Proteolytic cleavage of ataxin-7 promotes SCA7 retinal degeneration and neurological dysfunction.

The neurodegenerative disorder spinocerebellar ataxia type 7 (SCA7) is caused by a polyglutamine (polyQ) expansion in the ataxin-7 protein, categorizing SCA7 as one member of a large class of heritable neurodegenerative proteinopathies. Cleavage of ataxin-7 by the protease caspase-7 has been demonstrated in vitro, and the accumulation of proteolytic cleavage products in SCA7 patients and mouse models has been identified as an early pathological change. However, it remains unknown whether a causal relationship exists between ataxin-7 proteolysis and in vivo SCA7 disease progression. To determine whether caspase cleavage is a critical event in SCA7 disease pathogenesis, we generated transgenic mice expressing polyQ-expanded ataxin-7 with a second-site mutation (D266N) to prevent caspase-7 proteolysis. When we compared SCA7-D266N mice with SCA7 mice lacking the D266N mutation, we found that SCA7-D266N mice exhibited improved motor performance, reduced neurodegeneration and substantial lifespan extension. Our findings indicate that proteolysis at the D266 caspase-7 cleavage site is an important mediator of ataxin-7 neurotoxicity, suggesting that inhibition of caspase-7 cleavage of polyQ-ataxin-7 may be a promising therapeutic strategy for this untreatable disorder.

Dimethyl sulfoxide and dimethyl formamide increase lifespan of C. elegans in liquid.

Lifespan extension through pharmacological intervention may provide valuable tools to understanding the mechanisms of aging and could uncover new therapeutic approaches for the treatment of age-related disease. Although the nematode Caenorhabditis elegans is well known as a particularly suitable model for genetic manipulations, it has been recently used in a number of pharmacological studies searching for compounds with anti-aging activity. These compound screens are regularly performed in amphipathic solvents like dimethyl sulfoxide (DMSO), the solvent of choice for high-throughput drug screening experiments performed throughout the world. In this work, we report that exposing C. elegans to DMSO in liquid extends lifespan up to 20%. Interestingly, another popular amphipathic solvent, dimethyl formamide (DMF), produces a robust 50% increase in lifespan. These compounds work through a mechanism independent of insulin-like signaling and dietary restriction (DR). Additionally, the mechanism does not involve an increased resistance to free radicals or heat shock suggesting that stress resistance does not play a major role in the lifespan extension elicited by these compounds. Interestingly, we found that DMSO and DMF are able to decrease the paralysis associated with amyloid-ß3-42 aggregation, suggesting a role of protein homeostasis for the mechanism elicited by these molecules to increase lifespan.

Posttranslational modification of ataxin-7 at lysine 257 prevents autophagy-mediated turnover of an N-terminal caspase-7 cleavage fragment.

Polyglutamine (polyQ) expansion within the ataxin-7 protein, a member of the STAGA [SPT3-TAF(II)31-GCN5L acetylase] and TFTC (GCN5 and TRRAP) chromatin remodeling complexes, causes the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7). Proteolytic processing of ataxin-7 by caspase-7 generates N-terminal toxic polyQ-containing fragments that accumulate with disease progression and play an important role in SCA7 pathogenesis. To elucidate the basis for the toxicity of these fragments, we evaluated which posttranslational modifications of the N-terminal fragment of ataxin-7 modulate turnover and toxicity. Here, we show that mutating lysine 257 (K257), an amino acid adjacent to the caspase-7 cleavage site of ataxin-7 regulates turnover of the truncation product in a repeat-dependent manner. Modification of ataxin-7 K257 by acetylation promotes accumulation of the fragment, while unmodified ataxin-7 is degraded. The degradation of the caspase-7 cleavage product is mediated by macroautophagy in cell culture and primary neuron models of SCA7. Consistent with this, the fragment colocalizes with autophagic vesicle markers, and enhanced fragment accumulation increases in these lysosomal structures. We suggest that the levels of fragment accumulation within the cell is a key event in SCA7 neurodegeneration, and enhancing clearance of polyQ-containing fragments may be an effective target to reduce neurotoxicity in SCA7.

Calpain-1 cleaves and activates caspase-7.

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V(max) when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca(2+) dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.

Proteolytic cleavage of ataxin-7 by caspase-7 modulates cellular toxicity and transcriptional dysregulation.

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis.

Developmental shift in the apostat: comparison of neurones and astrocytes.

The intrinsic pathway of apoptosis was investigated in cell-free extracts of neurones and astrocytes at various stages of maturation. Neuronal extracts were activated 65-fold after 3 days, 9-fold after 7 days, and were not activated after 10 days in culture. In contrast, astrocyte extracts were activated to a similar extent at all stages, up to 60 days in culture. The co-incubation of neuronal and astrocyte extracts followed by addition of cytochrome c/2'-deoxyadenosine 5'-triphosphate led to a 40-fold activation, suggesting that the development-associated neuronal shift does not involve the appearance of a dominant inhibitor, but rather downregulation of some key component(s) involved in caspase activation.

Alternative, nonapoptotic programmed cell death: mediation by arrestin 2, ERK2, and Nur77.

Programmed cell death (pcd) may take the form of apoptosis or of nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here we report that alternative, nonapoptotic pcd induced by the neurokinin-1 receptor (NK(1)R) activated by its ligand Substance P, is mediated by a MAPK phosphorylation cascade recruited by the scaffold protein arrestin 2. The activation of the protein kinases Raf-1, MEK2, and ERK2 is essential for this form of nonapoptotic pcd, leading to the phosphorylation of the orphan nuclear receptor Nur77. NK(1)R-mediated cell death was inhibited by a dominant negative form of arrestin 2, Raf-1, or Nur77, by MEK1/2-specific inhibitors, and by RNA interference directed against ERK2 or MEK2 but not ERK1 or MEK1 and against Nur77. The MAPK pathway is also activated in neurons in primary culture undergoing NK(1)R-mediated death, since the MEK inhibitor PD98059 inhibited Substance P-induced death in primary striatal neurons. These results suggest that Nur77, which is regulated by a MAPK pathway activated via arrestin 2, modulates NK(1)R-mediated nonapoptotic pcd.