analyzing-parkinson-s-disease-mouse-model-induced-adeno-associated

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

universidad mayor

After an incomplete spinal cord injury (SCI), partial recovery of locomotion is accomplished with time. Previous studies have established a functional link between extension of axon collaterals from spared spinal tracts and locomotor recovery after SCI, but the tissular signals triggering collateral sprouting have not been identified. Here, we investigated whether axonal degeneration after SCI contributes to the sprouting of collaterals from axons spared after injury. To this end, we evaluated collateral sprouting from BDA-labeled uninjured corticospinal axons after spinal cord hemisection (SCI(H)) in wild type (WT) mouse and Wld(S) mouse strains, which shows a significant delay in Wallerian degeneration after injury. After SCI(H), spared fibers of WT mice extend collateral sprouts to both intact and denervated sides of the spinal cord distant from the injury site. On the contrary, in the Wld(S) mice collateral sprouting from spared fibers was greatly reduced after SCI(H). Consistent with a role for collateral sprouting in functional recovery after SCI, locomotor recovery after SCI(H) was impaired in Wld(S) mice compared to WT animals. In conclusion, our results identify axonal degeneration as one of the triggers for collateral sprouting from the contralesional uninjured fibers after an SCI(H). These results open the path for identifying molecular signals associated with tissular changes after SCI that promotes collateral sprouting and functional recovery. 

Sprouting of axonal collaterals after spinal cord injury is prevented by delayed axonal degeneration.

Tar DNA binding protein 43 (TDP-43) is the principal component of ubiquitinated protein inclusions present in nervous tissue of most cases of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies described a TDP-43 transgenic mouse model that develops progressive motor dysfunction in the absence of protein aggregation or significant motoneuron loss, questioning its validity to study ALS. Here we have further characterized the course of the disease in TDP-43 mice using a battery of tests and biochemical approaches. We confirmed that TDP-43 mutant mice develop impaired motor performance, accompanied by progressive body weight loss. Significant differences were observed in life span between genders, where females survived longer than males. Histopathological analysis of the spinal cord demonstrated a significant motoneurons loss, accompanied by axonal degeneration, astrogliosis and microglial activation. Importantly, histopathological alterations observed in TDP-43 mutant mice were similar to some characteristic changes observed in mutant SOD1 mice. Unexpectedly, we identified the presence of different species of disulfide-dependent TDP-43 aggregates in cortex and spinal cord tissue. Overall, this study indicates that TDP-43 transgenic mice develop key features resembling key aspects of ALS, highlighting its relevance to study disease pathogenesis.

Disulfide cross-linked multimers of TDP-43 and spinal motoneuron loss in a TDP-43 ALS/FTD mouse model.

Wallerian degeneration is an active program tightly associated with axonal degeneration, required for axonal regeneration and functional recovery after nerve damage. Here we provide a functional molecular foundation for our undertstanding of the complex non-cell autonomous role of glial cells in the regulation of axonal degeneration. To shed light on the complexity of the molecular machinery governing axonal degeneration we employ a multi-model, unbiased, in vivo approach combining morphological assesment and quantitative proteomics with in silico-based higher order functional clustering to genetically uncouple the intrinsic and extrinsic processes governing Wallerian degeneration. Highlighting a pivotal role for glial cells in the early stages fragmenting the axon by a cytokinesis-like process and a cell autonomous stage of axonal disintegration associated to mitochondrial dysfunction.

Molecular analysis of axonal-intrinsic and glial-associated co-regulation of axon degeneration.

Glial cells regulate a wide variety of neuronal functions during physiological and pathological conditions. Therefore, the study of glial cells and their association with axons is of paramount importance in order to understand the physiology of the nervous system. This chapter describes a detailed protocol to prepare and stain teased nerve fibers from peripheral nerves using fluorescent indirect immunolabeling and staining with vital dyes. For immunofluorescence analysis, we describe techniques to study the axonal compartment and the expression of cytoplasmic and plasma membrane proteins in Schwann cells.

Teased Fiber Preparation of Myelinated Nerve Fibers from Peripheral Nerves for Vital Dye Staining and Immunofluorescence Analysis.

Axonal degeneration, which contributes to functional impairment in several disorders of the nervous system, is an important target for neuroprotection. Several individual factors and subcellular events have been implicated in axonal degeneration, but researchers have so far been unable to identify an integrative signaling pathway activating this self-destructive process. Through pharmacological and genetic approaches, we tested whether necroptosis, a regulated cell-death mechanism implicated in the pathogenesis of several neurodegenerative diseases, is involved in axonal degeneration. Pharmacological inhibition of the necroptotic kinase RIPK1 using necrostatin-1 strongly delayed axonal degeneration in the peripheral nervous system and CNS of wild-type mice of either sex and protected  sensory axons from degeneration after mechanical and toxic insults. These effects were also observed after genetic knock-down of , a second key regulator of necroptosis, and the downstream effector  (). RIPK1 inhibition prevented mitochondrial fragmentation  and , a typical feature of necrotic death, and inhibition of mitochondrial fission by Mdivi also resulted in reduced axonal loss in damaged nerves. Furthermore, electrophysiological analysis demonstrated that inhibition of necroptosis delays not only the morphological degeneration of axons, but also the loss of their electrophysiological function after nerve injury. Activation of the necroptotic pathway early during injury-induced axonal degeneration was made evident by increased phosphorylation of the downstream effector MLKL. Our results demonstrate that axonal degeneration proceeds by necroptosis, thus defining a novel mechanistic framework in the axonal degenerative cascade for therapeutic interventions in a wide variety of conditions that lead to neuronal loss and functional impairment. We show that axonal degeneration triggered by diverse stimuli is mediated by the activation of the necroptotic programmed cell-death program by a cell-autonomous mechanism. This work represents a critical advance for the field since it identifies a defined degenerative pathway involved in axonal degeneration in both the peripheral nervous system and the CNS, a process that has been proposed as an early event in several neurodegenerative conditions and a major contributor to neuronal death. The identification of necroptosis as a key mechanism for axonal degeneration is an important step toward the development of novel therapeutic strategies for nervous-system disorders, particularly those related to chemotherapy-induced peripheral neuropathies or CNS diseases in which axonal degeneration is a common factor.

Axonal Degeneration Is Mediated by Necroptosis Activation.

Parkinson's disease (PD) is the second most common neurodegenerative condition, characterized by motor impairment due to the progressive degeneration of dopaminergic neurons in the substantia nigra and depletion of dopamine release in the striatum. Accumulating evidence suggest that degeneration of axons is an early event in the disease, involving destruction programs that are independent of the survival of the cell soma. Necroptosis, a programmed cell death process, is emerging as a mediator of neuronal loss in models of neurodegenerative diseases. Here, we demonstrate activation of necroptosis in postmortem brain tissue from PD patients and in a toxin-based mouse model of the disease. Inhibition of key components of the necroptotic pathway resulted in a significant delay of 6-hydroxydopamine-dependent axonal degeneration of dopaminergic and cortical neurons in vitro. Genetic ablation of necroptosis mediators MLKL and RIPK3, as well as pharmacological inhibition of RIPK1 in preclinical models of PD, decreased dopaminergic neuron degeneration, improving motor performance. Together, these findings suggest that axonal degeneration in PD is mediated by the necroptosis machinery, a process here referred to as necroaxoptosis, a druggable pathway to target dopaminergic neuronal loss.

The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease.

Peripheral nerve injuries result in motor and sensory dysfunction which can be recovered by compensatory or regenerative processes. In situations where axonal regeneration of injured neurons is hampered, compensation by collateral sprouting from uninjured neurons contributes to target reinnervation and functional recovery. Interestingly, this process of collateral sprouting from uninjured neurons has been associated with the activation of growth-associated programs triggered by Wallerian degeneration. Nevertheless, the molecular alterations at the transcriptomic level associated with these compensatory growth mechanisms remain to be fully elucidated. We generated a surgical model of partial sciatic nerve injury in mice to mechanistically study degeneration-induced collateral sprouting from spared fibers in the peripheral nervous system. Using next-generation sequencing and Ingenuity Pathway Analysis, we described the sprouting-associated transcriptome of uninjured sensory neurons and compare it with the activated by regenerating neurons. In vitro approaches were used to functionally assess sprouting gene candidates in the mechanisms of axonal growth. Using a novel animal model, we provide the first description of the sprouting transcriptome observed in uninjured sensory neurons after nerve injury. This collateral sprouting-associated transcriptome differs from that seen in regenerating neurons, suggesting a molecular program distinct from axonal growth. We further demonstrate that genetic upregulation of novel sprouting-associated genes activates a specific growth program in vitro, leading to increased neuronal branching. These results contribute to our understanding of the molecular mechanisms associated with collateral sprouting in vivo. The data provided here will therefore be instrumental in developing therapeutic strategies aimed at promoting functional recovery after injury to the nervous system.

Article	Year
Sprouting of axonal collaterals after spinal cord injury is prevented by delayed axonal degeneration. Experimental neurology\| PubMed ID: 25079366	2014
Disulfide cross-linked multimers of TDP-43 and spinal motoneuron loss in a TDP-43 ALS/FTD mouse model. Scientific reports\| PubMed ID: 29079747	2017
Molecular analysis of axonal-intrinsic and glial-associated co-regulation of axon degeneration. Cell death & disease\| PubMed ID: 29120410	2017
Teased Fiber Preparation of Myelinated Nerve Fibers from Peripheral Nerves for Vital Dye Staining and Immunofluorescence Analysis. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29546717	2018
Axonal Degeneration Is Mediated by Necroptosis Activation. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 30850513	2019
The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease. Cell death and differentiation\| PubMed ID: 31591470	2020
Correction: The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease. Cell death and differentiation\| PubMed ID: 32047275	2020
Collateral Sprouting of Peripheral Sensory Neurons Exhibits a Unique Transcriptomic Profile. Molecular neurobiology\| PubMed ID: 32696431	2020

Alejandra Catenaccio

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