Marianne K.O. Grant

Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration.

The microtubule-associated protein tau accumulates in Alzheimers and other fatal dementias, which manifest when forebrain neurons die. Recent advances in understanding these disorders indicate that brain dysfunction precedes neurodegeneration, but the role of tau is unclear. Here, we show that early tau-related deficits develop not from the loss of synapses or neurons, but rather as a result of synaptic abnormalities caused by the accumulation of hyperphosphorylated tau within intact dendritic spines, where it disrupts synaptic function by impairing glutamate receptor trafficking or synaptic anchoring. Mutagenesis of 14 disease-associated serine and threonine amino acid residues to create pseudohyperphosphorylated tau caused tau mislocalization while creation of phosphorylation-deficient tau blocked the mistargeting of tau to dendritic spines. Thus, tau phosphorylation plays a critical role in mediating tau mislocalization and subsequent synaptic impairment. These data establish that the locus of early synaptic malfunction caused by tau resides in dendritic spines.

Brain amyloid-β oligomers in ageing and Alzheimer’s disease

Alzheimers disease begins about two decades before the onset of symptoms or neuron death, and is believed to be caused by pathogenic amyloid-β aggregates that initiate a cascade of molecular events culminating in widespread neurodegeneration. The microtubule binding protein tau may mediate the effects of amyloid-β in this cascade. Amyloid plaques comprised of insoluble, fibrillar amyloid-β aggregates are the most characteristic feature of Alzheimers disease. However, the correspondence between the distribution of plaques and the pattern of neurodegeneration is tenuous. This discrepancy has stimulated the investigation of other amyloid-β aggregates, including soluble amyloid-β oligomers. Different soluble amyloid-β oligomers have been studied in several mouse models, but not systematically in humans. Here, we measured three amyloid-β oligomers previously described in mouse models-amyloid-β trimers, Aβ*56 and amyloid-β dimers-in brain tissue from 75 cognitively intact individuals, ranging from young children to the elderly, and 58 impaired subjects with mild cognitive impairment or probable Alzheimers disease. As in mouse models, where amyloid-β trimers appear to be the fundamental amyloid-β assembly unit of Aβ*56 and are present in young mice prior to memory decline, amyloid-β trimers in humans were present in children and adolescents; their levels rose gradually with age and were significantly above baseline in subjects in their 70s. Aβ*56 levels were negligible in children and young adults, rose significantly above baseline in subjects in their 40s and increased steadily thereafter. Amyloid-β dimers were undetectable until subjects were in their 60s; their levels then increased sharply and correlated with plaque load. Remarkably, in cognitively intact individuals we found strong positive correlations between Aβ*56 and two pathological forms of soluble tau (tau-CP13 and tau-Alz50), and negative correlations between Aβ*56 and two postsynaptic proteins (drebrin and fyn kinase), but none between amyloid-β dimers or amyloid-β trimers and tau or synaptic proteins. Comparing impaired with age-matched unimpaired subjects, we found the highest levels of amyloid-β dimers, but the lowest levels of Aβ*56 and amyloid-β trimers, in subjects with probable Alzheimers disease. In conclusion, in cognitively normal adults Aβ*56 increased ahead of amyloid-β dimers or amyloid-β trimers, and pathological tau proteins and postsynaptic proteins correlated with Aβ*56, but not amyloid-β dimers or amyloid-β trimers. We propose that Aβ*56 may play a pathogenic role very early in the pathogenesis of Alzheimers disease.

Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration

The accumulation of amyloid-β (Aβ) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimers disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aβ oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aβ oligomers in vivo.

Correlation of Specific Amyloid-β Oligomers With Tau in Cerebrospinal Fluid From Cognitively Normal Older Adults

IMPORTANCE To improve the ability to develop treatments that prevent incipient Alzheimer disease (AD) from progressing to overt AD, it is important to understand the molecular basis of the earliest pathophysiological abnormalities and to determine how amyloid-β (Aβ) is involved very early in its pathogenesis. OBJECTIVE To investigate 2 specific Aβ oligomers, Aβ trimers and Aβ*56, in human cerebrospinal fluid (CSF); evaluate the effects of aging and AD; and obtain support for the hypothesis that they may be pathogenic by determining their relationships to CSF tau. DESIGN A CSF sampling study. SETTINGS The University of Minnesota Medical School in Minneapolis, Minnesota, and the Salhgrenska University Hospital, Sweden. PARTICIPANTS Forty-eight older adults with mild cognitive impairment or AD (impaired group); 49 age-matched cognitively intact control subjects (unimpaired group); and 10 younger, normal control subjects. MAIN OUTCOME MEASURES Measurements of CSF Aβ trimers, Aβ*56, the 42-amino acid Aβ isoform (Aβ1-42), total tau (T-tau), and phospho-tau 181 (p-tau(181)). The hypothesis being tested was formulated after data collection. RESULTS We observed that Aβ trimers and Aβ*56 levels increased with age; within the unimpaired group, they were elevated in subjects with T-tau/Aβ1-42 ratios greater than a cutoff that distinguished the unimpaired group from subjects with AD. In the unimpaired group, T-tau and p-tau(181) were found to correlate strongly with Aβ trimers and Aβ*56 (r > 0.63), but not with Aβ1-42 (-0.10 < r < -0.01). The strong correlations were found to be attenuated in the impaired group. CONCLUSIONS AND RELEVANCE In cognitively intact older adults, CSF Aβ trimers and Aβ*56 were elevated in individuals at risk for AD, and they showed stronger relationships with tau than did Aβ1-42, a surrogate for Aβ fibril deposition. These findings suggest that prior to overt symptoms, 1 or both of the Aβ oligomers, but not fibrillar Aβ, is coupled to tau; however, this coupling is weakened or broken when AD advances to symptomatic stages. The uncoupling is interesting in light of the failure of experimental Aβ therapies to improve mild cognitive impairment/AD, which has prompted a shift in the timing of Aβ therapies to asymptomatic subjects. Knowing which Aβ species to target in asymptomatic subjects may enhance the success of future treatments for AD.

Endogenous expression of nNOS protein in several neuronal cell lines.

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma x glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells.

Long-Term Changes in the Muscarinic M1 Receptor Induced by Instantaneous Formation of Wash-Resistant Xanomeline-Receptor Complex

Unlike other M1 muscarinic acetylcholine receptor agonists, xanomeline demonstrates a unique mode of binding to the receptor. It not only binds reversibly to the receptors conventional orthosteric site but also binds persistently at a secondary binding domain(s) on the M1 receptor. This results in persistent activation of the receptor even after extensive washout, and allosteric modulation of the orthosteric site. In the current study, we investigated how the effects of very brief exposure (1 min) of intact Chinese hamster ovary cells expressing M1 receptors to xanomeline followed by washout change with time. Pretreatment with xanomeline for 1 min resulted in a concentration-dependent wash-resistant inhibition of [3H]N-methylscopolamine (NMS) binding, with a lower potency than that observed in the continuous presence of xanomeline in the binding assay medium. This effect was associated with wash-resistant receptor activation. Incubation of pretreated and washed cells in control medium for 24 h transformed the monophasic xanomeline wash-resistant binding curve to one that exhibits two distinct potencies. This was the result of the appearance of a new very high-potency binding component without a change in the low-potency state. The delayed effects of persistently bound xanomeline are mainly due to reduction of the maximal binding of [3H]NMS without a change in its affinity. These treatment conditions also reversed persistent receptor activation by xanomeline. Our results imply that brief exposure to xanomeline followed by washing and prolonged waiting may result in delayed receptor desensitization accompanied by internalization or down-regulation.

Long-term wash-resistant effects of brief interaction of xanomeline at the M1 muscarinic receptor.

Compared to other M(1) muscarinic acetylcholine receptor (M(1) mAChR) agonists, xanomeline demonstrates both reversible and persistent modes of binding to the receptor. In our study, we investigated the long-term consequences of brief incubation of Chinese hamster ovary cells expressing M(1) mAChR (M(1)-CHO) with low concentrations of xanomeline followed by washing off the free drug. Thus, M(1)-CHO cells were exposed to 100 nM xanomeline for 1h then washed extensively. Washed cells were either used immediately for binding assays or incubated for 23 h in the absence of free xanomeline. Only the latter treatment conditions resulted in marked attenuation of binding of the muscarinic radioligand [(3)H]N-methylscopolamine ([(3)H]NMS) to intact cells. Shortening the xanomeline pretreatment period to 1 min had the same trends as the 1h pretreatment, implying that xanomeline binds instantly to the receptor to elicit long-term wash-resistant effects. Presence of atropine during the brief period of xanomeline pretreatment did not markedly modulate xanomelines long-term effects, which suggests that persistent anchoring of the xanomeline molecule to the M(1) receptor takes place at a site distinct from the orthosteric binding domain. Our findings suggest the possibility of a time-dependent transition of the conformation of the muscarinic M(1) receptor-xanomeline complex between states that vary in their ability to bind [(3)H]NMS. However, possible involvement of other mechanisms of long-term receptor regulation cannot be discounted.

Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization.

Mechanisms of M3 muscarinic receptor regulation by wash-resistant xanomeline binding

Background/Aims: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect. Methods: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments. Results: The above treatment protocol with xanomeline resulted in similar effects of the binding of [3H]NMS and [3H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent. Conclusion: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.

Co-administration of resveratrol with doxorubicin in young mice attenuates detrimental late-occurring cardiovascular changes

Aims Doxorubicin (DOX) is among the most effective chemotherapies used in paediatric cancer patients. However, the clinical utility of DOX is offset by its well-known cardiotoxicity, which often does not appear until later in life. Since hypertension significantly increases the risk of late-onset heart failure in childhood cancer survivors, we investigated whether juvenile DOX exposure impairs the ability to adapt to angiotensin II (Ang II)-induced hypertension later in life and tested a treatment that could prevent this. Methods and results Five-week-old male mice were administered a low dose of DOX (4 mg/kg) or saline once a week for 3 weeks and then allowed to recover for 5 weeks. Following the 5-week recovery period, mice were infused with Ang II or saline for 2 weeks. In another cohort, mice were fed chow containing 0.4% resveratrol 1 week before, during, and 1 week after the DOX administrations. One week after the last DOX administration, p38 mitogen-activated protein kinase (MAPK) was activated in hearts of DOX-treated mice demonstrating molecular signs of cardiac stress; yet, there was no change in cardiac function between groups. However, DOX-treated mice failed to develop compensatory cardiac hypertrophy in response to Ang II-induced hypertension later in life. Of importance, mice receiving DOX with resveratrol co-administration displayed normalization in p38 MAPK activation in the heart and a restored capacity for cardiac hypertrophy in response to Ang II-induced hypertension. Conclusion We have developed a juvenile mouse model of DOX-induced cardiotoxicity that displays no immediate overt physiological dysfunction; but, leads to an impaired ability of the heart to adapt to hypertension later in life. We also show that co-administration of resveratrol during DOX treatment was sufficient to normalize molecular markers of cardiotoxicity and restore the ability of the heart to undergo adaptive remodelling in response to hypertension later in life.