Dopaminergic neuron loss in mice due to increased levels of wild-type human α-Synuclein only takes place under conditions of accelerated aging.

Understanding the intricate pathogenic mechanisms behind Parkinson's disease (PD) and its multifactorial nature presents a significant challenge in disease modeling. To address this, we explore genetic models that better capture the disease's complexity. Given that aging is the primary risk factor for PD, this study investigates the impact of aging in conjunction with overexpression of wild-type human α-synuclein (α-Syn) in the dopaminergic system. This is achieved by introducing a novel transgenic mouse strain overexpressing α-Syn under the TH-promoter within the senescence-accelerated SAMP8 (P8) genetic background. Behavioral assessments, conducted at both 10 and 16 months of age, unveil motor impairments exclusive to P8 α-SynTg mice, a phenomenon conspicuously absent in α-SynTg mice. These findings suggest a synergistic interplay between heightened α-Syn levels and the aging process, resulting in motor deficits. These motor disturbances correlate with reduced dopamine (DA) levels, increased DA turnover, synaptic terminal loss, and notably, the depletion of dopaminergic neurons in the substantia nigra and noradrenergic neurons in the locus coeruleus. Furthermore, P8 α-SynTg mice exhibit alterations in gut transit time, mirroring early PD symptoms. In summary, P8 α-SynTg mice effectively replicate parkinsonian phenotypes by combining α-Syn transgene expression with accelerated aging. This model offers valuable insights into the understanding of PD and serves as a valuable platform for further research.

IGF2 interacts with the imprinted gene Cdkn1c to promote terminal differentiation of neural stem cells.

Adult neurogenesis is supported by multipotent neural stem cells (NSCs) with unique properties and growth requirements. Adult NSCs constitute a reversibly quiescent cell population that can be activated by extracellular signals from the microenvironment in which they reside in vivo. Although genomic imprinting plays a role in adult neurogenesis through dose regulation of some relevant signals, the roles of many imprinted genes in the process remain elusive. Insulin-like growth factor 2 (IGF2) is encoded by an imprinted gene that contributes to NSC maintenance in the adult subventricular zone through a biallelic expression in only the vascular compartment. We show here that IGF2 additionally promotes terminal differentiation of NSCs into astrocytes, neurons and oligodendrocytes by inducing the expression of the maternally expressed gene cyclin-dependent kinase inhibitor 1c (Cdkn1c), encoding the cell cycle inhibitor p57. Using intraventricular infusion of recombinant IGF2 in a conditional mutant strain with Cdkn1c-deficient NSCs, we confirm that p57 partially mediates the differentiation effects of IGF2 in NSCs and that this occurs independently of its role in cell-cycle progression, balancing the relationship between astrogliogenesis, neurogenesis and oligodendrogenesis.

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2.

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Cholinergic Senescence in the Ts65Dn Mouse Model for Down Syndrome.

Down syndrome (DS) induces a variable phenotype including intellectual disabilities and early development of Alzheimer's disease (AD). Moreover, individuals with DS display accelerated aging that affects diverse organs, among them the brain. The Ts65Dn mouse is the most widely used model to study DS. Progressive loss of cholinergic neurons is one of the hallmarks of AD present in DS and in the Ts65Dn model. In this study, we quantify the number of cholinergic neurons in control and Ts65Dn mice, observing a general reduction in their number with age but in particular, a greater loss in old Ts65Dn mice. Increased expression of the m1 muscarinic receptor in the hippocampus counteracts this loss. Cholinergic neurons in the Ts65Dn mice display overexpression of the early expression gene c-fos and an increase in the expression of ß-galactosidase, a marker of senescence. A possible mechanism for senescence induction could be phosphorylation of the transcription factor FOXO1 and its retention in the cytoplasm, which we are able to confirm in the Ts65Dn model. In our study, using Ts65Dn mice, we observe increased cholinergic activity, which induces a process of early senescence that culminates in the loss of these neurons.

Aberrations of Genomic Imprinting in Glioblastoma Formation.

In human glioblastoma (GBM), the presence of a small population of cells with stem cell characteristics, the glioma stem cells (GSCs), has been described. These cells have GBM potential and are responsible for the origin of the tumors. However, whether GSCs originate from normal neural stem cells (NSCs) as a consequence of genetic and epigenetic changes and/or dedifferentiation from somatic cells remains to be investigated. Genomic imprinting is an epigenetic marking process that causes genes to be expressed depending on their parental origin. The dysregulation of the imprinting pattern or the loss of genomic imprinting (LOI) have been described in different tumors including GBM, being one of the earliest and most common events that occurs in human cancers. Here we have gathered the current knowledge of the role of imprinted genes in normal NSCs function and how the imprinting process is altered in human GBM. We also review the changes at particular imprinted loci that might be involved in the development of the tumor. Understanding the mechanistic similarities in the regulation of genomic imprinting between normal NSCs and GBM cells will be helpful to identify molecular players that might be involved in the development of human GBM.

Phenotypic characterization of MCP-1 expressing neurons in the rat cerebral cortex.

Chemokines are small, secreted molecules that mediate inflammatory reactions. Neurons and astrocytes constitutively express chemokines implicated in the process of neuroinflammation associated with neurodegenerative diseases. The monocyte chemoattractant protein-1 (MCP-1) has been widely related to this process. However, the constitutive expression of this molecule by neurons has not been elucidated so far. In this study, we set out to characterize the neurochemical phenotype of MCP-1-expressing neurons in the rat neocortex to infer its role in basal conditions. We observed the presence of two populations of neurons expressing MCP-1: One population of cells with weak expression of MCP-1 corresponding to principal neurons (Tbr-1 positive) and a second population with high expression of MCP-1 corresponding to inhibitory neurons (GAD-67 positive), in particular to CCK/CBR1 interneurons. Moreover, high MCP-1-expressing neurons were metabolically active (pCREB positive). The population of CCK interneurons that co-localizes with MCP-1 corresponds to the regular-spiking basket cells and is co-responsible for the perisomatic inhibition of principal pyramidal neurons. Previous studies have demonstrated that MCP-1 can alter the electric properties of neurons and a tonic function for this molecule has been postulated. As CCK-inhibitory neurons are affected in mood disorders, whether the expression of MCP-1 was maintained in humans could be part of the link between inflammatory responses and observed changes in mood state.

Cyclin-Dependent Kinase 4 Regulates Adult Neural Stem Cell Proliferation and Differentiation in Response to Insulin.

Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C ). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs. Stem Cells 2017;35:2403-2416.

MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin.

The identification of mechanisms that maintain stem cell niche architecture and homeostasis is fundamental to our understanding of tissue renewal and repair. Cell adhesion is a well-characterized mechanism for developmental morphogenetic processes, but its contribution to the dynamic regulation of adult mammalian stem cell niches is still poorly defined. We show that N-cadherin-mediated anchorage of neural stem cells (NSCs) to ependymocytes in the adult murine subependymal zone modulates their quiescence. We further identify MT5-MMP as a membrane-type metalloproteinase responsible for the shedding of the N-cadherin ectodomain in this niche. MT5-MMP is co-expressed with N-cadherin in adult NSCs and ependymocytes and, whereas MT5-MMP-mediated cleavage of N-cadherin is dispensable for the regulation of NSC generation and identity, it is required for proper activation of NSCs under physiological and regenerative conditions. Our results indicate that the proliferative status of stem cells can be dynamically modulated by regulated cleavage of cell adhesion molecules.

IRS2 signalling is required for the development of a subset of sensory spinal neurons.

Insulin and insulin-like growth factor-I play important roles in the development and maintenance of neurons and glial cells of the nervous system. Both factors activate tyrosine kinase receptors, which signal through adapter proteins of the insulin receptor substrate (IRS) family. Although insulin and insulin-like growth factor-I receptors are expressed in dorsal root ganglia (DRG), the function of IRS-mediated signalling in these structures has not been studied. Here we address the role of IRS2-mediated signalling in murine DRG. Studies in cultured DRG neurons from different embryonic stages indicated that a subset of nerve growth factor-responsive neurons is also dependent on insulin for survival at very early time points. Consistent with this, increased apoptosis during gangliogenesis resulted in a partial loss of trkA-positive neurons in DRG of Irs2 mutant embryos. Analyses in adult Irs2(-/-) mice revealed that unmyelinated fibre afferents, which express calcitonin gene-related peptide/substance P and isolectin B4, as well as some myelinated afferents to the skin were affected by the mutation. The diminished innervation of glabrous skin in adult Irs2(-/-) mice correlated with longer paw withdrawal latencies in the hot-plate assay. Collectively, these findings indicate that IRS2 signalling is required for the proper development of spinal sensory neurons involved in the perception of pain.

Perivascular nerve fiber α-synuclein regulates contractility of mouse aorta: a link to autonomic dysfunction in Parkinson's disease.

Parkinson's disease and other neurodegenerative disorders associated to changes in alpha-synuclein often result in autonomic dysfunction, most of the time accompanied by abundant expression of this synaptic protein in peripheral autonomic neurons. Given that expression of alpha-synuclein in vascular elements has been previously reported, the present study was undertaken to determine whether alpha-synuclein directly participates in the regulation of vascular responsiveness. We detected by immunohistochemistry perivascular nerve fibers containing alpha-synuclein in the aorta of mice while aortic endothelial cells and muscular fibers themselves did not exhibit detectable levels of this protein. To assess the effect of alpha-synuclein on vascular reactivity, aortic ring preparations obtained from alpha-synuclein-deficient knockout mice and from transgenic mice overexpressing human wild-type alpha-synuclein under the control of the tyrosine hydroxylase-promoter were mounted and equilibrated in organ baths for isometric tension recording. Lack of alpha-synuclein did not modify the relaxant responses to the endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasodilators, but resulted in a greater than normal norepinephrine-induced vasoconstriction along with a lowered response to dopamine, suggesting potential presynaptic changes in dopamine and norepinephrine releases in knockout mice. Overexpression of alpha-synuclein in TH-positive fibers resulted in complex abnormal responses, characterized by lowered acetylcholine-induced relaxation and lowered norepinephrin-induced contraction. Taken together, our data show for the first time that alpha-synuclein is present in sympathetic fibers supplying the murine aorta and provide evidence that changes in alpha-synuclein levels in perivascular fibers play a physiological role in the regulation of vascular function.

BDNF is essentially required for the early postnatal survival of nociceptors.

Neurotrophins promote the survival of specific types of neurons during development and ensure proper maintenance and function of mature responsive neurons. Significant effects of BDNF (Brain-Derived Neurotrophic Factor) on pain physiology have been reported but the contribution of this neurotrophin to the development of nociceptors has not been investigated. We present evidence that BDNF is required for the survival of a significant fraction of peptidergic and non-peptidergic nociceptors in dorsal root ganglia (DRG) postnatally. Bdnf homozygous mutant mice lose approximately half of all nociceptive neurons during the first 2 weeks of life and adult heterozygotes exhibit hypoalgesia and a loss of 25% of all nociceptive neurons. Our in vitro analyses indicate that BDNF-dependent nociceptive neurons also respond to NGF and GDNF. Expression analyses at perinatal times indicate that BDNF is predominantly produced within sensory ganglia and is more abundant than skin-derived NGF or GDNF. Function-blocking studies with BDNF specific antibodies in vitro or cultures of BDNF-deficient sensory neurons suggest that BDNF acts in an autocrine/paracrine way to promote the early postnatal survival of nociceptors that are also responsive to NGF and GDNF. Altogether, the data demonstrate an essential requirement for BDNF in the early postnatal survival of nociceptive neurons.

Metalloproteinase MT5-MMP is an essential modulator of neuro-immune interactions in thermal pain stimulation.

Peripheral interactions between nociceptive fibers and mast cells contribute to inflammatory pain, but little is known about mechanisms mediating neuro-immune communication. Here we show that metalloproteinase MT5-MMP (MMP-24) is an essential mediator of peripheral thermal nociception and inflammatory hyperalgesia. We report that MT5-MMP is expressed by CGRP-containing peptidergic nociceptors in dorsal root ganglia and that Mmp24-deficient mice display enhanced sensitivity to noxious thermal stimuli under basal conditions. Consistently, mutant peptidergic sensory neurons hyperinnervate the skin, a phenotype that correlates with changes in the regulated cleavage of the cell-cell adhesion molecule N-cadherin. In contrast to basal nociception, Mmp24(-/-) mice do not develop thermal hyperalgesia during inflammation, a phenotype that appears associated with alterations in N-cadherin-mediated cell-cell interactions between mast cells and sensory fibers. Collectively, our findings demonstrate an essential role of MT5-MMP in the development of dermal neuro-immune synapses and suggest that this metalloproteinase may be a target for pain control.