The Stereological Analysis and Spatial Distribution of Neurons in the Human Subthalamic Nucleus

The subthalamic nucleus (STN) is a small, ovoid structure, and an important site of deep brain stimulation (DBS) for the treatment of Parkinson’s disease. Although the STN is a clinically important structure, there are many unresolved issues with regard to it. These issues are especially related to the anatomical subdivision, neuronal phenotype, neuronal composition, and spatial distribution. In this study, we have examined the expression pattern of 8 neuronal markers [nNOS, NeuN, parvalbumin (PV), calbindin (CB), calretinin (CR), FOXP2, NKX2.1, and PAX6] in the adult human STN. All of the examined markers, except CB, were present in the STN. To determine the neuronal density, we have performed stereological analysis on Nissl-stained and immunohistochemical slides of positive markers. The stereology data were also used to develop a three-dimensional map of the spatial distribution of neurons within the STN. The nNOS population exhibited the largest neuronal density. The estimated total number of nNOS STN neurons is 281,308 ± 38,967 (± 13.85%). The STN neuronal subpopulations can be divided into two groups: one with a neuronal density of approximately 3,300 neurons/mm3 and the other with a neuronal density of approximately 2,200 neurons/mm3. The largest density of STN neurons was observed along the ventromedial border of the STN and the density gradually decreased toward the dorsolateral border. In this study, we have demonstrated the presence of 7 neuronal markers in the STN, three of which were not previously described in the human STN. The human STN is a collection of diverse, intermixed neuronal subpopulations, and our data, as far as the cytoarchitectonics is concerned, did not support the tripartite STN subdivision.

Diverse roles of MeCP2 in the specification and maintenance of midbrain dopamine phenotype.

Midbrain dopamine (DA) neurons are associated with locomotor and psychiatric disorders. DA neuronal phenotype is specified in ancestral progenitors and maintained throughout differentiation. Here we demonstrate that premature MeCP2 expression prevents DA progenitors from acquiring DA phenotype through interfering NURR1 transactivation. By contrast, the maintenance of DA phenotype is not affected by MeCP2 overexpression in DA neurons. By analyzing the DNA methylation and MeCP2 binding to the promoter of DA phenotype gene tyrosine hydroxylase (Th) along differentiation, we show that Th expression is determined by TET1-mediated de-methylation of NURR1 binding sites within Th promoter. Premature MeCP2 dominates the DNA binding of these sites thereby blocking TET1 function in DA progenitors, whereas TET1 prevents excessive MeCP2 binding in DA neurons. Finally, we show that targeted de-methylation in DA progenitors protects phenotype specification from premature MeCP2 expression, whereas targeted methylation disturbs phenotype maintenance in MeCP2-overexpressed DA neurons. These findings demonstrate MeCP2 as a novel determining factor for DA neuronal phenotype and function.

Direct induction of human neurons from fibroblasts carrying the neuropsychiatric 22q11.2 microdeletion reveals transcriptome- and epigenome-wide alterations

Standard methods for the creation of neuronal cells via direct induction from primary tissue use perinatal fibroblasts, which hinders the important study of patient specific genetic lesions such as those underlying neuropsychiatric disorders. To address this we developed a novel method for the direct induction of neuronal cells (induced neuronal cells, iN cells) from adult human fibroblast cells. Reprogramming fibroblasts into iN cells via recombinant virus resulted in cells that stain for markers such as MAP2 and PSA-NCAM and exhibit electrophysiological properties such as action potentials and voltage dependent sodium- and potassium currents that reveal a neuronal phenotype. Transcriptome and chromatin analysis using RNA-Seq, microRNA-Seq and ATAC-Seq, respectively, further confirm neuronal character. 22q11.2 Deletion-Syndrome (22q11DS) is caused by a large 3 million base-pair heterozygous deletion on human chromosome 22 and is strongly associated with neurodevelopmental, neuropsychiatric phenotypes such as schizophrenia and autism. We leverage the direct-iN cell model for the study of genetic neurodevelopmental conditions by presenting gene-by-gene as well as network-wide effects of the 22q11DS deletion on gene expression in human neuronal cells, on several levels of functional genomics analysis. Some of the genes within the 22q11DS deletion boundary exhibit unexpected cell-type-specific changes in transcript levels, and genome-wide we can detect dysregulation of calcium channel subunit genes and other genes known to be involved in autism or schizophrenia, such as NRXN1, as well synaptic pathways. This genome-wide effect on gene expression can also be observed at the microRNA and chromatin levels, showing that the iN cells have indeed converted to a neuronal phenotype at several regulatory levels: chromatin, protein-coding RNAs and microRNAs, revealing relevant disease pathways and genes. We present this model of inducing neurons from fibroblasts as a useful general resource to study the genetic and molecular basis of normal and abnormal brain development and brain function.

Age-Related Transcriptional Deregulation of Genes Coding Synaptic Proteins in Alzheimer's Disease Murine Model: Potential Neuroprotective Effect of Fingolimod

Alzheimer's disease (AD) induces time-dependent changes in sphingolipid metabolism, which may affect transcription regulation and neuronal phenotype. We, therefore, analyzed the influence of age, amyloid β precursor protein (AβPP), and the clinically approved, bioavailable sphingosine-1-phosphate receptor modulator fingolimod (FTY720) on the expression of synaptic proteins. RNA was isolated, reverse-transcribed, and subjected to real-time PCR. Expression of mutant (V717I) AβPP led to few changes at 3 months of age but reduced multiple mRNA coding for synaptic proteins in a 12-month-old mouse brain. Complexin 1 (Cplx1), SNAP25 (Snap25), syntaxin 1A (Stx1a), neurexin 1 (Nrxn1), neurofilament light (Nefl), and synaptotagmin 1 (Syt1) in the hippocampus, and VAMP1 (Vamp1) and neurexin 1 (Nrxn1) in the cortex were all significantly reduced in 12-month-old mice. Post mortem AD samples from the human hippocampus and cortex displayed lower expression of VAMP, synapsin, neurofilament light (NF-L) and synaptophysin. The potentially neuroprotective FTY720 reversed most AβPP-induced changes in gene expression (Cplx1, Stx1a, Snap25, and Nrxn1) in the 12-month-old hippocampus, which is thought to be most sensitive to early neurotoxic insults, but it only restored Vamp1 in the cortex and had no influence in 3-month-old brains. Further study may reveal the potential usefulness of FTY720 in the modulation of deregulated neuronal phenotype in AD brains.

Evidence for PKD2L1-positive neurons present distant from the central canal in the ventromedial spinal cord and Medulla of the adult mouse

Neurons in contact with the cerebrospinal fluid (CSF) are found around the medullo-spinal central canal (CC) in adult mice. These neurons (CSF-cNs), located within or below the ependymal cell layer known as the stem cell niche, present a characteristic morphology with a dendrite projecting to the CC and ending with a protrusion. They are GABAergic, characterized by an immature neuronal phenotype and selectively express PKD2L1, a channel member of the TRP channel superfamily with properties of sensory receptor. Using immunohistological techniques in mice, we characterize a new population of PKD2L1 positive cells that is observed around embryonic day 16 (E16), is present distant from the CC in a zone enriched with astrocytes and ependymal fibers of the ventro-medial spinal cord and medulla. With development, their number appears stable although smaller than that of CSF-cNs and they progressively become more distant from the CC with the reorganization of the CC region. These neurons share both functional and phenotypical properties with CSF-cNs, but they appear subdivided in two groups. One, present along the midline, has a bipolar morphology and extend a long dendrite along ependymal fibers and towards the CC. The second group, localized in more ventro-lateral regions, has a multipolar morphology and no apparent projection to the CC Altogether, we describe a novel population of PKD2L1+ neurons distant from the CC but with properties similar to CSF-cNs that might serve to sense modification in the composition of either CSF or interstitial liquid, a function that will need to be confirmed.

Inducible knockout of Clec16a in mice results in sensory neurodegeneration

CLEC16A has been shown to play a role in autophagy/mitophagy processes. Additionally, genetic variants in CLEC16A have been implicated in multiple autoimmune diseases. We generated an inducible whole-body knockout, Clec16aΔUBC mice, to investigate the loss of function of CLEC16A. The mice exhibited a neuronal phenotype including tremors and impaired gait that rapidly progressed to dystonic postures. Nerve conduction studies and pathological analysis revealed loss of sensory axons that are associated with this phenotype. Activated microglia and astrocytes were found in regions of the CNS. Several mitochondrial-related proteins were up- or down-regulated. Upregulation of interferon stimulated gene 15 (IGS15) were observed in neuronal tissues. CLEC16A expression inversely related to IGS15 expression. ISG15 may be the link between CLEC16A and downstream autoimmune, inflammatory processes. Our results demonstrate that a whole-body, inducible knockout of Clec16a in mice results in an inflammatory neurodegenerative phenotype resembling spinocerebellar ataxia.

INPP5K and SIL1 associated pathologies with overlapping clinical phenotypes converge through dysregulation of PHGDH

Marinesco-Sjögren syndrome (MSS) is a rare human disorder caused by biallelic mutations in SIL1 characterized by cataracts in infancy, myopathy and ataxia, symptoms that are also associated with a novel disorder caused by mutations in INPP5K. While these phenotypic similarities may suggest commonalties at a molecular level, an overlapping pathomechanism has not been established yet. In this study, we present six new INPP5K patients and expand the current mutational and phenotypical spectrum of the disease showing the clinical overlap between MSS and the INPP5K-phenotype. We applied unbiased proteomic profiling on cells derived from MSS- and INPP5K-patients and identified alterations in D-3-phosphoglycerate dehydrogenase as a common molecular feature. D-3-phosphoglycerate dehydrogenase modulates the production of L-serine and mutations in this enzyme were previously associated with a neurological phenotype, which clinically overlaps with MSS and INPP5K-disease. As, L-serine administration represents a promising therapeutic strategy for D-3-phosphoglycerate dehydrogenase patients, we tested the effect of L-serine in generated sil1, phgdh and inpp5k a + b zebrafish models which showed an improvement in their neuronal phenotype. Thus our study defines a core phenotypical feature underpinning a key common molecular mechanism in three rare diseases and reveals a common and novel therapeutic target for these patients.

Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs).

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.