Anti-IGLON5 disease: A new case without neuropathologic evidence of brainstem tauopathy.

OBJECTIVE: To describe the neuropathologic features and the molecular data of phosphorylated tau (pTau) in a new case of anti-IgLON5 disease. METHODS: Review of clinical data, postmortem neuropathologic examination. Biochemical analyses of pTau were performed in brain samples from the present case and from a previously described patient with anti-IgLON5 with the characteristic brainstem tauopathy. RESULTS: The patient was a 71-year-old man with a clinical syndrome consisting of sleep disturbance and bulbar symptoms. IgLON5 antibodies of predominant IgG4 subtype were detected in serum and CSF. He carried the HLA DRB1*10:01-DQB1*05:01 haplotype. Despite treatment with IV immunoglobulins, he unexpectedly died during sleep 2 years after disease onset. Histology showed neurofibrillary pathology and ß-amyloid deposits consistent with Alzheimer disease (AD) of intermediate severity. pTau deposits were absent in the brainstem. There were few perivascular CD8+ T-cell infiltrates in the posterior hypothalamus, amygdala, and brainstem with microglial activation. The pTau immunoblot showed a pattern of bands consistent with AD, which was different from that observed in the patient with anti-IgLON5 with brainstem tauopathy who presented a differential band around 56 KDa. CONCLUSION: The absence of pTau deposits in the brainstem of the present patient suggests that the tauopathy of patients with anti-IgLON5 disease may be a late, secondary event. The anti-IgLON5 brainstem tauopathy has a specific molecular signature different from primary tauopathies. pTau deposits restricted to the hippocampus/limbic regions of patients with anti-IgLON5 may represent an age-related comorbidity.

Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.

Olfactory bulb proteome dynamics during the progression of sporadic Alzheimer's disease: identification of common and distinct olfactory targets across Alzheimer-related co-pathologies.

Olfactory dysfunction is present in up to 90% of Alzheimer's disease (AD) patients. Although deposition of hyperphosphorylated tau and ß-amyloid substrates are present in olfactory areas, the molecular mechanisms associated with decreased smell function are not completely understood. We have applied mass spectrometry-based quantitative proteomics to probe additional molecular disturbances in postmortem olfactory bulbs (OB) dissected from AD cases respect to neurologically intact controls (n=20, mean age 82.1 years). Relative proteome abundance measurements have revealed protein interaction networks progressively disturbed across AD stages suggesting an early imbalance in splicing factors, subsequent interrupted cycling of neurotransmitters, alteration in toxic and protective mechanisms of ß-amyloid, and finally, a mitochondrial dysfunction together with disturbance in neuron-neuron adhesion. We also present novel molecular findings in the OB in an autopsy cohort composed by Lewy body disease (LBD), frontotemporal lobar degeneration (FTLD), mixed dementia, and progressive supranuclear palsy (PSP) cases (n = 41, mean age 79.7 years). Olfactory mediators deregulated during the progression of AD such as Visinin-like protein 1, RUFY3 protein, and Copine 6 were also differentially modulated in the OB in LBD, FTLD, and mixed dementia. Only Dipeptidyl aminopeptidase-like protein 6 showed a specific down-regulation in AD. However, no differences were observed in the olfactory expression of this protein panel in PSP subjects. This study demonstrates an olfactory progressive proteome modulation in AD, unveiling cross-disease similarities and differences especially for specific proteins involved in dendritic and axonic distributions that occur in the OB during the neurodegenerative process.

Anatomo-proteomic characterization of human basal ganglia: focus on striatum and globus pallidus.

BACKGROUND: The basal ganglia (BG) are a complex network of subcortical nuclei involved in the coordination and integration of the motor activity. Although these independent anatomical structures are functionally related, the proteome present in each isolated nucleus remains largely unexplored. In order to analyse the BG proteome in a large-scale format, we used a multi-dimensional fractionation approach which combines isolation of anatomically-defined nuclei, and protein/peptide chromatographic fractionation strategies coupled to mass spectrometry. RESULTS: Using this workflow, we have obtained a proteomic expression profile across striatum and globus pallidus structures among which 1681 proteins were located in caudate nucleus (CN), 1329 in putamen, 1419 in medial globus pallidus (GPi), and 1480 in lateral globus pallidus (GPe), establishing a BG reference proteome to a depth of 2979 unique proteins. Protein interactome mapping highlighted significant clustering of common proteins in striatal and pallidal structures, contributing to oxidative phosphorylation, protein degradation and neurotrophin signalling pathways. In silico analyses emphasized specific pathways represented in striatal and pallidal structures highlighting 5-hydroxytryptamine degradation, synaptic vesicle trafficking, and dopamine, metabotropic glutamate and muscarinic acetylcholine receptor pathways. Additional bioinformatic analyses also revealed that: i) nearly 4% of identified proteins have been previously associated to neurodegenerative syndromes, ii) 11% of protein set tends to localize to synaptic terminal, and iii) 20% of identified proteins were also localized in cerebrospinal fluid (CSF). CONCLUSIONS: Overall, the anatomo-proteomic profiling of BG complements the anatomical atlas of the human brain transcriptome, increasing our knowledge about the molecular basis of the BG and the etiology of the movement disorders.

A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool.

BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. METHODS: To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. RESULTS: We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. CONCLUSIONS: Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

Lewy bodies under atomic force microscope.

Lewy bodies are the hallmark of Parkinson disease and their sophisticated analysis will undoubtedly elucidate the pathogenic process. They have been studied by using different microscopic tools. The authors have used atomic force microscopy (AFM) to study the ultramicrotom cut postmortem brain tissue of Parkinson disease patients. Under the same preparation conditions, they have found aggregated fibrillary nanostructures in Lewy bodies, as well as a loss of connections between neurons located in other parts of the substantia nigra. Although these results are preliminary and descriptive in nature, this paper reports the application of a novel and intriguing technique. Further studies including the study of cortical LB and Lewy neurites will be needed to determine the full potential of AFM in the study of the pathogenesis of cell death in Parkinson disease and other synucleinopathies.

Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas.

While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14(ARF), and p16(INK4A)), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14(ARF) and p16(INK4A) did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14(ARF) and p16(INK4A), in which other alterations (mutations, homozygous deletions) are prevalent.

The search for a role of the caudal intralaminar nuclei in the pathophysiology of Parkinson's disease.

The situation of the caudal intralaminar thalamic nuclei within basal ganglia circuits has gained increased attention over the past few years. Although initially considered as a "non-specific" thalamic nuclei, tract-tracing studies carried out over the past two decades have demonstrated that the centromedian-parafascicular thalamic complex (CM-Pf) is connected to virtually all basal ganglia components and related nuclei. Although the anatomical basis sustaining the thalamic modulation of basal ganglia circuits has long been characterized, the functional significance of these transverse circuits still remain to be properly accommodated within the basal ganglia model, both under normal conditions as well as in situations of dopaminergic depletion. However, the recent demonstration of primary (e.g., non-dopamine related) neurodegenerative phenomena restricted to the CM-Pf in Parkinson's disease (PD) has renewed interest in the role played by the caudal intralaminar nuclei in the pathophysiology of PD. Concomitantly, evidence has become available of increased metabolic activity in the caudal intralaminar nuclei in rodent models of PD. Finally, CM-Pf neurosurgery in patients suffering from PD has produced contrasting outcomes, indicating that a consensus is still to be reached regarding the potential usefulness of targeting the caudal intralaminar nuclei to treat movement disorders of basal ganglia origin.

Expression of the mRNAs encoding for the vesicular glutamate transporters 1 and 2 in the rat thalamus.

Vesicular glutamate transporters (VGLUTs) are responsible for glutamate trafficking and for the subsequent regulated release of this excitatory neurotransmitter at the synapse. Three isoforms of the VGLUT have been identified, now known as VGLUT1, VGLUT2, and VGLUT3. Both VGLUT1 and VGLUT2 have been considered definitive markers of glutamatergic neurons, whereas VGLUT3 is expressed in nonglutamatergic neurons such as cholinergic striatal interneurons. It is widely believed that VGLUT1 and VGLUT2 are expressed in a complementary manner at the cortical and thalamic levels, suggesting that these glutamatergic neurons fulfill different physiological functions. In the present work, we analyzed the pattern of VGLUT1 and VGLUT2 mRNA expression at the thalamic level by using single and dual in situ hybridization. In accordance with current beliefs, we found significant expression of VGLUT2 mRNA in all the thalamic nuclei, while moderate expression of VGLUT1 mRNA was consistently found in both the principal relay and the association thalamic nuclei. Interestingly, individual neurons within these nuclei coexpressed both VGLUT1 and VGLUT2 mRNAs, suggesting that these individual thalamic neurons may have different ways of trafficking glutamate. These results call for a reappraisal of the previously held concept regarding the mutually exclusive distribution of VGLUT transporters in the central nervous system.

Detection of two different mRNAs in a single section by dual in situ hybridization: a comparison between colorimetric and fluorescent detection.

We have compared the performance of two methods designed to simultaneously detect two different mRNAs within a single brain section by dual ISH. Specific mRNA riboprobes labeled with biotin and digoxigenin were simultaneously hybridized and visualized using either brightfield or fluorescence microscopy. For brightfield visualization, the biotin-labeled riboprobe was detected with a peroxidase chromogen, whereas, an alkaline phosphatase substrate was used for the detection of the digoxigenin-labeled riboprobe. Dual fluorescent ISH involved the detection of the biotin-labeled riboprobe with an Alexa((R))488-conjugated streptavidin followed by the visualization of the digoxigenin-labeled riboprobe with the red fluorescent substrate HNPP. The dual ISH protocols presented here offer sensitive methods to detect the expression of two mRNAs of interest, with both colorimetric and fluorescent ISH each having its strengths and limitations. For example, dual colorimetric ISH has proven to be particularly useful to study the distribution of two mRNAs in different brain nuclei, whereas, dual fluorescent ISH has provided better results when studying the co-localization of two different mRNAs in single neurons. The comprehensive step-by-step procedure is presented, together with a troubleshooting section in which the advantages and limitations of these procedures are reviewed in depth. Moreover, alternative protocols for dual ISH were also compared to those presented here.

PTEN and DMBT1 homozygous deletion and expression in medulloblastomas and supratentorial primitive neuroectodermal tumors.

Medulloblastoma, which accounts for 20-25% of all childhood brain tumors, is defined as a primitive neuroectodermal tumor (PNET) located in the cerebellum. Supratentorial PNET are less frequent than medulloblastoma. But their clinical outcome is worse than in medulloblastomas. Chromosome 10q contains at least 2 tumor suppressor genes that might play a role in brain tumor development: PTEN and DMBT1. The aim of this study was to compare the status of homozygous deletion and expression of PTEN and DMBT1 genes in PNET primary tumor samples and cell lines. Homozygous deletions of PTEN and DMBT1 were studied in 32 paraffin-embedded PNET samples (23 medulloblastomas and 9 supratentorial PNET) and in 7 PNET cell lines, by differential PCR and by FISH. PTEN homozygous losses were demonstrated in 7 medulloblastomas (32%) and in no supratentorial PNET, while homozygous deletions of DMBT1 appeared in 1 supratentorial PNET (20%) and in 7 medulloblastomas (33%). No homozygous deletion of PTEN or DMBT1 was detected in any of the PNET cell lines either by differential PCR or by FISH. Expression study of the 2 genes was performed in the 7 PNET cell lines by RT-PCR. One PNET cell line lacked PTEN and DMBT1 expression, while 2 medulloblastoma cell lines did not express DMBT1. Our results add some positive data to the hypothesis that supratentorial PNETs and medulloblastomas might be genetically different.

Chromosomal abnormalities in human glioblastomas: gain in chromosome 7p correlating with loss in chromosome 10q.

Various genomic alterations have been detected in glioblastoma. Chromosome 7p, with the epidermal growth factor receptor locus, together with chromosome 10q, with the phosphatase and tensin homologue deleted in chromosome 10 and deleted in malignant brain tumors-1 loci, and chromosome 9p, with the cyclin-dependent kinase inhibitor 2A locus, are among the most frequently damaged chromosomal regions in glioblastoma. In this study, we evaluated the genetic status of 32 glioblastomas by comparative genomic hybridization; the sensitivity of comparative genomic hybridization versus differential polymerase chain reaction to detect deletions at the phosphatase and tensin homologue deleted in chromosome 10, deleted in malignant brain tumors-1, and cyclin-dependent kinase inhibitor 2A loci and amplifications at the cyclin-dependent kinase 4 locus; the frequency of genetic lesions (gain or loss) at 16 different selected loci (including oncogenes, tumor-suppressor genes, and proliferation markers) mapping on 13 different chromosomes; and the possible existence of a statistical association between any pair of molecular markers studied, to subdivide the glioblastoma entity molecularly. Comparative genomic hybridization showed that the most frequent region of gain was chromosome 7p, whereas the most frequent losses occurred on chromosomes 10q and 13q. The only statistically significant association was found for 7p gain and 10q loss.