John Maguire

α-Synuclein activates stress signaling protein kinases in THP-1 cells and microglia

Here we show that alpha-synuclein, a major constituent of Lewy bodies, induces inflammation in human microglial and human THP-1 cells. Secretions from such stimulated THP-1 cells contain increased levels of IL-1beta and TNF-alpha. When stimulated by alpha-synuclein in combination with IFN-gamma, secretions from the cells also become toxic towards SH-SY5Y neuroblastoma cells. The A30P, E46K and A53T alpha-synuclein mutations, which induce Parkinsons disease, are more potent than normal alpha-synuclein in the induction of such cytotoxicity. To investigate the signaling mechanisms evoked, protein phosphorylation profiling was applied. At least 81 target phospho-sites were identified. Large increases were induced in the three major mitogen-activated protein (MAP) kinase pathways: p38 MAP kinase, extracellular regulated protein-serine kinase (ERK)1/2 and c-Jun-N-terminal kinase (JNK). Upregulation occurred within minutes following exposure to alpha-synuclein, which is consistent with a receptor-mediated effect. These findings demonstrate that alpha-synuclein acts as a potent inflammatory stimulator of microglial cells, and that inhibitors of such stimulation might be beneficial in the treatment of Parkinsons disease and other synucleinopathies.

Alpha-synuclein and its disease-causing mutants induce ICAM-1 and IL-6 in human astrocytes and astrocytoma cells

Autosomal dominant Parkinson disease (PD) is caused by duplication or triplication of the α‐synuclein gene as well as by the A30P, E46K, and A53T mutations. The mechanisms are unknown. Reactive astrocytes in the substantia nigra of PD and MPTP‐treated monkeys display high levels of the inflammatory mediator intercellular adhesion molecule‐1 (ICAM‐1), indicating that chronic inflammation contributes to the degeneration. Here we report that α‐synuclein strongly stimulates human astrocytes as well as human U‐373 MG astrocytoma cells to up‐regulate both interleukin (IL)‐6 and ICAM‐1 (ED505 µg ml–1). The mutated forms are more potent stimulators than wild‐type (WT) α‐synuclein in these assays. We demonstrate by immunoblotting analysis that this up‐regulation is associated with activation of the major mitogen‐activated protein kinase (MAPK) pathways. It is also attenuated by PD 98059, an inhibitor of the MAPK/extracellular‐regulated kinase kinase MEK1/2, SP 600125, an inhibitor of c‐Jun N‐terminal kinase (JNK), and SB 202190, an inhibitor of p38 MAPK. The inhibitory effects on human astrocytes have IC50 values of 2, 5, and 1.5 M respectively. We hypothesize that the neuroinflammation stimulated by release of an excess of normal α‐synuclein or by release of its mutated forms can be involved in the pathobiology of PD.—Klegeris, A., Giasson, B. I., Zhang, H., Maguire, J., Pelech, S., McGeer, P. L. Alphaα‐synuclein and its disease‐causing mutants induce ICAM‐1 and IL‐6 in human astrocytes and astrocytoma cells. FASEB J. 20, 2000–2008 (2006)

Loss of heterozygosity for loci on chromosome arms 1p and 10q in oligodendroglial tumors: relationship to outcome and chemosensitivity

Oligodendroglial tumors frequently have deletions of chromosomal loci on 1p and 19q. Loss of heterozygosity (LOH) of chromosome 10 may be a negative prognostic factor. We reviewed 23 patients with oligodendroglial tumors, to evaluate the frequency of 1p and 10q LOH and correlate with clinical outcome. Three loci (D1S402, D1S1172, MCT118) on 1p and 2 loci (D10S520 and D10S521) on 10q were analyzed for LOH using PCR techniques. Sixteen oligodendrogliomas (6 low grade and 10 anaplastic) and 7 oligoastrocytomas (1 low grade and 6 anaplastic) were studied. Overall 14/22 (64%) showed 1p LOH and 7/23 (30%) showed 10q LOH. Of 7 patients with some response to chemotherapy, all showed 1p LOH and none had 10q LOH. Of 5 patients with stable or progressive disease, 1 had 1p LOH and 2 showed 10q LOH. The presence of 1p LOH was significantly associated with response to chemotherapy (p = 0.02). Median progression free survival (PFS) was 31 months for 1p intact patients and 118 months for the 1p LOH group (p = 0.014). Median PFS for 10q LOH patients was 31 and 118 months for patients with intact chromosome 10 (p = 0.016). 1p LOH is a predictor of response to chemotherapy and a good prognostic factor. 10q LOH is less common in oligodendroglial tumors but predicts for worse outcome. Molecular genotyping of oligodendroglial tumors is recommended as part of the standard diagnostic workup.

Expression of complement messenger RNAs and proteins by human oligodendroglial cells.

Neurons, astrocytes, microglia, and endothelial cells are capable of synthesizing most, if not all, of the complement proteins. Little is known, however, about the capacity of oligodendroglial cells to generate complement components. This study evaluated expression of complement mRNAs and their protein products by human oligodendrocytes. Cells were isolated and cultured from white matter of seven adult cases that had undergone surgical temporal lobe resection for epilepsy. Oligodendroglial cultures were characterized by the expression of such cell type‐specific mRNAs as myelin proteolipid protein (PLP), oligodendrocyte‐specific protein (OSP), and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and were further characterized by immunostaining for such differentiation markers as myelin basic protein (MBP), PLP, CNPase, and O4. RT‐PCR analysis showed that the oligodendroglial cells expressed detectable levels of complement mRNAs for the C1q B‐chain, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 γ subunit, and C9. Immunostaining was positive for C1q, C1s, C2, C3, C4, C5, C6, C7, C8, and C9. Double immunostaining for the oligodendrocyte marker O4 and the complement protein C3 demonstrated that all O4‐positive cells were also positive for C3, indicating constitutive C3 expression. These results indicate that oligodendroglial cells may be a source of complement proteins in human brain and thus could contribute to the pathogenesis of several neurodegenerative and inflammatory diseases of the CNS, such as Alzheimers disease, multiple sclerosis, and progressive supranuclear palsy, where complement‐activated oligodendrocytes are abundant. GLIA 42:417–423, 2003.

Decreased skeletal muscle mitochondrial DNA in patients with statin-induced myopathy

Statins are widely used to treat hyperlipidemia and lower cardiovascular disease risk. While statins are generally well tolerated, some patients experience statin-induced myopathy (SIM). Statin treatment has been associated with mitochondrial dysfunction and mitochondrial DNA (mtDNA) depletion. In this retrospective study, skeletal muscle biopsies from patients diagnosed with SIM were studied. These were compared with biopsies from patients clinically assessed as having statin-unrelated myopathy but whose biopsy showed no or negligible pathology. For each biopsy sample, mtDNA was quantified relative to nuclear DNA (mtDNA content) by qPCR, mtDNA deletions were investigated by long-template PCR followed by gel densitometry, and mtDNA oxidative damage was quantified using a qPCR-based assay. For a subset of matched samples, mtDNA heteroplasmy and mutations were investigated by cloning/sequencing. Skeletal muscle mtDNA content was significantly lower in SIM patients (N=23, mean±SD, 2036±1146) than in comparators (N=24, 3220±1594), p=0.006. There was no difference in mtDNA deletion score or oxidative mtDNA damage between the two groups, and no evidence of increased mtDNA heteroplasmy or somatic mutations was detected. The significant difference in skeletal muscle mtDNA suggests that SIM or statin treatments are associated with depletion of skeletal muscle mtDNA or that patients with an underlying predisposition to SIM have lower mtDNA levels. If statins induce mtDNA depletion, this would likely reflect decreased mitochondria biogenesis and/or increased mitochondria autophagy. Further work is necessary to distinguish between the lower mtDNA as a predisposition to SIM or an effect of SIM or statin treatment.

Diagnostic utility of a modified forearm ischemic exercise test and technical issues relevant to exercise testing.

The sensitivity and specificity of a modified forearm ischemic test (FIT) are described in the diagnosis of glycogen storage disease, myoadenylate deaminase deficiency, and mitochondrial disease. FIT and muscle biopsy results were reviewed from 99 patients (glycogen storage disease [GSD], myoadenylate deaminase deficiency [AMPD], mitochondrial disease [MITO], miscellaneous neuromuscular disorders, and controls). The influence of catheter placement and an antecedent sugar bolus were also assessed in healthy young men. The FIT had a sensitivity of 1.00 and a specificity of 1.00 for a diagnosis of GSD, whereas the corresponding values were 1.00 and 0.37 for AMPD deficiency. A baseline lactate of >2.5 mmol/L provided the highest sensitivity (0.62) and specificity (1.00) for MITO disease. A baseline and +1 min sample provided optimal sensitivity and specificity for GSD and AMPD deficiency. Catheter placement in any vein other than the ipsilateral antecubital resulted in attenuated lactate responses (P < 0.0001). A pre‐FIT sugar bolus did not alter the postexercise lactate or ammonia response. Thus, a modified FIT was helpful in the diagnosis of GSD and excluding AMPD deficiency, but not in the diagnosis of MITO disease. Catheter placement is critical to the interpretation of a FIT, whereas pretesting diet is less important. Muscle Nerve 27: 359–366, 2003

Prolyl endopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP-1 cell neurotoxicity

Reactive microglial cells may exacerbate the pathology in some neurodegenerative disorders. Supernatants of stimulated human microglial cells, or their surrogate THP‐1 cells, are lethal to cultured human neuroblastoma SH‐SY5Y cells. To explore this neurotoxicity, we examined the spectrum of proteins generated by THP‐1 cells using the technique of stable isotope labeling by amino acids in cell culture (SILAC). Unstimulated cells were grown in medium with light L‐[12C6] arginine while cells stimulated by lipopolysaccharide (LPS) plus interferon‐γ (IFN‐γ) were grown in medium with heavy L‐[13C6] arginine. Proteins isolated from the media were digested with trypsin, and relative concentrations of generated peptides determined by mass spectrometry. More than 1,500 proteins or putative proteins were identified. Of these, 174 were increased and 189 decreased by more than twofold in the stimulated cell supernatant. We selected one upregulated protein, prolyl endopeptidase (PEP), for further investigation of its potential contribution to neurotoxicity. We first confirmed its upregulation by comparing its enzymatic activity in stimulated and unstimulated cell supernatants. We then evaluated two specific PEP inhibitors, Boc‐Asn‐Phe‐Pro‐aldehyde and Z‐Pro‐Pro‐aldehyde‐dimethyl acetal, for their potential to reduce toxicity of stimulated THP‐1 cell and human microglia supernatants towards SH‐SY5Y cells. We found both to be partially protective in a concentration‐dependent manner. Inhibition of PEP may be a therapeutic approach to neurodegenerative disorders including Alzheimer and Parkinson diseases.

Cryptic t(X;18), ins(6;18), and SYT-SSX2 gene fusion in a case of intraneural monophasic synovial sarcoma.

A 54-year-old male presented with a spontaneous peroneal nerve palsy and a diagnosis of monophasic synovial sarcoma (SS) was rendered by histologic examination. Cytogenetic analysis revealed a complex abnormal karyotype without evidence of the typical t(X;18)(p11;q11) associated with SS. Subsequent reverse transcriptase polymerase chain reaction analysis showed the presence of an SYT/SSX2 fusion transcript, confirming the presence of a cyptic t(X;18). In light of -X, -18 and marker chromosomes evident in the G-band karyotype, it was suspected that a cryptic chromosomal rearrangement involving the marker chromosomes would harbor an X;18 fusion. Multi-colored karytotyping (M-FISH) revealed a previously unrecognized t(X;18) and t(5;19) in the marker chromosomes as well as unrecognized ins(6;18) and t(16;20). The addition of M-FISH analysis in this case led to the identification of complex inter-chromosomal rearrangements, thus providing an accurate karyotype.

Loss of 1p and 7p in radiation-induced meningiomas identified by comparative genomic hybridization

Cytogenetic and molecular studies of radiation-induced meningiomas (RIM) are rare and controversial. While comparative genomic hybridization (CGH) analysis identified monosomy 22 as the predominant change in RIM, occurring in frequencies comparable to those found in spontaneous meningioma (SM), molecular genetic analysis shows infrequent loss of chromosome 22 DNA markers. We have performed CGH analysis of six additional cases of RIM and detected an unbalanced genome in five of 6 cases. Loss of 1p and 7p was identified in the majority of RIM with an abnormal karyotype (4/5 cases), whereas loss of 6q occurred in three of five cases. Only one of five RIM had monosomy for chromosome 22. Loss of 7p is not frequently reported in SM and yet it was detected in four of 5 RIM with an abnormal karyotype in our study. Molecular and cytogenetic studies of chromosome 7 copy number should be attempted on a larger number of RIM to further investigate the role of 7p loss in RIM.

Neurosyphilitic gumma in a homosexual man with HIV infection confirmed by polymerase chain reaction

The brain gumma is a rare manifestation of the tertiary stage of syphilis. A case of neurosyphilitic gumma was confirmed by the Treponema pallidum polymerase chain reaction in a 46-year-old HIV-positive homosexual man. The patient presented with a severe headache and was hospitalized. A computed tomography scan was performed which revealed a left frontal lobe mass. Lymphoma was suspected. However, infectious disease diagnostics were performed on the cerebrospinal fluid that included investigations for syphilis and other microbiological agents such as Toxoplasma gondii. This revealed a reactive venereal disease research laboratory test, a reactive syphilis rapid plasma reagin and a reactive T. pallidum particle agglutination test. The patient was treated for syphilis till complete recovery.