which might lead to frontotemporal dementia (FTD). In this study, we aimed to perform analytical and clinical validation of a commercial progranulin kit for clinical use. Analytical validation parameters including assay precision, selectivity, measurement range, dilution linearity, interferences and sample stability were tested according to previously described procedures. For clinical validation, PGRN levels were measured in plasma from 32 cognitively healthy individuals, 52 confirmed GRN mutation carriers, 25 C9orf72 mutation carriers and 216 patients with different neurodegenerative diseases of which 70 were confirmed as non-mutation carriers. Among the analytical validation parameters, assay precision and repeatability were very stable (coefficients of variation <7 %). Spike recovery was 96 %, the measurement range was 6.25-400 μg/L and dilution linearity ranged from 1:50-1:200. Hemolysis did not interfere with progranulin levels, and these were resistant to freeze/thaw cycles and storage at different temperatures. For the clinical validation, the assay was capable of distinguishing GRN mutation carriers from controls and non-GRN mutation carriers with very good sensitivity and specificity at a cut-off of 57 μg/L (97 %, 100 %, respectively). In this study, we demonstrate robust analytical and diagnostic performance of this commercial progranulin kit for implementation in clinical laboratory practice. This easy-to-use test allows identification of potential GRN mutation carriers, which may guide further evaluation of the patient. This assay might also be used to evaluate the effect of novel PGRN-targeting drugs and therapies.

Haploinsufficiency of progranulin causes cell type specific impairments in PINK1/Parkin mitophagy

AbstractBackgroundGRN mutations that result in progranulin haploinsufficiency cause frontotemporal dementia (FTD). Mitophagy, the selective autophagy of damaged mitochondria, is impaired in several neurodegenerative diseases. A number of genes linked to FTD (e.g. OPTN, SQSTM, VCP and TBK1) are also known to play a role in mitophagy. Xenophagy, the selective autophagy of non‐host pathogens, relies on some of the same proteins as mitophagy (Parkin and TBK1) and is reduced in GRN knockout mice. Progranulin has also been found to play a role in mitophagy in mouse kidney cells. We therefore hypothesised that loss of progranulin could lead to defective mitophagy in neurons, astrocytes and microglia.MethodsWe investigated PINK1/Parkin mitophagy in astrocytic‐like H4 cells and neuron‐like Parkin overexpressing SHSY5Y cells (PoE‐5Ys) +/‐ siRNA against GRN. We also examined induced pluripotent stem cells (iPSCs) from four controls, three patients with FTD‐associated GRN mutations (R493X and C31fs) and a CRISPR series from the human iPSC Neurodegenerative Disease Initiative1,2,3 (iNDI) with an isogenic control, heterozygous and homozygous R493X mutation lines. The iPSCs were differentiated to cortical neurons, astrocytes and microglia. PINK1/Parkin mitophagy was induced using Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor). PINK1 accumulation, levels of S65 phosphorylated ubiquitin (pUb), other mitophagy markers as well as proteins hypothesised to play a role in the mechanism were examined using western blotting and immunofluorescence (ICC).ResultsLower levels of pUb were detected in PoE‐5Ys treated with oligomycin/antimycin following GRN knockdown. There was a significant reduction in mitophagy in GRN siRNA treated H4 cells, detected by ICC and western blotting of mitophagy markers. Initial results from iPSC neurons found no significant difference in mitophagy between control and patient lines. Work is ongoing in a larger set of iPSC neurons, astrocytes and microglia. Preliminary results suggest that reducing progranulin affects mitophagy in astrocytes but has a limited effect on mitophagy in neurons.ConclusionsThe results suggest that progranulin has a cell specific role in mitophagy with progranulin regulating stability and/or activity of PINK1. Current work aims to understand the mechanisms of this process and to dissect cell‐type specific contributions of progranulin to mitophagy.

FTD-associated behavioural and transcriptomic abnormalities in ‘humanized’ progranulin-deficient mice: A novel model for progranulin-associated FTD

Frontotemporal dementia (FTD) is an early onset dementia characterized by neuropathology and behavioural changes. A common genetic cause of FTD is haploinsufficiency of the gene progranulin (GRN). Mouse models of progranulin deficiency have provided insight into progranulin neurobiology, but the description of phenotypes with preclinical relevance has been limited in the currently available heterozygous progranulin-null mice. The identification of robust and reproducible FTD-associated behavioural, neuropathological, and biochemical phenotypes in progranulin deficient mice is a critical step in the preclinical development of therapies for FTD. In this work, we report the generation of a novel, ‘humanized’ mouse model of progranulin deficiency that expresses a single, targeted copy of human GRN in the absence of mouse progranulin. We also report the in-depth, longitudinal characterization of humanized progranulin-deficient mice and heterozygous progranulin-null mice over 18 months. Our analysis yielded several novel progranulin-dependent physiological and behavioural phenotypes, including increased marble burying, open field hyperactivity, and thalamic microgliosis in both models. RNAseq analysis of cortical tissue revealed an overlapping profile of transcriptomic dysfunction. Further transcriptomic analysis offers new insights into progranulin neurobiology. In sum, we have identified several consistent phenotypes in two independent mouse models of progranulin deficiency that are expected to be useful endpoints in the development of therapies for progranulin-deficient FTD. Furthermore, the presence of the human progranulin gene in the humanized progranulin-deficient mice will expedite the development of clinically translatable gene therapy strategies.

Antisense oligonucleotides targeting miR‐29b binding site increase translation of progranulin protein: potential therapeutic strategy for progranulin‐deficient frontotemporal dementia

AbstractBackground GRN mutations cause frontotemporal dementia (FTD) due to haploinsufficiency of progranulin. Several microRNAs (miRs), including miR‐29b, have been reported to negatively regulate progranulin protein levels. Here, we tested if antisense oligonucleotides (ASOs) – which are versatile modulators of target mRNA/protein levels – can be used to increase progranulin levels by sterically blocking the miR‐29b binding site.MethodWe designed 48 ASOs targeting the miR‐29b binding site in the 3’ UTR of the human GRN mRNA. We treated H4 neuroglioma cells and iPSC‐derived neurons with these ASOs and subsequently measured progranulin protein levels by western blot and ELISA. We performed further studies to determine the mechanism of action of these ASOs using ribosomal profiling, metabolic labeling, and FRET assays.ResultsWe identified 16 ASOs that increased progranulin protein levels in a dose‐dependent manner. Ribosomal profiling experiments revealed that cells treated with ASOs had marked enrichment in GRN mRNA in heavy polyribosome fractions, compared to cells treated with a scrambled control ASO, suggesting that the ASOs increase the rate of progranulin translation. Consistent with this, ASO treatment resulted in increased levels of newly synthesized progranulin protein. FRET‐based assays showed that ASOs can effectively compete miR‐29b from its binding site in the GRN 3’ UTR RNA under in vitro conditions.ConclusionsTogether, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This strategy may be therapeutically feasible for progranulin‐deficient FTD as well as other conditions of haploinsufficiency.

Overexpression of progranulin increases pathological protein accumulation by suppressing autophagic flux

Progranulin (PGRN) haploinsufficiency from autosomal dominant mutations in the PGRN gene causes frontotemporal lobar degeneration, which is characterized by cytoplasmic inclusions predominantly containing TDP-43 (FTLD-TDP). PGRN supplementation for patients with a PGRN gene mutation has recently been proposed as a therapeutic strategy to suppress FTLD-TDP. However, it currently remains unclear whether excessive amounts of PGRN are beneficial or harmful. We herein report the effects of PGRN overexpression on autophagic flux in a cultured cell model. PGRN overexpression increased the level of an autophagosome marker without promoting autophagosome formation and decreased the signal intensity of an autolysosome marker, indicating the suppression of autophagic flux due to reductions in the formation of autolysosomes. Assessments of lysosome numbers and biogenesis using LysoTracker and cells stably expressing TFEB-GFP, respectively, indicated that PGRN overexpression increased the lysosome numbers without lysosomal biogenesis. These results suggest that PGRN overexpression suppressed autophagic flux by inhibiting autophagosome-lysosome fusion. Moreover, PGRN overexpression enhanced polyglutamine aggregation and aggregate-prone TDP-43 accumulation, indicating that the suppression of autophagic flux by excessive amounts of PGRN worsens the pathology of neurodegenerative diseases.

A multifaceted role of progranulin in regulating amyloid-beta dynamics and responses.

Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN polymorphisms are associated with Alzheimer's disease. PGRN is highly expressed in the microglia near Aβ plaques and influences plaque dynamics and microglial activation. However, the detailed mechanisms remain elusive. Here we report that PGRN deficiency reduces human APP and Aβ levels in the young male but not female mice. PGRN-deficient microglia exhibit increased expression of markers associated with microglial activation, including CD68, galectin-3, TREM2, and GPNMB, specifically near Aβ plaques. In addition, PGRN loss leads to up-regulation of lysosome proteins and an increase in the nuclear localization of TFE3, a transcription factor involved in lysosome biogenesis. Cultured PGRN-deficient microglia show enhanced nuclear translocation of TFE3 and inflammation in response to Aβ fibril treatment. Taken together, our data revealed a sex- and age-dependent effect of PGRN on APP metabolism and a role of PGRN in regulating lysosomal activities and inflammation in plaque-associated microglia.

Neuropathological and behavioral characterization of aged Grn R493X progranulin-deficient frontotemporal dementia knockin mice

Frontotemporal lobar degeneration (FTLD) causes a spectrum of clinical presentations of frontotemporal dementia (FTD), including progressive changes in behavior, personality, executive function, and language. Up to 20% of familial FTLD cases are caused by progranulin (GRN) haploinsufficiency (FTD-GRN), with one of the most common causal variant being a nonsense mutation at arginine 493 (R493X). Recently, a genetic knockin FTD-GRN mouse model was generated bearing this GrnR493X mutation, at the analogous arginine in murine Grn. Aged, homozygous GrnR493X mice (GrnR493X/R493X) have been shown to phenotypically replicate several neuropathological hallmarks previously demonstrated in Grn null mice. We conducted a comprehensive neuropathological and behavioral assessment of 18 month old GrnR493X/R493X mice, observing a striking lysosomal dysfunction and thalamic neurodegeneration not previously described in this model, as well as a male-specific increase in generalized anxiety. These findings provide additional phenotypic markers of pathogenesis in aged GrnR493X/R493X mice that will contribute to better defining mechanisms underlying FTD-GRN, and offer relevant outcome measures for preclinical efficacy testing of novel therapeutics that target nonsense mutations leading to this devastating disease.

Anti-sortilin1 Antibody Up-Regulates Progranulin via Sortilin1 Down-Regulation.

Progranulin (PGRN) haploinsufficiency associated with loss-of-function mutations in the granulin gene causes frontotemporal dementia (FTD). This suggests that increasing PGRN levels could have promising therapeutic implications for patients carrying GRN mutations. In this study, we explored the therapeutic potential of sortilin1 (SORT1), a clearance receptor of PGRN, by generating and characterizing monoclonal antibodies against SORT1. Anti-SORT1 monoclonal antibodies were generated by immunizing Sort1 knockout mice with SORT1 protein. The antibodies were classified into 7 epitope bins based on their competitive binding to the SORT1 protein and further defined by epitope bin-dependent characteristics, including SORT1-PGRN blocking, SORT1 down-regulation, and binding to human and mouse SORT1. We identified a positive correlation between PGRN up-regulation and SORT1 down-regulation. Furthermore, we also characterized K1-67 antibody via SORT1 down-regulation and binding to mouse SORT1 in vivo and confirmed that K1-67 significantly up-regulated PGRN levels in plasma and brain interstitial fluid of mice. These data indicate that SORT1 down-regulation is a key mechanism in increasing PGRN levels via anti-SORT1 antibodies and suggest that SORT1 is a potential target to correct PGRN reduction, such as that in patients with FTD caused by GRN mutation.

Progranulin Adsorbs to Polypropylene Tubes and Disrupts Functional Assays: Implications for Research, Biomarker Studies, and Therapeutics.

Progranulin (PGRN) is a tightly regulated, secreted glycoprotein involved in a wide range of biological processes that is of tremendous interest to the scientific community due to its involvement in neoplastic, neurodevelopmental, and neurodegenerative diseases. In particular, progranulin haploinsufficiency leads to frontotemporal dementia. While performing experiments with a HIS-tagged recombinant human (rh) PGRN protein, we observed a measurable depletion of protein from solution due to its adsorption onto polypropylene (PPE) microcentrifuge tubes. In this study, we have quantified the extent of rhPGRN adsorption to PPE tubes while varying experimental conditions, including incubation time and temperature. We found that ∼25–35% of rhPGRN becomes adsorbed to the surface of PPE tubes even after a short incubation period. We then directly showed the deleterious impact of PGRN adsorption in functional assays and have recommended alternative labware to minimize these effects. Although the risk of adsorption of some purified proteins and peptides to polymer plastics has been characterized previously, this is the first report of rhPGRN adsorption. Moreover, since PGRN is currently being studied and utilized in both basic science laboratories to perform in vitro studies and translational laboratories to survey PGRN as a quantitative dementia biomarker and potential replacement therapy, the reported observations here are broadly impactful and will likely significantly affect the design and interpretation of future experiments centered on progranulin biology.

Haploinsufficiency of progranulin causes impairments in PINK/PARKIN mitophagy

AbstractBackgroundGRN mutations, resulting in haploinsufficiency of progranulin, cause frontotemporal dementia (FTD) with TDP‐43 pathology. Impairments in mitophagy, the selective autophagy of damaged mitochondria, have been identified in several neurodegenerative diseases, and multiple neurodegenerative disease genes, including PINK1, Parkin, VCP and TBK1, have been shown to play a role in this pathway. A role for progranulin in the regulation of neuronal mitophagy has not been explored, however progranulin deficient mice exhibit reduced xenophagy (selective clearance of bacteria). We therefore hypothesised that loss of progranulin could lead to defective mitophagy.MethodWe used two in vitro models to investigate PINK1/Parkin mitophagy: SHSY5Y cells overexpressing Parkin (PoE‐SHSY5Y) +/‐ siRNA against GRN, and induced pluripotent stem cell (iPSC) derived cortical neurons from two patients with FTD‐associated GRN mutations (R493X and C31fs). PINK1/Parkin mitophagy was induced using a combination of Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor), and PINK1 accumulation and levels of S65 phosphorylated ubiquitin (the substrate of PINK1 and a marker of its activity) were examined using western blot and immunofluorescence. Subsequent mitophagy was assessed by examining Mitofusin‐2 ubiquitination and degradation, and TIM23 levels by western blot.ResultA reduction in progranulin levels resulted in reduced accumulation of PINK1 and lower levels of mitochondrial phospho‐ubiquitin following oligomycin/antimycin treatment in both PoE‐SHSY5Y and iPSC‐neurons with GRN mutations. Ubiquitination and degradation of mitofusin‐2 was also decreased in cells with reduced progranulin.ConclusionThese results suggest that progranulin plays a role in mitophagy by regulating stability and/or activity of PINK1. Ongoing work aims to understand the mechanisms by which progranulin and/or individual granulins contribute to this process and to dissect cell‐type specific contributions of progranulin to mitophagy in iPSC‐ derived neurons, astrocytes and microglia.

Network analysis of the brain proteome of GRN knockout mice reveals pathogenic mechanisms shared in human frontotemporal dementia caused by GRN mutations

AbstractBackgroundMutations in the GRN gene, encoding the progranulin (PGRN) protein, are a common cause of Frontotemporal dementia (FTD) and induce haploinsufficiency of PGRN. PGRN is composed of 7.5 repeating domains called granulins (GRNs 1‐7). PGRN is processed into GRNs within the lysosome, where we hypothesize they are critical for lysosome homeostasis and function. However, it is unclear how decreased levels of lysosomal PGRN/GRNs cause FTD, Alzheimer’s disease, and related neurodegenerative disorders.MethodsWe performed quantitative proteomic analysis of whole‐brain tissue from wild‐type (Grn +/+) and PGRN knockout (Grn ‐/‐) mice at 3‐ and 19‐months of age using 10‐plex Tandem Mass Tag (TMT) isobaric labeling mass spectrometry. Protein changes were validated using immunobloting, immunohistochemistry, and ELISAs.ResultsDeep proteome analysis of mouse brains detected 8,695 proteins. Weighted correlation network analysis (WGCNA) of the brain proteome of Grn ‐/‐ compared to Grn +/+ mice identified 29 modules of highly co‐expressed proteins. In particular, 3 modules strongly correlated to Grn deficiency, increased with age, and were enriched with lysosomal proteins (Gpnmb, Ctsd, Tpp1) and inflammatory proteins (Lgals3, GFAP, CD44). Immunohistochemistry revealed age‐dependent increases in multiple lysosomal proteins and inflammation throughout the Grn ‐/‐ brain, particularly in the thalamus. Importantly, we discovered similar changes in the brain and cerebrospinal fluid (CSF) of individuals with GRN mutations that cause FTD (FTD‐GRN). GPNMB and galectin‐3 (LGALS3) were significantly elevated in the lysates of FTD‐GRN brain. Further, GPNMB levels were significantly increased in human FTD‐GRN CSF compared to controls and other genetic forms of FTD (MAPT and C9orf72).ConclusionsThese data suggest that common pathogenic pathways are dysregulated in human FTD cases with GRN haploinsufficiency and Grn ‐/‐ mice. Moreover, GPNMB is a novel, potential biomarker for FTD. Our findings support the idea that insufficiency of PGRN and GRNs in humans cause FTD, AD, and related neurodegenerative diseases through lysosomal dysfunction and neuroinflammation and suggest novel therapeutic approaches.

Fronto-temporal dementia risk gene TMEM106B has opposing effects in different lysosomal storage disorders.

TMEM106B is a transmembrane protein localized to the endo-lysosomal compartment. Genome-wide association studies have identified TMEM106B as a risk modifier of Alzheimer’s disease and frontotemporal lobar degeneration, especially with progranulin haploinsufficiency. We recently demonstrated that TMEM106B loss rescues progranulin null mouse phenotypes including lysosomal enzyme dysregulation, neurodegeneration and behavioural alterations. However, the reason whether TMEM106B is involved in other neurodegenerative lysosomal diseases is unknown. Here, we evaluate the potential role of TMEM106B in modifying the progression of lysosomal storage disorders using progranulin-independent models of Gaucher disease and neuronal ceroid lipofuscinosis. To study Gaucher disease, we employ a pharmacological approach using the inhibitor conduritol B epoxide in wild-type and hypomorphic Tmem106b−/− mice. TMEM106B depletion ameliorates neuronal degeneration and some behavioural abnormalities in the pharmacological model of Gaucher disease, similar to its effect on certain progranulin null phenotypes. In order to examine the role of TMEM106B in neuronal ceroid lipofuscinosis, we crossbred Tmem106b−/− mice with Ppt1−/−, a genetic model of the disease. In contrast to its conduritol B epoxide-rescuing effect, TMEM106B loss exacerbates Purkinje cell degeneration and motor deficits in Ppt1−/− mice. Mechanistically, TMEM106B is known to interact with subunits of the vacuolar ATPase and influence lysosomal acidification. In the pharmacological Gaucher disease model, the acidified lysosomal compartment is enhanced and TMEM106B loss rescues in vivo phenotypes. In contrast, gene-edited neuronal loss of Ppt1 causes a reduction in vacuolar ATPase levels and impairment of the acidified lysosomal compartment, and TMEM106B deletion exacerbates the mouse Ppt1−/− phenotype. Our findings indicate that TMEM106B differentially modulates the progression of the lysosomal storage disorders Gaucher disease and neuronal ceroid lipofuscinosis. The effect of TMEM106B in neurodegeneration varies depending on vacuolar ATPase state and modulation of lysosomal pH. These data suggest TMEM106B as a target for correcting lysosomal pH alterations, and in particular for therapeutic intervention in Gaucher disease and neuronal ceroid lipofuscinosis.

Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice.

Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). Loss of PGRN leads to lysosome dysfunction during aging. TMEM106B, a gene encoding a lysosomal membrane protein, is the main risk factor for FTLD with PGRN haploinsufficiency. But how TMEM106B affects FTLD disease progression remains to be determined. Here, we report that TMEM106B deficiency in mice leads to accumulation of lysosome vacuoles at the distal end of the axon initial segment in motor neurons and the development of FTLD‐related pathology during aging. Ablation of both PGRN and TMEM106B in mice results in severe neuronal loss and glial activation in the spinal cord, retina, and brain. Enlarged lysosomes are frequently found in both microglia and astrocytes. Loss of both PGRN and TMEM106B results in an increased accumulation of lysosomal vacuoles in the axon initial segment of motor neurons and enhances the manifestation of FTLD phenotypes with a much earlier onset. These results provide novel insights into the role of TMEM106B in the lysosome, in brain aging, and in FTLD pathogenesis.

Network Analysis of the Brain Proteome of Grn Knockout Mice Reveals Pathogenic Mechanisms Shared in Human Frontotemporal dementia caused by GRN mutations.

Mutations in the GRN gene, which encodes the progranulin (PGRN) protein, are one of the most common causes of Frontotemporal dementia (FTD) and cause disease through haploinsufficiency of PGRN. PGRN is composed of 7.5 repeating domains termed granulins (GRNs 1–7) that are joined by linker regions. We have recently found that PGRN is processed into ~6kDa GRNs within the lysosome, where we hypothesize they are critical for lysosome homeostasis (Holler et al, eNeuro, 2017 Aug 18;4(4)). However, the molecular mechanisms caused by decreased levels of lysosomal PGRN/GRNs that lead to FTD, Alzheimer’s disease, and other neurodegenerative disorders are still unknown. To gain insight into the mechanisms of FTD pathogenesis, we performed an unbiased quantitative proteomic analysis of whole‐brain tissue from wild‐type (WT) and Progranulin knockout (Grn−/−) mice at 3 and 19‐months of age. We utilized a 10‐plex Tandem Mass Tag (TMT) isobaric labeling mass spectrometry approach for peptide labeling and quantification that enabled a deep proteome analysis of mouse brain and detection of 8,695 proteins. Differential expression analysis of the brain proteome of 3‐month old Grn−/− versus WT mice revealed 29 up and 26 down‐regulated proteins, while 119 proteins were up and 20 proteins were down‐regulated in 19‐month old Grn−/− mice. Then we performed weighted correlation network analysis (WGCNA) on the brain proteome of Grn−/− vs WT mice and identified 29 modules of highly co‐expressed proteins. In particular, 3 modules were strongly correlated to Grn deficiency, increased with age, and were enriched with lysosomal proteins (Ctsd, Fuca2, Tpp1) and inflammatory proteins (Gpnmb, Lgals3, GFAP, CD44, S100a C1qa). Subsequent validation of changes identified through proteomics using western blotting and immunohistochemistry revealed age‐dependent increases in multiple lysosomal proteins (i.e. Cathepsin D and Z) and markers of gliosis (GFAP, IBA1) throughout the brain, with a particularly marked increase in the thalamus in old Grn−/− mice versus age‐matched WT. Next, we asked if similar changes occurred in the biofluids or post‐mortem tissue from individuals with GRN mutations associated with FTD. We found that GPNMB and galectin‐3 (LGALS3) were significantly elevated in the lysates of FTD‐GRN brain samples compared to healthy controls. Total levels of GPNMB were also elevated human FTD‐GRN cerebrospinal fluid and plasma samples compared to controls. These data suggest that common pathogenic pathways are dysregulated in human FTD cases with GRN haploinsufficiency and mice completely deficient for Grn. Moreover, our findings support the idea that insufficiency of PGRN and GRNs in humans may cause FTD, AD and related neurodegenerative diseases through lysosomal dysfunction and neuroinflammation and suggest novel therapeutic approaches.Support or Funding Information1) National Institute of Health NINDS grants (R01NS105971; R01NS093362)2) the Association for Frontotemporal Degeneration grant3) The Bluefield Project to Cure FTD