Depletion of TDP-43 decreases fibril and plaque ß-amyloid and exacerbates neurodegeneration in an Alzheimer's mouse model.

TDP-43 proteinopathy, initially associated with ALS and FTD, is also found in 30-60% of Alzheimer's disease (AD) cases and correlates with worsened cognition and neurodegeneration. A major component of this proteinopathy is depletion of this RNA-binding protein from the nucleus, which compromises repression of non-conserved cryptic exons in neurodegenerative diseases. To test whether nuclear depletion of TDP-43 may contribute to the pathogenesis of AD cases with TDP-43 proteinopathy, we examined the impact of depletion of TDP-43 in populations of neurons vulnerable in AD, and on neurodegeneration in an AD-linked context. Here, we show that some populations of pyramidal neurons that are selectively vulnerable in AD are also vulnerable to TDP-43 depletion in mice, while other forebrain neurons appear spared. Moreover, TDP-43 depletion in forebrain neurons of an AD mouse model exacerbates neurodegeneration, and correlates with increased prefibrillar oligomeric Aß and decreased Aß plaque burden. These findings support a role for nuclear depletion of TDP-43 in the pathogenesis of AD and provide strong rationale for developing novel therapeutics to alleviate the depletion of TDP-43 and functional antemortem biomarkers associated with its nuclear loss.

PTBP1 and PTBP2 Repress Nonconserved Cryptic Exons.

The fidelity of RNA splicing is maintained by a network of factors, but the molecular mechanisms that govern this process have yet to be fully elucidated. We previously found that TDP-43, an RNA-binding protein implicated in neurodegenerative disease, utilizes UG microsatellites to repress nonconserved cryptic exons and prevent their incorporation into mRNA. Here, we report that two well-characterized splicing factors, polypyrimidine tract-binding protein 1 (PTBP1) and polypyrimidine tract-binding protein 2 (PTBP2), are also nonconserved cryptic exon repressors. In contrast to TDP-43, PTBP1 and PTBP2 utilize CU microsatellites to repress both conserved tissue-specific exons and nonconserved cryptic exons. Analysis of these conserved splicing events suggests that PTBP1 and PTBP2 repression is titrated to generate the transcriptome diversity required for neuronal differentiation. We establish that PTBP1 and PTBP2 are members of a family of cryptic exon repressors.

Activity and circadian rhythm influence synaptic Shank3 protein levels in mice.

Various recent studies revealed that the proteins of the Shank family act as major scaffold organizing elements in the post-synaptic density of excitatory synapses and that their expression level is able to influence synapse formation, maturation and ultimately brain plasticity. An imbalance in Shank3 protein levels has been associated with a variety of neuropsychological and neurodegenerative disorders including autism spectrum disorders and Phelan-McDermid syndrome. Given that sleep disorders and low melatonin levels are frequently observed in autism spectrum disorders, and that circadian rhythms may be able to modulate Shank3 signaling and thereby synaptic function, here, we performed in vivo studies on CBA mice using protein biochemistry to investigate the synaptic expression levels of Shank3α during the day in different brain regions. Our results show that synaptic Shank3 protein concentrations exhibit minor oscillations during the day in hippocampal and striatal brain regions that correlate with changes in serum melatonin levels. Furthermore, as circadian rhythms are tightly connected to activity levels in mice, we increased physical activity using running wheels. The expression of Shank3α increases rapidly by induced activity in thalamus and cortex, but decreases in striatum, superimposing the circadian rhythms of different brain regions. We conclude that synaptic Shank3 proteins build highly dynamic platforms that are modulated by the light:dark cycles but even more so driven by activity. Using wild-type CBA mice, we show that Shank3 is a highly dynamic and activity-regulated protein at synapses. In the hippocampus, changes in synaptic Shank3 levels are influenced by circadian rhythm/melatonin concentration, while running activity increases and decreases levels of Shank3 in the cortex and striatum respectively.

Application of Polymeric Nanoparticles for CNS Targeted Zinc Delivery In Vivo.

A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinclevels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich zinc in the brain within a small amount of time.

Emerging Use of Nanotechnology in the Treatment of Neurological Disorders.

Neurological disorders represent one of the major health concerns worldwide. Yet currently employed treatment strategies have not been very successful in the treatment of many of these disorders. One of the root causes of this lack of success is that many pharmaceutically active compounds are unable to reach their target sites of action inside the body. The delivery of substances from systemic circulation to the desired site of action, namely central nervous system (CNS), is hindered by CNS extracellular and intracellular barriers. One promising approach to circumvent these barriers is the use of nanoscaled drug delivery systems. These nanosized drug carriers display various advantages over other conventional drug delivery methods such as high drug loading capacity, targeted action, reduced toxicity, and increased therapeutic effect. Nano-neuroscience is thereby emerging as an exciting field of study and a promising future direction for the delivery of therapeutics to their targeted site of action inside the CNS for the treatment of various neurological and psychiatric disorders. Here, we will first discuss the general pharmacokinetics of therapeutics depending on the route of administration, drawbacks of conventional drug delivery systems and challenges for CNS drug delivery, namely CNS barriers. Next, a short overview of the strategies to circumvent these barriers will be given. Finally, nanotechnology and its emerging use as drug delivery systems will be discussed. This includes the advantages of nanoparticles over other conventional drug delivery systems; production of nanoparticles and their designing as an effective drug carrier; various types of nanoparticles; and some examples of their efficient use in the delivery of bioactive substances, and in the treatment of neurological disorders mainly Alzheimer's disease, brain tumors and neuroAIDS. Lastly, a future perspective on the use of nanotechnology in CNS drug delivery will be highlighted.

Loss of COMMD1 and copper overload disrupt zinc homeostasis and influence an autism-associated pathway at glutamatergic synapses.

Recent studies suggest that synaptic pathology in autism spectrum disorder (ASD) might be caused by the disruption of a signaling pathway at excitatory glutamatergic synapses, which can be influenced by environmental factors. Some factors, such as prenatal zinc deficiency, dysfunction of metallothioneins as well as deletion of COMMD1, all affect brain metal-ion homeostasis and have been associated with ASD. Given that COMMD1 regulates copper levels and that copper and zinc have antagonistic properties, here, we followed the idea that copper overload might induce a local zinc deficiency affecting key players of a putative ASD pathway such as ProSAP/Shank proteins as reported before. Our results show that increased copper levels indeed interfere with intracellular zinc concentrations and affect synaptic ProSAP/Shank levels, which similarly are altered by manipulation of copper and zinc levels through overexpression and knockdown of COMMD1. In line with this, acute and prenatal copper overload lead to local zinc deficiencies in mice. Pups exposed to prenatal copper overload furthermore show a reduction in ProSAP/Shank protein levels in the brain as well as a decreased NMDAR subunit 1 concentration. Thus, it might be likely that brain metal ion status influences a distinct pathway in excitatory synapses associated with genetic forms of ASD.

Characterization of lysosome-destabilizing DOPE/PLGA nanoparticles designed for cytoplasmic drug release.

Polymeric nanoparticles (NPs) offer a promising approach for therapeutic intracellular delivery of proteins, conventionally hampered by short half-lives, instability and immunogenicity. Remarkably, NPs uptake occurs via endocytic internalization leading to NPs content's release within lysosomes. To overcome lysosomal degradation and achieve NPs and/or loaded proteins release into cytosol, we propose the formulation of hybrid NPs by adding 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as pH sensitive component in the formulation of poly-lactide-co-glycolide (PLGA) NPs. Hybrid NPs, featured by different DOPE/PLGA ratios, were characterized in terms of structure, stability and lipid organization within the polymeric matrix. Experiments on NIH cells and rat primary neuronal cultures highlighted the safety profile of hybrid NPs. Moreover, after internalization, NPs are able to transiently destabilize the integrity of lysosomes in which they are taken up, speeding their escape and favoring cytoplasmatic localization. Thus, these DOPE/PLGA-NPs configure themselves as promising carriers for intracellular protein delivery.

Insight on the fate of CNS-targeted nanoparticles. Part II: Intercellular neuronal cell-to-cell transport.

The application of polymeric nanoparticles (NPs) has a promising future for targeting and delivering drugs into the central nervous system (CNS). However, the fate of NPs once entered in the brain after crossing the blood-brain barrier (BBB) and taken up into neuronal cells is a neglected area of study. Thus, here, we investigate the possible mechanisms of a cell-to-cell transport of poly-lactide-co-glycolide (PLGA) NPs modified with a glycopeptide (g7-NPs), already demonstrated to be able to cross the BBB after in vivo administration in rodents. We also tested antibody (Ab) -modified g7-NPs both in vitro and in vivo to investigate the possibility of specific targeting. Our results show that g7-NPs can be transported intra- and inter-cellularly within vesicles after vesicular internalization. Moreover, cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. The transport is dependent on F-actin and can be increased by induction of TNT-like structures overexpressing M-Sec, a central factor and inducer of TNT formation. Moreover, cell-to-cell transport occurs independently from NP surface modification with antibodies. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration of NPs in mice.

The PSD protein ProSAP2/Shank3 displays synapto-nuclear shuttling which is deregulated in a schizophrenia-associated mutation.

Recently, mutations in ProSAP2/Shank3 have been discovered as one of the genetic factors for schizophrenia (SCZ). Here, we show that the postsynaptic density protein ProSAP2/Shank3 undergoes activity dependent synapse-to-nucleus shuttling in hippocampal neurons. Our study shows that the de novo mutation (R1117X) in ProSAP2/Shank3 that was identified in a patient with SCZ leads to an accumulation of mutated ProSAP2/Shank3 within the nucleus independent of synaptic activity. Furthermore, we identified novel nuclear ProSAP2/Shank3 interaction partners. Nuclear localization of mutated ProSAP2/Shank3 alters transcription of several genes, among them already identified genetic risk factors for SCZ such as Synaptotagmin 1 and LRRTM1. Comparing the SCZ mutation of ProSAP2/Shank3 to the knockdown of ProSAP2/Shank3 we found some shared features such as reduced synaptic density in neuronal cultures. However, hippocampal neurons expressing the ProSAP2/Shank3 SCZ mutation furthermore show altered E/I ratio and reduced dendritic branching. Thus, we conclude that the uncoupling of ProSAP2/Shank3 nuclear shuttling from synaptic activity may represent a molecular mechanism that contributes to the pathology of SCZ in patients with mutations in ProSAP2/Shank3.

Zinc deficiency dysregulates the synaptic ProSAP/Shank scaffold and might contribute to autism spectrum disorders.

Proteins of the ProSAP/Shank family act as major organizing scaffolding elements within the postsynaptic density of excitatory synapses. Deletions, mutations or the downregulation of these molecules has been linked to autism spectrum disorders, the related Phelan McDermid Syndrome or Alzheimer's disease. ProSAP/Shank proteins are targeted to synapses depending on binding to zinc, which is a prerequisite for the assembly of the ProSAP/Shank scaffold. To gain insight into whether the previously reported assembly of ProSAP/Shank through zinc ions provides a crossing point between genetic forms of autism spectrum disorder and zinc deficiency as an environmental risk factor for autism spectrum disorder, we examined the interplay between zinc and ProSAP/Shank in vitro and in vivo using neurobiological approaches. Our data show that low postsynaptic zinc availability affects the activity dependent increase in ProSAP1/Shank2 and ProSAP2/Shank3 levels at the synapse in vitro and that a loss of synaptic ProSAP1/Shank2 and ProSAP2/Shank3 occurs in a mouse model for acute and prenatal zinc deficiency. Zinc-deficient animals displayed abnormalities in behaviour such as over-responsivity and hyperactivity-like behaviour (acute zinc deficiency) and autism spectrum disorder-related behaviour such as impairments in vocalization and social behaviour (prenatal zinc deficiency). Most importantly, a low zinc status seems to be associated with an increased incidence rate of seizures, hypotonia, and attention and hyperactivity issues in patients with Phelan-McDermid syndrome, which is caused by haploinsufficiency of ProSAP2/Shank3. We suggest that the molecular underpinning of prenatal zinc deficiency as a risk factor for autism spectrum disorder may unfold through the deregulation of zinc-binding ProSAP/Shank family members.

Calpain cleavage and inactivation of the sodium calcium exchanger-3 occur downstream of Aß in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of ß-amyloid (Aß) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase-3, activity is a prominent feature of AD brain. In addition, we observe increased calpain-mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aß1-42. We also show that exposure of primary cortical neurons to oligomeric Aß1-42 results in calpain-dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aß toxicity. Our findings suggest that Aß mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.