Raffaele d’Isa | IRCCS Ospedale San Raffaele (original) (raw)

Papers by Raffaele d’Isa

Research paper thumbnail of Nuclear ERK1/2 signaling potentiation enhances neuroprotection and cognition via Importinα1/KPNA2

EMBO Molecular Medicine

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneratio... more Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5‐mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy roden...

Research paper thumbnail of Editorial: Animal-friendly methods for rodent behavioral testing in neuroscience research

Frontiers in behavioral neuroscience, Jun 25, 2024

Research paper thumbnail of The origin of the phrase comparative psychology: an historical overview

Frontiers in Psychology

Comparative psychology, in its narrow meaning, refers to the study of the similarities and differ... more Comparative psychology, in its narrow meaning, refers to the study of the similarities and differences in the psychology and behavior of different species. In a broader meaning, it includes comparisons between different biological and socio-cultural groups, such as species, sexes, developmental stages, ages, and ethnicities. This broader meaning originated by extension from the former narrow meaning, which historically was the original meaning of the phrase (interspecies psychological and behavioral comparisons) and which still today is the focus of the field. Currently, comparative psychology is a subject of study in hundreds of universities all over the world. Nevertheless, a question that is often asked but seldom answered is: when did the phrase comparative psychology first appear and where did it come from? In the present work, we tracked down the origins of the phrase comparative psychology. In order to do so, at first we described the origin of the word psychology, coined in ...

Research paper thumbnail of Designing animal-friendly behavioral tests for neuroscience research: The importance of an ethological approach

Frontiers in Behavioral Neuroscience

Research paper thumbnail of Visual Evoked Potentials to Monitor Myelin Cuprizone-Induced Functional Changes

Frontiers in Neuroscience

The visual system is one of the most accessible routes to study the central nervous system under ... more The visual system is one of the most accessible routes to study the central nervous system under pathological conditions, such as in multiple sclerosis (MS). Non-invasive visual evoked potential (VEP) and optical coherence tomography (OCT) were used to assess visual function and neuroretinal thickness in C57BL/6 taking 0.2% cuprizone for 7 weeks and at 5, 8, 12, and 15 days after returning to a normal diet. VEPs were significantly delayed starting from 4 weeks on cuprizone, with progressive recovery off cuprizone, becoming significant at day 8, complete at day 15. In contrast, OCT and neurofilament staining showed no significant axonal thinning. Optic nerve histology indicated that whilst there was significant myelin loss at 7 weeks on the cuprizone diet compared with healthy mice, at 15 days off cuprizone diet demyelination was significantly less severe. The number of Iba 1+ cells was found increased in cuprizone mice at 7 weeks on and 15 days off cuprizone. The combined use of VEP...

Research paper thumbnail of Optic nerve involvement in experimental autoimmune encephalomyelitis to homologous spinal cord homogenate immunization in the dark agouti rat

Journal of Neuroimmunology, 2018

Dark-Agouti rats were immunized with spinal cord homogenate to develop Experimental Autoimmune En... more Dark-Agouti rats were immunized with spinal cord homogenate to develop Experimental Autoimmune Encephalomyelitis, a model of multiple sclerosis. We assessed motor signs and recorded VEPs for five or eight weeks with epidural or epidermal electrodes, respectively, with final histopathology of optic nerves (ONs). Injected rats exhibited motor deficits a week after immunization. VEP delays arose from the 2nd to the 5th week, when a recovery occurred in epidermal-recorded rats. ON damage appeared in epidural-, but not in epidermal-recorded rats, probably due to a remyelination process. VEP could be exploited as neurophysiological marker to test novel treatments against neurodegeneration involving ONs.

Research paper thumbnail of Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model

Blood, 2010

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of ... more Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patien...

Research paper thumbnail of The 4‐Hole‐Board Test for Assessment of Long‐Term Spatial Memory in Mice

Current Protocols

The hole-board test has been used in rodents since the early 60s to measure exploratory behavior,... more The hole-board test has been used in rodents since the early 60s to measure exploratory behavior, locomotor activity and cognitive function. The test is based on rodents' natural curiosity and attraction for novelty. Basically, the hole-board consists of a small square arena with an extractable platform as floor, which has a set of equally spaced circular holes on its surface. In this article, we describe the protocol of a 4-hole-board test allowing the assessment of long-term spatial memory in mice without the employment of water or food restriction, painful stimuli (as electrical shocks) or any aversive condition (as forced swimming or exposure to intense light). Four holes are present on the floor of the square arena (one for each of its four quadrants). Mice released in the arena spontaneously approach the holes and explore them by briefly inserting the snout inside, a behavior defined as nose-poking (or head-dipping). If, after 24 hr, rodents are re-exposed to the hole-board, the novelty of the holes decreases. Animals with an intact long-term memory will show a reduction of the frequency of nose-poking into the holes. The total number of nose-pokes on day 1 is an index of exploration, while the percentage of decrease in nose-poking on day 2 represents an index of long-term spatial memory. Number of quadrant crossings is scored as a control measure for locomotor activity, which with the present protocol should remain stable across the days of testing. Indeed, the 4-hole-board test represents a stress-free and animal-friendly option to evaluate long-term spatial memory. In the present paper, we provide detailed description of the hole-board apparatus and step-by-step protocol for assessment of spatial memory in mice. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Validation of the 4-hole-board Basic Protocol 2: Evaluation of long-term spatial memory through the 4-hole-board test.

Research paper thumbnail of Dose‐dependent effect of myelin oligodendrocyte glycoprotein on visual function and optic nerve damage in experimental autoimmune encephalomyelitis

Journal of Neuroscience Research, 2022

Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprote... more Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprotein (MOG) to develop experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis. Typical EAE motor impairments were assessed daily and noninvasive visual evoked potentials (VEPs) were recorded at baseline and 5 weeks after immunization, with final histopathology of optic nerves (ONs). Immunized rats exhibited a relapsing‐remitting clinical course. Both VEP and histological abnormalities were detected in a MOG dose‐dependent gradient. Increasing MOG dosage augmented visual function impairment in EAE, which could be monitored with VEP recording to assess demyelination and axonal loss along ONs.

Research paper thumbnail of Non-invasive visual evoked potentials under sevoflurane versus ketamine-xylazine in rats

Research paper thumbnail of Apparatus design and behavioural testing protocol for the evaluation of spatial working memory in mice through the spontaneous alternation T-maze

Scientific Reports, 2021

Spatial working memory can be assessed in mice through the spontaneous alternation T-maze test. T... more Spatial working memory can be assessed in mice through the spontaneous alternation T-maze test. The T-maze is a T-shaped apparatus featuring a stem (start arm) and two lateral goal arms (left and right arms). The procedure is based on the natural tendency of rodents to prefer exploring a novel arm over a familiar one, which induces them to alternate the choice of the goal arm across repeated trials. During the task, in order to successfully alternate choices across trials, an animal has to remember which arm had been visited in the previous trial, which makes spontaneous alternation T-maze an optimal test for spatial working memory. As this test relies on a spontaneous behaviour and does not require rewards, punishments or pre-training, it represents a particularly useful tool for cognitive evaluation, both time-saving and animal-friendly. We describe here in detail the apparatus and the protocol, providing representative results on wild-type healthy mice.

Research paper thumbnail of Functional evolution of visual involvement in experimental autoimmune encephalomyelitis

Multiple Sclerosis Journal - Experimental, Translational and Clinical, 2020

Background Experimental autoimmune encephalomyelitis (EAE) is a common animal model of multiple s... more Background Experimental autoimmune encephalomyelitis (EAE) is a common animal model of multiple sclerosis (MS). C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein exhibit chronic disease course, together with optic neuritis, consisting of demyelination/axonal loss of the optic nerve. Objectives To characterize functional and structural visual damages in two different phases of EAE: pre- and post-motor onset. Methods Visual alterations were detected with Visual Evoked Potential (VEP), Electroretinogram (ERG) and Optical Coherence Tomography (OCT). Optic nerve histology was performed at 7 (pre-motor onset) or 37 (post-motor onset) days post-immunization (dpi). Results At 7 dpi, optic nerve inflammation was similar in EAE eyes with and without VEP latency delay. Demyelination was detected in EAE eyes with latency delay (p

Research paper thumbnail of A new electrophysiological non-invasive method to assess retinocortical conduction time in the Dark Agouti rat through the simultaneous recording of electroretinogram and visual evoked potential

Documenta Ophthalmologica, 2019

Purpose To develop a non-invasive method exploiting simultaneous recording of epidermal visual ev... more Purpose To develop a non-invasive method exploiting simultaneous recording of epidermal visual evoked potential (VEP) and epicorneal electroretinogram (ERG) to study retinocortical function and to evaluate its reliability and repeatability over time. Methods Female wild-type DA rats were anesthetized with ketamine/xylazine (40/5 mg/kg). Epidermal VEP (Ag/AgCl cup electrode on scalp) and epicorneal ERG (gold ring electrode on eye surface) were recorded simultaneously in response to flash stimulation. Results ANOVA for repeated measures showed that peak times of ERG b-wave and of VEP N1 and P2 were stable across 6 weekly time-points, as well as the corresponding amplitudes. Mean retinocortical time from b-wave to N1 (RCT1) was 7.6 ms and remained comparable across the 6 time-points. Mean retinocortical time from b-wave to P2 (RCT2) was 28.7 ms and did not show significant variations over time. Coefficient of variation (CoV%) and CoV% adjusted for sample size, namely relative standard error (RSE%), were calculated as indexes of repeatability. Good RSE% over time was obtained (< 5% for b-wave, N1 and P2 peak times; < 20% and < 7% for RCT1 and RCT2, respectively). Conclusions Simultaneous recording of ERG and VEP has been previously achieved through invasive methods requiring surgery. Here, we present a new non-invasive method, which allowed to obtain peak and retinocortical times that were constant across a long period and had a good repeatability over time. This method will ensure not only a gain in animal welfare, but will also avoid stress and eye or brain lesions which can interfere with experimental variables.

Research paper thumbnail of Modulation of ERK1/MAPK3 potentiates ERK nuclear signalling, facilitates neuronal cell survival and improves memory in mouse models of neurodegenerative disorders

Cell signalling mechanisms are central to neuronal activity and their dysregulation may lead to n... more Cell signalling mechanisms are central to neuronal activity and their dysregulation may lead to neurodegenerative processes and associated cognitive decline. So far, a major effort has been directed toward the dissection of disease specific pathways with the still unmet promise to develop precision medicine strategies. With a different approach, here we show that a selective genetic potentiation of neuronal ERK signalling prevents cell death in vitro and in vivo in the mouse brain while ERK attenuation does the opposite. This neuroprotective effect can also be induced pharmacologically by a cell permeable peptide mimicking the loss of ERK1 MAP kinase, leading to a selective enhancement of ERK2 mediated nuclear cell signalling. The drug treatment prevents neurodegeneration in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's disease (PD). Importantly, the selective potentiation of ERK2 signalling facilitates both structural and synaptic plasticity, enha...

Research paper thumbnail of Non‐invasive Visual Evoked Potentials to assess optic nerve involvement in the Dark Agouti rat model of Experimental Autoimmune Encephalomyelitis induced by Myelin Oligodendrocyte Glycoprotein

Research paper thumbnail of Semi-invasive and non-invasive recording of visual evoked potentials in mice

Documenta Ophthalmologica, 2019

PurposeVisual evoked potentials (VEPs) are used to assess visual function in preclinical models o... more PurposeVisual evoked potentials (VEPs) are used to assess visual function in preclinical models of neurodegenerative diseases. VEP recording with epidural screw electrodes is a common method to study visual function in rodents, despite being an invasive procedure that can damage the tissue under the skull. The present study was performed to test a semi-invasive (epicranial) and a non-invasive (epidermal) VEP recording technique, comparing them with the classic epidural acquisition method.MethodsFlash VEPs were recorded from C57BL/6 mice on three separate days within 2 weeks. Waveforms, latencies and amplitudes of the components were compared between the three different methods, utilizing coefficient of repeatability, coefficient of variation and intersession standard deviation to evaluate reproducibility.ResultsWhile epidural electrodes succeeded in recording two negative peaks (N1 and N2), epicranial and epidermal electrodes recorded a single peak (N1). Statistical indexes showed a comparable reproducibility between the three techniques, with a greater stability of N1 latency recorded through epicranial electrodes. Moreover, N1 amplitudes recorded with the new less-invasive methods were more reproducible compared to the invasive gold-standard technique.ConclusionsThese results demonstrate the reliability of semi- and non-invasive VEP recordings, which can be useful to evaluate murine models of neurological diseases.

Research paper thumbnail of Visual evoked potentials can be reliably recorded using noninvasive epidermal electrodes in the anesthetized rat

Documenta ophthalmologica. Advances in ophthalmology, Jun 1, 2018

Visual evoked potentials (VEPs) are a powerful tool to evaluate nervous conduction along the visu... more Visual evoked potentials (VEPs) are a powerful tool to evaluate nervous conduction along the visual pathways, both in humans and in animal models. Traditionally, epidural screw electrodes are used to record VEPs in preclinical research. Here we tested the feasibility in the preclinical setting of the same noninvasive technique used for clinical VEP acquisition, by using epidermal cup electrodes with no surgical procedures. Monocular flash VEPs were recorded bilaterally under sevoflurane anesthesia once a week for 6 weeks in 14 dark Agouti rats, 7 with implanted epidural screws and 7 with epidermal 6 mm Ø Ag/AgCl cups. VEP traces obtained with the two techniques were morphologically comparable. There were no significant differences in latency of the main visual component between screw-recorded VEPs (sVEPs) and cup-recorded VEPs (cVEPs). Amplitude values with epidermal cups were significantly lower than those with epidural screws. Both techniques provided latencies and amplitudes whic...

Research paper thumbnail of Severe Intellectual Disability and Enhanced GABAergic Synaptogenesis in a Novel Model of Rare RASopathies

Biological Psychiatry, 2017

Dysregulation of Ras-extracellular signal-related kinase (ERK) signaling gives rise to RASopathie... more Dysregulation of Ras-extracellular signal-related kinase (ERK) signaling gives rise to RASopathies, a class of neurodevelopmental syndromes associated with intellectual disability. Recently, much attention has been directed at models bearing mild forms of RASopathies whose behavioral impairments can be attenuated by inhibiting the Ras-ERK cascade in the adult. Little is known about the brain mechanisms in severe forms of these disorders. We performed an extensive characterization of a new brain-specific model of severe forms of RASopathies, the KRAS(12V) mutant mouse. The KRAS(12V) mutation results in a severe form of intellectual disability, which parallels mental deficits found in patients bearing mutations in this gene. KRAS(12V) mice show a severe impairment of both short- and long-term memory in a number of behavioral tasks. At the cellular level, an upregulation of ERK signaling during early phases of postnatal development, but not in the adult state, results in a selective enhancement of synaptogenesis in gamma-aminobutyric acidergic interneurons. The enhancement of ERK activity in interneurons at this critical postnatal time leads to a permanent increase in the inhibitory tone throughout the brain, manifesting in reduced synaptic transmission and long-term plasticity in the hippocampus. In the adult, the behavioral and electrophysiological phenotypes in KRAS(12V) mice can be temporarily reverted by inhibiting gamma-aminobutyric acid signaling but not by a Ras-ERK blockade. Importantly, the synaptogenesis phenotype can be rescued by a treatment at the developmental stage with Ras-ERK inhibitors. These data demonstrate a novel mechanism underlying inhibitory synaptogenesis and provide new insights in understanding mental dysfunctions associated to RASopathies.

Research paper thumbnail of MicelackingRas- GRF1 showcontextualfearcondition- ing butnotspatialmemoryimpair- ments:convergentevidencefromtwo independentlygeneratedmousemutant lines

Frontiers in Behavioral Neuroscience, 2011

Research paper thumbnail of Behavioral Methods for the Study of the Ras–ERK Pathway in Memory Formation and Consolidation: Passive Avoidance and Novel Object Recognition Tests

Methods in Molecular Biology, 2013

Memory is a high-level brain function that enables organisms to adapt their behavioral responses ... more Memory is a high-level brain function that enables organisms to adapt their behavioral responses to the environment, hence increasing their probability of survival. The Ras-ERK pathway is a key molecular intracellular signalling cascade for memory consolidation. In this chapter we will describe two main one-trial behavioral tests commonly used in the field of memory research in order to assess the role of Ras-ERK signalling in long-term memory: passive avoidance and object recognition. Passive avoidance (PA) is a fear-motivated instrumental learning task, designed by Jarvik and Essman in 1960, in which animals learn to refrain from emitting a behavioral response that has previously been associated with a punishment. We will describe here the detailed protocol and show some examples of how PA can reveal impairments or enhancements in memory consolidation following loss or gain of function genetic manipulations of the Ras-ERK pathway. The phenotypes of global mutants as Ras-GRF1 KO, GENA53, and ERK1 KO mice, as well as of conditional region-specific mutants (striatal K-CREB mice), will be illustrated as examples. Novel object recognition (NOR), developed by Ennaceur and Delacour in 1988, is instead a more recent and highly ecological test, which relies on the natural tendency of rodents to spontaneously approach and explore novel objects, representing hence a useful non-stressful tool for the study of memory in animals without the employment of punishments or starvation/water restriction regimens. Careful indications will be given on how to select the positions for the novel object, in order to counterbalance for individual side preferences among mice during the training. Finally, the methods for calculating two learning indexes will be described. In addition to the classical discrimination index (DI) that measures the ability of an animal to discriminate between two different objects which are presented at the same time, we will describe the formula of a new index that we present here for the first time, the recognition index (RI), which quantifies the ability of an animal to recognize a same object at different time points and that, by taking into account the basal individual preferences displayed during the training, can give a more accurate measure of an animal&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s actual recognition memory.

Research paper thumbnail of Nuclear ERK1/2 signaling potentiation enhances neuroprotection and cognition via Importinα1/KPNA2

EMBO Molecular Medicine

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneratio... more Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5‐mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy roden...

Research paper thumbnail of Editorial: Animal-friendly methods for rodent behavioral testing in neuroscience research

Frontiers in behavioral neuroscience, Jun 25, 2024

Research paper thumbnail of The origin of the phrase comparative psychology: an historical overview

Frontiers in Psychology

Comparative psychology, in its narrow meaning, refers to the study of the similarities and differ... more Comparative psychology, in its narrow meaning, refers to the study of the similarities and differences in the psychology and behavior of different species. In a broader meaning, it includes comparisons between different biological and socio-cultural groups, such as species, sexes, developmental stages, ages, and ethnicities. This broader meaning originated by extension from the former narrow meaning, which historically was the original meaning of the phrase (interspecies psychological and behavioral comparisons) and which still today is the focus of the field. Currently, comparative psychology is a subject of study in hundreds of universities all over the world. Nevertheless, a question that is often asked but seldom answered is: when did the phrase comparative psychology first appear and where did it come from? In the present work, we tracked down the origins of the phrase comparative psychology. In order to do so, at first we described the origin of the word psychology, coined in ...

Research paper thumbnail of Designing animal-friendly behavioral tests for neuroscience research: The importance of an ethological approach

Frontiers in Behavioral Neuroscience

Research paper thumbnail of Visual Evoked Potentials to Monitor Myelin Cuprizone-Induced Functional Changes

Frontiers in Neuroscience

The visual system is one of the most accessible routes to study the central nervous system under ... more The visual system is one of the most accessible routes to study the central nervous system under pathological conditions, such as in multiple sclerosis (MS). Non-invasive visual evoked potential (VEP) and optical coherence tomography (OCT) were used to assess visual function and neuroretinal thickness in C57BL/6 taking 0.2% cuprizone for 7 weeks and at 5, 8, 12, and 15 days after returning to a normal diet. VEPs were significantly delayed starting from 4 weeks on cuprizone, with progressive recovery off cuprizone, becoming significant at day 8, complete at day 15. In contrast, OCT and neurofilament staining showed no significant axonal thinning. Optic nerve histology indicated that whilst there was significant myelin loss at 7 weeks on the cuprizone diet compared with healthy mice, at 15 days off cuprizone diet demyelination was significantly less severe. The number of Iba 1+ cells was found increased in cuprizone mice at 7 weeks on and 15 days off cuprizone. The combined use of VEP...

Research paper thumbnail of Optic nerve involvement in experimental autoimmune encephalomyelitis to homologous spinal cord homogenate immunization in the dark agouti rat

Journal of Neuroimmunology, 2018

Dark-Agouti rats were immunized with spinal cord homogenate to develop Experimental Autoimmune En... more Dark-Agouti rats were immunized with spinal cord homogenate to develop Experimental Autoimmune Encephalomyelitis, a model of multiple sclerosis. We assessed motor signs and recorded VEPs for five or eight weeks with epidural or epidermal electrodes, respectively, with final histopathology of optic nerves (ONs). Injected rats exhibited motor deficits a week after immunization. VEP delays arose from the 2nd to the 5th week, when a recovery occurred in epidermal-recorded rats. ON damage appeared in epidural-, but not in epidermal-recorded rats, probably due to a remyelination process. VEP could be exploited as neurophysiological marker to test novel treatments against neurodegeneration involving ONs.

Research paper thumbnail of Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model

Blood, 2010

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of ... more Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patien...

Research paper thumbnail of The 4‐Hole‐Board Test for Assessment of Long‐Term Spatial Memory in Mice

Current Protocols

The hole-board test has been used in rodents since the early 60s to measure exploratory behavior,... more The hole-board test has been used in rodents since the early 60s to measure exploratory behavior, locomotor activity and cognitive function. The test is based on rodents' natural curiosity and attraction for novelty. Basically, the hole-board consists of a small square arena with an extractable platform as floor, which has a set of equally spaced circular holes on its surface. In this article, we describe the protocol of a 4-hole-board test allowing the assessment of long-term spatial memory in mice without the employment of water or food restriction, painful stimuli (as electrical shocks) or any aversive condition (as forced swimming or exposure to intense light). Four holes are present on the floor of the square arena (one for each of its four quadrants). Mice released in the arena spontaneously approach the holes and explore them by briefly inserting the snout inside, a behavior defined as nose-poking (or head-dipping). If, after 24 hr, rodents are re-exposed to the hole-board, the novelty of the holes decreases. Animals with an intact long-term memory will show a reduction of the frequency of nose-poking into the holes. The total number of nose-pokes on day 1 is an index of exploration, while the percentage of decrease in nose-poking on day 2 represents an index of long-term spatial memory. Number of quadrant crossings is scored as a control measure for locomotor activity, which with the present protocol should remain stable across the days of testing. Indeed, the 4-hole-board test represents a stress-free and animal-friendly option to evaluate long-term spatial memory. In the present paper, we provide detailed description of the hole-board apparatus and step-by-step protocol for assessment of spatial memory in mice. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Validation of the 4-hole-board Basic Protocol 2: Evaluation of long-term spatial memory through the 4-hole-board test.

Research paper thumbnail of Dose‐dependent effect of myelin oligodendrocyte glycoprotein on visual function and optic nerve damage in experimental autoimmune encephalomyelitis

Journal of Neuroscience Research, 2022

Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprote... more Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprotein (MOG) to develop experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis. Typical EAE motor impairments were assessed daily and noninvasive visual evoked potentials (VEPs) were recorded at baseline and 5 weeks after immunization, with final histopathology of optic nerves (ONs). Immunized rats exhibited a relapsing‐remitting clinical course. Both VEP and histological abnormalities were detected in a MOG dose‐dependent gradient. Increasing MOG dosage augmented visual function impairment in EAE, which could be monitored with VEP recording to assess demyelination and axonal loss along ONs.

Research paper thumbnail of Non-invasive visual evoked potentials under sevoflurane versus ketamine-xylazine in rats

Research paper thumbnail of Apparatus design and behavioural testing protocol for the evaluation of spatial working memory in mice through the spontaneous alternation T-maze

Scientific Reports, 2021

Spatial working memory can be assessed in mice through the spontaneous alternation T-maze test. T... more Spatial working memory can be assessed in mice through the spontaneous alternation T-maze test. The T-maze is a T-shaped apparatus featuring a stem (start arm) and two lateral goal arms (left and right arms). The procedure is based on the natural tendency of rodents to prefer exploring a novel arm over a familiar one, which induces them to alternate the choice of the goal arm across repeated trials. During the task, in order to successfully alternate choices across trials, an animal has to remember which arm had been visited in the previous trial, which makes spontaneous alternation T-maze an optimal test for spatial working memory. As this test relies on a spontaneous behaviour and does not require rewards, punishments or pre-training, it represents a particularly useful tool for cognitive evaluation, both time-saving and animal-friendly. We describe here in detail the apparatus and the protocol, providing representative results on wild-type healthy mice.

Research paper thumbnail of Functional evolution of visual involvement in experimental autoimmune encephalomyelitis

Multiple Sclerosis Journal - Experimental, Translational and Clinical, 2020

Background Experimental autoimmune encephalomyelitis (EAE) is a common animal model of multiple s... more Background Experimental autoimmune encephalomyelitis (EAE) is a common animal model of multiple sclerosis (MS). C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein exhibit chronic disease course, together with optic neuritis, consisting of demyelination/axonal loss of the optic nerve. Objectives To characterize functional and structural visual damages in two different phases of EAE: pre- and post-motor onset. Methods Visual alterations were detected with Visual Evoked Potential (VEP), Electroretinogram (ERG) and Optical Coherence Tomography (OCT). Optic nerve histology was performed at 7 (pre-motor onset) or 37 (post-motor onset) days post-immunization (dpi). Results At 7 dpi, optic nerve inflammation was similar in EAE eyes with and without VEP latency delay. Demyelination was detected in EAE eyes with latency delay (p

Research paper thumbnail of A new electrophysiological non-invasive method to assess retinocortical conduction time in the Dark Agouti rat through the simultaneous recording of electroretinogram and visual evoked potential

Documenta Ophthalmologica, 2019

Purpose To develop a non-invasive method exploiting simultaneous recording of epidermal visual ev... more Purpose To develop a non-invasive method exploiting simultaneous recording of epidermal visual evoked potential (VEP) and epicorneal electroretinogram (ERG) to study retinocortical function and to evaluate its reliability and repeatability over time. Methods Female wild-type DA rats were anesthetized with ketamine/xylazine (40/5 mg/kg). Epidermal VEP (Ag/AgCl cup electrode on scalp) and epicorneal ERG (gold ring electrode on eye surface) were recorded simultaneously in response to flash stimulation. Results ANOVA for repeated measures showed that peak times of ERG b-wave and of VEP N1 and P2 were stable across 6 weekly time-points, as well as the corresponding amplitudes. Mean retinocortical time from b-wave to N1 (RCT1) was 7.6 ms and remained comparable across the 6 time-points. Mean retinocortical time from b-wave to P2 (RCT2) was 28.7 ms and did not show significant variations over time. Coefficient of variation (CoV%) and CoV% adjusted for sample size, namely relative standard error (RSE%), were calculated as indexes of repeatability. Good RSE% over time was obtained (< 5% for b-wave, N1 and P2 peak times; < 20% and < 7% for RCT1 and RCT2, respectively). Conclusions Simultaneous recording of ERG and VEP has been previously achieved through invasive methods requiring surgery. Here, we present a new non-invasive method, which allowed to obtain peak and retinocortical times that were constant across a long period and had a good repeatability over time. This method will ensure not only a gain in animal welfare, but will also avoid stress and eye or brain lesions which can interfere with experimental variables.

Research paper thumbnail of Modulation of ERK1/MAPK3 potentiates ERK nuclear signalling, facilitates neuronal cell survival and improves memory in mouse models of neurodegenerative disorders

Cell signalling mechanisms are central to neuronal activity and their dysregulation may lead to n... more Cell signalling mechanisms are central to neuronal activity and their dysregulation may lead to neurodegenerative processes and associated cognitive decline. So far, a major effort has been directed toward the dissection of disease specific pathways with the still unmet promise to develop precision medicine strategies. With a different approach, here we show that a selective genetic potentiation of neuronal ERK signalling prevents cell death in vitro and in vivo in the mouse brain while ERK attenuation does the opposite. This neuroprotective effect can also be induced pharmacologically by a cell permeable peptide mimicking the loss of ERK1 MAP kinase, leading to a selective enhancement of ERK2 mediated nuclear cell signalling. The drug treatment prevents neurodegeneration in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's disease (PD). Importantly, the selective potentiation of ERK2 signalling facilitates both structural and synaptic plasticity, enha...

Research paper thumbnail of Non‐invasive Visual Evoked Potentials to assess optic nerve involvement in the Dark Agouti rat model of Experimental Autoimmune Encephalomyelitis induced by Myelin Oligodendrocyte Glycoprotein

Research paper thumbnail of Semi-invasive and non-invasive recording of visual evoked potentials in mice

Documenta Ophthalmologica, 2019

PurposeVisual evoked potentials (VEPs) are used to assess visual function in preclinical models o... more PurposeVisual evoked potentials (VEPs) are used to assess visual function in preclinical models of neurodegenerative diseases. VEP recording with epidural screw electrodes is a common method to study visual function in rodents, despite being an invasive procedure that can damage the tissue under the skull. The present study was performed to test a semi-invasive (epicranial) and a non-invasive (epidermal) VEP recording technique, comparing them with the classic epidural acquisition method.MethodsFlash VEPs were recorded from C57BL/6 mice on three separate days within 2 weeks. Waveforms, latencies and amplitudes of the components were compared between the three different methods, utilizing coefficient of repeatability, coefficient of variation and intersession standard deviation to evaluate reproducibility.ResultsWhile epidural electrodes succeeded in recording two negative peaks (N1 and N2), epicranial and epidermal electrodes recorded a single peak (N1). Statistical indexes showed a comparable reproducibility between the three techniques, with a greater stability of N1 latency recorded through epicranial electrodes. Moreover, N1 amplitudes recorded with the new less-invasive methods were more reproducible compared to the invasive gold-standard technique.ConclusionsThese results demonstrate the reliability of semi- and non-invasive VEP recordings, which can be useful to evaluate murine models of neurological diseases.

Research paper thumbnail of Visual evoked potentials can be reliably recorded using noninvasive epidermal electrodes in the anesthetized rat

Documenta ophthalmologica. Advances in ophthalmology, Jun 1, 2018

Visual evoked potentials (VEPs) are a powerful tool to evaluate nervous conduction along the visu... more Visual evoked potentials (VEPs) are a powerful tool to evaluate nervous conduction along the visual pathways, both in humans and in animal models. Traditionally, epidural screw electrodes are used to record VEPs in preclinical research. Here we tested the feasibility in the preclinical setting of the same noninvasive technique used for clinical VEP acquisition, by using epidermal cup electrodes with no surgical procedures. Monocular flash VEPs were recorded bilaterally under sevoflurane anesthesia once a week for 6 weeks in 14 dark Agouti rats, 7 with implanted epidural screws and 7 with epidermal 6 mm Ø Ag/AgCl cups. VEP traces obtained with the two techniques were morphologically comparable. There were no significant differences in latency of the main visual component between screw-recorded VEPs (sVEPs) and cup-recorded VEPs (cVEPs). Amplitude values with epidermal cups were significantly lower than those with epidural screws. Both techniques provided latencies and amplitudes whic...

Research paper thumbnail of Severe Intellectual Disability and Enhanced GABAergic Synaptogenesis in a Novel Model of Rare RASopathies

Biological Psychiatry, 2017

Dysregulation of Ras-extracellular signal-related kinase (ERK) signaling gives rise to RASopathie... more Dysregulation of Ras-extracellular signal-related kinase (ERK) signaling gives rise to RASopathies, a class of neurodevelopmental syndromes associated with intellectual disability. Recently, much attention has been directed at models bearing mild forms of RASopathies whose behavioral impairments can be attenuated by inhibiting the Ras-ERK cascade in the adult. Little is known about the brain mechanisms in severe forms of these disorders. We performed an extensive characterization of a new brain-specific model of severe forms of RASopathies, the KRAS(12V) mutant mouse. The KRAS(12V) mutation results in a severe form of intellectual disability, which parallels mental deficits found in patients bearing mutations in this gene. KRAS(12V) mice show a severe impairment of both short- and long-term memory in a number of behavioral tasks. At the cellular level, an upregulation of ERK signaling during early phases of postnatal development, but not in the adult state, results in a selective enhancement of synaptogenesis in gamma-aminobutyric acidergic interneurons. The enhancement of ERK activity in interneurons at this critical postnatal time leads to a permanent increase in the inhibitory tone throughout the brain, manifesting in reduced synaptic transmission and long-term plasticity in the hippocampus. In the adult, the behavioral and electrophysiological phenotypes in KRAS(12V) mice can be temporarily reverted by inhibiting gamma-aminobutyric acid signaling but not by a Ras-ERK blockade. Importantly, the synaptogenesis phenotype can be rescued by a treatment at the developmental stage with Ras-ERK inhibitors. These data demonstrate a novel mechanism underlying inhibitory synaptogenesis and provide new insights in understanding mental dysfunctions associated to RASopathies.

Research paper thumbnail of MicelackingRas- GRF1 showcontextualfearcondition- ing butnotspatialmemoryimpair- ments:convergentevidencefromtwo independentlygeneratedmousemutant lines

Frontiers in Behavioral Neuroscience, 2011

Research paper thumbnail of Behavioral Methods for the Study of the Ras–ERK Pathway in Memory Formation and Consolidation: Passive Avoidance and Novel Object Recognition Tests

Methods in Molecular Biology, 2013

Memory is a high-level brain function that enables organisms to adapt their behavioral responses ... more Memory is a high-level brain function that enables organisms to adapt their behavioral responses to the environment, hence increasing their probability of survival. The Ras-ERK pathway is a key molecular intracellular signalling cascade for memory consolidation. In this chapter we will describe two main one-trial behavioral tests commonly used in the field of memory research in order to assess the role of Ras-ERK signalling in long-term memory: passive avoidance and object recognition. Passive avoidance (PA) is a fear-motivated instrumental learning task, designed by Jarvik and Essman in 1960, in which animals learn to refrain from emitting a behavioral response that has previously been associated with a punishment. We will describe here the detailed protocol and show some examples of how PA can reveal impairments or enhancements in memory consolidation following loss or gain of function genetic manipulations of the Ras-ERK pathway. The phenotypes of global mutants as Ras-GRF1 KO, GENA53, and ERK1 KO mice, as well as of conditional region-specific mutants (striatal K-CREB mice), will be illustrated as examples. Novel object recognition (NOR), developed by Ennaceur and Delacour in 1988, is instead a more recent and highly ecological test, which relies on the natural tendency of rodents to spontaneously approach and explore novel objects, representing hence a useful non-stressful tool for the study of memory in animals without the employment of punishments or starvation/water restriction regimens. Careful indications will be given on how to select the positions for the novel object, in order to counterbalance for individual side preferences among mice during the training. Finally, the methods for calculating two learning indexes will be described. In addition to the classical discrimination index (DI) that measures the ability of an animal to discriminate between two different objects which are presented at the same time, we will describe the formula of a new index that we present here for the first time, the recognition index (RI), which quantifies the ability of an animal to recognize a same object at different time points and that, by taking into account the basal individual preferences displayed during the training, can give a more accurate measure of an animal&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s actual recognition memory.