The Impact of the hAPP695SW Transgene and Associated Amyloid-β Accumulation on Murine Hippocampal Biochemical Pathways

Background: Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) peptide in the brain. Objective: Gain a better insight into alterations in major biochemical pathways underlying AD. Methods: We compared metabolomic profiles of hippocampal tissue of 20-month-old female Tg2576 mice expressing the familial AD-associated hAPP695SW transgene with their 20-month-old wild type female littermates. Results: The hAPP695SW transgene causes overproduction and accumulation of Aβ in the brain. Out of 180 annotated metabolites, 54 metabolites differed (30 higher and 24 lower in Tg2576 versus wild-type hippocampal tissue) and were linked to the amino acid, nucleic acid, glycerophospholipid, ceramide, and fatty acid metabolism. Our results point to 1) heightened metabolic activity as indicated by higher levels of urea, enhanced fatty acid β-oxidation, and lower fatty acid levels; 2) enhanced redox regulation; and 3) an imbalance of neuro-excitatory and neuro-inhibitory metabolites in hippocampal tissue of aged hAPP695SW transgenic mice. Conclusion: Taken together, our results suggest that dysregulation of multiple metabolic pathways associated with a concomitant shift to an excitatory-inhibitory imbalance are contributing mechanisms of AD-related pathology in the Tg2576 mouse.

The Na+/Ca2+ Exchanger 3 Is Functionally Coupled With the NaV1.6 Voltage-Gated Channel and Promotes an Endoplasmic Reticulum Ca2+ Refilling in a Transgenic Model of Alzheimer’s Disease

The remodelling of neuronal ionic homeostasis by altered channels and transporters is a critical feature of the Alzheimer’s disease (AD) pathogenesis. Different reports converge on the concept that the Na+/Ca2+ exchanger (NCX), as one of the main regulators of Na+ and Ca2+ concentrations and signalling, could exert a neuroprotective role in AD. The activity of NCX has been found to be increased in AD brains, where it seemed to correlate with an increased neuronal survival. Moreover, the enhancement of the NCX3 currents (INCX) in primary neurons treated with the neurotoxic amyloid β 1–42 (Aβ1–42) oligomers prevented the endoplasmic reticulum (ER) stress and neuronal death. The present study has been designed to investigate any possible modulation of the INCX, the functional interaction between NCX and the NaV1.6 channel, and their impact on the Ca2+ homeostasis in a transgenic in vitro model of AD, the primary hippocampal neurons from the Tg2576 mouse, which overproduce the Aβ1–42 peptide. Electrophysiological studies, carried in the presence of siRNA and the isoform-selective NCX inhibitor KB-R7943, showed that the activity of a specific NCX isoform, NCX3, was upregulated in its reverse, Ca2+ influx mode of operation in the Tg2576 neurons. The enhanced NCX activity contributed, in turn, to increase the ER Ca2+ content, without affecting the cytosolic Ca2+ concentrations of the Tg2576 neurons. Interestingly, our experiments have also uncovered a functional coupling between NCX3 and the voltage-gated NaV1.6 channels. In particular, the increased NaV1.6 currents appeared to be responsible for the upregulation of the reverse mode of NCX3, since both TTX and the Streptomyces griseolus antibiotic anisomycin, by reducing the NaV1.6 currents, counteracted the increase of the INCX in the Tg2576 neurons. In agreement, our immunofluorescence analyses revealed that the NCX3/NaV1.6 co-expression was increased in the Tg2576 hippocampal neurons in comparison with the WT neurons. Collectively, these findings indicate that NCX3 might intervene in the Ca2+ remodelling occurring in the Tg2576 primary neurons thus emerging as a molecular target with a neuroprotective potential, and provide a new outcome of the NaV1.6 upregulation related to the modulation of the intracellular Ca2+ concentrations in AD neurons.

Oat Extract Avenanthramide-C Reverses Hippocampal Long-Term Potentiation Decline in Tg2576 Mice

Memory deterioration in Alzheimer’s disease (AD) is thought to be underpinned by aberrant amyloid β (Aβ) accumulation, which contributes to synaptic plasticity impairment. Avenanthramide-C (Avn-C), a polyphenol compound found predominantly in oats, has a range of biological properties. Herein, we performed methanolic extraction of the Avns-rich fraction (Fr. 2) from germinated oats using column chromatography, and examined the effects of Avn-C on synaptic correlates of memory in a mouse model of AD. Avn-C was identified in Fr. 2 based on 1H-NMR analysis. Electrophysiological recordings were performed to examine the effects of Avn-C on the hippocampal long-term potentiation (LTP) in a Tg2576 mouse model of AD. Avn-C from germinated oats restored impaired LTP in Tg2576 mouse hippocampal slices. Furthermore, Avn-C-facilitated LTP was associated with changes in the protein levels of phospho-glycogen synthase kinase-3β (p-GSK3β-S9) and cleaved caspase 3, which are involved in Aβ-induced synaptic impairment. Our findings suggest that the Avn-C extract from germinated oats may be beneficial for AD-related synaptic plasticity impairment and memory decline.

Metabolic Profiling of Suprachiasmatic Nucleus Reveals Multifaceted Effects in an Alzheimer’s Disease Mouse Model

Background: Circadian rhythm disturbance is commonly observed in Alzheimer’s disease (AD). In mammals, these rhythms are orchestrated by the superchiasmatic nucleus (SCN). Our previous study in the Tg2576 AD mouse model suggests that inflammatory responses, most likely manifested by low GABA production, may be one of the underlying perpetrators for the changes in circadian rhythmicity and sleep disturbance in AD. However, the mechanistic connections between SCN dysfunction, GABA modulation, and inflammation in AD is not fully understood. Objective: To reveal influences of amyloid pathology in Tg2576 mouse brain on metabolism in SCN and to identify key metabolic sensors that couple SCN dysfunction with GABA modulation and inflammation. Methods: High resolution magic angle spinning (HR-MAS) NMR in conjunction with multivariate analysis was applied for metabolic profiling in SCN of control and Tg2576 female mice. Immunohistochemical analysis was used to detect neurons, astrocytes, expression of GABA transporter 1 (GAT1) and Bmal1. Results: Metabolic profiling revealed significant metabolic deficits in SCN of Tg2576 mice. Reductions in glucose, glutamate, GABA, and glutamine provide hints toward an impaired GABAergic glucose oxidation and neurotransmitter cycling in SCN of AD mice. In addition, decreased redox co-factor NADPH and glutathione support a redox disbalance. Immunohistochemical examinations showed low expression of the core clock gene, Bmal1, especially in activated astrocytes. Moreover, decreased expression of GAT1 in astrocytes indicates low GABA recycling in this cell type. Conclusion: Our results suggest that redox disbalance and compromised GABA signaling are important denominators and connectors between neuroinflammation and clock dysfunction in AD.

Paraoxonase-1 and -3 Protein Expression in the Brain of the Tg2576 Mouse Model of Alzheimer’s Disease

Background: Brain oxidative lipid damage and inflammation are common in neurodegenerative diseases such as Alzheimer’s disease (AD). Paraoxonase-1 and -3 (PON1 and PON3) protein expression was demonstrated in tissue with no PON1 or PON3 gene expression. In the present study, we examine differences in PON1 and PON3 protein expression in the brain of a mouse model of AD. Methods: we used peroxidase- and fluorescence-based immunohistochemistry in five brain regions (olfactory bulb, forebrain, posterior midbrain, hindbrain and cerebellum) of transgenic (Tg2576) mice with the Swedish mutation (KM670/671NL) responsible for a familial form of AD and corresponding wild-type mice. Results: We found intense PON1 and PON3-positive staining in star-shaped cells surrounding Aβ plaques in all the studied Tg2576 mouse-brain regions. Although we could not colocalize PON1 and PON3 with astrocytes (star-shaped cells in the brain), we found some PON3 colocalization with microglia. Conclusions: These results suggest that (1) PON1 and PON3 cross the blood–brain barrier in discoidal high-density lipoproteins (HDLs) and are transferred to specific brain-cell types; and (2) PON1 and PON3 play an important role in preventing oxidative stress and lipid peroxidation in particular brain-cell types (likely to be glial cells) in AD pathology and potentially in other neurodegenerative diseases as well.

Investigating the role of the ghrelin axis in cognitive decline and dementia

Ghrelin is a 28-amino acid hormone that is generated in a wide number of tissues. Its active form, acyl-ghrelin is able to bind to its receptor GHS-R and exert a variety of functions. In the brain, acyl-ghrelin has been associated with neuroprotection, improved memory and adult hippocampal neurogenesis (AHN). However, the mechanisms controlling acyl-ghrelin-mediated AHN are still unknown. To elucidate this process, different markers of neurogenesis were assessed in a mouse model in which GHS-R+ neurones were ablated specifically from the rostral DG of the hippocampus (rDG), showing that rDG GHS-R+ neurones are essential for maintaining AHN. Acyl-ghrelin has also been shown to prevents the damage caused by neurodegeneration, at least in young animal models of disease. In this thesis, we demonstrated that in the geriatric Tg2576 mouse model of Alzheimer’s disease, acyl-ghrelin maintained an effect on β-amyloid (Aβ) plaques in the hippocampus, promoting a reduction of the Aβ plaques size in AD-like mouse model compared to WT mice. Collectively, research findings highlight the importance of circulating acyl-ghrelin in the brain. However, ghrelin exists in two distinct forms and acyl-ghrelin can be enzymatically modified to the ‘inactive' unacylated-ghrelin (UAG) by acyl-protein thioesterase 1 (APT1). Preventing APT1 mediated de-acylation and increasing acyl-ghrelin bio-availability may prevent the damage caused by neurodegeneration. Unpublished data from our group suggest that PalmostatinB, an APT1 inhibitor, increases levels of acyl-ghrelin in macrophage cells (that naturally produce ghrelin). Therefore, this and other APT1 inhibitors may be considered possible therapeutic agents for the treatment of cognitive decline and diseases associated with dementia. We confirmed that, among several APT1 inhibitors, PalmostatinB is able to increase the level of acyl-ghrelin in vitro. However, further research is warranted into APT1 inhibitors as a novel therapeutic approach to treating cognitive decline and dementia. Together, the data in this thesis support a role for the ghrelinergic system components in modulating brain functions.

Early changes in synaptic and intrinsic properties of dentate gyrus granule cells in a mouse model of Alzheimer’s disease neuropathology and atypical effects of the cholinergic antagonist atropine

ABSTRACTIt has been reported that hyperexcitability occurs in a subset of patients with Alzheimer’s disease (AD) and hyperexcitability could contribute to the disease. Several studies have suggested that the hippocampal dentate gyrus (DG) may be an important area where hyperexcitability occurs. Therefore, we tested the hypothesis that the principal DG cell type, granule cells (GCs), would exhibit changes at the single-cell level which would be consistent with hyperexcitability and might help explain it. We used the Tg2576 mouse, where it has been shown that hyperexcitability is robust at 2-3 months of age. GCs from 2-3-month-old Tg2576 mice were compared to age-matched wild type (WT) mice. Effects of muscarinic cholinergic antagonism were tested because previously we found that Tg2576 mice exhibited hyperexcitability in vivo that was reduced by the muscarinic cholinergic antagonist atropine, counter to the dogma that in AD one needs to boost cholinergic function. The results showed that GCs from Tg2576 mice exhibited increased frequency of spontaneous excitatory postsynaptic potentials/currents (sEPSP/Cs) and reduced frequency of spontaneous inhibitory synaptic events (sIPSCs) relative to WT, increasing the excitation:inhibition (E:I) ratio. There was an inward glutamatergic current that we defined here as a novel synaptic current (nsC) in Tg2576 mice because it was very weak in WT mice. Although not usually measured, intrinsic properties were distinct in Tg2576 GCs relative to WT. In summary, GCs of the Tg2576 mouse exhibit early electrophysiological alterations that are consistent with increased synaptic excitation, reduced inhibition, and muscarinic cholinergic dysregulation. The data support previous suggestions that the DG and cholinergic system contribute to hyperexcitability early in life in AD mouse models.HIGHLIGHTSGranule cells (GCs) in young Tg2576 mice had abnormal synaptic activity.Young Tg2576 GCs had increased excitatory and reduced inhibitory synaptic activity.GC intrinsic properties were altered in young Tg2576 mice.Muscarinic cholinergic regulation of GCs was altered in young Tg2576 mice.Atropine led to a novel spontaneous inward current that was very strong in Tg2576 GCs.

Dysregulated Gut Homeostasis Observed Prior to the Accumulation of the Brain Amyloid-β in Tg2576 Mice

Amyloid plaques in Alzheimer’s disease (AD) are associated with inflammation. Recent studies demonstrated the involvement of the gut in cerebral amyloid-beta (Aβ) pathogenesis; however, the mechanisms are still not well understood. We hypothesize that the gut bears the Aβ burden prior to brain, highlighting gut–brain axis (GBA) interaction in neurodegenerative disorders. We used pre-symptomatic (6-months) and symptomatic (15-months) Tg2576 mouse model of AD compared to their age-matched littermate WT control. We identified that dysfunction of intestinal epithelial barrier (IEB), dysregulation of absorption, and vascular Aβ deposition in the IEB occur before cerebral Aβ aggregation is detectible. These changes in the GBA were associated with elevated inflammatory plasma cytokines including IL-9, VEGF and IP-10. In association with reduced cerebral myelin tight junction proteins, we identified reduced levels of systemic vitamin B12 and decrease cubilin, an intestinal B12 transporter, after the development of cerebral Aβ pathology. Lastly, we report Aβ deposition in the intestinal autopsy from AD patients with confirmed cerebral Aβ pathology that is not present in intestine from non-AD controls. Our data provide evidence that gut dysfunction occurs in AD and may contribute to its etiology. Future therapeutic strategies to reverse AD pathology may involve the early manipulation of gut physiology and its microbiota.