A lysosomal enigma CLN5 and its significance in understanding neuronal ceroid lipofuscinosis.

Neuronal Ceroid Lipofuscinosis (NCL), also known as Batten disease, is an incurable childhood brain disease. The thirteen forms of NCL are caused by mutations in thirteen CLN genes. Mutations in one CLN gene, CLN5, cause variant late-infantile NCL, with an age of onset between 4 and 7 years. The CLN5 protein is ubiquitously expressed in the majority of tissues studied and in the brain, CLN5 shows both neuronal and glial cell expression. Mutations in CLN5 are associated with the accumulation of autofluorescent storage material in lysosomes, the recycling units of the cell, in the brain and peripheral tissues. CLN5 resides in the lysosome and its function is still elusive. Initial studies suggested CLN5 was a transmembrane protein, which was later revealed to be processed into a soluble form. Multiple glycosylation sites have been reported, which may dictate its localisation and function. CLN5 interacts with several CLN proteins, and other lysosomal proteins, making it an important candidate to understand lysosomal biology. The existing knowledge on CLN5 biology stems from studies using several model organisms, including mice, sheep, cattle, dogs, social amoeba and cell cultures. Each model organism has its advantages and limitations, making it crucial to adopt a combinatorial approach, using both human cells and model organisms, to understand CLN5 pathologies and design drug therapies. In this comprehensive review, we have summarised and critiqued existing literature on CLN5 and have discussed the missing pieces of the puzzle that need to be addressed to develop an efficient therapy for CLN5 Batten disease.

The impact of the immune system on the safety and efficiency of enzyme replacement therapy in lysosomal storage disorders.

In the light of clinical experience in infantile onset Pompe patients, the immunological impact on the tolerability and long-term efficacy of enzyme replacement therapy (ERT) for lysosomal storage disorders has come under renewed scrutiny. This article details the currently proposed immunological mechanisms involved in the development of anti-drug antibodies and the current therapies used in their treatment. Given the current understanding of the adaptive immune response, it focuses particularly on T cell dependent mechanisms and the paradigm of using lymphocytic negative selection as a predictor of antibody formation. This concept originally postulated in the 1970s, stipulated that the genotypically determined lack of production or production of a variant protein determines an individual's lymphocytic repertoire. This in turn is the key factor in determining the potential severity of an individual's immunological response to ERT. It also highlights the need for immunological assay standardization particularly those looking at describing the degree of functional impact, robust biochemical or clinical endpoints and detailed patient subgroup identification if the true evaluations of impact are to be realised.

Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb.

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity.

Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord.

Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting.

Cooperation between Shh and IGF-I in promoting myogenic proliferation and differentiation via the MAPK/ERK and PI3K/Akt pathways requires Smo activity.

Sonic Hedgehog (Shh) has been shown to promote adult myoblast proliferation and differentiation and affect Akt phosphorylation via its effector Smoothened (Smo). Here, the relationship between Shh and insulin-like growth factor I (IGF-I) was examined with regard to myogenic differentiation via signaling pathways which regulate this process. Each factor enhanced Akt and MAPK/ERK (p42/44) phosphorylation and myogenic factor expression levels in a dose-responsive manner, while combinations of Shh and IGF-I showed additive effects. Blockage of the IGF-I effects by neutralizing antibody partially reduced Shh's effects on signaling pathways, suggesting that IGF-I enhances, but is not essential for Shh effects. Addition of cyclopamine, a Smo inhibitor, reduced Shh- and IGF-I-induced Akt phosphorylation in a similar manner, implying that Shh affects gain of the IGF-I signaling pathway. This implication was also examined via a genetic approach. In cultures derived from Smo(mut) (MCre;Smo(flox/flox)) mice lacking Smo expression specifically in hindlimb muscles, IGF-I-induced Akt and p42/44 phosphorylation was significantly reduced compared to IGF-I's effect on Smo(cont) cells. Moreover, remarkable inhibition of the stimulatory effect of IGF-I on myogenic differentiation was observed in Smo(mut) cultures, implying that intact Smo is required for IGF-I effects in myoblasts. Immunoprecipitation assays revealed that tyrosine-phosphorylated proteins, including the regulatory unit of PI3K (p85), are recruited to Smo in response to Shh. Moreover, IGF-IR was found to associate with Smo in response to Shh and to IGF-I, suggesting that Shh and IGF-I are already integrated at the receptor level, a mechanism by which their signaling pathways interact in augmenting their effects on adult myoblasts.

Patterned Stimulation of the Chrimson Opsin in Glutamatergic Motor Thalamus Neurons Improves Forelimb Akinesia in Parkinsonian Rats.

Parkinson's disease (PD) is a motor disorder charactertised by altered neural activity throughout the basal ganglia-thalamocortical circuit. Electrical deep brain stimulation (DBS) is efficacious in alleviating motor symptoms, but has several notable side-effects, most likely reflecting the non-specific nature of electrical stimulation and/or the brain regions targeted. We determined whether specific optogenetic activation of glutamatergic motor thalamus (Mthal) neurons alleviated forelimb akinesia in a chronic rat model of PD. Parkinsonian rats (unilateral 6-hydroxydopamine injection) were injected with an adeno-associated viral vector (AAV5-CaMKII-Chrimson-GFP) to transduce glutamatergic Mthal neurons with the red-shifted Chrimson opsin. Optogenetic stimulation with orange light at 15 Hz tonic and a physiological pattern, previously recorded from a Mthal neuron in a control rat, significantly increased forelimb use in the reaching test (p < 0.01). Orange light theta burst stimulation, 15 Hz and control reaching patterns significantly reduced akinesia (p < 0.0001) assessed by the step test. In contrast, forelimb use in the cylinder test was unaffected by orange light stimulation with any pattern. Blue light (control) stimulation failed to alter behaviours. Activation of Chrimson using complex patterns in the Mthal may be an alternative treatment to recover movement in PD. These vector and opsin changes are important steps towards translating optogenetic stimulation to humans.

Anterior thalamic nuclei neurons sustain memory.

A hippocampal-diencephalic-cortical network supports memory function. The anterior thalamic nuclei (ATN) form a key anatomical hub within this system. Consistent with this, injury to the mammillary body-ATN axis is associated with examples of clinical amnesia. However, there is only limited and indirect support that the output of ATN neurons actively enhances memory. Here, in rats, we first showed that mammillothalamic tract (MTT) lesions caused a persistent impairment in spatial working memory. MTT lesions also reduced rhythmic electrical activity across the memory system. Next, we introduced 8.5 Hz optogenetic theta-burst stimulation of the ATN glutamatergic neurons. The exogenously-triggered, regular pattern of stimulation produced an acute and substantial improvement of spatial working memory in rats with MTT lesions and enhanced rhythmic electrical activity. Neither behaviour nor rhythmic activity was affected by endogenous stimulation derived from the dorsal hippocampus. Analysis of immediate early gene activity, after the rats foraged for food in an open field, showed that exogenously-triggered ATN stimulation also increased Zif268 expression across memory-related structures. These findings provide clear evidence that increased ATN neuronal activity supports memory. They suggest that ATN-focused gene therapy may be feasible to counter clinical amnesia associated with dysfunction in the mammillary body-ATN axis.

Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice.

Physical training regulates muscle metabolic and contractile properties by altering gene expression. Electrical activity evoked in muscle fiber membrane during physical activity is crucial for such regulation, but the subsequent intracellular pathway is virtually unmapped. Here we investigate the ability of myogenin, a muscle-specific transcription factor strongly regulated by electrical activity, to alter muscle phenotype. Myogenin was overexpressed in transgenic mice using regulatory elements that confer strong expression confined to differentiated post-mitotic fast muscle fibers. In fast muscles from such mice, the activity levels of oxidative mitochondrial enzymes were elevated two- to threefold, whereas levels of glycolytic enzymes were reduced to levels 0.3-0.6 times those found in wild-type mice. Histochemical analysis shows widespread increases in mitochondrial components and glycogen accumulation. The changes in enzyme content were accompanied by a reduction in fiber size, such that many fibers acquired a size typical of oxidative fibers. No change in fiber type-specific myosin heavy chain isoform expression was observed. Changes in metabolic properties without changes in myosins are observed after moderate endurance training in mammals, including humans. Our data suggest that myogenin regulated by electrical activity may mediate effects of physical training on metabolic capacity in muscle.

Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-1 astrocytes.

O-2A progenitor cells are bipotential glial precursors that give rise to both oligodendrocytes and type-2 astrocytes on a precise schedule in the rat CNS. Studies in culture suggest that oligodendrocyte differentiation occurs constitutively, while type-2 astrocyte differentiation requires an exogenous inducer such as fetal calf serum. Here we describe a rat brain cell culture system in which type-2 astrocytes develop on schedule in the absence of exogenous inducers. Coincident with type-2-astrocyte development, the cultures produce an approximately 20 kd type-2-astrocyte-inducing factor(s). Purified cultures of type-1 astrocytes can produce a similar factor(s). Under conditions where they produce type-2-astrocyte-inducing factor(s), both brain and type-1 astrocyte cultures produce a factor(s) with ciliary neurotrophic (CNTF)-like activity. Purified CNTF, like the inducers from brain and type-1 astrocyte cultures, prematurely induces type-2 astrocyte differentiation in brain cultures. These findings suggest that type-2 astrocyte development is initiated by a CNTF-like protein produced by type-1 astrocytes.

MAP1B is encoded as a polyprotein that is processed to form a complex N-terminal microtubule-binding domain.

Microtubule-associated protein 1B (MAP1B), an abundant developmentally regulated neuronal protein, is a stoichiometric complex of a heavy chain and two light chains (light chain 1 and light chain 3). We find that light chain 1 is encoded within the 3' end of a previously reported MAP1B heavy chain cDNA. Amino acid sequencing, epitope mapping, Northern blotting, and Southern blotting indicate that the light chain and heavy chain are encoded by the same mRNA within the same open reading frame. In addition, amino acid sequencing of a 120 kd microtubule-binding and light chain-binding fragment of the heavy chain reveals that light chain 1 binds near the heavy chain N-terminus. Together these data indicate that the heavy chain and light chain 1 are produced by proteolytic processing of a MAP1B polyprotein and form a complex microtubule-binding domain.

Adult-born dentate granule cell excitability depends on the interaction of neuron age, ontogenetic age and experience.

Early during their maturation, adult-born dentate granule cells (aDGCs) are particularly excitable, but eventually develop the electrophysiologically quiet properties of mature cells. However, the stability versus plasticity of this quiet state across time and experience remains unresolved. By birthdating two populations of aDGCs across different animal ages, we found for 10-month-old rats the expected reduction in excitability across cells aged 4-12 weeks, as determined by Egr1 immunoreactivity. Unexpectedly, cells 35 weeks old (after genesis at an animal age of 2 months) were as excitable as 4-week-old cells, in the dorsal hippocampus. This high level of excitability at maturity was specific for cells born in animals 2 months of age, as cells born later in life did not show this effect. Importantly, excitability states were not fixed once maturity was gained, but were enhanced by enriched environment exposure or LTP induction, indicating that any maturational decrease in excitability can be compensated by experience. These data reveal the importance of the animal's age for aDGC excitability, and emphasize their prolonged capability for plasticity during adulthood.

Muscle development: electrical control of gene expression.

The electrical activity resulting from stimulation by motor neurons regulates gene expression in skeletal muscle fibres. A recent study has suggested a mechanism by which distinct patterns of electrical stimulus might be integrated to control the contractile properties of these fibres.

Muscle development: reversal of the differentiated state.

Cell fate selection and cell cycle exit are fundamental features of differentiation during animal development. Accumulating data suggest that these processes are more readily reversible than previously supposed and are beginning to point at the underlying molecular mechanisms.

Temporal profile of subventricular zone progenitor cell migration following quinolinic acid-induced striatal cell loss.

A number of studies have demonstrated directed migration of neural progenitor cells to sites of brain injury and disease, however a detailed examination of when a cell is "born" in relation to injury induction and the migratory response of that cell has not previously been determined. This study therefore examined the temporal correlation between progenitor cell proliferation ("birth") and neuroblast migratory response into the damaged striatum following quinolinic acid (QA) lesioning of the adult rat striatum. Retroviral labeling of subventricular zone (SVZ)-derived progenitor cells demonstrated that cell loss in the QA-lesioned striatum increased progenitor cell migration through the rostral migratory stream (RMS) for up to 30 days. In addition, a population of dividing cells originating from the SVZ generated doublecortin positive neuroblasts that migrated into the damaged striatum in response to cell loss invoked by the QA lesion. Quantification of bromodeoxyuridine (BrdU)-labeled cells co-expressing doublecortin revealed that the majority of cells present in the damaged striatum were generated from progenitor cells dividing within 2 days either prior to or following the QA lesion. In contrast, cells dividing 2 or more days following QA lesioning, migrated into the striatum and exhibited a glial phenotype. These results demonstrate that directed migration of SVZ-derived cells and neuroblast differentiation in response to QA lesioning of the striatum is acute and transient. We propose this is predominantly due to a reduced capacity over time for newly generated neuroblasts to respond to the lesioned environment due to a loss or inhibition of migratory cues.

False-positive results on colorimetric carbon dioxide analysis in neonatal resuscitation: potential for serious patient harm.

A term infant requiring resuscitation was found to have a false-positive color change on a colorimetric carbon dioxide device as a result of administration of epinephrine via an endotracheal tube. Using models of direct application and vapor exposure with a test lung, we discovered that epinephrine, atropine, infasurf and naloxone may result in false-positive color change. This false-positive response may lead to delayed recognition of esophageal intubation.

Control of muscle fibre and motoneuron diversification.

Recent studies have elucidated both the mechanism of early formation of diverse muscle fibre types and the matching of diverse populations of motoneurons to their appropriate muscle targets. Highlights include the demonstration that distinct signals are necessary for the formation of several distinct myoblast populations in the vertebrate somite, the identification of motoneuron subtypes, studies of how motoneurons target appropriate muscles, and rapid progress on the Drosophila neuromuscular system. We propose a model in which four classes of decision control the patterning of both motoneurons and muscles.

Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts.

Patterning of fast and slow muscle fibres in limbs is regulated by signals from non-muscle cells. Myoblast lineage has, however, also been implicated in fibre type patterning. Here we test a founder cell hypothesis for the role of myoblast lineage, by implanting characterized fast and slow mouse myoblast clones into chick limb buds. In culture, late foetal mouse myoblast clones are committed to a probability (range 0-0.92) of slow myosin heavy chain (MyHC) expression. In contrast, when implanted into chick limbs, fast mouse myoblast clones express myosin characteristic of their new environment, without fusion to chick muscle cells and in the absence of innervation. Therefore, local signals exist within the chick limb bud during primary myogenesis that can override intrinsic commitment of at least some myoblasts, and induce slow MyHC.

MyoD protein is differentially accumulated in fast and slow skeletal muscle fibres and required for normal fibre type balance in rodents.

MyoD is a muscle-specific transcription factor involved in commitment of cells to myogenesis. MyoD mRNA levels differ between fast and slow muscles, suggesting that MyoD may regulate aspects of fibre type. Here we show that detectable MyoD protein becomes restricted during development to the nuclei of the fastest classes of fibres in fast muscles. myoDm1 mice, in which the myoD gene has been disrupted, show subtle shifts in fibre type of fast muscles toward a slower character, suggesting that MyoD is involved in the maintenance of the fast IIB/IIX fibre type. In contrast, slow muscle shifts to a faster phenotype in myoDm1. Moreover, MD6.0-lacZ transgenic mice with the myoD promoter driving lacZ, show highest beta-galactosidase activity in the fastest fibres of fast muscles, but also express low levels in slow fibres of slow, but not fast, muscles, suggesting distinct regulation of gene expression in slow fibres of fast and slow muscles.