Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II

In neurodegenerative disorders associated with primary or secondarymitochondrial defects such as Huntington's disease (HD), cellsof the striatum are particularly vulnerable to cell death, althoughthe mechanisms by which this cell death is induced are unclear.Dopamine, found in high concentrations in the striatum, mayplay a role in striatal cell death. We show that in primarystriatal cultures, dopamine increases the toxicity of an N-terminalfragment of mutated huntingtin (Htt-171-82Q). Mitochondrialcomplex II protein (mCII) levels are reduced in HD striatum,indicating that this protein may be important for dopamine-mediatedstriatal cell death. We found that dopamine enhances the toxicityof the selective mCII inhibitor, 3-nitropropionic acid. We alsodemonstrated that dopamine doses that are insufficient to producecell loss regulate mCII expression at the mRNA, protein andcatalytic activity level. We also show that dopamine-induceddown-regulation of mCII levels can be blocked by several dopamineD2 receptor antagonists. Sustained overexpression of mCII subunitsusing lentiviral vectors abrogated the effects of dopamine,both by high dopamine concentrations alone and neuronal deathinduced by low dopamine concentrations together with Htt-171-82Q.This novel pathway links dopamine signaling and regulation ofmCII activity and could play a key role in oxidative energymetabolism and explain the vulnerability of the striatum inneurodegenerative diseases.     

Aggregation of N-terminal huntingtin is dependent on the length of its glutamine repeats

Huntington's disease (HD) is caused by expansion of a glutamine repeat in huntingtin. Mutant huntingtin contains 36-55 repeats in adult HD patients and >60 repeats in juvenile HD patients. An N-terminal fragment of mutant huntingtin forms aggregates in neuronal nuclei in the brains of transgenic mice and HD patients. Aggregation of expanded polyglutamine is thought to be a common pathological mechanism in HD and other glutamine repeat diseases. It is not clear how the length of the repeats is correlated with formation of protein aggregates. By expressing a series of huntingtin constructs encoding various glutamine repeats (23-150 units) in cultured cells we observed N-terminal fragments of huntingtin (amino acids 1-67 and 1-212), but not full- length huntingtins, with glutamine repeats >/=66 units formed protein aggregates. Huntingtin aggregation was not induced when the repeat was </=49 units and was markedly promoted by very long repeats >/=120 units. This study suggests that various N-terminal fragments of mutant huntingtin can form aggregates and that aggregation is prompted by lengthening the glutamine repeat.      

Nuclear import of the Drosophila Rel protein Dorsal is regulated by phosphorylation

In Drosophila, dorsal-ventral polarity is determined by a maternally encoded signal transduction pathway that culminates in the graded nuclear localization of the Rel protein, Dorsal. Dorsal is retained in the cytoplasm by the IkappaB protein, Cactus. Signal-dependent phosphorylation of Cactus results in the degradation of Cactus and the nuclear targeting of Dorsal. We present an in-depth study of the functional importance of Dorsal phosphorylation. We find that Dorsal is phosphorylated by the ventral signal while associated with Cactus, and that Dorsal phosphorylation is essential for its nuclear import. In vivo phospholabeling of Dorsal is limited to serine residues in both ovaries and early embryos. A protein bearing mutations in six conserved serines abolishes Dorsal activity, is constitutively cytoplasmic, and appears to eliminate Dorsal phosphorylation, but still interacts with Cactus. Two individual serine-to-alanine mutations produce unexpected results. In a wild-type signaling background, a mutation in the highly conserved PKA site (S312) produces only a weak loss-of-function; however, it completely destabilizes the protein in a cactus mutant background. Significantly, the phosphorylation of another completely conserved serine (S317) regulates the high level of nuclear import found in ventral cells. We conclude that the formation of a wild-type Dorsal nuclear gradient requires the phosphorylation of both Cactus and Dorsal. The strong conservation of the serines suggests that phosphorylation of other Rel proteins is essential for their proper nuclear targeting.     

枸杞多糖通过泛素蛋白酶体途径降解突变亨廷顿蛋白

目的　探讨枸杞多糖（Lycium barbarum polysaccharide,LBP）降解突变亨廷顿蛋白（mutant huntingtin,mHtt）的途径。 方法　在稳定表达mHtt 160Q的HEK293细胞中使用不同浓度LBP,CCK8法检测细胞活力,caspase-3活性酶标法检测caspase-3活性;使用荧光显微镜检测、Image Pro Plus 6.0分析LBP对HEK293-160Q细胞中mHtt的影响并同时使用RT-PCR法检测LBP是否影响其mRNA水平;使用LBP、MG132及氯喹,分组处理HEK293-160Q细胞,通过Western Blot法检测不同组细胞中mHtt的变化。 结果　LBP能提高HEK293-160Q细胞活力,降低caspase-3活性;LBP能减少细胞中mHtt且不改变其mRNA;不同药物处理HEK293-160Q细胞后,发现与只使用LBP相比,同时使用LBP与MG132会显著降低mHtt的降解,而同时使用LBP与氯喹则对mHtt的降解没有影响。 结论　LBP能通过泛素蛋白酶体途径降解mHtt,减轻mHtt所引起的细胞毒性继而提高细胞活力、抑制细胞凋亡。     

Dynamic association of tau with neuronal membranes is regulated by phosphorylation

Tau is an abundant cytosolic protein which regulates cytoskeletal stability by associating with microtubules in a phosphorylation-dependent manner. We have found a significant proportion of tau is located in the membrane fraction of rat cortical neurons and is dephosphorylated, at least at Tau-1 (Ser199/Ser202), AT8 (Ser199/Ser202/Thr205) and PHF-1 (Ser396/Ser404) epitopes. Inhibition of tau kinases casein kinase 1 (CK1) or glycogen synthase kinase-3 decreased tau phosphorylation and significantly increased amounts of tau in the membrane fraction. Mutation of serine/threonine residues to glutamate to mimic phosphorylation in the N-terminal, but not C-terminal, region of tau prevented its membrane localization in transfected cells, demonstrating that the phosphorylation state of tau directly impacts its localization. Inhibiting CK1 in neurons lacking the tyrosine kinase fyn also induced tau dephosphorylation but did not affect its membrane association. Furthermore, inhibition of CK1 increased binding of neuronal tau to the fyn-SH3 domain. We conclude that trafficking of tau between the cytosol and the neuronal membrane is dynamically regulated by tau phosphorylation through a mechanism dependent on fyn expression.     

热休克蛋白40和热休克蛋白70抑制N2a细胞内突变亨廷顿蛋白聚积和毒性

目的 研究热休克蛋白40(HSP40)和热休克蛋白70(HSP70)对N2a细胞内突变亨廷顿蛋白(htt) 聚集物形成和毒性的影响. 方法 应用荧光显微镜术和免疫印迹技术检测单独或共同过表达HSP40和HSP70对N2a细胞内转染的突变htt(含有150个谷氨酰胺重复数,150Q)聚集物形成的影响;应用四甲基偶氮唑盐(MTT)法分析细胞活力和比色法检测细胞内活性氧(ROS)含量. 结果 单独过表达HSP40或HSP70,尤其是共同过表达HSP40和HSP70显著减少150Q htt在N2a细胞内的聚积,各组含有聚集物的细胞为(n=1 000):仅表达150Q htt 组约50%,过表达HSP40组约12%,过表达HSP70组约15%,共同过表达HSP40和HSP70组约5%.MTT分析结果显示,单独尤其是共同过表达HSP40和HSP70能显著增加150Q细胞的活力( P<0.01, n =3),同时减少ROS产生( P<0.01, n =3). 结论 HSP40和HSP70通过抑制细胞内突变htt聚积以及减少氧化应激而增加150Q N2a细胞活力.     

Dopamine D1 receptor-mediated aggregation of N-terminal fragments of mutant huntingtin and cell death in a neuroblastoma cell line

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by abnormal CAG repeat expansion in the IT15 gene encoding huntingtin protein (htt). Mutated htt is predicted to acquire toxic properties in specific brain regions. For instance, striatal neurons expressing dopamine receptors predominantly degenerate in HD patients. Although the basis of this specific vulnerability remains unclear, a great deal of evidence has documented the ability of the dopamine system to modulate the toxicity of expanded htt. To investigate the relationship between dopamine receptors and expanded htt, we transfected enhanced green fluorescent proteins (EGFP) tagged to normal (25 CAG) or mutant (103 CAG) htt in SK-N-MC neuroblastoma cells that endogenously express D1 receptors. Forming nuclear and cytoplasmic aggregates, mutant htt-EGFP was toxic to cells beyond 24 h post-transfection. Remarkably, low doses of a selective D1 receptors agonist or forskolin, an activator of adenylate cyclase, accelerated the formation of mutant htt nuclear aggregates, whereas the number of cytoplasmic aggregates was decreased. These effects were associated with a minor increase in cell death. Understanding the functional bases of these effects may further elucidate the role of dopamine receptors signaling in the complex pathophysiology of HD.     

Significantly differential diffusion of neuropathological aggregates in the brain of transgenic mice carrying N-terminal mutant huntingtin fused with green fluorescent protein

Huntington’s disease (HD) is a genetically neurodegenerative disease, affecting the central nervous system and leading to mental and motor dysfunctions. To date, there is no cure for HD; as a result, HD patients gradually suffer devastating symptoms, such as chorea, weight loss, depression and mood swings, until death. According to previous studies, the exon 1 region of the huntingtin (HTT) gene with expanded CAG trinucleotide repeats plays a critical role in causing HD. In vitro studies using exon 1 of HTT fused with green fluorescent protein (GFP) gene have facilitated discovering several mechanisms of HD. However, whether this chimera construct exerts similar functions in vivo is still not clear. Here, we report the generation of transgenic mice carrying GFP fused with mutant HTT exon 1 containing 84 CAG trinucleotide repeats, and the evaluation of phenotypes via molecular, neuropathological and behavioral analyses. Results show that these transgenic mice not only displayed neuropathological characteristics, observed either by green fluorescent signals or by immunohistochemical staining, but also progressively developed pathological and behavioral symptoms of HD. Most interestingly, these transgenic mice showed significantly differential expression levels of nuclear aggregates between cortex and striatum regions, highly mimicking selective expression of mutant HTT in HD patients. To the best of our knowledge, this is the first report showing different nuclear diffusion profiling in mouse models with transgenic mice carrying the exon 1 region of mutant HTT. Our model will be beneficial for tracing the expression of mutant HTT and accelerating the understanding of selective pathological progression in HD.     

Neuropeptide gene expression in brain is differentially regulated by midbrain dopamine neurons

Summary In situ hybridization was used to study the expression of prepro-neuropeptide Y (NPY), preprosomatostatin (SOM), preprotachykinin (PPT) and preprocholecystokinin (CCK) mRNA in caudate-putamen and frontoparietal cortex of rat brain with unilateral lesion of midbrain dopamine neurons. Neurons expressing NPY and SOM mRNA showed a similar distribution and the expression of both NPY and SOM appears to be regulated by dopamine in a similar fashion. Following a dopamine deafferentation, the numerical density of both NPY and SOM mRNA producing neurons almost doubled in the lesioned caudate-putamen with no change in the average grain density over positive neurons. Hence, in the intact caudate-putamen dopamine appears to suppress expression of these two neuropeptide genes leading to an activation of both NPY and SOM mRNA expression in many non- or low-expressing neurons when the level of dopamine is decreased. In the fronto-parietal cortex, on the other hand, dopamine appears to stimulate NPY and SOM gene expression. Thus, in the absence of dopamine about half of the NPY positive neurons disappeared. However, for SOM the number of positive neurons did not change, but rather most positive neurons appeared to have down-regulated their SOM mRNA expression. No evidence was found for a change in CCK mRNA expression by the dopamine deafferentation, while PPT mRNA expression decreased in the deafferented caudate-putamen. Consequently, dopamine exerts dissimilar effects on the expression of different neuropeptide genes, that in turn do not respond in the same way in different brain regions.     

Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice

Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated Hdh(Q111) knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four Hdh(Q111) phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.     

Wild type huntingtin reduces the cellular toxicity of mutant huntingtin in mammalian cell models of Huntington's disease

OBJECTIVES—Recent data suggest that wild type huntingtin can protect against apoptosis in the testis of mice expressing full length huntingtin transgenes with expanded CAG repeats. It is not clear if this protective effect was confined to particular cell types, or if wild type huntingtin exerted its protective effect in this model by simply reducing the formation of toxic proteolytic fragments from mutant huntingtin.
METHODS—We cotransfected neuronal (SK-N-SH, human neuroblastoma) and non-neuronal (COS-7, monkey kidney) cell lines with HD exon 1 (containing either 21 or 72 CAG repeats) construct DNA and either full length wild type huntingtin or pFLAG (control vector).
RESULTS—Full length wild type huntingtin significantly reduced cell death resulting from the mutant HD exon 1 fragments containing 72 CAG repeats in both cell lines. Wild type huntingtin did not significantly modulate cell death caused by transfection of HD exon 1 fragments containing 21 CAG repeats in either cell line.
CONCLUSIONS—Our results suggest that wild type huntingtin can significantly reduce the cellular toxicity of mutant HD exon 1 fragments in both neuronal and non-neuronal cell lines. This suggests that wild type huntingtin can be protective in different cell types and that it can act against the toxicity caused by a mutant huntingtin fragment as well as against a full length transgene.


Keywords: Huntington's disease; huntingtin; apoptosis     

Mouse p87 wee1 kinase is regulated by M-phase specific phosphorylation

We have cloned a mousewee1 kinase cDNA (mwee1). The clone is 2258 bp in length and its open reading frame corresponds to 646 amino acid residues. The molecular weight of this kinase is 87 kDa in SDS-PAGE, which is about 1.7-fold larger than the human p50 ^wee1 kinase reported previously. In a cell cycle, the mousewee1 kinase is phosphorylated at M-phase, and anin vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the humanwee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or entire molecule is extensively phosphorylated bycdc2-cyclin B kinase. Furthermore, the activity of thewee1 kinase was reduced by phosphorylation with the mitotic extract which containedcdc2-cyclin B kinase