Ulrika Beitnere

Neuroprotective Properties of Mildronate, a Small Molecule, in a Rat Model of Parkinson’s Disease

Previously, we have found that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a small molecule with charged nitrogen and oxygen atoms, protects mitochondrial metabolism that is altered by inhibitors of complex I and has neuroprotective effects in an azidothymidine-neurotoxicity mouse model. In the present study, we investigated the effects of mildronate in a rat model of Parkinson’s disease (PD) that was generated via a unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). We assessed the expression of cell biomarkers that are involved in signaling cascades and provide neural and glial integration: the neuronal marker TH (tyrosine hydroxylase); ubiquitin (a regulatory peptide involved in the ubiquitin-proteasome degradation system); Notch-3 (a marker of progenitor cells); IBA-1 (a marker of microglial cells); glial fibrillary acidic protein, GFAP (a marker of astrocytes); and inducible nitric oxide synthase, iNOS (a marker of inflammation). The data show that in the 6-OHDA-lesioned striatum, mildronate completely prevented the loss of TH, stimulated Notch-3 expression and decreased the expression of ubiquitin, GFAP and iNOS. These results provide evidence for the ability of mildronate to control the expression of an array of cellular proteins and, thus, impart multi-faceted homeostatic mechanisms in neurons and glial cells in a rat model of PD. We suggest that the use of mildronate provides a protective effect during the early stages of PD that can delay or halt the progression of this neurodegenerative disease.

A Novel 1,4-Dihydropyridine Derivative Improves Spatial Learning and Memory and Modifies Brain Protein Expression in Wild Type and Transgenic APPSweDI Mice

Ca2+ blockers, particularly those capable of crossing the blood-brain barrier (BBB), have been suggested as a possible treatment or disease modifying agents for neurodegenerative disorders, e.g., Alzheimer’s disease. The present study investigated the effects of a novel 4-(N-dodecyl) pyridinium group-containing 1,4-dihydropyridine derivative (AP-12) on cognition and synaptic protein expression in the brain. Treatment of AP-12 was investigated in wild type C57BL/6J mice and transgenic Alzheimer’s disease model mice (Tg APPSweDI) using behavioral tests and immunohistochemistry, as well as mass spectrometry to assess the blood-brain barrier (BBB) penetration. The data demonstrated the ability of AP-12 to cross the BBB, improve spatial learning and memory in both mice strains, induce anxiolytic action in transgenic mice, and increase expression of hippocampal and cortical proteins (GAD67, Homer-1) related to synaptic plasticity. The compound AP-12 can be seen as a prototype molecule for use in the design of novel drugs useful to halt progression of clinical symptoms (more specifically, anxiety and decline in memory) of neurodegenerative diseases, particularly Alzheimer’s disease.

Memory-enhancing and brain protein expression-stimulating effects of novel calcium antagonist in Alzheimer’s disease transgenic female mice

The prevalence of Alzheimers disease (AD) is higher in females than in males, and causes more severe cognitive, memory and behavioral impairments. Previously, in male transgenic (Tg) APPSweDI mice, we reported that the novel lipophilic 1,4-dihydropyridine (DHP) derivative AP-12 crossed the blood-brain barrier, blocked neuronal and vascular calcium channels, changed brain protein expression and improved behavior. In this study, we used female Tg APPSweDI mice to assess the effects of AP-12 on behavior, and brain protein expression, with a particular focus on those of the GABAergic system. The results showed that in female Tg mice, similar to male Tg mice, AP-12 improved spatial learning/memory performance in the water maze test and demonstrated anxiolytic effect in the elevated zero maze (after single administration of AP-12) and elevated plus maze (after chronic injections of AP-12). In addition, we demonstrated upregulated expression of glutamate decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) in the cingulate cortex and hippocampus, pointing to the role of the GABAergic system as one of the neural networks dysregulated in AD. In both female and male mice, AP-12 did not change the expression of hippocampal Homer-1, a protein which is involved in synaptic plasticity. However, in cingulate cortex, the staining density of Homer-1 was significantly increased in female mice. Further, female mice (similar to male mice) did not show changes in brain AChE expression and in the amyloid beta load in the hippocampus and cingulate cortex. In conclusion, the memory enhancing, anxiolytic and protein expression effects of AP-12 did not show sex specificity in APPSweDI mice. Considering the ability of AP-12 to block brain calcium channels and improve memory by enhancing the GABAergic and synaptic plasticity processes, AP-12 is a promising compound which merits further pre-clinical studies to investigate its usefulness in the treatment of AD.

Mildronate improves cognition and reduces amyloid-β pathology in transgenic Alzheimer's disease mice

Mildronate, a carnitine congener drug, previously has been shown to provide neuroprotection in an azidothymidine‐induced mouse model of neurotoxicity and in a Parkinsons disease rat model. The aim of this study was to investigate the effects of mildronate treatment on cognition and pathology in Alzheimers disease (AD) model mice (APPSweDI). Mildronate was administered i.p. daily at 50 or 100 mg/kg for 28 days. At the end of treatment, the animals were behaviorally and cognitively tested, and brains were assessed for AD‐related pathology, inflammation, synaptic markers, and acetylcholinesterase (AChE). The data show that mildronate treatment significantly improved animal performance in water maze and social recognition tests, lowered amyloid‐β deposition in the hippocampus, increased expression of the microglia marker Iba‐1, and decreased AChE staining, although it did not alter expression of proteins involved in synaptic plasticity (GAP‐43, synaptophysin, and GAD67). Taken together, these findings indicate mildronates ability to improve cognition and reduce amyloid‐β pathology in a mouse model of AD and its possible therapeutic utility as a disease‐modifying drug in AD patients.

Carnitine congener mildronate protects against stress- and haloperidol-induced impairment in memory and brain protein expression in rats.

The present study investigates the efficacy of mildronate, a carnitine congener, to protect stress and haloperidol-induced impairment of memory in rats and the expression of brain protein biomarkers involved in synaptic plasticity, such as brain-derived neurotrophic factor (BDNF), acetylcholine esterase and glutamate decarboxylase 67 (GAD67). Two amnesia models were used: 2h immobilization stress and 3-week haloperidol treatment. Stress caused memory impairment in the passive avoidance test and induced a significant 2-fold BDNF elevation in hippocampal and striatal tissues that was completely inhibited by mildronate. Mildronate decreased the level of GAD67 (but not acetylcholine esterase) expression by stress. Haloperidol decrease by a third hippocampal BDNF and acetylcholine esterase (but not GAD67) expression, which was normalized by mildronate; it also reversed the haloperidol-induced memory impairment in Barnes test. The results suggest the usefulness of mildronate as protector against neuronal disturbances caused by stress or haloperidol.

Very low doses of muscimol and baclofen ameliorate cognitive deficits and regulate protein expression in the brain of a rat model of streptozocin-induced Alzheimer’s disease

Abstract Recent studies devoted to neuroprotection have focused on the role of the gamma‐aminobutyric acid (GABA) system in regulating neuroinflammatory processes which play a key role in the neurodegenerative processes observed in Alzheimers disease (AD) by inducing glial cell overactivation and impairing neurotransmission. Data on the efficacy of classical GABA‐A and GABA‐B receptor agonists (muscimol and baclofen, respectively) in animal models of AD are not available. Moreover, no published studies have examined the ability of optimal doses of these compounds to prevent neuroinflammation, the alterations in neurotransmission and cognitive deficits. In the present study, we used a non‐transgenic rat model of AD obtained by intracerebroventricular streptozocin (STZ) injection and assessed the effects of muscimol and baclofen at very low doses (0.01–0.05 mg/kg) on spatial memory and the expression of cortical and hippocampal proteins related to neuroinflammation, namely proteins involved in astroglial functions (glial fibrillary acidic protein, GFAP), GABA synthesis (GABA synthesizing enzyme, glutamic acid decarboxylase 67, GAD67) and acetylcholine degradation (acetylcholine esterase). The presented study demonstrated that in a rat model of STZ‐induced AD both muscimol and baclofen at the tested doses exerted memory‐enhancing and anti‐inflammatory effects, as well as normalization of acetylcholine esterase and GABA expression. We suggested that the function of very low doses of GABA receptor agonists differs from typical GABA‐related inhibition and may be mediated by the allosteric sites of GABA receptors or other non‐specific cell regulatory pathways.

Spruce Needle Polyprenols Protect Against Atorvastatin-Induced Muscle Weakness and do not Influence Central Nervous System Functions in Rats

Abstract Polyprenols (PPs) have been identified in almost all living organisms. The richest source of PPs is the needles of conifer trees. Endogenously, PPs, similarly to cholesterol, are synthesised in human and animal cells via the mevalonate pathway. Previous studies have demonstrated the anti-oxidant properties of PPs. To our knowledge, no studies have been published on the influence of PPs on muscle strength. We hypothesised that administration of PPs could prevent changes in muscle functioning caused by statins (weakness, etc.). In the present study, atorvastatin (80 mg/kg) was used as a model compound. PPs at doses 1, 10 and 20 mg/kg were administered. Both drugs were given per os for 16 days. The influence of atorvastatin, PPs and their combination on behaviour, muscle strength, plasma cholesterol and creatine kinase activity was assessed in female Wistar rats. Our data demonstrated that atorvastatin considerably impaired muscle strength, whereas PPs protected that effect. Neither PPs, nor atorvastatin influenced plasma cholesterol levels, whereas PPs at dose 20 mg/kg elevated creatine kinase activity by about 25%. PPs at the tested doses did not alter behaviour, indicating safety of central nervous system functions. The obtained data suggest usefulness of PPs as a complement in statin therapy to reduce muscle-related side effects.

GABAA agonist muscimol ameliorates learning/memory deficits in streptozocin-induced Alzheimer’s disease non-transgenic rat model

Background: GABAergic inhibitory action regulates learning/memory processes and contributes to neurotransmission (Gong et al., 2009). Existing evidence suggests GABAergic system is involved in pathophysiology of Alzheimer’s disease (AD) via inhibitory interneuron deficits (Verret et al., 2012) and decrease in functional GABAA receptors (Limon et al., 2012). In vitro, GABA and muscimol (GABAA receptor agonist) blocked neuronal death induced by Aβ in rat hippocampal and cortical neurons (Paula-Lima et al., 2005). Our concept: low doses of muscimol may prevent learning/memory deficits in intracerebroventricular (icv) streptozocin (STZ)-induced AD non-transgenic rat model. Methods. Wistar male rats (280±20 g) were pre-treated with saline (control) or muscimol (0.01 and 0.05 mg/kg) for 3 days. On day 4, rats received icv STZ (100 μg/ml) or aCSF. From day 18, rats received muscimol or saline for 4 days; rat spatial learning and memory were assessed in water maze test (4 trials/day) by recording the time to reach the hidden platform (escape latency). A probe trial without platform was carried out 24 h after the training trials, and the number of platform zone crossings has been recorded. Results. STZ statistically increased the escape latency vs. control group (p<0.0001). Muscimol at both doses significantly decreased the escape latency in STZ rats vs. STZ, reversing STZ effect by about 90% on days 3 and 4 (p<0.0001). In probe trial, the number of platform crossings in muscimol+STZ rats’ was significantly increased vs. STZ rats. Muscimol at both doses per se showed values comparable to control. Conclusions. Obtained data suggest that icv STZ significantly decreased rat spatial learning and memory and learning ability. Muscimol at both low doses significantly improved rats’ learning and memory abilities in both normal and AD-type rats. One may suggest that intensification of GABAergic processes may be a useful pharmacotherapeutic strategy to halt early memory decline in AD.