Tau-Mediated Nuclear Depletion and Cytoplasmic Accumulation of SFPQ in Alzheimer's and Pick's Disease

Tau dysfunction characterizes neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). Here, we performed an unbiased SAGE (serial analysis of gene expression) of differentially expressed mRNAs in the amygdala of transgenic pR5 mice that express human tau carrying the P301L mutation previously identified in familial cases of FTLD. SAGE identified 29 deregulated transcripts including Sfpq that encodes a nuclear factor implicated in the splicing and regulation of gene expression. To assess the relevance for human disease we analyzed brains from AD, Pick's disease (PiD, a form of FTLD), and control cases. Strikingly, in AD and PiD, both dementias with a tau pathology, affected brain areas showed a virtually complete nuclear depletion of SFPQ in both neurons and astrocytes, along with cytoplasmic accumulation. Accordingly, neurons harboring either AD tangles or Pick bodies were also depleted of SFPQ. Immunoblot analysis of human entorhinal cortex samples revealed reduced SFPQ levels with advanced Braak stages suggesting that the SFPQ pathology may progress together with the tau pathology in AD. To determine a causal role for tau, we stably expressed both wild-type and P301L human tau in human SH-SY5Y neuroblastoma cells, an established cell culture model of tau pathology. The cells were differentiated by two independent methods, mitomycin C-mediated cell cycle arrest or neuronal differentiation with retinoic acid. Confocal microscopy revealed that SFPQ was confined to nuclei in non-transfected wild-type cells, whereas in wild-type and P301L tau over-expressing cells, irrespective of the differentiation method, it formed aggregates in the cytoplasm, suggesting that pathogenic tau drives SFPQ pathology in post-mitotic cells. Our findings add SFPQ to a growing list of transcription factors with an altered nucleo-cytoplasmic distribution under neurodegenerative conditions

Immunocytochemical visualization of muscarinic cholinoceptors in the human cerebral cortex

The monoclonal antibody M 35 was used to study the cellular and subcellular distribution of muscarinic acetylcholine receptors in the human cerebral cortex. M 35, raised against purified muscarinic receptor protein, exerts muscarinic agonist-like effects at cholinergic synapses. Applied to human cortical tissue immediately fixed upon neurosurgical removal, light microscopically M 35 revealed immunoreactive perikarya predominantly of the pyramidal cell type in layers II/III and V, their labeled apical dendrites extending into the superficial layers. Furthermore, a smaller number of neurons of various non-pyramidal morphology in layers VI, IV and III was immunopositive. At the ultrastructural level, immunoprecipitate decorated distinct regions of the perikaryal cytoplasm, numerous dendritic profiles and synapses of both symmetric and asymmetric appearance. In the perikarya immunoprecipitate was associated with ribosomes, the endoplasmic reticulum and the Golgi apparatus. In dendrites the mierotubular system, in synaptic complexes the postsynaptic membranes were decorated. The results suggest that novel informations about the cell type specific and subcellular distribution of human muscarinic cholinoceptors can be obtained by M 35 immunocytochemistry

Muscarinic Cholinoceptive Neurons in the Frontal Cortex in Alzheimer’s Disease

The cellular distribution of muscarinic acetylcholine receptor protein in the frontal cortex of Alzheimer (AD) patients, age-matched and middle-aged controls was assessed quantitatively by means of immunohistochemistry using the monoclonal antibody M35. As shown previously in biopsy cortices, mainly layer II/III and V pyramidal neurons were immunolabeled. Neither distribution nor numbers of labeled cells displayed significant differences between the groups investigated. This is in accordance with the results of ligand binding studies that mostly failed to reveal different binding characteristics in AD compared to controls. Muscarinic and nicotinic receptor proteins have been shown to be colocalized in many cholinoceptive pyramidal neurons. Since nicotinic receptors-in contrast to muscarinic receptor proteins-are severely reduced in AD, this indicates a selective impairment of nicotinic receptor expression and not a significant death of cholinoceptive neurons per se.

SAGE (Serial Analysis of Gene Expression) analysis in the amygdala of P301L tau mutant pR5 mice identifies 29 deregulated genes that includes <i>Sfpq</i>.

<p>SAGE was used to obtain a total of 92,000 sequence tags from pooled amygdalae dissected from ten 10 month-old male pR5 mice (P301L) and ten wild-type (WT) littermate controls. Numbers of counted tags are listed for the two genotypes; ‘combined’ indicates that more than one tag was obtained per deregulated gene (for these, the individual tags are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035678#pone-0035678-t002" target="_blank">Table 2</a>). 29 genes presented a significant (p<0.01) regulation, including nuclear and mitochondrial encoded mitochondrial genes that are shown in light grey (Gene ID for mt-Co1: 17708; mt-Co3: 17710; mt-Atp8: 17706). Of these, 11 were up- (in white) and 14 down-regulated (in grey), with mitochondrial genes indicated (in <b>bold</b>).</p

Deregulation of the nuclear factor <i>Sfpq</i> in tau transgenic mice.

<p>(A) Transcriptomic SAGE analysis of P301L tau mutant pR5 (TG) compared to wild-type (WT) amygdala identified differentially expressed genes within several functional categories (pie chart). The strongly deregulated ‘transcription’ genes (green) included <i>Sfpq</i>. (B) Scheme of the domain structure of the 707 amino acid-long nuclear protein SFPQ. The amino-terminal glutamic acid (E)/glutamine(Q)/proline(P)-rich domain is followed by two RNA/DNA-binding domains (RRMs).</p

Tau transfection causes SFPQ aggregation in postmitotic cells.

<p>(A) Mitomycin C (Mito C)-mediated cell cycle arrest or neuronal differentiation with retinoic acid (RA) of V5-tagged wild-type or P301L tau-expressing SH-SY5Y compared to untransfected (CT) SH-SY5Y neuroblastoma cells reveals SFPQ aggregates (arrows) in the cytoplasm that are not seen in CT. Insets: detailed view of vesicular SFPQ in the cytoplasm. Nuclear staining: DAPI (blue). (B) Western blotting reveals that total levels of SFPQ are not altered under any of these conditions. Actin has been used for normalisation.</p

Neuronal and glial expression of SFPQ revealed in non-transgenic wild-type (CT) control mouse brain shown for the amygdala (A) and the hippocampus (B,C).

<p>Double immunofluorescence for SFPQ (green)/MAP2 (red) (A,B) and SFPQ (green)/GFAP (red) (C) reveals an exclusively nuclear localization in both neurons and astrocytes of WT mice.</p