Background ALS is a heterogeneous disease in which different factors such as mitochondrial phenot... more Background ALS is a heterogeneous disease in which different factors such as mitochondrial phenotypes act in combination with a genetic predisposition. This study addresses the question of whether homoplasmic (total mitochondrial genome of a sample is affected) and/or heteroplasmic mutations (wildtype and mutant mitochondrial DNA molecules coexist) might play a role in familial ALS. Blood was drawn from familial ALS patients with a possible maternal pattern of inheritance according to their pedigrees, which was compared to blood of ALS patients without maternal association as well as age-matched controls. In two cohorts, we analyzed the mitochondrial genome from whole blood or isolated white blood cells and platelets using a resequencing microarray (Affymetrix MitoChip v2.0) that is able to detect homoplasmic and heteroplasmic mitochondrial DNA mutations and allows the assessment of low-level heteroplasmy. Results We identified an increase in homoplasmic ND5 mutations, a subunit of respiratory chain complex I, in whole blood of ALS patients that allowed maternal inheritance. This effect was more pronounced in patients with bulbar onset. Heteroplasmic mutations were significantly increased in different mitochondrial genes in platelets of patients with possible maternal inheritance. No increase of low-level heteroplasmy was found in maternal ALS patients. Conclusion Our results indicate a contribution of homoplasmic ND5 mutations to maternally associated ALS with bulbar onset. Therefore, it might be conceivable that specific maternally transmitted rather than randomly acquired mitochondrial DNA mutations might contribute to the disease process. This stands in contrast with observations from Alzheimer's and Parkinson's diseases showing an age-dependent accumulation of unspecific mutations in mitochondrial DNA.
Astrocytes play a key role in modulating synaptic transmission by controlling the available extra... more Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A 1 (A 1 R) and A 2A (A 2A R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A 1 R-A 2A R that signal via two different G-proteins, G s and G i/o , and either enhances (A 2A R) or inhibits (A 1 R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glianeuron) synapse.
Circadian rhythms play a fundamental role in mammalian physiology. Circadian rhythms are synchron... more Circadian rhythms play a fundamental role in mammalian physiology. Circadian rhythms are synchronized through communication with the master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN synchronizes peripheral clocks in various organs. Nevertheless, isolated peripheral tissues and even cultured cells maintain circadian rhythmicity in the absence of input from the SCN. The molecular clock components (Bmal1, CLOCK, Cry and Period genes) are responsible for this cell autonomous rhythmicity. This core clock machinery is found in most tissues and has been estimated to mediate the circadian transcription of 20% of active genes. Circadian dysregulation has been implicated in aging, and in several disorders, such as metabolic diseases, mood disorders, and neurodegenerative diseases. Thus, the circadian clock itself may become a therapeutic target for age-related diseases. Indeed, some behavioral and pharmacological strategies ("clock drugs") change clock gene expression and rhythms and may restore circadian rhythms in patients, hence mitigate disease symptoms and age-related decline. Furthermore, circadian systems have been shown to influence pharmacokinetics and pharmacodynamics. And interestingly, some randomized clinical trials showed that circadian-based treatments (chronotherapy) had relevant patient outcomes. Integration of chronotherapeutics to clinical trial design may improve the success of drug candidates.
Glutamate-induced excitotoxicity is responsible for neuronal death in acute neurological conditio... more Glutamate-induced excitotoxicity is responsible for neuronal death in acute neurological conditions as well as in chronic neurodegeneration. In this review, we give an overview of the contribution of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis (ALS). The selective motor neuron death that is the hallmark of this neurodegenerative disease seems to be related to a number of intrinsic characteristics of these neurons. Most of these characteristics relate to calcium entry and calcium handling in the motor neurons as intracellular free calcium concentrations increase quickly due to a high glutamate-induced calcium influx in combination with a low calcium-buffering capacity. The high calcium influx is because of the presence of GluR2 lacking, calcium-permeable AMPA receptors while a low expression of calcium-binding proteins explains the low calcium-buffering capacity. In the absence of these proteins, mitochondria play an important role to remove calcium from the cytoplasm. While all of these characteristics make at least a subpopulation of motor neurons intrinsically very prone to AMPA receptor mediated excitotoxicity, this vulnerability is further increased by the disease process. Mutated genes as well as unknown factors do not only influence the intrinsic characteristics of the motor neurons, but also the properties of the surrounding astrocytes. In conclusion, excitotoxicity remains an intriguing pathological pathway that could not only explain the selectivity of the motor neuron death but also the role of surrounding non-neuronal cells in ALS. In addition, excitotoxicity is also an interesting drug-able target as indicated by the only FDA-approved drug, riluzole, as well as by a number of ongoing clinical trials.
The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in t... more The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the cerebral cortex, are regulated by specific high-affinity Na+/Cl− dependent plasma membrane transporters. Three GABA transporters (GATs), named GAT-1, GAT-2, and GAT-3 appear to play an important role in determining GABA’s effects. Studies on the distribution, cellular and subcellular localization, ontogeny, relationships of GATs with GABA-releasing elements using a variety of light and electron microscopic immunocytochemical techniques, as well as on their physiological effects performed over the last 25 years in our laboratory have contributed to unveil their organizational plan and at least some of their physiological roles. Although many details are missing, the anatomy and physiology of the cortical GABA uptake system in the adult (and developing) cerebral cortex appears to be sufficiently understood to allow the study of its dynamic physiological features, as well as its role in neuropsychiatric diseases.
The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in t... more The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the cerebral cortex, are regulated by specific high-affinity Na+/Cl− dependent plasma membrane transporters. Three GABA transporters (GATs), named GAT-1, GAT-2, and GAT-3 appear to play an important role in determining GABA’s effects. Studies on the distribution, cellular and subcellular localization, ontogeny, relationships of GATs with GABA-releasing elements using a variety of light and electron microscopic immunocytochemical techniques, as well as on their physiological effects performed over the last 25 years in our laboratory have contributed to unveil their organizational plan and at least some of their physiological roles. Although many details are missing, the anatomy and physiology of the cortical GABA uptake system in the adult (and developing) cerebral cortex appears to be sufficiently understood to allow the study of its dynamic physiological features, as well as its role in ...
Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interv... more Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interventions in patients with Mild Cognitive Impairment (MCI) is crucial. Cerebral Brain Derived Neurotrophic Factor (BDNF) alterations were evidenced in Alzheimer's disease, but the value of blood BDNF in MCI is unclear, especially because of the incomplete/incorrect management of the numerous confounding factors unrelated to the disease. The present study, applying a multidisciplinary methodological approach, aimed to clarify whether blood BDNF can really mirror the cognitive symptoms of MCI, thus supporting the evaluation of clinical protocols' effectiveness as well as the definition of the conversion rate to dementia. Healthy elderly subjects (HE) and MCI patients were assessed for socio-demographic, neuropsychological, pharmacological and lifestyle data, and plasma BDNF was measured (baseline); then, in the MCI cohort, the biomarker was tested in a comprehensive cognitive stimulation intervention (CS) as well as in a 2-year follow-up period. Plasma BDNF, cleansed from all the interfering factors, i) did not discriminate HE and MCI patients, ii) in MCI patients reflected mood, social engagement, and subjective memory complaints but not cognition, iii) changed due to CS, although with no correlations to cognitive performances, iv) predicted no functional deterioration. Our data indicate that the possible biased use of plasma BDNF in MCI is critically risky.
In the version of this article initially published, values for 12-week Iba-1 (blue curve in colum... more In the version of this article initially published, values for 12-week Iba-1 (blue curve in column 3, row 3: Fig. 2f) and 1-year GFAP (blue curve in column 4, row 2: Fig. 2f) of the control samples (thin film) were inadvertently mixed up, resulting in incorrect data shown in Figure 2h,i; Figure 2 source data; and the P values in the corresponding Supplementary Tables. Additionally, in Extended Data Figure 6a, the traces at 11 months were accidentally plotted for all months. The figures, source data and supplementary information have been updated in the HTML and PDF versions of the article. These changes do not affect the results or conclusions of the study.
Advances in Experimental Medicine and Biology, 1999
The brain consists of a complex network in which neurones and glial cells are structurally and fu... more The brain consists of a complex network in which neurones and glial cells are structurally and functionally interwoven. Astrocytes, the most numerous member of the glial family, were originally considered, along with the whole glial population, to be only of structural importance (Virchow, 1846). For example, during development the radial glia, the precursors of astrocytes, serve as a scaffold at which neurones migrate to form the layered structure of different brain regions such as the cortex, the hippocampus or the cerebellum. During the last two decades, considerable knowledge about astrocytes has accumulated regarding their physiological function. One exciting function is their contribution to the regulation of the extracellular space and, thereby, also of brain excitability (Walz, 1989). Qualities such as their capacity for uptake and metabolism of transmitters, buffering capacity of ions and ability to convey external signals via surface receptors to biological responses within the cells indicate an intimate crosstalk between glial cells and neurones. The other major glial population in the brain are the oligodendrocytes. As small cells with few processes they form the myelin sheath, a highly lipid enriched stack of cell membranes enwrapping 50 to 300αm long axonal segments to enhance the conduction of electrical signals and to inhibit electrical crosstalk between individual axons. Oligodendrocytes are capable of myelinating up to 50 axonal segments simultaneously. Mature oligodendrocytes develop from progenitors originating from the subventricular zone as the germinative layer (Miller, 1996). In vertebrates, progenitors start to migrate to their final destination regions, the presumptive white matter, during the first postnatal week.
Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interv... more Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interventions in patients with Mild Cognitive Impairment (MCI) is crucial. Cerebral Brain Derived Neurotrophic Factor (BDNF) alterations were evidenced in Alzheimer's disease, but the value of blood BDNF in MCI is unclear, especially because of the incomplete/incorrect management of the numerous confounding factors unrelated to the disease. The present study, applying a multidisciplinary methodological approach, aimed to clarify whether blood BDNF can really mirror the cognitive symptoms of MCI, thus supporting the evaluation of clinical protocols' effectiveness as well as the definition of the conversion rate to dementia. Healthy elderly subjects (HE) and MCI patients were assessed for socio-demographic, neuropsychological, pharmacological and lifestyle data, and plasma BDNF was measured (baseline); then, in the MCI cohort, the biomarker was tested in a comprehensive cognitive stimulation intervention (CS) as well as in a 2-year follow-up period. Plasma BDNF, cleansed from all the interfering factors, i) did not discriminate HE and MCI patients, ii) in MCI patients reflected mood, social engagement, and subjective memory complaints but not cognition, iii) changed due to CS, although with no correlations to cognitive performances, iv) predicted no functional deterioration. Our data indicate that the possible biased use of plasma BDNF in MCI is critically risky.
Metabotropic glutamate (Glu) receptors (mGluRs) and GABA B receptors are highly expressed at pres... more Metabotropic glutamate (Glu) receptors (mGluRs) and GABA B receptors are highly expressed at presynaptic sites. To verify the possibility that the two classes of metabotropic receptors contribute to axon terminals heterogeneity, we studied the localization of mGluR1α, mGluR5, mGluR2/3, mGluR7, and GABA B1 in VGLUT1-, VGLUT2-, and VGAT-positive terminals in the cerebral cortex of adult rats. VGLUT1-positive puncta expressed mGluR1α (∼5%), mGluR5 (∼6%), mGluR2/3 (∼22%), mGluR7 (∼17%), and GABA B1 (∼40%); VGLUT2-positive terminals expressed mGluR1α (∼10%), mGluR5 (∼11%), mGluR2/3 (∼20%), mGluR7 (∼28%), and GABA B1 (∼25%); whereas VGAT-positive puncta expressed mGluR1α (∼27%), mGluR5 (∼24%), mGluR2/3 (∼38%), mGluR7 (∼31%), and GABA B1 (∼19%). Control experiments ruled out the possibility that postsynaptic mGluRs and GABA B1 might have significantly biased our results. We also performed functional assays in synaptosomal preparations, and showed that all agonists modify Glu and GABA levels, which return to baseline upon exposure to antagonists. Overall, these findings indicate that mGluR1α, mGluR5, mGluR2/3, mGluR7, and GABA B1 expression differ significantly between glutamatergic and GABAergic axon terminals, and that the robust expression of heteroreceptors may contribute to the homeostatic regulation of the balance between excitation and inhibition.
The plasticity of glutamatergic transmission in the Ventral Tegmental Area (VTA) represents a fun... more The plasticity of glutamatergic transmission in the Ventral Tegmental Area (VTA) represents a fundamental mechanism in the modulation of dopamine neuron burst firing and the phasic dopamine release at VTA target regions. These processes encode basic behavioral responses, including locomotor activity, learning and motivated-behaviors. Here we describe a hitherto unidentified mechanism of long-lasting potentiation of glutamatergic synapses on DA neurons. We found that VTA astrocytes respond to dopamine neuron bursts with Ca 2+ elevations that require activation of endocannabinoid CB1 and dopamine D2 receptors colocalized at the same astrocytic process. Astrocytes, in turn, release glutamate that, through presynaptic metabotropic glutamate receptor activation coupled with neuronal nitric oxide production, induces long-lasting potentiation of excitatory synapses on adjacent dopamine neurons. Consistent with this finding, selective activation of VTA astrocytes increases dopamine neuron bursts in vivo and induces locomotor hyperactivity. Astrocytes play, therefore, a key role in the modulation of VTA dopamine neuron activity.
Background: Numerous mouse models of Alzheimer’s disease (AD) are available, but all suffer from ... more Background: Numerous mouse models of Alzheimer’s disease (AD) are available, but all suffer from certain limitations, thus prompting further attempts. To date, no one model exists with amyloidopathy in a BALB/c strain. Objective: To generate and characterize the C.B6/J-APPswe mouse, a model of AD with a mutated human gene for the amyloid-β protein precursor (AβPP) inserted in a BALB/c background. Methods: We analyzed five groups at different ages (3, 6, 9, 12, and 16–18 months) of C.B6/J-APPswe and wild-type mice (50% males and 50% females) for the main hallmarks of AD by western blotting, amyloid-β (Aβ) ELISA, immunocytochemistry, electrophysiology, and behavioral tests. Results: The C.B6/J-APPswe mouse displays early AβPP and Aβ production, late amyloid plaques formation, high level of Tau phosphorylation, synaptic deficits (reduced density and functional impairment due to a reduced post-synaptic responsiveness), neurodegeneration caused by apoptosis and necroptosis/necrosis, micr...
Background Recent clinical and experimental studies have highlighted the involvement of Ventral T... more Background Recent clinical and experimental studies have highlighted the involvement of Ventral Tegmental Area (VTA) dopamine (DA) neurons for the early pathogenesis of Alzheimer’s Disease (AD). We have previously described a progressive and selective degeneration of these neurons in the Tg2576 mouse model of AD, long before amyloid-beta plaque formation. The degenerative process in DA neurons is associated with an autophagy flux impairment, whose rescue can prevent neuronal loss. Impairments in autophagy can be the basis for accumulation of damaged mitochondria, leading to disturbance in calcium (Ca2+) homeostasis, and to functional and structural deterioration of DA neurons. Methods In Tg2576 mice, we performed amperometric recordings of DA levels and analysis of dopaminergic fibers in the Nucleus Accumbens – a major component of the ventral striatum precociously affected in AD patients – together with retrograde tracing, to identify the most vulnerable DA neuron subpopulations in...
Migraine is a common but poorly understood sensory circuit disorder. Mouse models of familial hem... more Migraine is a common but poorly understood sensory circuit disorder. Mouse models of familial hemiplegic migraine (FHM, a rare monogenic form of migraine with aura) show increased susceptibility to cortical spreading depression (CSD, the phenomenon that underlies migraine aura and can activate migraine headache mechanisms), allowing an opportunity to investigate the mechanisms of CSD and migraine onset. In FHM type 2 (FHM2) knock-in mice with reduced expression of astrocytic Na + , K +-ATPases, the reduced rate of glutamate uptake into astrocytes can account for the facilitation of CSD initiation. Here, we investigated the underlying mechanisms and show that the reduced rate of glutamate clearance in FHM2 mice results in increased amplitude and slowing of rise time and decay of the NMDA receptor (NMDAR) excitatory postsynaptic current (EPSC) elicited in layer 2/3 pyramidal cells by stimulation of neuronal afferents in somatosensory cortex slices. The relative increase in NMDAR activation in FHM2 mice is activity-dependent, being larger after high-frequency compared to low-frequency afferent activity. Inhibition of GluN1-N2B NMDARs, which hardly affected the NMDAR EPSC in wild-type mice, rescued the increased and prolonged activation of NMDARs as well as the facilitation of CSD induction and propagation in FHM2 mice. Our data suggest that the enhanced susceptibility to CSD in FHM2 is mainly due to specific activation of extrasynaptic GluN1-N2B NMDARs and point to these receptors as possible therapeutic targets for prevention of CSD and migraine.
Failure of anti-amyloid- peptide (A) therapies against Alzheimer's disease (AD), a neurodegener... more Failure of anti-amyloid- peptide (A) therapies against Alzheimer's disease (AD), a neurodegenerative disorder characterized by high amounts of the peptide in the brain, raised the question of the physiological role of A released at low concentrations in the healthy brain. To address this question, we studied the presynaptic and postsynaptic mechanisms underlying the neuromodulatory action of picomolar amounts of oligomeric A 42 (oA 42) on synaptic glutamatergic function in male and female mice. We found that 200 pM oA 42 induces an increase of frequency of miniature EPSCs and a decrease of paired pulse facilitation, associated with an increase in docked vesicle number, indicating that it augments neurotransmitter release at presynaptic level. oA 42 also produced postsynaptic changes as shown by an increased length of postsynaptic density, accompanied by an increased expression of plasticity-related proteins such as cAMPresponsive element binding protein phosphorylated at Ser133, calcium-calmodulin-dependent kinase II phosphorylated at Thr286, and brain-derived neurotrophic factor, suggesting a role for A in synaptic tagging. These changes resulted in the conversion of early into late long-term potentiation through the nitric oxide/cGMP/protein kinase G intracellular cascade consistent with a cGMP-dependent switch from short-to long-term memory observed in vivo after intrahippocampal administration of picomolar amounts of oA 42. These effects were present upon extracellular but not intracellular application of the peptide and involved ␣7 nicotinic acetylcholine receptors. These observations clarified the physiological role of oA 42 in synaptic function and memory formation providing solid fundamentals for investigating the pathological effects of high A levels in the AD brains.
Background ALS is a heterogeneous disease in which different factors such as mitochondrial phenot... more Background ALS is a heterogeneous disease in which different factors such as mitochondrial phenotypes act in combination with a genetic predisposition. This study addresses the question of whether homoplasmic (total mitochondrial genome of a sample is affected) and/or heteroplasmic mutations (wildtype and mutant mitochondrial DNA molecules coexist) might play a role in familial ALS. Blood was drawn from familial ALS patients with a possible maternal pattern of inheritance according to their pedigrees, which was compared to blood of ALS patients without maternal association as well as age-matched controls. In two cohorts, we analyzed the mitochondrial genome from whole blood or isolated white blood cells and platelets using a resequencing microarray (Affymetrix MitoChip v2.0) that is able to detect homoplasmic and heteroplasmic mitochondrial DNA mutations and allows the assessment of low-level heteroplasmy. Results We identified an increase in homoplasmic ND5 mutations, a subunit of respiratory chain complex I, in whole blood of ALS patients that allowed maternal inheritance. This effect was more pronounced in patients with bulbar onset. Heteroplasmic mutations were significantly increased in different mitochondrial genes in platelets of patients with possible maternal inheritance. No increase of low-level heteroplasmy was found in maternal ALS patients. Conclusion Our results indicate a contribution of homoplasmic ND5 mutations to maternally associated ALS with bulbar onset. Therefore, it might be conceivable that specific maternally transmitted rather than randomly acquired mitochondrial DNA mutations might contribute to the disease process. This stands in contrast with observations from Alzheimer's and Parkinson's diseases showing an age-dependent accumulation of unspecific mutations in mitochondrial DNA.
Astrocytes play a key role in modulating synaptic transmission by controlling the available extra... more Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A 1 (A 1 R) and A 2A (A 2A R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A 1 R-A 2A R that signal via two different G-proteins, G s and G i/o , and either enhances (A 2A R) or inhibits (A 1 R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glianeuron) synapse.
Circadian rhythms play a fundamental role in mammalian physiology. Circadian rhythms are synchron... more Circadian rhythms play a fundamental role in mammalian physiology. Circadian rhythms are synchronized through communication with the master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN synchronizes peripheral clocks in various organs. Nevertheless, isolated peripheral tissues and even cultured cells maintain circadian rhythmicity in the absence of input from the SCN. The molecular clock components (Bmal1, CLOCK, Cry and Period genes) are responsible for this cell autonomous rhythmicity. This core clock machinery is found in most tissues and has been estimated to mediate the circadian transcription of 20% of active genes. Circadian dysregulation has been implicated in aging, and in several disorders, such as metabolic diseases, mood disorders, and neurodegenerative diseases. Thus, the circadian clock itself may become a therapeutic target for age-related diseases. Indeed, some behavioral and pharmacological strategies ("clock drugs") change clock gene expression and rhythms and may restore circadian rhythms in patients, hence mitigate disease symptoms and age-related decline. Furthermore, circadian systems have been shown to influence pharmacokinetics and pharmacodynamics. And interestingly, some randomized clinical trials showed that circadian-based treatments (chronotherapy) had relevant patient outcomes. Integration of chronotherapeutics to clinical trial design may improve the success of drug candidates.
Glutamate-induced excitotoxicity is responsible for neuronal death in acute neurological conditio... more Glutamate-induced excitotoxicity is responsible for neuronal death in acute neurological conditions as well as in chronic neurodegeneration. In this review, we give an overview of the contribution of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis (ALS). The selective motor neuron death that is the hallmark of this neurodegenerative disease seems to be related to a number of intrinsic characteristics of these neurons. Most of these characteristics relate to calcium entry and calcium handling in the motor neurons as intracellular free calcium concentrations increase quickly due to a high glutamate-induced calcium influx in combination with a low calcium-buffering capacity. The high calcium influx is because of the presence of GluR2 lacking, calcium-permeable AMPA receptors while a low expression of calcium-binding proteins explains the low calcium-buffering capacity. In the absence of these proteins, mitochondria play an important role to remove calcium from the cytoplasm. While all of these characteristics make at least a subpopulation of motor neurons intrinsically very prone to AMPA receptor mediated excitotoxicity, this vulnerability is further increased by the disease process. Mutated genes as well as unknown factors do not only influence the intrinsic characteristics of the motor neurons, but also the properties of the surrounding astrocytes. In conclusion, excitotoxicity remains an intriguing pathological pathway that could not only explain the selectivity of the motor neuron death but also the role of surrounding non-neuronal cells in ALS. In addition, excitotoxicity is also an interesting drug-able target as indicated by the only FDA-approved drug, riluzole, as well as by a number of ongoing clinical trials.
The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in t... more The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the cerebral cortex, are regulated by specific high-affinity Na+/Cl− dependent plasma membrane transporters. Three GABA transporters (GATs), named GAT-1, GAT-2, and GAT-3 appear to play an important role in determining GABA’s effects. Studies on the distribution, cellular and subcellular localization, ontogeny, relationships of GATs with GABA-releasing elements using a variety of light and electron microscopic immunocytochemical techniques, as well as on their physiological effects performed over the last 25 years in our laboratory have contributed to unveil their organizational plan and at least some of their physiological roles. Although many details are missing, the anatomy and physiology of the cortical GABA uptake system in the adult (and developing) cerebral cortex appears to be sufficiently understood to allow the study of its dynamic physiological features, as well as its role in neuropsychiatric diseases.
The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in t... more The extracellular levels of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the cerebral cortex, are regulated by specific high-affinity Na+/Cl− dependent plasma membrane transporters. Three GABA transporters (GATs), named GAT-1, GAT-2, and GAT-3 appear to play an important role in determining GABA’s effects. Studies on the distribution, cellular and subcellular localization, ontogeny, relationships of GATs with GABA-releasing elements using a variety of light and electron microscopic immunocytochemical techniques, as well as on their physiological effects performed over the last 25 years in our laboratory have contributed to unveil their organizational plan and at least some of their physiological roles. Although many details are missing, the anatomy and physiology of the cortical GABA uptake system in the adult (and developing) cerebral cortex appears to be sufficiently understood to allow the study of its dynamic physiological features, as well as its role in ...
Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interv... more Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interventions in patients with Mild Cognitive Impairment (MCI) is crucial. Cerebral Brain Derived Neurotrophic Factor (BDNF) alterations were evidenced in Alzheimer's disease, but the value of blood BDNF in MCI is unclear, especially because of the incomplete/incorrect management of the numerous confounding factors unrelated to the disease. The present study, applying a multidisciplinary methodological approach, aimed to clarify whether blood BDNF can really mirror the cognitive symptoms of MCI, thus supporting the evaluation of clinical protocols' effectiveness as well as the definition of the conversion rate to dementia. Healthy elderly subjects (HE) and MCI patients were assessed for socio-demographic, neuropsychological, pharmacological and lifestyle data, and plasma BDNF was measured (baseline); then, in the MCI cohort, the biomarker was tested in a comprehensive cognitive stimulation intervention (CS) as well as in a 2-year follow-up period. Plasma BDNF, cleansed from all the interfering factors, i) did not discriminate HE and MCI patients, ii) in MCI patients reflected mood, social engagement, and subjective memory complaints but not cognition, iii) changed due to CS, although with no correlations to cognitive performances, iv) predicted no functional deterioration. Our data indicate that the possible biased use of plasma BDNF in MCI is critically risky.
In the version of this article initially published, values for 12-week Iba-1 (blue curve in colum... more In the version of this article initially published, values for 12-week Iba-1 (blue curve in column 3, row 3: Fig. 2f) and 1-year GFAP (blue curve in column 4, row 2: Fig. 2f) of the control samples (thin film) were inadvertently mixed up, resulting in incorrect data shown in Figure 2h,i; Figure 2 source data; and the P values in the corresponding Supplementary Tables. Additionally, in Extended Data Figure 6a, the traces at 11 months were accidentally plotted for all months. The figures, source data and supplementary information have been updated in the HTML and PDF versions of the article. These changes do not affect the results or conclusions of the study.
Advances in Experimental Medicine and Biology, 1999
The brain consists of a complex network in which neurones and glial cells are structurally and fu... more The brain consists of a complex network in which neurones and glial cells are structurally and functionally interwoven. Astrocytes, the most numerous member of the glial family, were originally considered, along with the whole glial population, to be only of structural importance (Virchow, 1846). For example, during development the radial glia, the precursors of astrocytes, serve as a scaffold at which neurones migrate to form the layered structure of different brain regions such as the cortex, the hippocampus or the cerebellum. During the last two decades, considerable knowledge about astrocytes has accumulated regarding their physiological function. One exciting function is their contribution to the regulation of the extracellular space and, thereby, also of brain excitability (Walz, 1989). Qualities such as their capacity for uptake and metabolism of transmitters, buffering capacity of ions and ability to convey external signals via surface receptors to biological responses within the cells indicate an intimate crosstalk between glial cells and neurones. The other major glial population in the brain are the oligodendrocytes. As small cells with few processes they form the myelin sheath, a highly lipid enriched stack of cell membranes enwrapping 50 to 300αm long axonal segments to enhance the conduction of electrical signals and to inhibit electrical crosstalk between individual axons. Oligodendrocytes are capable of myelinating up to 50 axonal segments simultaneously. Mature oligodendrocytes develop from progenitors originating from the subventricular zone as the germinative layer (Miller, 1996). In vertebrates, progenitors start to migrate to their final destination regions, the presumptive white matter, during the first postnatal week.
Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interv... more Availability of reliable prognostic biomarkers also able to monitor preventive/therapeutic interventions in patients with Mild Cognitive Impairment (MCI) is crucial. Cerebral Brain Derived Neurotrophic Factor (BDNF) alterations were evidenced in Alzheimer's disease, but the value of blood BDNF in MCI is unclear, especially because of the incomplete/incorrect management of the numerous confounding factors unrelated to the disease. The present study, applying a multidisciplinary methodological approach, aimed to clarify whether blood BDNF can really mirror the cognitive symptoms of MCI, thus supporting the evaluation of clinical protocols' effectiveness as well as the definition of the conversion rate to dementia. Healthy elderly subjects (HE) and MCI patients were assessed for socio-demographic, neuropsychological, pharmacological and lifestyle data, and plasma BDNF was measured (baseline); then, in the MCI cohort, the biomarker was tested in a comprehensive cognitive stimulation intervention (CS) as well as in a 2-year follow-up period. Plasma BDNF, cleansed from all the interfering factors, i) did not discriminate HE and MCI patients, ii) in MCI patients reflected mood, social engagement, and subjective memory complaints but not cognition, iii) changed due to CS, although with no correlations to cognitive performances, iv) predicted no functional deterioration. Our data indicate that the possible biased use of plasma BDNF in MCI is critically risky.
Metabotropic glutamate (Glu) receptors (mGluRs) and GABA B receptors are highly expressed at pres... more Metabotropic glutamate (Glu) receptors (mGluRs) and GABA B receptors are highly expressed at presynaptic sites. To verify the possibility that the two classes of metabotropic receptors contribute to axon terminals heterogeneity, we studied the localization of mGluR1α, mGluR5, mGluR2/3, mGluR7, and GABA B1 in VGLUT1-, VGLUT2-, and VGAT-positive terminals in the cerebral cortex of adult rats. VGLUT1-positive puncta expressed mGluR1α (∼5%), mGluR5 (∼6%), mGluR2/3 (∼22%), mGluR7 (∼17%), and GABA B1 (∼40%); VGLUT2-positive terminals expressed mGluR1α (∼10%), mGluR5 (∼11%), mGluR2/3 (∼20%), mGluR7 (∼28%), and GABA B1 (∼25%); whereas VGAT-positive puncta expressed mGluR1α (∼27%), mGluR5 (∼24%), mGluR2/3 (∼38%), mGluR7 (∼31%), and GABA B1 (∼19%). Control experiments ruled out the possibility that postsynaptic mGluRs and GABA B1 might have significantly biased our results. We also performed functional assays in synaptosomal preparations, and showed that all agonists modify Glu and GABA levels, which return to baseline upon exposure to antagonists. Overall, these findings indicate that mGluR1α, mGluR5, mGluR2/3, mGluR7, and GABA B1 expression differ significantly between glutamatergic and GABAergic axon terminals, and that the robust expression of heteroreceptors may contribute to the homeostatic regulation of the balance between excitation and inhibition.
The plasticity of glutamatergic transmission in the Ventral Tegmental Area (VTA) represents a fun... more The plasticity of glutamatergic transmission in the Ventral Tegmental Area (VTA) represents a fundamental mechanism in the modulation of dopamine neuron burst firing and the phasic dopamine release at VTA target regions. These processes encode basic behavioral responses, including locomotor activity, learning and motivated-behaviors. Here we describe a hitherto unidentified mechanism of long-lasting potentiation of glutamatergic synapses on DA neurons. We found that VTA astrocytes respond to dopamine neuron bursts with Ca 2+ elevations that require activation of endocannabinoid CB1 and dopamine D2 receptors colocalized at the same astrocytic process. Astrocytes, in turn, release glutamate that, through presynaptic metabotropic glutamate receptor activation coupled with neuronal nitric oxide production, induces long-lasting potentiation of excitatory synapses on adjacent dopamine neurons. Consistent with this finding, selective activation of VTA astrocytes increases dopamine neuron bursts in vivo and induces locomotor hyperactivity. Astrocytes play, therefore, a key role in the modulation of VTA dopamine neuron activity.
Background: Numerous mouse models of Alzheimer’s disease (AD) are available, but all suffer from ... more Background: Numerous mouse models of Alzheimer’s disease (AD) are available, but all suffer from certain limitations, thus prompting further attempts. To date, no one model exists with amyloidopathy in a BALB/c strain. Objective: To generate and characterize the C.B6/J-APPswe mouse, a model of AD with a mutated human gene for the amyloid-β protein precursor (AβPP) inserted in a BALB/c background. Methods: We analyzed five groups at different ages (3, 6, 9, 12, and 16–18 months) of C.B6/J-APPswe and wild-type mice (50% males and 50% females) for the main hallmarks of AD by western blotting, amyloid-β (Aβ) ELISA, immunocytochemistry, electrophysiology, and behavioral tests. Results: The C.B6/J-APPswe mouse displays early AβPP and Aβ production, late amyloid plaques formation, high level of Tau phosphorylation, synaptic deficits (reduced density and functional impairment due to a reduced post-synaptic responsiveness), neurodegeneration caused by apoptosis and necroptosis/necrosis, micr...
Background Recent clinical and experimental studies have highlighted the involvement of Ventral T... more Background Recent clinical and experimental studies have highlighted the involvement of Ventral Tegmental Area (VTA) dopamine (DA) neurons for the early pathogenesis of Alzheimer’s Disease (AD). We have previously described a progressive and selective degeneration of these neurons in the Tg2576 mouse model of AD, long before amyloid-beta plaque formation. The degenerative process in DA neurons is associated with an autophagy flux impairment, whose rescue can prevent neuronal loss. Impairments in autophagy can be the basis for accumulation of damaged mitochondria, leading to disturbance in calcium (Ca2+) homeostasis, and to functional and structural deterioration of DA neurons. Methods In Tg2576 mice, we performed amperometric recordings of DA levels and analysis of dopaminergic fibers in the Nucleus Accumbens – a major component of the ventral striatum precociously affected in AD patients – together with retrograde tracing, to identify the most vulnerable DA neuron subpopulations in...
Migraine is a common but poorly understood sensory circuit disorder. Mouse models of familial hem... more Migraine is a common but poorly understood sensory circuit disorder. Mouse models of familial hemiplegic migraine (FHM, a rare monogenic form of migraine with aura) show increased susceptibility to cortical spreading depression (CSD, the phenomenon that underlies migraine aura and can activate migraine headache mechanisms), allowing an opportunity to investigate the mechanisms of CSD and migraine onset. In FHM type 2 (FHM2) knock-in mice with reduced expression of astrocytic Na + , K +-ATPases, the reduced rate of glutamate uptake into astrocytes can account for the facilitation of CSD initiation. Here, we investigated the underlying mechanisms and show that the reduced rate of glutamate clearance in FHM2 mice results in increased amplitude and slowing of rise time and decay of the NMDA receptor (NMDAR) excitatory postsynaptic current (EPSC) elicited in layer 2/3 pyramidal cells by stimulation of neuronal afferents in somatosensory cortex slices. The relative increase in NMDAR activation in FHM2 mice is activity-dependent, being larger after high-frequency compared to low-frequency afferent activity. Inhibition of GluN1-N2B NMDARs, which hardly affected the NMDAR EPSC in wild-type mice, rescued the increased and prolonged activation of NMDARs as well as the facilitation of CSD induction and propagation in FHM2 mice. Our data suggest that the enhanced susceptibility to CSD in FHM2 is mainly due to specific activation of extrasynaptic GluN1-N2B NMDARs and point to these receptors as possible therapeutic targets for prevention of CSD and migraine.
Failure of anti-amyloid- peptide (A) therapies against Alzheimer's disease (AD), a neurodegener... more Failure of anti-amyloid- peptide (A) therapies against Alzheimer's disease (AD), a neurodegenerative disorder characterized by high amounts of the peptide in the brain, raised the question of the physiological role of A released at low concentrations in the healthy brain. To address this question, we studied the presynaptic and postsynaptic mechanisms underlying the neuromodulatory action of picomolar amounts of oligomeric A 42 (oA 42) on synaptic glutamatergic function in male and female mice. We found that 200 pM oA 42 induces an increase of frequency of miniature EPSCs and a decrease of paired pulse facilitation, associated with an increase in docked vesicle number, indicating that it augments neurotransmitter release at presynaptic level. oA 42 also produced postsynaptic changes as shown by an increased length of postsynaptic density, accompanied by an increased expression of plasticity-related proteins such as cAMPresponsive element binding protein phosphorylated at Ser133, calcium-calmodulin-dependent kinase II phosphorylated at Thr286, and brain-derived neurotrophic factor, suggesting a role for A in synaptic tagging. These changes resulted in the conversion of early into late long-term potentiation through the nitric oxide/cGMP/protein kinase G intracellular cascade consistent with a cGMP-dependent switch from short-to long-term memory observed in vivo after intrahippocampal administration of picomolar amounts of oA 42. These effects were present upon extracellular but not intracellular application of the peptide and involved ␣7 nicotinic acetylcholine receptors. These observations clarified the physiological role of oA 42 in synaptic function and memory formation providing solid fundamentals for investigating the pathological effects of high A levels in the AD brains.
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