Spinal muscular atrophy (SMA) is a pediatric genetic disease, characterized by motor neuron (MN) ... more Spinal muscular atrophy (SMA) is a pediatric genetic disease, characterized by motor neuron (MN) death, leading to progressive muscle weakness, respiratory failure, and, in the most severe cases, to death. Abnormalities at the neuromuscular junction (NMJ) have been reported in SMA, including neurofilament (NF) accumulation at presynaptic terminals, immature and smaller than normal endplates, reduced transmitter release, and, finally, muscle denervation. Here we have studied the role of agrin in SMAΔ7 mice, the experimental model of SMAII. We observed a 50% reduction in agrin expression levels in quadriceps of P10 SMA mice compared to age-matched WT controls. To counteract such condition, we treated SMA mice from birth onwards with therapeutic agrin biological NT-1654, an active splice variant of agrin retaining synaptogenic properties, which is also resistant to proteolytic cleavage by neurotrypsin. Mice were analyzed for behavior, muscle and NMJ histology, and survival. Motor behav...
Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous m... more Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer...
Proceedings of the National Academy of Sciences of the United States of America, Feb 14, 2017
Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degenera... more Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and...
VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic La... more VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic st...
Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington&#... more Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington's disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7-NPs-Chol), which per se is not blood-brain barrier (BBB) permeable, to obtain high-rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7-NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7-NPs-Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonst...
Despite knowledge on the molecular basis of ALS having quickly progressed over the last few years... more Despite knowledge on the molecular basis of ALS having quickly progressed over the last few years, such discoveries have not yet translated into new therapeutics. With the advancement of stem cell technologies there is hope for stem cell therapeutics as novel treatments for ALS. Areas Covered: we discuss in detail the therapeutic potential of different types of stem cells in pre-clinical and clinical works. Moreover we address many open questions in clinical translation. Expert Opinion: SC therapy is a potentially promising new treatment for ALS and the need to better understand how to develop cell-based experimental treatments, and how to implement them in clinical trials, becomes more pressing. Mesenchymal Stem Cells and Neural Fetal Stem Cells have emerged as safe and potentially effective cell types but there is a need to carry out appropriately designed experimental studies to verify their long-term safety and possibly efficacy. Moreover the cost-benefit analysis of the results must take into account the quality of life of the patients as a major endpoint. It is our opinion that a multicenter international clinical program aimed at fine-tuning and coordinating transplantation procedures and protocols is mandatory.
Several functional connectivity approaches require the definition of a set of ROIs that act as ne... more Several functional connectivity approaches require the definition of a set of ROIs that act as network nodes. Different methods have been developed to define these nodes and to derive their functional and effective connections, most of which are rather complex. Here we aim to propose a relatively simple “one-step” border detection and ROI estimation procedure employing the fuzzy c-mean clustering algorithm. To test this procedure and to explore insular connectivity beyond the two/three-region model currently proposed in the literature, we parcellated the insular cortex of a group of twenty healthy right-handed volunteers (10 females) scanned in a resting state condition. Employing a high-dimensional functional connectivity-based clustering process, we confirmed the two patterns of connectivity previously described. This method revealed a complex pattern of functional connectivity where the two previously detected insular clusters are subdivided into several other networks, some of w...
The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injur... more The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The d-retro-inverso form of c-Jun N-terminal kinase-inhibitor (D-JNKI1), a cell-permeable inhibitor of JNK, powerfully reduces neuronal death induced by permanent and transient ischemia, even when administered 6 h after the ischemic insult, offering a clinically relevant window. We investigated the JNK molecular cascade activation in rat cerebral ischemia and the effects of D-JNKI1 on this cascade. c-Jun activation starts after 3 h after ischemia and peaks at 6 h in the ischemic core and in the penumbra at 1 h and at 6 h respectively. The 6 h c-Jun activation peak correlates well with that of P-JNK. We also examined the activation of the two direct JNK activators, MAP kinase kinase 4 (MKK4) and MAP kinase kinase 7 (MKK7). MKK4 showed the same time course as JNK in both core and penumbra, reaching peak activation at 6 h. MKK7 did not show any significant increase of phosphorylation in either core or penumbra. D-JNKI1 markedly prevented the increase of P-c-Jun in both core and penumbra and powerfully inhibited caspase-3 activation in the core. These results confirm that targeting the JNK cascade using the TAT cell-penetrating peptide offers a promising therapeutic approach for ischemia, raising hopes for human neuroprotection, and elucidates the molecular pathways leading to and following JNK activation.
The human insula is hidden in the depth of the cerebral hemisphere by the overlying frontal and t... more The human insula is hidden in the depth of the cerebral hemisphere by the overlying frontal and temporal opercula, and consists of three cytoarchitectonically distinct regions: the anterior agranular area, posterior granular area, and the transitional dysgranular zone; each has distinct histochemical staining patterns and specific connectivity. Even though there are several studies reporting the functional connectivity of the insula with the cingulated cortex, its relationships with other brain areas remain elusive in humans. Therefore, we decided to use resting state functional connectivity to elucidate in details its connectivity, in terms of cortical and subcortical areas, and also of lateralization. We investigated correlations in BOLD fluctuations between specific regions of interest of the insula and other brain areas of right-handed healthy volunteers, on both sides of the brain. Our findings document two major complementary networks involving the ventral-anterior and dorsal-...
The human insula has been parcellated on the basis of resting state functional connectivity and d... more The human insula has been parcellated on the basis of resting state functional connectivity and diffusion tensor imaging. Little is known about the organization of the insula when involved in active tasks. We explored this issue using a novel meta-analytic clustering approach. We queried the BrainMap database asking for papers involving normal subjects that recorded activations in the insular cortex, retrieving 1305 papers, involving 22,872 subjects and a total of 2957 foci. Data were analyzed with several different methodologies, some of which expressly designed for this work. We used meta-analytic connectivity modeling and meta-analytic clustering of data obtained from the BrainMap database. We performed cluster analysis to subdivide the insula in areas with homogeneous connectivity, and density analysis of the activated foci using Voronoi tessellation. Our results confirm and extend previous findings obtained investigating the resting state connectivity of the anterior-posterior ...
Spinal muscular atrophy (SMA) is a pediatric genetic disease, characterized by motor neuron (MN) ... more Spinal muscular atrophy (SMA) is a pediatric genetic disease, characterized by motor neuron (MN) death, leading to progressive muscle weakness, respiratory failure, and, in the most severe cases, to death. Abnormalities at the neuromuscular junction (NMJ) have been reported in SMA, including neurofilament (NF) accumulation at presynaptic terminals, immature and smaller than normal endplates, reduced transmitter release, and, finally, muscle denervation. Here we have studied the role of agrin in SMAΔ7 mice, the experimental model of SMAII. We observed a 50% reduction in agrin expression levels in quadriceps of P10 SMA mice compared to age-matched WT controls. To counteract such condition, we treated SMA mice from birth onwards with therapeutic agrin biological NT-1654, an active splice variant of agrin retaining synaptogenic properties, which is also resistant to proteolytic cleavage by neurotrypsin. Mice were analyzed for behavior, muscle and NMJ histology, and survival. Motor behav...
Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous m... more Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer...
Proceedings of the National Academy of Sciences of the United States of America, Feb 14, 2017
Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degenera... more Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and...
VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic La... more VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic st...
Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington&#... more Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington's disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7-NPs-Chol), which per se is not blood-brain barrier (BBB) permeable, to obtain high-rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7-NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7-NPs-Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonst...
Despite knowledge on the molecular basis of ALS having quickly progressed over the last few years... more Despite knowledge on the molecular basis of ALS having quickly progressed over the last few years, such discoveries have not yet translated into new therapeutics. With the advancement of stem cell technologies there is hope for stem cell therapeutics as novel treatments for ALS. Areas Covered: we discuss in detail the therapeutic potential of different types of stem cells in pre-clinical and clinical works. Moreover we address many open questions in clinical translation. Expert Opinion: SC therapy is a potentially promising new treatment for ALS and the need to better understand how to develop cell-based experimental treatments, and how to implement them in clinical trials, becomes more pressing. Mesenchymal Stem Cells and Neural Fetal Stem Cells have emerged as safe and potentially effective cell types but there is a need to carry out appropriately designed experimental studies to verify their long-term safety and possibly efficacy. Moreover the cost-benefit analysis of the results must take into account the quality of life of the patients as a major endpoint. It is our opinion that a multicenter international clinical program aimed at fine-tuning and coordinating transplantation procedures and protocols is mandatory.
Several functional connectivity approaches require the definition of a set of ROIs that act as ne... more Several functional connectivity approaches require the definition of a set of ROIs that act as network nodes. Different methods have been developed to define these nodes and to derive their functional and effective connections, most of which are rather complex. Here we aim to propose a relatively simple “one-step” border detection and ROI estimation procedure employing the fuzzy c-mean clustering algorithm. To test this procedure and to explore insular connectivity beyond the two/three-region model currently proposed in the literature, we parcellated the insular cortex of a group of twenty healthy right-handed volunteers (10 females) scanned in a resting state condition. Employing a high-dimensional functional connectivity-based clustering process, we confirmed the two patterns of connectivity previously described. This method revealed a complex pattern of functional connectivity where the two previously detected insular clusters are subdivided into several other networks, some of w...
The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injur... more The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The d-retro-inverso form of c-Jun N-terminal kinase-inhibitor (D-JNKI1), a cell-permeable inhibitor of JNK, powerfully reduces neuronal death induced by permanent and transient ischemia, even when administered 6 h after the ischemic insult, offering a clinically relevant window. We investigated the JNK molecular cascade activation in rat cerebral ischemia and the effects of D-JNKI1 on this cascade. c-Jun activation starts after 3 h after ischemia and peaks at 6 h in the ischemic core and in the penumbra at 1 h and at 6 h respectively. The 6 h c-Jun activation peak correlates well with that of P-JNK. We also examined the activation of the two direct JNK activators, MAP kinase kinase 4 (MKK4) and MAP kinase kinase 7 (MKK7). MKK4 showed the same time course as JNK in both core and penumbra, reaching peak activation at 6 h. MKK7 did not show any significant increase of phosphorylation in either core or penumbra. D-JNKI1 markedly prevented the increase of P-c-Jun in both core and penumbra and powerfully inhibited caspase-3 activation in the core. These results confirm that targeting the JNK cascade using the TAT cell-penetrating peptide offers a promising therapeutic approach for ischemia, raising hopes for human neuroprotection, and elucidates the molecular pathways leading to and following JNK activation.
The human insula is hidden in the depth of the cerebral hemisphere by the overlying frontal and t... more The human insula is hidden in the depth of the cerebral hemisphere by the overlying frontal and temporal opercula, and consists of three cytoarchitectonically distinct regions: the anterior agranular area, posterior granular area, and the transitional dysgranular zone; each has distinct histochemical staining patterns and specific connectivity. Even though there are several studies reporting the functional connectivity of the insula with the cingulated cortex, its relationships with other brain areas remain elusive in humans. Therefore, we decided to use resting state functional connectivity to elucidate in details its connectivity, in terms of cortical and subcortical areas, and also of lateralization. We investigated correlations in BOLD fluctuations between specific regions of interest of the insula and other brain areas of right-handed healthy volunteers, on both sides of the brain. Our findings document two major complementary networks involving the ventral-anterior and dorsal-...
The human insula has been parcellated on the basis of resting state functional connectivity and d... more The human insula has been parcellated on the basis of resting state functional connectivity and diffusion tensor imaging. Little is known about the organization of the insula when involved in active tasks. We explored this issue using a novel meta-analytic clustering approach. We queried the BrainMap database asking for papers involving normal subjects that recorded activations in the insular cortex, retrieving 1305 papers, involving 22,872 subjects and a total of 2957 foci. Data were analyzed with several different methodologies, some of which expressly designed for this work. We used meta-analytic connectivity modeling and meta-analytic clustering of data obtained from the BrainMap database. We performed cluster analysis to subdivide the insula in areas with homogeneous connectivity, and density analysis of the activated foci using Voronoi tessellation. Our results confirm and extend previous findings obtained investigating the resting state connectivity of the anterior-posterior ...
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Papers by Alessandro Vercelli