Defective dentate nucleus GABA receptors in essential tremor.

The development of new treatments for essential tremor, the most frequent movement disorder, is limited by a poor understanding of its pathophysiology and the relative paucity of clinicopathological studies. Here, we report a post-mortem decrease in GABA(A) (35% reduction) and GABA(B) (22-31% reduction) receptors in the dentate nucleus of the cerebellum from individuals with essential tremor, compared with controls or individuals with Parkinson's disease, as assessed by receptor-binding autoradiography. Concentrations of GABA(B) receptors in the dentate nucleus were inversely correlated with the duration of essential tremor symptoms (r(2) = 0.44, P < 0.05), suggesting that the loss of GABA(B) receptors follows the progression of the disease. In situ hybridization experiments also revealed a diminution of GABA(B(1a+b)) receptor messenger RNA in essential tremor (↓27%). In contrast, no significant changes of GABA(A) and GABA(B) receptors (protein and messenger RNA), GluN2B receptors, cytochrome oxidase-1 or GABA concentrations were detected in molecular or granular layers of the cerebellar cortex. It is proposed that a decrease in GABA receptors in the dentate nucleus results in disinhibition of cerebellar pacemaker output activity, propagating along the cerebello-thalamo-cortical pathways to generate tremors. Correction of such defective cerebellar GABAergic drive could have a therapeutic effect in essential tremor.

Transforming growth factor: beta signaling is essential for limb regeneration in axolotls.

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-beta). In the present study, the full length sequence of the axolotl TGF-beta1 cDNA was isolated. The spatio-temporal expression pattern of TGF-beta1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-beta signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-beta type I receptor, SB-431542, we show that TGF-beta signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-beta signaling are down-regulated. These data directly implicate TGF-beta signaling in the initiation and control of the regeneration process in axolotls.

The axolotl limb: a model for bone development, regeneration and fracture healing.

Among vertebrates, urodele amphibians (e.g., axolotls) have the unique ability to perfectly regenerate complex body parts after amputation. The limb has been the most widely studied due to the presence of three defined axes and its ease of manipulation. Hence, the limb has been chosen as a model to study the process of skeletogenesis during axolotl development, regeneration and to analyze this animal's ability to heal bone fractures. Extensive studies have allowed researchers to gain some knowledge of the mechanisms controlling growth and pattern formation in regenerating and developing limbs, offering an insight into how vertebrates are able to regenerate tissues. In this study, we report the cloning and characterization of two axolotl genes; Cbfa-1, a transcription factor that controls the remodeling of cartilage into bone and PTHrP, known for its involvement in the differentiation and maturation of chondrocytes. Whole-mount in situ hybridization and immunohistochemistry results show that Cbfa-1, PTHrP and type II collagen are expressed during limb development and regeneration. These genes are expressed during specific stages of limb development and regeneration which are consistent with the appearance of skeletal elements. The expression pattern for Cbfa-1 in late limb development was similar to the expression pattern found in the late stages of limb regeneration (i.e. re-development phase) and it did not overlap with the expression of type II collagen. It has been reported that the molecular mechanisms involved in the re-development phase of limb regeneration are a recapitulation of those used in developing limbs; therefore the detection of Cbfa-1 expression during regeneration supports this assertion. Conversely, PTHrP expression pattern was different during limb development and regeneration, by its intensity and by the localization of the signal. Finally, despite its unsurpassed abilities to regenerate, we tested whether the axolotl was able to regenerate non-union bone fractures. We show that while the axolotl is able to heal a non-stabilized union fracture, like other vertebrates, it is incapable of healing a bone gap of critical dimension. These results suggest that the axolotl does not use the regeneration process to repair bone fractures.

Biochemical characterization of Abeta and tau pathologies in mild cognitive impairment and Alzheimer's disease.

We report a post mortem biochemical analysis of amyloid-beta (Abeta) (ELISA) and tau (Western immunoblots) in the temporo-parietal neocortex of subjects with a clinical diagnosis of mild cognitive impairment (MCI, n=12), Alzheimer's disease (AD, n=12) or no cognitive impairment (NCI, n=12). Levels of Abeta _{42} in the detergent-insoluble protein fractions were significantly higher in persons with AD but did not differentiate individuals with MCI. Conversion of tau into its insoluble form (soluble/insoluble tau ratio) or into paired helical filament tau (PHF_{tau}) were the biochemical variables most closely related to clinical and neuropathological diagnoses, but they did not distinguished MCI from the two other groups. Interestingly, soluble/insoluble total tau ratio, PHF_{tau} and insoluble Abeta_{42} concentrations in the cortex correlated strongly with global cognition scores proximate to death and with immunohistochemical and histological quantification of Abeta and tau pathologies. Our data suggest that 1) insoluble Abeta _{42} and insoluble tau (total or PHF_{tau}) show a significant relationship with the clinical and neuropathological diagnosis of AD; 2) Although MCI appears to represent an intermediate stage between NCI and AD, the quantification of cortical Abeta and tau pathologies did not significantly distinguish subjects with MCI from either group.

Expression of heat-shock protein 70 during limb development and regeneration in the axolotl.

Urodele amphibians (e.g., axolotls) have the unique ability, among vertebrates, to regenerate perfectly many parts of their body after amputation. The limb has been the most widely studied regenerating structure in these organisms and provides an ideal model in which to understand how vertebrates can regenerate complex tissues. The present study focuses on Hsp-70, a member of the stress-related heat-shock protein family. This protein is normally induced after a stress or trauma such as heat-shock, ultraviolet irradiation, or wounding. Thus, studying its expression during axolotl limb regeneration, a response to an important traumatic event (amputation), is of great interest to further understand how the regenerative process is mediated. Using molecular biology and biochemical techniques, we have characterized both the spatiotemporal and quantitative expression patterns of Hsp-70 in axolotl development and regeneration. Our results show that Hsp-70 is expressed and regulated during axolotl development as in other vertebrates. Our data also demonstrate an up-regulation of the RNA transcript for Hsp-70 during limb regeneration as early as 24 hr after amputation that is maintained up to early differentiation. We also demonstrate a similar pattern of expression for the protein during regeneration. Finally, we show that axolotl Hsp-70 is induced threefold after heat-shock as observed in other vertebrates.