Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.

Altered maturation of the primary somatosensory cortex in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and results from the loss of the fragile X mental retardation protein (FMRP). Many fragile X-related cognitive and behavioral features emerge during childhood and are associated with abnormal synaptic and cellular organization of the cerebral cortex. Identifying the roles of FMRP in cortical development will provide a basis for understanding the pathogenesis of the syndrome. However, how the loss of FMRP influences the developmental trajectory of cortical maturation remains unclear. We took advantage of the stereotyped and well-characterized development of the murine primary somatosensory cortex to examine cortical maturation during a time-window that corresponds to late embryonic and early postnatal development in the human. In the Fmr1 knockout mouse, we find a delay in somatosensory map formation, alterations in the morphology profile of dendrites and spines of layer 4 neurons and a decrease in the synaptic levels of proteins involved in glutamate receptor signaling at times corresponding to the highest levels of FMRP expression. In contrast, cortical arealization, synaptic density in layer 4 and early postnatal regulation of mRNAs encoding synaptic proteins are not altered in Fmr1 knockout mice. The specificity of the developmental delay in Fmr1 knockout mice indicates that the loss of FMRP does not result in a general stalling of cerebral cortex maturation. Instead, our results suggest that inaccurate timing of developmental processes caused by the loss of FMRP may lead to alterations in neural circuitry that underlie behavioral and cognitive dysfunctions associated with FXS.

ApoE isoform-specific regulation of regeneration in the peripheral nervous system.

Apolipoprotein E (apoE) is a 34 kDa glycoprotein with three distinct isoforms in the human population (apoE2, apoE3 and apoE4) known to play a major role in differentially influencing risk to, as well as outcome from, disease and injury in the central nervous system. In general, the apoE4 allele is associated with poorer outcomes after disease or injury, whereas apoE3 is associated with better responses. The extent to which different apoE isoforms influence degenerative and regenerative events in the peripheral nervous system (PNS) is still to be established, and the mechanisms through which apoE exerts its isoform-specific effects remain unclear. Here, we have investigated isoform-specific effects of human apoE on the mouse PNS. Experiments in mice ubiquitously expressing human apoE3 or human apoE4 on a null mouse apoE background revealed that apoE4 expression significantly disrupted peripheral nerve regeneration and subsequent neuromuscular junction re-innervation following nerve injury compared with apoE3, with no observable effects on normal development, maturation or Wallerian degeneration. Proteomic isobaric tag for relative and absolute quantitation (iTRAQ) screens comparing healthy and regenerating peripheral nerves from mice expressing apoE3 or apoE4 revealed significant differences in networks of proteins regulating cellular outgrowth and regeneration (myosin/actin proteins), as well as differences in expression levels of proteins involved in regulating the blood-nerve barrier (including orosomucoid 1). Taken together, these findings have identified isoform-specific roles for apoE in determining the protein composition of peripheral nerve as well as regulating nerve regeneration pathways in vivo.

Murine cathepsin D deficiency is associated with dysmyelination/myelin disruption and accumulation of cholesteryl esters in the brain.

Cathepsin D (CTSD) deficiencies are fatal neurological diseases that in human infants and in sheep are characterized by extreme loss of neurons and myelin. To date, similar morphological evidence for myelin disruption in CTSD knockout mice has not been reported. Here, we show that CTSD deficiency leads to pronounced myelin changes in the murine brain: myelin-related proteolipid protein and myelin basic protein were both markedly reduced at postnatal day 24, and the amount of lipids characteristically high in myelin (e.g. plasmalogen-derived alkenyl chains and glycosphingolipid-derived 20- and 24-carbon acyl chains) were significantly lowered compared with controls. These changes were accompanied by ultrastructural alterations of myelin, including significant thinning of myelin sheaths. Furthermore, in CTSD knockout brains there was a pronounced accumulation of cholesteryl esters and abnormal levels of proteins related to cholesterol transport, with an increased content of apolipoprotein E and a reduced content of ATP-binding cassette transporter A1. These results provide evidence for dysmyelination and altered trafficking of cholesterol in brains of CTSD knockout mice, and warrant further studies on the role of lipid metabolism in the pathogenesis of CTSD deficiencies.

Slowly progressive dopamine cell loss--a model on which to test neuroprotective strategies for Parkinson's disease?

Making animal models of human disease is a very flawed process. Aspects of the disease can be imitated but models do not necessarily give reliable leads for treatment strategies. When Ungerstedt in Sweden first described the 6-hydroxydopamine (6-OHDA) treated rat model of Parkinson's disease /89/ we knew that the symptoms would not map readily to those of the human disease--rats have four legs after all. On the other hand, the neuropathology looked exactly like end-stage Parkinsonian pathology. That remained true even as we explored other types of neuropathology in the rats /24,43-46,80/. Many of today's treatments for Parkinsonism are developed from pharmacological studies on that model of rats with a chemically induced lesion. However, the 6-OHDA model does not address the important issue of a cure for the disease. The triggers and the time-course of dopamine (DA) cell death in rats are known for nearly every disease model - but for the human disease there is no equivalent knowledge. In the human, the neurons have been dying for a considerable time before the symptoms become obvious and they go on dying even with adequate symptomatic relief /94/, but after intracerebral administration of 6-OHDA to an animal the cells die quickly; all cells are destroyed in less than 5 days /42,88,89/. Thus, we were interested in developing an animal model of DA cell death with a slower time-course. After ibotenic acid injections into rat globus pallidus (GP), DA cells are lost from the ipsilateral substantia nigra over the slower time scale of about six weeks. This time scale has allowed us to test some interventions to prevent the cells from dying. Although some attempts have succeeded, cell death is prevented only for three weeks -beyond that treatments fail and DA cells die. At the moment, this model has at least opened a window into causes of neuronal death in a slower time scale /94/ than previous rodent models.

Microglial activation is not prevented by tacrolimus but dopamine neuron damage is reduced in a rat model of Parkinson's disease progression.

A progressive model of Parkinson's disease has been recently developed in the rat where a unilateral excitotoxic injection into the globus pallidus leads to a gradual loss of dopaminergic neurons in the ipsilateral substantia nigra over a period of at least 6 weeks. In this model microglial activation is observed in the ipsilateral substantia nigra 3 weeks after the lesion and could contribute to neuronal death at this time. The immunosuppressant drug tacrolimus (FK506) reduces dopamine cell death at 3 weeks following a globus pallidus lesion, but not thereafter. Tacrolimus-mediated neuroprotection could result from suppression of microglial activation but the microglial activation at three weeks post-lesion was not much reduced. Microglial activation was observed even in the apparent absence of neuronal death, prompting the suggestion that tacrolimus may prevent, or at least delay, the release of toxic cytokines from activated microglia. By 6 weeks after the GP lesion, even this mechanism fails to protect the dopamine cells from damage.

Delayed synaptic degeneration in the CNS of Wlds mice after cortical lesion.

Therapies that might delay degeneration of synapses offer an appealing strategy for treatment of neurodegenerative diseases, including Alzheimer's disease and related dementias, prion diseases, schizophrenia and amyotrophic lateral sclerosis. Analysis of mouse mutants provides one possible avenue towards identifying relevant mechanisms. Here, we used quantitative and serial section electron microscopy to find out whether the onset and time course of pre-synaptic nerve terminal degeneration is delayed in the striatum of Wallerian degeneration slow (Wld(s)) mutant mice. Synaptic degeneration was observed within 48 h of cortical ablation in wild-type mice but was delayed by approximately 1 week in Wld(s) mice. However, the morphological characteristics of degenerating nerve terminals in wild-type and Wld(s) mice were indistinguishable, in contrast to the differences reported previously in studies of the PNS. Surprisingly, the delayed onset of synaptic degeneration was accompanied by an increased incidence of complex synaptic morphologies on post-synaptic spines in the denervated Wld(S) striatum indicating an enhanced plastic response at both injured and uninjured synapses. The data suggest that targeting Wallerian-like mechanisms of synaptic degeneration could lead to the development of new therapies for the treatment of CNS disorders where synapse loss is a primary feature.

Post-mortem brain analyses of the Lothian Birth Cohort 1936: extending lifetime cognitive and brain phenotyping to the level of the synapse.

INTRODUCTION: Non-pathological, age-related cognitive decline varies markedly between individuals andplaces significant financial and emotional strain on people, their families and society as a whole.Understanding the differential age-related decline in brain function is critical not only for the development oftherapeutics to prolong cognitive health into old age, but also to gain insight into pathological ageing suchas Alzheimer's disease. The Lothian Birth Cohort of 1936 (LBC1936) comprises a rare group of people forwhom there are childhood cognitive test scores and longitudinal cognitive data during older age, detailedstructural brain MRI, genome-wide genotyping, and a multitude of other biological, psycho-social, andepidemiological data. Synaptic integrity is a strong indicator of cognitive health in the human brain;however, until recently, it was prohibitively difficult to perform detailed analyses of synaptic and axonalstructure in human tissue sections. We have adapted a novel method of tissue preparation at autopsy toallow the study of human synapses from the LBC1936 cohort in unprecedented morphological andmolecular detail, using the high-resolution imaging techniques of array tomography and electronmicroscopy. This allows us to analyze the brain at sub-micron resolution to assess density, proteincomposition and health of synapses. Here we present data from the first donated LBC1936 brain andcompare our findings to Alzheimer's diseased tissue to highlight the differences between healthy andpathological brain ageing. RESULTS: Our data indicates that compared to an Alzheimer's disease patient, the cognitively normalLBC1936 participant had a remarkable degree of preservation of synaptic structures. However,morphological and molecular markers of degeneration in areas of the brain associated with cognition(prefrontal cortex, anterior cingulate cortex, and superior temporal gyrus) were observed. CONCLUSIONS: Our novel post-mortem protocol facilitates high-resolution neuropathological analysis of the well-characterized LBC1936 cohort, extending phenotyping beyond cognition and in vivo imaging to nowinclude neuropathological changes, at the level of single synapses. This approach offers an unprecedentedopportunity to study synaptic and axonal integrity during ageing and how it contributes to differences in agerelatedcognitive change.

Erratum to: Post-mortem brain analyses of the Lothian Birth Cohort 1936: extending lifetime cognitive and brain phenotyping to the level of the synapse.

The original version of this article [1] unfortunately contained several mistakes. The presentation of Table 2 and 3 was incorrect, in the HTML and PDF versions of this article. The corrected Tables 2 and 3 are given below.

A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton.

Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.

Studying synapses in human brain with array tomography and electron microscopy.

Postmortem studies of synapses in human brain are problematic because of the axial resolution limit of light microscopy and the difficulty in preserving and analyzing ultrastructure with electron microscopy (EM). Array tomography (AT) overcomes these problems by embedding autopsy tissue in resin and cutting ribbons of ultrathin serial sections. Ribbons are imaged with immunofluorescence, allowing high-throughput imaging of tens of thousands of synapses to assess synapse density and protein composition. The protocol takes ~3 d per case, excluding image analysis, which is done at the end of the study. Parallel processing for transmission electron microscopy (TEM) using a protocol modified to preserve the structure in human samples allows complementary ultrastructural studies. Incorporation of AT and TEM into brain banking is a potent way of phenotyping synapses in well-characterized clinical cohorts in order to develop clinicopathological correlations at the synapse level. This will be important for research in neurodegenerative disease, developmental disease and psychiatric illness.

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 presymptomatic CTSD knockout (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 (mEPSCs) with no effect on paired-pulse modulation of the evoked excitatory post synaptic potentials in the hippocampus of Ctsd mice. The reduced mEPSCs frequency was observed before the appearance of epilepsy or any morphologic sign of synaptic degeneration. Taken together, these data indicate that CTSD is required for normal synaptic function and that a failure in synaptic trafficking or recycling may bean early and important pathologic mechanism in Ctsd mice; these presynaptic abnormalities may initiate synaptic degeneration in advance of subsequent neuronal loss.

Synaptic protection in the brain of WldS mice occurs independently of age but is sensitive to gene-dose.

BACKGROUND: Disruption of synaptic connectivity is a significant early event in many neurodegenerative conditions affecting the aging CNS, including Alzheimer's disease and Parkinson's disease. Therapeutic approaches that protect synapses from degeneration in the aging brain offer the potential to slow or halt the progression of such conditions. A range of animal models expressing the slow Wallerian Degeneration (Wld(S)) gene show robust neuroprotection of synapses and axons from a wide variety of traumatic and genetic neurodegenerative stimuli in both the central and peripheral nervous systems, raising that possibility that Wld(S) may be useful as a neuroprotective agent in diseases with synaptic pathology. However, previous studies of neuromuscular junctions revealed significant negative effects of increasing age and positive effects of gene-dose on Wld(S)-mediated synaptic protection in the peripheral nervous system, raising doubts as to whether Wld(S) is capable of directly conferring synapse protection in the aging brain. METHODOLOGY/PRINCIPAL FINDINGS: We examined the influence of age and gene-dose on synaptic protection in the brain of mice expressing the Wld(S) gene using an established cortical lesion model to induce synaptic degeneration in the striatum. Synaptic protection was found to be sensitive to Wld(S) gene-dose, with heterozygous Wld(S) mice showing approximately half the level of protection observed in homozygous Wld(S) mice. Increasing age had no influence on levels of synaptic protection. In contrast to previous findings in the periphery, synapses in the brain of old Wld(S) mice were just as strongly protected as those in young mice. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that Wld(S)-mediated synaptic protection in the CNS occurs independently of age, but is sensitive to gene dose. This suggests that the Wld(S) gene, and in particular its downstream endogenous effector pathways, may be potentially useful therapeutic agents for conferring synaptic protection in the aging brain.