Gideon M. Shaked

Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease

Profound neuronal dysfunction in the entorhinal cortex contributes to early loss of short-term memory in Alzheimers disease. Here we show broad neuroprotective effects of entorhinal brain-derived neurotrophic factor (BDNF) administration in several animal models of Alzheimers disease, with extension of therapeutic benefits into the degenerating hippocampus. In amyloid-transgenic mice, BDNF gene delivery, when administered after disease onset, reverses synapse loss, partially normalizes aberrant gene expression, improves cell signaling and restores learning and memory. These outcomes occur independently of effects on amyloid plaque load. In aged rats, BDNF infusion reverses cognitive decline, improves age-related perturbations in gene expression and restores cell signaling. In adult rats and primates, BDNF prevents lesion-induced death of entorhinal cortical neurons. In aged primates, BDNF reverses neuronal atrophy and ameliorates age-related cognitive impairment. Collectively, these findings indicate that BDNF exerts substantial protective effects on crucial neuronal circuitry involved in Alzheimers disease, acting through amyloid-independent mechanisms. BDNF therapeutic delivery merits exploration as a potential therapy for Alzheimers disease.

Mechanisms of Hybrid Oligomer Formation in the Pathogenesis of Combined Alzheimer's and Parkinson's Diseases

Background Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimers disease (AD) and Parkinsons disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear. Methodology/Principal Findings In order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels. Conclusion/Significance These results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

Aβ induces cell death by direct interaction with its cognate extracellular domain on APP (APP 597–624)

Amyloid β‐peptide (Aβ) is postulated to play a central role in the pathogenesis of Alzheimers disease. We recently proposed a pathway of Aβ‐induced toxicity that is APP dependent and involves the facilitation of APP complex formation by Aβ. The APP‐dependent component requires cleavage of APP at position 664 in the cytoplasmic domain, presumably by caspases or caspase‐like proteases, with release of a potentially cytotoxic C31 peptide. In this study we show that Aβ interacted directly and specifically with membrane‐bound APP to facilitate APP homo‐oligomerization. Using chimeric APP molecules, this interaction was shown to take place between Aβ and its homologous sequence on APP. Consistent with this finding, we demonstrated that Aβ also facilitated the oligomerization of β‐secretase cleaved APP C‐terminal fragment (C99). We found that the YENPTY domain in the APP cytoplasmic tail and contained within C31 is critical for this cell death pathway. Deletion or alanine‐ scanning mutagenesis through this domain significantly attenuated cell death apparently without affecting either APP dimerization or cleavage at position 664. This indicated that sequences within C31 are required after its release from APP. As the YENPTY domain has been shown to interact with a number of cytosolic adaptor molecules, it is possible that the interaction of APP, especially dimeric forms of APP, with these molecules contribute to cell death.—Shaked, G. M., Kummer, M. P., Lu, D. C., Galvan, V., Bredesen, D. E., Koo, E. H. Aβ induces cell death by direct interaction with its cognate extracellular domain on APP (APP 597–624). FASEB J. 20, E546‐E555 (2006)

Amyloid β protein toxicity mediated by the formation of amyloid‐β protein precursor complexes

The amyloid‐β protein precursor, a type 1 transmembrane protein, gives rise to the amyloid β‐protein, a neurotoxic peptide postulated to be involved in the pathogenesis of Alzheimers disease. Here, we show that soluble amyloid β protein accelerates amyloid precursor protein complex formation, a process that contributes to neuronal cell death. The mechanism of cell death involves the recruitment of caspase‐8 to the complex, followed by intracytoplasmic caspase cleavage of amyloid precursor protein. In vivo, the levels of soluble amyloid β protein correlated with caspase‐cleaved fragments of the amyloid precursor protein in brains of Alzheimers disease subjects. These findings suggest that soluble amyloid β protein–induced multimerization of the amyloid precursor protein may be another mechanism by which amyloid β protein contributes to synapse loss and neuronal cell death seen in Alzheimers disease. Ann Neurol 2003;54:781–789

Rapid, concurrent alterations in pre- and postsynaptic structure induced by naturally-secreted amyloid-beta protein.

In Alzheimers disease increasing evidence attributes synaptic and cognitive deficits to soluble oligomers of amyloid beta protein (Abeta), even prior to the accumulation of amyloid plaques, neurofibrillary tangles, and neuronal cell death. Here we show that within 1-2 h picomolar concentrations of cell-derived, soluble Abeta induce specific alterations in pre- and postsynaptic morphology and connectivity in cultured hippocampal neurons. Clusters of presynaptic vesicle markers decreased in size and number at glutamatergic but not GABAergic terminals. Dendritic spines also decreased in number and became dysmorphic, as spine heads collapsed and/or extended long protrusions. Simultaneous time-lapse imaging of axon-dendrite pairs revealed that shrinking spines sometimes became disconnected from their presynaptic varicosity. Concomitantly, miniature synaptic potentials decreased in amplitude and frequency. Spine changes were prevented by blockers of nAChRs and NMDARs. Washout of Abeta within the first day reversed these spine changes. Further, spine changes reversed spontaneously by 2 days, because neurons acutely developed resistance to continuous Abeta exposure. Thus, rapid Abeta-induced synapse destabilization may underlie transient behavioral impairments in animal models, and early cognitive deficits in Alzheimers patients.

Rapid, Concurrent Alterations in Pre- and Postsynaptic Structure Induced by Soluble Natural Amyloid-β Protein

In Alzheimers disease increasing evidence attributes synaptic and cognitive deficits to soluble oligomers of amyloid beta protein (Abeta), even prior to the accumulation of amyloid plaques, neurofibrillary tangles, and neuronal cell death. Here we show that within 1-2 h picomolar concentrations of cell-derived, soluble Abeta induce specific alterations in pre- and postsynaptic morphology and connectivity in cultured hippocampal neurons. Clusters of presynaptic vesicle markers decreased in size and number at glutamatergic but not GABAergic terminals. Dendritic spines also decreased in number and became dysmorphic, as spine heads collapsed and/or extended long protrusions. Simultaneous time-lapse imaging of axon-dendrite pairs revealed that shrinking spines sometimes became disconnected from their presynaptic varicosity. Concomitantly, miniature synaptic potentials decreased in amplitude and frequency. Spine changes were prevented by blockers of nAChRs and NMDARs. Washout of Abeta within the first day reversed these spine changes. Further, spine changes reversed spontaneously by 2 days, because neurons acutely developed resistance to continuous Abeta exposure. Thus, rapid Abeta-induced synapse destabilization may underlie transient behavioral impairments in animal models, and early cognitive deficits in Alzheimers patients.

Mechanism of cytotoxicity mediated by the C31 fragment of the amyloid precursor protein

The cytoplasmic tail of the amyloid precursor protein (APP) contains two putatively cytotoxic peptides, Jcasp and C31, derived by caspase cleavage of APP. Jcasp is a fragment starting from the epsilon-secretase site to position 664, while C31 is a fragment from position 665 to the C-terminus. Our studies now showed that compared to C31, Jcasp appeared to play a minor role in cytotoxicity. In particular, inhibition of Jcasp generation by treatment of gamma-secretase inhibitor did not lead to any attenuation of C31-induced toxicity. Secondly, because C31 toxicity is largely absent in cells lacking endogenous APP, we determined, using a split beta-galactosidase complementary assay to monitor protein-protein interactions, the presence of APP associated complexes. Our results demonstrated that both APP homomeric and C31/APP heteromeric complexes were correlated with cell death, indicating that C31 complexes with APP to recruit the interacting partners that initiate the signals related to cellular toxicity.

Interactions between the amyloid precursor protein C-terminal domain and G proteins mediate calcium dysregulation and amyloid β toxicity in Alzheimer’s disease

Alzheimer’s disease is characterized by neuropathological accumulations of amyloid β(1–42) [Aβ(1–42)], a cleavage product of the amyloid precursor protein (APP). Recent studies have highlighted the role of APP in Aβ‐mediated toxicity and have implicated the G‐protein system; however, the exact mechanisms underlying this pathway are as yet undetermined. In this context, we sought to investigate the role of calcium upregulation following APP‐dependent, Aβ‐mediated G‐protein activation. Initial studies on the interaction between APP, Aβ and Go proteins demonstrated that the interaction between APP, specifically its C‐terminal ‐YENPTY‐ region, and Go was reduced in the presence of Aβ. Cell death and calcium influx in Aβ‐treated cells were shown to be APP dependent and to involve G‐protein activation because these effects were blocked by use of the G‐protein inhibitor, pertussis toxin. Collectively, these results highlight a role for the G‐protein system in APP‐dependent, Aβ‐induced toxicity and calcium dysregulation. Analysis of the APP:Go interaction in human brain samples from Alzheimer’s disease patients at different stages of the disease revealed a decrease in the interaction, correlating with disease progression. Moreover, the reduced interaction between APP and Go was shown to correlate with an increase in membrane Aβ levels and G‐protein activity, showing for first time that the APP:Go interaction is present in humans and is responsive to Aβ load. The results presented support a role for APP in Aβ‐induced G‐protein activation and suggest a mechanism by which basal APP binding to Go is reduced under pathological loads of Aβ, liberating Go and activating the G‐protein system, which may in turn result in downstream effects including calcium dysregulation. These results also suggest that specific antagonists of G‐protein activity may have a therapeutic relevance in Alzheimer’s disease.

Rapid, concurrent alterations in pre- and postsynaptic structure induced by naturally-secreted amyloid-β protein

In Alzheimers disease increasing evidence attributes synaptic and cognitive deficits to soluble oligomers of amyloid beta protein (Abeta), even prior to the accumulation of amyloid plaques, neurofibrillary tangles, and neuronal cell death. Here we show that within 1-2 h picomolar concentrations of cell-derived, soluble Abeta induce specific alterations in pre- and postsynaptic morphology and connectivity in cultured hippocampal neurons. Clusters of presynaptic vesicle markers decreased in size and number at glutamatergic but not GABAergic terminals. Dendritic spines also decreased in number and became dysmorphic, as spine heads collapsed and/or extended long protrusions. Simultaneous time-lapse imaging of axon-dendrite pairs revealed that shrinking spines sometimes became disconnected from their presynaptic varicosity. Concomitantly, miniature synaptic potentials decreased in amplitude and frequency. Spine changes were prevented by blockers of nAChRs and NMDARs. Washout of Abeta within the first day reversed these spine changes. Further, spine changes reversed spontaneously by 2 days, because neurons acutely developed resistance to continuous Abeta exposure. Thus, rapid Abeta-induced synapse destabilization may underlie transient behavioral impairments in animal models, and early cognitive deficits in Alzheimers patients.

Valproic acid treatment results in increased accumulation of prion proteins

PrPSc, the only identified component of the prion, is an aberrant isoform of PrPC, a glycoprotein of unknown function. In this study, it was shown that valproic acid, a widely used antiepileptic drug, can cause an increase of several orders of magnitude in the accumulation of PrPC in normal neuroblastoma cells (N2a), and of both PrP isoforms in scrapie infected neuroblastoma cells (ScN2a). Although preliminary results indicate that valproic acid administration to hamsters inoculated with prions had no significant effect on disease incubation time, it is suggested that administration of valproic acid to humans at risk of developing Creutzfeldt‐Jakob disease should be evaluated with caution.