Cahir J. O'Kane

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease.

Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.

Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and the ensuing loss of neurotransmitter exocytosis has implicated synaptobrevin in this process. To further the study of synaptic function in a genetically tractable organism and to generate a tool to disable neuronal communication for behavioural studies, we have expressed a gene encoding tetanus toxin light chain in Drosophila. Toxin expression in embryonic neurons removes detectable synaptobrevin and eliminates evoked, but not spontaneous, synaptic vesicle release. No other developmental or morphological defects are detected. Correspondingly, only synaptobrevin (n-syb), but not the ubiquitously expressed syb protein, is cleaved by tetanus toxin in vitro. Targeted expression of toxin can produce specific behavioral defects; in one case, the olfactory escape response is reduced.

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntingtons disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntingtons disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.

Associative Learning Disrupted by Impaired Gs Signaling in Drosophila Mushroom Bodies

Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3′,5′ monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein α subunit (Gαs*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Gαs* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Gαs* did not affect gross MB morphology, and wild-type Gαs expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.

α-Synuclein impairs macroautophagy: implications for Parkinson's disease.

α-Synuclein impairs autophagosome formation and mislocalizes Atg9 by inhibiting Rab1a.

Dynein mutations impair autophagic clearance of aggregate-prone proteins.

Mutations that affect the dynein motor machinery are sufficient to cause motor neuron disease. It is not known why there are aggregates or inclusions in affected tissues in mice with such mutations and in most forms of human motor neuron disease. Here we identify a new mechanism of inclusion formation by showing that decreased dynein function impairs autophagic clearance of aggregate-prone proteins. We show that mutations of the dynein machinery enhanced the toxicity of the mutation that causes Huntington disease in fly and mouse models. Furthermore, loss of dynein function resulted in premature aggregate formation by mutant huntingtin and increased levels of the autophagosome marker LC3-II in both cell culture and mouse models, compatible with impaired autophagosome-lysosome fusion.

Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila.

In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.

Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease

Huntington disease (HD) is caused by a polyglutamine-expansion mutation in huntingtin (HTT) that makes the protein toxic and aggregate-prone. The subcellular localisation of huntingtin and many of its interactors suggest a role in endocytosis, and recently it has been shown that huntingtin interacts indirectly with the early endosomal protein Rab5 through HAP40. Here we show that Rab5 inhibition enhanced polyglutamine toxicity, whereas Rab5 overexpression attenuated toxicity in our cell and fly models of HD. We tried to identify a mechanism for the Rab5 effects in our HD model systems, and our data suggest that Rab5 acts at an early stage of autophagosome formation in a macromolecular complex that contains beclin 1 (BECN1) and Vps34. Interestingly chemical or genetic inhibition of endocytosis also impeded macroautophagy, and enhanced aggregation and toxicity of mutant huntingtin. However, in contrast to Rab5, inhibition of endocytosis by various means suppressed autophagosome-lysosome fusion (the final step in the macroautophagy pathway) similar to bafilomycin A1. Thus, Rab5, which has previously been thought to be exclusively involved in endocytosis, has a new role in macroautophagy. We have previously shown that macroautophagy is an important clearance route for several aggregate-prone proteins including mutant huntingtin. Thus, better understanding of Rab5-regulated autophagy might lead to rational therapeutic targets for HD and other protein-conformation diseases.

The Partner of Inscuteable/Discs-Large Complex Is Required to Establish Planar Polarity during Asymmetric Cell Division in Drosophila

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Hereditary spastic paraplegias: membrane traffic and the motor pathway

Voluntary movement is a fundamental way in which animals respond to, and interact with, their environment. In mammals, the main CNS pathway controlling voluntary movement is the corticospinal tract, which encompasses connections between the cerebral motor cortex and the spinal cord. Hereditary spastic paraplegias (HSPs) are a group of genetic disorders that lead to a length-dependent, distal axonopathy of fibres of the corticospinal tract, causing lower limb spasticity and weakness. Recent work aimed at elucidating the molecular cell biology underlying the HSPs has revealed the importance of basic cellular processes — especially membrane trafficking and organelle morphogenesis and distribution — in axonal maintenance and degeneration.