Huntington's disease




IkB Kinase complex regulates cleavage of huntingtin protein

            Ali Khoshnan, Jan Ko            

Cleavage of huntingtin (Htt) protein plays a key role in the pathogenesis of Huntington’s disease (HD). However, the environmental signals and molecular pathways that regulate this event are not well characterized.  One potential factor is the accumulation of DNA damage that is known to occur in HD neurons. We have discovered that the DNA damaging agent etoposide stimulates cleavage of endogenous Htt in cultured human neurons, generating N-terminal fragments of ~85 kDa. Etoposide also stimulates cleavage of full-length mutant Htt, which could lead to accumulation of neurotoxic fragments.  Moreover, we find that etoposide-induced Htt cleavage is regulated by the IkB kinase b (IKKb). Silencing IKKb using a small hairpin RNA, or small molecule inhibition of its activity, blocks Htt cleavage and promotes neuronal survival. In terms of mechanism, IKKb phosphorylates the pro-survival protein Bcl-xL, promotes its degradation and leads to the activation of caspase-3 and subsequent Htt cleavage. Inhibition of IKKb prevents etoposide-stimulated degradation of Bcl-xL. Moreover, neurons engineered to express elevated levels of Bcl-xL resist etposide-induced caspase-3 activation and Htt cleavage. These data indicate that IKKb regulates stress-induced Htt cleavage and is therefore a potential target for regulating Htt turnover.

Validation of IKKb as a therapeutic target for HD

            Ali Khoshnan, Jan Ko

            The role of the IkB-kinase complex (IKK) in neuronal survival and degeneration is not well understood.  In non-neuronal cells, IKK regulates the activity of the transcription factor NF-kB.  The core components of the IKK complex include two serine-threonine kinases, IKKa (IKK1) and IKKb (IKK2), and a regulatory, non-catalytic module, IKKg (NEMO).  IKKa and IKKg also have NF-kB independent functions.  We previously showed that binding of mutant Htt activates IKKb in neuronal and animal models of HD (Khoshnan et al., 2004).  Moreover, IKKb promotes stress-induced cleavage of full length Htt, which could result in build up of oligomeric neurotoxic fragments. Therefore, Htt cleavage and IKKb activation can form a positive feedback loop that could perpetuate neuronal degeneration.  We are testing the hypothesis that reduction of IKKb expression could ameliorate HD pathology.  To this end, we have deleted its expression in the CNS and are crossing these knockout mice with an HD mouse model.

Characterization of the neuroprotective functions of IKKa

Ali Khoshnan

Our recent studies indicate that IKKa expression in neurons promotes survival and imparts protection against DNA damage induced by genotoxic stress. Further investigations of IKKa in neurons have led to discovery that IKKa also promotes BDNF expression and enhances neurite sprouting. IKKa appears to regulate the activity of MeCP2, and CREB binding proteins, which are important modulators of BDNF expression.  We also find that IKKa regulates expression of microRNAs that have neuroprotective properties.  Studies are in progress to identify key regulatory targets of IKKa in neurons, dissect its role in BDNF expression, and understand how it promotes neuroprotection in stressed neurons.

Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity

            Amber L. Southwell, Ali Khoshnan, Paul H. Patterson

            While expanded polyQ repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected.  The polyQ expansion that causes Huntington’s disease (HD) is in the first exon (HDx1) of huntingtin (Htt).  However, other parts of the protein including the 17 N-terminal amino acids (AA) and two proline (polyP) repeat domains regulate the toxicity of mutant Htt.  The role of the P-rich domain that is flanked by the polyP domains has not been explored.  Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in the toxicity, aggregation, localization and turnover of HDx1. Three domains in the proline rich region (PRR) of HDx1 are defined by intrabodies: MW7 binds the two polyP domains, and Happs1 and 3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as VL12.3, which binds the N-terminal 17 AA, decrease the toxicity and aggregation of mutant Htt, but they do so by different mechanisms.  The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which VL12.3 does not change. In contrast, expression of VL12.3 increases the nuclear localization of Htt.  We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD.  We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells.

Preliminary in vivo studies in adult mice suggest that intrastriatal injection of a VL12.3-expressing adeno-associated virus (AAV) reduces aggregation of mHtt and ameliorates the reduction of neuronal size caused by injection of mHtt-lentivirus.  AAV-VL12.3 also improves the amphetamine-induced rotation bias seen with unilateral mHtt lentivirus injection.

 

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This page last updated August, 2008 by C. Patterson.