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Background A mutation in the C9orf72 gene is the most common genetic mutation of familial and sporadic ALS, as well as familial FTD. While prior studies have focused on elucidating the mechanisms of neuronal dysfunction and neurodegeneration associated with this genetic mutation, the contribution of microglia to disease pathogenesis in the ALS/FTD disease spectrum remains poorly understood. Methods Here, we generated a new disease model consisting of cultured C9orf72 ALS/FTD patient-derived induced pluripotent stem cells differentiated into microglia (iPSC-MG). We used this model to study the intrinsic cellular and molecular phenotypes of microglia triggered by the C9orf72 gene mutation. Results We show that C9orf72 ALS/FTD iPSC-MG have a similar transcriptional profile compared to control iPSC-MG, despite the presence of C9orf72-associated phenotypes including reduced C9orf72 protein levels and dipeptide-repeat protein translation. Interestingly, C9orf72 ALS/FTD iPSC-MG exhibit intrinsic dysfunction of phagocytic activity upon exposure to Aβ or brain synaptoneurosomes and display a heightened inflammatory response. Detailed analysis of the endosomal and lysosomal pathways revealed altered expression of endosomal marker early endosome antigen 1 and lysosomal associated membrane protein 1 in C9orf72 ALS/FTD iPSC-MG, which was confirmed in patient postmortem tissues. Conclusions These findings demonstrate that unstimulated C9orf72 iPSC-MG mono-cultures share a largely similar transcriptome profile with control microglia, despite the presence of C9orf72 disease phenotypes. The dysfunction of the endosomal-lysosomal pathway as demonstrated by aberrant microglia phagocytosis and engulfment of cellular debris and brain pathogens suggests that disease-related microglia phenotypes are not intrinsic but instead require microglia to be activated. In summary, the C9orf72 iPSC-MG culture system provides a novel human disease model to study the role of microglia in C9orf72 ALS/FTD disease pathogenesis.

The noncoding genome is substantially larger than the protein-coding genome but has been largely unexplored by genetic association studies. Here, we performed region-based rare variant association analysis of25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole genomes and the whole genomes of 70,403 non-ALS controls. We identified interleukin-18 receptor accessory protein (IL18RAP) 3' untranslated region (3'UTR) variants as significantly enriched in non-ALS genomes and associated with a fivefold reduced risk of developing ALS, and this was replicated in an independent cohort. These variants in the IL18RAP 3'UTR reduce mRNA stability and the binding of double-stranded RNA (dsRNA)-binding proteins. Finally, the variants of the IL18RAP 3'UTR confer a survival advantage for motor neurons because they dampen neurotoxicity of human induced pluripotent stem cell (iPSC)-derived microglia bearing an ALS-associated expansion in C9orf72, and this depends on NF-κB signaling. This study reveals genetic variants that protect against ALS by reducing neuroinflammation and emphasizes the importance of noncoding genetic association studies.

BACKGROUND The P1 /P2 phenotypic polymorphism is one of the earliest blood groups discovered in humans. These blood groups have been connected to different levels of expression of the A4GALT gene in P1 and P2 red blood cells; however, the detailed molecular genetic mechanism that leads to these two phenotypes has not been established. STUDY DESIGN AND METHODS After our previous identification of an association between the single-nucleotide polymorphisms (SNPs) rs2143918 and rs5751348 in A4GALT gene and the P1 /P2 phenotype, we conduct a survey of transcription factors that might connect these SNPs with the differential expression of the P1 -A4GALT and P2 -A4GALT alleles. An in silico analysis of potential transcription factor binding motifs within the polymorphic SNPs rs2143918 and rs5751348 genomic regions was performed, and this was followed by reporter assays examining the candidate transcription factors, gene expression profiling, electrophoretic mobility shift assays, and P1 -A4GALT and P2 -A4GALT allelic expression analysis. RESULTS The results revealed that the differential binding of transcription factor early growth response 1 to the SNP rs5751348 genomic region with the different genotypes in the A4GALT gene leads to differential activation of P1 -A4GALT and P2 -A4GALT expression. CONCLUSION The present investigation, together with our previous study (Lai et al., Transfusion 2014;54:3222-31), have elucidated the molecular genetic details associated with the P1 /P2 blood groups.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with diverse etiologies. Therefore, the identification of common disease mechanisms and therapeutics targeting these mechanisms could dramatically improve clinical outcomes. To this end, we developed induced motor neuron (iMN) models from C9ORF72 and sporadic ALS (sALS) patients to identify targets that are effective against these types of cases, which together comprise ~90% of patients. We find that iMNs from C9ORF72 and several sporadic ALS patients share two common defects - impaired autophagosome formation and the aberrant accumulation of glutamate receptors. Moreover, we show that an anticoagulation-deficient form of activated protein C, 3K3A-APC, rescues these defects in both C9ORF72 and sporadic ALS iMNs. As a result, 3K3A-APC treatment lowers C9ORF72 dipeptide repeat protein (DPR) levels, restores nuclear TDP-43 localization, and rescues the survival of both C9ORF72 and sporadic ALS iMNs. Importantly, 3K3A-APC also lowers glutamate receptor levels and rescues proteostasis in vivo in C9ORF72 gain- and loss-of-function mouse models. Thus, motor neurons from C9ORF72 and at least a subset of sporadic ALS patients share common, early defects in autophagosome formation and glutamate receptor homeostasis and a single therapeutic approach may be efficacious against these disease processes.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

The PAfter our previous identification of an association between the single-nucleotide polymorphisms (SNPs) rs2143918 and rs5751348 in A4GALT gene and the PThe results revealed that the differential binding of transcription factor early growth response 1 to the SNP rs5751348 genomic region with the different genotypes in the A4GALT gene leads to differential activation of PThe present investigation, together with our previous study (Lai et al., Transfusion 2014;54:3222-31), have elucidated the molecular genetic details associated with the P

The major challenge in current chemotherapy is to increase local effective therapeutic concentration of drugs as well as to minimize toxicity and side effects for patients. The targeted delivery of drugs to their desired site of action in a controlled manner plays an essential role in the development of drug formulations. A photocage refers to a caged molecule rendered biologically inert by a photolabile protecting group. Molecules are illuminated with light to liberate the caged group and then become active forms. In this study, we formulate upconversion nanoparticles (UCNPs) as the NIR-triggered targeting and drug delivery vehicles that successfully deliver in vitro and in vivo for near-infrared light photocontrolled targeting, bioimaging, and chemotherapy. It is noted that there has been no report on the systemic administration UCNP-based drug delivery agents for evaluation of bioimaging and chemotherapy. To achieve phototargeting, the tumor-homing agent (i.e., folic acid) has been constructed as a photoresponsive molecule. For the chemotherapeutic effect, the antitumor drug doxorubicin is thiolated on the surface of UCNPs, forming a disulfide bond that can be cleaved by lysosomal enzymes within the cells. The caged UNCPs can serve as a platform for the improvement of selective targeting and possible reduction of adverse side effects from chemotherapy.

We synthesize a NIR MHI-148 dye, a lipophilic heptamethine cyanine, with capability in tumor-targeting property to accumulate in the mitochondria of tumor. In the context of MHI-148 dye, we demonstrate effective tumor targeting and NIR fluorescence in vitro and in vivo for MHI-148 as compared to ICG. A series of porous Gd silicates related nanoparticles, i.e. Gd silicate, Gd silicate@mSiO(2) (mSiO(2): mesoporous silica shell), and Gd(3+)-chelated Gd silicate@mSiO(2) (Gd(3+)-DOTA chelated on the mSiO(2)) are fabricated to demonstrate their magnetic resonance (MR) contrast imaging effects. Those Gd silicates related nanoparticles exhibit dual MR effect, expressing T(1)-brightened and T(2)-darkened effects, in lower magnetic field. In high magnetic field, an abnormal enhanced transverse relaxivity (r(2)) appears, showing an effective T(2)-lowering effect, possibly due to concentrated Gd amount and porous architecture. The r(2) value increases 4-5 times as the field strength increased from 3T to 7T. The Gd(3+)-chelated Gd silicate@mSiO(2) has given large r(2) (T(2)-lowering effect) up to 343.8 s(-1) mM(-1), which is even larger than the reported magnetic Fe(3)O(4) nanoparticles measured at the same field. Using a 9.4T animal micro MRI system we have seen effectively darken in signal for those porous Gd silicates related NPs, while no such phenomenon appears in commercial Gd-DOTA agent. The MHI-148 is then conjugated on the porous Gd silicate@mSiO(2) nanoparticles for a new paradigm with three functionalities for in vivo tumor targeting, near-infrared fluorescent and MR imaging by means of only using MHI-148 dye.

IgA is the most abundant antibody in mammals. However, the mechanism of its class switching is still not clear. The formation of the R-loops, as the target for AID, has been proposed to play a crucial role during mammalian class switch recombination. Here, we provide a systematic evaluation of R-loops at Sα (IgA) in CH12F3-2A cells, which is a unique cell model system for class switch recombination because of its consistent switching to IgA upon stimulation. The results of R-loop analysis demonstrate distinct features specific to Sα. Some R-loops may initiate from the end of Iα, but all terminate exclusively within Sα. Time-course analysis also indicates that the percentage of R-loops peaks prior to the occurrence of class switch recombination. This is the first demonstration that R-loops form at Sα in vitro and in situ, despite variable G density and relatively few GGGG clusters in Sα. The short distance from the promoter to Sα may compensate for the less robust R-loop-forming factors at Sα relative to other switch regions. In conclusion, R-loops at the Sα region further support R-loop formation as a general feature of all stimulated switch regions.