Progranulin Deficiency Research Articles

PGRN Inhibits Early B-cell Activation and IgE Production Through the IFITM3-STAT1 Signaling Pathway in Asthma.

Progranulin (PGRN) plays a critical role in bronchial asthma and the function of various immune cells. However, the mechanisms by which PGRN influences B-cell receptor (BCR) signaling and immunoglobulin E(IgE) production are not fully understood. The study aimed to elucidate the molecular mechanisms through which PGRN affects BCR signaling, B-cell differentiation, and IgE production. A PGRN knockout mouse model, along with techniques including flow cytometry, the creation of a bone marrow chimeric mouse model, total internal reflection fluorescence (TIRF), and Western blot (WB) analysis is employed, to investigate the link between PGRN and various aspects of B-cell biology. It is discovered that the absence of PGRN in mice alters peripheral B-cell subpopulations, promotes IgE class switching in a cell-intrinsic manner, and affects B-cell subpopulations. Additionally, PGRN modulates B-cell functions by regulating BCR signaling pathways, metabolic processes, and the actin cytoskeleton during early B-cell activation. Significantly, PGRN deficiency results in diminished production of NP-specific antibodies. Moreover, it is found that PGRN inhibits B-cell activation and IgE production through the PGRN-IFITM3-STAT1 signaling pathway. The findings provide new strategies for the targeted treatment of bronchial asthma, highlighting the crucial role of PGRN in B-cell signaling and IgE production.

Selective neuronal expression of progranulin is sufficient to provide neuroprotective and anti-inflammatory effects after traumatic brain injury

Progranulin (PGRN), which is produced in neurons and microglia, is a neurotrophic and anti-inflammatory glycoprotein. Human loss-of-function mutations cause frontotemporal dementia, and PGRN knockout (KO) mice are a model for dementia. In addition, PGRN KO mice exhibit severe phenotypes in models of traumatic or ischemic central nervous system (CNS) disorders, including traumatic brain injury (TBI). It is unknown whether restoration of progranulin expression in neurons (and not in microglia) might be sufficient to prevent excessive TBI-evoked brain damage. To address this question, we generated mice with Nestin-Cre-driven murine PGRN expression in a PGRN KO line (PGRN-KONestinGrn) to rescue PGRN in neurons. PGRN expression analysis in primary CNS cell cultures from naïve mice and in (non-) injured brain tissue from PGRN-KONestinGrn revealed expression of PGRN in neurons but not in microglia. After experimental TBI, examination of the structural brain damage at 5 days post-injury (dpi) showed that the TBI-induced loss of brain tissue and hippocampal neurons was exacerbated in PGRN-KOGrnflfl mice (PGRN knockout with the mGrn fl-STOP-fl allele, Cre-negative), as expected, whereas the tissue damage in PGRN-KONestinGrn mice was similar to that in PGRN-WT mice. Analysis of CD68+ immunofluorescent microglia and Cd68 mRNA expression showed that excessive microglial activation was rescued in PGRN-KONestinGrn mice, and the correlation of brain injury with Cd68 expression suggested that Cd68 was a surrogate marker for excessive brain injury caused by PGRN deficiency. The results show that restoring neuronal PGRN expression was sufficient to rescue the exacerbated neuropathology of TBI caused by PGRN deficiency, even in the absence of microglial PGRN. Hence, endogenous microglial PGRN expression was not essential for the neuroprotective or anti-inflammatory effects of PGRN after TBI in this study.Graphical

Sortilin is associated with progranulin deficiency and autism-like behaviors in valproic acid-induced autism rats.

Neuroinflammation and microglial activation-related dendritic injury contribute to the pathogenesis of Autism Spectrum Disorder (ASD). Previous studies show that Progranulin (PGRN) is a growth factor associated with inflammation and synaptic development, but the role of PGRN in autism and the mechanisms underlying changes in PGRN expression remain unclear. To investigate the impact of PGRN in autism, we stereotactically injected recombinant PGRN into the hippocampus of ASD model rats. Additionally, we explored the possibility that sortilin may be the factor behind the alterations in PGRN by utilizing SORT1 knockdown. Ultimately, we aimed to identify potential targets for the treatment of autism. PGRN could alleviate inflammatory responses, protect neuronal dendritic spines, and ameliorate autism-like behaviors. Meanwhile, elevated expression of sortilin and decreased levels of PGRN were observed in both ASD patients and rats. Enhanced sortilin levels facilitated PGRN internalization into lysosomes. Notably, suppressing SORT1 expression amplified PGRN levels, lessened microglial activation, and mitigated inflammation, thereby alleviating autism-like behaviors. Collectively, our findings highlight elevated sortilin levels in ASD rat brains, exacerbating dendrite impairment by affecting PGRN expression. PGRN supplementation and SORT1 knockdown hold potential as therapeutic strategies for ASD.

Abstract 12: Deficiency of Progranulin Decreases Mitochondrial Function and Induces a Whitening Phenotype in Perivascular Adipose Tissue, Suppressing Its Anti-Contractile Effects

Perivascular adipose tissue (PVAT) is recognized as an endocrine organ that secretes a variety of adipokines, which negatively regulate vascular contraction. Progranulin (PGRN), a glycoprotein encoded by the GRN gene, is highly expressed in adipocytes, and is implicated in inflammation, cellular proliferation, and repair mechanisms. The relationship between PGRN and PVAT remains unclear. We hypothesized that the lack of PGRN negatively affects PVAT quality. We used male C57BL/6J wild-type (WT) and global PGRN knockout (KO) mice, aged 10-12 weeks. Energy expenditure was analyzed via the Comprehensive Lab Animal Monitoring System (CLAMS). Vascular function was studied in endothelium-intact aortae with PVAT [PVAT(+)] and without PVAT [PVAT(-)]. Additionally, 3T3-L1 cells were used to evaluate the molecular mechanisms. The KO mice showed a decreased respiratory exchange ratio (RER) and a whitening phenotype of PVAT, which was accompanied by a loss of anti-contractile effects [(mN) WT_PVAT(-): 5.2±0.3 vs PVAT(+): 3.4±0.5*, *P<0.05 and KO_PVAT(-): 6.1±0.7 vs KO_PVAT(+): 5.7±0.4]. Additionally, PVAT from KO displayed a significant reduction in nitric oxide production [(RFU) WT_PVAT: 223.1± 12.3 vs KO_PVAT: 67.3±9.1*, *P<0.05] and endothelial NO synthase phosphorylation. Additionally, the lack of PGRN attenuated the expression of mitochondrial complexes [(AU) WT_PVAT: 1.3± 0.03 vs KO_PVAT: 0.6±0.06*, *P<0.05] measured by the OXPHOS cocktail antibody. Via high-resolution respirometry, we found that isolated mitochondria from KO_PVAT showed impaired succinate-driven respiration or complex II activity. To understand if the whitening phenotype of PVAT was leading to its dysfunction, we induced a browning phenotype of PVAT by treating the mice with a β3-agonist for 7 days (CL 316243, 1mg/kg/day). This treatment increased RER restored the PVAT phenotype, and improved PVAT anti-contractile effect and mitochondrial respiration. Using 3T3-L1 cells, we engineered the overexpression and knockdown of PGRN via lentivirus (LV) infection and siRNA, respectively. Suppression of PGRN increased mRNA of whitening markers (1.5-fold increase vs scramble siRNA), while overexpression elevated mRNA of browning and mitochondrial markers (1.3-fold increase vs scramble LV). In summary, our findings reveal that PGRN is crucial for maintaining the phenotype and anti-contractile function of PVAT and highlight PGRN as a pharmacological target for vascular outcomes in cardiometabolic conditions.

Benzoxazole-derivatives enhance progranulin expression and reverse the aberrant lysosomal proteome caused by GRN haploinsufficiency

Heterozygous loss-of-function mutations in the GRN gene are a major cause of hereditary frontotemporal dementia. The mechanisms linking frontotemporal dementia pathogenesis to progranulin deficiency are not well understood, and there is currently no treatment. Our strategy to prevent the onset and progression of frontotemporal dementia in patients with GRN mutations is to utilize small molecule positive regulators of GRN expression to boost progranulin levels from the remaining functional GRN allele, thus restoring progranulin levels back to normal within the brain. This work describes a series of blood-brain-barrier-penetrant small molecules which significantly increase progranulin protein levels in human cellular models, correct progranulin protein deficiency in Grn+/− mouse brains, and reverse lysosomal proteome aberrations, a phenotypic hallmark of frontotemporal dementia, more efficiently than the previously described small molecule suberoylanilide hydroxamic acid. These molecules will allow further elucidation of the cellular functions of progranulin and its role in frontotemporal dementia and will also serve as lead structures for further drug development.

CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.

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 and Lysosomal pH: Implications for the Proinflammatory Response in Neurodegenerative Diseases

Progranulin (PGRN) deficiency and lysosomal dysfunction have been independently linked to Alzheimer's disease. Several lines of evidence suggest that there is a link between PGRN and lysosomal function. PGRN is a pleiotropic signaling molecule whose activity depends upon differential proteolytic processing. The different PGRN subunits generated during processing can regulate inflammation, lysosomal function, and/or growth. Not only is the lysosome a site of PGRN processing, the resulting PGRN subunits promote the function of several lysosomal proteases. In addition, in models of PGRN insuffciency, increasing the levels of PGRN restores lysosomal function and reduces inflammation. As microglial inflammation is key to the development of neurodegenerative diseases, this work investigates the link between lysosomal function, PGRN, and inflammation utilizing two cell lines, each for a distinct set of experiments. The SW13 human adrenal carcinoma cell line that exists in two epigenetically distinct subtypes was used to focus on differences in lysosomal pH and PGRN processing. The HMC3 human microglial cell line was used to measure microglial activation and cytokine production. We hypothesize that PGRN, when appropriately processed, will restore lysosomal function to reduce pro-inflammatory cytokine production. Results suggest that differential processing of PGRN is correlated to both differences in rate of growth, inflammatory cytokine production, and lysosomal pH as PGRN expression and processing varies when cells are stimulated which may be related to observed changes in lysosomal pH and proteases. Ongoing experiments address the possibility that decreasing lysosomal pH may shift the processing of PGRN, influencing the biological activities associated with this versatile signaling molecule. As mutations in the PGRN gene are implicated in both dysfunctional lysosomal pH and a variety of neurodegenerative diseases, these results may provide a novel approach to regulate proper intracellular functions to slow or prevent the development of disease. Midwestern University Biomedical Sciences Program Funds awarded to AZY and PB; Kenneth A. Suarez Research Fellowship Funding awarded to PB; Arizona Alzheimer's Consortium Pilot Grant Funding awarded to EEH and KJL; Midwestern Arizona Alzheimer’s Consortium (MAAC) Grant Funding awarded to EEH and KJL; Midwestern University Intramural Funding awarded to EEH and KJL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multiomics Evaluation of Human iPSCs and iPSC-Derived Neurons.

Human induced pluripotent stem cells (iPSCs) can be differentiated into neurons, providing living human neurons to model brain diseases. However, it is unclear how different types of molecules work together to regulate stem cell and neuron biology in healthy and disease states. In this study, we conducted integrated proteomics, lipidomics, and metabolomics analyses with confident identification, accurate quantification, and reproducible measurements to compare the molecular profiles of human iPSCs and iPSC-derived neurons. Proteins, lipids, and metabolites related to mitosis, DNA replication, pluripotency, glycosphingolipids, and energy metabolism were highly enriched in iPSCs, whereas synaptic proteins, neurotransmitters, polyunsaturated fatty acids, cardiolipins, and axon guidance pathways were highly enriched in neurons. Mutations in the GRN gene lead to the deficiency of the progranulin (PGRN) protein, which has been associated with various neurodegenerative diseases. Using this multiomics platform, we evaluated the impact of PGRN deficiency on iPSCs and neurons at the whole-cell level. Proteomics, lipidomics, and metabolomics analyses implicated PGRN's roles in neuroinflammation, purine metabolism, and neurite outgrowth, revealing commonly altered pathways related to neuron projection, synaptic dysfunction, and brain metabolism. Multiomics data sets also pointed toward the same hypothesis that neurons seem to be more susceptible to PGRN loss compared to iPSCs, consistent with the neurological symptoms and cognitive impairment from patients carrying inherited GRN mutations.

Progranulin deficiency associates with postmenopausal osteoporosis via increasing ubiquitination of estrogen receptor α

Estrogen deficiency is considered the most important cause of postmenopausal osteoporosis. However, the underlying mechanism is still not completely understood. In this study, progranulin (PGRN) was isolated as a key regulator of bone mineral density in postmenopausal women through high throughput proteomics screening. In addition, PGRN-deficient mice exhibited significantly lower bone mass than their littermates in an ovariectomy-induced osteoporosis model. Furthermore, estrogen-mediated inhibition of osteoclastogenesis and bone resorption as well as its protection against ovariectomy-induced bone loss largely depended on PGRN. Mechanistic studies revealed the existence of a positive feedback regulatory loop between PGRN and estrogen signaling. In addition, loss of PGRN led to the reduction of estrogen receptor α, the important estrogen receptor involved in estrogen regulation of osteoporosis, through enhancing its degradation via K48-linked ubiquitination. These findings not only provide a previously unrecognized interplay between PGRN and estrogen signaling in regulating osteoclastogenesis and osteoporosis but may also present a new therapeutic approach for the prevention and treatment of postmenopausal osteoporosis by targeting PGRN/estrogen receptor α.

Progranulin deficiency attenuates tubulointerstitial injury in a mouse unilateral ureteral obstruction model.

Progranulin (PGRN) may have two opposing effects-inflammation and anti-inflammation-in different diseases. Although previous studies have reported that PGRN is involved in liver fibrosis, its involvement in tubulointerstitial fibrosis remains to be fully elucidated. Herein, we investigated these issues using PGRN-knockout (KO) mice treated with unilateral ureteral obstruction (UUO). Eight-week-old male PGRN-KO and wild-type (WT) mice were euthanized 3 and 7 days following UUO, and their kidneys were harvested for histopathological analysis. The renal expression of PGRN was evaluated by immunohistochemical and/or western blot analyses. The renal mRNA levels of markers related to inflammation (Il1b, Tnf, Il6, Ccl2, and Adgre1) and fibrosis (Tgfb1, Acta2, Fn1, and Col1a2) were evaluated using quantitative PCR. Histological changes such as renal tubular atrophy, urinary casts, and tubulointerstitial fibrosis were significantly improved in UUO-KO mice compared with UUO-WT mice. Quantitative PCR revealed that the mRNA expression levels of all inflammation- and fibrosis-related markers were lower in UUO-KO mice than in UUO-WT mice at 3 and/or 7 days after UUO. Moreover, PGRN and GRN protein levels were higher in the kidneys of UUO-WT mice than in mice that did not undergo UUO. Elevated GRN levels associated with excess PGRN levels may be involved in the occurrence of renal inflammation and fibrosis in UUO mice.

Progranulin-deficient macrophages cause cardiotoxicity under hypoxic conditions

Myocardial infarction (MI) induces structural and electrical cardiac remodeling in response to ischemic insult, causing lethal arrhythmias and sudden death. Progranulin (PGRN) is a glycoprotein mainly expressed in macrophages that modulates the immune responses. In this study, we investigated the direct influence of PGRN knockout (Grn−/−) macrophages on post-MI pathophysiology. An MI mouse model was established by ligating the left coronary artery for RNA sequencing and electrocardiographic analysis. Bone marrow-derived macrophages (BMDMs) were injected into mice and supernatant was collected for the measurement of reactive oxygen species (ROS) levels and extracellular flux analysis. Administration of Grn−/− BMDMs prolonged the QT intervals in the MI mouse model. Moreover, genes highly expressed in macrophages were upregulated in Grn−/− heart after MI. Post-hypoxic supernatant of Grn−/− BMDMs increased the oxygen–glucose deprivation-induced cardiomyocyte death. Grn−/− BMDMs exhibited increased ROS production, oxygen consumption, and extracellular acidification under hypoxia and inflammatory conditions. These findings suggest that PGRN deficiency causes cardiotoxicity via secretory components of macrophages that exhibit metabolic abnormalities under hypoxia.

Multi-modal proteomic characterization of lysosomal function and proteostasis in progranulin-deficient neurons

BackgroundProgranulin (PGRN) is a lysosomal glycoprotein implicated in various neurodegenerative diseases, including frontotemporal dementia and neuronal ceroid lipofuscinosis. Over 70 mutations discovered in the GRN gene all result in reduced expression of the PGRN protein. Genetic and functional studies point toward a regulatory role for PGRN in lysosome functions. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomes remain unclear.MethodsWe developed multifaceted proteomic techniques to characterize the dynamic lysosomal biology in living human neurons and fixed mouse brain tissues. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactome in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in human i3Neurons for the first time.ResultsLeveraging the multi-modal proteomics and live-cell imaging techniques, we comprehensively characterized how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. We found that PGRN loss impairs the lysosome’s degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased hydrolases within the lysosome, altered protein regulations related to lysosomal transport, and elevated lysosomal pH. Consistent with impairments in lysosomal function, GRN-null i3Neurons and frontotemporal dementia patient-derived i3Neurons carrying GRN mutation showed pronounced alterations in protein turnover, such as cathepsins and proteins related to supramolecular polymerization and inherited neurodegenerative diseases.ConclusionThis study suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which influences global proteostasis in neurons. Beyond the study of progranulin deficiency, these newly developed proteomic methods in neurons and brain tissues provided useful tools and data resources for the field to study the highly dynamic neuronal lysosome biology.Graphical

Progranulin deficiency exacerbates cardiac remodeling after myocardial infarction.

Myocardial infarction (MI) is a lethal disease that causes irreversible cardiomyocyte death and subsequent cardiovascular remodeling. We have previously shown that the administration of recombinant progranulin (PGRN) protects against myocardial ischemia and reperfusion injury. However, the post-MI role of PGRN remains unclear. In the present study, we investigated the effects of PGRN deficiency on cardiac remodeling after MI. Wild-type and PGRN-knockout mice were subjected to MI by ligation of the left coronary artery for histological, electrophysiological, and protein expression analysis. Cardiac macrophage subpopulations were analyzed by flow cytometry. Bone marrow-derived macrophages (BMDMs) were acquired and treated with LPS + IFN-γ and IL-4 to evaluate mRNA levels and phagocytic ability. PGRN expression was gradually increased in the whole heart at 1, 3, and 7 days after MI. Macrophages abundantly expressed PGRN at the border areas at 3 days post-MI. PGRN-knockout mice showed higher mortality, increased LV fibrosis, and severe arrhythmia following MI. PGRN deficiency increased the levels of CD206 and MerTK expression and macrophage infiltration in the infarcted myocardium, which was attributed to a larger subpopulation of cardiac CCR2+ Ly6Clow CD11b+ macrophages. PGRN-deficient BMDMs exhibited higher TGF-β, IL-4R, and lower IL-1β, IL-10 and increased acute phagocytosis following stimulation of LPS and IFN-γ. PGRN deficiency reduced survival and increased cardiac fibrosis following MI with the induction of abnormal subpopulation of cardiac macrophages early after MI, thereby providing insight into the relationship between properly initiating cardiac repair and macrophage polarization after MI.

Progranulin deficiency results in sex-dependent alterations in microglia in response to demyelination.

Heterozygous mutations in the granulin (GRN) gene, resulting in the haploinsufficiency of the progranulin (PGRN) protein, is a leading cause of frontotemporal lobar degeneration (FTLD). Complete loss of the PGRN protein causes neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. Polymorphisms in the GRN gene have also been associated with several other neurodegenerative diseases, including Alzheimer's disease (AD), and Parkinson's disease (PD). PGRN deficiency has been shown to cause myelination defects previously, but how PGRN regulates myelination is unknown. Here, we report that PGRN deficiency leads to a sex-dependent myelination defect with male mice showing more severe demyelination in response to cuprizone treatment. This is accompanied by exacerbated microglial proliferation and activation in the male PGRN-deficient mice. Interestingly, both male and female PGRN-deficient mice show sustained microglial activation after cuprizone removal and a defect in remyelination. Specific ablation of PGRN in microglia results in similar sex-dependent phenotypes, confirming a microglial function of PGRN. Lipid droplets accumulate in microglia specifically in male PGRN-deficient mice. RNA-seq analysis and mitochondrial function assays reveal key differences in oxidative phosphorylation in male versus female microglia under PGRN deficiency. A significant decrease in myelination and accumulation of myelin debris and lipid droplets in microglia were found in the corpus callosum regions of FTLD patients with GRN mutations. Taken together, our data support that PGRN deficiency leads to sex-dependent alterations in microglia with subsequent myelination defects.

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.

Progranulin Deficiency Induces Mitochondrial Dysfunction in Frontotemporal Lobar Degeneration with TDP-43 Inclusions

Loss-of-function (LOF) mutations in GRN gene, which encodes progranulin (PGRN), cause frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). FTLD-TDP is one of the most common forms of early onset dementia, but its pathogenesis is not fully understood. Mitochondrial dysfunction has been associated with several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Here, we have investigated whether mitochondrial alterations could also contribute to the pathogenesis of PGRN deficiency-associated FTLD-TDP. Our results showed that PGRN deficiency induced mitochondrial depolarization, increased ROS production and lowered ATP levels in GRN KD SH-SY5Y neuroblastoma cells. Interestingly, lymphoblasts from FTLD-TDP patients carrying a LOF mutation in the GRN gene (c.709-1G > A) also demonstrated mitochondrial depolarization and lower ATP levels. Such mitochondrial damage increased mitochondrial fission to remove dysfunctional mitochondria by mitophagy. Interestingly, PGRN-deficient cells showed elevated mitochondrial mass together with autophagy dysfunction, implying that PGRN deficiency induced the accumulation of damaged mitochondria by blocking its degradation in the lysosomes. Importantly, the treatment with two brain-penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27), known for preventing the phosphorylation and cytosolic accumulation of TDP-43, rescued mitochondrial function in PGRN-deficient cells. Taken together, these results suggest that mitochondrial function is impaired in FTLD-TDP associated with LOF GRN mutations and that the TDP-43 pathology linked to PGRN deficiency might be a key mechanism contributing to such mitochondrial dysfunction. Furthermore, our results point to the use of drugs targeting TDP-43 pathology as a promising therapeutic strategy for restoring mitochondrial function in FTLD-TDP and other TDP-43-related diseases.

Progranulin deficiency suppresses allergic asthma and enhances efferocytosis via PPAR-γ/MFG-E8 regulation in macrophages.

Efferocytosis can resolve airway inflammation and enhance airway tolerance in allergic asthma. While previous work has reported that progranulin (PGRN) regulated macrophage efferocytosis, but it is unclear whether PGRN-mediated efferocytosis is associated with asthma. Here, we found that in an ovalbumin (OVA)-induced allergic asthma model, the airway inflammation was suppressed and the apoptosis in lung tissues was ameliorated in PGRN-deficient mice. In contrast, PGRN knockdown in human bronchial epithelial cellsincreased apoptosis in vitro. Furthermore, PGRN-deficient macrophages had significantly stronger efferocytosis ability than wild type (WT) macrophages both in vitro and in vivo. PGRN-deficient peritoneal macrophages (PMs) exhibited increased expression of genes associated with efferocytosis including milk fat globule-epidermal growth factor 8 (MFG-E8), peroxisome proliferator-activated receptor gamma (PPAR-γ) and sirtuin1 (SIRT1) and increased capacity to produce the anti-inflammatory mediator interleukin (IL)-10 during efferocytosis. GW9662, the inhibitor of PPAR-γ, abolished increased efferocytosis and MFG-E8 expression in PGRN-deficient PMs suggesting that PGRN deficiency enhanced MFG-E8-mediated efferocytosis through PPAR-γ. Correspondingly, efferocytosis genes were increased in the lungs of OVA-induced PGRN-deficient mice. GW9662 treatment reduced MFG-E8 expression but did not significantly affect airway inflammation. Our results demonstrated that PGRN deficiency enhanced efferocytosis via the PPAR-γ/MFG-E8 pathway and this may be one of the reasons PGRN deficiency results in inhibition of airway inflammation in allergic asthma.

Progranulin from different gliocytes in the nucleus accumbens exerts distinct roles in FTD- and neuroinflammation-induced depression-like behaviors

BackgroundNeuroinflammation in the nucleus accumbens (NAc) is well known to influence the progression of depression. However, the molecular mechanisms triggering NAc neuroinflammation in depression have not been fully elucidated. Progranulin (PGRN) is a multifunctional growth factor that is linked to the innate immune response and inflammation, and PGRN plays a key role in neurodegenerative diseases such as frontotemporal dementia (FTD). Here, the purpose of this study was to validate whether PGRN was involved in the NAc neuroinflammation-promoted depressive-like phenotype.MethodsA NAc neuroinflammation-relevant depression-like model was established using wild-type (WT) and PGRN-knockout (KO) mice after NAc injection with lipopolysaccharide (LPS), and various behavioral tests related to cognition, social recognition, depression and anxiety were performed with WT and PGRNKO mice with or without NAc immune challenge. RT‒PCR, ELISA, western blotting and immunofluorescence staining were used to determine the expression and function of PGRN in the neuroinflammatory reaction in the NAc after LPS challenge. The morphology of neurons in the NAc from WT and PGRNKO mice under conditions of NAc neuroinflammation was analyzed using Golgi–Cox staining, followed by Sholl analyses. The potential signaling pathways involved in NAc neuroinflammation in PGRNKO mice were investigated by western blotting.ResultsUnder normal conditions, PGRN deficiency induced FTD-like behaviors in mice and astrocyte activation in the NAc, promoted the release of the inflammatory cytokines interleukin (IL)-6 and IL-10 and increased dendritic complexity and synaptic protein BDNF levels in the NAc. However, NAc neuroinflammation enhanced PGRN expression, which was located in astrocytes and microglia within the NAc, and PGRN deficiency in mice alleviated NAc neuroinflammation-elicited depression-like behaviors, seemingly inhibiting astrocyte- and microglia-related inflammatory reactions and neuroplasticity complexity in the NAc via the p38 and nuclear factor of kappa (NF-κB) signaling pathways present in the NAc after neuroinflammation.ConclusionsOur results suggest that PGRN exerts distinct function on different behaviors, showing protective roles in the FTD-like behavior and detrimental effects on the neuroinflammation-related depression-like behavior, resulting from mediating astrocyte and microglial functions from the NAc in different status.

PGRN deficiency exacerbates, whereas a brain penetrant PGRN derivative protects, GBA1 mutation-associated pathologies and diseases

Mutations in GBA1, encoding glucocerebrosidase (GCase), cause Gaucher disease (GD) and are also genetic risks in developing Parkinson's disease (PD). Currently, the approved therapies are only effective for directly treating visceral symptoms, but not for primary neuronopathic involvement in GD (nGD). Progranulin (PGRN), encoded by GRN, is a novel modifier of GCase, but the impact of PGRN in GBA1 mutation-associated pathologies invivo remains unknown. Herein, Grn-/- mice crossed into Gba9v/9v mice, a Gba1 mutant line homozygous for the Gba1 D409V mutation, generating Grn-/-Gba9v/9v (PG9V) mice. PG9V mice exhibited neurobehavioral deficits, early onset, and more severe GD phenotypes compared to Grn-/- and Gba9v/9v mice. Moreover, PG9V mice also displayed PD-like phenotype. Mechanistic analysis revealed that PGRN deficiency caused severe neuroinflammation with microgliosis and astrogliosis, along with impaired autophagy associated with the Gba1 mutation. A PGRN-derived peptide, termed ND7, ameliorated the disease phenotype in GD patient fibroblasts exvivo. Unexpectedly, ND7 penetrated the blood-brain barrier (BBB) and effectively ameliorated the nGD manifestations and PD pathology in Gba9v/null and PG9V mice. Collectively, this study not only provides the first line of invivo but also exvivo evidence demonstrating the crucial role of PGRN in GBA1/Gba1 mutation-related pathologies, as well as a clinically relevant mouse model for mechanistic and potential therapeutics studies for nGD and PD. Importantly, a BBB penetrant PGRN-derived biologic was developed that may provide treatment for rare lysosomal storage diseases and common neurodegenerative disorders, particularly nGD and PD.