LETC inhibits α-Syn aggregation and ameliorates motor deficiencies in the L62 mouse model of synucleinopathy.

Alpha-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). Here, we explored the efficacy of N,N,N',N'-tetraethyl-10H-phenothiazine-3,7-diamine dihydrochloride (LETC), a protein aggregation inhibitor, on α-Syn aggregation. In both cellular models and transgenic mice, α-Syn aggregation was achieved by the overexpression of full-length human α-Syn fused with a signal sequence peptide. α-Syn accumulated in transfected DH60.21 neuroblastoma cells and α-Syn aggregation was inhibited by LETC with an EC50 of 0.066 ± 0.047 µM. Full-length human α-Syn overexpressing Line 62 (L62) mice accumulated neuronal α-Syn that was associated with a decreased motor performance in the open field and automated home cage. LETC, administered orally for 6 weeks at 10 mg/kg significantly decreased α-Syn-positive neurons in multiple brain regions and this resulted in a rescue of movement deficits in the open field in these mice. LETC however, did not improve activity deficits of L62 mice in the home cage environment. The results suggest that LETC may provide a potential disease modification therapy in synucleinopathies through the inhibition of α-Syn aggregation.

Parvalbumin-containing GABA cells and schizophrenia: experimental model based on targeted gene delivery through adeno-associated viruses.

Understanding the contribution of transmitter systems in behavioural pharmacology has a long tradition. Multiple techniques such as transmitter-specific lesions, and also localized administration of pharmacological toxins including agonists and antagonists of selected receptors have been applied. More recently, modern genetic tools have permitted cell-type selective interferences, for example by expression of light-sensitive channels followed by optogenetic stimulation in behaviourally meaningful settings or by engineered channels termed DREADDS that respond to peripherally administered drugs. We here took a similar approach and employed a Cre recombinase-dependent viral delivery system (adeno-associated virus) to express tetanus toxin light chain (TeLc) and thus, block neural transmission specifically in parvalbumin-positive (PV+) neurons of the limbic and infralimbic prefrontal circuitry. PV-TeLc cohorts presented with normal circadian activity as recorded in PhenoTyper home cages, but a reproducible increase in anxiety was extracted in both the open field and light-dark box. Interestingly, working memory assessed in a spontaneous alternation Y-maze task was impaired in PV-TeLc mice. We also recorded local field potentials from a separate cohort and found no global changes in brain activity, but found a behaviourally relevant lack of modulation in the gamma spectral band. These anomalies are reminiscent of endophenotypes of schizophrenia and appear to be critically dependent on GABAergic signalling through PV neurones. At the same time, these observations validate the use of viral vector delivery and its expression in Cre-lines as a useful tool for understanding the role of selective components of the brain in behaviour and the underpinning physiology.

A Protein Aggregation Inhibitor, Leuco-Methylthioninium Bis(Hydromethanesulfonate), Decreases α-Synuclein Inclusions in a Transgenic Mouse Model of Synucleinopathy.

α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). We have tested whether N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate) (leuco-methylthioninium bis(hydromethanesulfonate); LMTM), a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn) fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 µM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62) transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK)-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.

In-vivo evidence that high mobility group box 1 exerts deleterious effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model and Parkinson's disease which can be attenuated by glycyrrhizin.

High-mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that is released during tissue damage from immune and non-immune cells - including microglia and neurons. HMGB1 can contribute to progression of numerous chronic inflammatory and autoimmune diseases which is mediated in part by interaction with the receptor for advanced glycation endproducts (RAGE). There is increasing evidence from in vitro studies that HMGB1 may link the two main pathophysiological components of Parkinson's disease (PD), i.e. progressive dopaminergic degeneration and chronic neuroinflammation which underlie the mechanistic basis of PD progression. Analysis of tissue and biofluid samples from PD patients, showed increased HMGB1 levels in human postmortem substantia nigra specimens as well as in the cerebrospinal fluid and serum of PD patients. In a mouse model of PD induced by sub-acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), systemic administration of neutralizing antibodies to HMGB1 partly inhibited the dopaminergic cell death, and reduced the increase of RAGE and tumour necrosis factor-alpha. The small natural molecule glycyrrhizin, a component from liquorice root which can directly bind to HMGB1, both suppressed MPTP-induced HMGB1 and RAGE upregulation while reducing MPTP-induced dopaminergic cell death in a dose dependent manner. These results provide first in vivo evidence that HMGB1 serves as a powerful bridge between progressive dopaminergic neurodegeneration and chronic neuroinflammation in a model of PD, suggesting that HMGB1 is a suitable target for neuroprotective trials in PD.

Evidence for a role of adaptive immune response in the disease pathogenesis of the MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease and results from the loss of dopaminergic neurons of the nigrostriatal pathway. The pathogenesis of PD is poorly understood, but inflammatory processes have been implicated. Indeed increases in the number of major histocompatibility complex II (MHC II) reactive cells have long been recognised in the brains of PD patients at post-mortem. However whether cells expressing MHC II play an active role in PD pathogenesis has not been delineated. This was addressed utilising a transgenic mouse null for MHC II and the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In wild-type mice MHC II levels in the ventral midbrain were upregulated 1-2 days after MPTP treatment and MHC II was localized in both astrocytes and microglia. MHC II null mice showed significant reductions in MPTP-induced dopaminergic neuron loss and a significantly reduced invasion of astrocytes and microglia in MHC II null mice receiving MPTP compared with controls. In addition, MHC II null mice failed to show increases in interferon-γ or tumour necrosis factor-α in the brain after MPTP treatment, as was found in wild-type mice. However, interleukin-1ß was significantly increased in both wild-type and MHC II null mice. These data indicate that in addition to microglial cell/myeloid cell activation MHC Class II-mediated T cell activation is required for the full expression of pathology in this model of PD.

Polymeric alkylpyridinium salts permit intracellular delivery of human Tau in rat hippocampal neurons: requirement of Tau phosphorylation for functional deficits.

Patients suffering from tauopathies including frontotemporal dementia (FTD) and Alzheimer's disease (AD) present with intra-neuronal aggregation of microtubule-associated protein Tau. During the disease process, Tau undergoes excessive phosphorylation, dissociates from microtubules and aggregates into insoluble neurofibrillary tangles (NFTs), accumulating in the soma. While many aspects of the disease pathology have been replicated in transgenic mouse models, a region-specific non-transgenic expression model is missing. Complementing existing models, we here report a novel region-specific approach to modelling Tau pathology. Local co-administration of the pore-former polymeric 1,3-alkylpyridinium salts (Poly-APS) extracted from marine sponges, and synthetic full-length 4R recombinant human Tau (hTau) was performed in vitro and in vivo. At low doses, Poly-APS was non-toxic and cultured cells exposed to Poly-APS (0.5 µg/ml) and hTau (1 µg/ml; ~22 µM) had normal input resistance, resting-state membrane potentials and Ca(2+) transients induced either by glutamate or KCl, as did cells exposed to a low concentration of the phosphatase inhibitor Okadaic acid (OA; 1 nM, 24 h). Combined hTau loading and phosphatase inhibition resulted in a collapse of the membrane potential, suppressed excitation and diminished glutamate and KCl-stimulated Ca(2+) transients. Stereotaxic infusions of Poly-APS (0.005 µg/ml) and hTau (1 µg/ml) bilaterally into the dorsal hippocampus at multiple sites resulted in hTau loading of neurons in rats. A separate cohort received an additional 7-day minipump infusion of OA (1.2 nM) intrahippocampally. When tested 2 weeks after surgery, rats treated with Poly-APS+hTau+OA presented with subtle learning deficits, but were also impaired in cognitive flexibility and recall. Hippocampal plasticity recorded from slices ex vivo was diminished in Poly-APS+hTau+OA subjects, but not in other treatment groups. Histological sections confirmed the intracellular accumulation of hTau in CA1 pyramidal cells and along their processes; phosphorylated Tau was present only within somata. This study demonstrates that cognitive, physiological and pathological symptoms reminiscent of tauopathies can be induced following non-mutant hTau delivery into CA1 in rats, but functional consequences hinge on increased Tau phosphorylation. Collectively, these data validate a novel model of locally infused recombinant hTau protein as an inducer of Tau pathology in the hippocampus of normal rats; future studies will provide insights into the pathological spread and maturation of Tau pathology.

Comparison of automated home-cage monitoring systems: emphasis on feeding behaviour, activity and spatial learning following pharmacological interventions.

BACKGROUND: Different automated systems have been developed to facilitate long-term and continuous assessment of behaviours including locomotor activity, feeding behaviour and circadian activity. NEW METHOD: This study assessed the effectiveness of three different observation systems as methods for determining strain and pharmacological induced differences in locomotor activity, feeding behaviour and spatial learning. The effect of the CB1 antagonist AM251 on feeding behaviour was determined in the PhenoMaster and PhenoTyper. Next, effects of cholinergic (scopolamine) and glutamatergic (Phenylcyclidine, PCP) receptor antagonism and dopaminergic agonism (apomorphine) on activity were assessed in the PhenoTyper and IntelliCage. Finally, the IntelliCage was utilised to determine differences in activity and spatial learning of C57BL/6 and DBA/2 mouse strains following pharmacological intervention. RESULTS: AM251 induced a suppression of food intake, feeding behaviour and a reduction in body weight in both the PhenoTyper and PhenoMaster. Apomorphine reduced activity in both the PhenoTyper and IntelliCage. Whereas, decreased activity was evident with PCP in the PhenoTyper, but not IntelliCage and Scopolamine induced a trend towards elevated levels of activity in the IntelliCage but not PhenoTyper. Strain differences in activity and spatial learning were also evident, with increased corner visits and drug induced impairments only observed with C57BL/6 mice. COMPARISON WITH EXISTING METHOD: The automated home cage observation systems determined similar drug and strain effects on behaviour to those observed using traditional methods. CONCLUSIONS: All three observation systems reported drug-induced changes in behaviour however, they differ in their application of spatial learning tasks and utilisation of single versus group housed recordings.

In vitro modelling of Alzheimer's disease: degeneration and cell death induced by viral delivery of amyloid and tau.

With increasing life expectancy, Alzheimer's disease (AD) and other dementias pose an increasing and as yet unresolved health problem. A variety of cellular models of AD has helped to decipher some key aspects of amyloid and tau related degeneration. The initial approach of extracellular applications of synthetic peptides has now been replaced by the introduction of amyloid precursor protein (APP) and tau genes. In the present study adenoviral transductions were exploited for gene delivery into primary rat hippocampal and dorsal root ganglion (DRG) cultures to enable comparative and mechanistic studies at the cellular level and subsequent drug testing. Time lapse experiments revealed a different pattern of cell death: apoptotic-like for APP whereas tau positive cells joined and formed clusters. Mutated human APP or tau expression caused accelerated neuronal damage and cell death (cf. EGFP: -50% for APP at 5 days; -40% for tau at 3 days). This reduction in viability was preceded by decreased excitability, monitored via responses to depolarising KCl-challenges in Ca(2+) imaging experiments. Additionally, both transgenes reduced neurite outgrowth in DRG neurones. Treatment studies confirmed that APP induced-damage can be ameliorated by ß- and γ-secretase inhibitors (providing protection to 60-100% of control levels), clioquinol (80%) and lithium (100%); while anti-aggregation treatments were beneficial for tau-induced damage (60-90% recovery towards controls). Interestingly, caffeine was the most promising drug candidate for therapeutic intervention with high efficacy in both APP (77%) and tau-induced models (72% recovery). Overall, these cellular models offer advantages for mechanistic studies and target identification in AD and related disorders.

Bi-directional alterations of LTP after acute homocysteine exposure.

Homocysteine (HCY) is a known risk factor for neuronal diseases. We here report that HCY (10-1000 microM) interfered bi-directionally with long-term potentiation (LTP) in hippocampal slices, causing an impairment at concentrations <100 microM, and enhancement > or =500 microM. By comparison, NMDA unidirectionally reduced LTP, whereas l-cysteine led to facilitated LTP. Such HCY-induced alterations in neuronal communication may contribute to cognitive failure in dementia.

Scopolamine and MK801-induced working memory deficits in rats are not reversed by CBD-rich cannabis extracts.

Smoking marijuana causes working and short-term memory deficits, an effect that is mediated by cannabinoid receptor (CB1) activation in the brain. While this may be due to the main psychoactive constituent Delta9-tetrahydrocannabinol (Delta9-THC), plant extracts also contain other cannabinoid and terpenoid compounds with unknown properties. Towards this end, we have recently shown that high concentrations of plant extracts rich in cannabidiol (CBD) can reverse working memory deficits induced by Delta9-THC which is a remaining contaminant of this extract [Fadda P, Robinson L, Fratta W, Pertwee RG, Riedel G. Differential effects of THC- and CBD-rich cannabis-extracts on working memory in rats. Neuropahrmacology 2004;47:1170-9]. Since this effect was dose-dependent and indicative of memory enhancing qualities of the CBD-rich extract, this prompted a wider investigation into the effects of CBD on other forms of amnesia in order to determine the mechanism of action and to reveal its potency against anticholinergic and antiglutamatergic agents. We employed a spatial delayed matching to position task in the open-field water maze. Both scopolamine (0.2 mg/kg i.p.) and dizocilpine (MK801: 0.1mg/kg i.p.) impaired working memory at delays of 30 s and 4 h. Two doses of CBD-rich extracts (5 and 10 mg/kg), which did not affect working memory when given alone, were unable to reverse these deficits when co-administered with scopolamine or MK801. These data suggest that reversal of working memory deficits by CBD-rich extracts are specific to the cannabinoid system and do not compensate for acutely induced cholinergic or glutamatergic receptor hypoactivity.

Altered cellular distribution of phospho-tau proteins coincides with impaired retrograde axonal transport in neurons of aged rats.

We hypothesize that the age-related degeneration of cytoskeleton in basal forebrain cholinergic neurons renders the NGF-TrkA signaling system non-functional and thereby impairs trophic support. Comparing young (4 months) and aged (28 months) rat brain, we examined immunohistochemically the compartmentalization of phosphorylated Tau protein using antibodies phospho-Tau404 and phospho-Tau231 of the GSK3beta kinase, known to phosphorylate Tau, the neurotrophin NGF, and its receptor P-TrkA. Retrograde labeling of basal forebrain cholinergic cells after injection of fluorogold into multiple sites in cortex and hippocampus revealed a significantly lower number of fluorogold-positive cells in aged brain. Despite a lower density of P-TrkA immunoreactivity in cortex and hippocampus of aged rats, there was no difference in NGF expression. In young animals phospho-Tau404, phospho-Tau231, and GSK3 immunoreactivity was observed mainly in neuronal fibers with lower staining in somata both in cortex and hippocampus. By contrast, Tau and GSK3 labeling were confined to the cell bodies in aged rats. This is confirmation that aging leads to a redistribution of cytoskeletal proteins. Since a somatic localization of phospho-Tau is indicative of cytoskeletal breakdown, we suggest that failure of axonal trafficking may be responsible for the lack of trophic support in aged cholinergic neurons of the basal forebrain.

Long-term study of chronic oral aluminum exposure and spatial working memory in rats.

The authors report an effort to advance animal models that mimic the cognitive decline of Alzheimer's disease. Rats were trained and repeatedly tested in a spatial delayed matching-to-position paradigm in the water maze, with the location of the submerged platform changing between, but not within, days. After Trial 1 (random search) and intertrial intervals of 30 s or 1 hr, memory was tested in Trial 2. Young rats quickly acquired this task and were repeatedly tested after different intervals over 7 months, with a slight increase in performance toward the end of testing, but no difference in latencies between delays. Oral long-term treatment of 1 group with 0.1% aluminum caused no delay-dependent working memory deficit. This testing protocol may enable between- and within-subject long-term assessment of spatial working memory before and after drug treatment and may prove useful in animal models of progressive cognitive decline.