Cm Fernandez-Martos

Differential Expression of Wnts after Spinal Cord Contusion Injury in Adult Rats

Background Spinal cord injury is a major cause of disability that has no clinically accepted treatment. Functional decline following spinal cord injury is caused by mechanical damage, secondary cell death, reactive gliosis and a poor regenerative capacity of damaged axons. Wnt proteins are a family of secreted glycoproteins that play key roles in different developmental processes although little is known of the expression patterns and functions of Wnts in the adult central nervous system in normal or diseased states. Findings Using qRT-PCR analysis, we demonstrate that mRNA encoding most Wnt ligands and soluble inhibitors are constitutively expressed in the healthy adult spinal cord. Strikingly, contusion spinal cord injury induced a time-dependent increase in Wnt mRNA expression from 6 hours until 28 days post-injury, and a narrow peak in the expression of soluble Wnt inhibitors between 1 and 3 days post-injury. These results are consistent with the increase in the migration shift, from day 1 to 7, of the intracellular Wnt signalling component, Dishevelled-3. Moreover, after an initial decrease by 1 day, we also found an increase in phosphorylation of the Wnt co-receptor, low-density lipoprotein receptor-related protein 6, and an increase in active β-catenin protein, both of which suffer a dramatic change, from a homogeneous expression pattern in the grey matter to a disorganized injury-induced pattern. Conclusions Our results suggest a role for Wnts in spinal cord homeostasis and injury. We demonstrate that after injury Wnt signalling is activated via the Wnt/β-catenin and possibly other pathways. These findings provide an important foundation to further address the function of individual Wnt proteins in vivo and the pathophysiology of spinal cord injury.

Acute leptin treatment enhances functional recovery after spinal cord injury.

Background Spinal cord injury is a major cause of long-term disability and has no current clinically accepted treatment. Leptin, an adipocyte-derived hormone, is best known as a regulator of food intake and energy expenditure. Interestingly, several studies have demonstrated that leptin has significant effects on proliferation and cell survival in different neuropathologies. Here, we sought to evaluate the role of leptin after spinal cord injury. Findings Based on its proposed neuroprotective role, we have evaluated the effects of a single, acute intraparenchymal injection of leptin in a clinically relevant animal model of spinal cord injury. As determined by quantitative Real Time-PCR, endogenous leptin and the long isoform of the leptin receptor genes show time-dependent variations in their expression in the healthy and injured adult spinal cord. Immunohistochemical analysis of post-injury tissue showed the long isoform of the leptin receptor expression in oligodendrocytes and, to a lesser extent, in astrocytes, microglia/macrophages and neurons. Moreover, leptin administered after spinal cord injury increased the expression of neuroprotective genes, reduced caspase-3 activity and decreased the expression of pro-inflammatory molecules. In addition, histological analysis performed at the completion of the study showed that leptin treatment reduced microglial reactivity and increased caudal myelin preservation, but it did not modulate astroglial reactivity. Consequently, leptin improved the recovery of sensory and locomotor functioning. Conclusions Our data suggest that leptin has a prominent neuroprotective and anti-inflammatory role in spinal cord damage and highlights leptin as a promising therapeutic agent.

Gene expression of axon growth promoting factors in the deer antler.

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deers normal and pedicle skin.

Spatio-Temporal Expression Pattern of Frizzled Receptors after Contusive Spinal Cord Injury in Adult Rats

Background Wnt proteins are a large family of molecules that are critically involved in multiple central nervous system (CNS) developmental processes. Experimental evidences suggest a role for this family of proteins in many CNS disorders, including spinal cord injury (SCI), which is a major neuropathology owing to its high prevalence and chronic sensorimotor functional sequelae. Interestingly, most Wnt proteins and their inhibitors are expressed in the uninjured spinal cord, and their temporal expression patterns are dramatically altered after injury. However, little is known regarding the expression of their better-known receptors, the Frizzled family, after SCI. Thus, the aim of the present study was to evaluate the expression of Frizzled receptors in the damaged spinal cord. Findings Based on the evidence that Wnts are expressed in the spinal cord and are transcriptionally regulated by SCI in adulthood, we analysed the spatio-temporal mRNA and protein expression patterns of Frizzled receptors after contusive SCI using quantitative RT-PCR and single and double immunohistochemistry, respectively. Our results show that almost all of the 10 known Frizzled receptors were expressed in specific spatial patterns in the uninjured spinal cords. Moreover, the Frizzled mRNAs and proteins were expressed after SCI, although their expression patterns were altered during the temporal progression of SCI. Finally, analysis of cellular Frizzled 5 expression pattern by double immunohistochemistry showed that, in the uninjured spinal cord, this receptor was expressed in neurons, oligodendrocytes, astrocytes, microglia and NG2+ glial precursors. After injury, Frizzled 5 was not only still expressed in oligodendrocytes, astrocytes and NG2+ glial precursors but also in axons at all evaluated time points. Moreover, Frizzled 5 was expressed in reactive microglia/macrophages from 3 to 14 days post-injury. Conclusions Our data suggest the involvement of Frizzled receptors in physiological spinal cord function and in the cellular and molecular events that characterise its neuropathology.

Neurofilament light gene deletion exacerbates amyloid, dystrophic neurite, and synaptic pathology in the APP/PS1 transgenic model of Alzheimer's disease.

Alzheimers disease (AD) is a progressive neurodegenerative disease associated with the loss of cognitive function. Neurofilament (NF) triplet proteins, the major structural (intermediate filament) proteins of neurons, are expressed in a subset of pyramidal cells that show a high degree of vulnerability to degeneration in AD. Alterations in the NF triplet proteins in amyloid-beta (Aβ) plaque-associated dystrophic neurites (DNs) represent the first cytoskeletal aberration to occur in the neocortex in the earliest stages of AD. We generated transgenic APP/PS1 (APPswe/PSEN1dE9) mice on the neurofilament light knockout (NFL KO) background to explore the role of NFL deletion in the context of DN formation, synaptic changes, and other neuropathologic features. Our analysis demonstrated that NFL deficiency significantly increased neocortical DN pathology, Aβ deposition, synapse vulnerability, and microgliosis in APP/PS1 mice. Thus, NFs may have a role in protecting neurites from dystrophy and in regulating cellular pathways related to the generation of Aβ plaques.

Mid-life environmental enrichment increases synaptic density in CA1 in a mouse model of Aβ-associated pathology and positively influences synaptic and cognitive health in healthy ageing

Early‐life cognitive enrichment may reduce the risk of experiencing cognitive deterioration and dementia in later‐life. However, an intervention to prevent or delay dementia is likely to be taken up in mid to later‐life. Hence, we investigated the effects of environmental enrichment in wildtype mice and in a mouse model of Aβ neuropathology (APPSWE/PS1dE9) from 6 months of age. After 6 months of housing in standard laboratory cages, APPSWE/PS1dE9 (n = 27) and healthy wildtype (n = 21) mice were randomly assigned to either enriched or standard housing. At 12 months of age, wildtype mice showed altered synaptic protein levels and relatively superior cognitive performance afforded by environmental enrichment. Environmental enrichment was not associated with alterations to Aβ plaque pathology in the neocortex or hippocampus of APPSWE/PS1dE9 mice. However, a significant increase in synaptophysin immunolabeled puncta in the hippocampal subregion, CA1, in APPSWE/PS1dE9 mice was detected, with no significant synaptic density changes observed in CA3, or the Fr2 region of the prefrontal cortex. Moreover, a significant increase in hippocampal BDNF was detected in APPSWE/PS1dE9 mice exposed to EE, however, no changes were detected in neocortex or between Wt animals. These results demonstrate that mid to later‐life cognitive enrichment has the potential to promote synaptic and cognitive health in ageing, and to enhance compensatory capacity for synaptic connectivity in pathological ageing associated with Aβ deposition.

The Ryk Receptor Is Expressed in Glial and Fibronectin-Expressing Cells after Spinal Cord Injury

Wnt proteins play a critical role in central nervous system development and have been implicated in several neuropathologies, including spinal cord injury (SCI). Ryk, an unconventional Wnt receptor, regulates axonal regeneration after SCI, although its expression pattern in this neuropathology remains unclear. Therefore, we sought to define the spatiotemporal and cellular pattern of Ryk expression after a contusive SCI in adult rats using quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analysis. Under physiological conditions, Ryk is expressed in neurons, astrocytes, and blood vessels, but not in oligodendrocytes, microglia, NG2+ glial precursor cells, or axonal projections. Following SCI, we observed an increase in Ryk mRNA expression from 24 h post-injury until 7 days post-injury, whereas its protein levels were significantly augmented at 7 and 14 days post-injury. Moreover, the spatial and cellular Ryk expression pattern was altered in the damaged tissue, where this receptor was observed in reactive astrocytes and microglia/macrophages, NG2+ glial precursors, fibronectin+ cells, oligodendrocytes, and axons. In conclusion, we demonstrate that Ryk is expressed in the unlesioned spinal cord and that, after SCI, its spatiotemporal and cellular expression pattern changed dramatically, being expressed in cells involved in the spinal cord response to damage.

Defining the earliest pathological changes of Alzheimer’s disease

The prospects for effectively treating well-established dementia, such as Alzheimer’s disease (AD), are slim, due to the destruction of key brain pathways that underlie higher cognitive function. There has been a substantial shift in the field towards detecting conditions such as AD in their earliest stages, which would allow preventative or therapeutic approaches to substantially reduce risk and/or slow the progression of disease. AD is characterized by hallmark pathological changes such as extracellular Aβ plaques and intracellular neurofibrillary pathology, which selectively affect specific subclasses of neurons and brain circuits. Current evidence indicates that Aβ plaques begin to form many years before overt dementia, a gradual and progressive pathology which offers a potential target for early intervention. Early Aβ changes in the brain result in localized damage to dendrites, axonal processes and synapses, to which excitatory synapses and the processes of projection neurons are highly vulnerable. Aβ pathology is replicated in a range of transgenic models overexpressing mutant human familial AD genes (eg APP and presenilin 1). Studying the development of aberrant regenerative and degenerative changes in neuritic processes associated with Aβ plaques may represent the best opportunity to understand the relationship between the pathological hallmarks of AD and neuronal damage, and to develop early interventions to prevent, slow down or mitigate against Aβ pathology and/or the neuronal alterations that leads to cognitive impairment.

Changes in TDP-43 expression in development, aging, and in the neurofilament light protein knockout mouse

The transactive response DNA-binding protein 43 (TDP-43) has been identified as a neurofilament light (NF-L) messenger RNA (mRNA)-binding protein. Abnormally increased levels of TDP-43 are detected in patients with amyotrophic lateral sclerosis and a downregulation of NF-L mRNA. However, links between NF-L and TDP-43 expressions are unclear. In this study, we investigated whether the deficiency of NF-L protein can result in alterations in TDP-43 localization or protein expression and whether this is altered with aging. There was a significant increase in TDP-43 protein levels in the cortex and lumbar spinal cord in 12-month-old NF-L knockout (NF-L KO) mice, compared with wild-type (WT) C57BL/6 mice. However, there was no difference in either the phosphorylation of TDP-43 between WT and NF-L KO mice or the abnormal mislocalization of TDP-43 to the cytoplasm in NF-L KO animals. Our findings suggest that NF-L protein or mRNA may negatively affect the expression of TDP-43 in the central nervous system. However, altered phosphorylation of TDP-43 may be more highly associated with aging than the levels of TDP-43 expression.

Combination treatment with leptin and pioglitazone in a mouse model of Alzheimer's disease

Combination therapy approaches may be necessary to address the many facets of pathologic change in the brain in Alzheimers disease (AD). The drugs leptin and pioglitazone have previously been shown individually to have neuroprotective and anti‐inflammatory actions, respectively, in animal models.