Granulins rescue inflammation, lysosome dysfunction, and neuropathology in a mouse model of progranulin deficiency.

Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases.

Regulation of TFEB nuclear localization by HSP90AA1 promotes autophagy and longevity.

TFEB (transcription factor EB) regulates multiple genes involved in the process of macroautophagy/autophagy and plays a critical role in lifespan determination. However, the detailed mechanisms that regulate TFEB activity are not fully clear. In this study, we identified a role for HSP90AA1 in modulating TFEB. HSP90AA1 was phosphorylated by CDK5 at Ser 595 under basal condition. This phosphorylation inhibited HSP90AA1, disrupted its binding to TFEB, and impeded TFEB's nuclear localization and subsequent autophagy induction. Pro-autophagy signaling attenuated CDK5 activity and enhanced TFEB function in an HSP90AA1-dependent manner. Inhibition of HSP90AA1 function or decrease in its expression significantly attenuated TFEB's nuclear localization and transcriptional function following autophagy induction. HSP90AA1-mediated regulation of a TFEB ortholog was involved in the extended lifespan of Caenorhabditis elegans in the absence of its food source bacteria. Collectively, these findings reveal that this regulatory process plays an important role in modulation of TFEB, autophagy, and longevity.Abbreviations : AL: autolysosome; AP: autophagosome; ATG: autophagy related; BafA1: bafilomycin A1; CDK5: cyclin-dependent kinase 5; CDK5R1: cyclin dependent kinase 5 regulatory subunit 1; CR: calorie restriction; FUDR: 5-fluorodeoxyuridine; HSP90AA1: heat shock protein 90 alpha family class A member 1; MAP1LC3: microtubule associated protein 1 light chain 3; NB: novobiocin sodium; SQSTM1: sequestosome 1; TFEB: transcription factor EB; WT: wild type.

Proximity-based labeling reveals DNA damage-induced phosphorylation of fused in sarcoma (FUS) causes distinct changes in the FUS protein interactome.

Accumulation of cytoplasmic inclusions containing fused in sarcoma (FUS), an RNA/DNA-binding protein, is a common hallmark of frontotemporal lobar degeneration and amyotrophic lateral sclerosis neuropathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 aired with mass spectrometry to investigate whether N-terminal phosphorylation alters the FUS protein-protein interaction network (interactome), and subsequently, FUS function. We report the first analysis comparing the interactomes of three FUS variants: homeostatic wildtype FUS (FUS WT), phosphomimetic FUS (FUS PM; a proxy for N-terminally phosphorylated FUS), and the toxic FUS proline 525 to leucine mutant (FUS P525L) that causes juvenile amyotrophic lateral sclerosis. We found that the phosphomimetic FUS interactome is uniquely enriched for a group of cytoplasmic proteins that mediate mRNA metabolism and translation, as well as nuclear proteins involved in the spliceosome and DNA repair functions. Furthermore, we identified and validated the RNA-induced silencing complex RNA helicase MOV10 as a novel interacting partner of FUS. Finally, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady-state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the interactome and function of FUS.

Lysosome dysfunction as a cause of neurodegenerative diseases: Lessons from frontotemporal dementia and amyotrophic lateral sclerosis.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative disorders that are thought to exist on a clinical and pathological spectrum. FTD and ALS are linked by shared genetic causes (e.g. C9orf72 hexanucleotide repeat expansions) and neuropathology, such as inclusions of ubiquitinated, misfolded proteins (e.g. TAR DNA-binding protein 43; TDP-43) in the CNS. Furthermore, some genes that cause FTD or ALS when mutated encode proteins that localize to the lysosome or modulate endosome-lysosome function, including lysosomal fusion, cargo trafficking, lysosomal acidification, autophagy, or TFEB activity. In this review, we summarize evidence that lysosomal dysfunction, caused by genetic mutations (e.g. C9orf72, GRN, MAPT, TMEM106B) or toxic-gain of function (e.g. aggregation of TDP-43 or tau), is an important pathogenic disease mechanism in FTD and ALS. Further studies into the normal function of many of these proteins are required and will help uncover the mechanisms that cause lysosomal dysfunction in FTD and ALS. Mutations or polymorphisms in genes that encode proteins important for endosome-lysosome function also occur in other age-dependent neurodegenerative diseases, including Alzheimer's (e.g. APOE, PSEN1, APP) and Parkinson's (e.g. GBA, LRRK2, ATP13A2) disease. A more complete understanding of the common and unique features of lysosome dysfunction across the spectrum of neurodegeneration will help guide the development of therapies for these devastating diseases.

Lysosomal Dysfunction and Other Pathomechanisms in FTLD: Evidence from Progranulin Genetics and Biology.

It has been more than a decade since heterozygous loss-of-function mutations in the progranulin gene (GRN) were first identified as an important genetic cause of frontotemporal lobar degeneration (FTLD). Due to the highly diverse biological functions of the progranulin (PGRN) protein, encoded by GRN, multiple possible disease mechanisms have been proposed. Early work focused on the neurotrophic properties of PGRN and its role in the inflammatory response. However, since the discovery of homozygous GRN mutations in patients with a lysosomal storage disorder, investigation into the possible roles of PGRN and its proteolytic cleavage products granulins, in lysosomal function and dysfunction, has taken center stage. In this chapter, we summarize the GRN mutational spectrum and its associated phenotypes followed by an in-depth discussion on the possible disease mechanisms implicated in FTLD-GRN. We conclude with key outstanding questions which urgently require answers to ensure safe and successful therapy development for GRN mutation carriers.

Network analysis of the progranulin-deficient mouse brain proteome reveals pathogenic mechanisms shared in human frontotemporal dementia caused by GRN mutations.

Heterozygous, loss-of-function mutations in the granulin gene (GRN) encoding progranulin (PGRN) are a common cause of frontotemporal dementia (FTD). Homozygous GRN mutations cause neuronal ceroid lipofuscinosis-11 (CLN11), a lysosome storage disease. PGRN is a secreted glycoprotein that can be proteolytically cleaved into seven bioactive 6 kDa granulins. However, it is unclear how deficiency of PGRN and granulins causes neurodegeneration. To gain insight into the mechanisms of FTD pathogenesis, we utilized Tandem Mass Tag isobaric labeling mass spectrometry to perform an unbiased quantitative proteomic analysis of whole-brain tissue from wild type (Grn+/+) and Grn knockout (Grn-/-) mice at 3- and 19-months of age. At 3-months lysosomal proteins (i.e. Gns, Scarb2, Hexb) are selectively increased indicating lysosomal dysfunction is an early consequence of PGRN deficiency. Additionally, proteins involved in lipid metabolism (Acly, Apoc3, Asah1, Gpld1, Ppt1, and Naaa) are decreased; suggesting lysosomal degradation of lipids may be impaired in the Grn-/- brain. Systems biology using weighted correlation network analysis (WGCNA) of the Grn-/- brain proteome identified 26 modules of highly co-expressed proteins. Three modules strongly correlated to Grn deficiency and were enriched with lysosomal proteins (Gpnmb, CtsD, CtsZ, and Tpp1) and inflammatory proteins (Lgals3, GFAP, CD44, S100a, and C1qa). We find that lysosomal dysregulation is exacerbated with age in the Grn-/- mouse brain leading to neuroinflammation, synaptic loss, and decreased markers of oligodendrocytes, myelin, and neurons. In particular, GPNMB and LGALS3 (galectin-3) were upregulated by microglia and elevated in FTD-GRN brain samples, indicating common pathogenic pathways are dysregulated in human FTD cases and Grn-/- mice. GPNMB levels were significantly increased in the cerebrospinal fluid of FTD-GRN patients, but not in MAPT or C9orf72 carriers, suggesting GPNMB could be a biomarker specific to FTD-GRN to monitor disease onset, progression, and drug response. Our findings support the idea that insufficiency of PGRN and granulins in humans causes neurodegeneration through lysosomal dysfunction, defects in autophagy, and neuroinflammation, which could be targeted to develop effective therapies.

Divergent FUS phosphorylation in primate and mouse cells following double-strand DNA damage.

Fused in sarcoma (FUS) is a RNA/DNA protein involved in multiple nuclear and cytoplasmic functions including transcription, splicing, mRNA trafficking, and stress granule formation. To accomplish these many functions, FUS must shuttle between cellular compartments in a highly regulated manner. When shuttling is disrupted, FUS abnormally accumulates into cytoplasmic inclusions that can be toxic. Disrupted shuttling of FUS into the nucleus is a hallmark of ~10% of frontotemporal lobar degeneration (FTLD) cases, the neuropathology that underlies frontotemporal dementia (FTD). Multiple pathways are known to disrupt nuclear/cytoplasmic shuttling of FUS. In earlier work, we discovered that double-strand DNA breaks (DSBs) trigger DNA-dependent protein kinase (DNA-PK) to phosphorylate FUS (p-FUS) at N-terminal residues leading to the cytoplasmic accumulation of FUS. Therefore, DNA damage may contribute to the development of FTLD pathology with FUS inclusions. In the present study, we examined how DSBs effect FUS phosphorylation in various primate and mouse cellular models. All cell lines derived from human and non-human primates exhibit N-terminal FUS phosphorylation following calicheamicin γ1 (CLM) induced DSBs. In contrast, we were unable to detect FUS phosphorylation in mouse-derived primary neurons or immortalized cell lines regardless of CLM treatment, duration, or concentration. Despite DNA damage induced by CLM treatment, we find that mouse cells do not phosphorylate FUS, likely due to reduced levels and activity of DNA-PK compared to human cells. Taken together, our work reveals that mouse-derived cellular models regulate FUS in an anomalous manner compared to primate cells. This raises the possibility that mouse models may not fully recapitulate the pathogenic cascades that lead to FTLD with FUS pathology.

Chimeric Peptide Species Contribute to Divergent Dipeptide Repeat Pathology in c9ALS/FTD and SCA36.

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.

Molecular Signatures of Neuroinflammation Induced by αSynuclein Aggregates in Microglial Cells.

Alpha-synuclein (αSynAgg) are pathological hallmarks of Parkinson's disease (PD) and other synucleinopathies that induce microglial activation and immune-mediated neurotoxicity, but the molecular mechanisms of αSynAgg-induced immune activation are poorly defined. We performed quantitative proteomics by mass spectrometry coupled with PCR, immunohistochemical and functional validations studies to define the molecular characteristics of alpha synuclein mediated microglial activation. In mouse microglia, αSynAgg induced robust pro-inflammatory activation (increased expression of 864 genes including Irg1, Ifit1, and Pyhin) and increased nuclear proteins involved in RNA synthesis, splicing, and anti-viral defense mechanisms. Conversely, αSynAgg decreased expression several proteins (including Cdc123, Sod1, and Grn), which were predominantly cytosolic and involved in metabolic, proteasomal and lysosomal mechanisms. Pathway analyses and confirmatory in vitro studies suggested that αSynAgg partly mediates its effects via Stat3 activation. As predicted by our proteomic findings, we verified that αSynAgg induces mitochondrial dysfunction in microglia. Twenty-six proteins differentially expressed by αSynAgg were also identified as PD risk genes in genome-wide association studies (upregulated: Brd2, Clk1, Siglec1; down-regulated: Memo1, Arhgap18, Fyn, and Pgrn/Grn). We validated progranulin (PGRN) as a lysosomal PD-associated protein that is downregulated by αSynAgg in microglia in-vivo and is expressed by microglia in post-mortem PD brain, congruent with our in vitro findings. Conclusion: Together, proteomics approach both reveals novel molecular insights into αSyn-mediated neuroinflammation in PD and other synucleinopathies.

TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD.

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.

Short Aß peptides attenuate Aß42 toxicity in vivo.

Processing of amyloid-ß (Aß) precursor protein (APP) by γ-secretase produces multiple species of Aß: Aß40, short Aß peptides (Aß37-39), and longer Aß peptides (Aß42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aß42 but increase the relative abundance of short Aß peptides. To evaluate the pathological relevance of these peptides, we expressed Aß36-40 and Aß42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aß42 toxicity. In contrast to Aß42, the short Aß peptides were not toxic and, when coexpressed with Aß42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aß38 and Aß40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aß42 deposition, Aß38 and Aß40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aß42 by raising the levels of short Aß peptides could attenuate the toxic effects of Aß42.

Intracellular Proteolysis of Progranulin Generates Stable, Lysosomal Granulins that Are Haploinsufficient in Patients with Frontotemporal Dementia Caused by GRN Mutations.

Homozygous or heterozygous mutations in the GRN gene, encoding progranulin (PGRN), cause neuronal ceroid lipofuscinosis (NCL) or frontotemporal dementia (FTD), respectively. NCL and FTD are characterized by lysosome dysfunction and neurodegeneration, indicating PGRN is important for lysosome homeostasis in the brain. PGRN is trafficked to the lysosome where its functional role is unknown. PGRN can be cleaved into seven 6-kDa proteins called granulins (GRNs); however, little is known about how GRNs are produced or if levels of GRNs are altered in FTD-GRN mutation carriers. Here, we report the identification and characterization of antibodies that reliably detect several human GRNs by immunoblot and immunocytochemistry. Using these tools, we find that endogenous GRNs are present within multiple cell lines and are constitutively produced. Further, extracellular PGRN is endocytosed and rapidly processed into stable GRNs within lysosomes. Processing of PGRN into GRNs is conserved between humans and mice and is modulated by sortilin expression and mediated by cysteine proteases (i.e. cathpesin L). Induced lysosome dysfunction caused by alkalizing agents or increased expression of transmembrane protein 106B (TMEM106B) inhibit processing of PGRN into GRNs. Finally, we find that multiple GRNs are haploinsufficient in primary fibroblasts and cortical brain tissue from FTD-GRN patients. Taken together, our findings raise the interesting possibility that GRNs carry out critical lysosomal functions and that loss of GRNs should be explored as an initiating factor in lysosomal dysfunction and neurodegeneration caused by GRN mutations.

Disulfide bonds and disorder in granulin-3: An unusual handshake between structural stability and plasticity.

Granulins (GRNs) are a family of small (∼6 kDa) proteins generated by the proteolytic processing of their precursor, progranulin (PGRN), in many cell types. Both PGRN and GRNs are implicated in a plethora of biological functions, often in opposing roles to each other. Lately, GRNs have generated significant attention due to their implicated roles in neurodegenerative disorders. Despite their physiological and pathological significance, the structure-function relationships of GRNs are poorly defined. GRNs contain 12 conserved cysteines forming six intramolecular disulfide bonds, making them rather exceptional, even among a few proteins with high disulfide bond density. Solution NMR investigations in the past have revealed a unique structure containing putative interdigitated disulfide bonds for several GRNs, but GRN-3 was unsolvable due to its heterogeneity and disorder. In our previous report, we showed that abrogation of disulfide bonds in GRN-3 renders the protein completely disordered (Ghag et al., Prot Eng Des Sel 2016). In this study, we report the cellular expression and biophysical analysis of fully oxidized, native GRN-3. Our results indicate that both E. coli and human embryonic kidney (HEK) cells do not exclusively make GRN-3 with homogenous disulfide bonds, likely due to the high cysteine density within the protein. Biophysical analysis suggests that GRN-3 structure is dominated by irregular loops held together only by disulfide bonds, which induced remarkable thermal stability to the protein despite the lack of regular secondary structure. This unusual handshake between disulfide bonds and disorder within GRN-3 could suggest a unique adaptation of intrinsically disordered proteins towards structural stability.

Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: a novel therapeutic lead to treat frontotemporal dementia.

BACKGROUND: Progranulin (PGRN) is a secreted growth factor important for neuronal survival and may do so, in part, by regulating lysosome homeostasis. Mutations in the PGRN gene (GRN) are a common cause of frontotemporal lobar degeneration (FTLD) and lead to disease through PGRN haploinsufficiency. Additionally, complete loss of PGRN in humans leads to neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Importantly, Grn-/- mouse models recapitulate pathogenic lysosomal features of NCL. Further, GRN variants that decrease PGRN expression increase the risk of developing Alzheimer's disease (AD) and Parkinson's disease (PD). Together these findings demonstrate that insufficient PGRN predisposes neurons to degeneration. Therefore, compounds that increase PGRN levels are potential therapeutics for multiple neurodegenerative diseases. RESULTS: Here, we performed a cell-based screen of a library of known autophagy-lysosome modulators and identified multiple novel activators of a human GRN promoter reporter including several common mTOR inhibitors and an mTOR-independent activator of autophagy, trehalose. Secondary cellular screens identified trehalose, a natural disaccharide, as the most promising lead compound because it increased endogenous PGRN in all cell lines tested and has multiple reported neuroprotective properties. Trehalose dose-dependently increased GRN mRNA as well as intracellular and secreted PGRN in both mouse and human cell lines and this effect was independent of the transcription factor EB (TFEB). Moreover, trehalose rescued PGRN deficiency in human fibroblasts and neurons derived from induced pluripotent stem cells (iPSCs) generated from GRN mutation carriers. Finally, oral administration of trehalose to Grn haploinsufficient mice significantly increased PGRN expression in the brain. CONCLUSIONS: This work reports several novel autophagy-lysosome modulators that enhance PGRN expression and identifies trehalose as a promising therapeutic for raising PGRN levels to treat multiple neurodegenerative diseases.

FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage.

Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS.

Complex relationships between substrate sequence and sensitivity to alterations in γ-secretase processivity induced by γ-secretase modulators.

γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-ß (Aß) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer's disease modify the profile of Aß isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aß42 and in some circumstances Aß43. Compounds termed γ-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of Aß42, respectively. As GSMs lower the level of production of pathogenic forms of long Aß isoforms, they are of great interest as potential Alzheimer's disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the γ-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate γ-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.

Steroids as γ-secretase modulators.

Aggregation and accumulation of Aß42 play an initiating role in Alzheimer's disease (AD); thus, selective lowering of Aß42 by γ-secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aß production, we screened 170 steroids at 10 µM for effects on Aß42 secreted from human APP-overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aß42, whereas many nonacidic steroids actually raised Aß42. Studies on the more potent compounds showed that Aß42-lowering steroids were bonafide GSMs and Aß42-raising steroids were inverse GSMs. The most potent steroid GSM identified was 5ß-cholanic acid (EC50=5.7 µM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4-androsten-3-one-17ß-carboxylic acid ethyl ester (EC50=6.25 µM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aß production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.

Progranulin does not bind tumor necrosis factor (TNF) receptors and is not a direct regulator of TNF-dependent signaling or bioactivity in immune or neuronal cells.

Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in the GRN gene causing haploinsufficiency result in a familial form of frontotemporal dementia (FTD). Recently, a direct interaction between PGRN and tumor necrosis factor receptors (TNFR I/II) was reported and proposed to be a mechanism by which PGRN exerts anti-inflammatory activity, raising the possibility that aberrant PGRN-TNFR interactions underlie the molecular basis for neuroinflammation in frontotemporal lobar degeneration pathogenesis. Here, we report that we find no evidence for a direct physical or functional interaction between PGRN and TNFRs. Using coimmunoprecipitation and surface plasmon resonance (SPR) we replicated the interaction between PGRN and sortilin and that between TNF and TNFRI/II, but not the interaction between PGRN and TNFRs. Recombinant PGRN or transfection of a cDNA encoding PGRN did not antagonize TNF-dependent NFκB, Akt, and Erk1/2 pathway activation; inflammatory gene expression; or secretion of inflammatory factors in BV2 microglia and bone marrow-derived macrophages (BMDMs). Moreover, PGRN did not antagonize TNF-induced cytotoxicity on dopaminergic neuroblastoma cells. Last, co-addition or pre-incubation with various N- or C-terminal-tagged recombinant PGRNs did not alter lipopolysaccharide-induced inflammatory gene expression or cytokine secretion in any cell type examined, including BMDMs from Grn+/- or Grn-/- mice. Therefore, the neuroinflammatory phenotype associated with PGRN deficiency in the CNS is not a direct consequence of the loss of TNF antagonism by PGRN, but may be a secondary response by glia to disrupted interactions between PGRN and Sortilin and/or other binding partners yet to be identified.

Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis.

BACKGROUND: Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS) can cause familial and sporadic amyotrophic lateral sclerosis (ALS) and rarely frontotemproal dementia (FTD). FUS accumulates in neuronal cytoplasmic inclusions (NCIs) in ALS patients with FUS mutations. FUS is also a major pathologic marker for a group of less common forms of frontotemporal lobar degeneration (FTLD), which includes atypical FTLD with ubiquitinated inclusions (aFTLD-U), neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD). These diseases are now called FUS proteinopathies, because they share this disease marker. It is unknown how FUS mutations cause disease and the role of FUS in FTD-FUS cases, which do not have FUS mutations. In this paper we report the development of somatic brain transgenic (SBT) mice using recombinant adeno-associated virus (rAAV) to investigate how FUS mutations lead to neurodegeneration. RESULTS: We compared SBT mice expressing wild-type human FUS (FUSWT), and two ALS-linked mutations: FUSR521C and FUSΔ14, which lacks the nuclear localization signal. Both FUS mutants accumulated in the cytoplasm relative to FUSWT. The degree of this shift correlated with the severity of the FUS mutation as reflected by disease onset in humans. Mice expressing the most aggressive mutation, FUSΔ14, recapitulated many aspects of FUS proteinopathies, including insoluble FUS, basophilic and eosiniphilic NCIs, and other pathologic markers, including ubiquitin, p62/SQSTM1, α-internexin, and the poly-adenylate(A)-binding protein 1 (PABP-1). However, TDP-43 did not localize to inclusions. CONCLUSIONS: Our data supports the hypothesis that ALS or FTD-linked FUS mutations cause neurodegeneration by increasing cyotplasmic FUS. Accumulation of FUS in the cytoplasm may retain RNA targets and recruit additional RNA-binding proteins, such as PABP-1, into stress-granule like aggregates that coalesce into permanent inclusions that could negatively affect RNA metabolism. Identification of mutations in other genes that cause ALS/FTD, such as C9ORF72, sentaxin, and angiogenin, lends support to the idea that defective RNA metabolism is a critical pathogenic pathway. The SBT FUS mice described here will provide a valuable platform for dissecting the pathogenic mechanism of FUS mutations, define the relationship between FTD and ALS-FUS, and help identify therapeutic targets that are desperately needed for these devastating neurodegenerative disorders.

Transient pharmacologic lowering of Aß production prior to deposition results in sustained reduction of amyloid plaque pathology.

BACKGROUND: Alzheimer's disease (AD) is the leading cause of dementia among the elderly. Disease modifying therapies targeting Aß that are in development have been proposed to be more effective if treatment was initiated prior to significant accumulation of Aß in the brain, but optimal timing of treatment initiation has not been clearly established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain Aß with a γ-secretase inhibitor (GSI ) for 1-3 months (M) treatment windows in APP Tg2576 mice and subsequent aging of the mice to either 15M or 18M. RESULTS: These data show that reducing Aß production in a 2-3M windows both initiated and discontinued before detectable Aß deposition has the most significant impact on Aß loads up to 11M after treatment discontinuation. In contrast, initiation of treatment for 3M windows from 7-10M or 12-15M shows progressively decreasing efficacy. CONCLUSIONS: These data have major implications for clinical testing of therapeutics aimed at lowering Aß production, indicating that; i) these therapies may have little efficacy unless tested as prophylactics or in the earliest preclinical stage of AD where there is no or minimal Aß accumulation and ii) lowering Aß production transiently during a critical pre-deposition window potentially provides long-lasting efficacy after discontinuation of the treatment.