DJ-1 can form ß-sheet structured aggregates that co-localize with pathological amyloid deposits.

The loss of native function of the DJ-1 protein has been linked to the development of Parkinson's (PD) and other neurodegenerative diseases. Here we show that DJ-1 aggregates into ß-sheet structured soluble and fibrillar aggregates in vitro under physiological conditions and that this process is promoted by the oxidation of its catalytic Cys106 residue. This aggregation resulted in the loss of its native biochemical glyoxalase function and in addition oxidized DJ-1 aggregates were observed to localize within Lewy bodies, neurofibrillary tangles and amyloid plaques in human PD and Alzheimer's (AD) patients' post-mortem brain tissue. These findings suggest that the aggregation of DJ-1 may be a critical player in the development of the pathology of PD and AD and demonstrate that loss of DJ-1 function can happen through DJ-1 aggregation. This could then contribute to AD and PD disease onset and progression.

Deregulation of the protocadherin gene FAT1 alters muscle shapes: implications for the pathogenesis of facioscapulohumeral dystrophy.

Generation of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD.

Plasticity versus specificity in RTK signalling modalities for distinct biological outcomes in motor neurons.

BACKGROUND: Multiple growth factors are known to control several aspects of neuronal biology, consecutively acting as morphogens to diversify neuronal fates, as guidance cues for axonal growth, and as modulators of survival or death to regulate neuronal numbers. The multiplicity of neuronal types is permitted by the combinatorial usage of growth factor receptors, each of which is expressed in distinct and overlapping subsets of neurons, and by the multitasking role of growth factor receptors, which recruit multiple signalling cascades differentially required for distinct biological outcomes. We have explored signalling robustness in cells where a given receptor tyrosine kinase (RTK) elicits qualitatively distinct outcomes. As the HGF/Met system regulates several biological responses in motor neurons (MN) during neuromuscular development, we have investigated the signalling modalities through which the HGF/Met system impacts on MN biology, and the degree of robustness of each of these functions, when challenged with substitutions of signalling pathways. RESULTS: Using a set of mouse lines carrying signalling mutations that change the Met phosphotyrosine binding preferences, we have asked whether distinct functions of Met in several MN subtypes require specific signalling pathways, and to which extent signalling plasticity allows a pleiotropic system to exert distinct developmental outcomes. The differential ability of signalling mutants to promote muscle migration versus axonal growth allowed us to uncouple an indirect effect of HGF/Met signalling on nerve growth through the regulation of muscle size from a direct regulation of motor growth via the PI3 kinase (PI3K), but not Src kinase, pathway. Furthermore, we found that HGF/Met-triggered expansion of Pea3 expression domain in the spinal cord can be accomplished through several alternative signalling cascades, differentially sensitive to the Pea3 dosage. Finally, we show that the regulation of MN survival by HGF/Met can equally be achieved in vitro and in vivo by alternative signalling cascades involving either PI3K-Akt or Src and Mek pathways. CONCLUSIONS: Our findings distinguish MN survival and fate specification, as RTK-triggered responses allowing substitutions of the downstream signalling routes, from nerve growth patterning, which depends on a selective, non-substitutable pathway.

Generation of diverse neuronal subtypes in cloned populations of stem-like cells.

BACKGROUND: The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material. RESULTS: In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons. CONCLUSION: The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification.

Astroglia genesis in vitro: distinct effects of retinoic acid in different phases of neural stem cell differentiation.

In the developing CNS, the manifestation of the macro-glial phenotypes is delayed behind the formation of neurons. The "neurons first--glia second" principle seems to be valid for neural tissue differentiation throughout the neuraxis, but the reasons behind are far from clear. In the presented study, the mechanisms of this timing were investigated in vitro, in the course of the neural differentiation of one cell derived NE-4C neuroectodermal stem and P19 embryonic carcinoma cells. The data demonstrated that astrocyte formation coincided in time with the maturation of postmitotic neurons, but the close vicinity of neurons did not initiate astrocyte formation before schedule. All-trans retinoic acid, a well-known inducer of neuronal differentiation, on the other hand, blocked effectively the astroglia production if present in defined stages of the in vitro neuroectodermal cell differentiation. According to the data, retinoic acid plays at least a dual role in astrogliogenesis: while it is needed for committing neural progenitors for a future production of astrocytes, it prevents premature astrogliogenesis by inhibiting the differentiation of primed glial progenitors.

Changes of KCl sensitivity of proliferating neural progenitors during in vitro neurogenesis.

The effects of KCl-treatment on the survival and proliferation of NE-4C self-renewing neural progenitor cells were investigated during early phases of in vitro induced neurogenesis. NE-4C cells, derived from the anterior brain vesicles of embryonic mouse (E9), divided continuously under non-inducing conditions, but acquired neuronal features within 6 days, if induced by all-trans retinoic acid (RA). During the first 2 days of induction, the cells went on proliferating and did not show signs of morphological differentiation. In this stage, the resting membrane potential of RA-induced cells adopted more negative values in comparison to non-induced ones. Despite the increased membrane polarity and K+ conductance, addition of 20-50 mM KCl failed to elicit inward Na+ currents and did not induce an increase in the intracellular Ca+ level. Long-term treatment with 25 mM KCl, on the other hand, resulted in a selective loss of cells committed to neuronal fate by both decreasing the rate of cell proliferation and increasing the rate of cell death. The data indicate that the viability and proliferation of neural progenitors are influenced by extracellular K+-level in a differentiation stage-dependent manner.