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1.
Cells ; 13(7)2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38607016

ABSTRACT

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.


Subject(s)
Nervous System Diseases , Spastic Paraplegia, Hereditary , Animals , Humans , Membrane Proteins/metabolism , Membrane Transport Proteins/genetics , GTP-Binding Proteins/metabolism , Spastic Paraplegia, Hereditary/genetics , Mammals/metabolism
2.
Acta Neuropathol Commun ; 10(1): 40, 2022 03 28.
Article in English | MEDLINE | ID: mdl-35346366

ABSTRACT

Hereditary spastic paraplegias (HSPs) are a group of inherited, progressive neurodegenerative conditions characterised by prominent lower-limb spasticity and weakness, caused by a length-dependent degeneration of the longest corticospinal upper motor neurons. While more than 80 spastic paraplegia genes (SPGs) have been identified, many cases arise from mutations in genes encoding proteins which generate and maintain tubular endoplasmic reticulum (ER) membrane organisation. The ER-shaping proteins are essential for the health and survival of long motor neurons, however the mechanisms by which mutations in these genes cause the axonopathy observed in HSP have not been elucidated. To further develop our understanding of the ER-shaping proteins, this study outlines the generation of novel in vivo and in vitro models, using CRISPR/Cas9-mediated gene editing to knockout the ER-shaping protein ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1), mutations in which give rise to the HSP subtype SPG61. Loss of Arl6IP1 in Drosophila results in progressive locomotor deficits, emulating a key aspect of HSP in patients. ARL6IP1 interacts with ER-shaping proteins and is required for regulating the organisation of ER tubules, particularly within long motor neuron axons. Unexpectedly, we identified physical and functional interactions between ARL6IP1 and the phospholipid transporter oxysterol-binding protein-related protein 8 in both human and Drosophila model systems, pointing to a conserved role for ARL6IP1 in lipid homeostasis. Furthermore, loss of Arl6IP1 from Drosophila neurons results in a cell non-autonomous accumulation of lipid droplets in axonal glia. Importantly, treatment with lipid regulating liver X receptor-agonists blocked lipid droplet accumulation, restored axonal ER organisation, and improved locomotor function in Arl6IP1 knockout Drosophila. Our findings indicate that disrupted lipid homeostasis contributes to neurodegeneration in HSP, identifying a potential novel therapeutic avenue for the treatment of this disorder.


Subject(s)
Liver X Receptors , Spastic Paraplegia, Hereditary , Animals , Disease Models, Animal , Drosophila/metabolism , Endoplasmic Reticulum/metabolism , Humans , Liver X Receptors/agonists , Membrane Transport Proteins/genetics , Spastic Paraplegia, Hereditary/drug therapy , Spastic Paraplegia, Hereditary/genetics
3.
Front Neurosci ; 15: 784987, 2021.
Article in English | MEDLINE | ID: mdl-34867178

ABSTRACT

Optic atrophy (OA) with autosomal inheritance is a form of optic neuropathy characterized by the progressive and irreversible loss of vision. In some cases, this is accompanied by additional, typically neurological, extra-ocular symptoms. Underlying the loss of vision is the specific degeneration of the retinal ganglion cells (RGCs) which form the optic nerve. Whilst autosomal OA is genetically heterogenous, all currently identified causative genes appear to be associated with mitochondrial organization and function. However, it is unclear why RGCs are particularly vulnerable to mitochondrial aberration. Despite the relatively high prevalence of this disorder, there are currently no approved treatments. Combined with the lack of knowledge concerning the mechanisms through which aberrant mitochondrial function leads to RGC death, there remains a clear need for further research to identify the underlying mechanisms and develop treatments for this condition. This review summarizes the genes known to be causative of autosomal OA and the mitochondrial dysfunction caused by pathogenic mutations. Furthermore, we discuss the suitability of available in vivo models for autosomal OA with regards to both treatment development and furthering the understanding of autosomal OA pathology.

4.
BMC Bioinformatics ; 22(1): 427, 2021 Sep 08.
Article in English | MEDLINE | ID: mdl-34496765

ABSTRACT

BACKGROUND: In mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm. This organelle is of particular interest to biologists, as its dysfunction is associated with numerous diseases, which often manifest themselves as changes to the structure and organisation of the reticular network. Due to its complex morphology, image analysis methods to quantitatively describe this organelle, and importantly any changes to it, are lacking. RESULTS: In this work we detail a methodological approach that utilises automated high-content screening microscopy to capture images of cells fluorescently-labelled for various ER markers, followed by their quantitative analysis. We propose that two key metrics, namely the area of dense ER and the area of polygonal regions in between the reticular elements, together provide a basis for measuring the quantities of rough and smooth ER, respectively. We demonstrate that a number of different pharmacological perturbations to the ER can be quantitatively measured and compared in our automated image analysis pipeline. Furthermore, we show that this method can be implemented in both commercial and open-access image analysis software with comparable results. CONCLUSIONS: We propose that this method has the potential to be applied in the context of large-scale genetic and chemical perturbations to assess the organisation of the ER in adherent cell cultures.


Subject(s)
Endoplasmic Reticulum , Image Processing, Computer-Assisted , Animals , Cell Line , Humans , Software
5.
Brain Sci ; 10(9)2020 Sep 17.
Article in English | MEDLINE | ID: mdl-32957716

ABSTRACT

Mitochondrial morphology, distribution and function are maintained by the opposing forces of mitochondrial fission and fusion, the perturbation of which gives rise to several neurodegenerative disorders. The large guanosine triphosphate (GTP)ase dynamin-related protein 1 (Drp1) is a critical regulator of mitochondrial fission by mediating membrane scission, often at points of mitochondrial constriction at endoplasmic reticulum (ER)-mitochondrial contacts. Hereditary spastic paraplegia (HSP) subtype SPG61 is a rare neurodegenerative disorder caused by mutations in the ER-shaping protein Arl6IP1. We have previously reported defects in both the ER and mitochondrial networks in a Drosophila model of SPG61. In this study, we report that knockdown of Arl6IP1 lowers Drp1 protein levels, resulting in reduced ER-mitochondrial contacts and impaired mitochondrial load at the distal ends of long motor neurons. Increasing mitochondrial fission, by overexpression of wild-type Drp1 but not a dominant negative Drp1, increases ER-mitochondrial contacts, restores mitochondrial load within axons and partially rescues locomotor deficits. Arl6IP1 knockdown Drosophila also demonstrate impaired autophagic flux and an accumulation of ubiquitinated proteins, which occur independent of Drp1-mediated mitochondrial fission defects. Together, these findings provide evidence that impaired mitochondrial fission contributes to neurodegeneration in this in vivo model of HSP.

6.
Front Neurosci ; 13: 1051, 2019.
Article in English | MEDLINE | ID: mdl-31680803

ABSTRACT

The hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative conditions whose characteristic feature is degeneration of the longest axons within the corticospinal tract which leads to progressive spasticity and weakness of the lower limbs. Though highly genetically heterogeneous, the majority of HSP cases are caused by mutations in genes encoding proteins that are responsible for generating and organizing the tubular endoplasmic reticulum (ER). Despite this, the role of the ER within neurons, particularly the long axons affected in HSP, is not well understood. Throughout axons, ER tubules make extensive contacts with other organelles, the cytoskeleton and the plasma membrane. At these ER contacts, protein complexes work in concert to perform specialized functions including organelle shaping, calcium homeostasis and lipid biogenesis, all of which are vital for neuronal survival and may be disrupted by HSP-causing mutations. In this article we summarize the proteins which mediate ER contacts, review the functions these contacts are known to carry out within neurons, and discuss the potential contribution of disruption of ER contacts to axonopathy in HSP.

7.
J Genet Genomics ; 44(10): 493-501, 2017 Oct 20.
Article in English | MEDLINE | ID: mdl-29037990

ABSTRACT

The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), and extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, we infer that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, we find that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo.


Subject(s)
Carrier Proteins/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/physiology , Neurons/physiology , Valosin Containing Protein/metabolism , Animals , Carrier Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Gene Knockdown Techniques , Neurons/metabolism
8.
Elife ; 62017 07 25.
Article in English | MEDLINE | ID: mdl-28742022

ABSTRACT

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.


Subject(s)
Axons/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Endoplasmic Reticulum/metabolism , Membrane Transport Proteins/genetics , Spastic Paraplegia, Hereditary/genetics , Animals , Axonal Transport , Axons/ultrastructure , Disease Models, Animal , Drosophila Proteins/deficiency , Drosophila melanogaster/classification , Drosophila melanogaster/cytology , Drosophila melanogaster/ultrastructure , Endoplasmic Reticulum/ultrastructure , Gene Expression , Humans , Larva/cytology , Larva/genetics , Larva/metabolism , Larva/ultrastructure , Membrane Transport Proteins/deficiency , Mutation , Phylogeny , Protein Isoforms/deficiency , Protein Isoforms/genetics , Spastic Paraplegia, Hereditary/metabolism , Spastic Paraplegia, Hereditary/pathology
9.
Hum Mol Genet ; 25(13): 2827-2837, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27170313

ABSTRACT

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative disorders characterized by degeneration of the longest motor neurons in the corticospinal tract, leading to muscle weakness and spasticity of the lower limbs. Pathogenic variants in genes encoding proteins that shape the endoplasmic-reticulum (ER) network are a leading cause of HSP, however, the mechanisms by which loss of ER-shaping proteins underpin degeneration of selective neurons in HSP remain poorly understood. To begin to address this, we have generated a novel in vivo model of HSP in Drosophila melanogaster by targeted knockdown of the ER-shaping protein Arl6IP1 Variants in the human homolog of this gene have recently been linked to HSP subtype SPG61. Arl6IP1 RNAi flies display progressive locomotor deficits without a marked reduction in lifespan, recapitulating key features of HSP in human patients. Loss of Arl6IP1 leads to fragmentation of the smooth ER and disrupted mitochondrial network organization within the distal ends of long motor neurons. Furthermore, genetically increasing mitochondrial fission, by overexpression of dynamin-related protein 1 (Drp1), restores mitochondrial network organization and rescues locomotor deficits in two independent Drosophila models of HSP. Taken together, these results propose a role for ER-shaping proteins in mitochondrial network organization in vivo and suggest that impaired mitochondrial organization may be a common mechanism underpinning some forms of HSP.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Membrane Proteins/metabolism , Motor Neurons/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adenosine Triphosphatases/metabolism , Animals , Disease Models, Animal , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Endoplasmic Reticulum/metabolism , Humans , Membrane Proteins/genetics , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , RNA Interference , Spastic Paraplegia, Hereditary/genetics
10.
J Rare Dis Res Treat ; 1(3): 17-21, 2016.
Article in English | MEDLINE | ID: mdl-28603788

ABSTRACT

Hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative conditions characterised by retrograde degeneration of the longest motor neurons in the corticospinal tract, resulting in muscle weakness and spasticity of the lower limbs. To date more than 70 genetic loci have been associated with HSP, however the majority of cases are caused by mutations that encode proteins responsible for generating and maintaining tubular endoplasmic reticulum (ER) structure. These ER-shaping proteins are vital for the long-term survival of axons, however the mechanisms by which mutations in these proteins give rise to HSP remain poorly understood. To begin to address this we have characterized in vivo loss of function models of two very rare forms of HSP caused by loss of the ER-shaping proteins ARL6IP1 (SPG61) and RTN2 (SPG12). These models display progressive locomotor defects, disrupted organisation of the tubular ER and length-dependant defects in the axonal mitochondrial network. Here we compare our findings with those associated with more common forms HSP including: Spastin, Atlastin-1 and REEP 1 which together account for over half of all cases of autosomal dominant HSP. Furthermore, we discuss recent observations in other HSP models which are directly implicated in mitochondrial function and localization. Overall, we highlight the common features of our rare models of HSP and other models of disease which could indicate shared mechanisms underpinning neurodegeneration in these disorders.

11.
Front Cell Neurosci ; 8: 233, 2014.
Article in English | MEDLINE | ID: mdl-25161610

ABSTRACT

Several cytokines and chemokines are now known to play normal physiological roles in the brain where they act as key regulators of communication between neurons, glia, and microglia. In particular, cytokines and chemokines can affect cardinal cellular and molecular processes of hippocampal-dependent long-term memory consolidation including synaptic plasticity, synaptic scaling and neurogenesis. The chemokine, CX3CL1 (fractalkine), has been shown to modulate synaptic transmission and long-term potentiation (LTP) in the CA1 pyramidal cell layer of the hippocampus. Here, we confirm widespread expression of CX3CL1 on mature neurons in the adult rat hippocampus. We report an up-regulation in CX3CL1 protein expression in the CA1, CA3 and dentate gyrus (DG) of the rat hippocampus 2 h after spatial learning in the water maze task. Moreover, the same temporal increase in CX3CL1 was evident following LTP-inducing theta-burst stimulation in the DG. At physiologically relevant concentrations, CX3CL1 inhibited LTP maintenance in the DG. This attenuation in dentate LTP was lost in the presence of GABAA receptor/chloride channel antagonism. CX3CL1 also had opposing actions on glutamate-mediated rise in intracellular calcium in hippocampal organotypic slice cultures in the presence and absence of GABAA receptor/chloride channel blockade. Using primary dissociated hippocampal cultures, we established that CX3CL1 reduces glutamate-mediated intracellular calcium rises in both neurons and glia in a dose dependent manner. In conclusion, CX3CL1 is up-regulated in the hippocampus during a brief temporal window following spatial learning the purpose of which may be to regulate glutamate-mediated neurotransmission tone. Our data supports a possible role for this chemokine in the protective plasticity process of synaptic scaling.

12.
J Genet Genomics ; 40(6): 297-306, 2013 Jun 20.
Article in English | MEDLINE | ID: mdl-23790629

ABSTRACT

At least 25 genes, many involved in trafficking, localisation or shaping of membrane organelles, have been identified as causative genes for the neurodegenerative disorder hereditary spastic paraplegia (HSP). One of the most commonly mutated HSP genes, atlastin-1, encodes a dynamin-like GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes. However, the molecular mechanisms of atlastin-1-related membrane fusion and axonopathy remain unclear. To better understand its mode of action, we used affinity purification coupled with mass spectrometry to identify protein interactors of atlastin in Drosophila. Analysis of 72 identified proteins revealed that the atlastin interactome contains many proteins involved in protein processing and transport, in addition to proteins with roles in mRNA binding, metabolism and mitochondrial proteins. The highest confidence interactor from mass spectrometry analysis, the ubiquitin-selective AAA-ATPase valosin-containing protein (VCP), was validated as an atlastin-interacting protein, and VCP and atlastin showed overlapping subcellular distributions. Furthermore, VCP acted as a genetic modifier of atlastin: loss of VCP partially suppressed an eye phenotype caused by atlastin overexpression, whereas overexpression of VCP enhanced this phenotype. These interactions between atlastin and VCP suggest a functional relationship between these two proteins, and point to potential shared mechanisms between HSP and other forms of neurodegeneration.


Subject(s)
Adenosine Triphosphatases/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila/enzymology , GTP Phosphohydrolases/metabolism , Protein Interaction Mapping , Spastic Paraplegia, Hereditary/enzymology , Adenosine Triphosphatases/genetics , Animals , Disease Models, Animal , Drosophila/genetics , Drosophila/growth & development , Drosophila Proteins/genetics , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/genetics , Eye/enzymology , Eye/growth & development , Female , GTP Phosphohydrolases/genetics , Humans , Male , Protein Binding , Spastic Paraplegia, Hereditary/genetics , Valosin Containing Protein
13.
Brain Behav Immun ; 27(1): 71-9, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23036922

ABSTRACT

A range of adverse, early life environmental influences such as viral infection and social deprivation are thought to increase risk of psychiatric illness later in life. Here, we used peripheral administration of the viral infection mimic polyriboinosinic-polyribocytidylic acid (polyI:C) to compare the consequences of peripubertal infection and isolation rearing. Isolation rearing induced deficits in sensorimotor gating and recognition memory while no changes in social interaction or spatial learning were observed. PolyI:C injection during the peripubertal period markedly increased expression of interferon-stimulated genes (Ifit2, Prkr, Mx2 and Irf7) in the hippocampal dentate gyrus demonstrating that peripheral administration of the viral mimic in the adolescent animal does have direct effects in the brain. Peripubertal infection mimicry induced a similar but later emerging behavioural deficit in prepulse inhibition implying the existence of a peripubertal window of opportunity for viral-mediated cytokine increases to impact brain development and function. PolyI:C treatment also impaired novel object recognition but did not alter spatial reference memory or social interaction. Combining the polyI:C challenge with social isolation did not exacerbate the behavioural deficits seen with isolation rearing alone. Using Irf7 as a marker, peripubertal viral infection mimicry, isolation rearing and a combination of both were all seen to produce a long-lasting molecular imprint on the interferon-associated signalling pathway in the principal neuron population of the hippocampal dentate gyrus. The data suggest that the sensitivity of brain structure and function to disruption by viral infection extends into the peripubertal period. Moreover, augmented interferon signalling in hippocampus may represent a common molecular imprint of environmental insults associated with neuropsychiatric illnesses like schizophrenia.


Subject(s)
Behavior, Animal , Dentate Gyrus , Interferon Inducers/pharmacology , Interferon Regulatory Factor-7 , Interferons/metabolism , Poly I-C/pharmacology , Virus Diseases/metabolism , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Dentate Gyrus/drug effects , Dentate Gyrus/metabolism , GTP-Binding Proteins/drug effects , GTP-Binding Proteins/metabolism , Interferon Regulatory Factor-7/drug effects , Interferon Regulatory Factor-7/metabolism , Male , Myxovirus Resistance Proteins , Rats , Rats, Wistar , Sensory Gating/drug effects , Sensory Gating/physiology , Sexual Maturation/physiology , Social Isolation
14.
Hum Mol Genet ; 21(15): 3356-65, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22543973

ABSTRACT

Several causative genes for hereditary spastic paraplegia encode proteins with intramembrane hairpin loops that contribute to the curvature of the endoplasmic reticulum (ER), but the relevance of this function to axonal degeneration is not understood. One of these genes is reticulon2. In contrast to mammals, Drosophila has only one widely expressed reticulon orthologue, Rtnl1, and we therefore used Drosophila to test its importance for ER organization and axonal function. Rtnl1 distribution overlapped with that of the ER, but in contrast to the rough ER, was enriched in axons. The loss of Rtnl1 led to the expansion of the rough or sheet ER in larval epidermis and elevated levels of ER stress. It also caused abnormalities specifically within distal portions of longer motor axons and in their presynaptic terminals, including disruption of the smooth ER (SER), the microtubule cytoskeleton and mitochondria. In contrast, proximal axon portions appeared unaffected. Our results provide direct evidence for reticulon function in the organization of the SER in distal longer axons, and support a model in which spastic paraplegia can be caused by impairment of axonal the SER. Our data provide a route to further understanding of both the role of the SER in axons and the pathological consequences of the impairment of this compartment.


Subject(s)
Drosophila Proteins/genetics , Drosophila/metabolism , Endoplasmic Reticulum, Smooth/metabolism , Spastic Paraplegia, Hereditary/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Animals , Animals, Genetically Modified , Axons/metabolism , Disease Models, Animal , Drosophila Proteins/metabolism , Spastic Paraplegia, Hereditary/metabolism
15.
Proteomics ; 11(21): 4189-201, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22002935

ABSTRACT

Information storage in the brain depends on the ability of neurons to alter synaptic connectivity within key circuitries such as the hippocampus. Memory-associated synaptic plasticity is mediated by a temporal cascade of de novo protein synthesis and altered protein processing. Here, we have used two-dimensional difference in gel electrophoresis (2-D DIGE) to investigate memory-specific protein changes in the hippocampal dentate gyrus at increasing times following spatial learning. We identified 42 proteins that were significantly regulated in the first 24 h of spatial memory consolidation. Two distinct waves of protein expression regulation were evident, at 3 and 12 h post-learning and this is in agreement with studies employing inhibitors of global translation. Functional classification of the memory-associated proteins revealed that the majority of regulated proteins contributed either to cellular structure or cellular metabolism. For example, actins, tubulins and intermediate filament proteins, core proteins of the three major cytoskeletal components, were dynamically regulated at times that suggest a role in memory-associated synaptic reorganization. Increased proteasome-mediated protein degradation was evident in the early post-training period including the down-regulation of phosphoprotein enriched in astrocytes 15 kDa, a key inhibitor of extracellular signal-regulated kinase signaling. Some of the most substantial protein expression changes were observed for secreted carrier proteins including transthyretin and serum albumin at 6-12 h post-learning, regulations that could serve an important role in increasing the supply of retinoic acid and thyroid hormone, key synaptic plasticity-promoting signals in the adult brain. Together these observations provide further insight into protein level regulations occurring in the hippocampus during spatial memory consolidation.


Subject(s)
Dentate Gyrus/metabolism , Maze Learning , Proteome/metabolism , Proteomics , Animals , Apoptosis Regulatory Proteins , Gene Expression Regulation , Male , Memory , Phosphoproteins/genetics , Phosphoproteins/metabolism , Prealbumin/genetics , Prealbumin/metabolism , Proteome/genetics , Rats , Rats, Wistar , Serum Albumin/genetics , Serum Albumin/metabolism , Two-Dimensional Difference Gel Electrophoresis
16.
J Neurochem ; 113(3): 601-14, 2010 May.
Article in English | MEDLINE | ID: mdl-20096092

ABSTRACT

The critical sequence of molecular, neurotransmission and synaptic disruptions that underpin the emergence of psychiatric disorders like schizophrenia remain to be established with progress only likely using animal models that capture key features of such disorders. We have related the emergence of behavioural, neurochemical and synapse ultrastructure deficits to transcriptional dysregulation in the medial prefrontal cortex of Wistar rats reared in isolation. Isolation reared animals developed sensorimotor deficits at postnatal day 60 which persisted into adulthood. Analysis of gene expression prior to the emergence of the sensorimotor deficits revealed a significant disruption in transcriptional control, notably of immediate early and interferon-associated genes. At postnatal day 60 many gene transcripts relating particularly to GABA transmission and synapse structure, for example Gabra4, Nsf, Syn2 and Dlgh1, transiently increased expression. A subsequent decrease in genes such as Gria2 and Dlgh2 at postnatal day 80 suggested deficits in glutamatergic transmission and synapse integrity, respectively. Microdialysis studies revealed decreased extracellular glutamate suggesting a state of hypofrontality while ultrastructural analysis showed total and perforated synapse complement in layer III to be significantly reduced in the prefrontal cortex of postnatal day 80 isolated animals. These studies provide a molecular framework to understand the developmental emergence of the structural and behavioural characteristics that may in part define psychiatric illness.


Subject(s)
Cerebral Cortex/metabolism , Gene Expression Regulation/physiology , Social Isolation/psychology , Animals , Behavior, Animal/physiology , Cerebral Cortex/chemistry , Cerebral Cortex/ultrastructure , Computational Biology , DNA/biosynthesis , DNA/genetics , Male , Microdialysis , Motor Activity/physiology , Multigene Family , Oligonucleotide Array Sequence Analysis , RNA/biosynthesis , RNA/genetics , RNA, Complementary/biosynthesis , RNA, Complementary/genetics , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Stress, Psychological/genetics , Stress, Psychological/psychology , Synapses/physiology , Transcription Factors
17.
Brain Res Bull ; 81(4-5): 385-90, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-19909798

ABSTRACT

Activity of the transcription factor NF-kappaB is required for memory formation, but the identity and function of the genes it may regulate in this context remain obscure. Here, we comprehensively characterise NF-kappaB throughout the rat hippocampus following passive avoidance training and report significant subregion-specific increased activity across the dorsoventral axis 3h post-learning. Moreover, putative NF-kappaB binding motifs predominated in structural genes previously shown to regulate 3h following avoidance conditioning, the protein products of which may be involved in the subsequent synaptic remodelling required for consolidation. Finally, we assessed the influence of NF-kappaB-mediated transcription on neuritic structure and report that inhibition of NF-kappaB significantly decreases growth and branching of primary hippocampal neurons. These results suggest that NF-kappaB activity following hippocampal learning may contribute to consolidation-associated synaptic reorganisation.


Subject(s)
Hippocampus/physiology , Learning/physiology , NF-kappa B/metabolism , Neuronal Plasticity/physiology , Neurons/physiology , Synapses/physiology , Animals , Avoidance Learning/physiology , Cells, Cultured , Fluorescent Antibody Technique , Hippocampus/drug effects , Male , Memory/physiology , NF-kappa B/antagonists & inhibitors , Neurites/drug effects , Neurites/physiology , Neurons/drug effects , Polymerase Chain Reaction , Rats , Rats, Wistar , Synapses/drug effects , Time Factors
18.
Cereb Cortex ; 20(8): 1915-25, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20016002

ABSTRACT

Expressed throughout the central nervous system, the myocardin-related, megakaryoblastic acute leukemia 1 and 2 (Mkl1/2) are transcriptional cofactors that can be found tethered in the cytoplasm to monomeric actin but on synaptic activation translocate to the nucleus and associate with transcription factors such as serum response factor (SRF) to regulate expression of structural genes. This implies a potential role for Mkls in linking synaptic activity, through gene-expression control, to neuronal structural plasticity. Here, we present evidence that Mkls, particularly Mkl2, are powerful regulators of neuronal structure in vitro. Moreover, using the passive avoidance-conditioning paradigm, we identify learning-associated alterations of neuronal Mkl expression that appear to contribute to 2 phases of gene regulation during memory consolidation in the hippocampus. Gene regulation immediately after learning includes Egr2 and may be facilitated by downregulation of Mkls likely releasing ternary complex factor-regulated SRF activity. The second transcriptional phase occurs later at the 3-h postavoidance time point when Mkl accumulates in the nucleus of hippocampal neurons and there is enhanced transcription of Mkl-dependent structural genes that may contribute to the elaboration of new, memory-associated synapses known to appear over the subsequent 3-h period.


Subject(s)
Hippocampus/metabolism , Kinesins/physiology , Neuronal Plasticity/physiology , Neurons/metabolism , Transcription Factors/physiology , Animals , Avoidance Learning/physiology , Cell Differentiation/physiology , Early Growth Response Protein 2/genetics , Early Growth Response Protein 2/metabolism , Gene Expression Regulation, Developmental/genetics , Hippocampus/cytology , Male , Memory/physiology , Neuronal Plasticity/genetics , Neurons/cytology , Protein Transport/genetics , Rats , Rats, Wistar , Synaptic Transmission/genetics , Transcription Factors/genetics
19.
J Neurochem ; 112(4): 991-1004, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20002519

ABSTRACT

Long-term memory is formed by alterations in glutamate-dependent excitatory synaptic transmission, which is in turn regulated by synaptosomal protein of 25 kDa (SNAP-25), a key component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex essential for exocytosis of neurotransmitter-filled synaptic vesicles. Both reduced and excessive SNAP-25 activity has been implicated in various disease states that involve cognitive dysfunctions such as attention deficit hyperactivity disorder, schizophrenia and Alzheimer's disease. Here, we over-express SNAP-25 in the adult rat dorsal hippocampus by infusion of a recombinant adeno-associated virus vector, to evaluate the consequence of late adolescent-adult dysfunction of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein in the absence of developmental disruption. We report a specific and significant increase in the levels of extracellular glutamate detectable by microdialysis and a reduction in paired-pulse facilitation in the hippocampus. In addition, SNAP-25 over-expression produced cognitive deficits, delaying acquisition of a spatial map in the water maze and impairing contextual fear conditioning, both tasks known to be dorsal hippocampal dependent. The high background transmission state and pre-synaptic dysfunction likely result in interference with requisite synapse selection during spatial and fear memory consolidation. Together these studies provide the first evidence that excess SNAP-25 activity, restricted to the adult period, is sufficient to mediate significant deficits in the memory formation process.


Subject(s)
Gene Expression Regulation/physiology , Hippocampus/metabolism , Memory Disorders , Neuronal Plasticity/physiology , Synaptosomal-Associated Protein 25/metabolism , Animals , Avoidance Learning/physiology , Biophysics/methods , Cell Line, Transformed , Conditioning, Classical/physiology , Dependovirus/genetics , Dependovirus/metabolism , Disease Models, Animal , Electric Stimulation/methods , Exploratory Behavior/physiology , Flow Cytometry/methods , Glutamic Acid/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hippocampus/physiology , Humans , In Vitro Techniques , Male , Maze Learning/physiology , Memory Disorders/metabolism , Memory Disorders/pathology , Memory Disorders/physiopathology , Microdialysis/methods , Neural Inhibition/physiology , Rats , Rats, Wistar , Synaptosomal-Associated Protein 25/genetics , Transduction, Genetic/methods , Transfection/methods
20.
Neurobiol Learn Mem ; 88(3): 342-51, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17543552

ABSTRACT

Recent evidence has suggested a role for Notch in memory consolidation but the means by which this evolutionarily conserved mechanism serves these plasticity-related processes remains to be established. We have examined a role for this signalling pathway in the hippocampal dentate gyrus of Wistar rats at increasing times following passive avoidance conditioning. Our principal finding is that a transient attenuation of Notch signalling occurs at the 10-12h post-training time. In this period, extracellular Notch-1 protein fragment exhibited a significant 2- to 3-fold increase but, by contrast, Notch-1 mRNA levels were significantly reduced. Moreover, transient inactivation of Notch-1 signalling was further suggested by concomitant reductions in the Notch ligand Jagged-1 and Notch-1 target protein Hes-1 mRNA levels. The C-terminal fragment of PS-1, necessary for gamma-secretase activity, was also significantly reduced at the 12h post-training time. These events were commensurate with the increase of a Notch immunoreactive fragment of 66 kDa in the nuclear fraction of the dentate gyrus. This fragment, identified with two different Notch-1 antisera, was not the expected NICD polypeptide of approximately 110 kDa and its accumulation was found to correlate with a significantly reduced expression of the Hes-1 transcriptional repressor. During the period of reduced Notch activity, a transient increase in soluble beta-catenin and GSK-3beta phosphorylation was observed, indicating a reciprocal activation of the Wnt signalling pathway. As down-regulation of Notch signalling promotes differentiation and neurite outgrowth in post-mitotic neurons, it is proposed that this pathway regulates the integration of synapses transiently produced during memory consolidation.


Subject(s)
Avoidance Learning/physiology , Dentate Gyrus/metabolism , Memory/physiology , Presenilin-1/metabolism , Receptor, Notch1/metabolism , Amyloid Precursor Protein Secretases/metabolism , Analysis of Variance , Animals , Extracellular Fluid/metabolism , Gene Expression Regulation/physiology , Male , Neuronal Plasticity/physiology , RNA, Messenger/analysis , Rats , Rats, Wistar , Receptor, Notch1/genetics , Signal Transduction/physiology , Statistics, Nonparametric , Time Factors
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