Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction

HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation

The peripheral immune response is altered in Huntington's disease, but the underlying mechanisms are unclear. Using RNA interference to lower huntingtin levels in leucocytes from patients, Träger et al. reverse disease-associated phenotypes including cytokine elevation and transcriptional dysregulation, and argue for a direct effect of mutant huntingtin on NFκΒ signallin

Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre‐clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non‐human or non‐neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra‐nuclear inclusions in a polyQ length‐dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction

HTT-lowering reverses Huntington's disease immune dysfunction caused by NF kappa B pathway dysregulation

Huntington’s disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington’s disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington’s disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function–the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington’s disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington’s disease

In vivo studies on the dopamine re-uptake mechanism in the striatum of the rat: effects of benztropine, sodium and ouabain

We used the push-pull perfusion technique to study the in vivo changes in dopamine (DA) levels in the rat striatum in response to treatments which could affect DA re-uptake into the nigrostriatal DA terminals. Benztropine (10-6 M), a potent DA uptake inhibitor induced a 1.7-fold increase in DA levels in the perfusates compared to basal levels. Perfusion with a Na+-free medium in which Na+ was replaced with either Tris-Cl or choline-Cl in equimolar proportions induced respectively 6.5- and 8.5-fold increases in DA levels in the perfusates. Perfusion of media containing NaCl:Tris-Cl (50:50) or NaCl:choline-Cl (50:50) did not significantly alter the levels of DA in the perfusates. Ouabain (10-6 M) did not significantly alter DA levels but at a concentration of 10-4 M, there was a 5.3-fold increase in DA levels in the perfusates compared to basal levels. These results thus demonstrate that the raised DA levels in the extracellular space in response to benztropine is due to the action of the drug in blocking the uptake of DA. The dependence of the uptake mechanism on the presence of Na+ in the external medium and hence on metabolic energy (Na pump) is clearly demonstrated. However, the massive elevation of DA levels under these conditions cannot be due solely to an inhibition of DA uptake but to the carrier-mediated DA exit from cytoplasmic stores resulting from a running down of the ionic gradient