Shohreh Majd

Effects of proNGF on neuronal viability, neurite growth and amyloid-beta metabolism.

Alzheimer’s disease (AD) is characterized pathologically by the deposition of amyloid-β peptides (Aβ), neurofibrillary tangles, distinctive neuronal loss and neurite dystrophy. Nerve growth factor (NGF) has been suggested to be involved in the pathogenesis of AD, however, the role of its precursor (proNGF) in AD remains unknown. In this study, we investigated the effect of proNGF on neuron death, neurite growth and Aβ production, in vitro and in vivo. We found that proNGF promotes the death of different cell lines and primary neurons in culture, likely dependent on the expression of p75NTR. We for the first time found that proNGF has an opposite role in neurite growth to that of mature NGF, retarding neurite growth in both cell lines and primary neurons. proNGF is localized to the Aβ plaques in AD mice brain, however, it had no significant effect on Aβ production in vitro and in vivo. Our findings suggest that proNGF is an important factor involving AD pathogenesis.

Neuronal response in Alzheimer’s and Parkinson’s disease: the effect of toxic proteins on intracellular pathways

Accumulation of protein aggregates is the leading cause of cellular dysfunction in neurodegenerative disorders. Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, Prion disease and motor disorders such as amyotrophic lateral sclerosis, present with a similar pattern of progressive neuronal death, nervous system deterioration and cognitive impairment. The common characteristic is an unusual misfolding of proteins which is believed to cause protein deposition and trigger degenerative signals in the neurons. A similar clinical presentation seen in many neurodegenerative disorders suggests the possibility of shared neuronal responses in different disorders. Despite the difference in core elements of deposits in each neurodegenerative disorder, the cascade of neuronal reactions such as activation of glycogen synthase kinase-3 beta, mitogen-activated protein kinases, cell cycle re-entry and oxidative stress leading to a progressive neurodegeneration are surprisingly similar. This review focuses on protein toxicity in two neurodegenerative diseases, AD and PD. We reviewed the activated mechanisms of neurotoxicity in response to misfolded beta-amyloid and α-synuclein, two major toxic proteins in AD and PD, leading to neuronal apoptosis. The interaction between the proteins in producing an overlapping pathological pattern will be also discussed.

Reciprocal Induction Between α-Synuclein and β-Amyloid in Adult Rat Neurons

In spite of definite roles for β-amyloid (Aβ) in familial Alzheimer’s disease (AD), the cause of sporadic AD remains unknown. Amyloid senile plaques and Lewy body pathology frequently coexist in neocortical and hippocampal regions of AD and Parkinson’s diseases. However, the relationship between Aβ and α-synuclein (α-Syn), the principle components in the pathological structures, in neuronal toxicity and the mechanisms of their interaction are not well studied. As Aβ and α-Syn accumulate in aging patients, the biological functions and toxicity of these polypeptides in the aging brain may be different from those in young brain. We examined the neurotoxicity influences of Aβ1-42 or α-Syn on mature neurons and the effects of Aβ1-42 or α-Syn on the production of endogenous α-Syn or Aβ1-40 reciprocally using a model of culture enriched with primary neurons from the hippocampus of adult rats. Treatment of neurons with high concentrations of Aβ1-42 or α-Syn caused significant apoptosis of neurons. Following Aβ1-42 treatment at sub apoptotic concentrations, both intra- and extra-cellular α-Syn levels were significantly increased. Reciprocally, the non-toxic levels of α-Syn treatment also increased intra- and extra-cellular Aβ1-40 levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, suppressed α-Syn-induced Aβ1-40 elevation, as well as Aβ1-42-induced α-Syn elevation. Thus, high concentrations of Aβ1-42 and α-Syn exert toxic effects on mature neurons; however, non-toxic concentration treatment of these polypeptides induced the production of each other reciprocally with possible involvement of PI3K pathway.

Early Glycogen Synthase Kinase‐3β (GSK‐3β) and Protein Phosphatase 2A (PP2A) independent tau dephosphorylation during global brain ischemia and reperfusion following cardiac arrest and the role of the Adenosine Monophosphate Kinase (AMPK) pathway

Abnormal tau phosphorylation (p‐tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p‐tau is controlled by Glycogen Synthase Kinase (GSK)‐3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p‐tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser396 and Ser262after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK‐3β sensitive but AMPK insensitive residues, Ser202/Thr205 (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK‐3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p‐AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p‐tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK‐3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation.

The impact of tau hyperphosphorylation at Ser262 on memory and learning after global brain ischaemia in a rat model of reversible cardiac arrest

An increase in phosphorylated tau (p-tau) is associated with Alzheimers disease (AD), and brain hypoxia. Investigation of the association of residue-specific tau hyperphosphorylation and changes in cognition, leads to greater understanding of its potential role in the pathology of memory impairment. The aims of this study are to investigate the involvement of the main metabolic kinases, Liver Kinase B1 (LKB1) and Adenosine Monophosphate Kinase Protein Kinase (AMPK), in tau phosphorylation-derived memory impairment, and to study the potential contribution of the other tau kinases and phosphatases including Glycogen Synthase Kinase (GSK-3β), Protein kinase A (PKA) and Protein Phosphatase 2A (PP2A). Spatial memory and learning were tested in a rat global brain ischemic model of reversible cardiac arrest (CA). The phosphorylation levels of LKB1, AMPK, GSK-3β, PP2A, PKA and tau-specific phosphorylation were assessed in rats, subjected to ischaemia/reperfusion and in clinically diagnosed AD and normal human brains. LKB1 and AMPK phosphorylation increased 4 weeks after CA as did AMPK related p-tau (Ser262). The animals showed unchanged levels of GSK-3β specific p-tau (Ser202/Thr205), phospho-PP2A (Tyr307), total GSK-3β, PP2A, phospho-cAMP response element-binding protein (CREB) which is an indicator of PKA activity, and no memory deficits. AD brains had hyperphosphorylated tau in all the residues of Ser262, Ser202 and Thr205, with increased phosphorylation of both AMPK (Thr172) and GSK-3β (Ser9), and reduced PP2A levels. Our data suggests a crucial role for a combined activation of tau kinases and phosphatases in adversely affecting memory and that hyperphosphorylation of tau in more than one specific site may be required to create memory deficits.

Compound C enhances tau phosphorylation at Serine396 via PI3K activation in an AMPK and rapamycin independent way in differentiated SH-SY5Y cells

Aggregation of hyperphosphorylated tau (p-tau) in the form of neurofibrillary tangles (NFT) is a main hallmark for Alzheimers disease (AD). Activation of cellular metabolic axis, made of adenosine monophosphate kinase protein kinase (AMPK) and mammalian target of rapamycin (mTOR) have been implicated in generating tau pathology of AD. Thus, blocking either of these two proteins or both, are suggested as the future therapeutic approaches for AD. How and to what level these approaches could be applied, however are not entirely clear. By using Compound C (CC) in this study, we showed a substantial decrease in mTOR activity in a rapamycin-independent way without blocking AMPK. This decline in mTOR activity was accompanied by an increase in phosphoinositide 3 kinase (PI3K)/Akt activity and a parallel increase in p-tau (Ser396) but not p-tau (Ser262) in differentiated SH-SY5Y neuroblastoma cells. This elevation was blocked when the cells were treated with 15 μM of LY294002, a specific PI3K inhibitor, suggesting PI3K involvement in CC-mediated tau hyperphosphorylation at Ser396. For all groups the activity levels of glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase-5 (cdk5) and protein phosphatase 2A (PP2A), the other main kinases and phosphatase responsible for tau phosphorylation/dephosphorylation remained unchanged. Collectively, our results demonstrate that rapamycin-independent blocking of mTOR enhances p-tau (Ser396) in a PI3K-dependent way, suggesting the careful consideration of future therapeutic approaches for AD, which will be based on mTOR inhibition.

Beta estradiol and norepinephrine treatment of differentiated SH-SY5Y cells enhances tau phosphorylation at (Ser396) and (Ser262) via AMPK but not mTOR signaling pathway

&NA; Hyperphosphorylation of tau is one of the main hallmarks for Alzheimers disease (AD) and many other tauopathies. Norepinephrine (NE), a stress‐related hormone and 17‐&bgr;‐estradiol (E2) thought to influence tau phosphorylation (p‐tau) and AD pathology. The controversy around the impact of NE and E2 requires further clarification. Moreover, the combination effect of physiological and psychological stress and estrogen alteration during menopause, which affect p‐tau, has not been addressed. Exposure to E2 is believed to reduce NE release, however, the link between these two hormones and AD at cellular level was also remained unknown. Here, we examined whether NE and E2 treatment of differentiated SH‐SY5Y cells affected tau phosphorylation. The involvement of adenosine monophosphate kinase protein kinase (AMPK) and target of Rapamycin (mTOR) as the possible mechanisms, underlying this effect was also investigated. Subsequent to SH‐SY5Y differentiation to mature neurons, we treated the cells with NE, E2 and NE plus E2 in presence and absence of Compound C and Rapamycin. Cell viability was not affected by our treatment while our Western blot and immunofluorescent findings showed that exposure to NE and E2 separately, and in combination enhanced p‐tau (Ser396) and (Ser262)/tau but not (Ser202/Thr205)/tau. Blocking AMPK by Compound C reduced p‐tau (Ser396) and (Ser262), while GSK‐3&bgr; and PP2A activities were remained unchanged. We also found that blocking mTOR by Rapamycin did not change increased p‐tau (Ser396) and (Ser262) due to NE + E2 treatment. Collectively, our results suggested that tau hyperphosphorylation due to exposure to NE/E2 was mediated by AMPK, the main energy regulator of cells during stress with no significant involvement of mTOR, GSK‐3&bgr; and PP2A. HighlightsNE and E2 separately and in combination increase p‐tau at Ser396/Ser262 (AMPK‐sensitive) but not Ser202/Thr205 (AMPK‐insensitive) residues.Compound C inhibits AMPK activity and blocks p‐tau (Ser396, 262) elevation due to NE and E2 treatment.Rapamycin (m‐TOR inhibitor) has no effect on NE+ E2 dependent p‐tau elevation at (Ser396, 262).

Tau Positive Neurons Show Marked Mitochondrial Loss and Nuclear Degradation in Alzheimer's Disease

BACKGROUND Alzheimers disease (AD) pathology consists of intraneuronal neurofibrillary tangles, made of hyperphosphorylated tau and extracellular accumulation of beta amyloid (Aβ) in Aβ plaques. There is an extensive debate as to which pathology initiates and is responsible for cellular loss in AD. METHODS Using confocal and light microscopy, post mortem brains from control and AD cases, an antibody to SOD2 as a marker for mitochondria and an antibody to all forms of tau, we analyzed mitochondrial density in tau positive neurons along with nuclear degradation by calculating the raw integrative density. RESULTS Our findings showed an extensive staining of aggregated tau in cell bodies, dystrophic neurites and neurofilaments in AD with minimal staining in control tissue, along with a marked decrease in mitochondria in tau positive (tau+) neurons. The control or tau negative (tau-) neurons in AD contained an even distribution of mitochondria, which was greatly diminished in tau+ neurons by 40%. There were no significant differences between control and tau- neurons in AD. Tau+ neurons showed marked nuclear degradation which appeared to progress with the extent of tau aggregation. The aggregated tau infiltrated and appeared to break the nuclear envelope with progressively more DNA exiting the nucleus and associating with the aggregated intracellular tau. CONCLUSION We report that the mitochondrial decrease is likely due to a decrease in the protein synthesis rather than a redistribution of mitochondria because of the decreased axonal transport. We suggest that the decrease in mitochondria and nuclear degradation are key mechanisms for the neuronal loss seen in AD.