Labeling of P15 WT anddtTg4DRGs for TUNEL shows an increase in TUNEL reactivity indtTg4DRGs relative to WT (Supplemental Figure S1). (ER) in sensory neurons corresponding to in vivo induction of ER stress proteins. ER stress subsequently leads to sensory neurodegeneration NVP-BHG712 through induction of a proapoptotic caspase cascade.dtsensory neurons display neurodegenerative pathologies, including Ca2+dyshomeostasis, unfolded protein response (UPR) induction, caspase activation, and apoptosis. Isoform-specific loss-of-function analysis attributes these neurodegenerative pathologies to specific loss of dystonin-a2. Inhibition of either UPR or caspase signaling promotes the viability of cells deficient in dystonin. This study provides insight into the mechanism ofdtneuropathology and proposes a role for dystonin-a2 as AF1 a mediator of normal ER structure and function. == INTRODUCTION == The eukaryotic cytoskeleton comprises three cytoskeletal networks, including microtubules, actin microfilaments, and intermediate filaments. These filaments rely on cytoskeletal cross-linking proteins to facilitate their multifaceted functions, ranging from vesicular transport and maintenance of organelle integrity to mitosis (Fuchs and Karakesisoglou, 2001). As such, loss of cytoskeletal-linking proteins can have grave implications on cellular functioning and viability (Sonnenberg and Liem, 2007). In particular, loss of the neuronal cytoskeletal linking protein dystonin has been attributed to degeneration of both sensory and motor neurons (Brownet al., 1995;Guoet al., 1995;De Repentignyet al., 2011). Dystonia musculorum (dt) is an inherited homozygous recessive sensory neuropathy caused by mutations in the dystonin gene (Dst;Duchenet al., 1964). At approximately 2 wk postnatal development,dtmice exhibit loss of control of the fore limbs, hind limbs, and trunk, and they die shortly thereafter of unknown causes (Duchen, 1976). Severaldtalleles exist through spontaneous mutations (dt27), chemically induced mutations, targeted alleles (dttm1Efu), and transgenic insertions (dtTg4;Poolet al., 2005). Although only threedtmutations (dtTg4, dttm1Efu, anddtAlb) have been characterized at the DNA level,dtTg4anddt27are allelic and do not complement (Kotharyet al., 1988;Guoet al., 1995). To date,dtpathologies have been recorded in motor neurons, skeletal muscle, NVP-BHG712 and Schwann cells, but degeneration is most prominent in the sensory neurons of the dorsal root ganglia (DRG;Dowlinget al., 1997;Bernieret al., 1998;Dalpeet al., 1999;De Repentignyet al., 2011). TheDstgene is exceptionally large (400 kb in mice) and gives rise to three tissue-specific dystonin isoformsdystonin-e (epithelial isoform, 315 kDa), dystonin-b (muscle isoform, 834 kDa), and dystonin-a (neuronal isoform, 615 kDa;Sawamuraet al., 1991;Brownet al., 1995;Leunget al., 2001;Okumuraet al., 2002). Whereas dystonin-e serves as an autoantigen in the skin blistering disease bullous pemphigoid, loss of function of the dystonin-a isoform or isoforms is causal in thedtdisorder (Kotharyet al., 1988;Sawamuraet al., 1991;Brownet al., 1995;Poolet al., 2005). Like muscle, three major neuronal isoforms are derived through alternative splicing, namely dystonin-a1, dystonin-a2, and dystonin-a3 (Young and Kothary, 2007). These isoforms share an N-terminal actin-binding domain, an extensive coiled-coil region, and a C-terminal microtubule-binding domain, allowing for interactions with cytoskeletal filaments (Leunget al., 2001). Although the dystonin-a isoforms share similar domain architecture, it is the unique N-terminal regions that NVP-BHG712 differentiate them and dictate their subcellular localization. Specifically, dystonin-a1 encodes a short N-terminal domain that includes an actin-binding domain, whereas dystonin-a2 possesses a transmembrane domain and dystonin-a3 possesses a putative myristoylation domain, aiding their membrane localization to the nuclear envelope and perinuclear membranes and to the plasma membrane, respectively (Jeffersonet al., 2006;Younget al., 2006). Recently it has been demonstrated that the lack of one copy of theDstgene or the presence of truncated forms of the mutant protein in humans can lead to a partial disorganization of the sensory and motor circuits. Giorda and collaborators described a human subject with a translocation in theDstgene that specifically disrupted neuron- and muscle-specific isoforms and was associated with a profound delay in the acquisition of cognitive and motor skills, as well as in visual maturation (Giordaet al., 2004). Thus, although no mutations inDsthave been identified in humans, haploinsufficiency or altered expression can result in phenotypic abnormalities reminiscent of the inherited disorder in mice. A common cellular pathology precedingdtsensory neuron degeneration is axonal swelling, characterized by the accumulation of organelles and vacuoles withindtsensory neurons (Janota, 1972). These accumulations are accompanied by disorganized neurofilament, actin and microtubule networks indtsensory neurons (Yanget al., 1996,1999;Leunget al., 1999a), which in turn lead to bidirectional impairment of fast axonal transport (Dalpeet al., 1998;De Repentignyet al., 2003). Although neurofilament defects do exist indtmice, they are not causal in initiation ofdtpathology (Bernier and Kothary, 1998;Eyeret al., 1998). Moreover, the microtubule cytoskeleton ofdtand wild-type (WT) sensory neurons grown in culture displays no ultrastructural defects, and local trafficking of mitochondrial organelles proceeds normally (Poolet al., 2006). Nonetheless, enhanced apoptosis is observed in these cultures (Poolet al., 2006). Consequently, it is plausible that abnormalities aside from axonal cytoskeleton defects may initiate or contribute todtsensory neuron pathology. Some studies suggest that the dystonin-a2 isoform is involved in nuclear envelope structuring, nuclear tethering, and organization of membranous structures surrounding the nucleus (Younget al., 2003,2006;Young and Kothary, 2008). Indeed,dtTg4sensory neurons (devoid of dystonin-a1 and dystonin-a2 expression) display perikaryal defects and show altered expression of the microsomal enzyme protein disulfide isomers (Young.
