CED-4 CARD domain residues can modulate non-apoptotic neuronal regeneration functions independently from apoptosis.

A major challenge in regenerative medicine is the repair of injured neurons. Regeneration of laser-cut C. elegans neurons requires early action of core apoptosis activator CED-4/Apaf1 and CED-3/caspase. While testing models for CED-4 as a candidate calcium-sensitive activator of repair, we unexpectedly discovered that amino acid substitutions affecting alpha-helix-6 within the CED-4 caspase recruitment domain (CARD) confer a CED-4 gain-of-function (gf) activity that increases axonal regrowth without disrupting CED-4 apoptosis activity. The in vivo caspase reporter CA-GFP reveals a rapid localized increase in caspase activity upon axotomy, which is absent in ced-4 and ced-3 loss-of-function mutants but present in the ced-4(gf) mutant. The ced-3 loss-of-function mutation can significantly suppress the axonal regrowth of the ced-4(gf) mutant, indicating that CED-4(gf) regeneration depends on CED-3 caspase. Thus, we identified a subdomain within the CED-4 CARD that regulates the dynamic and controlled caspase activity required for efficient regeneration.

Neuronal repair: Apoptotic proteins make good.

The potential of the central nervous system (CNS) to regenerate is regulated by a complex interaction of neuronal intrinsic and extrinsic factors that remain poorly understood. Significant research has been dedicated to identifying these factors to facilitate design of therapies that will treat the functional impairment associated with CNS injuries. Over the last decade, the development of in vivo laser severing of single axons in C. elegans has established an invaluable model for the genetic identification of novel regeneration factors. In a recent study we report the unexpected identification of the core apoptotic proteins CED-4/Apaf-1 and the executioner caspase CED-3 as important factors that promote early events in regeneration in C. elegans. Other upstream regulators of apoptosis do not influence regeneration, indicating the existence of a novel mechanism for activation of CED-4 and CED-3 in neuronal repair. CED-4 and CED-3 function downstream of injury-induced calcium transients and appear to act through the conserved DLK-1 pathway to promote regeneration. We propose a working model for calcium-dependent localized activation of CED-4 and CED-3 caspase and discuss questions raised including mechanisms for spatially regulating activated CED-3 and the possible substrates that it might cleave to initiate regeneration.

Basal and stress-induced Hsp70 are modulated by ataxin-3.

Regulation of basal and induced levels of hsp70 is critical for cellular homeostasis. Ataxin-3 is a deubiquitinase with several cellular functions including transcriptional regulation and maintenance of protein homeostasis. While investigating potential roles of ataxin-3 in response to cellular stress, it appeared that ataxin-3 regulated hsp70. Basal levels of hsp70 were lower in ataxin-3 knockout (KO) mouse brain from 2 to 63 weeks of age and hsp70 was also lower in fibroblasts from ataxin-3 KO mice. Transfecting KO cells with ataxin-3 rescued basal levels of hsp70 protein. Western blots of representative chaperones including hsp110, hsp90, hsp70, hsc70, hsp60, hsp40/hdj2, and hsp25 indicated that only hsp70 was appreciably altered in KO fibroblasts and KO mouse brain. Turnover of hsp70 protein was similar in wild-type (WT) and KO cells; however, basal hsp70 promoter reporter activity was decreased in ataxin-3 KO cells. Transfecting ataxin-3 restored hsp70 basal promoter activity in KO fibroblasts to levels of promoter activity in WT cells; however, mutations that inactivated deubiquitinase activity or the ubiquitin interacting motifs did not restore full activity to hsp70 basal promoter activity. Hsp70 protein and promoter activity were higher in WT compared to KO cells exposed to heat shock and azetidine-2-carboxylic acid, but WT and KO cells had similar levels in response to cadmium. Heat shock factor-1 had decreased levels and increased turnover in ataxin-3 KO fibroblasts. Data in this study are consistent with ataxin-3 regulating basal level of hsp70 as well as modulating hsp70 in response to a subset of cellular stresses.

The core apoptotic executioner proteins CED-3 and CED-4 promote initiation of neuronal regeneration in Caenorhabditis elegans.

A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms.

Proteotoxic stress increases nuclear localization of ataxin-3.

Spinocerebellar ataxia type 3 (SCA3)/Machado Joseph disease results from expansion of the polyglutamine domain in ataxin-3 (Atx3). Atx3 is a transcriptional co-repressor, as well as a deubiquitinating enzyme that appears to function in cellular pathways involved in protein homeostasis. In this study, we show that interactions of Atx3 with valosin-containing protein and hHR23B are dynamic and modulated by proteotoxic stresses. Heat shock, a general proteotoxic stress, also induced wild-type and pathogenic Atx3 to accumulate in the nucleus. Mapping studies showed that two regions of Atx3, the Josephin domain and the C-terminus, regulated heat shock-induced nuclear localization. Heat shock-induced nuclear localization of Atx3 was not affected by a casein kinase-2 inhibitor or by mutating a predicted nuclear localization signal. However, serine-111 of Atx3 was required for nuclear localization of the Josephin domain and regulated nuclear localization of full-length Atx3. Atx3 null cells were more sensitive to toxic effects of heat shock suggesting that Atx3 had a protective function in the cellular response to heat shock. Importantly, we found that oxidative stress also induced nuclear localization of Atx3; both wild-type and pathogenic Atx3 accumulated in the nucleus of SCA3 patient fibroblasts following oxidative stress. Heat shock and oxidative stress are the first processes identified that increase nuclear localization of Atx3. Observations in this study provide new and important insights for understanding SCA3 pathology as the nucleus is likely a key site for early pathogenesis.