Ana Knezovic

What have we learned from the streptozotocin-induced animal model of sporadic Alzheimer’s disease, about the therapeutic strategies in Alzheimer’s research

Experimental models that faithfully mimic the developmental pathology of sporadic Alzheimer’s disease (sAD) in humans are important for testing the novel therapeutic approaches in sAD treatment. Widely used transgenic mice AD models have provided valuable insights into the molecular mechanisms underlying the memory decline but, due to the particular β-amyloid-related gene manipulation, they resemble the familial but not the sporadic AD form, and are, therefore, inappropriate for this purpose. In line with the recent findings of sAD being recognised as an insulin resistant brains state (IRBS), a new, non-transgenic, animal model has been proposed as a representative model of sAD, developed by intracerebroventricular application of the betacytotoxic drug streptozotocin (STZ-icv). The STZ-icv-treated animals (mostly rats and mice) develop IRBS associated with memory impairment and progressive cholinergic deficits, glucose hypometabolism, oxidative stress and neurodegeneration that share many features in common with sAD in humans. The therapeutic strategies (acetylcholinesterase inhibitors, antioxidants and many other drugs) that have been tested until now on the STZ-icv animal model have been reviewed and the comparability of the drugs’ efficacy in this non-transgenic sAD model and the results from clinical trials on sAD patients, evaluated.

Long-term oral galactose treatment prevents cognitive deficits in male Wistar rats treated intracerebroventricularly with streptozotocin

Basic and clinical research has demonstrated that dementia of sporadic Alzheimers disease (sAD) type is associated with dysfunction of the insulin-receptor (IR) system followed by decreased glucose transport via glucose transporter GLUT4 and decreased glucose metabolism in brain cells. An alternative source of energy is d-galactose (the C-4-epimer of d-glucose) which is transported into the brain by insulin-independent GLUT3 transporter where it might be metabolized to glucose via the Leloir pathway. Exclusively parenteral daily injections of galactose induce memory deterioration in rodents and are used to generate animal aging model, but the effects of oral galactose treatment on cognitive functions have never been tested. We have investigated the effects of continuous daily oral galactose (200 mg/kg/day) treatment on cognitive deficits in streptozotocin-induced (STZ-icv) rat model of sAD, tested by Morris Water Maze and Passive Avoidance test, respectively. One month of oral galactose treatment initiated immediately after the STZ-icv administration, successfully prevented development of the STZ-icv-induced cognitive deficits. Beneficial effect of oral galactose was independent of the rat age and of the galactose dose ranging from 100 to 300 mg/kg/day. Additionally, oral galactose administration led to the appearance of galactose in the blood. The increase of galactose concentration in the cerebrospinal fluid was several times lower after oral than after parenteral administration of the same galactose dose. Oral galactose exposure might have beneficial effects on learning and memory ability and could be worth investigating for improvement of cognitive deficits associated with glucose hypometabolism in AD.

Multi-target iron-chelators improve memory loss in a rat model of sporadic Alzheimer's disease

AIM Novel effective treatment is urgently needed for sporadic Alzheimers disease (sAD). M30 ([5-(N-methyl-N-propargylaminomethyl)-8-hydroxyquinoline]) and HLA-20 (5-{4-propargylpiperazin-1-ylmethyl}-8-hydroxyquinoline) are brain permeable, iron chelating compounds with antioxidant activity, showing also neuroprotective activity in animal models of neurodegeneration.Weaimed to explore their therapeutic potential in non-transgenic (non-Tg) rat model of sAD developed by intracerebroventricular administration of streptozotocin (STZ-icv). MAIN METHODS Therapeutic effects of chronic oral M30 (2 and 10 mg/kg) and HLA20 (5 and 10 mg/kg) treatment on cognitive impairment in STZ-icv rat model were explored by Morris Water Maze (MWM) and Passive Avoidance (PA) tests in neuropreventive and neurorescue paradigms. Data were analysed by Kruskal–Wallis and Mann–Whitney U test (p b 0.05). KEY FINDINGS Five-day oral pre-treatment with M30 and HLA20 dose-dependently prevented development of spatial memory impairment (MWM probe trial-time +116%/M30; +60%/HLA20) in STZ-icv rat model (p b 0.05). Eleven-week oral treatment with M30 (3×/week), initiated 8 days after STZ-icv administration dosedependently ameliorated already developed cognitive deficits in MWM test (reduced number of mistakes 3 months after the STZ-icv treatment — 59%; p b 0.05) and fully restored them in PA test (+314%; p b 0.05). Chronic M30 treatment fully restored (−47%/PHF1;−65%/AT8; p b 0.05) STZ-induced hyperphosphorylation of tau protein and normalized decreased expression of insulin degrading enzyme (+37%; p b 0.05) in hippocampus. SIGNIFICANCE The results provide first evidence of therapeutic potential of M30 and HLA20 in STZ-icv rat model of sAD with underlying molecular mechanism, further supporting the important role of multi-target ironchelators in sAD treatment.

Characterization of cognitive deficits in spontaneously hypertensive rats, accompanied by brain insulin receptor dysfunction.

BackgroundThe spontaneously hypertensive rat (SHR) has been used to model changes in the central nervous system associated with cognitive-related disorders. Recent human and animal studies indicate a possible relationship between cognitive deficits, insulin resistance and hypertension. We aimed to investigate whether cognitively impaired SHRs develop central and/or peripheral insulin resistance and how their cognitive performance is influenced by the animal’s sex and age as well as strains used for comparison (Wistar and Wistar-Kyoto/WKY).MethodsThree and seven-month-old SHR, Wistar, and WKY rats were studied for their cognitive performance using Morris Water Maze (MWM) and Passive Avoidance tests (PAT). Plasma glucose and insulin were obtained after oral glucose tolerance tests. Cerebral cortex, hippocampus, and striatum status of insulin-receptor (IR) β-subunit and glycogen synthase kinase-3β (GSK3β) and their phosphorylated forms were obtained via ELISA.ResultsSHRs performed poorly in MWM and PAT in comparison to both control strains but more pronouncedly compared to WKY. Females performed poorer than males and 7-month-old SHRs had poorer MWM performance than 3-month-old ones. Although plasma glucose levels remained unchanged, plasma insulin levels were significantly increased in the glucose tolerance test in 7-month-old SHRs. SHRs demonstrated reduced expression and increased activity of IRβ-subunit in cerebral cortex, hippocampus, and striatum with different regional changes in phospho/total GSK3β ratio, as compared to WKYs.ConclusionResults indicate that cognitive deficits in SHRs are accompanied by both central and peripheral insulin dysfunction, thus allowing for the speculation that SHRs might additionally be considered as a model of insulin resistance-induced type of dementia.

Long-Term Effects of Intracerebroventricular Streptozotocin Treatment on Adult Neurogenesis in the Rat Hippocampus

Altered adult hippocampal neurogenesis (AN) plays a role in the etiopathology of Alzheimers disease (AD), a disorder characterized by a progressive loss of memory and spatial orientation impairment. Diabetes is shown to be one risk factor for the development of the sporadic form of AD (sAD), which affects >95% of AD patients. Streptozotocin intracerebroventricularily (STZ icv) treated rats, which develop an insulin-resistant brain state and learning and memory deficits preceding amyloid beta and tau pathology, may act as an appropriate animal model for sAD. The goal of our quantitative immunohistochemistry study was to compare short-term (1 month) and long-term (3 months) effects of STZ icv treatment on different AN stages. Applying MCM2 antibodies we quantified cell (e.g. stem cell) proliferation, by the use of NeuroD and DCX antibodies we analyzed immature neurons. BrdU incorporation with approximately 27 days of survival before sacrifice allowed us to quantify and identify surviving newborn cells. Performing co-localization studies with antibodies detecting BrdU and cell-type specific markers we could confirm that STZ treatment does not affect the differentiation fate of newly generated cells. Whereas STZ icv treatment does not seem to considerably influence cell proliferation over a shortterm period (1 month), in the long-term (3 months) it significantly decreased generation of immature and mature neurons. This reduction seen after 3 months was specific for the septal hippocampus, discussed to be important for spatial learning. Moreover, AN changes display the same timeline as the development of amyloid beta pathology in this animal model of sAD.

Glucagon-like peptide-1 mediates effects of oral galactose in streptozotocin-induced rat model of sporadic Alzheimer’s disease

&NA; Insulin resistance and metabolic dysfunction in the brain are considered to be the pathophysiological core of sporadic Alzheimers disease (sAD). In line with that fact, nutrients that could have therapeutic effects at this level have been investigated as possible targets in AD therapy. Galactose, an epimer of glucose, may serve as an alternative source of energy, and given orally may stimulate secretion of the incretin hormone glucagon‐like peptide‐1 (GLP‐1). Our preliminary research indicated that oral galactose might prevent development of memory impairment in a rat model of sAD generated by intracerebroventricular administration of streptozotocin (STZ‐icv). Here, we explored whether chronic oral galactose treatment could have beneficial effects on cognitive deficits already manifested at the time of initiation of galactose treatment in adult STZ‐icv rats (treatment initiated 1 month after STZ‐icv injection). The results clearly show that a 2‐month exposure to oral galactose (200 mg/kg/day administered in a drink ad libitum) normalises impaired learning and memory functions. Memory improvement was accompanied by an improvement in brain glucose hypometabolism measured by 18fluorodeoxyglucose‐positron emission tomography neuroimaging and by increments in active GLP‐1 plasma levels as well as by an increased expression of GLP‐1 receptors in the hippocampus and hypothalamus. Our findings provide strong evidence of beneficial effects of oral galactose treatment in the STZ‐icv rat model of sAD and present possible underlying mechanisms including both direct effects of galactose within the brain and indirect GLP‐1‐induced neuroprotective effects that might open a new, dietary‐based strategy in sAD treatment. HighlightsOral galactose normalises cognitive deficit in STZ‐icv rat AD model.Oral galactose might serve as an alternative source of energy in the brain.Oral galactose improves glucose hypometabolism in the brain of STZ‐icv rat.Oral galactose increases plasma level of active GLP‐1 in STZ‐icv rat.Oral galactose increases the expression of GLP‐1 receptor in the brain of STZ‐icv rat.

Therapeutic potential of a novel multifunctional iron chelator on cognitive decicits and insulin degrading enzyme expression in a rat model of sporadic Alzheimer's disease

Background There is a need in modern pharmacology for a representative animal model which should accurately mimic sporadic Alzheimer’s disease (sAD), the prevailing type of dementia in humans, and thus could be suitable for novel drug testing. Rats treated intracerebroventricularly with the betacytotoxic agent streptozotocin (STZ-icv), have been proposed recently as a non-transgenic sAD model which demonstrates AD-like pathology features at cognitive, neurochemical and structural level. In addition to the cognitive deficits, pathological accumulation of amyloid b (Ab) peptide is one of the neuropathological hallmarks of sAD, and a growing body of evidence suggests the involvement of insulin degrading enzyme (IDE), responsible for Ab degradation, in sAD pathophysiology. We have explored the time course of cognitive deficits and hippocampal (HPC) IDE expression in the STZ-icv rat model of sAD, and the therapeutic potential of the novel multifunctional iron-chelating drug M30 to improve these deficits.

Early changes of cholinergic receptors in hippocampus and parietotemporal cortex of the rat model of sporadic Alzheimer's disease

disease (AD) and neurodegeneration. VitaminD has been shown to down-regulate the L-type voltage-sensitive calcium channels, LVSCC-A1C and LVSCC-A1D, and up-regulate (nerve growth factor) NGF. However, expression of these proteins when VDR is repressed is unknown. The aim of this study is to investigate LVSCC-A1C, LVSCC-A1D, calbindin-D28k, inducible nitric oxide synthase (iNOS) expressions and NGF release in VDR-silenced primary cortical neurons. Methods: qRT-PCR and western blots were performed to determine VDR, LVSCC-A1C, LVSCC-A1D, calbindin-D28k and iNOS expression levels. NGF and cytotoxicity levels were determined by ELISA. Apoptosis was determined by TUNEL. Results: Our findings illustrate that LVSCC-A1C and iNOS expressions increased rapidly in cortical neurons when VDR is down-regulated, whereas, LVSCC-A1D levels did not change, NGF release calbindin-D28k expression decreased in response to VDR down-regulation. Although vitamin D regulates LVSCC-A1C through VDR, it may not regulate LVSCC-A1D through VDR. Conclusions: Our results indicate that suppression of VDR or vitamin D-VDR pathway disruption disrupts LVSCC-A1C, calbindin-D28k and NGF production and increase oxidative stress. Thus it can make neurons vulnerable to aging and neurodegeneration, and when combined with As toxicity, it is possible to explain some of the events that occur during neurodegeneration.