Éva M. Szegő

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.

The NAD‐dependent deacetylase sirtuin 2 is a suppressor of microglial activation and brain inflammation

Deleterious sustained inflammation mediated by activated microglia is common to most of neurologic disorders. Here, we identified sirtuin 2 (SIRT2), an abundant deacetylase in the brain, as a major inhibitor of microglia‐mediated inflammation and neurotoxicity. SIRT2‐deficient mice (SIRT2−/−) showed morphological changes in microglia and an increase in pro‐inflammatory cytokines upon intracortical injection of lipopolysaccharide (LPS). This response was associated with increased nitrotyrosination and neuronal cell death. Interestingly, manipulation of SIRT2 levels in microglia determined the response to Toll‐like receptor (TLR) activation. SIRT2 overexpression inhibited microglia activation in a process dependent on serine 331 (S331) phosphorylation. Conversely, reduction of SIRT2 in microglia dramatically increased the expression of inflammatory markers, the production of free radicals, and neurotoxicity. Consistent with increased NF‐κB‐dependent transcription of inflammatory genes, NF‐κB was found hyperacetylated in the absence of SIRT2, and became hypoacetylated in the presence of S331A mutant SIRT2. This finding indicates that SIRT2 functions as a ‘gatekeeper’, preventing excessive microglial activation through NF‐κB deacetylation. Our data uncover a novel role for SIRT2 opening new perspectives for therapeutic intervention in neuroinflammatory disorders.

A mouse model of anxiety molecularly characterized by altered protein networks in the brain proteome

Recently, several attempts have been made to describe changes related to certain anxiety states in the proteome of experimental animal models. However, these studies are restricted by limitations regarding the number and correct identification of separated proteins. Moreover, the application of a systems biology approach to discover the molecular mechanisms of anxiety requires genetically homogenous inbred animal models. Therefore, we developed a novel mouse model of anxiety using a combination of crossbreeding (inbred for 35 generations) and behavioral selection. We found significant changes in 82 proteins in the total brain proteome compared to the control proteome. Thirty-four of these proteins had been previously identified in other anxiety, depression or repeated psychosocial stress studies. The identified proteins are associated with different cellular functions, including synaptic transmission, metabolism, proteolysis, protein biosynthesis and folding, cytoskeletal proteins, brain development and neurogenesis, oxidative stress, signal transduction. Our proteomics data suggest that alterations in serotonin receptor-associated proteins, in the carbohydrate metabolism, in the cellular redox system and in synaptic docking are all involved in anxiety.

α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner

Alpha-synuclein (αS) misfolding is associated with Parkinsons disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS-Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.

Altered Functional Protein Networks in the Prefrontal Cortex and Amygdala of Victims of Suicide

Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on post mortem brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of post mortem tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard t test and 1.3 fold change as cut off for significance. Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the q-values for all the t tests performed and by using 0.06 and 0.4 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively. From these we identified 59 proteins in the cortex and 11 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies. The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis.

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.

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.

Action of estrogen on survival of basal forebrain cholinergic neurons: Promoting amelioration

Extensive studies during the past two decades provide compelling evidence that the gonadal steroid, estrogen, has the potential to affect the viability of basal forebrain cholinergic neurons. These observations reflect a unique ameliorative feature of estrogen as it restores and protects the cholinergic neurons against noxious stimuli or neurodegenerative processes. Hence, we first address the ameliorative function of estrogen on basal forebrain cholinergic neurons such as the actions of estrogen on neuronal plasticity of cholinergic neurons, estrogen-induced memory enhancement and the ameliorative role of estrogen on cholinergic neuron related neurodegenerative processes such as Alzheimers disease. Second, we survey recent data as to possible mechanisms underlying the ameliorative actions of estrogen; influencing the amyloid precursor protein processing, enhancement in neurotrophin receptor signaling and estrogen-induced non-classical actions on second messenger systems. In addition, clinical relevance, pitfalls and future aspects of estrogen therapy on basal forebrain cholinergic neurons will be discussed.

Idebenone and resveratrol extend lifespan and improve motor function of HtrA2 knockout mice

Heterozygous loss-of-function mutation of the human gene for the mitochondrial protease HtrA2 has been associated with increased risk to develop mitochondrial dysfunction, a process known to contribute to neurodegenerative disorders such as Huntingtons disease (HD) and Parkinsons disease (PD). Knockout of HtrA2 in mice also leads to mitochondrial dysfunction and to phenotypes that resemble those found in neurodegenerative disorders and, ultimately, lead to death of animals around postnatal day 30. Here, we show that Idebenone, a synthetic antioxidant of the coenzyme Q family, and Resveratrol, a bioactive compound extracted from grapes, are both able to ameliorate this phenotype. Feeding HtrA2 knockout mice with either compound extends lifespan and delays worsening of the motor phenotype. Experiments conducted in cell culture and on brain tissue of mice revealed that each compound has a different mechanism of action. While Idebenone acts by downregulating the integrated stress response, Resveratrol acts by attenuating apoptosis at the level of Bax. These activities can account for the delay in neuronal degeneration in the striata of these mice and illustrate the potential of these compounds as effective therapeutic approaches against neurodegenerative disorders such as HD or PD.

A30P α-synuclein impairs dopaminergic fiber regeneration and interacts with L-DOPA replacement in MPTP-treated mice.

Parkinsons disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta (SNpc). α-synuclein (αsyn) has been linked to the pathophysiology of PD, because of its mutations causing familial PD and its accumulation in brains of patients with familial and sporadic PD. Dopamine (DA) replacement is the most effective therapy for ameliorating the motor symptoms of PD; however, it remains controversial whether DA-replacement boosts regeneration in the dopaminergic system or accelerates disease progression and enhances neuronal loss. Here, we studied the effect of chronic L-DOPA treatment on dopaminergic neurons in wild-type (WT) and A30P αsyn transgenic mice after MPTP treatment. Acute MPTP intoxication induced degeneration of dopaminergic neurons in both WT and A30P αsyn transgenic mice. A strong regeneration of dopaminergic fibers at 90 days after MPTP was observed in WT mice. In contrast, regeneration was less pronounced in A30P αsyn mice. Chronic L-DOPA treatment after MPTP intoxication did not only reduce the regeneration of nigrostriatal fibers but also led to an increased apoptotic gene-expression profile in the SNpc and to a decline of TH-positive neurons in A30P αsyn. Our findings reveal that the presence of A30P αsyn inhibits the regeneration of nigrostriatal dopaminergic fibers, and that L-DOPA treatment might interact with the pathogenesis in PD.