The human brainome: network analysis identifies HSPA2 as a novel Alzheimer’s disease target.

Our hypothesis is that changes in gene and protein expression are crucial to the development of late-onset Alzheimer’s disease. Previously we examined how DNA alleles control downstream expression of RNA transcripts and how those relationships are changed in late-onset Alzheimer’s disease. We have now examined how proteins are incorporated into networks in two separate series and evaluated our outputs in two different cell lines. Our pipeline included the following steps: (i) predicting expression quantitative trait loci; (ii) determining differential expression; (iii) analysing networks of transcript and peptide relationships; and (iv) validating effects in two separate cell lines. We performed all our analysis in two separate brain series to validate effects. Our two series included 345 samples in the first set (177 controls, 168 cases; age range 65–105; 58% female; KRONOSII cohort) and 409 samples in the replicate set (153 controls, 141 cases, 115 mild cognitive impairment; age range 66–107; 63% female; RUSH cohort). Our top target is heat shock protein family A member 2 (HSPA2), which was identified as a key driver in our two datasets. HSPA2 was validated in two cell lines, with overexpression driving further elevation of amyloid-β40 and amyloid-β42 levels in APP mutant cells, as well as significant elevation of microtubule associated protein tau and phosphorylated-tau in a modified neuroglioma line. This work further demonstrates that studying changes in gene and protein expression is crucial to understanding late onset disease and further nominates HSPA2 as a specific key regulator of late-onset Alzheimer’s disease processes.10.1093/brain/awy215_video1awy215media15824729224001.

Association of Genetic Variants With Primary Open-Angle Glaucoma Among Individuals With African Ancestry.

Importance: Primary open-angle glaucoma presents with increased prevalence and a higher degree of clinical severity in populations of African ancestry compared with European or Asian ancestry. Despite this, individuals of African ancestry remain understudied in genomic research for blinding disorders. Objectives: To perform a genome-wide association study (GWAS) of African ancestry populations and evaluate potential mechanisms of pathogenesis for loci associated with primary open-angle glaucoma. Design, Settings, and Participants: A 2-stage GWAS with a discovery data set of 2320 individuals with primary open-angle glaucoma and 2121 control individuals without primary open-angle glaucoma. The validation stage included an additional 6937 affected individuals and 14 917 unaffected individuals using multicenter clinic- and population-based participant recruitment approaches. Study participants were recruited from Ghana, Nigeria, South Africa, the United States, Tanzania, Britain, Cameroon, Saudi Arabia, Brazil, the Democratic Republic of the Congo, Morocco, Peru, and Mali from 2003 to 2018. Individuals with primary open-angle glaucoma had open iridocorneal angles and displayed glaucomatous optic neuropathy with visual field defects. Elevated intraocular pressure was not included in the case definition. Control individuals had no elevated intraocular pressure and no signs of glaucoma. Exposures: Genetic variants associated with primary open-angle glaucoma. Main Outcomes and Measures: Presence of primary open-angle glaucoma. Genome-wide significance was defined as P < 5 × 10-8 in the discovery stage and in the meta-analysis of combined discovery and validation data. Results: A total of 2320 individuals with primary open-angle glaucoma (mean [interquartile range] age, 64.6 [56-74] years; 1055 [45.5%] women) and 2121 individuals without primary open-angle glaucoma (mean [interquartile range] age, 63.4 [55-71] years; 1025 [48.3%] women) were included in the discovery GWAS. The GWAS discovery meta-analysis demonstrated association of variants at amyloid-ß A4 precursor protein-binding family B member 2 (APBB2; chromosome 4, rs59892895T>C) with primary open-angle glaucoma (odds ratio [OR], 1.32 [95% CI, 1.20-1.46]; P = 2 × 10-8). The association was validated in an analysis of an additional 6937 affected individuals and 14 917 unaffected individuals (OR, 1.15 [95% CI, 1.09-1.21]; P < .001). Each copy of the rs59892895*C risk allele was associated with increased risk of primary open-angle glaucoma when all data were included in a meta-analysis (OR, 1.19 [95% CI, 1.14-1.25]; P = 4 × 10-13). The rs59892895*C risk allele was present at appreciable frequency only in African ancestry populations. In contrast, the rs59892895*C risk allele had a frequency of less than 0.1% in individuals of European or Asian ancestry. Conclusions and Relevance: In this genome-wide association study, variants at the APBB2 locus demonstrated differential association with primary open-angle glaucoma by ancestry. If validated in additional populations this finding may have implications for risk assessment and therapeutic strategies.

Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis.

OBJECTIVE: Hippocampal sclerosis is the most common neuropathologic finding in cases of medically intractable mesial temporal lobe epilepsy. In this study, we analyzed the gene expression profiles of dentate granule cells of patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from RNA collected from small homogenous cell populations, and to shed light on the transcriptional changes associated with hippocampal sclerosis. METHODS: RNA was extracted, and complementary DNA (cDNA) was prepared and amplified from dentate granule cells that had been harvested by laser capture microdissection from surgically resected hippocampi from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis. Sequencing libraries were sequenced, and the resulting sequencing reads were aligned to the reference genome. Differential expression analysis was used to ascertain expression differences between patients with and without hippocampal sclerosis. RESULTS: Greater than 90% of the RNA-Seq reads aligned to the reference. There was high concordance between transcriptional profiles obtained for duplicate samples. Principal component analysis revealed that the presence or absence of hippocampal sclerosis was the main determinant of the variance within the data. Among the genes up-regulated in the hippocampal sclerosis samples, there was significant enrichment for genes involved in oxidative phosphorylation. SIGNIFICANCE: By analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis, we have demonstrated the utility of next-generation sequencing methods for producing biologically relevant results from small populations of homogeneous cells, and have provided insight on the transcriptional changes associated with this pathology.

Allopregnanolone levels are reduced in temporal cortex in patients with Alzheimer's disease compared to cognitively intact control subjects.

The neurosteroid allopregnanolone has pronounced neuroprotective actions, increases myelination, and enhances neurogenesis. Evidence suggests that allopregnanolone dysregulation may play a role in the pathophysiology of Alzheimer's disease (AD) and other neurodegenerative disorders. Our prior data demonstrate that allopregnanolone is reduced in prefrontal cortex in male patients with AD compared to male cognitively intact control subjects, and inversely correlated with neuropathological disease stage (Braak and Braak). We therefore determined if allopregnanolone levels are also reduced in AD patients compared to control subjects in temporal cortex, utilizing a larger set of samples from both male and female patients. In addition, we investigated if neurosteroids are altered in subjects who are APOE4 allele carriers. Allopregnanolone, dehydroepiandrosterone (DHEA), and pregnenolone levels were determined in temporal cortex postmortem samples by gas chromatography/mass spectrometry, preceded by high performance liquid chromatography (40 subjects with AD/41 cognitively intact control subjects). Allopregnanolone levels are reduced in temporal cortex in patients with AD (median 2.68 ng/g, n=40) compared to control subjects (median 5.64 ng/g, n=41), Mann-Whitney p=0.0002, and inversely correlated with Braak and Braak neuropathological disease stage (Spearman r=-0.38, p=0.0004). DHEA and pregnenolone are increased in patients with AD compared to control subjects. Patients carrying an APOE4 allele demonstrate reduced allopregnanolone levels in temporal cortex (Mann-Whitney p=0.04). In summary, our findings indicate that neurosteroids are altered in temporal cortex in patients with AD and related to neuropathological disease stage. In addition, the APOE4 allele is associated with reduced allopregnanolone levels. Neurosteroids may be relevant to the neurobiology and therapeutics of AD.

The effect of SNCA 3' region on the levels of SNCA-112 splicing variant.

Genetic variability at the 3' region of SNCA locus has been repeatedly associated with susceptibility to sporadic Parkinson's disease (PD). Accumulated evidence emphasizes the importance of SNCA dosage and expression levels in PD pathogenesis. However, the mechanism through which the 3' region of SNCA gene modulates the risk to develop sporadic PD remained elusive. We studied the effect of PD risk-associated variants at SNCA 3' regions on SNCA112-mRNA (exon 5 in-frame skipping) levels in vivo in 117 neuropathologically normal, human brain frontal cortex samples. SNPs tagging the SNCA 3' showed significant effects on the relative levels of SNCA112-mRNA from total SNCA transcripts levels. The "risk" alleles were correlated with increased expression ratio of SNCA112-mRNA from total. We provide evidence for functional consequences of PD-associated SNCA gene variants at the 3' region, suggesting that genetic regulation of SNCA splicing plays an important role in the development of the disease. Further studies to determine the definite functional variant/s within SNCA 3'and to establish their association with PD pathology are necessary.

Tissue-specific genetic control of splicing: implications for the study of complex traits.

Numerous genome-wide screens for polymorphisms that influence gene expression have provided key insights into the genetic control of transcription. Despite this work, the relevance of specific polymorphisms to in vivo expression and splicing remains unclear. We carried out the first genome-wide screen, to our knowledge, for SNPs that associate with alternative splicing and gene expression in human primary cells, evaluating 93 autopsy-collected cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononucleated cell samples collected from living healthy donors. We identified 23 high confidence associations with total expression and 80 with alternative splicing as reflected by expression levels of specific exons. Fewer than 50% of the implicated SNPs however show effects in both tissue types, reflecting strong evidence for distinct genetic control of splicing and expression in the two tissue types. The data generated here also suggest the possibility that splicing effects may be responsible for up to 13 out of 84 reported genome-wide significant associations with human traits. These results emphasize the importance of establishing a database of polymorphisms affecting splicing and expression in primary tissue types and suggest that splicing effects may be of more phenotypic significance than overall gene expression changes.

Alzheimer's disease is associated with reduced expression of energy metabolism genes in posterior cingulate neurons.

Alzheimer's disease (AD) is associated with regional reductions in fluorodeoxyglucose positron emission tomography (FDG PET) measurements of the cerebral metabolic rate for glucose, which may begin long before the onset of histopathological or clinical features, especially in carriers of a common AD susceptibility gene. Molecular evaluation of cells from metabolically affected brain regions could provide new information about the pathogenesis of AD and new targets at which to aim disease-slowing and prevention therapies. Data from a genome-wide transcriptomic study were used to compare the expression of 80 metabolically relevant nuclear genes from laser-capture microdissected non-tangle-bearing neurons from autopsy brains of AD cases and normal controls in posterior cingulate cortex, which is metabolically affected in the earliest stages; other brain regions metabolically affected in PET studies of AD or normal aging; and visual cortex, which is relatively spared. Compared with controls, AD cases had significantly lower expression of 70% of the nuclear genes encoding subunits of the mitochondrial electron transport chain in posterior cingulate cortex, 65% of those in the middle temporal gyrus, 61% of those in hippocampal CA1, 23% of those in entorhinal cortex, 16% of those in visual cortex, and 5% of those in the superior frontal gyrus. Western blots confirmed underexpression of those complex I-V subunits assessed at the protein level. Cerebral metabolic rate for glucose abnormalities in FDG PET studies of AD may be associated with reduced neuronal expression of nuclear genes encoding subunits of the mitochondrial electron transport chain.

Metabolomic changes in autopsy-confirmed Alzheimer's disease.

BACKGROUND: Metabolomics, the global science of biochemistry, provides powerful tools to map perturbations in the metabolic network and enables simultaneous quantification of several metabolites to identify metabolic perturbances that might provide insights into disease. METHODS: In this pilot study, we took a targeted electrochemistry-based metabolomics approach where liquid chromatography followed by coulometric array detection enables quantification of over 30 metabolites within key neurotransmitter pathways (dopamine and serotonin) and pathways involved in oxidative stress. RESULTS: Using samples from postmortem ventricular cerebrospinal fluid (15 Alzheimer's disease [AD] and 15 nondemented subjects with autopsy-confirmed diagnoses) and by using regression models, correlations, Wilcoxon rank-sum tests, and t-tests we identified alterations in tyrosine, tryptophan, purine, and tocopherol pathways in patients with AD. Reductions in norepinephrine and its related metabolites were also seen, consistent with previously published data. CONCLUSIONS: These data support further investigation of metabolomics in larger samples of clinical AD as well as in those with preclinical disease for use as biomarkers.

Adult cases of leukoencephalopathy, cerebral calcifications, and cysts: expanding the spectrum of the disorder.

Leukoencephalopathy with cerebral calcifications and cysts (LCC) was first reported in children who developed cognitive decline and variable extrapyramidal, cerebellar, and pyramidal signs, with or without seizures. Leukoencephalopathy with cerebral calcifications and cysts is characterized by progressive formation of brain cysts that can generate a mass effect simulating a neoplasm. Retinal changes that overlap with Coats disease, a microangiopathy with retinal telangiectasias and exudates, may also occur. We and others have reported LCC cases in adults. Neuroimaging shows diffuse leukoencephalopathy, multifocal calcifications especially of deep gray and white matter, multifocal enhancement, and variably sized cysts that may require surgical decompression. Biopsies adjacent to cysts have shown angiomatous and/or severely hyalinized blood vessels surrounded by myelin loss and gliosis, calcifications, and Rosenthal fibers. We report 2 additional adult-onset cases of LCC. Case 1 is a 40-year-old man who developed neurological symptoms and cirrhosis and died of acute gastrointestinal bleeding; he had numerous retinal microinfarcts at autopsy. Case 2 is a 55-year-old woman who was found by chance to have LCC; one and a half years later, her course remains benign. These cases expand the spectrum of adult-onset LCC, the etiology of which is unknown.

Cerebrovascular smooth muscle actin is increased in nondemented subjects with frequent senile plaques at autopsy: implications for the pathogenesis of Alzheimer disease.

We previously found that vascular smooth muscle actin (SMA) is reduced in the brains of patients with late stage Alzheimer disease (AD) compared with brains of nondemented, neuropathologically normal subjects. To assess the pathogenetic significance and disease specificity of this finding, we studied 3 additional patient groups: nondemented subjects without significant AD type pathology ("Normal"; n = 20), nondemented subjects with frequent senile plaques at autopsy ("Preclinical AD"; n = 20), and subjects with frontotemporal dementia ("FTD"; n = 10). The groups were matched for sex and age with those previously reported; SMA immunohistochemistry and image analysis were performed as previously described. Surprisingly, SMA expression in arachnoid, cerebral cortex, and white matter arterioles was greater in the Preclinical AD group than in the Normal and FTD groups. The plaques were not associated with amyloid angiopathy or other vascular disease in this group. Smooth muscle actin expression in the brains of the Normal group was intermediate between the Preclinical AD and FTD groups. All 3 groups exhibited much greater SMA expression than in our previous report. The presence of frequent plaques and increased arteriolar SMA expression in the brains of nondemented subjects suggest that increased SMA expression might represent a physiological response to neurodegeneration that could prevent or delay overt expression dementia in AD.

A novel human disease with abnormal prion protein sensitive to protease.

OBJECTIVE: To report a novel prion disease characterized by distinct histopathological and immunostaining features, and associated with an abnormal isoform of the prion protein (PrP) that, contrary to the common prion diseases, is predominantly sensitive to protease digestion. METHODS: Eleven subjects were investigated at the National Prion Disease Pathology Surveillance Center for clinical, histopathological, immunohistochemical, genotypical, and PrP characteristics. RESULTS: Patients presented with behavioral and psychiatric manifestations on average at 62 years, whereas mean disease duration was 20 months. The type of spongiform degeneration, the PrP immunostaining pattern, and the presence of microplaques distinguished these cases from those with known prion diseases. Typical protease-resistant PrP was undetectable in the cerebral neocortex with standard diagnostic procedures. After enrichment, abnormal PrP was detected at concentrations 16 times lower than common prion diseases; it included nearly 4 times less protease-resistant PrP, which formed a distinct electrophoretic profile. The subjects examined comprised about 3% of sporadic cases evaluated by the National Prion Disease Pathology Surveillance Center. Although several subjects had family histories of dementia, no mutations were found in the PrP gene open reading frame. INTERPRETATION: The distinct histopathological, PrP immunohistochemical, and physicochemical features, together with the homogeneous genotype, indicate that this is a previously unidentified type of disease involving the PrP, which we designated "protease-sensitive prionopathy" (or PSPr). Protease-sensitive prionopathy is not rare among prion diseases, and it may be even more prevalent than our data indicate because protease-sensitive prionopathy cases are likely also to be classified within the group of non-Alzheimer's dementias.

Altered neuronal gene expression in brain regions differentially affected by Alzheimer's disease: a reference data set.

Alzheimer's Disease (AD) is the most widespread form of dementia during the later stages of life. If improved therapeutics are not developed, the prevalence of AD will drastically increase in the coming years as the world's population ages. By identifying differences in neuronal gene expression profiles between healthy elderly persons and individuals diagnosed with AD, we may be able to better understand the molecular mechanisms that drive AD pathogenesis, including the formation of amyloid plaques and neurofibrillary tangles. In this study, we expression profiled histopathologically normal cortical neurons collected with laser capture microdissection (LCM) from six anatomically and functionally discrete postmortem brain regions in 34 AD-afflicted individuals, using Affymetrix Human Genome U133 Plus 2.0 microarrays. These regions include the entorhinal cortex, hippocampus, middle temporal gyrus, posterior cingulate cortex, superior frontal gyrus, and primary visual cortex. This study is predicated on previous parallel research on the postmortem brains of the same six regions in 14 healthy elderly individuals, for which LCM neurons were similarly processed for expression analysis. We identified significant regional differential expression in AD brains compared with control brains including expression changes of genes previously implicated in AD pathogenesis, particularly with regard to tangle and plaque formation. Pinpointing the expression of factors that may play a role in AD pathogenesis provides a foundation for future identification of new targets for improved AD therapeutics. We provide this carefully phenotyped, laser capture microdissected intraindividual brain region expression data set to the community as a public resource.

Cerebral cortical arteriolar angiopathy, vascular beta-amyloid, smooth muscle actin, Braak stage, and APOE genotype.

BACKGROUND AND PURPOSE: We examined the associations among the vascular beta-amyloid levels, smooth muscle actin, wall thickness, and lumen diameter to achieve greater understanding of the arteriolar changes that accompany Alzheimer disease (AD). METHODS: Post-mortem pathology brain specimens from 76 patients with AD and 19 non-AD age control subjects were studied. We analyzed arterioles of the frontal cortex (Brodmann area 10) by immunohistochemistry and morphometry, and derived measures of vascular beta-amyloid level, smooth muscle actin (SMA) volume, and arteriolar wall thickness and lumen diameter. APOE genotype was determined for each case. RESULTS: Overall, there was a striking reciprocal relationship between arteriolar beta-amyloid volume and smooth muscle actin (P<0.0001). In addition, there was a strong positive association between progressively accumulating vascular beta-amyloid and augmentations in both wall thickness (P<0.0001) and lumen width (P<0.0001). In comparison with non-AD control subjects, smooth muscle actin was decreased in patients clinically diagnosed with AD and was reduced >10-fold in cases with AD pathology (Braak I to VI) compared with those lacking AD neuropathology. Significantly altered composition and structure of cortical vessels in pre-Braak stages corroborated our hypothesis that arterioles are devastated early in the AD pathological process. Smooth muscle actin, arteriolar wall thickness, and luminal diameter did not vary with Braak stage severity (P>0.05), indicating that substantial arteriolar damage may precede at least some of the interstitial plaques and neuronal tangles. Moreover, the structural and biochemical arteriolar abnormalities did not vary as a function of APOE genotype (P>0.05). CONCLUSIONS: We postulate that in elderly patients, the continually progressing beta-amyloid-associated angiopathy, at the arteriolar level, harms the contractile apparatus and cerebral blood flow autoregulation, thereby making the downstream capillaries vulnerable to damage. Collectively, our observations lend further support to the idea that microvascular damage has a role, perhaps relatively early, in the onset of major AD pathology.

Cerebrospinal fluid dehydroepiandrosterone levels are correlated with brain dehydroepiandrosterone levels, elevated in Alzheimer's disease, and related to neuropathological disease stage.

OBJECTIVE: It is currently unknown whether cerebrospinal fluid (CSF) neurosteroid levels are related to brain neurosteroid levels in humans. CSF and brain dehydroepiandrosterone (DHEA) levels are elevated in patients with Alzheimer's disease (AD), but it is unclear whether CSF DHEA levels are correlated with brain DHEA levels within the same subject cohort. We therefore determined DHEA and pregnenolone levels in AD patients (n = 25) and cognitively intact control subjects (n = 16) in both CSF and temporal cortex. DESIGN: DHEA and pregnenolone levels were determined by gas chromatography/mass spectrometry preceded by HPLC. Frozen CSF and temporal cortex specimens were provided by the Alzheimer's Disease Research Center at Duke University Medical Center. Data were analyzed by Mann-Whitney U test statistic and Spearman correlational analyses. RESULTS: CSF DHEA levels are positively correlated with temporal cortex DHEA levels (r = 0.59, P < 0.0001) and neuropathological disease stage (Braak and Braak) (r = 0.42, P = 0.007). CSF pregnenolone levels are also positively correlated with temporal cortex pregnenolone levels (r = 0.57, P < 0.0001) and tend to be correlated with neuropathological disease stage (Braak) (r = 0.30, P = 0.06). CSF DHEA levels are elevated (P = 0.032), and pregnenolone levels tend to be elevated (P = 0.10) in patients with AD, compared with cognitively intact control subjects. CONCLUSIONS: These findings indicate that CSF DHEA and pregnenolone levels are correlated with temporal cortex brain levels of these neurosteroids and that CSF DHEA is elevated in AD and related to neuropathological disease stage. Neurosteroids may thus be relevant to the pathophysiology of AD.

Fatal Granulomatous Amebic Encephalitis in a Heart Transplant Patient: Clinical, Radiographic, and Autopsy Findings.

Granulomatous amebic encephalitis is a rare necrotizing infection of the CNS that occurs most commonly in immunocompromised individuals and is usually fatal. It is difficult to diagnose as the clinical symptoms and radiographic findings are often mistaken for other bacterial, viral, fungal, or protozoan infections. Herein, we present the case of a 69-year-old heart transplant recipient who suffered fulminant neurological decline ∼5 months after transplant. Extensive radiographic and laboratory testing did not provide a definite anatomic diagnosis and, despite aggressive clinical treatment, he died. An autopsy examination demonstrated numerous brain abscesses which contained amebic trophozoites and cysts. An indirect immunofluorescence assay performed at the Centers for Disease Control confirmed the presence of Acanthamoeba species. To the best of our knowledge, only 13 other cases of Acanthamoeba amebic encephalitis have been reported in patients who have received solid organ transplants and this is the second case reported in a heart transplant recipient. This case emphasizes that amebic encephalitis should be in the differential diagnosis for immunocompromised patients with new brain lesions found on radiographic imaging.

Analysis of shared heritability in common disorders of the brain.

Disorders of the brain can exhibit considerable epidemiological comorbidity and often share symptoms, provoking debate about their etiologic overlap. We quantified the genetic sharing of 25 brain disorders from genome-wide association studies of 265,218 patients and 784,643 control participants and assessed their relationship to 17 phenotypes from 1,191,588 individuals. Psychiatric disorders share common variant risk, whereas neurological disorders appear more distinct from one another and from the psychiatric disorders. We also identified significant sharing between disorders and a number of brain phenotypes, including cognitive measures. Further, we conducted simulations to explore how statistical power, diagnostic misclassification, and phenotypic heterogeneity affect genetic correlations. These results highlight the importance of common genetic variation as a risk factor for brain disorders and the value of heritability-based methods in understanding their etiology.

A novel cerebral microangiopathy with endothelial cell atypia and multifocal white matter lesions: a direct mycoplasmal infection?

We present 3 sporadic cases of a subacute to chronic, progressive motor (i.e. weakness, ataxia, spasticity, dysarthria, and dysphagia) and cognitive disorder in adults of both sexes, without proven immunocompromise or malignancy. Neuroimaging studies revealed tiny calcifications with atrophy of the cerebrum, pons, and midbrain in 1 patient, cerebral atrophy in another, and cerebral atrophy and periventricular white matter hyperintensities in the third. Clinical diagnoses included cortico-pontine-cerebellar degeneration, mixed neurodegenerative disorder, progressive supranuclear palsy, diffuse Lewy body disease, and Lyme disease. One atrophic brain revealed widely disseminated, millimeter-sized gray lesions in cerebral white matter and obscured anatomic markings of the basis pontis. The most conspicuous microscopic feature in all was capillaries with focally piled up endothelial nuclei, some of which appeared to be multinucleated, or enlarged, hyperchromatic crescentic single nuclei. Although seen mostly without associated damage, they were also noted with white matter lesions displaying vacuolation, demyelination, spheroids, necrosis, vascular fibrosis, and mineralization; these were most severe in the basis pontis. Immunostains and probes to herpes simplex virus-I, -II, and -8; adenovirus, cytomegalovirus, varicella-zoster, Epstein-Barr virus, measles, JC virus, and herpes hominis virus-6 were negative. Electron microscopy revealed no virions in endothelial cells with multilobed or multiple nuclei and duplicated basal laminae. However, mycoplasma-like bodies, mostly 400 to 600 nm in size, were found in endothelial cell cytoplasm and capillary lumina. Platelets adhered to affected endothelial cells. Polymerase chain reaction and immunohistochemistry of fixed samples for Mycoplasma fermentans were negative; other species of Mycoplasma remain viable pathogenic candidates.

Postmortem delay has minimal effect on brain RNA integrity.

The Bryan Alzheimer Disease Research Center obtains postmortem human brain tissue from patients with Alzheimer disease (AD) and cognitively normal control subjects for molecular and genetic research programs. A growing body of research suggests that variations in gene transcript levels may contribute to the onset and progression of disease. Identifying how the regulation of gene expression may affect AD requires the use of high-quality mRNA from banked human brains. The present study was conducted to establish the quality and suitability of available banked brain tissue for future gene expression studies. We chose 32 AD cases with Braak stage IV, V, or VI. These AD cases were matched to 36 normal control cases by age and sex when possible. Multiple regions from each brain were sampled, including frontal cortex, temporal cortex, occipital cortex, and cerebellum. Hippocampus was also available for study from 14 control cases. A comparison of several antemortem and postmortem variables, such as postmortem interval, agonal state, ventricular cerebrospinal fluid pH, and cause of death were analyzed. RNA was isolated from at least 1 area from every brain and most brains yielded intact RNA from all regions tested. Analysis of the clinical variables did not reveal any features that correlated with the ability to recover intact mRNA. We conclude that undegraded mRNA may be isolated from most brain regions many hours postmortem and that neither the pH of ventricular fluid nor postmortem interval is predictive of mRNA integrity.

RB1CC1 insufficiency causes neuronal atrophy through mTOR signaling alteration and involved in the pathology of Alzheimer's diseases.

RB1-inducible Coiled-Coil 1 (RB1CC1) has been shown to be a novel tumor suppressor regulating RB1 expression. Neuronal abundance of RB1CC1 is reported to contribute to the non-proliferating enlarged cell phenotype through the maintenance of RB1 and mTOR. To clarify whether RB1CC1 insufficiency is involved in neuronal atrophy and Alzheimer's pathology, we investigated modifications of RB1CC1 as a possible cause of atrophy or death through the disturbance of mTOR signaling in Neuro-2a neuroblastoma cells. We also evaluated the correlation between RB1CC1 and mTOR signaling in a series of Alzheimer's brain tissues. Though RB1CC1 introduction enhanced neurite growth, RNAi-mediated knockdown of RB1CC1 or rapamycin treatment caused neurite atrophy and apoptosis due to mTOR signaling reduction in the differentiated Neuro-2a cells. Both TSC1 and RB1CC1 were equally functional and maintained mTOR signaling, indicated by phospho-S6 (Ser240/244) expression in 69% of Alzheimer's (9/13 cases) and 100% of normal brains (6/6 cases). However, scanty RB1CC1 expression, less than TSC1, caused phospho-S6 disappearance in 31% of Alzheimer's tissues (4/13 cases). These findings suggest that RB1CC1 insufficiency may result in mTOR signaling repression through unbalanced TSC1 abundance and may induce neuronal atrophy. These observations may have implications for the pathogenesis of Alzheimer's disease.

Somatic uniparental disomy of Chromosome 16p in hemimegalencephaly.

Hemimegalencephaly (HME) is a heterogeneous cortical malformation characterized by enlargement of one cerebral hemisphere. Somatic variants in mammalian target of rapamycin (mTOR) regulatory genes have been implicated in some HME cases; however, ∼70% have no identified genetic etiology. Here, we screened two HME patients to identify disease-causing somatic variants. DNA from leukocytes, buccal swabs, and surgically resected brain tissue from two HME patients were screened for somatic variants using genome-wide genotyping arrays or sequencing of the protein-coding regions of the genome. Functional studies were performed to evaluate the molecular consequences of candidate disease-causing variants. Both HME patients evaluated were found to have likely disease-causing variants in DNA extracted from brain tissue but not in buccal swab or leukocyte DNA, consistent with a somatic mutational mechanism. In the first case, a previously identified disease-causing somatic single nucleotide in MTOR was identified. In the second case, we detected an overrepresentation of the alleles inherited from the mother on Chromosome 16 in brain tissue DNA only, indicative of somatic uniparental disomy (UPD) of the p-arm of Chromosome 16. Using methylation analyses, an imprinted locus on 16p spanning ZNF597 was identified, which results in increased expression of ZNF597 mRNA and protein in the brain tissue of the second case. Enhanced mTOR signaling was observed in tissue specimens from both patients. We speculate that overexpression of maternally expressed ZNF597 led to aberrant hemispheric development in the patient with somatic UPD of Chromosome 16p possibly through modulation of mTOR signaling.