Jan C. Koch

Mechanisms of acute axonal degeneration in the optic nerve in vivo

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson’s disease

Axonal degeneration is one of the earliest features of Parkinson’s disease pathology, which is followed by neuronal death in the substantia nigra and other parts of the brain. Inhibition of axonal degeneration combined with cellular neuroprotection therefore seem key to targeting an early stage in Parkinson’s disease progression. Based on our previous studies in traumatic and neurodegenerative disease models, we have identified rho kinase as a molecular target that can be manipulated to disinhibit axonal regeneration and improve survival of lesioned central nervous system neurons. In this study, we examined the neuroprotective potential of pharmacological rho kinase inhibition mediated by fasudil in the in vitro 1-methyl-4-phenylpyridinium cell culture model and in the subchronic in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. Application of fasudil resulted in a significant attenuation of dopaminergic cell loss in both paradigms. Furthermore, dopaminergic terminals were preserved as demonstrated by analysis of neurite network in vitro, striatal fibre density and by neurochemical analysis of the levels of dopamine and its metabolites in the striatum. Behavioural tests demonstrated a clear improvement in motor performance after fasudil treatment. The Akt survival pathway was identified as an important molecular mediator for neuroprotective effects of rho kinase inhibition in our paradigm. We conclude that inhibition of rho kinase using the clinically approved small molecule inhibitor fasudil may be a promising new therapeutic strategy for Parkinson’s disease.

Axonal degeneration as a therapeutic target in the CNS

Degeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory and degenerative neurological diseases. Increasing evidence suggests that axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. We review the evidence for axonal destruction from pathological findings and animal models with particular emphasis on neurodegenerative and neurotraumatic disorders. We discuss the basic morphological and temporal modalities of axonal degeneration (acute, chronic and focal axonal degeneration and Wallerian degeneration). Based on the mechanistic concepts, we then delineate in detail the major molecular mechanisms that underlie the degenerative cascade, such as calcium influx, axonal transport, protein aggregation and autophagy. We finally concentrate on putative therapeutic targets based on the mechanistic prerequisites.

ROCKing Regeneration: Rho Kinase Inhibition as Molecular Target for Neurorestoration

Regenerative failure in the CNS largely depends on pronounced growth inhibitory signaling and reduced cellular survival after a lesion stimulus. One key mediator of growth inhibitory signaling is Rho-associated kinase (ROCK), which has been shown to modulate growth cone stability by regulation of actin dynamics. Recently, there is accumulating evidence the ROCK also plays a deleterious role for cellular survival. In this manuscript we illustrate that ROCK is involved in a variety of intracellular signaling pathways that comprise far more than those involved in neurite growth inhibition alone. Although ROCK function is currently studied in many different disease contexts, our review focuses on neurorestorative approaches in the CNS, especially in models of neurotrauma. Promising strategies to target ROCK by pharmacological small molecule inhibitors and RNAi approaches are evaluated for their outcome on regenerative growth and cellular protection both in preclinical and in clinical studies.

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells.

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinsons disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson’s disease progression, particularly in the context of bioenergetic dysfunction.

AAV.shRNA-mediated downregulation of ROCK2 attenuates degeneration of dopaminergic neurons in toxin-induced models of Parkinson's disease in vitro and in vivo

Parkinsons disease (PD) is a neurodegenerative disorder with prominent neuronal cell death in the substantia nigra (SN) and other parts of the brain. Previous studies in models of traumatic and neurodegenerative CNS disease showed that pharmacological inhibition of Rho-associated kinase (ROCK), a molecule involved in inhibitory signaling in the CNS, by small-molecule inhibitors improves neuronal survival and increases regeneration. Most small-molecule inhibitors, however, offer only limited target specificity and also inhibit other kinases, including both ROCK isoforms. To establish the role of the predominantly brain-expressed ROCK2 isoform in models of regeneration and PD, we used adeno-associated viral vectors (AAV) to specifically knockdown ROCK2 in neurons. Rat primary midbrain neurons (PMN) were transduced with AAV expressing short-hairpin-RNA (shRNA) against ROCK2 and LIM-domain kinase 1 (LIMK1), one of the downstream targets of ROCK2. While knock-down of ROCK2 and LIMK1 both enhanced neurite regeneration in a traumatic scratch lesion model, only ROCK2-shRNA protected PMN against 1-methyl-4-phenylpyridinium (MPP+) toxicity. Moreover, AAV.ROCK2-shRNA increased levels of the pro-survival markers Bcl-2 and phospho-Erk1. In vivo, AAV.ROCK2-shRNA vectors were injected into the ipsilateral SN and a unilateral 6-OHDA striatal lesion was performed. After four weeks, behavioral, immunohistochemical and biochemical alterations were investigated. Downregulation of ROCK2 protected dopaminergic neurons in the SN from 6-OHDA-induced degeneration and resulted in significantly increased TH-positive neuron numbers. This effect, however, was confined to nigral neuronal somata as striatal terminal density, dopamine and metabolite levels were not significantly preserved. Interestingly, motor behavior was improved in the ROCK2-shRNA treated animals compared to control after four weeks. Our studies thus confirm ROCK2 as a promising therapeutic target in models of PD and demonstrate that neuron-specific inhibition of ROCK2 promotes survival of lesioned dopaminergic neurons.

TGF-β 1 enhances neurite outgrowth via regulation of proteasome function and EFABP

Malfunction of the ubiquitin-proteasome system has been implicated as a causal factor in the pathogenesis of aggregation-related disorders, e.g. Parkinsons disease. We show here that Transforming growth factor-beta 1 (TGF-beta), a multifunctional cytokine and trophic factor for dopaminergic (DAergic) neurons modulates proteasome function in primary midbrain neurons. TGF-beta differentially inhibited proteasomal subactivities with a most pronounced time-dependent inhibition of the peptidyl-glutamyl peptide hydrolyzing-like and chymotrypsin-like subactivity. Regulation of proteasomal activity could be specifically quantified in the DAergic subpopulation. Protein blot analysis revealed an accumulation of ubiquitinated proteins after TGF-beta treatment. The identity of these enriched proteins was further analyzed by 2D-gel electrophoresis and mass spectrometry. We found epidermal fatty acid binding protein (EFABP) to be strongly increased and ubiquitinated after TGF-beta treatment and confirmed this finding by co-immunoprecipitation. While application of TGF-beta increased neurite regeneration in a scratch lesion model, downregulation of EFABP by siRNA significantly decreased this effect. We thus postulate that a differential regulation of proteasomal function, as demonstrated for TGF-beta, can result in an enrichment of proteins, such as EFABP, that mediate physiological functions, such as neurite regeneration.

X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons

Transition metals have been suggested to play a pivotal role in the pathogenesis of Parkinsons disease. X‐ray microscopy combined with a cryogenic setup is a powerful method for elemental imaging in low concentrations and high resolution in intact cells, eliminating the need for fixation and sectioning of the specimen. Here, we performed an elemental distribution analysis in cultured primary midbrain neurons with a step size in the order of 300 nm and ~ 0.1 ppm sensitivity under cryo conditions by using X‐ray fluorescence microscopy. We report the elemental mappings on the subcellular level in primary mouse dopaminergic (DAergic) and non‐DAergic neurons after treatment with transition metals. Application of Fe2+ resulted in largely extracellular accumulation of iron without preference for the neuronal transmitter subtype. A quantification of different Fe oxidation states was performed using X‐ray absorption near edge structure analysis. After treatment with Mn2+, a cytoplasmic/paranuclear localization of Mn was observed preferentially in DAergic neurons, while no prominent signal was detectable after Mn3+ treatment. Immunocytochemical analysis correlated the preferential Mn uptake to increased expression of voltage‐gated calcium channels in DAergic neurons. We discuss the implications of this differential elemental distribution for the selective vulnerability of DAergic neurons and Parkinsons disease pathogenesis.

Upregulation of reggie-1/flotillin-2 promotes axon regeneration in the rat optic nerve in vivo and neurite growth in vitro.

The ability of fish retinal ganglion cells (RGCs) to regenerate their axons was shown to require the re-expression and function of the two proteins reggie-1 and -2. RGCs in mammals fail to upregulate reggie expression and to regenerate axons after lesion suggesting the possibility that induced upregulation might promote regeneration. In the present study, RGCs in adult rats were induced to express reggie-1 by intravitreal injection of adeno-associated viral vectors (AAV2/1) expressing reggie-1 (AAV.R1-EGFP) 14d prior to optic nerve crush. Four weeks later, GAP-43-positive regenerating axons had crossed the lesion and grown into the nerve at significantly higher numbers and length (up to 5mm) than the control transduced with AAV.EGFP. Consistently, after transduction with AAV.R1-EGFP as opposed to AAV.EGFP, primary RGCs in vitro grew long axons on chondroitin sulfate proteoglycan (CSPG) and Nogo-A, both glial cell-derived inhibitors of neurite growth, suggesting that reggie-1 can provide neurons with the ability to override inhibitors of neurite growth. This reggie-1-mediated enhancement of growth was reproduced in mouse hippocampal and N2a neurons which generated axons 40-60% longer than their control counterparts. This correlates with the reggie-1-dependent activation of Src and PI3 kinase (PI3K), of the Rho family GTPase Rac1 and downstream effectors such as cofilin. This increased growth also depends on TC10, the GTPase involved in cargo delivery to the growth cone. Thus, the upregulation of reggie-1 in mammalian neurons provides nerve cells with neuron-intrinsic properties required for axon growth and successful regeneration in the adult mammalian CNS.

Rho kinase inhibition by fasudil in the striatal 6-hydroxydopamine lesion mouse model of Parkinson disease.

Chronic degeneration of nigrostriatal projections, followed by nigral dopaminergic cell death, is a key feature of Parkinson disease (PD). This study examines the neuroprotective potential of the rho kinase inhibitor fasudil in the 6-hydroxydopamine (6-OHDA) mouse model of PD in vivo. C57Bl/6 mice were lesioned by striatal stereotactic injections with 4 μg of 6-OHDA and treated with fasudil 30 or 100 mg/kg body weight via drinking water. Motor behavior was tested biweekly; histologic and biochemical analyses were performed at 4 and 12 weeks after lesion. Motor behavior was severely impaired after 6-OHDA lesion and was not improved by fasudil treatment. Fasudil 100 mg/kg did not significantly increase the number of dopaminergic cells in the substantia nigra after 12 weeks versus lesion controls. Interestingly, however, high-performance liquid chromatography analysis of dopamine metabolites revealed that striatal levels of 3,4-dihydroxyphenylacetic acid were significantly increased after 12 weeks, suggesting a regenerative response. In contrast to recent findings in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin model, fasudil effects seem limited in this severe 6-OHDA model of PD. Nevertheless, high therapeutic concentrations of fasudil are suggestive of a proregenerative potential for dopaminergic neurons, making further evaluations of rho kinase inhibition as a proregenerative therapeutic strategy in PD promising.