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Understanding the biology of Waldenström macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secretes human immunoglobulin M (h-IgM) with κ-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2), with a consistent amplification of 14q32 (immunoglobulin heavy chain; IgH) identical to its founding tumor sample. The clonal relationship is confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in the MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Overall, RPCI-WM1 represents a valuable model to study Waldenström macroglobulinemia.

Objective: Autosomal dominant parkinsonism, hypoventilation, depression and severe weight loss (Perry syndrome) is an early-onset rapidly progressive disease. At autopsy, previous studies have found severe neuronal loss in the substantia nigra without Lewy bodies. Transactive response DNA-binding protein of 43 kDa (TDP-43) has recently been identified as a major ubiquitinated constituent of neuronal and glial inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis. This study reports clinical, genetic and neuropathologic investigations of Perry syndrome., Methods: Clinical data and autopsy brain tissue samples were collected from eight patients from four genealogically unrelated kindreds with Perry syndrome. Brain tissue was studied with immunohistochemistry and biochemistry for TDP-43. Patients were screened for mutations in the progranulin (GRN) and TDP-43 (TARDBP) genes., Results: The mean age at onset was 47 years (range 40-56), and the mean age at death was 52 years (range 44-64). In all patients, we identified TDP-43-positive neuronal inclusions, dystrophic neurites and axonal spheroids in a predominantly pallidonigral distribution, and we demonstrated changes in solubility and electrophoretic mobility of TDP-43 in brain tissue. The inclusions were highly pleomorphic and predominated in the extrapyramidal system, sparing the cortex, hippocampus and motor neurons. There were no mutations in GRN or TARDBP., Interpretation: Perry syndrome displays unique TDP-43 pathology that is selective for the extrapyramidal system and spares the neocortex and motor neurons.

Cholinergic cell lines were established by fusion of embryonic day 17 wild-type neurons from rat basal forebrain (BF) and upper brainstem (BS) with N18tg neuroblastoma cells. Isolated clones expressed choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) activities that were increased upon differentiation with retinoic acid. Clones from the BF expressed high levels of the tyrosine kinase type A (TrkA) receptor expression and activation of the mitogen-activated kinase ERK2 upon treatment with nerve growth factor. Like wild-type cholinergic populations, the six clones studied were variably resistant to nitric oxide (NO) excess from addition of S-nitroso-N-acetyl-D, L-penicillamine (SNAP). Of these, the BS2 clone exhibited resistance like in vivo BS cholinergic neurons, while the MS10 clone mimicked in vivo BF vulnerability. Apoptosis in response to NO excess was preceded by increases in mitochondrial responses bax/bcl-2 ratios, but cytochrome C was not released. Mitochondrial levels of apoptosis initiating factor (AIF) were either unchanged or increased, and only in MS clones was endonuclease G (EndoG) released. Microarray data indicated the existence of endoplasmic reticular (ER) stress and caspase-4 and caspase-12 were involved in the pathway to DNA fragmentation. The array data also indicated a survival role for mdm2, and its blockade rendered vulnerable the brainstem survivor clone BS2. Akt and ERK1/2 pathways were activated in response to NO and their blockade increased DNA fragmentation. Blockade of GSK-3 alpha/beta, a downstream target of Akt, reduced SNAP toxicity and this was more prominent in basal forebrain clones. We have identified two cholinergic cell lines useful for molecular studies of cholinergic vulnerability. We hypothesize that, in cholinergic neurons, control of ER stress signaling may be a major factor in differential vulnerability.

We developed a simplified protocol for sensitive quantitation of mRNA using polymerase chain reaction (PCR) amplification of cDNA made by reverse transcriptase (RT), as resolved with capillary electrophoresis (CE) and detected with laser-induced fluorescence (LIF). The conditions required for adequate accuracy of the simplified version of the RT/PCR quantitation, in which a single concentration of external standard and amplification to within or near the plateau phase are used, were established for assay of mRNAs expressed at high, moderate, and low abundance. The mRNAs for the cytosolic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH) and the growth-associated protein GAP-43 in cultured SN49 neuroblastoma cells were used as target genes for high and moderate levels of expression, respectively. Using cultured mouse microglial cells (BV-2), we demonstrated the utility of this RT/PCR/CE/LIF protocol to quantitate a low-abundance mRNA, encoding a form of nitric oxide synthase (i-NOS) induced by treatment with endotoxin. The appearance of i-NOS mRNA after endotoxin treatment of BV-2 cells was confirmed by Northern blot analysis and in situ hybridization histochemistry, and functional enzyme activity was followed by release of nitric oxide (as nitrite) into the medium. The many advantages of the 'single-point' RT/PCR/CE/LIF protocol for quantitating mRNAs of interest include: simplified protocol, elimination of the use of radiotracers, high sensitivity and precision, and semi-automation of the quantitation phase of analysis.