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SummaryObjective Epilepsy affects 60 million people worldwide. Despite the development of antiepileptic drugs, up to 35% of patients are drug refractory with uncontrollable seizures. Toll-like receptors (TLRs) are central components of the nonspecific innate inflammatory response. Because TLR3 was recently implicated in neuronal plasticity, we hypothesized that it may contribute to the development of epilepsy after status epilepticus (SE). Methods To test the involvement of TLR3 in epileptogenesis, we used the pilocarpine model for SE in TLR3-deficient mice and their respective wild-type controls. In this model, a single SE event leads to spontaneous recurrent seizures (SRS). Two weeks after SE, mice were implanted with wireless electroencephalography (EEG) transmitters for up to 1 month. The impact of TLR3 deficiency on SE was assessed using separate cohorts of mice regarding EEG activity, seizure progression, hippocampal microglial distribution, and expression of the proinflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)β. Results Our data indicate that TLR3 deficiency reduced SRS, microglial activation, and the levels of the proinflammatory cytokines TNFα and IFNβ, and increased survival following SE. Significance This study reveals novel insights into the pathophysiology of epilepsy and the contribution of TLR3 to disease progression. Our results identify the TLR3 pathway as a potential future therapeutic target in SE.

Autism spectrum disorders (ASD) are characterized by social communication deficits, cognitive rigidity, and repetitive stereotyped behaviors. Mesenchymal stem cells (MSC) have a paracrine regenerative effect, and were speculated to be a potential therapy for ASD. The BTBR inbred mouse strain is a commonly used model of ASD as it demonstrates robust behavioral deficits consistent with the diagnostic criteria for ASD. BTBR mice also exhibit decreased brain-derived neurotrophic factor (BDNF) signaling and reduced hippocampal neurogenesis. In the current study, we evaluated the behavioral and molecular effects of intracerebroventricular MSC transplantation in BTBR mice. Transplantation of MSC resulted in a reduction of stereotypical behaviors, a decrease in cognitive rigidity and an improvement in social behavior. Tissue analysis revealed elevated BDNF protein levels in the hippocampus accompanied by increased hippocampal neurogenesis in the MSC-transplanted mice compared with sham treated mice. This might indicate a possible mechanism underpinning the behavioral improvement. Our study suggests a novel therapeutic approach which may be translatable to ASD patients in the future.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons and skeletal muscle atrophy. The majority of ALS cases are acquired spontaneously, with inherited disease accounting for only 10 % of all cases. Recent studies provide compelling evidence that aggregates of misfolded proteins underlie both types of ALS. Small molecules such as artificial chaperones can prevent or even reverse the aggregation of proteins associated with various human diseases. However, their very high active concentration (micromolar range) severely limits their utility as drugs. We synthesized several ester and amide derivatives of chemical chaperones. The lead compound 14, 3-((5-((4,6-dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-amine oxide shows, in the micromolar concentration range, both neuronal and astrocyte protective effects in vitro; at daily doses of 10 mg kg(-1) 14 improved the neurological functions and delayed body weight loss in ALS mice. Members of this new chemical chaperone derivative class are strong candidates for the development of new drugs for ALS patients.

The discovery of neurogenesis in the adult brain has created new possibilities for therapeutics in neurodegenerative diseases. Neural precursor cells, which have been found in various parts of the brain, e.g., the subventricular zone (SVZ) and substantia nigra (SN), have promising potential to replace the extensive loss of neurons occurring in neurodegenerative disorders. In Parkinson’s disease (PD) the degeneration of nigral dopaminergic neurons and consequently the nigrostriatal pathway, which has been found to innervate proximally to the SVZ, causes motor and cognitive impairments. There is strong evidence that neurogenesis is impaired in PD, which has been related to the nonmotor symptoms of the disease. Recent evidence supports that this impairment in neurogenesis is partially caused by the lack of dopamine in one of the adult neurogenic niches, the SVZ. Thus, restoring the dopaminergic pathway in PD patients may have implications not only for motor function improvement, but for other cognitive and autonomic symptoms. Currently, there are no effective treatments that can stop or reverse the neurodegeneration process in the brain. Here we review the neurogenic process and observed alterations found in PD animal models and postmortem brains of PD patients. Finally, we review several attempts to stimulate the neurogenic process for nigral and/or striatal dopaminergic restoration by transgenic expression, exercise, or cell therapy. J. Comp. Neurol. 000:000–000, 2014.

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca(2+)), are essential ingredients for life. Despite the tremendous importance of Ca(2+) for the well-being of living systems, the noninvasive determination of the changes in Ca(2+) levels in general, and extracellular Ca(2+) levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca(2+) levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca(2+) concentrations in the range of 250 µm to 2.5 mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca(2+) , which, in turn, affects the T2 relaxation of the water protons in their vicinity. The new alginate-coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca(2+) levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca(2+) levels in vivo. These results demonstrate that alginate-coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca(2+) levels in myriad in vitro and in vivo biomedical applications.

Amyotrophic Lateral Sclerosis (ALS) is a fatal, rapidly progressive, neurodegenerative disease caused by motor neuron degeneration. Despite extensive efforts, the underlying cause of ALS and the path of neurodegeneration remain elusive. Astrocyte activation occurs in response to central nervous system (CNS) insult and is considered a double edged sword in many pathological conditions. We propose that reduced glutamatergic and trophic response of astrocytes to activation may, over time, lead to accumulative CNS damage, thus facilitating neurodegeneration. We found that astrocytes derived from the SOD1 G93A ALS mouse model exhibit a reduced glutamatergic and trophic response to specific activations compared to their wild-type counterparts. Wild-type astrocytes exhibited a robust response when activated with lipopolysaccharide (LPS), G5 or treated with ceftriaxone in many parameters evaluated. These parameters include increased expression of GLT-1 and GLAST the two major astrocytic glutamate transporters, accompanied by a marked increase in the astrocytic glutamate clearance and up-regulation of neurtrophic factor expression. However, not only do un-treated SOD1 G93A astrocytes take up glutamate less efficiently, but in response to activation they show no further increase in any of the glutamatergic parameters evaluated. Furthermore, activation of wild-type astrocytes, but not SOD1 G93A astrocytes, improved their ability to protect the motor neuron cell line NSC-34 from glutamate induced excitotoxicity. Our data indicates that altered astrocyte activation may well be pivotal to the pathogenesis of ALS. V C 2012 Wiley Periodicals, Inc.

Conventional diffusion MRI methods are mostly capable of portraying microarchitectural elements such as fiber orientation in white matter from detection of diffusion anisotropy, which arises from the coherent organization of anisotropic compartments. Double-pulsed-field-gradient MR methods provide a means for obtaining microstructural information such as compartment shape and microscopic anisotropies even in scenarios where macroscopic organization is absent. Here, we apply angular double-pulsed-gradient-spin-echo MRI in the rat brain both ex vivo and in vivo for the first time. Robust angular dependencies are detected in the brain at long mixing time (t(m) ). In many pixels, the oscillations seem to originate from residual directors in randomly oriented media, i.e., from residual ensemble anisotropy, as corroborated by quantitative simulations. We then developed an analysis scheme that enables one to map of structural indices such as apparent eccentricity (aE) and residual phase (φ) that enables characterization of the rat brain in general, and especially the rat gray matter. We conclude that double-pulsed-gradient-spin-echo MRI may in principle become important in characterizing gray matter morphological features and pathologies in both basic and applied neurosciences.

Neural stem cells give rise to new hippocampal neurons throughout adulthood. Defects in neurogenesis are associated with cognitive dysfunctions, such as Alzheimer disease (AD). Our understanding of the signals controlling this process is limited. The present in vitro study explored the manner in which the Wnt signaling pathway regulates the differentiation of hippocampal progenitors (HPs) into neurons under the influence of amyloid β(42) (Aβ(42) ). The results showed that oligomeric Aβ(42) reduced neuronal differentiation. This process was accompanied by a reduction in active β-catenin levels and proneural gene expression. The addition of Wnt3a increased the neuronal differentiation of Aβ(42) -treated HPs, at the expense of astrocyte differentiation. The effect of Wnt signaling was attributable to progenitor cell differentiation to the neuronal lineage, and not to increased proliferation or rescue of neurons. The interruption of Wnt signaling by oligomeric Aβ(42) may have clinical implications for the treatment of impaired neurogenesis in AD.

The design and synthesis of a novel nuclear factor erythroid 2-related factor 2 (Nrf2) enhancer is reported. Using a structure-based virtual screening approach, several commercially available compounds were identified as having high probability to interact with the Nrf2-binding pocket in the Kelch-like ECH-associated protein 1 (Keap1). Keap1 is an adaptor protein that recruits Nrf2 to a cullin-3-dependent ubiquitin ligase complex. The identified compounds were tested against rat pheochromocytoma PC-12 cells for their cytoprotective activity, and one compound (SKT359126) demonstrated an Nrf2-mediated cell-protective effect. Based on the structure of SKT359126, 23 novel derivatives were synthesized and evaluated. Of the screened derivatives, 1-{4-[(3,4-dihydroxybenzylidene)amino]phenyl}-5-oxopyrrolidine-3-carboxylic acid demonstrated better activity than the parent molecules in activating the Nrf2 transduction pathway in a dose- and time-dependent manner. This compound represents a promising starting point for the development of therapeutics for the treatment of oxidative-stress-related diseases.

Quinolinic acid (QA) induced striatal lesion is an important model for excitotoxicity that is also used for efficacy studies. To date, the morphological and spectroscopic indices of this model have not been studied longitudinally by MRI; therefore the objectives of this study were aimed at following the lesion progression and changes in N-acetyl aspartate (NAA) as viewed by MRI and MRSI, respectively, in-vivo over a period of 49 days. We found that the affected areas exhibited both high and low apparent diffusion coefficients (ADC) even 49 days post QA injection in three of the six tested animals. MRI-guided histological analysis correlated areas characterized by high ADCs on day 49 with cellular loss, while areas characterized by lower ADCs were correlated with macrophage infiltration (CD68 positive stain). Our MRSI study revealed an initial reduction of NAA levels in the lesioned striatum, which significantly recovered with time, although not to control levels. Total-striatum normalized NAA levels recovered from 0.67 +/- 0.15 (of the contralateral row) on day 1 to 0.90 +/- 0.12 on day 49. Our findings suggest that NAA should be considered as a marker for neuronal dysfunction, in addition to neuronal viability. Some behavioral indices could be correlated to permanent neuronal damage while others demonstrated a spontaneous recovery parallel to the NAA recovery. Our findings may have implications in efficacy-oriented studies performed on the QA model.

It is believed that oxidative stress (OS) plays an important role in the loss of dopaminergic nigrostriatal neurons in Parkinson's disease (PD) and that treatment with antioxidants might be neuroprotective. However, most currently available antioxidants cannot readily penetrate the blood brain barrier after systemic administration. We now report that AD4, the novel low molecular weight thiol antioxidant and the N-acytel cysteine (NAC) related compound, is capable of penetrating the brain and protects neurons in general and especially dopaminergic cells against various OS-generating neurotoxins in tissue cultures. Moreover, we found that treatment with AD4 markedly decreased the damage of dopaminergic neurons in three experimental models of PD. AD4 suppressed amphetamine-induced rotational behaviour in rats with unilateral 6-OHDA-induced nigral lesion. It attenuated the reduction in striatal dopamine levels in mice treated with 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP). It also reduced the dopaminergic neuronal loss following chronic intrajugular administration of rotenone in rats. Our findings suggest that AD4 is a novel potential new neuroprotective drug that might be effective at slowing down nigral neuronal degeneration and illness progression in patients with PD.

Parkinson's disease is a chronic neurodegenerative disease of the extrapyramidal system associated with dopaminergic neuronal loss in the basal ganglia. However, several other neurotransmitters, such as serotonin, gamma-amino-butyric acid and glutamate, are also related to the symptoms of Parkinson's disease patients and their response to levodopa treatment. The co-expression of cannabinoid and dopamine receptors in the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement in Parkinson's disease. In the present study we treated unilaterally 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine)-lesioned rats with the enantiomers of the synthetic cannabinoid 7-hydroxy-delta6-tetrahydrocannabinol 1,1-dimethylheptyl. Treatment with its (-)- (3R, 4R) enantiomer (code-name HU-210), a potent cannabinoid receptor type 1 agonist, reduced the rotations induced by levodopa/carbidopa or apomorphine by 34% and 44%, respectively. In contrast, treatment with the (+)- (3S, 4S) enantiomer (code-name HU-211), an N-methyl-D-aspartate antagonist, as well as the psychotropically inactive cannabis constituent: cannabidiol and its primary metabolite, 7-hydroxy-cannabinol, did not show any reduction of rotational behavior. Our results indicate that activation of the CB1 stimulates the dopaminergic system ipsilaterally to the lesion, and may have implications in the treatment of Parkinson's disease.

Bcl-2 is an antiapoptotic protein located in the outer mitochondrial membrane. Cellular perturbations associated with programmed cell death may be the consequence of disrupted mitochondrial function as well as excessive production of reactive oxygen species (ROS). Numerous studies indicate that Bcl-2 is involved in opposing cell death induced by oxidative stimuli, but its mode of action is uncertain. We reexamined the role of Bcl-2 by using a loss-of-function model, Bcl-2 knockout mice. Brains from Bcl-2-deficient mice had a 43% higher content of oxidized proteins and 27% lower number of cells in the cerebellum relative to wild-type mice. Incubation of cerebellar neurons from Bcl-2 +/+ brains with 0.5 mM dopamine caused 25% cell death, whereas in Bcl-2-deficient cells, it resulted in 52% death; glial cells provided protection in both cultures. Splenocytes from Bcl-2-deficient mice were also killed more effectively by dopamine as well as paraquat. Bcl-2-deficient mice did not survive intraperitoneal injection of MPTP, which caused a decrease in dopamine level in the striatum of Bcl-2 +/- brains, which was more significant than in wild-type mice. When compared with Bcl-2 +/+ brains, brains of 8-day-old Bcl-2-deficient mice had higher activities of the antioxidant enzymes GSH reductase (192%) and GSH transferase (142%), whereas at the age of 30 days, GSH peroxidase was significantly lower (66%). Activities of GSH transferase and GSH reductase increased significantly (158 and 262%, respectively) from day 8 to day 30 in Bcl-2 +/+ mice, whereas GSH peroxidase decreased (31%) significantly in Bcl-2 -/- animals. In summary, our results demonstrated enhanced oxidative stress and susceptibility to oxidants as well as altered levels of antioxidant enzymes in brains of Bcl-2-deficient mice. It is concluded that Bcl-2 affects cellular levels of ROS, which may be due to an effect either on their production or on antioxidant pathways.

The etiology of Parkinson's disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF-kappaB) is activated by oxidative stress, we studied the involvement of NF-kappaB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1-0.5 m M) treatment increased the phosphorylation of the IkappaB protein, the inhibitory subunit of NF-kappaB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF-kappaB-p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine-induced NF-kappaB activation was also evidenced by electromobility shift assay using radioactive labeled NF-kappaB consensus DNA sequence. Cell-permeable NF-kappaB inhibitor SN-50 rescued the cells from dopamine-induced apoptosis and showed the importance of NF-kappaB activation to the induction of apoptosis. Furthermore, flow cytometry assay demonstrated a higher level of translocated NF-kappaB-p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our findings suggest that NF-kappaB activation is essential to dopamine-induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinson's disease.

Many in vitro studies have shown that levodopa is a potent toxin which is lethal to various cultured neuronal and non-neuronal cells. The in vitro toxicity of levodopa is linked mainly to its auto-oxidation, which generates a variety of harmful free radical species including superoxide, hydrogen peroxide, and hydroxyl radicals, and also semiquinones and quinones produced via the dopa-melanin metabolic route. Such toxic effects of levodopa can be blocked by co-treatment with antioxidants, particularly thiol-containing compounds. Several studies have shown that levodopa kills cells by triggering apoptosis, an active, intrinsic cell suicide program. Exposure of cultured neurons to levodopa induced the characteristic apoptotic cascade, including cell shrinkage, membrane blebbing, and nuclear and DNA fragmentation. Although levodopa is extremely toxic in vitro, there is no evidence that it damages nigrostriatal dopaminergic neurons in vivo in experimental animals and in patients with Parkinson's disease (PD). Likewise, although there is some evidence for the occurrence of apoptosis in the parkinsonian substantia nigra, it is not known whether levodopa administration is capable of inducing or accelerating programmed cell death of residual pigmented nigral neurons in PD.