Ubiquitin-specific protease-14 reduces cellular aggregates and protects against mutant huntingtin-induced cell degeneration: involvement of the proteasome and ER stress-activated kinase IRE1α.

Huntington's disease (HD) is an autosomal inherited neurological disease caused by a CAG-repeat expansion in the first exon of huntingtin gene encoding for the huntingtin protein (Htt). In HD, there is an accumulation of intracellular aggregates of mutant Htt that negatively influence cellular functions. The aggregates contain ubiquitin, and part of the HD pathophysiology could result from an imbalance in cellular ubiquitin levels. Deubiquitinating enzymes are important for replenishing the ubiquitin pool, but less is known about their roles in brain diseases. We show here that overexpression of the ubiquitin-specific protease-14 (Usp14) reduces cellular aggregates in mutant Htt-expressing cells mainly via the ubiquitin proteasome system. We also observed that the serine-threonine kinase IRE1 involved in endoplasmic reticulum (ER) stress responses is activated in mutant Htt-expressing cells in culture as well as in the striatum of mutant Htt transgenic (BACHD) mice. Usp14 interacted with IRE1 in control cells but less in mutant Htt-expressing cells. Overexpression of Usp14 in turn was able to inhibit phosphorylation of IRE1α in mutant Htt-overexpressing cells and to protect against cell degeneration and caspase-3 activation. These results show that ER stress-mediated IRE1 activation is part of mutant Htt toxicity and that this is counteracted by Usp14 expression. Usp14 effectively reduced cellular aggregates and counteracted cell degeneration indicating an important role of this protein in mutant Htt-induced cell toxicity.

Autocrine endocannabinoid signaling through CB1 receptors potentiates OX1 orexin receptor signaling.

It has been proposed that OX(1) orexin receptors and CB(1) cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX(1) receptors and potentiate OX(1) receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX(1) receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX(1)-CB(1) receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX(1) receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB(1) receptor-expressing cells. When OX(1) and CB(1) receptors were expressed in the same cells, OX(1) stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX(1) response as such was fully abolished by specific inhibition of CB(1) receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX(1)-CB(1) heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX(1)-CB(1) synergism and thus suggest a functional rather than a molecular interaction of OX(1) and CB(1) receptors.

GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway.

Increased protein aggregation and altered cell signaling accompany many neurodegenerative diseases including Huntington's disease (HD). Cell stress is counterbalanced by signals mediating cell repair but the identity of these are not fully understood. We show here that the mammalian target of rapamycin (mTOR) pathway is inhibited and cytoprotective autophagy is activated in neuronal PC6.3 cells at 24 h after expression of mutant huntingtin proteins. Tuberous sclerosis complex (TSC) 1/2 interacted with growth arrest and DNA damage protein 34 (GADD34), which caused TSC2 dephosphorylation and induction of autophagy in mutant huntingtin expressing cells. However, GADD34 and autophagy decreased at later time points, after 48 h of transfection with the concomitant increase in mTOR activity. Overexpression of GADD34 counteracted these effects and increased cytoprotective autophagy and cell survival. These results show that GADD34 plays an important role in cell protection in mutant huntingtin expressing cells. Modulation of GADD34 and the TSC pathway may prove useful in counteracting cell degeneration accompanying HD and other neurodegenerative diseases.

Downregulation of NF-kappaB signaling by mutant huntingtin proteins induces oxidative stress and cell death.

Accumulation of abnormal proteins and endoplasmic reticulum stress accompany neurodegenerative diseases including Huntington's disease. We show that the expression of mutant huntingtin proteins with extended polyglutamine repeats differentially affected endoplasmic reticulum signaling cascades linked to the inositol-requiring enzyme-1 (IRE1) pathway. Thus, the p38 and c-Jun N-terminal kinase pathways were activated, while the levels of the nuclear factor-kappaB-p65 (NF-kappaB-p65) protein decreased. Downregulation of NF-kappaB signaling was linked to decreased antioxidant levels, increased oxidative stress, and enhanced cell death. Concomitantly, calpain was activated, and treatment with calpain inhibitors restored NF-kappaB-p65 levels and increased cell viability. The calpain regulator, calpastatin, was low in cells expressing mutant huntingtin, and overexpression of calpastatin counteracted the deleterious effects caused by N-terminal mutant huntingtin proteins. These results show that calpastatin and an altered NF-kappaB-p65 signaling are crucial factors involved in oxidative stress and cell death mediated by mutant huntingtin proteins.

Endoplasmic reticulum stress inhibition protects against excitotoxic neuronal injury in the rat brain.

Elevated brain glutamate with activation of neuronal glutamate receptors accompanies neurological disorders, such as epilepsy and brain trauma. However, the mechanisms by which excitotoxicity triggers neuronal injury are not fully understood. We have studied the glutamate receptor agonist kainic acid (KA) inducing seizures and excitotoxic cell death. KA caused the disintegration of the endoplasmic reticulum (ER) membrane in hippocampal neurons and ER stress with the activation of the ER proteins Bip, Chop, and caspase-12. Salubrinal, inhibiting eIF2alpha (eukaryotic translation initiation factor 2 subunit alpha) dephosphorylation, significantly reduced KA-induced ER stress and neuronal death in vivo and in vitro. KA-induced rise in intracellular calcium was not affected by Salubrinal. The results show that ER responses are essential parts of excitotoxicity mediated by glutamate receptor activation and that Salubrinal decreases neuronal death in vivo. Inhibition of ER stress by small molecular compounds may be beneficial for treatment of various neuronal injuries and brain disorders.

Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins.

Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.