Comparative proteomic profiling reveals mechanisms for early spinal cord vulnerability in CLN1 disease

CLN1 disease is a fatal inherited neurodegenerative lysosomal storage disease of early childhood, caused by mutations in the CLN1 gene, which encodes the enzyme Palmitoyl protein thioesterase-1 (PPT-1). We recently found significant spinal pathology in Ppt1-deficient (Ppt1−/−) mice and human CLN1 disease that contributes to clinical outcome and precedes the onset of brain pathology. Here, we quantified this spinal pathology at 3 and 7 months of age revealing significant and progressive glial activation and vulnerability of spinal interneurons. Tandem mass tagged proteomic analysis of the spinal cord of Ppt1−/−and control mice at these timepoints revealed a significant neuroimmune response and changes in mitochondrial function, cell-signalling pathways and developmental processes. Comparing proteomic changes in the spinal cord and cortex at 3 months revealed many similarly affected processes, except the inflammatory response. These proteomic and pathological data from this largely unexplored region of the CNS may help explain the limited success of previous brain-directed therapies. These data also fundamentally change our understanding of the progressive, site-specific nature of CLN1 disease pathogenesis, and highlight the importance of the neuroimmune response. This should greatly impact our approach to the timing and targeting of future therapeutic trials for this and similar disorders

The association between retinal vascular geometry changes and diabetic retinopathy and their role in prediction of progression: an exploratory study

Background: The study describes the relationship of retinal vascular geometry (RVG) to severity of diabetic retinopathy (DR), and its predictive role for subsequent development of proliferative diabetic retinopathy (PDR). Methods. The research project comprises of two stages. Firstly, a comparative study of diabetic patients with different grades of DR. (No DR: Minimal non-proliferative DR: Severe non-proliferative DR: PDR) (10:10: 12: 19). Analysed RVG features including vascular widths and branching angles were compared between patient cohorts. A preliminary statistical model for determination of the retinopathy grade of patients, using these features, is presented. Secondly, in a longitudinal predictive study, RVG features were analysed for diabetic patients with progressive DR over 7 years. RVG at baseline was examined to determine risk for subsequent PDR development. Results: In the comparative study, increased DR severity was associated with gradual vascular dilatation (p = 0.000), and widening of the bifurcating angle (p = 0.000) with increase in smaller-child-vessel branching angle (p = 0.027). Type 2 diabetes and increased diabetes duration were associated with increased vascular width (p = <0.05 In the predictive study, at baseline, reduced small-child vascular width (OR = 0.73 (95 CI 0.58-0.92)), was predictive of future progression to PDR. Conclusions: The study findings suggest that RVG alterations can act as novel markers indicative of progression of DR severity and establishment of PDR. RVG may also have a potential predictive role in determining the risk of future retinopathy progression. © 2014 Habib et al.; licensee BioMed Central Ltd

Aberrant crossed corticospinal facilitation in muscles distant from a spinal cord injury.

Crossed facilitatory interactions in the corticospinal pathway are impaired in humans with chronic incomplete spinal cord injury (SCI). The extent to which crossed facilitation is affected in muscles above and below the injury remains unknown. To address this question we tested 51 patients with neurological injuries between C2-T12 and 17 age-matched healthy controls. Using transcranial magnetic stimulation we elicited motor evoked potentials (MEPs) in the resting first dorsal interosseous, biceps brachii, and tibialis anterior muscles when the contralateral side remained at rest or performed 70% of maximal voluntary contraction (MVC) into index finger abduction, elbow flexion, and ankle dorsiflexion, respectively. By testing MEPs in muscles with motoneurons located at different spinal cord segments we were able to relate the neurological level of injury to be above, at, or below the location of the motoneurons of the muscle tested. We demonstrate that in patients the size of MEPs was increased to a similar extent as in controls in muscles above the injury during 70% of MVC compared to rest. MEPs remained unchanged in muscles at and within 5 segments below the injury during 70% of MVC compared to rest. However, in muscles beyond 5 segments below the injury the size of MEPs increased similar to controls and was aberrantly high, 2-fold above controls, in muscles distant (>15 segments) from the injury. These aberrantly large MEPs were accompanied by larger F-wave amplitudes compared to controls. Thus, our findings support the view that corticospinal degeneration does not spread rostral to the lesion, and highlights the potential of caudal regions distant from an injury to facilitate residual corticospinal output after SCI

Deletion of the Pluripotency-Associated Tex19.1 Gene Causes Activation of Endogenous Retroviruses and Defective Spermatogenesis in Mice

As genetic information is transmitted through successive generations, it passes between pluripotent cells in the early embryo and germ cells in the developing foetus and adult animal. Tex19.1 encodes a protein of unknown function, whose expression is restricted to germ cells and pluripotent cells. During male spermatogenesis, Tex19.1 expression is highest in mitotic spermatogonia and diminishes as these cells differentiate and progress through meiosis. In pluripotent stem cells, Tex19.1 expression is also downregulated upon differentiation. However, it is not clear whether Tex19.1 has an essential function in germ cells or pluripotent stem cells, or what that function might be. To analyse the potential role of Tex19.1 in pluripotency or germ cell function we have generated Tex19.1−/− knockout mice and analysed the Tex19.1−/− mutant phenotype. Adult Tex19.1−/− knockout males exhibit impaired spermatogenesis. Immunostaining and histological analysis revealed defects in meiotic chromosome synapsis, the persistence of DNA double-strand breaks during meiosis, and a loss of post-meiotic germ cells in the testis. Furthermore, expression of a class of endogenous retroviruses is upregulated during meiosis in the Tex19.1−/− testes. Increased transposition of endogenous retroviruses in the germline of Tex19.1−/− mutant mice, and the concomitant increase in DNA damage, may be sufficient to disrupt the normal processes of recombination and chromosome synapsis during meiosis and cause defects in spermatogenesis. Our results suggest that Tex19.1 is part of a specialised mechanism that operates in the germline to repress transposable genetic elements and maintain genomic stability through successive generations

Distinct Early Molecular Responses to Mutations Causing vLINCL and JNCL Presage ATP Synthase Subunit C Accumulation in Cerebellar Cells

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6nclf/nclf cerebellar cells and compared them to wild-type and CbCln3Δex7/8/Δex7/8 cerebellar cells. CbCln6nclf/nclf cells and CbCln3Δex7/8/Δex7/8 cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6nclf/nclf and CbCln3Δex7/8/Δex7/8 cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3Δex7/8 and Cln6nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival

Chemical genetics strategies for identification of molecular targets

Chemical genetics is an emerging field that can be used to study the interactions of chemical compounds, including natural products, with proteins. Usually, the identification of molecular targets is the starting point for studying a drug’s mechanism of action and this has been a crucial step in understanding many biological processes. While a great variety of target identification methods have been developed over the last several years, there are still many bioactive compounds whose target proteins have not yet been revealed because no routine protocols can be adopted. This review contains information concerning the most relevant principles of chemical genetics with special emphasis on the different genomic and proteomic approaches used in forward chemical genetics to identify the molecular targets of the bioactive compounds, the advantages and disadvantages of each and a detailed list of successful examples of molecular targets identified with these approaches

Large-Scale Phenotyping of an Accurate Genetic Mouse Model of JNCL Identifies Novel Early Pathology Outside the Central Nervous System

Cln3Δex7/8 mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3Δex7/8 mice. Homozygous Cln3Δex7/8 mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10–14 weeks of age. Homozygous Cln3Δex7/8 mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12–13 week old homozygous Cln3Δex7/8mice, which were also seen to a lesser extent in heterozygous Cln3Δex7/8 mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15–16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3Δex7/8 mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3Δex7/8 neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3Δex7/8 mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3Δex7/8 mice that merit further study for JNCL biomarker development

Identification of calcium-binding proteins associated with the human sperm plasma membrane

<p>Abstract</p> <p>Background</p> <p>The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.</p> <p>Methods</p> <p>Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.</p> <p>Results</p> <p>Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.</p> <p>Conclusion</p> <p>The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.</p

Sex Differences in the Brain: A Whole Body Perspective

Most writing on sexual differentiation of the mammalian brain (including our own) considers just two organs: the gonads and the brain. This perspective, which leaves out all other body parts, misleads us in several ways. First, there is accumulating evidence that all organs are sexually differentiated, and that sex differences in peripheral organs affect the brain. We demonstrate this by reviewing examples involving sex differences in muscles, adipose tissue, the liver, immune system, gut, kidneys, bladder, and placenta that affect the nervous system and behavior. The second consequence of ignoring other organs when considering neural sex differences is that we are likely to miss the fact that some brain sex differences develop to compensate for differences in the internal environment (i.e., because male and female brains operate in different bodies, sex differences are required to make output/function more similar in the two sexes). We also consider evidence that sex differences in sensory systems cause male and female brains to perceive different information about the world; the two sexes are also perceived by the world differently and therefore exposed to differences in experience via treatment by others. Although the topic of sex differences in the brain is often seen as much more emotionally charged than studies of sex differences in other organs, the dichotomy is largely false. By putting the brain firmly back in the body, sex differences in the brain are predictable and can be more completely understood

Neural Circuits Underlying Rodent Sociality: A Comparative Approach

All mammals begin life in social groups, but for some species, social relationships persist and develop throughout the course of an individual’s life. Research in multiple rodent species provides evidence of relatively conserved circuitry underlying social behaviors and processes such as social recognition and memory, social reward, and social approach/avoidance. Species exhibiting different complex social behaviors and social systems (such as social monogamy or familiarity preferences) can be characterized in part by when and how they display specific social behaviors. Prairie and meadow voles are closely related species that exhibit similarly selective peer preferences but different mating systems, aiding direct comparison of the mechanisms underlying affiliative behavior. This chapter draws on research in voles as well as other rodents to explore the mechanisms involved in individual social behavior processes, as well as specific complex social patterns. Contrasts between vole species exemplify how the laboratory study of diverse species improves our understanding of the mechanisms underlying social behavior. We identify several additional rodent species whose interesting social structures and available ecological and behavioral field data make them good candidates for study. New techniques and integration across laboratory and field settings will provide exciting opportunities for future mechanistic work in non-model species