The awakening of dormant neuronal precursors in the adult and aged brain.

Beyond the canonical neurogenic niches, there are dormant neuronal precursors in several regions of the adult mammalian brain. Dormant precursors maintain persisting post-mitotic immaturity from birth to adulthood, followed by staggered awakening, in a process that is still largely unresolved. Strikingly, due to the slow rate of awakening, some precursors remain immature until old age, which led us to question whether their awakening and maturation are affected by aging. To this end, we studied the maturation of dormant precursors in transgenic mice (DCX-CreERT2 /flox-EGFP) in which immature precursors were labelled permanently in vivo at different ages. We found that dormant precursors are capable of awakening at young age, becoming adult-matured neurons (AM), as well as of awakening at old age, becoming late AM. Thus, protracted immaturity does not prevent late awakening and maturation. However, late AM diverged morphologically and functionally from AM. Moreover, AM were functionally most similar to neonatal-matured neurons (NM). Conversely, late AM were endowed with high intrinsic excitability and high input resistance, and received a smaller amount of spontaneous synaptic input, implying their relative immaturity. Thus, late AM awakening still occurs at advanced age, but the maturation process is slow.

Depolarization and Hyperexcitability of Cortical Motor Neurons after Spinal Cord Injury Associates with Reduced HCN Channel Activity.

A spinal cord injury (SCI) damages the axonal projections of neurons residing in the neocortex. This axotomy changes cortical excitability and results in dysfunctional activity and output of infragranular cortical layers. Thus, addressing cortical pathophysiology after SCI will be instrumental in promoting recovery. However, the cellular and molecular mechanisms of cortical dysfunction after SCI are poorly resolved. In this study, we determined that the principal neurons of the primary motor cortex layer V (M1LV), those suffering from axotomy upon SCI, become hyperexcitable following injury. Therefore, we questioned the role of hyperpolarization cyclic nucleotide gated channels (HCN channels) in this context. Patch clamp experiments on axotomized M1LV neurons and acute pharmacological manipulation of HCN channels allowed us to resolve a dysfunctional mechanism controlling intrinsic neuronal excitability one week after SCI. Some axotomized M1LV neurons became excessively depolarized. In those cells, the HCN channels were less active and less relevant to control neuronal excitability because the membrane potential exceeded the window of HCN channel activation. Care should be taken when manipulating HCN channels pharmacologically after SCI. Even though the dysfunction of HCN channels partakes in the pathophysiology of axotomized M1LV neurons, their dysfunctional contribution varies remarkably between neurons and combines with other pathophysiological mechanisms.

Immune-mediated platelet depletion augments Alzheimer's disease neuropathological hallmarks in APP-PS1 mice.

In Alzheimer's disease (AD), platelets become dysfunctional and might contribute to amyloid beta deposition. Here, we depleted platelets in one-year-old APP Swedish PS1 dE9 (APP-PS1) transgenic mice for five days, using intraperitoneal injections of an anti-CD42b antibody, and assessed changes in cerebral amyloidosis, plaque-associated neuritic dystrophy and gliosis. In APP-PS1 female mice, platelet depletion shifted amyloid plaque size distribution towards bigger plaques and increased neuritic dystrophy in the hippocampus. In platelet-depleted females, plaque-associated Iba1+ microglia had lower amounts of fibrillar amyloid beta cargo and GFAP+ astrocytic processes showed a higher overlap with thioflavin S+ amyloid plaques. In contrast to the popular hypothesis that platelets foster plaque pathology, our data suggest that platelets might limit plaque growth and attenuate plaque-related neuritic dystrophy at advanced stages of amyloid plaque pathology in APP-PS1 female mice. Whether the changes in amyloid plaque pathology are due to a direct effect on amyloid beta deposition or are a consequence of altered glial function needs to be further elucidated.

Transcriptomic Profiling Identifies CD8+ T Cells in the Brain of Aged and Alzheimer's Disease Transgenic Mice as Tissue-Resident Memory T Cells.

Peripheral immune cell infiltration into the brain is a prominent feature in aging and various neurodegenerative diseases such as Alzheimer's disease (AD). As AD progresses, CD8+ T cells infiltrate into the brain parenchyma, where they tightly associate with neurons and microglia. The functional properties of CD8+ T cells in the brain are largely unknown. To gain further insights into the putative functions of CD8+ T cells in the brain, we explored and compared the transcriptomic profile of CD8+ T cells isolated from the brain and blood of transgenic AD (APPswe/PSEN1dE9, line 85 [APP-PS1]) and age-matched wild-type (WT) mice. Brain CD8+ T cells of APP-PS1 and WT animals had similar transcriptomic profiles and substantially differed from blood circulating CD8+ T cells. The gene signature of brain CD8+ T cells identified them as tissue-resident memory (Trm) T cells. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis on the significantly upregulated genes revealed overrepresentation of biological processes involved in IFN-ß signaling and the response to viral infections. Furthermore, brain CD8+ T cells of APP-PS1 and aged WT mice showed similar differentially regulated genes as brain Trm CD8+ T cells in mouse models with acute virus infection, chronic parasite infection, and tumor growth. In conclusion, our profiling of brain CD8+ T cells suggests that in AD, these cells exhibit similar adaptive immune responses as in other inflammatory diseases of the CNS, potentially opening the door for immunotherapy in AD.

Phenotypic Characterization and Brain Structure Analysis of Calcium Channel Subunit α2δ-2 Mutant (Ducky) and α2δ Double Knockout Mice.

Auxiliary α2δ subunits of voltage-gated calcium channels modulate channel trafficking, current properties, and synapse formation. Three of the four isoforms (α2δ-1, α2δ-2, and α2δ-3) are abundantly expressed in the brain; however, of the available knockout models, only α2δ-2 knockout or mutant mice display an obvious abnormal neurological phenotype. Thus, we hypothesize that the neuronal α2δ isoforms may have partially specific as well as redundant functions. To address this, we generated three distinct α2δ double knockout mouse models by crossbreeding single knockout (α2δ-1 and -3) or mutant (α2δ-2/ducky) mice. Here, we provide a first phenotypic description and brain structure analysis. We found that genotypic distribution of neonatal litters in distinct α2δ-1/-2, α2δ-1/-3, and α2δ-2/-3 breeding combinations did not conform to Mendel's law, suggesting premature lethality of single and double knockout mice. Notably, high occurrences of infant mortality correlated with the absence of specific α2δ isoforms (α2Δ-2 > α2δ-1 > α2δ-3), and was particularly observed in cages with behaviorally abnormal parenting animals of α2δ-2/-3 cross-breedings. Juvenile α2δ-1/-2 and α2δ-2/-3 double knockout mice displayed a waddling gate similar to ducky mice. However, in contrast to ducky and α2δ-1/-3 double knockout animals, α2δ-1/-2 and α2δ-2/-3 double knockout mice showed a more severe disease progression and highly impaired development. The observed phenotypes within the individual mouse lines may be linked to differences in the volume of specific brain regions. Reduced cortical volume in ducky mice, for example, was associated with a progressively decreased space between neurons, suggesting a reduction of total synaptic connections. Taken together, our findings show that α2δ subunits differentially regulate premature survival, postnatal growth, brain development, and behavior, suggesting specific neuronal functions in health and disease.

A novel humanized mouse model to study the function of human cutaneous memory T cells in vivo in human skin.

Human skin contains a population of memory T cells that supports tissue homeostasis and provides protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tissue environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rγnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES was generated from human keratinocytes and fibroblasts and was initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC, this reductionist system allowed us to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

Suicídio em Mato Grosso - Brasil: 1996 a 2015 / Suicide in Mato Grosso - Brazil: 1996 to 2015

Introdução: O suicídio é uma das dez principais causas de mortalidade no mundo; é considerado um grande problema de saúde pública seja por sua magnitude, complexidade, seja pelo forte impacto social, econômico e pessoal. O Brasil é o oitavo país em número de suicídios, com média de 24 suicídios por dia. Objetivo: descrever a mortalidade por suicídios em Mato Grosso, no período de 1996 a 2015. Métodos: Realizou-se um estudo descritivo a partir de dados secundários do Sistema de Informações sobre Mortalidade/Ministério da Saúde. Os dados foram analisados em termos de frequência relativa (proporções, razão e taxas). Resultados: Os resultados revelam estabilidade da taxa de suicídio em Mato Grosso - média 6,8 óbitos/100.000 habitantes, mas com valores superiores ao Brasil - média 5,7 óbitos/100.000 habitantes. Observa-se a sobremortalidade masculina (Razão de sexo = 3,47), e taxas de suicídio mais elevadas em idosos (9,8/100.000 habitantes). As lesões autoprovocadas prevaleceram em relação à autointoxicação como método utilizado para o suicídio em uma razão de 4,83; porém, com comportamento diferente entre os sexos: 6,69 e 2,11, para o sexo masculino e feminino respectivamente. Entre as lesões autoprovocadas, enforcamento (62,6%) e arma de fogo (24,6%) foram as mais frequentes, enquanto as intoxicações por pesticidas (56,4%) predominaram entre as autointoxicações. Conclusão: O estudo revela informações pouco exploradas sobre o suicídio no estado de Mato Grosso e, portanto, relevantes para ampliar o conhecimento sobre o problema e sua prevenção.(AU)

Introduction: Suicide is one of the ten leading causes of mortality worldwide and considered a major problem for public health either due to its magnitude, complexity or by a strong social, economic and personal impact. Brazil is the eighth country in number of suicides, with an average of 24 suicides a day. Objective: describing mortality due to a suicide case in Mato Grosso from 1996 to 2015. Methods: A descriptive study from secondary data of the Mortality Data System/Ministry of Health was carried out. The data were analyzed in terms of relative frequency (proportions, ratio and rates). Results: The results show stability of the suicide rate in Mato Grosso - mean 6.8 deaths /100,000 inhabitants, but with values higher than in Brazil - 5.7 deaths/100,000 inhabitants. Excessive male mortality and higher suicide rates in the elderly (9,8/100,000) have been noted. Self-inflicted injuries prevailed in relation to intentional intoxication as a method used for suicide in a ratio of 4.83, however with different behavior between genders: 6.69 and 2.11, for males and females respectively. Among the self-inflicted injuries, hanging (62.6%) and firearm (24.6%) were the most frequent, while pesticide poisonings (56.4%) predominated among autointoxications. Conclusion: The study reveals little explored information on the suicide in the state of Mato Grosso and, hence, relevant to increase the understanding of the problem and its prevention.(AU)

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation.

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (CaV1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth.

Restricting calcium currents is required for correct fiber type specification in skeletal muscle.

Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease.

Loss of the calcium channel ß4 subunit impairs parallel fibre volley and Purkinje cell firing in cerebellum of adult ataxic mice.

The auxiliary voltage-gated calcium channel subunit ß4 supports targeting of calcium channels to the cell membrane, modulates ionic currents and promotes synaptic release in the central nervous system. ß4 is abundant in cerebellum and its loss causes ataxia. However, the type of calcium channels and cerebellar functions affected by the loss of ß4 are currently unknown. We therefore studied the structure and function of Purkinje cells in acute cerebellar slices of the ß4 (-/-) ataxic (lethargic) mouse, finding that loss of ß4 affected Purkinje cell input, morphology and pacemaker activity. In adult lethargic cerebellum evoked postsynaptic currents from parallel fibres were depressed, while paired-pulse facilitation and spontaneous synaptic currents were unaffected. Because climbing fibre input was spared, the parallel fibre/climbing fibre input ratio was reduced. The dendritic arbor of adult lethargic Purkinje cells displayed fewer and shorter dendrites, but a normal spine density. Accordingly, the width of the molecular and granular layers was reduced. These defects recapitulate the impaired cerebellar maturation observed upon Cav 2.1 ataxic mutations. However, unlike Cav 2.1 mutations, lethargic Purkinje cells also displayed a striking decrease in pacemaker firing frequency, without loss of firing regularity. All these deficiencies appear in late development, indicating the importance of ß4 for the normal differentiation and function of mature Purkinje cells networks. The observed reduction of the parallel fibre input, the altered parallel fibre/climbing fibre ratio and the reduced Purkinje cell output can contribute to the severe motor impairment caused by the loss of the calcium channel ß4 subunit in lethargic mice.

Differential neuronal targeting of a new and two known calcium channel ß4 subunit splice variants correlates with their regulation of gene expression.

The ß subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ß subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of ß isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual ß variants in specific neuronal functions. In the present study, an alternatively spliced ß4 subunit lacking the variable N terminus (ß4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length ß4 variants (ß4a and ß4b), ß4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of ß4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (ß4-null) mice individually reconstituted with ß4a, ß4b, and ß4e. Notably, the number of genes regulated by each ß4 splice variant correlated with the rank order of their nuclear-targeting properties (ß4b > ß4a > ß4e). Together, these findings support isoform-specific functions of ß4 splice variants in neurons, with ß4b playing a dual role in channel modulation and gene regulation, whereas the newly detected ß4e variant serves exclusively in calcium-channel-dependent functions.