Wallerian degeneration of injured neuronal axons and synapses is blocked in Wld super(S) mutant mice by expression of an nicotinamide mononucleotide adenylyl transferase 1 (Nmnat-1)/truncated-Ube4b ...chimeric gene. The protein product of the Wld super(S) gene localizes to neuronal nuclei. Here we show that Wld super(S) protein expression selectively alters mRNA levels of other genes in Wld super(S) mouse cerebellum in vivo and following transfection of human embryonic kidney (HEK293) cells in vitro. The largest changes, identified by microarray analysis and quantitative real-time polymerase chain reaction of cerebellar mRNA, were an approximate 10-fold down-regulation of pituitary tumour-transforming gene-1 (pttg1) and an approximate 5-fold up-regulation of a structural homologue of erythroid differentiation regulator-1 (edr1l-EST). Transfection of HEK293 cells with a Wld super(S)-eGFP construct produced similar changes in mRNA levels for these and seven other genes, suggesting that regulation of gene expression by Wld super(S) is conserved across different species, including humans. Similar modifications in mRNA levels were mimicked for some of the genes (including pttg1) by 1 mM nicotinamide adenine dinucleotide (NAD). However, expression levels of most other genes (including edr1l-EST) were insensitive to NAD. Pttg1 super(-/-) mutant mice showed no neuroprotective phenotype. Transfection of HEK293 cells with constructs comprising either full-length Nmnat-1 or the truncated Ube4b fragment (N70-Ube4b) demonstrated selective effects of Nmnat-1 (down-regulated pttg1) and N70-Ube4b (up-regulated edr1l-EST) on mRNA levels. Similar changes in pttg1 and edr1l-EST were observed in the mouse NSC34 motor neuron-like cell line following stable transfection with Wld super(S). Together, the data suggest that the Wld super(S) protein co-regulates expression of a consistent subset of genes in both mouse neurons and human cells. Targeting Wld super(S)-induced gene expression may lead to novel therapies for neurodegeneration induced by trauma or by disease in humans.
Slow Wallerian degeneration (
Wld
S
) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD
+
synthesizing ...enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld
S
targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld
S
lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD
+
synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld
S
protein influence the intranuclear location of both ubiquitin proteasome and NAD
+
synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.
Slow Wallerian degeneration (Wld
S
) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD
+
synthesizing ...enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld
S
targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld
S
lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD
+
synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld
S
protein influence the intranuclear location of both ubiquitin proteasome and NAD
+
synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.
Slow Wallerian degeneration (WlduS mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C- terminal region, derived from NAD super(+) synthesizing ...enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N- terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld super(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld super(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD super(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld super(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD super(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.
The slow Wallerian degeneration phenotype, WldsuperS, which delays Wallerian degeneration and axon pathology for several weeks, has so far been studied only in mice. A rat model would have several ...advantages. First, rats model some human disorders better than mice. Second, the larger body size of rats facilitates more complex surgical manipulations. Third, rats provide a greater yield of tissue for primary culture and biochemical investigations. We generated transgenic WldsuperS rats expressing the Ube4b-Nmnat1 chimeric gene in the central and peripheral nervous system. As in WldsuperS mice, their axons survive up to 3 weeks after transection and remain functional for at least 1 week. Protection of axotomized nerve terminals is stronger than in mice, particularly in one line, where 95-100% of neuromuscular junctions remained intact and functional after 5 days. Furthermore, the loss of synaptic phenotype with age was much less in rats than in mice. Thus, the slow Wallerian degeneration phenotype can be transferred to another mammalian species and synapses may be more effectively preserved after axotomy in species with longer axons.
Activation of M
1
muscarinic acetylcholine receptors (M
1
mAChR) inhibits M-type potassium currents (
I
K(M)
) and N-type calcium currents (
I
Ca
) in mammalian sympathetic ganglia. Previous ...antisense experiments suggested that, in rat superior cervical ganglion (SCG) neurons, both effects were partly mediated by the G-protein Gα
q
(Delmas et al., 1998a; Haley et al., 1998a), but did not eliminate a contribution by other pertussis toxin (PTX)-insensitive G-proteins. We have tested this further using mice deficient in the Gα
q
gene.
PTX-insensitive M
1
mAChR inhibition of
I
Ca
was strongly reduced in Gα
q
−/− mouse SCG neurons and was fully restored by acute overexpression of Gα
q
. In contrast, M
1
mAChR inhibition of
I
K(M)
persisted in Gα
q
−/− mouse SCG cells. However, unlike rat SCG neurons, muscarinic inhibition of
I
K(M)
was partly PTX-sensitive. Residual (PTX-insensitive)
I
K(M)
inhibition was slightly reduced in Gα
q
−/− neurons, and the remaining response was then suppressed by anti-Gα
q/11
antibodies.
Bradykinin (BK) also inhibits
I
K(M)
in rat SCG neurons via a PTX-insensitive G-protein (G
q
and/or G
11
; Jones et al., 1995). In mouse SCG neurons,
I
K(M)
inhibition by BK was fully PTX-resistant. It was unchanged in Gα
q
−/− mice but was abolished by anti-Gα
q/11
antibody.
We conclude that, in mouse SCG neurons (1) M
1
mAChR inhibition of
I
Ca
is mediated principally by G
q
, (2) M
1
mAChR inhibition of
I
K(M)
is mediated partly by G
q
, more substantially by G
11
, and partly by a PTX-sensitive G-protein(s), and (3) BK-induced inhibition of
I
K(M)
is mediated wholly by G
11
.
Activation of M(1) muscarinic acetylcholine receptors (M(1) mAChR) inhibits M-type potassium currents (I(K(M))) and N-type calcium currents (I(Ca)) in mammalian sympathetic ganglia. Previous ...antisense experiments suggested that, in rat superior cervical ganglion (SCG) neurons, both effects were partly mediated by the G-protein Galpha(q) (Delmas et al., 1998a; Haley et al., 1998a), but did not eliminate a contribution by other pertussis toxin (PTX)-insensitive G-proteins. We have tested this further using mice deficient in the Galpha(q) gene. PTX-insensitive M(1) mAChR inhibition of I(Ca) was strongly reduced in Galpha(q) -/- mouse SCG neurons and was fully restored by acute overexpression of Galpha(q). In contrast, M(1) mAChR inhibition of I(K(M)) persisted in Galpha(q)-/- mouse SCG cells. However, unlike rat SCG neurons, muscarinic inhibition of I(K(M)) was partly PTX-sensitive. Residual (PTX-insensitive) I(K(M)) inhibition was slightly reduced in Galpha(q) -/- neurons, and the remaining response was then suppressed by anti-Galpha(q/11) antibodies. Bradykinin (BK) also inhibits I(K(M)) in rat SCG neurons via a PTX-insensitive G-protein (G(q) and/or G(11); Jones et al., 1995). In mouse SCG neurons, I(K(M)) inhibition by BK was fully PTX-resistant. It was unchanged in Galpha(q) -/- mice but was abolished by anti-Galpha(q/11) antibody. We conclude that, in mouse SCG neurons (1) M(1) mAChR inhibition of I(Ca) is mediated principally by G(q), (2) M(1) mAChR inhibition of I(K(M)) is mediated partly by G(q), more substantially by G(11), and partly by a PTX-sensitive G-protein(s), and (3) BK-induced inhibition of I(K(M)) is mediated wholly by G(11).
Summary
Project Synergy aims to test the potential of new and emerging technologies to enhance the quality of mental health care provided by traditional face‐to‐face services. Specifically, it seeks ...to ensure that consumers get the right care, first time (delivery of effective mental health care early in the course of illness).
Using co‐design with affected individuals, Project Synergy has built, implemented and evaluated an online platform to assist the assessment, feedback, management and monitoring of people with mental disorders. It also promotes the maintenance of wellbeing by collating health and social information from consumers, their supportive others and health professionals. This information is reported back openly to consumers and their service providers to promote genuine collaborative care.
The online platform does not provide stand‐alone medical or health advice, risk assessment, clinical diagnosis or treatment; instead, it supports users to decide what may be suitable care options.
Using an iterative cycle of research and development, the first four studies of Project Synergy (2014–2016) involved the development of different types of online prototypes for young people (i) attending university; (ii) in three disadvantaged communities in New South Wales; (iii) at risk of suicide; and (iv) attending five headspace centres. These contributed valuable information concerning the co‐design, build, user testing and evaluation of prototypes, as well as staff experiences during development and service quality improvements following implementation.
Through ongoing research and development (2017–2020), these prototypes underpin one online platform that aims to support better multidimensional mental health outcomes for consumers; more efficient, effective and appropriate use of health professional knowledge and clinical skills; and quality improvements in mental health service delivery.
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