Trinucleotide expansions cause disease by both protein-and RNAmediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.T ranslation of mRNA into protein is an exquisitely regulated, almost error-free process. Well-established rules of translational initiation have been used as a cornerstone in biology to understand gene expression and to predict the consequences of disease-causing mutations (1). For microsatellite expansion disorders, mutations within or outside ATG-initiated ORFs are thought to cause disease either by protein gain-of-function, protein loss-of-function, or RNA gain-of-function mechanisms (2, 3).Microsatellite expansion mutations that express polyglutamine (polyGln) expansion proteins include Huntington disease (Huntingtin, HD), spinal bulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17. Since the discovery of these CAG·CTG expansion mutations, efforts to understand disease mechanisms have focused on elucidating the molecular effects of the polyGln proteins expressed from these loci. Although these polyGln expansion proteins bear no similarity to each other apart from the polyGln tract, a hallmark of these diseases is protein accumulation and aggregation in nuclear or cytoplasmic inclusions. Surprisingly, although the polyGln expansion proteins are widely expressed in the CNS and other tissues, only restricted populations of neurons are vulnerable in each disease (3).Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are the best-characterized examples of RNA-mediated expansion disorders (2). The mutation causing DM1 is a CTG-repeat expansion located in the 3′ UTR of the dystrophia myotonica-protein kinase (DMPK) gene. Although DM1 can be clinically more severe than DM2, the discovery of the DM2 mutation and several mouse models provide strong support that many features of these diseases result from RNA gain-of-function effects in which the dysregulation of RNA-binding proteins is mediated by the expression of CUG and CCUG transcripts (4). Additionally, RNA gain-of-function effects have been reported for CGG and CAG expansion RNAs (5, 6).Both RNA and protein mechanisms appear to operate...

Umbilical cord blood (UCB) is a rich source of hematopoetic stem cells (HSCs). We have isolated a novel cell line population of stem cells from human UCB that exhibit properties of self-renewal, but do not have cell-surface markers that are typically found on HSCs. Analysis of transcripts revealed that these cells express transcription factors Oct-4, Rex-1, and Sox-2 that are typically expressed by stem cells. We refer to these novel cells as nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs). Previous studies have shown that the intravenous infusion of UCBCs can ameliorate neurological deficits arising from ischemic brain injury. The identity of the cells that mediate this restorative effect, however, has yet to be determined. We postulate that nh-UCBSCs may be a source of the UCB cells that can mediate these effects. To test this hypothesis, we intravenously injected one million human nh-UCBSCs into rats 48 h after transient unilateral middle cerebral artery occlusion. Animals in other experimental groups received either saline injections or injections of RN33b neural stem cells. Animals were tested for neurological function before the infusion of nh-UCBSCs and at various time periods afterwards using a battery of behavioral tests. In limb placement tests, animals treated with nh-UCBSCs exhibited mean scores that were significantly better than animals treated with RN33b neural stem cells or saline. Similarly, in stepping tests, nh-UCBSC-treated animals again exhibited significantly better performance than the other experimental groups of animals. Analysis of infarct volume revealed that ischemic animals treated with nh-UCBSCs exhibited a 50% reduction in lesion volume in comparison to saline-treated controls. Histological analysis of brain tissue further revealed the presence of cells that stained for human nuclei. Some human nuclei-positive cells were also co-labeled for NeuN, indicating that the transplanted cells expressed markers of a neuronal phenotype. Cells expressing the human nuclei marker within the brain, however, were rather scant, suggesting that the restorative effects of nh-UCBSCs may be mediated by mechanisms other than cell replacement. To test this hypothesis, nh-UCBSCs were directly transplanted into the brain parenchyma after ischemic brain injury. Sprouting of nerve fibers from the nondamaged hemisphere into the ischemically damaged side of the brain was assessed by anterograde tracing using biotinylated dextran amine (BDA). Animals with nh-UCBSC transplants exhibited significantly greater densities of BDA-positive cells in the damaged side of the brain compared to animals with intraparenchymal saline injections. These results suggest that restorative effects observed with nh-UCBSC treatment following ischemic brain injury may be mediated by trophic actions that result in the reorganization of host nerve fiber connections within the injured brain.

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Intracerebral hemorrhage (ICH) is a devastating acute neurological disorder, without effective treatment, in which a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we evaluated whether TUDCA can reduce brain injury and improve neurological function after ICH in rats. Administration of TUDCA before or up to 6 h after stereotaxic collagenase injection into the striatum reduced lesion volumes at 2 days by as much as 50%. Apoptosis was Ϸ50% decreased in the area immediately surrounding the hematoma and was associated with a similar inhibition of caspase activity. These changes were also associated with improved neurobehavioral deficits as assessed by rotational asymmetry, limb placement, and stepping ability. Furthermore, TUDCA treatment modulated expression of certain Bcl-2 family members, as well as NF-B activity. In addition to its protective action at the mitochondrial membrane, TUDCA also activated the Akt-1͞protein kinase B␣ survival pathway and induced Bad phosphorylation at Ser-136. In conclusion, reduction of brain injury underlies the wide-range neuroprotective effects of TUDCA after ICH. Thus, given its clinical safety, TUDCA may provide a potentially useful treatment in patients with hemorrhagic stroke and perhaps other acute brain injuries associated with cell death by apoptosis.

Umbilical cord blood is a rich source of hematopoietic stem cells. It is routinely used for transplantation to repopulate cells of the immune system. Recent studies, however, have demonstrated that intravenous infusions of umbilical cord blood can ameliorate neurologic deficits associated with ischemic brain injury in rodents. Moreover, the infused cells penetrate into the parenchyma of the brain and adopt phenotypic characteristics typical of neural cells. In the present study we tested the hypothesis that the administration of umbilical cord blood can also diminish neurologic deficits caused by intracerebral hemorrhage (ICH). Intracerebral hemorrhage is a major cause of morbidity and mortality, and at the present time there are no adequate therapies that can minimize the consequences of this cerebrovascular event. ICH was induced in rats by intrastriatal injections of collagenase to cause bleeding in the striatum. Twenty-four hours after the induction of ICH rats received intravenous saphenous vein infusions of human umbilical cord blood (2.4 x 10(6) to 3.2 to 10(6) cells). Animals were evaluated using a battery of tests at day 1 after ICH, but before the administration of umbilical cord blood, and at days 7, and 14 after ICH (days 6 and 13, respectively, after cord blood administration). These tests included a neurological severity test, a stepping test, and an elevated body-swing test. Animals with umbilical cord blood infusions exhibited significant improvements in (1) the neurologic severity test at 6 and 13 days after cord blood infusion in comparison to saline-treated animals (P < 0.05); (2) the stepping test at day 6 (P < 0.05); and (3) the elevated body-swing test at day 13 (P< 0.05). These results demonstrate that the administration of human umbilical cord blood cells can ameliorate neurologic deficits associated with intracerebral hemorrhage.