Background: The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (Wld S ) mutant mice.

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The slow Wallerian degeneration phenotype, Wld(S), which delays Wallerian degeneration and axon pathology for several weeks, has so far been studied only in mice. A rat model would have several advantages. First, rats model some human disorders better than mice. Second, the larger body size of rats facilitates more complex surgical manipulations. Third, rats provide a greater yield of tissue for primary culture and biochemical investigations. We generated transgenic Wld(S) rats expressing the Ube4b/Nmnat1 chimeric gene in the central and peripheral nervous system. As in Wld(S) mice, their axons survive up to 3 weeks after transection and remain functional for at least 1 week. Protection of axotomized nerve terminals is stronger than in mice, particularly in one line, where 95-100% of neuromuscular junctions remained intact and functional after 5 days. Furthermore, the loss of synaptic phenotype with age was much less in rats than in mice. Thus, the slow Wallerian degeneration phenotype can be transferred to another mammalian species and synapses may be more effectively preserved after axotomy in species with longer axons.

Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders.

Slow Wallerian degeneration (Wld S ) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD؉ synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld S targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld S lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD ؉ synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld S protein influence the intranuclear location of both ubiquitin proteasome and NAD ؉ synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP. INTRODUCTIONThe E4 ubiquitination factor Ube4b (or Ufd2a) has a 123-amino acid N-terminal region that is essential for ubiquitination activity (Mahoney et al., 2002). It is unclear why this region is essential because it does not contain the U box, and it appears to be absent in invertebrate orthologues that ubiquitinate effectively (Koegl et al., 1999;Hatakeyama et al., 2001;Mahoney et al., 2002;Hoppe et al., 2004;Richly et al., 2005). It is important to understand the molecular mechanism of Ube4b because it has a key role in the ubiquitin proteasome system (UPS; Hoppe, 2005), it is neuroprotective in polyglutamine disorders and an important candidate gene for neuroblastoma (Krona et al., 2003). Information on the substrates of Ube4b is beginning to emerge (Hoppe et al., 2004;Okumura et al., 2004;Spinette et al., 2004;Richly et al., 2005) but there is much still to learn about its regulation.In the slow Wallerian degeneration mutant mouse (Wld S ), 70 amino acids of this essential domain of Ube4b form the N-terminus of a chimeric protein that delays Wallerian degeneration of injured axons in mice and rats by 10-fold (see Figure 1A; Lunn et al., 1989;Mack et al., 2001;Adalbert et al., 2005). The chimeric protein is absent in wild-type mice. This sequence (N70) is fused in Wld S protein to the full coding sequence of nicotinamide mononucleotide adenylyltransferase (Nmnat1; Conforti et al., 2000;Emanuelli et al., 2001;Mack et al., 2001) in regulating axon degeneration. Wld S also delays axon degeneration in a wide range of neurodegenerative disorders and acute retrograde axonal degeneration after spinal injury, indicating that axon degeneration mechanisms are more closely related th...

Scaffold-mediated tissue engineering has become a golden solution for the regeneration of damaged bone tissues that lack self-regeneration capability. A successful scaffold in bone tissue engineering comprises a multitude of suitable biological, microarchitectural, and mechanical properties acting as different signaling cues for the cells to mediate the new tissue formation. Therefore, careful design of bioactive scaffold macro-and microstructures in multiple length scales and biophysical properties fulfilling the tissue repair demands are necessary yet challenging to achieve. Herein, we have developed an antibacterial and biocompatible silica-silk fibroin (SF) gel-based ink through novel yet simple chemical approaches of sol−gel and self-assembly followed by processing the obtained gels as three-dimensional (3D) hybrid aerogel-based scaffolds exploiting the advanced materials design approaches of micro-extrusion-based 3D printing, and directional freeze-casting/drying approaches. As the main constituent of the hybrid biocompatible scaffold of this study, we used the SF extracted from Bombyx mori silkworm cocoon. However, to increase the cell responsivity and bactericidal efficiency of the final scaffold, thiol-ended antimicrobial and cell adhesive peptide sequence (SH-CM-RGD) was conjugated to silica-SF hybrid gels via covalent attachment using a spacer molecule through either preprint (prior to sol−gel) or during the post-printing steps on the previously printed silica-SF gel. In the next step, the hybrid Silica-SF-CM-RGD hydrogel ink was 3D-printed into the construct with interconnected porous structure with hierarchically organized porosity and a combination of several promising properties. Namely, due to the covalent linkage of the antibacterial peptide to the SF, the scaffold shows potent bactericidal efficiency toward Gram-positive and Gram-negative bacteria. Moreover, nanostructured silica components in the 3D-printed composites could intertwine with SF-CM-RGD to support the mechanical properties in the final scaffold and the final osteoconductivity of the scaffold. This study supports the promising properties of 3D-printed silica-SF-based hybrid aerogels constructs for repairing bone defect.

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Although the crucial role of professional phagocytes for the clearance of S. aureus infections is well-established, several studies indicate an adverse role of leukocytes in the dissemination of S. aureus during infection. Since only little is known about macrophages in this context, we analyzed the role of macrophages, and in particular reactive oxygen species deficiency, for the seeding of S. aureus metastases. Infection of bone marrow-derived macrophages (BMDM) with S. aureus revealed that NADPH oxidase 2 (NOX2-) deficient, but not NOX1- or NOX4-deficient, BMDM failed to clear intracellular S. aureus. Despite of larger intracellular bacterial burden, NOX2-deficient BMDM showed significantly improved survival. Intravenous injection of mice with in vitro-infected BMDMs carrying intracellular viable S. aureus led to higher bacterial loads in kidney and liver of mice compared to injection with plain S. aureus. An even higher frequency of liver abscesses was observed in mice infected with S. aureus-loaded nox2−/− BMDM. Thus, the improved intracellular survival of S. aureus and improved viability of NOX2-deficient BMDM is associated with an aggravated metastatic dissemination of S. aureus infection. A combination of vancomycin and the intracellularly active antibiotic rifampicin led to complete elimination of S. aureus from liver within 48 h, which was not achieved with vancomycin treatment alone, underscoring the impact of intracellular S. aureus on the course of disease. The results of our study indicate that intracellular S. aureus carried by macrophages are sufficient to establish a systemic infection. This suggests the inclusion of intracellularly active antibiotics in the therapeutic regimen of invasive S. aureus infections, especially in patients with NADPH oxidase deficiencies such as chronic granulomatous disease.