Morphologic and functional correlates of synaptic pathology in the cathepsin D knockout mouse model of congenital neuronal ceroid lipofuscinosis

Mutations in the cathepsin D (CTSD) gene cause an aggressive neurodegenerative disease (congenital neuronal ceroid lipofuscinosis) that leads to early death. Recent evidence suggests that presynaptic abnormalities play a major role in the pathogenesis of CTSD deficiencies. To identify the early events that lead to synaptic alterations, we investigated synaptic ultrastructure and function in pre-symptomatic CTSD knock-out (Ctsd(−/−)) mice. Electron microscopy revealed that there were significantly greater numbers of readily releasable synaptic vesicles present in Ctsd(−/−) mice than in wild-type control mice as early as postnatal day 16. The size of this synaptic vesicle pool continued to increase with disease progression in the hippocampus and thalamus of the Ctsd(−/−) mice. Electrophysiology revealed a markedly decreased frequency of miniature excitatory postsynaptic currents (EPSCs) with no effect on pair-pulse modulation of the evoked EPSPs in the hippocampus of Ctsd(−/−) mice. The reduced miniature EPSC frequency was observed before the appearance of epilepsy or any morphological sign of synaptic degeneration. Taken together, the data indicate that CTSD is required for normal synaptic function, and that a failure in synaptic trafficking or recycling may be an early and important pathological mechanism in Ctsd(−/−) mice; these presynaptic abnormalities may initiate synaptic degeneration in advance of subsequent neuronal loss

On the question of human life safety in geologically active zones

Today geological active zones unite active faults of lithosphere especially earth’s crust and caused by them zones of increased permeability such as paleo-valleys and underground water flows, karst and geological bodies, that are different in terms of composition and structure from the enclosing rocks. There is an evidence that mortality in geologically active zones increases dramatically, mental instability is detected and road accidents are more frequent. The purpose of this paper is to estimate the frequency of suicides among the residents of Saint Petersburg living above the geologically active zones and outside these zones and the influence of geomagnetic and gravitational disturbances on them. The dynamics of suicides among residents of 446 high-rise building in the territory of the Kalininsky and Vasileostrovsky districts of the city of Saint Petersburg is analyzed. Geological structure of those buildings was most studied. From 1999 to 2003 there were 268 suicides among the residents of such buildings. The group A included homes that were at least 40 m above the nearest tectonic fault. Group B included residential buildings located above or in the immediate vicinity of the faults. During the geomagnetic storms, full moon and new moon periods the number of suicides in a group A decreased. Magnetic storms and gravitational disturbances did not affect the frequency of suicides in a group B. There is also no significant correlation between dynamics of suicides and daily values of the K-index of the geomagnetic field, as well as between dynamics of suicides and the 3-hour geomagnetic activity in both groups. Results of studies presented in this paper show that there is no evidence of a significant negative impact of tectonic faults on people living above them

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.Peer reviewe

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.Peer reviewe

Expression of GluK1c underlies the developmental switch in presynaptic kainate receptor function

Peer reviewe

PGC-1 alpha Signaling Increases GABA(A) Receptor Subunit alpha 2 Expression, GABAergic Neurotransmission and Anxiety-Like Behavior in Mice

Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) is a master regulator of mitochondria biogenesis and cell stress playing a role in metabolic and degenerative diseases. In the brain PGC-1 alpha expression has been localized mainly to GABAergic interneurons but its overall role is not fully understood. We observed here that the protein levels of gamma-aminobutyric acid (GABA) type A receptor-alpha 2 subunit (GABAR alpha 2) were increased in hippocampus and brain cortex in transgenic (Tg) mice overexpressing PGC-1 alpha in neurons. Along with this, GABAR alpha 2 expression was enhanced in the hippocampus of the PGC-1 alpha Tg mice, as shown by quantitative PCR. Double immunostaining revealed that GABAR alpha 2 co-localized with the synaptic protein gephyrin in higher amounts in the striatum radiatum layer of the hippocampal CA1 region in the Tg compared with Wt mice. Electrophysiology revealed that the frequency of spontaneous and miniature inhibitory postsynaptic currents (mIPSCs) was increased in the CA1 region in the Tg mice, indicative of an augmented GABAergic transmission. Behavioral tests revealed an increase for anxiety-like behavior in the PGC-1 alpha Tg mice compared with controls. To study whether drugs acting on PPAR gamma can affect GABAR alpha 2, we employed pioglitazone that elevated GABAR alpha 2 expression in primary cultured neurons. Similar results were obtained using the specific PPAR gamma agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino) ethyl]-L-tyrosine hydrate (GW1929). These results demonstrate that PGC-1 alpha regulates GABAR alpha 2 subunits and GABAergic neurotransmission in the hippocampus with behavioral consequences. This indicates further that drugs like pioglitazone, widely used in the treatment of type 2 diabetes, can influence GABAR alpha 2 expression via the PPAR gamma/PGC-1 alpha system.Peer reviewe

Dominant mutations in ITPR3 cause Charcot-Marie-Tooth disease

Objective ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence thatITPR3is a Charcot-Marie-Tooth disease gene. Methods Whole-exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease-causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca(2+)imaging. Results Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygousITPR3p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified ade novo ITPR3variant p.Arg2524Cys. Altered Ca2+-transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant-negative effect on inositol 1,4,5-trisphosphate receptor type 3 function. Interpretation Together with two previously identified variants, our report adds further evidence thatITPR3is a disease-causing gene for CMT and indicates altered Ca(2+)homeostasis in disease pathogenesis.Peer reviewe

Alpha2-Containing Glycine Receptors Promote Neonatal Spontaneous Activity of Striatal Medium Spiny Neurons and Support Maturation of Glutamatergic Inputs

Glycine receptors (GlyRs) containing the α2 subunit are highly expressed in the developing brain, where they regulate neuronal migration and maturation, promote spontaneous network activity and subsequent development of synaptic connections. Mutations in GLRA2 are associated with autism spectrum disorder, but the underlying pathophysiology is not described yet. Here, using Glra2-knockout mice, we found a GlyR-dependent effect on neonatal spontaneous activity of dorsal striatum medium spiny neurons (MSNs) and maturation of the incoming glutamatergic innervation. Our data demonstrate that functional GlyRs are highly expressed in MSNs of one-week-old mice, but they do not generate endogenous chloride-mediated tonic or phasic current. Despite of that, knocking out the Glra2 severely affects the shape of action potentials and impairs spontaneous activity and the frequency of miniature AMPA receptor-mediated currents in MSNs. This reduction in spontaneous activity and glutamatergic signaling can attribute to the observed changes in neonatal behavioral phenotypes as seen in ultrasonic vocalizations and righting reflex. In adult Glra2-knockout animals, the glutamatergic synapses in MSNs remain functionally underdeveloped. The number of glutamatergic synapses and release probability at presynaptic site remain unaffected, but the amount of postsynaptic AMPA receptors is decreased. This deficit is a consequence of impaired development of the neuronal circuitry since acute inhibition of GlyRs by strychnine in adult MSNs does not affect the properties of glutamatergic synapses. Altogether, these results demonstrate that GlyR-mediated signaling supports neonatal spontaneous MSN activity and, in consequence, promotes the functional maturation of glutamatergic synapses on MSNs. The described mechanism might shed light on the pathophysiological mechanisms in GLRA2-linked autism spectrum disorder cases

Release and neuromodulatory effects of taurine in the rodent striatum

Tauriini on rikkipitoinen aminohappo, jonka on arveltu mahdollisesti olevan estävä neuromodulaattori nisäkäsaivoissa. Sitä on ehdotettu apulääkkeeksi monissa neurologisissa sairauksissa, kuten aivohalvauksessa ja epilepsiassa. Kuitenkin tauriinin solunulkoisesta pitoisuudesta aivoissa ja sen vaikutuskohteista molekyylitasolla tiedetään hyvin vähän. Tämä tiedon puute haittaa tämän myrkyttömän ja halvan aineen käyttöä lääkkeenä. Tutkimukseni tarkoituksena oli selvittää tauriinin toimintaa neuromodulaattorina ja arvioida, suojeleeko se hermosoluja iskemiassa. Kaikki kokeet tehtiin jyrsijöiden isoaivokuoren ja aivojuovion muodostamalla hermojärjestelmällä. Tauriinin solunulkoisen pitoisuuden osoitin olevan melko korkea ja tarkoin hermoaktiviteetin säätelemä. Hermosolujen aktivoituminen ja iskemia kohottivat huomattavasti tauriinin solunulkoista pitoisuutta, mikä kuvannee sen hermosoluja suojelevaa vaikutusta. Iskemiassa tauriini vähensi soluista glutamaatin vapautumista, mikä on merkitsevin patologinen tekijä iskemiassa ja aivohalvauksessa. Solukalvon kloridikanavien aktivoituminen ja solunsisäisen kalsiumionin pitoisuuden muutokset aiheuttivat tämän vaikutuksen. Yhteenvetona: tulokset osoittavat tauriinin todella olevan neuromodulaattori ja selvittävät sen hermosolujen suojelevaa mekanismia iskemiassa.Taurine is a sulfur-containing amino acid, thought to be a possible inhibitory neuromodulator in the mammalian brain. It is suggested to be the potent supplementary medication in treatment of various neurological diseases, including stroke and epilepsy. However, little is known regarding the regulation of extracellular concentrations of taurine in the brain and the molecular target(s) of its action. This lack of knowledge hampers the clinical usage of this non-toxic and cheap drug. This study was aimed to characterize taurine as a neuromodulator in the brain of mammals and to evaluate whether it is neuroprotective in ischemia. All experiments were done on the corticostriatal system of the rodent brain. It was shown that the extracellular concentration of taurine is large and strictly regulated by changes in neuronal activity. Neuronal depolarization and ischemia significantly elevate the extracellular concentration of taurine, which may reflect its intrinsic protective effect. Under ischemic conditions, taurine suppresses the release of glutamate, which is the main pathological factor in ischemia and stroke. The effect is mediated by activation of membrane chloride channels and regulation of concentration of calcium ions inside the cells. In overall, the findings show that taurine indeed is a neuromodulator and characterize its neuroprotective mechanisms in ischemia