α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, includingParkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

The ataxia (ax J ) mutation is a spontaneous recessive mutation that results in reduced expression of ubiquitinspecific protease 14, Usp14. Mice homozygous for the ax J mutation are retarded for growth and exhibit several behavioral disorders, including a resting tremor and hindlimb paralysis. Although pathological defects appear to be limited to the central nervous system, reduction of Usp14 expression was widespread in the ax J mice. Usp14 co-fractionated with proteasomes isolated from livers and brains of wildtype mice. Proteasomes isolated from the ax J brains still possessed deubiquitinating activity and were functionally competent to hydrolyze 20S proteasomal substrates in vitro. However, the levels of monomeric ubiquitin were reduced approximately 35% in most of the ax J tissues examined.These results indicate that Usp14 functions to maintain the cellular levels of monomeric ubiquitin in mammalian cells, and that alterations in the levels of ubiquitin may contribute to neurological disease. Keywords: ataxia, mutation, neurological, proteasome, ubiquitin, ubiquitin-specific protease 14. . These disorders are associated with changes in protein degradation that are manifested through the production of protein aggregates. However, it is unclear whether these alterations in the UPS are the primary cause or a secondary consequence of these disorders. Our recent studies on ataxia (ax J ) mice indicate that primary alterations in the UPS can lead to neurological dysfunction (Wilson et al. 2002). The ax J mutation is a spontaneously arising recessive neurological mutation (D'Amato and Hicks 1965). The ax J mice suffer from progressive motor system abnormalities that first appear as a resting tremor when the mice are 2-3 weeks old. At 4 weeks of age, ax J mice exhibit severe hind muscle wasting and ataxia. These mice are completely immobile by 6 weeks of age and death occurs at about 8 weeks of age. The neurological phenotypes observed in the ax J mice are the result of a genetic lesion in the gene encoding the deubiquitinating enzyme Usp14 (Wilson et al. 2002). The insertion of an intracisternal A-particle into intron 5 of Usp14 reduces the expression of Usp14 in the ax J mice to only 5-10% of the levels found in wild-type mice, indicating that the ax J mutation represents a hypomorphic allele of Usp14. A better understanding of the deficiencies in the ax J mice, and in other mice that harbor mutations in the UPS, should therefore provide valuable insights into how the UPS functions in the nervous system and how alterations in these pathways lead to disease.

The ataxia mutation (ax J ) is a recessive neurological mutation that results in reduced growth, ataxia, and hindlimb muscle wasting in mice. The ax J gene encodes ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme (DUB) that associates with the proteasome via its ubiquitin-like (Ubl) domain and is involved in processing ubiquitin chains. Analysis of Usp14 gene products demonstrated that Usp14 undergoes alternative pre-mRNA splicing to produce a full-length form of Usp14 that is capable of binding proteasomes and a form that contains a deletion in the Ubl domain. The full-length form of Usp14 is the only form that appears to be reduced in the ax J mice. Transgenic rescue of the ax J mice with neuronal-specific expression of Usp14 demonstrated that the full-length form of Usp14 was sufficient to restore viability and motor system function to the ax J mice. Biochemical analysis showed that the ubiquitin hydrolyase activity of this form of Usp14 is dependent on the presence of proteasomes, and neuronal expression of full-length Usp14 was able to restore the levels of monomeric ubiquitin in the brains of ax J mice. However, the ax J -rescued mice still displayed the Purkinje cell axonal swellings that are seen in the ax J mice, indicating that this cerebellar alteration is not the primary cause of the ax J movement disorders. These results show that the motor defects observed in the ax J mice are attributable to a neuropathic disease rather than to a muscular disorder and suggest that changes in proteasomal function may contribute to neurological dysfunction in the ax J mice.

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Homozygous ataxia (axJ) mice have reduced expression of ubiquitin-specific protease 14 (Usp14), resulting in severe neuromuscular defects and death by 2 months of age. Transgenic expression of Usp14 exclusively in the nervous system of axJ mice (axJ-Tg) prevents early lethality and restores motor system function to the axJ mice, enabling an analysis of the reproductive capabilities of Usp14-deficient mice. Although female axJ-Tg mice had a 75% reduction of Usp14 in the ovaries, they were able to produce normal litters. Ovary transfer experiments also demonstrated that the ovaries of axJ mice were capable of producing viable pups. In contrast, male axJ and axJ-Tg mice displayed a 50% reduction in testicular Usp14 levels and were infertile, indicating that Usp14 is required for development and function of the male reproductive system. Immunohistochemistry experiments showed that Usp14 is found in the redundant nuclear envelope and cytoplasmic droplet of epididymal spermatozoa. Analysis of axJ testes demonstrated a 50% reduction in testis weight, a 100-fold reduction in sperm number and the presence of abnormal spermatozoa in the epididymis. Histological examination of the Usp14-deficient testes revealed abnormal spermatogenesis and the presence of degenerating germ cells, indicating that Usp14 and the ubiquitin proteasome system are required for spermatid differentiation during spermiogenesis.

Background Reports show that stressful events before injury exacerbates post-injury pain. The mechanism underlying stress-induced heightened thermal pain is unclear. Here, we examined the effects of chronic intermittent stress (CIS) on nociceptive behaviors and brain-derived nerve growth factor (BDNF) system in the prefrontal cortex (PFC) and hypothalamus of rats with and without thermal injury. Results Unstressed rats showed transient mechanical allodynia during stress exposure. Stressed rats with thermal injury displayed persistent exacerbated mechanical allodynia ( P < 0.001). Increased expression of BDNF mRNA in the PFC ( P < 0.05), and elevated TrkB and p-TrkB ( P < 0.05) protein levels in the hypothalamus were observed in stressed rats with thermal injury but not in stressed or thermally injured rats alone. Furthermore, administration of CTX-B significantly reduced stress-induced exacerbated mechanical allodynia in thermally injured rats ( P < 0.001). Conclusion These results indicate that BDNF-TrkB signaling in PFC and hypothalamus contributes to CIS-induced exacerbated mechanical allodynia in thermal injury state. Electronic supplementary material The online version of this article (10.1186/s12868-019-0500-1) contains supplementary material, which is available to authorized users.

BackgroundGastroparesis is a significant co-morbidity affecting up to 50% of patients with diabetes and is disproportionately found in women. Prior studies have suggested that loss of interstitial cells of Cajal, hyperglycemia, and nitric oxide dysfunction are potential causes of gastroparesis. Since diabetic gastroparesis affects more women than men, we performed an exploratory study with a diabetic rat model to determine if sex hormone signaling is altered in those where gastroparesis develops.MethodsWe injected male rats with streptozotocin (STZ) to model type I diabetes, as confirmed by blood glucose levels. Gastroparesis was determined by acetaminophen gavage and serum acetaminophen levels. Rats were grouped based on acetaminophen and blood glucose data: diabetic (DM), diabetic and gastroparetic (DM + GP), and control (CM). Serum levels of testosterone, estrogen, and insulin were determined as well as aromatase expression in pyloric tissue and serum. Androgen receptor and estrogen receptor α (ERα) and β (ERβ) were also measured in the pylorus.ResultsCompared to CM, estrogen increased and testosterone decreased in both DM and DM + GP rats. Sex hormone levels were not different between DM and DM + GP. Serum aromatase was increased in DM and DM + GP rats; however, pyloric tissue levels were not significantly different from controls. ERα was unchanged and androgen receptor decreased in DM and DM + GP. ERβ was increased only in DM + GP animals.ConclusionOur study implicates increased pyloric ERβ in the development of gastroparesis in STZ-induced male diabetic rats. Increased serum aromatase is likely responsible for altered sex hormone levels. Our study supports the implication of sex hormone signaling in diabetic development and demonstrates a potential unique role for pyloric ERβ in male diabetic gastroparesis.

Ketamine is the recommended analgesic on the battlefield for Soldiers with hemorrhage, despite a lack of supportive evidence from laboratory or clinical studies. Hence, this study determined the effects of ketamine analgesia on cardiorespiratory responses and survival to moderate (37% blood volume; n=8/group) or severe hemorrhage (50% blood volume; n=10/group) after trauma in rats. We used a conscious hemorrhage model with extremity trauma (fibular fracture + soft tissue injury) while measuring mean arterial pressure (MAP), heart rate (HR), and body temperature (Tb) by telemetry, and respiration rate (RR), minute volume (MV), and tidal volume (TV) via whole body plethysmography . Male rats received saline (S) or 5.0 mg/kg ketamine (K) (100 µl/100 gram body weight) intra-arterially after trauma and hemorrhage. All rats survived 37% hemorrhage. For 50% hemorrhage, neither survival times (180 min (SD 78) vs 209 min (SD 66) nor percent survival (60% vs 80%) differed between S and K-treated rats. After 37% hemorrhage, K (compared with S) increased MAP, and decreased Tb and MV. After 50% hemorrhage, K (compared with S) increased MAP but decreased HR and MV. K effects on cardiorespiratory function were time-dependent, significant but modest, and transient at the analgesic dose given. K effects on Tb were also significant but modest, and more prolonged. Using this rat model, our data support the use of K as an analgesic in injured, hypovolemic patients.