Melita Salkovic-Petrisic

Brain insulin system dysfunction in streptozotocin intracerebroventricularly treated rats generates hyperphosphorylated tau protein

The intracerebroventricular (icv) application of streptozotocin (STZ) in low dosage was used in 3‐month‐old rats to explore brain insulin system dysfunction. Three months following STZ icv treatment, the expression of insulin‐1 and ‐2 mRNA was significantly reduced to 11% in hippocampus and to 28% in frontoparietal cerebral cortex, respectively. Insulin receptor (IR) mRNA expression decreased significantly in frontoparietal cerebral cortex and hippocampus (16% and 33% of control). At the protein/activity level, different abnormalities of protein tyrosine kinase activity (increase in hippocampus), total IR β‐subunit (decrease in hypothalamus) and phosphorylated IR tyrosine residues (increase) became apparent. The STZ‐induced disturbance in learning and memory capacities was not abolished by icv application of glucose transport inhibitors known to prevent STZ‐induced diabetes mellitus. The discrepancy between reduced IR gene expression and increase in both phosphorylated IR tyrosine residues/protein tyrosine kinase activity may indicate imbalance between phosphorylation/dephosphorylation of the IR β‐subunit causing its dysfunction. These abnormalities may point to a complex brain insulin system dysfunction after STZ icv application, which may lead to an increase in hyperphosphorylated tau‐protein concentration. Brain insulin system dysfunction is discussed as possible pathological core in the generation of hyperphosphorylated tau protein as a morphological marker of sporadic Alzheimer’s disease.

Alzheimer-like changes in protein kinase B and glycogen synthase kinase-3 in rat frontal cortex and hippocampus after damage to the insulin signalling pathway

The insulin‐resistant brain state is related to late‐onset sporadic Alzheimers disease, and alterations in the insulin receptor (IR) and its downstream phosphatidylinositol‐3 kinase signalling pathway have been found in human brain. These findings have not been confirmed in an experimental model related to sporadic Alzheimers disease, for example rats showing a neuronal IR deficit subsequent to intracerebroventricular (i.c.v.) treatment with streptozotocin (STZ). In this study, western blot analysis performed 1 month after i.c.v. injection of STZ showed an increase of 63% in the level of phosphorylated glycogen synthase kinase‐3α/β (pGSK‐3α/β) protein in the rat hippocampus, whereas the levels of the unphosphorylated form (GSK‐3α/β) and protein kinase B (Akt/PKB) remained unchanged. Three months after STZ treatment, pGSK‐3α/β and Akt/PKB levels tended to decrease (by 8 and 9% respectively). The changes were region specific, as a different pattern was found in frontal cortex. Structural alterations were also found, characterized by β‐amyloid peptide‐like aggregates in brain capillaries of rats treated with STZ. Similar neurochemical changes and cognitive deficits were recorded in rats treated with i.c.v. 5‐thio‐d‐glucose, a blocker of glucose transporter (GLUT)2, a transporter that is probably involved in brain glucose sensing. The IR signalling cascade alteration and its consequences in rats treated with STZ are similar to those found in humans with sporadic Alzheimers disease, and our results suggest a role for GLUT2 in Alzheimers pathophysiology.

Central insulin resistance as a trigger for sporadic Alzheimer-like pathology: an experimental approach

A growing body of evidence implicates impairments in brain insulin signaling in early sporadic Alzheimer disease (sAD) pathology. However, the most widely accepted hypothesis for AD aetiology stipulates that pathological aggregations of the amyloid beta (Abeta) peptide are the cause of all forms of Alzheimers disease. Streptozotocin-intracerebroventricularly (STZ-icv) treated rats are proposed as a probable experimental model of sAD. The current work reviews evidence obtained from this model indicating that central STZ administration induces brain pathology and behavioural alterations resembling those in sAD patients. Recently, alterations of the brain insulin system resembling those in sAD have been found in the STZ-icv rat model and are associated with tau protein hyperphosphorylation and Abeta-like aggregations in meningeal vessels. In line with these findings the hypothesis has been proposed that insulin resistance in the brain might be the primary event which precedes the Abeta pathology in sAD.

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.

Botulinum toxin type A reduces pain supersensitivity in experimental diabetic neuropathy: Bilateral effect after unilateral injection

We investigated antinociceptive activity of botulinum toxin type A (BTX-A) in a model of diabetic neuropathic pain in rats. Male Wistar rats were made diabetic by a single intraperitoneal injection of streptozotocin (80mg/kg). Sensitivity to mechanical and thermal stimuli was measured with the paw-pressure and hot-plate test, respectively. The formalin test was used to measure sensitivity to chemical stimuli. Diabetic animals with pain thresholds lower for at least 25% compared to the non-diabetic group were considered neuropathic and were injected with BTX-A either subcutaneously (3, 5 and 7U/kg) or intrathecally (1U/kg). Mechanical and thermal sensitivity was measured at several time-points. After peripheral application, BTX-A (5 and 7U/kg) reduced mechanical and thermal hypersensitivity not only on ipsilateral, but on contralateral side, too. The antinociceptive effect started 5days following BTX-A injection and lasted at least 15days. Formalin-induced hypersensitivity in diabetic animals was abolished as well. When applied intrathecally, BTX-A (1U/kg) reduced diabetic hyperalgesia within 24h supporting the assumption of retrograde axonal transport of BTX-A from the peripheral site of injection to central nervous system. The results presented here demonstrate the long-lasting pain reduction after single BTX-A injection in the animals with diabetic neuropathy. The bilateral pain reduction after unilateral toxin application and the effectiveness of lower dose with the faster onset after the intrathecal injection suggest the involvement of the central nervous system in the antinociceptive action of BTX-A in painful diabetic neuropathy.

Insulin-Resistant Brain State after Intracerebroventricular Streptozotocin Injection Exacerbates Alzheimer-like Changes in Tg2576 AβPP-Overexpressing Mice

For studying rare hereditary Alzheimers disease (AD), transgenic (Tg) animal models overexpressing amyloid-beta protein precursor (AbetaPP) followed by increased amyloid-beta (Abeta) formation are used. In contrast, sporadic AD has been proposed to start with an insulin-resistant brain state (IRBS).We investigated the effect of IRBS induced by intracerebroventricularly (icv) administered streptozotocin (STZ) on behavior, glycogen synthase kinase-3 (GSK) alpha/beta content, and the formation of AD-like morphological hallmarks Abeta and tau protein in AbetaPP Tg2576 mice. Nine-month-old Tg mice were investigated 6 months after a single icv injection of STZ or placebo. Spatial cognition was analyzed using the Morris water maze test. Soluble and aggregated Abeta40/42 fragments, total and phosphorylated tau protein, and GSK-3alpha/beta were determined by ELISA. Cerebral (immuno)histological analyses were performed. In Tg mice, STZ treatment increased mortality, reduced spatial cognition, and increased cerebral aggregated Abeta fragments, total tau protein, and congophilic amyloid deposits. These changes were associated with decreased GSK-3alpha/beta ratio (phosphorylated/total). A linear negative correlation was detected between Abeta42 and cognition, and between GSK-3alpha/beta ratio and aggregated Abeta40+42. No marked necrotic and apoptotic changes were observed. In conclusion, IRBS may aggravate AD-like changes such as behavioral and increase the formation of pathomorphological AD hallmarks via GSK-3alpha/beta pathway in AbetaPP-overexpressing mice.

Modeling Sporadic Alzheimer's Disease: The Insulin Resistant Brain State Generates Multiple Long-Term Morphobiological Abnormalities Including Hyperphosphorylated Tau Protein and Amyloid-β

Nosologically, Alzheimers disease (AD) is not a single disorder. Missense gene mutations involved in increased formation of the amyloid-beta protein precursor derivatives amyloid-beta (Abeta(1-40) and Abeta(1-42/43) lead to autosomal dominant familial AD, found in the minority of AD cases. However, millions of subjects suffer from sporadic AD (sAD) of late onset, for which no convincing evidence suggests Abeta as the primary disease-generating compound. Environmental factors operating during pregnancy and postnatally may affect susceptibility genes and stress factors (e.g., cortisol), consequently affecting brain development both structurally and functionally, causing diseases that only becoming manifest late in life. With aging, a desynchronization of biological systems may result, increasing further brain entropy/declining criticality. In sAD, this desynchronization may involve stress components, cortisol and noradrenaline, reactive oxygen species, and membrane damage as major candidates causing an insulin resistant brain state with decreased glucose/energy metabolism. This further leads to a derangement of ATP-dependent cellular and molecular work, of the cell function in general, as well as derangements in the endoplasmic reticulum/Golgi apparatus, axon, synapses, and membranes, in particular. A self-propagating process is thus generated, including the increased formation of hyperphosphorylated tau-protein and Abeta as abnormal terminal events in sAD rather than causing the disorder, as elaborated in the review.

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.

Dementia, diabetes, Alzheimer's disease, and insulin resistance in the brain: progress, dilemmas, new opportunities, and a hypothesis to tackle intersecting epidemics.

Dementia is an increasingly prevalent condition that intersects worldwide with the epidemic of type 2 diabetes mellitus (DM2). It would seem logical to expect that the occurrence of DM2 increases the likelihood of developing dementia, due to its deleterious effect on the cerebral vasculature and the associated hormonal and metabolic changes. Many reports indicate that it also increases the risk of developing Alzheimers disease (AD). However, other studies suggest that diabetes might have a relatively strong protective effect against AD, whereas genetically engineered animal models of the condition deteriorate more severely when there is a concomitant insulin resistant brain state (IRBS). Furthermore, IRBS alone is associated with anatomical, behavioral, and molecular changes that justify the proposal that AD may be due to an IRBS. This is explored in the context of accumulating evidence that the IRBS need not be related to peripheral insulin resistance, and that administration of insulin directly to the brain improves selected cognitive parameters targeted in AD. This view is consistent with the Damage Signals hypothesis of AD pathogenesis, which can help unifying the pleiotropic effects of agents toxic to insulin-producing/secreting (e.g., pancreatic β) cells, as well as IRBS caused by different mechanisms in AD. Such approach may help tackling the Innovation Gap, which results from a host of factors slowing down progress towards innovative palliation and prevention of AD, as well as dementia due to complications of diabetes distinct from AD, and both conditions combined with their commonly associated metabolic and hormonal alterations.

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.