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Physical Exercise As A Possible Therapy For Charcot-Marie-Tooth Type 1X NeuropathyDennis Klein, Xidi Yuan, Janos Groh, Rudolf Martini Department of Neurology, Developmental Neurobiology, University Hospital Würzburg, Germany

2020 PNS Virtual Event27 June - 30 June 2020

Virtual poster number 1250

Neuroinflammation as a common disease modifier in primary genetically disorders

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Introduction:The dominant, X-chromosome-linked form of Charcot-Marie-Tooth disorders is primarily caused by a plethora of

distinct mutations in the GJB1 gene (also designated CX32) that mostly result in loss of function. Despite

rehabilitative strategies or surgeries there is still no causative cure for patients. Thus, there are presently no

perspectives to ameliorate the typical symptoms reducing quality of life, such as weakness, sensory dysfunctions,

muscle atrophy, and often painful bone deformities. We identified detrimental neuroinflammation as a potentially

targetable disease amplifier in Cx32-deficient (Cx32def) mice and other CMT1 models1,2 (see also slide 3). As

secondary inflammation also appears to be involved in human CMTs, including CMT1X3, dampening inflammation

in peripheral nerves might be an option for alleviating the disease.

Since physical exercise has been shown to dampen neuroinflammation in the CNS4,5 , we here tested whether a

corresponding approach attenuates detrimental neuroinflammation and disease outcome in a mouse model for

CMT1X.

Pareyson and Marchesi, Lancet Neurol., 2009

Charcot-Marie-Tooth (CMT) diseases

Characterized by:- demyelination- axonopathy- reduced NCV - muscle atrophy- progressive distal muscle weakness - skeletal abnormalities and food deformities - sensory dysfunction

1. Klein, D. and Martini, R. (2016). 2. Klein, D. et al. (2015). 3. Groh, J. et al. (2012). 4. Einstein, O. et al. (2018). 5. Mandolesi, G. et al. (2019).

Neuroinflammation as a potentially targetable disease amplifier in distinct CMT1 mouse models

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Can physical exercise mitigate detrimental neuroinflammationand improve disease outcome in a mouse model for CMT1X?

Summary of the identified interplay of cells and secreted molecules during secondary inflammation inperipheral nerves of CMT1 models. A pivotal key molecule is the colony stimulating factor-1 (CSF-1;“Activator 2”) which is expressed by endoneurial fibroblasts (Fi) and drives macrophages (Mφ) into apathogenic state leading to myelin phagocytosis and Schwann cell (SC) dedifferentiation. Adapted fromKlein and Martini, Brain Res., 2016 .

Physical exercise

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Research question: Does VWR modulate neuroinflammation and ameliorate the disease outcome in CMT1X?

Methods:Cx32-deficient (Cx32def) mice were allowed to run in a commercially available running wheel for 18 hours/day for 4x/week. Wheels were equipped with a counter to

record individual activities. Methods for analysis: Rotarod and grip test were performed longitudinally. Immunocytochemistry of femoral nerves characterized

inflammation, electrophysiological recordings and electron microscopy scored nerve function and structure.

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A. Commercially available running wheel. Note the counter (arrowhead) that records training activity of the respective mouse.

B. Schematic representation of the training regimen. 3-month-old wild-type (WT) and Cx32def mice were allowed to run for six months (VWR) andanalyzed in comparison to control animals devoid of training (Ctrl).

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VWR only mildly reduces the numbers of macrophages, but alters their activation/polarization in Cx32def mice significantly

A. Immunohistochemical staining against the pan-macrophage marker F4/80 in cross-sections of femoral nerves from Cx32def Ctrl (left) and Cx32def VWR mice (right). Nuclei are labeled with DAPI.The boundaries of the investigated femoral quadriceps nerves are indicated by white dashed circles.B. Corresponding quantification of F4/80-positive profiles in affected femoral quadriceps nerves. Note a significant increase in numbers of macrophages in Cx32def Ctrl compared to WT Ctrl mice.Physical exercise only mildly reduces macrophage numbers in Cx32def VWR compared to Cx32def Ctrl mice. (n = 5 - 9). Individual mice and corresponding mean with SD are indicated. One-WayANOVA with post-hoc Tukey test.C. Immunohistochemical staining against the macrophage-activation marker CD206 in cross-sections of femoral nerves from Cx32def control (left) and Cx32def VWR mice (right). Nuclei are labeledwith DAPI. The boundaries of the investigated femoral quadriceps nerves are indicated by white dashed circles.D. Corresponding quantification of CD206-positive macrophages in affected femoral quadriceps nerves. Note a significant increase in the percentage of CD206-positive macrophages in Cx32def VWRcompared to Cx32def Ctrl mice (red circle; n = 5 - 9). Individual mice and corresponding mean with SD are indicated. One-Way ANOVA with post-hoc Tukey test.

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VWR attenuates axon perturbation in peripheral nerves of Cx32def mice

A. Representative electron micrograph of a femoral quadriceps nerve from a 9 months old Cx32def VWR mouse. Asterisks indicate thinly myelinated axons. Periaxonal vacuoles (v) and Büngnerbands (arrow) are also detectable.B. - G. Quantification of the number of axons (B), foamy (phagocytosing) macrophages (C), abnormally myelinated (comprising de- and thinly myelinated) fibers (D), periaxonal vacuoles (E) anddegenerating axons/Büngner bands. VWR has no effect on myelin integrity, but on axon preservation in Cx32def mice (n = 5 - 9). Individual mice and corresponding mean with SD are indicated.Kruskal-Wallis test with Bonferroni-Holm correction.G. + H. Quantification of axonal damage (comprising periaxonal vacuoles, degenerating axons and Büngner bands; G) and regression analysis correlating axonal damage with running distance (H).VWR significantly mitigates axonal damage (Two-tailed Student’s t-test) and may follow an optimum training pattern (Pearson r correlation). Red circle marks values from Cx32def Ctrl mice (devoid oftraining).

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Clinical consequences of VWR: functional improvement in Cx32def mice

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A. Nerve conduction studies. Quantification of nerve conduction velocity (NCV, left) and minimum F-wave latency (right) at 9 months of age. Cx32def Ctrl mice show significantly reduced NCV andprolonged F-wave latency compared to WT Ctrl mice. VWR improves NCV and significantly attenuates F-wave slowing (n = 8 - 10). Individual mice and corresponding mean with SD are indicated.One-Way ANOVA with post-hoc Tukey test.B. Longitudinal measurement of rotarod performance (latency to fall from turning rod [s]) at 3, 6 and 9 months of age. Percentage change compared to baseline measurement at 3M is shown. Note aconstant and significant decline of rotarod performance in Cx32def Ctrl mice (red dashed line) compared to WT Ctrl mice. This decline is significantly attenuated in Cx32def VWR mice (green dashedline). Note also improved motor outcome in WT VWR mice (n = 6 - 10). Individual mice and corresponding mean with SD are indicated. Two-Way ANOVA with Sidak’s multiple comparisons test.C. Longitudinal measurement of hind limb grip strength [N] at 3, 6 and 9 months of age. Percentage change compared to baseline measurement at 3M is shown. Note a constant and significant declinein grip strength in Cx32def Ctrl mice (red dashed line) compared to WT Ctrl mice. This decline is attenuated in Cx32def VWR mice (green dashed line), while VWR has no influence in WT mice (n = 6 -10). Individual mice and corresponding mean with SD are indicated. Two-Way ANOVA with Sidak’s multiple comparisons test.

Summary and Conclusion

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VWR from 3 - 9M in Cx32def mice

led only to a mild reduction in number of F4/80+ nerve macrophages, but substantially alteredtheir activation/polarization

mitigated axonal perturbation with a potential optimum outcome regarding running distance

improved nerve conduction, muscle strength and motor outcome

VWR might be a safe therapeutic option to mitigate clinical symptoms of CMT1X

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This study is supported by:

Open questions and future aspects with high clinical relevance

Can VWR also mitigate axonal damage and disease outcome when physical exercise starts atmore progressed disease stages?

Optimizing physical exercise (voluntary running) intensity to further modulate neuroinflammation,improve nerve structure and function, as well as the clinical outcome in a model for CMT1X.

Selected References1. Klein, D. and Martini, R. (2016). Myelin and macrophages in the PNS: An intimate relationship in trauma and disease. Brain Res 1641:130-138.

2. Klein, D. et al. (2015). Targeting the colony stimulating factor 1 receptor alleviates two forms of Charcot-Marie-Tooth disease in mice. Brain 138:3193-205.

3. Groh, J et al. (2012). Colony-stimulating factor-1 mediates macrophage-related neural damage in a model for Charcot-Marie-Tooth disease type 1X. Brain 135: 88-104.

4. Einstein, O. et al. (2018). Exercise training attenuates experimental autoimmune encephalomyelitis by peripheral immunomodulation rather than direct neuroprotection. Exp Neurol 299:56-64.

5. Mandolesi, G. et al. (2019). Voluntary Running Wheel Attenuates Motor Deterioration and Brain Damage in Cuprizone-Induced Demyelination. Neurobiol Dis 129:102-117.