Mitochondrial Ca2+ Signaling and Bioenergetics in Alzheimer's Disease.

Alzheimer's disease (AD) is a hereditary and sporadic neurodegenerative illness defined by the gradual and cumulative loss of neurons in specific brain areas. The processes that cause AD are still under investigation and there are no available therapies to halt it. Current progress puts at the forefront the "calcium (Ca2+) hypothesis" as a key AD pathogenic pathway, impacting neuronal, astrocyte and microglial function. In this review, we focused on mitochondrial Ca2+ alterations in AD, their causes and bioenergetic consequences in neuronal and glial cells, summarizing the possible mechanisms linking detrimental mitochondrial Ca2+ signals to neuronal death in different experimental AD models.

Accelerated Aging Characterizes the Early Stage of Alzheimer's Disease.

For Alzheimer's disease (AD), aging is the main risk factor, but whether cognitive impairments due to aging resemble early AD deficits is not yet defined. When working with mouse models of AD, the situation is just as complicated, because only a few studies track the progression of the disease at different ages, and most ignore how the aging process affects control mice. In this work, we addressed this problem by comparing the aging process of PS2APP (AD) and wild-type (WT) mice at the level of spontaneous brain electrical activity under anesthesia. Using local field potential recordings, obtained with a linear probe that traverses the posterior parietal cortex and the entire hippocampus, we analyzed how multiple electrical parameters are modified by aging in AD and WT mice. With this approach, we highlighted AD specific features that appear in young AD mice prior to plaque deposition or that are delayed at 12 and 16 months of age. Furthermore, we identified aging characteristics present in WT mice but also occurring prematurely in young AD mice. In short, we found that reduction in the relative power of slow oscillations (SO) and Low/High power imbalance are linked to an AD phenotype at its onset. The loss of SO connectivity and cortico-hippocampal coupling between SO and higher frequencies as well as the increase in UP-state and burst durations are found in young AD and old WT mice. We show evidence that the aging process is accelerated by the mutant PS2 itself and discuss such changes in relation to amyloidosis and gliosis.

Lighting Up Ca2+ Dynamics in Animal Models.

Calcium (Ca2+) signaling coordinates are crucial processes in brain physiology. Particularly, fundamental aspects of neuronal function such as synaptic transmission and neuronal plasticity are regulated by Ca2+, and neuronal survival itself relies on Ca2+-dependent cascades. Indeed, impaired Ca2+ homeostasis has been reported in aging as well as in the onset and progression of neurodegeneration. Understanding the physiology of brain function and the key processes leading to its derangement is a core challenge for neuroscience. In this context, Ca2+ imaging represents a powerful tool, effectively fostered by the continuous amelioration of Ca2+ sensors in parallel with the improvement of imaging instrumentation. In this review, we explore the potentiality of the most used animal models employed for Ca2+ imaging, highlighting their application in brain research to explore the pathogenesis of neurodegenerative diseases.

Spontaneous Afferent Activity Carves Olfactory Circuits.

Electrical activity has a key role in shaping neuronal circuits during development. In most sensory modalities, early in development, internally generated spontaneous activity sculpts the initial layout of neuronal wiring. With the maturation of the sense organs, the system relies more on sensory-evoked electrical activity. Stimuli-driven neuronal discharge is required for the transformation of immature circuits in the specific patterns of neuronal connectivity that subserve normal brain function. The olfactory system (OS) differs from this organizational plan. Despite the important role of odorant receptors (ORs) in shaping olfactory topography, odor-evoked activity does not have a prominent role in refining neuronal wiring. On the contrary, afferent spontaneous discharge is required to achieve and maintain the specific diagram of connectivity that defines the topography of the olfactory bulb (OB). Here, we provide an overview of the development of olfactory topography, with a focus on the role of afferent spontaneous discharge in the formation and maintenance of the specific synaptic contacts that result in the topographic organization of the OB.

Familial Alzheimer's disease presenilin-2 mutants affect Ca2+ homeostasis and brain network excitability.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aß) peptides, providing genetic support to the hypothesis of Aß pathogenicity. However, clinical trials focused on the Aß pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.

A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals.

Mitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

Presenilin-2 and Calcium Handling: Molecules, Organelles, Cells and Brain Networks.

Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-ß (Aß) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca2+ handling to Ca2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).

Intracellular Calcium Dysregulation by the Alzheimer's Disease-Linked Protein Presenilin 2.

Alzheimer's disease (AD) is the most common form of dementia. Even though most AD cases are sporadic, a small percentage is familial due to autosomal dominant mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes. AD mutations contribute to the generation of toxic amyloid ß (Aß) peptides and the formation of cerebral plaques, leading to the formulation of the amyloid cascade hypothesis for AD pathogenesis. Many drugs have been developed to inhibit this pathway but all these approaches currently failed, raising the need to find additional pathogenic mechanisms. Alterations in cellular calcium (Ca2+) signaling have also been reported as causative of neurodegeneration. Interestingly, Aß peptides, mutated presenilin-1 (PS1), and presenilin-2 (PS2) variously lead to modifications in Ca2+ homeostasis. In this contribution, we focus on PS2, summarizing how AD-linked PS2 mutants alter multiple Ca2+ pathways and the functional consequences of this Ca2+ dysregulation in AD pathogenesis.

Axonal Odorant Receptors Mediate Axon Targeting.

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine-binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons. Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting.

Ethnic influences on the perceptual properties of human chemosignals.

Individuals of African and Caucasian descent show different chemical signatures in their body odors (BO). Does such biological difference have a perceptual correlate? We tested BO donors and raters of Afro-Portuguese (AP) and Caucasian (C) descent to investigate whether olfactory ratings reveal an ethnic bias and whether olfactory ethnic discrimination is possible. C (vs. AP) women rated the C BO as more pleasant, even when controlling for intensity. The C BO labelled as AP was rated as more intense by C raters. Although discriminability of ethnicity and sex is at chance, a nominal advantage for AP vs. C BO emerges.

Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling.

The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.

Oligophrenin-1 regulates number, morphology and synaptic properties of adult-born inhibitory interneurons in the olfactory bulb.

Among the X-linked genes associated with intellectual disability, Oligophrenin-1 (OPHN1) encodes for a Rho GTPase-activating protein, a key regulator of several developmental processes, such as dendrite and spine formation and synaptic activity. Inhibitory interneurons play a key role in the development and function of neuronal circuits. Whether a mutation of OPHN1 can affect morphology and synaptic properties of inhibitory interneurons remains poorly understood. To address these open questions, we studied in a well-established mouse model of X-linked intellectual disability, i.e. a line of mice carrying a null mutation of OPHN1, the development and function of adult generated inhibitory interneurons in the olfactory bulb. Combining quantitative morphological analysis and electrophysiological recordings we found that the adult generated inhibitory interneurons were dramatically reduced in number and exhibited a higher proportion of filopodia-like spines, with the consequences on their synaptic function, in OPHN1 ko mice. Furthermore, we found that olfactory behaviour was perturbed in OPHN1 ko mice. Chronic treatment with a Rho kinase inhibitor rescued most of the defects of the newly generated neurons. Altogether, our data indicated that OPHN1 plays a key role in regulating the number, morphology and function of adult-born inhibitory interneurons and contributed to identify potential therapeutic targets.

Role of the odorant receptor in neuronal connectivity in the olfactory bulb.

Olfaction is a highly sophisticated sensory modality able to detect and discriminate thousands of different odours, even at very low concentration. How such a challenging task is achieved remains to be fully understood. A unique feature of the olfactory system is the dual role of the odorant receptor: it does detect odours in the olfactory epithelium but it also contributes to neuronal circuit formation in the olfactory bulb. The odorant receptors are indeed expressed on the cilia that protrude in the nasal cavity, where they bind odorants, and at the axon termini, where they could act as axon guidance molecules. In this review we discuss findings that show how the odorant receptor contributes in regulating neuronal connectivity.

Circuit formation and function in the olfactory bulb of mice with reduced spontaneous afferent activity.

The type of neuronal activity required for circuit development is a matter of significant debate. We addressed this issue by analyzing the topographic organization of the olfactory bulb in transgenic mice engineered to have very little afferent spontaneous activity due to the overexpression of the inwardly rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Kir2.1 mice). In these conditions, the topography of the olfactory bulb was unrefined. Odor-evoked responses were readily recorded in glomeruli with reduced spontaneous afferent activity, although the functional maps were coarser than in controls and contributed to altered olfactory discrimination behavior. In addition, overexpression of Kir2.1 in adults induced a regression of the already refined connectivity to an immature (i.e., coarser) status. Our data suggest that spontaneous activity plays a critical role not only in the development but also in the maintenance of the topography of the olfactory bulb and in sensory information processing.