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.

Ionizing Radiation induction of cholesterol biosynthesis in Lung tissue.

While evidence supporting the notion that exposures to heavy ion radiation increase the risk for cancer and other disease development is accumulating, the underlying biological mechanisms remain poorly understood. To identify novel phenotypes that persist over time that may be related to increased disease development risk, we performed a quantitative global proteome analysis of immortalized human bronchial epithelial cells (HBEC3-KT) at day 7 post exposure to 0.5 Gy Fe ion (600 MeV/nucleon, Linear Energy Transfer (LET) = 175 keV/µm). The analysis revealed a significant increase in the expression of 4 enzymes of the cholesterol biosynthesis pathway. Elevated expression of enzymes of the cholesterol pathway was associated with increased cholesterol levels in irradiated cells and in lung tissue measured by a biochemical method and by filipin staining of cell-bound cholesterol. While a 1 Gy dose of Fe ion was sufficient to induce a robust response, a dose of 5 Gy X-rays was necessary to induce a similar cholesterol accumulation in HBEC3-KT cells. Radiation-increased cholesterol levels were reduced by treatment with inhibitors affecting the activity of enzymes in the biosynthesis pathway. To examine the implications of this finding for radiotherapy exposures, we screened a panel of lung cancer cell lines for cholesterol levels following exposure to X-rays. We identified a subset of cell lines that increased cholesterol levels in response to 5 Gy X-rays. Survival studies revealed that statin treatment is radioprotective, suggesting that cholesterol increases are associated with cytotoxicity. In summary, our findings uncovered a novel radiation-induced response, which may modify radiation treatment outcomes and contribute to risk for radiation-induced cardiovascular disease and carcinogenesis.

Specific Proteomes of Hippocampal Regions CA2 and CA1 Reveal Proteins Linked to the Unique Physiology of Area CA2.

The hippocampus is well established as an essential brain center for learning and memory. Within the hippocampus, recent studies show that area CA2 is important for social memory and is an anomaly compared to its better-understood neighboring region, CA1. Unlike CA1, CA2 displays a lack of typical synaptic plasticity, enhanced calcium buffering and extrusion, and resilience to cell death following injury. Although recent studies have identified multiple molecular markers of area CA2, the proteins that mediate the unique physiology, signaling, and resilience of this region are unknown. Using a transgenic GFP-reporter mouse line that expresses eGFP in CA2, we were able to perform targeted dissections of area CA2 and CA1 for proteomic analysis. We identified over 100 proteins with robustly enriched expression in area CA2 compared to CA1. Many of these proteins, including RGS14 and NECAB2, have already been shown to be enriched in CA2 and important for its function, while many more merit further study in the context of enhanced expression in this enigmatic brain region. Furthermore, we performed a comprehensive analysis of the entire data set (>2300 proteins) using a weighted protein co-expression network analysis. This identified eight distinct co-expressed patterns of protein co-enrichment associated with increased expression in area CA2 tissue (compared to CA1). The novel data set we present here reveals a specific CA2 hippocampal proteome, laying the groundwork for future studies and a deeper understanding of area CA2 and the proteins mediating its unique physiology and signaling.

Trimethylation of Elongation Factor-Tu by the Dual Thermoregulated Methyltransferase EftM Does Not Impact Its Canonical Function in Translation.

The Pseudomonas aeruginosa methyltransferase EftM trimethylates elongation factor-Tu (EF-Tu) on lysine 5 to form a post-translational modification important for initial bacterial adherence to host epithelial cells. EftM methyltransferase activity is directly temperature regulated. The protein stability of EftM is tuned with a melting temperature (Tm) around 37 °C such that the enzyme is stable and active at 25 °C, but is completely inactivated by protein unfolding at higher temperatures. This leads to higher observable levels of EF-Tu trimethylation at the lower temperature. Here we report an additional layer of thermoregulation resulting in lower eftM mRNA transcript level at 37 °C compared to 25 °C and show that this regulation occurs at the level of transcription initiation. To begin to define the impact of this system on P. aeruginosa physiology, we demonstrate that EF-Tu is the only observable substrate for EftM. Further, we interrogated the proteome of three different wild-type P. aeruginosa strains, their eftM mutants, and these mutants complemented with eftM and conclude that trimethylation of EF-Tu by EftM does not impact EF-Tu's canonical function in translation. In addition to furthering our knowledge of this Pseudomonas virulence factor, this study provides an intriguing example of a protein with multiple layers of thermoregulation.

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.

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.

A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation.

BACKGROUND: Kv1.3 potassium channels regulate microglial functions and are overexpressed in neuroinflammatory diseases. Kv1.3 blockade may selectively inhibit pro-inflammatory microglia in neurological diseases but the molecular and cellular mechanisms regulated by Kv1.3 channels are poorly defined. METHODS: We performed immunoblotting and flow cytometry to confirm Kv1.3 channel upregulation in lipopolysaccharide (LPS)-activated BV2 microglia and in brain mononuclear phagocytes freshly isolated from LPS-treated mice. Quantitative proteomics was performed on BV2 microglia treated with control, LPS, ShK-223 (highly selective Kv1.3 blocker), and LPS+ShK-223. Gene ontology (GO) analyses of Kv1.3-dependent LPS-regulated proteins were performed, and the most representative proteins and GO terms were validated. Effects of Kv1.3-blockade on LPS-activated BV2 microglia were studied in migration, focal adhesion formation, reactive oxygen species production, and phagocytosis assays. In vivo validation of protein changes and predicted molecular pathways were performed in a model of systemic LPS-induced neuroinflammation, employing antigen presentation and T cell proliferation assays. Informed by pathway analyses of proteomic data, additional mechanistic experiments were performed to identify early Kv1.3-dependent signaling and transcriptional events. RESULTS: LPS-upregulated cell surface Kv1.3 channels in BV2 microglia and in microglia and CNS-infiltrating macrophages isolated from LPS-treated mice. Of 144 proteins differentially regulated by LPS (of 3141 proteins), 21 proteins showed rectification by ShK-223. Enriched cellular processes included MHCI-mediated antigen presentation (TAP1, EHD1), cell motility, and focal adhesion formation. In vitro, ShK-223 decreased LPS-induced focal adhesion formation, reversed LPS-induced inhibition of migration, and inhibited LPS-induced upregulation of EHD1, a protein involved in MHCI trafficking. In vivo, intra-peritoneal ShK-223 inhibited LPS-induced MHCI expression by CD11b+CD45low microglia without affecting MHCI expression or trafficking of CD11b+CD45high macrophages. ShK-223 inhibited LPS-induced MHCI-restricted antigen presentation to ovalbumin-specific CD8+ T cells both in vitro and in vivo. Kv1.3 co-localized with the LPS receptor complex and regulated LPS-induced early serine (S727) STAT1 phosphorylation. CONCLUSIONS: We have unraveled novel molecular and functional roles for Kv1.3 channels in pro-inflammatory microglial activation, including a Kv1.3 channel-regulated pathway that facilitates MHCI expression and MHCI-dependent antigen presentation by microglia to CD8+ T cells. We also provide evidence for neuro-immunomodulation by systemically administered ShK peptides. Our results further strengthen the therapeutic candidacy of microglial Kv1.3 channels in neurologic diseases.

A Multi-network Approach Identifies Protein-Specific Co-expression in Asymptomatic and Symptomatic Alzheimer's Disease.

Here, we report proteomic analyses of 129 human cortical tissues to define changes associated with the asymptomatic and symptomatic stages of Alzheimer's disease (AD). Network analysis revealed 16 modules of co-expressed proteins, 10 of which correlated with AD phenotypes. A subset of modules overlapped with RNA co-expression networks, including those associated with neurons and astroglial cell types, showing altered expression in AD, even in the asymptomatic stages. Overlap of RNA and protein networks was otherwise modest, with many modules specific to the proteome, including those linked to microtubule function and inflammation. Proteomic modules were validated in an independent cohort, demonstrating some module expression changes unique to AD and several observed in other neurodegenerative diseases. AD genetic risk loci were concentrated in glial-related modules in the proteome and transcriptome, consistent with their causal role in AD. This multi-network analysis reveals protein- and disease-specific pathways involved in the etiology, initiation, and progression of AD.

Changes in the detergent-insoluble brain proteome linked to amyloid and tau in Alzheimer's Disease progression.

Despite a key role of amyloid-beta (Aß) in Alzheimer's disease (AD), mechanisms that link Aß plaques to tau neurofibrillary tangles and cognitive decline still remain poorly understood. The purpose of this study was to quantify proteins in the sarkosyl-insoluble brain proteome correlated with Aß and tau insolubility in the asymptomatic phase of AD (AsymAD) and through mild cognitive impairment (MCI) and symptomatic AD. Employing label-free mass spectrometry-based proteomics, we quantified 2711 sarkosyl-insoluble proteins across the prefrontal cortex from 35 individual cases representing control, AsymAD, MCI and AD. Significant enrichment of Aß and tau in AD was observed, which correlated with neuropathological measurements of plaque and tau tangle density, respectively. Pairwise correlation coefficients were also determined for all quantified proteins to Aß and tau, across the 35 cases. Notably, six of the ten most correlated proteins to Aß were U1 small nuclear ribonucleoproteins (U1 snRNPs). Three of these U1 snRNPs (U1A, SmD and U1-70K) also correlated with tau consistent with their association with tangle pathology in AD. Thus, proteins that cross-correlate with both Aß and tau, including specific U1 snRNPs, may have potential mechanistic roles in linking Aß plaques to tau tangle pathology during AD progression.

The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA.

Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate post-transcriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, we investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This work reveals that nab2 mutant cells accumulate intron-containing pre-mRNA in vivo We extend this analysis to identify genetic interactions between mutant alleles of nab2 and genes encoding the splicing factor, MUD2, and the RNA exosome, RRP6, with in vivo consequences of altered pre-mRNA splicing and poly(A) tail length control. As further evidence linking Nab2 proteins to splicing, an unbiased proteomic analysis of vertebrate Nab2, ZC3H14, identifies physical interactions with numerous components of the spliceosome. We validated the interaction between ZC3H14 and U2AF2/U2AF65 Taking all the findings into consideration, we present a model where Nab2/ZC3H14 interacts with spliceosome components to allow proper coupling of splicing with subsequent mRNA processing steps contributing to a kinetic proofreading step that allows properly processed mRNA to exit the nucleus and escape Rrp6-dependent degradation.

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.

Postsynaptic GABAB receptor activity regulates excitatory neuronal architecture and spatial memory.

Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABAB receptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.

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.

Astrocytic activation of A1 receptors regulates the surface expression of NMDA receptors through a Src kinase dependent pathway.

Chemical transmitters released from astrocytes, termed gliotransmitters, modulate synaptic transmission and neuronal function. Using astrocyte-specific inducible transgenicmice (dnSNARE mice), we have demonstrated that inhibiting gliotransmission leads to reduced activation of adenosine A1 receptors (A1R) and impaired sleep homeostasis (Halassa et al. (2009) Neuron 61:213-219); Pascual et al. (2005) Science 310:113-116). Additionally, synaptic N-methyl-D-aspartate receptor (NMDAR) currents are reduced in these astrocyte-specific transgenic animals (Fellin et al. (2009) Proc Natl Acad Sci USA 106:15037-15042). Because of the importance of adenosine and NMDA receptors to sleep processes we asked whether there is a causal linkage between changes in A1R activation and synaptic NMDA receptors. We show that astrocytic dnSNARE expression leads to reduced tyrosine phosphorylation of Srckinase and NR2 subunits concomitant with the decreased surface expression of the NR2 subunits. To test the role of A1R signaling in mediating these actions, we show that incubation of wildtype (WT) slices with an A1R antagonist reduces tyrosine phosphorylation of Src kinase and NR2B, decreases the surface expression of the NR2B subunits and leads to smaller NMDA component of miniature EPSCs. In dnSNARE mice we could rescue WT phenotype by incubation in an A1R agonist:activation of A1 receptor led to increased tyrosine phosphorylation of Src kinase and NR2B subunits as well as increased the surface expression of the NR2B subunit and increased NMDA component of the synaptic mEPSC. These results provide the first demonstration that astrocytes can affect neuronal excitability on a long time scale by regulating the surface expression of NMDA receptors through the activation of specific intracellular signaling pathways.

Physiological properties of direction-selective ganglion cells in early postnatal and adult mouse retina.

Selective responses of retinal ganglion cells (RGCs) to the direction of motion have been recorded extracellularly from the rabbit and the mouse retina at eye opening. Recently, it has been shown that the development of this circuitry is light independent. Using whole-cell patch clamp recording, we report here that mouse early postnatal direction-selective ganglion cells (DSGCs) showed lower membrane excitability, lower reliability of synaptic transmission and much slower kinetics of light responses compared with adult DSGCs. However, the degree of direction selectivity of early postnatal DSGCs measured by the direction-selective index and the width of the directional tuning curve was almost identical to that of adult DSGCs. The DSGCs exhibited a clear selectivity for the direction of motion at the onset of light sensitivity. Furthermore, the degree of direction selectivity was not affected by rearing in complete darkness from birth to postnatal day 11 or 30. The formation of the retinal neurocircuitry for coding motion direction is completely independent of light.

ON direction-selective ganglion cells in the mouse retina.

Two types of ganglion cells (RGCs) compute motion direction in the retina: the ON-OFF direction-selective ganglion cells (DSGCs) and the ON DSGCs. The ON DSGCs are much less studied mostly due to the low encounter rate. In this study, we investigated the physiology, dendritic morphology and synaptic inputs of the ON DSGCs in the mouse retina. When a visual stimulus moved back and forth in the preferred-null axis, we found that the ON DSGCs exhibited a larger EPSC when the visual stimulus moved in the preferred direction and a larger IPSC in the opposite, or null direction, similar to what has been found in ON-OFF DSGCs. This similar synaptic input pattern is in contrast to other well-known differences, namely: profile of velocity sensitivity, distribution of preferred directions, and different central projection of the axons. Immunohistochemical staining showed that the dendrites of ON DSGCs exhibited tight cofasciculation with the cholinergic plexus. These findings suggest that cholinergic amacrine cells may play an important role in generating direction selectivity in the ON DSGCs, and that the mechanism for coding motion direction is probably similar for the two types of DSGCs in the retina.

Long-term potentiation of neuron-glia synapses mediated by Ca2+-permeable AMPA receptors.

Interactions between neurons and glial cells in the brain may serve important functions in the development, maintenance, and plasticity of neural circuits. Fast neuron-glia synaptic transmission has been found between hippocampal neurons and NG2 cells, a distinct population of macroglia-like cells widely distributed in the brain. We report that these neuron-glia synapses undergo activity-dependent modifications analogous to long-term potentiation (LTP) at excitatory synapses, a hallmark of neuronal plasticity. However, unlike the induction of LTP at many neuron-neuron synapses, both induction and expression of LTP at neuron-NG2 synapses involve Ca2+-permeable AMPA receptors on NG2 cells.