14 research outputs found
THE ROLE OF ACTIN CYTOSKELETON IN REGULATING NEURONAL FUNCTION IN PHYSIOLOGY AND DISEASE: ZOOM IN ON THE CYCLASE-ASSOCIATED PROTEIN 2
No full textLa malattia di Alzheimer (AD) è una patologia neurodegenerativa progressiva caratterizzata dalla
deposizione di placche extracellulari di beta amiloide (Aβ) e dall'accumulo intracellulare di proteina tau
iperfosforilata. Recenti studi indicano la perdita di sinapsi come il più significativo indicatore del declino
cognitivo nell'AD, portando alla classificazione dell'AD come “sinaptopatia”. Il citoscheletro di actina
svolge un ruolo critico nella plasticità sinaptica e le alterazioni delle dinamiche di actina contribuiscono
al deterioramento sinaptico nell'AD. Il mio progetto si è focalizzato sul ruolo della proteina Cyclase
associated protein 2 (CAP2), un modulatore dell'actina essenziale per l'architettura delle spine e la
trasmissione sinaptica e che risulta alterato nei pazienti con AD. In effetti, l'espressione di CAP2 è
ridotta nell'AD e, di conseguenza, i livelli sinaptici della forma dimerica di CAP2 sono ridotti, alterando
quindi l'associazione di cofilin con il dimero/monomero di CAP2. Inoltre, l'actina e le proteine ad essa
associate regolano la dinamica mitocondriale, per cui la disfunzione del citoscheletro di actina può
contribuire ulteriormente alla patologia dell'AD, aggiungendo un altro livello di complessità alla
malattia.
In questa tesi abbiamo riportato che i pazienti affetti da AD presentano livelli di CAP2 nel liquor più
elevati rispetto ai controlli e ai pazienti affetti da altre patologie neurodegenerative, che correlano
positivamente con la tau totale e la tau fosforilata in Thr181 (p-tau181). I livelli di mRNA di CAP2 sono
ridotti nell'ippocampo dei pazienti con AD. Pertanto, per evidenziare un potenziale legame biologico tra
CAP2 e tau, abbiamo ridotto l'espressione di CAP2 nei neuroni ippocampali e abbiamo misurato un
aumento significativo di p-tau181 e un concomitante aumento dell'attivazione della caspasi-3, senza
influire sulla vitalità cellulare. Inoltre, abbiamo dimostrato una maggiore variabilità, che suggerisce una
maggiore instabilità, del citoscheletro di actina delle spine dendritiche in seguito al silenziamento di
CAP2.
Inoltre, abbiamo dimostrato il coinvolgimento di CAP2 nei meccanismi rilevanti per la dinamica
mitocondriale actina-dipendente. Infatti, la delezione di CAP2 innesca un'evidente alterazione della
morfologia mitocondriale, mediata dalla inibizione delle dinamiche di fissione Drp1-dipendenti, e una
contemporanea perdita di massa mitocondriale causata da alterazioni nei processi di biogenesi.
Data la ridotta espressione di CAP2 nell'AD, abbiamo verificato gli effetti di un'aumentata espressione
di CAP2 nei topi AD e abbiamo osservato che questo approccio ripristina le vie sinaptiche mediate dal
complesso CAP2/cofilin e le vie di plasticità sinaptica, preservando così la funzione cognitiva.
L’overespressione di CAP2 riduce la formazione di cofilin-actin rods e attenua le anomalie della proteina
tau. CAP2 si accumula all'interno dei cofilin-actin rods specificamente indotti dagli oligomeri di Aβ.
Inoltre, la dimerizzazione di CAP2 è necessaria per prevenire la perdita di sinapsi indotta da Aβ, ma non
per proteggere i neuroni dalla formazione degli actin rods.
Nel complesso, questi risultati rafforzano il ruolo della disfunzione sinaptica nelle prime fasi dell'AD e
supportano la correlazione del deterioramento sinaptico dell'AD con le alterazioni del citoscheletro di
actina, evidenziando che CAP2 si trova al crocevia di molteplici vie biologiche alla base della patogenesi
dell'AD.Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the deposition
of extracellular amyloid-beta (Aβ) plaques and intracellular accumulation of hyperphosphorylated tau
protein. Recent studies point out synaptic loss as the strongest indicator of cognitive impairment in AD,
leading to the classification of AD as a “synaptopathy”. The actin cytoskeleton plays a critical role in
synaptic plasticity, and disruptions in actin dynamics contribute to synaptic failure in AD. We focused
on cyclase-associated protein 2 (CAP2), an actin-modifier essential for spine architecture and synaptic
transmission and altered in AD patients. Indeed, CAP2 expression is reduced in AD and, thereby, the
synaptic availability of CAP2 dimer is decreased, having an impact of cofilin association with the CAP2
dimer/monomer. Additionally, actin and its associated proteins regulate mitochondrial dynamics,
therefore actin cytoskeleton dysfunction may further contribute to AD pathology, adding another layer
of complexity to the disease pathology.
Here, we reported that AD patients show higher CAP2 CSF levels compared to controls and non-AD
patients, that positively correlate with total and tau phosphorylated at Thr181 (p-tau181). CAP2 mRNA
levels are reduced in the hippocampus of AD patients. Therefore, to assess a potential biological link
between CAP2 and tau, we downregulated CAP2 expression in hippocampal neurons and we measured
a significant increase in p-tau181 and a concomitant increase of caspase-3 activation without affecting
cell viability. In addition, we demonstrated a higher variance, suggesting increased instability, of
dendritic spine actin cytoskeleton upon CAP2 down-regulation.
Moreover, we show the involvement of CAP2 in those mechanisms relevant for actin-dependent
mitochondrial dynamics. Indeed, the ablation of CAP2 triggers an evident alteration of mitochondrial
morphology, mediated by the suppression of Drp1-dependent fission dynamics, and a simultaneous
loss of mitochondrial mass caused by alterations in mitobiogenesis processes.
Given the downregulation of CAP2 in AD, we described that CAP2 gene delivery in AD mice restores
synaptic CAP2/cofilin and plasticity pathways, thus preserving cognitive function. CAP2 overexpression
reduces cofilin actin rods formation and mitigates tau abnormalities. CAP2 is accumulated within
cofilin-actin rods specifically induced by Aβ oligomers. Furthermore, CAP2 dimerization is required for
preventing Aβ-induced synaptic loss but not to protect neurons from cofilin-actin rods formation.
Taken together, these findings strengthen the role of synaptic pathology in the early stages of AD and
support the correlation of AD synaptic failure with actin cytoskeleton dysfunction, highlighting CAP2 at
the crossroad of multiple pathways underlying AD pathogenesis
Nonlinear mixed-effects models to analyze actin dynamics in dendritic spines
No full textAbstract Fluorescence recovery after photobleaching (FRAP) allows to study actin-turnover in dendritic spines by providing recovery trajectories over time within a nested data structure (i.e. spine/neuron/culture). Statistical approaches to FRAP usually consider one-phase association models to estimate recovery-curve-specific parameters and test statistical hypotheses on curve parameters either at the spine or neuron level, ignoring the nested data structure. However, this approach leads to pseudoreplication concerns. We propose a nonlinear mixed-effects model to integrate the one-phase association model estimate with the nested data structure of FRAP experiments; this also allows us to model heteroscedasticity and time dependence in the data. We used this approach to evaluate the effect of the downregulation of the actin-binding protein CAP2 on actin dynamics. Our model allows the additional modelling of the variance function across experimental conditions, which may represent a novel parameter of interest in FRAP experiments. Indeed, the detected differential effect of the experimental condition on the variance component captures the increased instability of time-specific observations around the spine-specific trajectory for the CAP2-downregulated spines compared to the control spines. We hypothesise that this parameter reflects the increased instability of the actin cytoskeleton in dendritic spines upon CAP2 downregulation. We developed an R-based Shiny application, termed FRApp, to fit the statistical models introduced without requiring programming expertise
Dendritic spines in alzheimer’s disease : How the actin cytoskeleton contributes to synaptic failure
No full textAlzheimer’s disease (AD) is a neurodegenerative disorder characterized by Aβ-driven synaptic dysfunction in the early phases of pathogenesis. In the synaptic context, the actin cytoskeleton is a crucial element to maintain the dendritic spine architecture and to orchestrate the spine’s morphology remodeling driven by synaptic activity. Indeed, spine shape and synaptic strength are strictly correlated and precisely governed during plasticity phenomena in order to convert short-term alterations of synaptic strength into long-lasting changes that are embedded in stable structural modification. These functional and structural modifications are considered the biological basis of learning and memory processes. In this review we discussed the existing evidence regarding the role of the spine actin cytoskeleton in AD synaptic failure. We revised the physiological function of the actin cytoskeleton in the spine shaping and the contribution of actin dynamics in the endocytosis mechanism. The internalization process is implicated in different aspects of AD since it controls both glutamate receptor membrane levels and amyloid generation. The detailed understanding of the mechanisms controlling the actin cytoskeleton in a unique biological context as the dendritic spine could pave the way to the development of innovative synapse-tailored therapeutic interventions and to the identification of novel biomarkers to monitor synaptic loss in AD
The development of ADAM10 endocytosis inhibitors for the treatment of Alzheimer's disease
No full textThe development of new therapeutic avenues that target the early stages of Alzheimer???s disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-b. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and ac-tivity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treat-ment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted syn-aptic plasticity, as revealed by changes in the molecular compo-sitions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety is-sues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD
Cerebrospinal fluid cyclase-associated protein 2 is increased in Alzheimer’s disease and correlates with tau pathology
No full textSynaptic dysfunction represents an early pathological event that precedes neurodegeneration in Alzheimer’s disease (AD), even though the molecular mechanisms that underlie synaptic dysfunction remain to be completely understood. Nonetheless, in vivo synaptic biomarkers are highly relevant as they have the potential to reveal early-stage changes and to track target engagement of specific disease-modifying strategies
An association study of cyclase‐associated protein 2 and frailty
No full textFrailty is a geriatric syndrome that results from multisystem impairment caused by age-associated accumulation of deficits. The frailty index is used to define the level of frailty. Several studies have searched for molecular biomarkers associated with frailty, to meet the needs for personalized care. Cyclase-associated protein 2 (CAP2) is a multifunctional actin-binding protein involved in various physiological and pathological processes, that might reflect frailty's intrinsic complexity. This study aimed to investigate the association between frailty index and circulating CAP2 concentration in 467 community-dwelling older adults (median age: 79; range: 65-92 years) from Milan, Italy. The selected robust regression model showed that circulating CAP2 concentration was not associated with chronological age, as well as sex and education. However, circulating CAP2 concentration was significantly and inversely associated with the frailty index: a 0.1-unit increase in frailty index leads to similar to 0.5-point mean decrease in CAP2 concentration. Furthermore, mean CAP2 concentration was significantly lower in frail participants (i.e., frailty index & GE;0.25) than in non-frail participants. This study shows the association between serum CAP2 concentration and frailty status for the first time, highlighting the potential of CAP2 as a biomarker for age-associated accumulation of deficits
Looking at Alzheimer’s Disease Pathogenesis from the Nuclear Side
No full textAlzheimer’s disease (AD) is a neurodegenerative disorder representing the most common form of dementia. It is biologically characterized by the deposition of extracellular amyloid-β (Aβ) senile plaques and intracellular neurofibrillary tangles, constituted by hyperphosphorylated tau protein. The key protein in AD pathogenesis is the amyloid precursor protein (APP), which is cleaved by secretases to produce several metabolites, including Aβ and APP intracellular domain (AICD). The greatest genetic risk factor associated with AD is represented by the Apolipoprotein E ε4 (APOE ε4) allele. Importantly, all of the above-mentioned molecules that are strictly related to AD pathogenesis have also been described as playing roles in the cell nucleus. Accordingly, evidence suggests that nuclear functions are compromised in AD. Furthermore, modulation of transcription maintains cellular homeostasis, and alterations in transcriptomic profiles have been found in neurodegenerative diseases. This report reviews recent advancements in the AD players-mediated gene expression. Aβ, tau, AICD, and APOE ε4 localize in the nucleus and regulate the transcription of several genes, part of which is involved in AD pathogenesis, thus suggesting that targeting nuclear functions might provide new therapeutic tools for the disease
Functional interdependence of the actin regulators CAP1 and cofilin1 in control of dendritic spine morphology
No full textThe vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines, and therefore, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission, and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00018-022-04593-8
Glucose-derived glutamate drives neuronal terminal differentiation in vitro
No full textISSN:1469-221XISSN:1469-3178ISSN:1469-317
Nonlinear mixed-effects models to analyze actin dynamics in dendritic spines
No full textFluorescence recovery after photobleaching (FRAP) is used to study actin-turnover in dendritic spines providing recovery_trajectories over time within a nested data structure (i.e. spine/neuron/culture). Statistical approaches to FRAP usually_consider one-phase association models to estimate recovery-curve-specific parameters and test statistical hypotheses on_curve parameters either at the spine or neuron level, ignoring the nested data structure. However, this approach leads to_pseudoreplication concerns. We propose a nonlinear mixed-effects model to integrate the one-phase association model_estimate with nested data structure of FRAP experiments; this allowed us to model heteroscedasticity with an exponential_variance function, accounting for the different effects of the experimental condition, and autocorrelation structure over time with_an autoregressive model. We used this approach to evaluate the effect of the downregulation of the actin-binding protein CAP2_on actin dynamics. Our model was able to capture the higher variance, thus increased instability, of actin cytoskeleton upon_CAP2 down-regulation. We developed an R-based Shiny application, termed FRApp, to fit the statistical models introduced_without requiring programming expertise.
