Extracellular vesicle-mediated suicide mRNA/protein delivery inhibits glioblastoma tumor growth in vivo.

Extracellular vesicles (EVs) are considered as important mediators of intercellular communication, which carry a diverse repertoire of genetic information between cells. This feature of EVs can be used and improved to advance their therapeutic potential. We have previously shown that genetically engineered EVs carrying the suicide gene mRNA and protein-cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT)-inhibited schwannoma tumor growth in vivo. To further examine whether this approach can be applied to other cancer types, we established a subcutaneous xenograft glioblastoma tumor model in mice, as glioblastoma represents the most common primary brain tumor, which is highly aggressive compared with the original schwannoma tumor model. U87-MG glioblastoma cells were implanted into the flanks of nude SCID mice, and the animals were intratumorally injected with the EVs isolated from the cells expressing EGFP or CD-UPRT. After the intraperitoneal administration of the prodrug 5-fluorocytosine, the tumor growth was assessed by regular caliper measurements. Our data revealed that the treatment with the CD-UPRT-enriched EVs significantly reduced the tumor growth in mice. Taken together, our findings suggest that EVs uploaded with therapeutic CD-UPRT mRNA/protein may be a useful tool for glioblastoma treatment.

17q21.31 duplication causes prominent tau-related dementia with increased MAPT expression.

To assess the role of rare copy number variations in Alzheimer's disease (AD), we conducted a case-control study using whole-exome sequencing data from 522 early-onset cases and 584 controls. The most recurrent rearrangement was a 17q21.31 microduplication, overlapping the CRHR1, MAPT, STH and KANSL1 genes that was found in four cases, including one de novo rearrangement, and was absent in controls. The increased MAPT gene dosage led to a 1.6-1.9-fold expression of the MAPT messenger RNA. Clinical signs, neuroimaging and cerebrospinal fluid biomarker profiles were consistent with an AD diagnosis in MAPT duplication carriers. However, amyloid positon emission tomography (PET) imaging, performed in three patients, was negative. Analysis of an additional case with neuropathological examination confirmed that the MAPT duplication causes a complex tauopathy, including prominent neurofibrillary tangle pathology in the medial temporal lobe without amyloid-ß deposits. 17q21.31 duplication is the genetic basis of a novel entity marked by prominent tauopathy, leading to early-onset dementia with an AD clinical phenotype. This entity could account for a proportion of probable AD cases with negative amyloid PET imaging recently identified in large clinical series.

The c.65-2A>G splice site mutation is associated with a mild phenotype in Danon disease due to the transcription of normal LAMP2 mRNA.

Danon disease (DD) is a rare X-linked multisystem disorder caused by mutations of the LAMP2 gene and characterized by intellectual disability, skeletal myopathy and cardiomyopathy. The survival time is severely reduced. Contrasting with the usual disease course, we report on a family with an exceptionally mild phenotype of DD despite having two potentially damaging LAMP2 mutations. Using RNA-Seq analysis, we showed that a c.65-2A>G splice site mutation results in the tissue-specific production of four different transcripts including the full-length mRNA in muscle tissue but not in leukocytes. We confirmed our results by immunohistochemistry and immunoblotting, showing the detection of LAMP2 protein only in muscle. The second mutation (c.586A>T, p.T196S) has been reported before to have an uncertain clinical significance. In our patients, however, neither of the two mutations seem to have a high enough functional impact to cause a severe phenotype. Overall, our study reveals that alternative splicing is a potential mechanism in DD with underlying splice site mutations of the LAMP2 gene in order to rescue the full-length mRNA. Moreover, our report of a mild phenotype complements the DD spectrum, which is of great importance for a rare disease suspected to be underdiagnosed.

miR-200a-mediated suppression of non-muscle heavy chain IIb inhibits meningioma cell migration and tumor growth in vivo.

miR-200a has been implicated in the pathogenesis of meningiomas, one of the most common central nervous system tumors in humans. To identify how miR-200a contributes to meningioma pathogenesis at the molecular level, we used a comparative protein profiling approach using Gel-nanoLC-MS/MS and identified approximately 130 dysregulated proteins in miR-200a-overexpressing meningioma cells. Following the bioinformatic analysis to identify potential genes targeted by miR-200a, we focused on the non-muscle heavy chain IIb (NMHCIIb), and showed that miR-200a directly targeted NMHCIIb. Considering the key roles of NMHCIIb in cell division and cell migration, we aimed to identify whether miR-200a regulated these processes through NMHCIIb. We found that NMHCIIb overexpression partially rescued miR-200a-mediated inhibition of cell migration, as well as cell growth in vitro and in vivo. Moreover, siRNA-mediated silencing of NMHCIIb expression resulted in a similar migration phenotype in these cells and inhibited meningioma tumor growth in mice. Taken together, these results suggest that NMHCIIb might serve as a novel therapeutic target in meningiomas.

Depletion of minichromosome maintenance protein 7 inhibits glioblastoma multiforme tumor growth in vivo.

Minichromosome maintenance (MCM) proteins are key elements that function as a part of the pre-replication complex to initiate DNA replication in eukaryotes. Consistent with their roles in initiating DNA replication, overexpression of MCM family members has been observed in several malignancies. Through bioinformatic analysis of The Cancer Genome Atlas's data on glioblastoma multiforme (GBM), we found that the genomic region containing MCM7 gene was amplified in more than 80% of the present cases. To validate this finding and to identify the possible contribution of the remaining members of the MCM family to GBM progression, we used quantitative real-time PCR to analyze the gene expression profiles of all MCM family members in Grade IV (GBM) tissue samples and observed a significant upregulation in GBM samples compared with normal white matter tissues. In addition, we compared the observed gene expression profiles with those of Grade II and Grade III astrocytoma samples and determined that the observed upregulation was restricted and specific to Grade IV. MCM7 was the most upregulated gene in the gene set we analyzed, and therefore we wanted to identify the role of MCM7 in GBM progression. We determined that siRNA-mediated knockdown of MCM7 expression reduced GBM cell proliferation and also inhibited tumor growth in both xenograft and orthotopic mouse models of GBM. Taken together, our data suggest that MCM7 can be a potential prognostic marker and a novel therapeutic target in GBM therapy.

Unclassifiable tauopathy associated with an A152T variation in MAPT exon 7.

Mutations in the microtubule-associated tau (MAPT) gene are associated clinically with frontotemporal dementia with or without supranuclear palsy, corticobasal syndrome or parkinsonism. Here we present clinical, neuropathological, genetic and biochemical data on a patient with an A152T variation in exon 7 of MAPT. A 63-year-old man presented with memory disturbance and later speech disorder, followed by progressive dementia and terminally myoclonus together with periodic sharp waves in EEG. Duration of illness was 5 years. Similar neuropsychiatric symptoms were reported in the patient's father. Neuropathological evaluation revealed neuronal loss mainly in the frontal and temporal cortices and substantia nigra. Abundant phospho-tau immunoreactive thread-like structures and diffuse staining of neuronal cytoplasm predominated in the frontal and temporal cortex, and hippocampus. There was a lack of astrocytic plaques and tufted astrocytes, and only a moderate number of oligodendroglial coiled bodies were seen. Tau pathology was characterized by the 4R tau isoform; immunoblot revealed bands at 64 and 68 kDa, and ultrastructure of filaments was compatible with twisted ribbons. Pathogenic mutations have not been reported in exon 7. Our observation of an apparently familial disorder with a novel neuropathological phenotype suggests a possible pathogenic role of this MAPT gene variation, which might be different from mutations affecting the microtubule binding.

Alzheimer-type neuropathology in a 28 year old patient with iatrogenic Creutzfeldt-Jakob disease after dural grafting.

We report the case of a 28 year old man who had received a cadaverous dura mater graft after a traumatic open skull fracture with tearing of the dura at the age of 5 years. A clinical suspicion of Creutzfeldt-Jakob disease (CJD) was confirmed by a brain biopsy 5 months prior to death and by autopsy, thus warranting the diagnosis of iatrogenic CJD (iCJD) according to WHO criteria. Immunohistochemistry showed widespread cortical depositions of disease associated prion protein (PrP(sc)) in a synaptic pattern, and western blot analysis identified PrP(sc) of type 2A according to Parchi et al. Surprisingly, we found Alzheimer-type senile plaques and cerebral amyloid angiopathy in widespread areas of the brain. Plaque-type and vascular amyloid was immunohistochemically identified as deposits of beta-A4 peptide. CERAD criteria for diagnosis of definite Alzheimer's disease (AD) were met in the absence of neurofibrillar tangles or alpha-synuclein immunoreactive inclusions. There was no family history of AD, CJD, or any other neurological disease, and genetic analysis showed no disease specific mutations of the prion protein, presenilin 1 and 2, or amyloid precursor protein genes. This case represents (a) the iCJD case with the longest incubation time after dural grafting reported so far, (b) the youngest documented patient with concomitant CJD and Alzheimer-type neuropathology to date, (c) the first description of Alzheimer-type changes in iCJD, and (d) the second case of iCJD in Austria. Despite the young patient age, the Alzheimer-type changes may be an incidental finding, possibly related to the childhood trauma.

Increased incidence of genetic human prion disease in Hungary.

The authors performed analysis of the prion protein gene (PRNP) in 27 out of 109 confirmed prion disease patients between 1994 and 2004. E200K mutation was found in 17 cases. Another 10 patients, lacking PRNP analysis, showed positive family history. The mean annual incidence (0.27/million) and proportion (25.6%) of genetic prion disease is unusually high in Hungary and might be related to the migration of ancestors from the Slovakian focus.