Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants

Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs) are promising candidates for this type of therapy, because they (1) have migratory properties, enabling migration after transplantation, (2) can differentiate into sensory neurons and glial cells, and (3) can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP). Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair

Impact of Age on the Cerebrovascular Proteomes of Wild-Type and Tg-SwDI Mice

The structural integrity of cerebral vessels is compromised during ageing. Abnormal amyloid (Aβ) deposition in the vasculature can accelerate age-related pathologies. The cerebrovascular response associated with ageing and microvascular Aβ deposition was defined using quantitative label-free shotgun proteomic analysis. Over 650 proteins were quantified in vessel-enriched fractions from the brains of 3 and 9 month-old wild-type (WT) and Tg-SwDI mice. Sixty-five proteins were significantly increased in older WT animals and included several basement membrane proteins (nidogen-1, basement membrane-specific heparan sulfate proteoglycan core protein, laminin subunit gamma-1 precursor and collagen alpha-2(IV) chain preproprotein). Twenty-four proteins were increased and twenty-one decreased in older Tg-SwDI mice. Of these, increases in Apolipoprotein E (APOE) and high temperature requirement serine protease-1 (HTRA1) and decreases in spliceosome and RNA-binding proteins were the most prominent. Only six shared proteins were altered in both 9-month old WT and Tg-SwDI animals. The age-related proteomic response in the cerebrovasculature was distinctly different in the presence of microvascular Aβ deposition. Proteins found differentially expressed within the WT and Tg-SwDI animals give greater insight to the mechanisms behind age-related cerebrovascular dysfunction and pathologies and may provide novel therapeutic targets

HtrA1-dependent proteolysis of TGF-beta controls both neuronal maturation and developmental survival

Transforming growth factor-β (TGF-β) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-β accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice overexpressing a TGF-β-responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-β signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-β1, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-β signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-β could be one of the critical events controlling both neuronal maturation and developmental survival

HtrA1-dependent proteolysis of TGF-beta controls both neuronal maturation and developmental survival

Transforming growth factor-β (TGF-β) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-β accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice overexpressing a TGF-β-responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-β signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-β1, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-β signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-β could be one of the critical events controlling both neuronal maturation and developmental survival

Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed

Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc