James Raymick

Neuroprotective effect of the chemical chaperone, trehalose in a chronic MPTP-induced Parkinson's disease mouse model

Parkinsons disease (PD) is a progressive motor disease of unknown etiology in the majority of cases. The clinical features of PD emerge due to selective degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release DA. In the current in vivo mouse model study, we tested trehalose for its ability to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage to DA neurons. Trehalose is a naturally occurring disaccharide present in plants and animals and appears capable of protecting cells against various environmental stresses. The effect of trehalose is likely due to its action as a pharmacological chaperone which promotes protein stability. In the present study, there were four treatment groups: saline only (control); probenecid only; MPTP+probenecid; and trehalose+MPTP+probenecid. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the ventral midbrain SNc and CPu were significantly reduced by trehalose. Decreases in CPu dopamine levels produced by MPTP were also blocked by trehalose. Microglial activation and astrocytic hypertrophy induced by MPTP were greatly reduced by trehalose, indicating protection against neuroinflammation. These effects are commensurate with the observed trehalose sparing of motor deficits produced by MPTP in this mouse model. Two tight junctional proteins, ZO-1 and occludin, are downregulated following MPTP treatment and trehalose blocks this effect. Likewise, the glucose transporter-1 that is expressed in brain endothelial cells is also protected by trehalose from MPTP-induced down-regulation. This study is the first to demonstrate using fluoro-turoquoise FT gel perfusion techniques, the protection afforded by trehalose from MPTP-induced damage to microvessels and endothelial and suggests that trehalose therapy may have the potential to slow or ameliorate PD pathology.

Neuroprotective and Therapeutic Strategies against Parkinson's Disease: Recent Perspectives.

Parkinsonism is a progressive motor disease that affects 1.5 million Americans and is the second most common neurodegenerative disease after Alzheimer’s. Typical neuropathological features of Parkinson’s disease (PD) include degeneration of dopaminergic neurons located in the pars compacta of the substantia nigra that project to the striatum (nigro-striatal pathway) and depositions of cytoplasmic fibrillary inclusions (Lewy bodies) which contain ubiquitin and α-synuclein. The cardinal motor signs of PD are tremors, rigidity, slow movement (bradykinesia), poor balance, and difficulty in walking (Parkinsonian gait). In addition to motor symptoms, non-motor symptoms that include autonomic and psychiatric as well as cognitive impairments are pressing issues that need to be addressed. Several different mechanisms play an important role in generation of Lewy bodies; endoplasmic reticulum (ER) stress induced unfolded proteins, neuroinflammation and eventual loss of dopaminergic neurons in the substantia nigra of mid brain in PD. Moreover, these diverse processes that result in PD make modeling of the disease and evaluation of therapeutics against this devastating disease difficult. Here, we will discuss diverse mechanisms that are involved in PD, neuroprotective and therapeutic strategies currently in clinical trial or in preclinical stages, and impart views about strategies that are promising to mitigate PD pathology.

Efficacy and toxicity of clioquinol treatment and A-beta42 inoculation in the APP/PSI mouse model of Alzheimer's disease.

Alzheimers disease (AD), the most common human neurodegenerative disease, is characterized pathologically by numerous deposits of amyloid plaques in the brain. Systemic administration of clioquinol (CQ) and inoculation with amyloid-beta42 (Aβ42) vaccines have been demonstrated to significantly inhibit deposits of amyloid in AD brains. However, each of these treatments has also been reported to be neurotoxic. The generation of transgenic mice models of AD has made it possible to study aspects of this disease employing experimental animals. In the present study, we investigated the efficacy and toxicity of CQ and Aβ42 vaccine in a transgenic AD (APP/PS1) mouse model. Our results confirmed that both CQ and Aβ42 vaccine were effective in significantly reducing the deposits of amyloid in the brains of transgenic AD mice. We also report here that systemic CQ induces myelinopathies in the dorsal lateral geniculate nucleus (DLG), which was almost devoid of amyloid plaques and is the primary site of retinal efferent projections via the optic nerve. This is the first report that systemic administration of CQ causes myelinopathies in the central nervous system (CNS) of a transgenic AD mouse model as well as wild-type mice. Inoculation with an Aβ42 vaccine was also found, for the first time, to result in a significant increase in plaque-independent astrocytic hyperplasia in the dorsal part of the lateral septal nucleus (LSD) which was also devoid of plaques, reflecting potential brain inflammatory processes.

Characterization of myelin pathology in the hippocampal complex of a transgenic mouse model of Alzheimer's disease.

We have characterized the myelin changes observed within the hippocampal complex (HC) of a transgenic (Tg) mouse model of Alzheimers disease (AD). Individual myelinated fibers were labeled with Black-Gold II while amyloid plaques were labeled with either Congo Red or Pan-A-beta immunofluoresence. Myelinated fibers were never seen passing through amyloid plaques in any region, while conspicuous myelin pathology was seen within, and immediately adjacent to, the amyloid plaques in the HC of the AD-Tg mouse. This pathology consisted of a complete disruption of myelinated fibers passing through the plaque and the region immediately adjacent to the plaques exhibited an edematous swelling of the fibers. This pathology was most frequently observed within the molecular and polymorph layers of the dentate gyrus and the molecular layer of Ammons horn. The remaining layers of Ammons horn exhibited minimal myelin pathology, while moderate myelinopathy was observed in the subiculum. Since the HC is integral for memory function, these findings may help account for the memory problems so characteristic of the disease process. Because the molecular layers of the dentate gyrus and Ammons horn are the sites of inputs to the HC, the extensive myelin pathology observed in these regions would imply functional deafferentation of the HC. The appearance of some Black-Gold II positive debris within the plaques may reflect a possible cascade mechanism whereby the presence of plaques results in myelin degeneration, some of which is incorporated within the plaque, causing it to further expand in a self-perpetuating fashion.

Introducing Amylo-Glo, a novel fluorescent amyloid specific histochemical tracer especially suited for multiple labeling and large scale quantification studies

One of the hallmark pathologies associated with Alzheimers disease (AD) is the conspicuous deposition of extracellular amyloid plaques within the forebrain. These plaques are primarily composed of fibrular aggregates of the A-beta peptide. Traditional methods for the histological localization of these plaques typically rely on the use of the tracers Congo Red or Thioflavin S. This study describes the characterization of a novel fluorescent histochemical probe, Amylo-Glo, for the high resolution and contrast localization of amyloid plaques in brain tissue sections. Potential advantages over conventional amyloid plaque stains such as Congo Red or Thioflavin S can be attributed to its unique chemical and spectral properties. Specifically, it results in a very bright blue UV excitable stain under physiological conditions that will not bleed through when illuminated with other filters. Its brightness makes it ideal for low magnification quantification studies, while its unique excitation/emission profile and mild staining conditions makes it ideal for combination with multiple immunofluorescent labeling studies.

Combination therapy with lenalidomide and nanoceria ameliorates CNS autoimmunity

OBJECTIVE Multiple sclerosis (MS) is a debilitating neurological disorder involving an autoimmune reaction to oligodendrocytes and degeneration of the axons they ensheath in the CNS. Because the damage to oligodendrocytes and axons involves local inflammation and associated oxidative stress, we tested the therapeutic efficacy of combined treatment with a potent anti-inflammatory thalidomide analog (lenalidomide) and novel synthetic anti-oxidant cerium oxide nanoparticles (nanoceria) in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. METHODS C57BL/6 mice were randomly assigned to a control (no EAE) group, or one of the four myelin oligodendrocyte glycoprotein-induced EAE groups: vehicle, lenalidomide, nanoceria, or lenalidomide plus nanoceria. During a 23 day period, clinical EAE symptoms were evaluated daily, and MRI brain scans were performed at 11-13 days and 20-22 days. Histological and biochemical analyses of brain tissue samples were performed to quantify myelin loss and local inflammation. RESULTS Lenalidomide treatment alone delayed symptom onset, while nanoceria treatment had no effect on symptom onset or severity, but did promote recovery; lenalidomide and nanoceria each significantly attenuated white matter pathology and associated inflammation. Combined treatment with lenalidomide and nanoceria resulted in a near elimination of EAE symptoms, and reduced white matter pathology and inflammatory cell responses to a much greater extent than either treatment alone. INTERPRETATION By suppressing inflammation and oxidative stress, combined treatment with lenalidomide and nanoceria can reduce demyelination and associated neurological symptoms in EAE mice. Our preclinical data suggest a potential application of this combination therapy in MS.

Histopathological and electrophysiological indices of rotenone-evoked dopaminergic toxicity: Neuroprotective effects of acetyl-l-carnitine

Exposure to the natural pesticide, rotenone, a potent mitochondrial toxin, leads to degeneration in striatal nerve terminals and nigral neurons. Rotenone-induced behavioral, neurochemical and neuropathological changes in rats mimic those observed in Parkinsons disease (PD). Here, protective effects of acetyl-L-carnitine (ALC) in the brain dopaminergic toxicity after a prolonged exposure to rotenone were evaluated using electrophysiological and immunolabeling methods. Adult, male Sprague-Dawley rats were injected i.p. with rotenone alone (1 mg/kg) or rotenone with ALC (either 10 or 100 mg/kg; ALC10 or ALC100, respectively) once daily on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, 31, 33 and 37. Control rats received either 100mg/kg ALC or vehicle (30% Solutol HS 15 in 0.9% saline) injections. Animals were weighed on injection days and monitored daily. Motor nerve conduction velocity (MCV) was assessed within two days after treatment using compound muscle action potentials (CMAP) detected from the tail muscle through surface receiver electrodes installed around the distal part of the tail. Rats were perfused immediately after testing with 4% paraformaldehyde and immunohistochemical analysis of dopamine transporter (DAT), tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and microglial CD11b marker was performed in the caudate-putamen (CPu) and the substantia nigra pars compacta (SNc) in order to estimate dopaminergic neuronal and transporter damage. Additionally, effects of ALC on preventing microglial or astrocytic hypertrophy were also evaluated. In rats exposed to rotenone and rotenone/ACL10, a significant increases in both proximal (S1) and distal (S2) motor latency and a decrease in MCV were detected in tail nerves (p<0.05). The conduction parameters in rats co-treated with rotenone/ACL100 were not different from control. It was found that 100 mg/kg ALC prevented loss of TH and a decline of DAT level in the midbrain and also prevented the activation of both microglia and astroglia after rotenone treatment. Data indicate neuroprotective effects of ALC in rotenone-evoked dopaminergic neurotoxicity.

In situ demonstration of Fluoro-Turquoise conjugated gelatin for visualizing brain vasculature and endothelial cells and their characterization in normal and kainic acid exposed animals.

The present study describes a new method for the visualization of the vasculature lumen and endothelial cells and characterizes their morphology in the brains of normal and kainic acid (KA) treated rats. Herein, labeling was accomplished using Fluoro-Turquoise (FT), a novel reactive blue fluorochrome conjugated to gelatin. Strong blue fluorescence was observed throughout the brain vasculature following intra-cardiac perfusion with FT-gel in normal animals. However, in the brains of KA treated rats (hippocampus, midline and ventral thalamus, piriform cortex), the vascular lumen was typically constricted, sclerotic and only faintly stained. The advantages of FT-gel over other markers can be attributed to its unique chemical and spectral properties. Specifically, Fluoro-Turquoise is a very bright blue UV excitable dye that does not bleed through when visualized using other filters, making it ideal for multiple immunofluorescent labeling studies. Its brightness at low magnification also makes it ideal for low magnification whole brain imaging. Compared to alternative techniques for visualizing blood vessels, such as India ink, fluorescent dye-conjugated dextran, the corrosion technique, endothelial cell markers and lectins, the present method results in excellent visualization of blood vessels.

Temporal Progression of Kainic Acid Induced Changes in Vascular Laminin Expression in Rat Brain with Neuronal and Glial Correlates

We recently demonstrated a dramatic up regulation of laminin expression within the blood vessels of brain regions vulnerable to excitotoxin mediated neuronal degeneration. Although this effect was clearly demonstrable at 2 days post kainic acid exposure, its expression at shorter and longer post-dosing intervals has not been reported. Therefore, a primary goal of the present study was to characterize the laminin labeling at intervals ranging from 4 hours to 2 months following i.p. injection of kainic acid. To better characterize the nature and possible underlying mechanism of action of the changes in laminin expression, both Fluoro-Jade C and GFAP immunohistochemistry were employed respectively at all survival intervals. At the shortest intervals examined (4hr, 8hr), Fluoro-Jade C positive cells could be detected in the hippocampus, thalamus, and piriform cortex. In these same regions, both vascular laminin and astrocytic GFAP expression were up regulated. At intermediate survival intervals (2, 5, 14, and 21 days), the respective labeling of degenerating neurons, astrocytes and capillaries were all maximal. Morphologically, Fluoro-Jade C labeled degenerating neurons were labeled in their entirety, GFAP positive astrocytes appeared hypertrophic and blood vessels took on a fragmented appearance. Hypertrophied GFAP positive astrocytes were conspicuous around periphery of the lesion but absent within the core of the lesion at these times. At longer survival intervals (1-2 months), the number of FJ-C labeled degenerating neurons was greatly reduced, while GFAP staining essentially returned to base line and laminin expression remained noticeably elevated, although the vessels appeared to be intact morphologically. These data allow for speculation on possible mechanisms underlying these events.

K114 Inhibits A-beta Aggregation and Inflammation In Vitro and In Vivo in AD/Tg Mice

Alzheimers disease (AD) is the most common age related human neurodegenerative disorder. The major histopathological characteristics of the AD brain are extracellular amyloid-beta (Aβ) peptide loaded plaques and intraneuronal neurofibrillary tangles made of phosphorylated tau proteins. Amyloid plaques consist primarily of aggregated Aβ1-42 and Aβ1-40 peptides. The aim of our current study was to test novel ligands/agents with the potential to disrupt or inhibit the aggregation of Aβ peptide, specifically K114, (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene, which was initially developed as a potential positron emission tomography (PET) ligand for the in vivo detection of amyloid plaques. Systemic administration of K114 has been shown in the AD/transgenic (Tg) mouse model to be capable of crossing the blood-brain barrier (BBB) and be colocalized with amyloid plaques. In this study we determined whether K114 has the potential to inhibit Aβ aggregation in vitro in AD/Tg mice and also tested, in vivo, whether chronic daily orally administered K114 has any therapeutic potential as evidenced by inhibition or reduction of the deposits of amyloid aggregates in the brains of AD/Tg mice. Our results demonstrated that K114 strongly blocked, in vitro, the aggregation of Aβ peptide in the amyloid plaques of AD/Tg mouse brain. Systemic treatment with K114 was also effective in significantly reducing the deposits of amyloid plaques in the brains of living transgenic AD mice. Additionally, K114 significantly inhibited the typically observed plaque associated astrocytic activation, as revealed by GFAP immunohistochemistry, suggesting possible anti-inflammatory properties.