Large-Scale RNA Interference Screening in Mammalian Cells Identifies Novel Regulators of Mutant Huntingtin Aggregation

In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms

Rationale and Design of a Prospective, Multicentre, Stop Tyrosine Kinase Inhibitor Trial of Paediatric Patients with Chronic Myeloid Leukaemia with Sustained Complete Molecular Response (STKI-14)

Chronic myeloid leukaemia (CML) is a relatively rare disease in children, accounting for 2–3% of all paediatric leukaemia cases. Generally, children with CML can avoid hematopoietic stem cell transplantation and achieve molecular responses with tyrosine kinase inhibitors (TKI). However, CML stem cells are thought to survive in many patients, even after TKI treatment. Many aspects of the toxic effects of prolonged exposure to TKIs during childhood remain unclear, particularly those regarding growth impairment. This lack of clarity underscores the importance of the present clinical trial, which aims to clarify the feasibility of treatment-free remission (TFR) in children following TKI treatment. We aim to examine the long-term out-comes and complications of TKIs before and after cessation to better understand the unknown complications that could arise in adulthood. This trial targets patients who were diagnosed with CML at an age younger than 20 years, were in the chronic or accelerated phase at initial diagnosis and remained in complete molecular remission for at least 2 years after TKI administration. We will examine the utility of TKI cessation and assess the treatment results of patients who resumed TKI therapy after losing a major molecular response. We will also investigate factors related to the feasibility of a TFR after TKI cessation

Loss of aPKCλ in Differentiated Neurons Disrupts the Polarity Complex but Does Not Induce Obvious Neuronal Loss or Disorientation in Mouse Brains

<div><p>Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.</p></div

Differential roles of NF-Y transcription factor in ER chaperone expression and neuronal maintenance in the CNS

The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons, and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression

Rapid dissemination of alpha-synuclein seeds through neural circuits in an in-vivo prion-like seeding experiment

Abstract Accumulating evidence suggests that the lesions of Parkinson’s disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5–10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit–dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state

NeuN and GFAP staining of aPKCλ deletion mouse cerebrum.

<p>Immunohistochemical analysis of 7-month-old aPKCλ flox/−; S1-cre (S1-cko) or flox/+ (Cont) female mice (left two panels), or 15-month-old aPKCλ flox/flox; C2-cre (C2-cko) or flox/+; C2-cre (Cont) male mice (right two panels). (A) Staining of coronal sections with anti-NeuN, a neuronal marker. (B) Magnified images of boxed regions shown in (A). No distinct reduction of NeuN-positive cells in these aPKCλ deletion mice was found. (C) Staining of coronal sections with anti-GFAP, an astrocyte marker. (D) Magnified images of boxed regions shown in (C). No distinct induction of astrogliosis in these aPKCλ deletion mice. Cor (cortex) and Hpc (hippocampus). Bars are 1 mm (A, C) and 0.4 mm (B, D).</p

Cell orientation of cortical layer V neurons in aPKCλ deletion mice.

<p>(A) Coronal sections of 7-month-old aPKCλ flox/-; S1-cre (S1-cko) or flox/+ (Cont) female mice (left two panels), or 15-month-old aPKCλ flox/flox; C2-cre (C2-cko) or flox/+; C2-cre (Cont) male mice (right two panels) were stained with a Golgi marker GM130 (red) and an axon initial segment (AIS) marker Nav1.6 (green). Nuclei were stained with TOTO-3. Cortical layer V neurons are shown. Note that Golgi was abundant at superior part of the neurons (arrows) whereas AIS was detected in inferior region (arrowheads) in both control and aPKCλ deletion mice. (B) Immunofluorescence data in (A) were used for quantification of relative GM130 fluorescence intensities in superior region to those in whole cell region (n means number of analyzed cells). Values are means ± SD. A majority of the layer V neurons showed superior accumulation of GM130, which was not significantly affected in aPKCλ deletion mice. Bar is 20 µm (A).</p

Western blot analysis of aPKCλ and its interacting proteins in the brain of aPKCλ deletion mice.

<p>(A) Brains of 7-month-old female mice harboring aPKCλ flox/−; S1-cre (S1-cko) or flox/+ (Cont) were separated into five regions: striatum (Str), hippocampus (Hpc), cortex (Cor), other remaining cerebrum regions (Other) and cerebellum (Cbl). These tissue regions were subjected to Western blot analysis using antibody specific to aPKCλ (BD, 610175) or antibody recognizing both aPKCλ/ζ (Santa Cruz (SC), sc-216). Antibodies for PAR-6β, p62 and β-actin were also used for the analysis. (B) Brains of 13-month-old female mice harboring aPKCλ flox/+ (Cont) or flox/flox; C2-cre (C2-cko) were separated and analyzed as in (A). (C) Total cerebrum of 11-month-old female mice harboring aPKCλ flox/flox; S1-cre (S1-cko; n = 3) or flox/flox (Cont; n = 3) were subjected to Western blot analysis using anti-aPKCλ (BD) or anti-aPKCλ/ζ (SC). An alternative isoform of aPKCζ, PKMζ was also detected by anti-aPKCλ/ζ (SC). Antibodies for PAR-6β, PAR-3, Lgl-1, p62 and β-actin were also used for the analysis. (D) Bands in (C) were quantified and plotted. (E) Total cerebrum of 20-month-old female mice harboring aPKCλ flox/flox; C2-cre (C2-cko; n = 3) or flox/flox (Cont; n = 3) were subjected to Western blot analysis as in (C). (F) Bands in (E) were quantified and plotted. Values are means ±SD (*P<0.05, **P<0.01, ***P<0.001).</p

Immunoprecipitation assay using aPKCλ conditional deletion mouse brains.

<p>(A) Cerebra of 11-month-old male mice harboring aPKCλ flox/flox (Cont; n = 3) or aPKCλ flox/flox; S1-cre (aPKCλ S1-cko; n = 3) were lysed (Input) and subjected to immunoprecipitation (IP) with anti-Lgl-1 antisera. IP without antisera (-) was used as a negative control. The input and IP samples were analyzed by Western blotting using antibodies for Lgl-1 and aPKCλ. (B) Bands of IP samples in (A) were quantified and plotted. (C) Cerebra of 20-month-old male mice harboring aPKCλ flox/flox (Cont; n = 3) or aPKCλ flox/flox; C2-cre (aPKCλ C2-cko; n = 3) were subjected to IP and analyzed as in (A). (D) Bands of IP samples in (C) were quantified and plotted. Note the significant reduction of aPKCλ co-immunoprecipitated with Lgl-1 in these aPKCλ deletion mouse cerebra. Values are means ±SD (*P<0.05, ***P<0.001).</p

Quantitative RT-PCR of aPKCλ and its related genes in aPKCλ conditional deletion mice.

<p>(A, B) Specificity of primer sets for aPKCλ and aPKCζ for quantitative PCR. Plasmid DNA for mouse aPKCλ cDNA (A) or aPKCζ cDNA (B) at indicated relative concentrations was subjected to real-time PCR using primers of aPKCλ and aPKCζ (RD or KD). Crossing point (Cp) means the cycle number of first detection of positive signal for PCR product. Low Cp indicates efficient detection of template cDNA, whereas high Cp without inverse correlation with the amount of the input indicates no significant detection. (C) Data in (A) and (B) were used for quantification of relative amounts of cDNA. Specific amplifications by each primer set were confirmed. (D, E) Quantitative RT-PCR of aPKCλ and indicated genes in cerebra of 11-month-old male mice harboring aPKCλ flox/flox (Cont) or aPKCλ flox/flox; S1-cre (aPKCλ S1-cko) (D, n = 3 for each), or 20-month-old male mice harboring aPKCλ flox/flox (Cont) or aPKCλ flox/flox; C2-cre (aPKCλ C2-cko) (E, n = 3 for each). Amounts relative to control are shown. (F) Quantitative RT-PCR of aPKCζ in cerebra of aPKCλ flox/flox (Cont) male mice at 11 months or 20 months of age (n = 3 for each). aPKCζ plasmid DNA was used as standard for quantification. Primer sets for aPKCζ RD and KD were used to detect full-length aPKCζ and both aPKCζ/PKMζ, respectively. The values obtained by aPKCζ KD primer set were taken as 1. (G) Quantitative RT-PCR of aPKCζ and PKMζ in cerebra of 11-month-old male mice harboring aPKCλ flox/flox (Cont) or aPKCλ flox/flox; S1-cre (aPKCλ S1-cko) (n = 3 for each). Amount relative to control is shown. Values are means ±SD.</p