Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

Progranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and macrophages and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein mainly expressed in hematopoietic cells, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin-proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other's protein homeostasis.

A role of the frontotemporal lobar degeneration risk factor TMEM106B in myelination.

TMEM106B encodes a lysosomal membrane protein and was initially identified as a risk factor for frontotemporal lobar degeneration. Recently, a dominant D252N mutation in TMEM106B was shown to cause hypomyelinating leukodystrophy. However, how TMEM106B regulates myelination is still unclear. Here we show that TMEM106B is expressed and localized to the lysosome compartment in oligodendrocytes. TMEM106B deficiency in mice results in myelination defects with a significant reduction of protein levels of proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), the membrane proteins found in the myelin sheath. The levels of many lysosome proteins are significantly decreased in the TMEM106B-deficient Oli-neu oligodendroglial precursor cell line. TMEM106B physically interacts with the lysosomal protease cathepsin D and is required to maintain proper cathepsin D levels in oligodendrocytes. Furthermore, we found that TMEM106B deficiency results in lysosome clustering in the perinuclear region and a decrease in lysosome exocytosis and cell surface PLP levels. Moreover, we found that the D252N mutation abolished lysosome enlargement and lysosome acidification induced by wild-type TMEM106B overexpression. Instead, it stimulates lysosome clustering near the nucleus as seen in TMEM106B-deficient cells. Our results support that TMEM106B regulates myelination through modulation of lysosome function in oligodendrocytes.

The lysosomal function of progranulin, a guardian against neurodegeneration.

Progranulin (PGRN), encoded by the GRN gene in humans, is a secreted growth factor implicated in a multitude of processes ranging from regulation of inflammation to wound healing and tumorigenesis. The clinical importance of PGRN became especially evident in 2006, when heterozygous mutations in the GRN gene, resulting in haploinsufficiency, were found to be one of the main causes of frontotemporal lobar degeneration (FTLD). FTLD is a clinically heterogenous disease that results in the progressive atrophy of the frontal and temporal lobes of the brain. Despite significant research, the exact function of PGRN and its mechanistic relationship to FTLD remain unclear. However, growing evidence suggests a role for PGRN in the lysosome-most striking being that homozygous GRN mutation leads to neuronal ceroid lipofuscinosis, a lysosomal storage disease. Since this discovery, several links between PGRN and the lysosome have been established, including the existence of two independent lysosomal trafficking pathways, intralysosomal processing of PGRN into discrete functional peptides, and direct and indirect regulation of lysosomal hydrolases. Here, we summarize the cellular functions of PGRN, its roles in the nervous system, and its link to multiple neurodegenerative diseases, with a particular focus dedicated to recent lysosome-related mechanistic developments.

A recommended laparoscopic procedure for implantation of microcapsules in the peritoneal cavity of non-human primates.

BACKGROUND: The anatomical spatial distribution of microencapsulated islets transplanted into the peritoneal cavity of large animals remains a relatively unexplored area of study. In this study, we developed a new implantation approach using laparoscopy in order to avoid microcapsule amalgamation. This approach constitutes a clinically relevant method, which can be used to evaluate the distribution and in vivo biocompatibility of various types of transplanted microcapsules in the future. MATERIALS AND METHODS: Two healthy baboons were implanted intraperitoneally with microencapsulated islets through mini-laparotomy and observed at 76 d after implantation. Nine baboons underwent laparoscopic implantation of approximately 80,000 empty microcapsules. Microcapsule distribution was observed by laparoscopic camera during and after implantation at 1, 2, and 4 wk. At each time point, microcapsules were retrieved and evaluated with brightfield microscopy and histologic analysis. RESULTS: Mini-laparotomic implantation resulted in microcapusle aggregation in both baboons. In contrast, laparoscopic implantation resulted in even distribution of microcapsules throughout the peritoneum without sedimentation to the Douglas space in all animals. In eight out of nine animals, retrieved microcapsules were evenly distributed in the peritoneal cavity and presented with no pericapsular overgrowth and easily washed out during laparoscopic procedure. The one exception was attributed to microcapsule contamination with blood from the abdominal wall following trocar insertion. CONCLUSIONS: Laparoscopic implantation of microcapsules in non-human primates can be successfully performed and prevents microcapsule aggregation. Given the current widespread clinical application of laparoscopy, we propose that this presented laparoscopy technique could be applied in future clinical trials of microencapsulated islet transplantation.

Progranulin deficiency leads to reduced glucocerebrosidase activity.

Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GCase), and is essential for proper GCase activity. GCase activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.

The Interaction Between Progranulin with Sortilin and the Lysosome.

Accumulating evidence suggests that progranulin is essential for proper lysosomal function. Progranulin is a lysosomal resident protein and sortilin has been demonstrated to be the lysosomal trafficking receptor for progranulin. Here we describe the methods used to study the interaction between progranulin and sortilin, as well as the critical role of sortilin in mediating the lysosomal delivery of progranulin.

Lysosomal processing of progranulin.

BACKGROUND: Mutations resulting in progranulin (PGRN) haploinsufficiency cause frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), a devastating neurodegenerative disease. PGRN is localized to the lysosome and important for proper lysosome function. However, the metabolism of PGRN in the lysosome is still unclear. RESULTS: Here, we report that PGRN is processed into ~10 kDa peptides intracellularly in multiple cell types and tissues and this processing is dependent on lysosomal activities. PGRN endocytosed from the extracellular space is also processed in a similar manner. We further demonstrated that multiple cathepsins are involved in PGRN processing and cathepsin L cleaves PGRN in vitro. CONCLUSIONS: Our data support that PGRN is processed in the lysosome through the actions of cathepsins.

The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway.

Hexanucleotide repeat expansion in the C9orf72 gene is a leading cause of frontotemporal lobar degeneration (FTLD) with amyotrophic lateral sclerosis (ALS). Reduced expression of C9orf72 has been proposed as a possible disease mechanism. However, the cellular function of C9orf72 remains to be characterized. Here we report the identification of two binding partners of C9orf72: SMCR8 and WDR41. We show that WDR41 interacts with the C9orf72/SMCR8 heterodimer and WDR41 is tightly associated with the Golgi complex. We further demonstrate that C9orf72/SMCR8/WDR41 associates with the FIP200/Ulk1 complex, which is essential for autophagy initiation. C9orf72 deficient mice, generated using the CRISPR/Cas9 system, show severe inflammation in multiple organs, including lymph node, spleen and liver. Lymph node enlargement and severe splenomegaly are accompanied with macrophage infiltration. Increased levels of autophagy and lysosomal proteins and autophagy defects were detected in both the spleen and liver of C9orf72 deficient mice, supporting an in vivo role of C9orf72 in regulating the autophagy/lysosome pathway. In summary, our study elucidates potential physiological functions of C9orf72 and disease mechanisms of ALS/FTLD.

Histidine-tryptophan-ketoglutarate and University of Wisconsin solution demonstrate equal effectiveness in the preservation of human pancreata intended for islet isolation: a large-scale, single-center experience.

We previously reported a small-scale study on the efficacy of histidine-tryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution on pancreas preservation for islet isolation. In this large-scale, retrospective analysis (n = 252), we extend our initial description of the impact of HTK on islet isolation outcomes and include pancreatic digestion efficacy, purification outcomes, and islet size distribution. Multivariable linear regression analysis, adjusted for donor age, sex, BMI, cold ischemia time, and enzyme, demonstrated similar results for the HTK group (n = 95) and the UW group (n = 157), including postpurification islet yields (HTK: 289,702 IEQ vs. UW: 283,036 IEQ; p = 0.76), percentage of digested pancreatic tissue (HTK: 66.9% vs. UW: 64.1%; p = 0.18), and islet loss from postdigestion to postpurification (HTK: 24,972 IEQ vs. UW: 39,551 IEQ; p = 0.38). Changes in islet size between the postdigestion and postpurification stages were comparable within each islet size category for HTK and UW (p = 0.14-0.99). Tissue volume distribution across purification fractions and islet purity in the top fractions were similar between the groups; however, the HTK group had significantly higher islet purity in the middle fractions (p = 0.003-0.008). Islet viability and stimulation indices were also similar between the HTK and the UW groups. In addition, we analyzed a small sample of patients transplanted either with HTK (n = 7) or UW (n = 8) preserved islets and found no significant differences in posttransplant HbA1c, ß-score, and frequency of insulin independence. This study demonstrates that HTK and UW solutions offer comparable pancreas preservation for islet transplantation. More in vivo islet outcome data are needed for a complete analysis of the effects of HTK on islet transplantation.

Implication of mitochondrial cytoprotection in human islet isolation and transplantation.

Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and long-term insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes.

Highly purified versus filtered crude collagenase: comparable human islet isolation outcomes.

This study was designed to retrospectively compare the impact of crude Sigma V collagenase (Sigma V, n = 52) with high-purified Serva NB1 collagenase (Serva NB1, n = 42) on human islet isolation outcomes. A three-step filtration was applied to the crude Sigma V to remove endotoxin contamination and impurities; in addition, this process was used as a lot prescreening tool. Isolation outcomes were determined by digestion efficacy, islet yields, purity, viability, glucose-stimulated insulin release, and endotoxin content. The digestion efficacy between Sigma V and Serva NB1 was statistically significant (Sigma V: 64.71% vs. Serva NB1: 69.71%, p = 0.0014). However, the islet yields were similar (Sigma V: 23422.58 vs. Serva NB1: 271097 IEq, p = 0.23) between groups. There was no significant purity difference observed in fractions with purities greater than 75%. Viability (Sigma V: 93.3% vs. Serva NB1: 94.8%, p = 0.061) and stimulation indexes (Sigma V: 3.41 vs. Serva NB1: 2.74, p = 0.187) were also similar between the two groups. The impact of cold ischemia and age on the isolation outcome in the Sigma V group was comparable to the Serva NB1 group. The endotoxin content of the final products in the filtered Sigma V group was significantly less than that in the high-purified Serva NB1 group (0.022 vs. 0.052 EU/ml, p = 0.003). Additionally, in the Sigma V group there was minimal lot to lot variation and no significant loss of enzymatic activity after filtration. These findings indicate that the use of Sigma V or other crude enzyme blends for research pancreata is warranted to reduce isolation costs and increase the amount of islets available for critical islet research. These findings also validate the need for a systematic enzyme analysis to resolve these inconsistencies in overall enzyme quality once and for all.