SUMMARY The most common inherited form of Fronto-Temporal Lobar Degeneration (FTLD) known stems from Progranulin (GRN) mutation, and exhibits TDP-43 plus ubiquitin aggregates. Despite the causative role of GRN haploinsufficiency in FTLD-TDP, the neurobiology of this secreted glycoprotein is unclear. Here, we examined PGRN binding to the cell surface. PGRN binds to cortical neurons via its C-terminus, and unbiased expression cloning identifies Sortilin (Sort1) as a binding site. Sort1−/− neurons exhibit reduced PGRN binding. In the CNS, Sortilin is expressed by neurons and PGRN is most strongly expressed by activated microglial cells after injury. Sortilin rapidly endocytoses and delivers PGRN to lysosomes. Mice lacking Sortilin have elevations in brain and serum PGRN levels of 2.5- to 5-fold. The 50% PGRN decrease causative in FTLD-TDP cases is mimicked in GRN+/− mice, and is fully normalized by Sort1 ablation. Sortilin-mediated PGRN endocytosis is likely to play a central role in FTLD-TDP pathophysiology.

Haploinsufficiency of Progranulin (PGRN), a gene encoding a secreted glycoprotein, is a major cause of frontotemporal lobar degeneration with ubiquitin (FTLD-U) positive inclusions. Single nucleotide polymorphisms in the TMEM106B gene were recently discovered as a risk factor for FTLD-U, especially in patients with PGRN mutations. TMEM106B is also associated with cognitive impairment in amyotrophic lateral sclerosis patients. Despite these studies, little is known about TMEM106B at molecular and cellular levels and how TMEM106B contributes to FTLD. Here, we show that TMEM106B is localized in the late endosome/lysosome compartments and TMEM106B levels are regulated by lysosomal activities. Ectopic expression of TMEM106B induces morphologic changes of lysosome compartments and delays the degradation of endocytic cargoes by the endolysosomal pathway. Furthermore, overexpression of TMEM106B correlates with elevated levels of PGRN, possibly by attenuating lysosomal degradation of PGRN. These results shed light on the cellular functions of TMEM106B and the roles of TMEM106B in the pathogenesis of FTLD-U with PGRN mutations.

TAR DNA-binding protein-43 (TDP-43) proteinopathy has been linked to several neurodegenerative diseases, such as frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Phosphorylated and ubiquitinated TDP-43 C-terminal fragments have been found in cytoplasmic inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis patients. However, the factors and pathways that regulate TDP-43 aggregation are still not clear. We found that the C-terminal 15 kDa fragment of TDP-43 is sufficient to induce aggregation but the aggregation phenotype is modified by additional sequences. Aggregation is accompanied by phosphorylation at serine residues 409/410.Mutation of 409/410 to phosphomimetic aspartic acid residues significantly reduces aggregation. Inhibition of either proteasome or autophagy dramatically increases TDP-43 aggregation. Furthermore, TDP-43 aggregates colocalize with markers of autophagy and the adaptor protein p62/SQSTM1. Over-expression of p62/SQSTM1 reduces TDP-43 aggregation in an autophagy and proteasome-dependent manner. These studies suggest that aggregation of TDP-43 C-terminal fragments is regulated by phosphorylation events and both the autophagy and proteasome-mediated degradation pathways. Keywords: aggregation, autophagy, p62/SQSTM1, phosphorylation, proteasome, TDP-43. J. Neurochem. (2011) 116, 248-259. | 2011 | 116 | 248-259 doi: 10.1111/j.1471-4159.2010 Here, we show that the C-terminal fragment of TDP-43 mediates its aggregation. The 15 kDa fragment at the Cterminus of TDP-43 is sufficient to induce TDP-43 aggregation but the aggregation phenotype is modified by additional sequences. When over-expressed in mammalian cells, the C-terminal TDP-43 fragments are phosphorylated at serine residues 409/410. Phosphorylation plays an important role in regulating the aggregation of TDP-43 fragments. Furthermore, we show that these fragments are subject to autophagy and proteasome-mediated degradation pathways regulated by the adaptor protein p62/SQSTM1. These findings could have important implications for TDP-43 aggregation seen in many neurodegenerative diseases. JOURNAL OF NEUROCHEMISTRY Materials and methods AntibodiesThe following antibodies were used in our studies for western blot, immunoprecipitations, and immunofluorescence: rabbit anti-TDP-43 (ProteinTech, Chicago, IL, USA), rabbit anti-phospho TDP-43 pS409/410 (Cosmo Bio Co., Tokyo, Japan), mouse anti-green fluorescent protein (anti-GFP) (Covance, Princeton, NJ, USA), rabbit anti-GFP antiserum (provided by Dr Anthony Bretscher and Dr Scott Emr), mouse anti-c-Myc clone 9E10 (Sigma, St Louis, MO, USA), mouse anti-HA.11 clone 16B12 (Covance), mouse antip62/SQSTM1 (BD Biosciences, San Jose, CA, USA), mouse anti-GAPDH (Novus Biologicals, Littleton, CO, USA), rabbit anti-ATG5 (Epitomics, Burlingame, CA, USA, and Novus Biologicals) and goat anti-LC3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). PlasmidsHuman TDP-43, mouse p62, and mous...

Progranulin haplo-insufficiency is a main cause of frontotemporal lobar degeneration (FTLD) with TDP-43 aggregates. Previous studies have shown that sortilin regulates progranulin trafficking and is a main determinant of progranulin level in the brain. In this study, we mapped the binding site between progranulin and sortilin. Progranulin binds to the beta-propeller region of sortilin through its C-terminal tail. The C-terminal progranulin fragment is fully sufficient for sortilin binding and progranulin C-terminal peptide displaces progranulin binding to sortilin. Deletion of the last 3 residues of progranulin (QLL) abolishes its binding to sortilin and also sortilin dependent regulation of progranulin trafficking. Since progranulin haplo-insufficiency results in FTLD, these results may provide important insights into future studies of progranulin trafficking and signaling and progranulin based therapy for FTLD.

Objective —Increasing evidence suggests that contractile dysfunction in smooth muscle cells (SMCs) plays a critical role in aortic biomechanical dysfunction and aortic aneurysm and dissection (AAD) development. However, the mechanisms underlying SMC contractile dysfunction in sporadic AAD are poorly understood. In this study, we examined the role of the NLRP3–caspase-1 inflammasome, a key inflammatory cascade, in SMC contractile dysfunction in AAD. Approach and Results —We observed significant SMC contractile protein degradation in aortas from patients with sporadic thoracic AAD. The contractile protein degradation was associated with activation of the NLRP3–caspase-1 inflammasome cascade. In SMCs, caspase-1 bound and directly cleaved and degraded contractile proteins, leading to contractile dysfunction. Furthermore, Nlrp3 or caspase-1 deficiency in mice significantly reduced angiotensin II–induced contractile protein degradation, biomechanical dysfunction, and AAD formation in both thoracic and abdominal aortas. Finally, blocking this cascade with the inflammasome inhibitor, glyburide (an antidiabetic medication), reduced angiotensin II–induced AAD formation. Conclusions —Inflammasome-caspase-1–mediated degradation of SMC contractile proteins may contribute to aortic biomechanical dysfunction and AAD development. This cascade may be a therapeutic target in AAD formation. Additionally, glyburide may have protective effects against AAD development.