The mouse median nerve experimental model in regenerative research

Sciatic nerve crush injury in rat animal model is one of the most common experimental models used in regenerative research. However, the availability of transgenic mouse for nerve regeneration studies is constantly increasing and, therefore, the shift from rat model to mouse model is, in some cases, necessary. Moreover, since most of the human nerve lesions occur in the upper limb, it is also advantageous to shift from sciatic nerve to median nerve. In this study we described an experimental model which involves lesions of the median nerve in the mouse. Data showed that the finger flexor muscle contraction strength, assessed to evaluate the motor function recovery, and reached values not different from the control already 20 days after injury. The degree of nerve regeneration evaluated with stereological methods in light microscopy showed that, 25 days after injury, the number of regenerated myelinated fibers was comparable to the control, but they were smaller with a thinner myelin thickness. Stereological analysis made in electron microscopy confirmed these results, although the total number of fibers quantified was significantly higher compared to light microscopy analysis, due to the very small size of some fibers that can be detected only in electron microscopy

The Mouse Median Nerve Experimental Model in Regenerative Research

Sciatic nerve crush injury in rat animal model is one of the most common experimental models used in regenerative research. However, the availability of transgenic mouse for nerve regeneration studies is constantly increasing and, therefore, the shift from rat model to mouse model is, in some cases, necessary. Moreover, since most of the human nerve lesions occur in the upper limb, it is also advantageous to shift from sciatic nerve to median nerve. In this study we described an experimental model which involves lesions of the median nerve in the mouse. Data showed that the finger flexor muscle contraction strength, assessed to evaluate the motor function recovery, and reached values not different from the control already 20 days after injury. The degree of nerve regeneration evaluated with stereological methods in light microscopy showed that, 25 days after injury, the number of regenerated myelinated fibers was comparable to the control, but they were smaller with a thinner myelin thickness. Stereological analysis made in electron microscopy confirmed these results, although the total number of fibers quantified was significantly higher compared to light microscopy analysis, due to the very small size of some fibers that can be detected only in electron microscopy

The Prion-Like Spreading of Alpha-Synuclein in Parkinson’s Disease: Update on Models and Hypotheses

The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson’s disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn accumulation in the CNS are not fully understood, growing evidence suggests that de novo α-syn misfolding and/or neuronal internalization of aggregated α-syn facilitates conformational templating of endogenous α-syn monomers in a mechanism reminiscent of prions. A refined understanding of the biochemical and cellular factors mediating the pathological neuron-to-neuron propagation of misfolded α-syn will potentially elucidate the etiology of PD and unravel novel targets for therapeutic intervention. Here, we discuss recent developments on the hypothesis regarding trans-synaptic propagation of α-syn pathology in the context of neuronal vulnerability and highlight the potential utility of novel experimental models of synucleinopathies

Trans-synaptic spreading of alpha-synuclein pathology through sensory afferents leads to sensory nerve degeneration and neuropathic pain

Pain is a common non-motor symptom of Parkinsons disease (PD), with current limited knowledge of its pathophysiology. Here, we show that peripheral inoculation of mouse alpha-synuclein (alpha-Syn) pre-formed fibrils, in a transgenic mouse model of PD, elicited retrograde trans-synaptic spreading of alpha-Syn pathology (pSer129) across sensory neurons and dorsal nerve roots, reaching central pain processing regions, including the spinal dorsal horn and the projections of the anterolateral system in the central nervous system (CNS). Pathological peripheral to CNS propagation of alpha-Syn aggregates along interconnected neuronal populations within sensory afferents, was concomitant with impaired nociceptive response, reflected by mechanical allodynia, reduced nerve conduction velocities (sensory and motor) and degeneration of small- and medium-sized myelinated fibers. Our findings show a link between the transneuronal propagation of alpha-Syn pathology with sensory neuron dysfunction and neuropathic impairment, suggesting promising avenues of investigation into the mechanisms underlying pain in PD.Funding Agencies|Independent Research Fund Denmark (IRFD) [8020-00118B]; Lundbeck FoundationLundbeckfonden [R223-2015-4222, R313-2019-606, R248-2016-2518, R171-2014-591]</p

Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs

The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. Here we use novel fluorescently tagged cocaine analogs to visualize DAT and DAT trafficking in cultured live midbrain dopaminergic neurons. The fluorescent tags were extended from the tropane N-position of 2β-carbomethoxy-3β-(3,4-dichlorophenyl)tropane using an ethylamino-linker. The rhodamine-, OR Green-, or Cy3-labeled ligands had high binding affinity for DAT and enabled specific labeling of DAT in live neurons and visualization by confocal imaging. In the dopaminergic neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. FRAP (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than ∼30%). DAT was constitutively internalized into vesicular structures likely representing intracellular transporter pools. The internalization was blocked by lentiviral-mediated expression of dominant-negative dynamin and internalized DAT displayed partial colocalization with the early endosomal marker EGFP-Rab5 and with the transferrin receptor. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC

Comparative transcriptional analysis of satellite glial cell injury response

Background: Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNA sequencing (scRNAseq) technologies has enabled a new approach to investigate SGCs. Methods: In this study we used scRNAseq to investigate SGCs from mice subjected to sciatic nerve injury. We used a meta-analysis approach to compare the injury response with that found in other published datasets.  Furthermore, we also used scRNAseq to investigate how cells from the dorsal root ganglion (DRG) change after 3 days in culture. Results: From our meta-analysis of the injured conditions, we find that SGCs share a common signature of 18 regulated genes following sciatic nerve crush or sciatic nerve ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state while others start resembling Schwann cell-like precursors. Conclusion: By using integrated analyses of new and previously published scRNAseq datasets, this study provides a consensus view of which genes are most robustly changed in SGCs after injury. Our results are available via the Broad Institute Single Cell Portal, so that readers can explore and search for genes of interest

Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport

SummaryEfflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIα bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIα stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIα phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIα. A mutation of the DAT C terminus impairing CaMKIIα binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIα binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux