The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice.

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.

The Wlds mutation delays robust loss of motor and sensory axons in a genetic model for myelin-related axonopathy.

Mice deficient in the peripheral myelin component P0 mimic severe forms of inherited peripheral neuropathies in humans, with defective myelin formation and consequent axonal loss. We cross-bred these mice with the spontaneous mutant C57BL/Wld(s) typically showing protection from Wallerian degeneration because of fusion of the ubiquitination factor E4B (Ube4b) and nicotinamide mononucleotide adenylyltransferase (Nmnat) genes. We found that in the double mutants, the robust myelin-related axonal loss is reduced at 6 weeks and 3 months of age. Moreover, retrograde labeling from plantar nerves revealed an increased survival of motor axons. These motor axons appeared functionally active because both the amplitude of compound muscle action potentials and muscle strength were less reduced in the double mutants. At 6 months of age, reduction of axonal loss was no longer detectable in the double mutants when compared with littermates carrying the P0 null mutation only, although the Wld(s) gene was not reduced in its expression at this age. We conclude that myelin-related axonal loss is a process having some features in common with Wallerian degeneration. Introducing the Wld(s) gene would be a promising approach to delaying detrimental axonal loss in myelin disorders.

Genotyping methods to detect a unique neuroprotective factor (Wld(s)) for axons.

The slow Wallerian degeneration mouse, C57BL/Wld(s), carries a dominant mutation that delays Wallerian degeneration in the distal stump of an injured axon. The protective gene has been identified and also found to protect axons from the neurotoxin vincristine. Therefore, it is important to determine whether it has a widespread application to protect axons in neurological disease. In principle, this can be done by crossing Wld(s) to neurological mutant mice, but first a method is needed to track the inheritance of the neuroprotective Wld(s) allele. Due to the complex nature of the mutation, there is no simple method to distinguish Wld(s) homozygotes, heterozygotes and wild-type mice. Therefore, we report a genotyping method for Wld(s) based upon pulsed field gel electrophoresis (PFGE) and compare it with the alternatives of PCR and Southern blotting. The effect of the Wld(s) mutation on axon degeneration in diverse inherited pathologies, and the consequence for symptoms, can now be investigated.

Stable inheritance of an 85-kb triplication in C57BL/WldS mice.

The slow Wallerian degeneration mouse, C57BL/Wld(S), carries a dominant mutation that delays Wallerian degeneration in the distal stump of an injured axon. A highly unusual mutation, an 85-kb tandem triplication in the Wld(S) mouse was identified. Since two duplication cases have been identified before, pulsed field gel electrophoresis (PFGE) can be used to look for the instability of triplication at the chromosomal level. One hundred and eighty chromosomes of Wld(S) from three divergent breeding colonies have been examined and all found to carry the triplication. Thus, the triplication mutation is stable during both mitosis and meiosis, and the previously observed duplication is likely to have been surviving alleles of the original mutation rather than a partial reversion. The triplication has now been shown to be the causative mutation, acting through an Ube4b/Nmnat chimeric gene, indicating the possibility of Wld(S) preventing axon degeneration in diverse pathologies and altering the symptoms. The fact that triplication is stable rules out instability as a source of phenotypic variation. Thus, this result is essential for accurate interpretation of studies the effect of Wld(S) on neurodegenerative phenotypes.

Nutritional approaches in the risk reduction and management of Alzheimer's disease.

Alzheimer's disease (AD) is a heterogeneous and devastating neurodegenerative disease with increasing socioeconomic burden for society. In the past 30 y, notwithstanding advances in the understanding of the pathogenesis of the disease and consequent development of therapeutic approaches to novel pathogenic targets, no cure has so far emerged. This contribution focuses on recent nutritional approaches in the risk reduction and management of AD with emphasis on factors providing a rationale for nutritional approaches in AD, including compromised nutritional status, altered nutrient uptake and metabolism, and nutrient requirements for synapse formation. Collectively these factors are believed to result in specific nutritional requirement in AD. The chapter also emphasizes investigated nutritional interventions in patients with AD, including studies with single nutrients and with the specific nutrient combination Fortasyn Connect and discusses the current shift of paradigm to intervene in earlier stages of AD, which offers opportunities for investigating nutritional strategies to reduce the risk for disease progression. Fortasyn Connect was designed to enhance synapse formation and function in AD by addressing the putative specific nutritional requirements and contains docosahexaenoic acid, eicosapentaenoic acid, uridine-5'-mono-phosphate, choline, phospholipids, antioxidants, and B vitamins. Two randomized controlled trials (RCTs) with the medical food Souvenaid, containing Fortasyn Connect, showed that this intervention improved memory performance in mild, drug-naïve patients with AD. Electroencephalography outcome in one of these clinical studies suggests that Souvenaid has an effect on brain functional connectivity, which is a derivative of changed synaptic activity. Thus, these studies suggest that nutritional requirements in AD can be successfully addressed and result in improvements in behavioral and neuro-physiological alterations that are characteristic to AD. The recent advance of methodologies and techniques for early diagnosis of AD facilitates the investigation of strategies to reduce the risk for AD progression in the earliest stages of the disease. Nutrition-based approaches deserve further investigation as an integral part of such strategies due to their low risk for side effects and their potential to affect pathological processes of very early AD.

Protective mechanisms by cystatin C in neurodegenerative diseases.

Neurodegeneration occurs in acute pathological conditions such as stroke, ischemia, and head trauma and in chronic disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. While the cause of neuronal death is different and not always known in these varied conditions, hindrance of cell death would be beneficial in the prevention of, slowing of, or halting disease progression. Enhanced cystatin C (CysC) expression in these conditions caused a debate as to whether CysC up-regulation facilitates neurodegeneration or it is an endogenous neuroprotective attempt to prevent the progression of the pathology. However, recent in vitro and in vivo data have demonstrated that CysC plays protective roles via pathways that are dependent on inhibition of cysteine proteases, such as cathepsin B, or by induction of autophagy, induction of proliferation, and inhibition of amyloid-beta aggregation. Here we review the data demonstrating the protective roles of CysC under conditions of neuronal challenge and the protective pathways induced under various conditions. These data suggest that CysC is a therapeutic candidate that can potentially prevent brain damage and neurodegeneration.

Cystatin C protects neuronal cells from amyloid-beta-induced toxicity.

Multiple studies suggest that cystatin C (CysC) has a role in Alzheimer's disease (AD) and a decrease in CysC secretion is linked to the disease in patients with a polymorphism in the CysC gene. CysC binds amyloid-beta (Abeta) and inhibits formation of Abeta fibrils and oligomers both in vitro and in mouse models of amyloid deposition. Here we studied the effect of CysC on cultured primary hippocampal neurons and a neuronal cell line exposed to either oligomeric or fibrillar cytotoxic forms of Abeta. The extracellular addition of the secreted human CysC together with preformed either oligomeric or fibrillar Abeta increased cell survival. While CysC inhibits Abeta aggregation, it does not dissolve preformed Abeta fibrils or oligomers. Thus, CysC has multiple protective effects in AD, by preventing the formation of the toxic forms of Abeta and by direct protection of neuronal cells from Abeta toxicity. Therapeutic manipulation of CysC levels, resulting in slightly higher concentrations than physiological could protect neuronal cells from cell death in AD.

Complexes of amyloid-beta and cystatin C in the human central nervous system.

A role for cystatin C (CysC) in the pathogenesis of Alzheimer's disease (AD) has been suggested by the genetic linkage of a CysC gene (CST3) polymorphism with late-onset AD, the co-localization of CysC with amyloid-beta (Abeta) in AD brains, and binding of CysC to soluble Abeta in vitro and in mouse models of AD. This study investigates the binding between Abeta and CysC in the human central nervous system. While CysC binding to soluble Abeta was observed in AD patients and controls, a SDS-resistant CysC/Abeta complex was detected exclusively in brains of neuropathologically normal controls, but not in AD cases. The association of CysC with Abeta in brain from control individuals and in cerebrospinal fluid reveals an interaction of these two polypeptides in their soluble form. The association between Abeta and CysC prevented Abeta accumulation and fibrillogenesis in experimental systems, arguing that CysC plays a protective role in the pathogenesis of AD in humans and explains why decreases in CysC concentration caused by the CST3 polymorphism or by specific presenilin 2 mutations can lead to the development of the disease. Thus, enhancing CysC expression or modulating CysC binding to Abeta have important disease-modifying effects, suggesting a novel therapeutic intervention for AD.

Cystatin C inhibits amyloid-beta deposition in Alzheimer's disease mouse models.

Using transgenic mice expressing human cystatin C (encoded by CST3), we show that cystatin C binds soluble amyloid-beta peptide and inhibits cerebral amyloid deposition in amyloid-beta precursor protein (APP) transgenic mice. Cystatin C expression twice that of the endogenous mouse cystatin C was sufficient to substantially diminish amyloid-beta deposition. Thus, cystatin C has a protective role in Alzheimer's disease pathogenesis, and modulation of cystatin C concentrations may have therapeutic implications for the disease.