Immunolabelling of non‐phosphorylated neurofilament indicates damage of spinal cord axons in TSE‐infected goats

Nadeem, Muhammad; Spitzbarth, Ingo; Haist, Verena; Rohn, Karl; Tauscher, Kerstin; Bossers, Alex; Langeveld, J.P.M.; Papasavva-Stylianou, Penelope; Groschup, Martin H.; Baumgärtner, Wolfgang; Gerhauser, Ingo; Fast, Christine

doi:10.1136/vr.103425

Cited by 6 publications

(11 citation statements)

References 23 publications

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“…In order to establish the progression of hippocampal neurodegeneration throughout aging we used three groups of mice of different ages: adult (3-6 months), old (12-15 months) and aged (more To further evaluate age-associated axonal degeneration in the hippocampus, we studied the expression of two phosphorylated forms of neurofilaments (NFs), the main proteins shaping the axonal cytoskeleton. Most NFs are highly phosphorylated under physiological conditions (Petzold 2005), while increased non-phosphorylated NF (non-pNF) immunoreactivity is associated with inflammation, neurodegenerative diseases and brain injury (Tsunoda & Fujinami 2002;Seehusen & Baumgärtner 2010;Bock et al 2013;Nadeem et al 2016). Interestingly, almost undetectable immunoreactivity of non-pNF was observed in the DG of adult mice, while a progressive increase was evidenced during aging (Fig 1c,d).…”

Section: Resultsmentioning

confidence: 93%

Necroptosis inhibition counteracts neurodegeneration, memory decline and key hallmarks of aging, promoting brain rejuvenation

Arrázola

Lira

Quiroz³

et al. 2021

Preprint

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Age is the main risk factor for cognitive impairment and the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction and brain disabilities in humans, primates, rodents, and invertebrates. Necroptosis activation mediates degeneration of mechanical and chemically injured axons, but whether this pathway triggers axonal degeneration and cognitive impairment during brain aging has not been studied. Here we show that necroptosis is activated in the hippocampus during aging, especially in axonal tracts. Loss of the main necroptotic effector, Mlkl, was sufficient to delay age-associated axonal degeneration. Accordingly, aged Mlkl-KO mice also displayed a youthful phenotype at the synaptic and functional level, protecting against decreased synaptic transmission and memory decline. Short-term pharmacologic inhibition of necroptosis by targeting RIPK3 in aged mice, proved to be extraordinarily effective at reverting axonal degeneration and hippocampal-dependent functional impairment at the electrophysiological and behavioral level. Remarkably, a comprehensive quantitative proteomic analysis uncovered a set of aging hallmarks that were recovered in both, the genetic and pharmacologic models of necroptosis inhibition, including molecular biofunctions associated with brain rejuvenation. Taken together, these findings demonstrate that necroptosis contributes to the age-associated deterioration of axonal integrity, affecting hippocampal neuronal connectivity and cognitive function in aged individuals. We therefore propose necroptosis as an attractive target for the future development of geroprotective tools to treat age-related disabilities.

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Section: Resultsmentioning

confidence: 93%

Necroptosis inhibition counteracts neurodegeneration, memory decline and key hallmarks of aging, promoting brain rejuvenation

Arrázola

Lira

Quiroz³

et al. 2021

Preprint

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“…Therefore, it can be assumed that their immunoreactivity indicates their axonal localization. These findings indicate that the accumulation of lysosomal storage material might influence axonal transport and neurofilament phosphorylation status [75,76,88,89,103]. A disturbance in the kinesin-mediated transport of dynein could also affect dynein distribution, because dynein transport is also partly dependent on kinesin (direct transport) [104].…”

Section: Discussionmentioning

confidence: 93%

“…NF phosphorylation also controls the association of NFs with kinesin and dynein, and thereby their own axonal transport [100,101]. Thus, alterations in the amount and distribution of β-APP, pNF, nNF, kinesin, and dynein can be used as surrogate markers for axonal damage and disturbed axonal transport [75,76,88,102,103]. Furthermore, the antibodies are restricted to axons only in the CNS.…”

Section: Discussionmentioning

confidence: 99%

“…Immunohistochemistry was performed on paraffin sections applying the avidin-biotin-peroxidase complex method (Vector Laboratories Inc., Burlingame, CA, USA) with the chromogen 3.3 -diaminobenzidine-tetrahydrochloride (DAB) as previously described [75][76][77][78]. Primary antibodies directed against GFAP, CNPase, APP, MBP, Iba1, and PRX were used to investigate mice of all ages (two females and two males/time point/group) ( Table 1).…”

Section: Immunohistochemistrymentioning

confidence: 99%

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Axonopathy and Reduction of Membrane Resistance: Key Features in a New Murine Model of Human GM1-Gangliosidosis

Eikelberg¹,

Lehmbecker²,

Brogden³

et al. 2020

JCM

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GM1-gangliosidosis is caused by a reduced activity of β-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1−/− mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1−/− mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1−/− mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon–glial interactions and therapy trials of lysosomal storage diseases.

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“…Moreover, the present study demonstrated that G M1 and NGF supplementation results in an increased expression of pNF and nNF within neuronal processes, which was accompanied by a decrease in the pNF/nNF ratio possibly reflecting changes in the NF phosphorylation status. Normal axons are characterized by highly phosphorylated NFs, whereas damaged axons found in different inflammatory, degenerative and traumatic CNS diseases [114][115][116] and disturbed axonal transport 117,118 contain nNFs. Thus, supplementation of canine DRG neurons with G M1 and NGF might also cause degenerative changes in neuronal processes.…”

mentioning

confidence: 99%

Neurotrophic effects of GM1 ganglioside, NGF, and FGF2 on canine dorsal root ganglia neurons in vitro

Schwarz¹,

Lehmbecker²,

Tongtako

et al. 2020

Sci Rep

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certain growth factors 23. Gangliosides are sialic acid-containing glycosphingolipids that can be found in different cellular membranes. In the CNS gangliosides account for up to 10% of the total lipid content in neurons 24. They are involved in intercellular communication, cellular differentiation, neuronal development, and regeneration 25-28 due to their capacity to modulate Ca 2+ influx and their influence on the function of receptors for muscarinic acetylcholine, serotonin, glutamine, neurotransmitters, and neurotrophic factors 29. Cellular gangliosides concentrate in lipid microdomains termed lipid rafts, which are signaling platforms rich in cholesterol and glycosphingolipids and contain amongst others high affinity tropomyosin-related kinase (Trk) and low affinity neurotrophin receptors (p75 NTR) 30-32. These receptors are activated by nerve growth factor (NGF) and other neurotrophic factors and have a strong impact on neuronal development, maintenance, and survival as well as memory formation and storage 33,34. Anti-G M1 antibodies modulate the membrane-associated sphingomyelin metabolism by altering neutral sphingomyelinase activity thereby inhibiting NGF action on rat pheochromocytoma (PC12) cells 35,36. G M1 and NGF protect primary cultured rat embryonic dorsal root ganglia (DRG) and spinal cord neurons from glutamate-induced excitotoxicity 37,38. Both molecules may function by modulating Ca 2+ homeostasis, maintaining normal mitochondrial membrane potential or by promoting the mRNA expression of neuronal proteins such as growth associated protein 43 and neurofilaments (NFs) 38,39. G M1 also protects rats against high altitude cerebral edema by suppressing oxidative stress and production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α 40. Nevertheless, another study using an ischemia/reperfusion model in rats demonstrated that G M1 neuroprotection seems to be dependent on p75 NTR 41. Exogenous gangliosides also directly interact with growth factors such as basic fibroblast growth factor (FGF2) and inhibit FGF receptor binding 42,43. Moreover, G M1 can inhibit FGF2-mediated effects by acting on the same intracellular signaling molecules. For instance, FGF2 stimulates the activity of glycogen synthase kinase 3 (GSK3) in proliferating adult rat hippocampal progenitor cells, whereas G M1 prevents GSK3 activation in organotypic hippocampal slice cultures 44,45. In contrast, cell-associated G M1 has been described to act as a functional co-receptor for FGF2 43. Despite its name, FGF2 plays an essential role in neurogenesis, differentiation, axonal branching, and neuron survival in various types of brain and peripheral nerve lesions 46,47. FGF2 also stimulates the proliferation of neuronal precursor cells isolated from postnatal murine DRGs 48. The ability of adult rat ganglion cells to regrow axons in vitro can be influenced by FGF2 and G M1 49. Neurotrophic effects of FGF2 seem to be also mediated by soluble mediators released from glial cells 50. Summarized, NGF, FGF2 and gangliosides such as G M1 ...

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Immunolabelling of non‐phosphorylated neurofilament indicates damage of spinal cord axons in TSE‐infected goats

Cited by 6 publications

References 23 publications

Necroptosis inhibition counteracts neurodegeneration, memory decline and key hallmarks of aging, promoting brain rejuvenation

Necroptosis inhibition counteracts neurodegeneration, memory decline and key hallmarks of aging, promoting brain rejuvenation

Axonopathy and Reduction of Membrane Resistance: Key Features in a New Murine Model of Human GM1-Gangliosidosis

Neurotrophic effects of GM1 ganglioside, NGF, and FGF2 on canine dorsal root ganglia neurons in vitro

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