Faculty Bibliography

Dopamine transmission is critical for exploratory motor behaviour. A key regulator is acetylcholine; forebrain acetylcholine regulates striatal dopamine release, whereas brainstem cholinergic inputs regulate the transition of dopamine neurons from tonic to burst firing modes. How these sources of cholinergic activity combine to control dopamine efflux and exploratory motor behaviour is unclear. Here we show that mice lacking total forebrain acetylcholine exhibit enhanced frequency-dependent striatal dopamine release and are hyperactive in a novel environment, whereas mice lacking rostral brainstem acetylcholine are hypoactive. Exploratory motor behaviour is normalized by the removal of both cholinergic sources. Involvement of dopamine in the exploratory motor phenotypes observed in these mutants is indicated by their altered sensitivity to the dopamine D2 receptor antagonist raclopride. These results support a model in which forebrain and brainstem cholinergic systems act in tandem to regulate striatal dopamine signalling for proper control of motor activity.

A mutation in the BRI2/ITM2b gene causes loss of BRI2 protein leading to familial Danish dementia (FDD). BRI2 deficiency of FDD provokes an increase in amyloid-beta precursor protein (APP) processing since BRI2 is an inhibitor of APP proteolysis, and APP mediates the synaptic/memory deficits in FDD. APP processing is linked to Alzheimer disease (AD) pathogenesis, which is consistent with a common mechanism involving toxic APP metabolites in both dementias. We show that inhibition of APP cleavage by beta-secretase rescues synaptic/memory deficits in a mouse model of FDD. beta-cleavage of APP yields amino-terminal-soluble APPbeta (sAPPbeta) and beta-carboxyl-terminal fragments (beta-CTF). Processing of beta-CTF by gamma-secretase releases amyloid-beta (Abeta), which is assumed to cause AD. However, inhibition of gamma-secretase did not ameliorate synaptic/memory deficits of FDD mice. These results suggest that sAPPbeta and/or beta-CTF, rather than Abeta, are the toxic species causing dementia, and indicate that reducing beta-cleavage of APP is an appropriate therapeutic approach to treating human dementias. Our data and the failures of anti-Abeta therapies in humans advise against targeting gamma-secretase cleavage of APP and/or Abeta.

Decades of research have correlated increased levels of amyloid-beta peptide (Abeta) with neuropathological progression in Alzheimer's disease (AD) patients and transgenic models. Abeta precipitates synaptic and neuronal anomalies by perturbing intracellular signaling, which, in turn, may underlie cognitive impairment. Abeta also alters lipid metabolism, notably causing a deficiency of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], a phospholipid that regulates critical neuronal functions. Haploinsufficiency of the gene encoding synaptojanin 1 (Synj1), a major PI(4,5)P(2) phosphatase in the brain, provided protection against PI(4,5)P(2) breakdown and electrophysiological deficits attributable to Abeta. Based on these data, we tested whether reduction of Synj1 could rescue cognitive deficits and Abeta-induced morphological alterations of synapses. We found that hemizygous deletion of Synj1 in the context of a mouse model expressing the Swedish mutant of amyloid precursor protein rescues deficits in learning and memory without affecting amyloid load. Synj1 heterozygosity also rescued PI(4,5)P(2) deficiency in a synaptosome-enriched fraction from the brain of Tg2576 mice. Genetic disruption of Synj1 attenuated Abeta oligomer-induced changes in dendritic spines of cultured hippocampal neurons, sparing mature spine classes, which corroborates the protective role for Synj1 reduction against Abeta insult at the synapse. These results indicate that Synj1 reduction ameliorates AD-associated behavioral and synaptic deficits, providing evidence that Synj1 and, more generally, phosphoinositide metabolism may be promising therapeutic targets. Our work expands on recent studies identifying lipid metabolism and lipid-modifying enzymes as targets of AD-associated synaptic and behavioral impairment.

The type 2 ryanodine receptor/calcium release channel (RyR2), required for excitation-contraction coupling in the heart, is abundant in the brain. Chronic stress induces catecholamine biosynthesis and release, stimulating beta-adrenergic receptors and activating cAMP signaling pathways in neurons. In a murine chronic restraint stress model, neuronal RyR2 were phosphorylated by protein kinase A (PKA), oxidized, and nitrosylated, resulting in depletion of the stabilizing subunit calstabin2 (FKBP12.6) from the channel complex and intracellular calcium leak. Stress-induced cognitive dysfunction, including deficits in learning and memory, and reduced long-term potentiation (LTP) at the hippocampal CA3-CA1 connection were rescued by oral administration of S107, a compound developed in our laboratory that stabilizes RyR2-calstabin2 interaction, or by genetic ablation of the RyR2 PKA phosphorylation site at serine 2808. Thus, neuronal RyR2 remodeling contributes to stress-induced cognitive dysfunction. Leaky RyR2 could be a therapeutic target for treatment of stress-induced cognitive dysfunction.

The conventional view of AD (Alzheimer's disease) is that much of the pathology is driven by an increased load of beta-amyloid in the brain of AD patients (the 'Amyloid Hypothesis'). Yet, many therapeutic strategies based on lowering beta-amyloid have so far failed in clinical trials. This failure of beta-amyloid-lowering agents has caused many to question the Amyloid Hypothesis itself. However, AD is likely to be a complex disease driven by multiple factors. In addition, it is increasingly clear that beta-amyloid processing involves many enzymes and signalling pathways that play a role in a diverse array of cellular processes. Thus the clinical failure of beta-amyloid-lowering agents does not mean that the hypothesis itself is incorrect; it may simply mean that manipulating beta-amyloid directly is an unrealistic strategy for therapeutic intervention, given the complex role of beta-amyloid in neuronal physiology. Another possible problem may be that toxic beta-amyloid levels have already caused irreversible damage to downstream cellular pathways by the time dementia sets in. We argue in the present review that a more direct (and possibly simpler) approach to AD therapeutics is to rescue synaptic dysfunction directly, by focusing on the mechanisms by which elevated levels of beta-amyloid disrupt synaptic physiology.

Serotonin 4 (5-HT4) receptor signaling does not only have the physiological function of improving cognition, but might also be helpful in the therapy of Alzheimer's disease (AD) through regulation of the production of soluble amyloid-beta protein precursor alpha (sAbetaPPalpha). To analyze the relationship between 5-HT4 receptor signaling and sAbetaPPalpha production, we stably transfected H4 cells with AbetaPP and 5-HT4 receptor (H4/AbetaPP/5-HT4 cells). We found that 24-h incubation with the 5-HT4 receptor agonist RS-67333 upregulates matrix metalloproteinase-9 (MMP-9). Furthermore, MMP-9 overexpression enhanced sAbetaPPalpha levels, whereas knockdown with MMP-9 siRNA decreased sAbetaPPalpha levels. When RS-67333 was injected for 10 days in Tg2576 mice, a model of amyloid-beta peptide (Abeta) deposition, there was an increase in hippocampal levels of sAbetaPPalpha, C-terminal fragment alpha, and MMP-9, as well as a decrease in hippocampal senile plaque number and levels of the 40 amino acid peptide, Abeta40. Taken all together, these experiments demonstrate that 5-HT4 receptor stimulation induces expression of MMP-9 which cleaves AbetaPP through alpha-secretase-like activity, leading to an increase of sAbetaPPalpha levels and a reduction of Abeta load.

Learning and memory deficits in Alzheimer's disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA(A) receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen-glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD.

Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied by HPLC-MS/MS analysis of reaction products. Attenuated reactivity unmasked the cytoprotective actions of NO in contrast to the cytotoxic actions of higher NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce NO(2)(-), but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatment with a sGC inhibitor, ODQ, thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in hippocampal slices treated with oligomeric amyloid-beta peptide (Abeta) and was shown to restore synaptic function. The novel observation of furoxan activity of potential therapeutic use in the CNS warrants further studies.

Neurophysiological evidence suggests that a specialized cortical network is involved in the visual perception of biological motion; however, the temporal dynamics underlying this network is largely unexplored. We used magnetoencephalography to determine the spatial distribution and task-related temporal dynamics of the oscillatory activity of random and human motion. We recorded cortical responses in healthy adults while they passively viewed point-light displays of static dots, random, and human motion. By analyzing differences in the time-frequency distributions between pairs of conditions, we found that: (a) the perception of both motion conditions resulted in a significant decrease in the alpha/beta band in the right superior occipital gyrus and a significant decrease in the beta band in the right insula and (b) the human motion condition was associated with specific alterations in alpha, beta, and gamma bands with significant reductions in the alpha band in the right superior temporal gyrus, right precuneus, and left inferior parietal lobule, significant reductions in the beta band in the bilateral superior temporal gyrus, together with a significant increase in the gamma band in the left inferior parietal lobule and superior temporal regions. These data suggest that although the perception of both random and human motion involves desynchronization of oscillatory activity in alpha and beta bands in similar cortical regions, only human motion is associated with a larger network and significant alterations in the alpha/beta band particularly in the right hemisphere.