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Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 10 11 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 10 12 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 10 12 GC in total, and TransIt yielded 2.4 x 10 13 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Adeno-associated viral vectors are widely used as vehicles for gene transfer to the nervous system. The promoter and viral vector serotype are two key factors that determine the expression dynamics of the transgene. A previous comparative study has demonstrated that AAV1 displays efficient transduction of layer V corticospinal neurons, but the optimal promoter for transgene expression in corticospinal neurons has not been determined yet. In this paper, we report a side-by-side comparison between four commonly used promoters: the short CMV early enhancer/chicken β actin (sCAG), human cytomegalovirus (hCMV), mouse phosphoglycerate kinase (mPGK) and human synapsin (hSYN) promoter. Reporter constructs with each of these promoters were packaged in AAV1, and were injected in the sensorimotor cortex of rats and mice in order to transduce the corticospinal tract. Transgene expression levels and the cellular transduction profile were examined after 6 weeks. The AAV1 vectors harbouring the hCMV and sCAG promoters resulted in transgene expression in neurons, astrocytes and oligodendrocytes. The mPGK and hSYN promoters directed the strongest transgene expression. The mPGK promoter did drive expression in cortical neurons and oligodendrocytes, while transduction with AAV harbouring the hSYN promoter resulted in neuron-specific expression, including perineuronal net expressing interneurons and layer V corticospinal neurons. This promoter comparison study contributes to improve transgene delivery into the brain and spinal cord. The optimized transduction of the corticospinal tract will be beneficial for spinal cord injury research.

Background: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain., Methods: A plasmid expressing CERT L , the long isoform of CERTs, was used to study the interaction of CERT L with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERT L protein was employed to study interaction of CERT L with amyloid-β (Aβ), Aβ aggregation process in presence of CERT L , and the resulting changes in Aβ toxicity in neuroblastoma cells. CERT L was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay., Results: Here, we report that CERT L binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERT L , decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERT L in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype., Conclusion: Our results demonstrate a crucial role of CERT L in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques in any well-equipped molecular neurobiology laboratory. With this protocol we typically obtain research batches of 100-200 μL that range in titer from 5 × 10 12 to 2 × 10 13 genomic copies/mL.

Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson's disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson's disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson's disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option. SIGNIFICANCE STATEMENT Parkinson's disease (PD) is a neurodegenerative disease characterized by severe motor dysfunction due to progressive degeneration of mesencephalic dopaminergic (DA) neurons in the substantia nigra. To date, there is no regenerative treatment available. We previously showed that repulsive guidance molecule member a (RGMa) is upregulated in the substantia nigra of PD patients. Adeno-associated virus-mediated targeting of RGMa to mouse DA neurons showed that overexpression of this repulsive axon guidance and cell patterning cue models the behavioral and neuropathological characteristics of PD in a remarkable way. These findings have implications for therapy development as interfering with the function of this specific axon guidance cue may be beneficial to the survival of DA neurons., (Copyright © 2017 the authors 0270-6474/17/379361-19$15.00/0.)

Terminal Schwann cells (TSCs) are specialized cells that envelop the motor nerve terminal, and play a role in the maintenance and regeneration of neuromuscular junctions (NMJs). The chemorepulsive protein semaphorin 3A (SEMA3A) is selectively up-regulated in TSCs on fast-fatigable muscle fibers following experimental denervation of the muscle (BotoxA-induced paralysis or crush injury to the sciatic nerve) or in the motor neuron disease amyotrophic lateral sclerosis (ALS). Re-expression of SEMA3A in this subset of TSCs is thought to play a role in the selective plasticity of nerve terminals as observed in ALS and following BotoxA-induced paralysis. Using a mouse model expressing a mutant SEMA3A with diminished signaling capacity, we studied the influence of SEMA3A signaling at the NMJ with two denervation paradigms; a motor neuron disease model (the G93A-hSOD1 ALS mouse line) and an injury model (BotoxA-induced paralysis). ALS mice that either expressed 1 or 2 mutant SEMA3A alleles demonstrated no difference in ALS-induced decline in motor behavior. We also investigated the effects of BotoxA-induced paralysis on the sprouting capacity of NMJs in the K108N-SEMA3A mutant mouse, and observed no change in the differential neuronal plasticity found at NMJs on fast-fatigable or slow muscle fibers due to the presence of the SEMA3A mutant protein. Our data may be explained by the residual repulsive activity of the mutant SEMA3A, or it may imply that SEMA3A alone is not a key component of the molecular signature affecting NMJ plasticity in ALS or BotoxA-induced paralysis. Interestingly, we did observe a sex difference in motor neuron sprouting behavior after BotoxA-induced paralysis in WT mice which we speculate may be an important factor in the sex dimorphic differences seen in ALS., Competing Interests: The authors have declared that no competing interests exist.

Numerous lines of evidence suggest that DNA damage contributes to the initiation, progression, and severity of neurodegenerative diseases. However, the molecular mechanisms responsible for this relationship remain unclear. This review integrates historical data with contemporary findings to propose that DNA damage exacerbates neurodegenerative diseases by inducing transcription errors. First, we describe the scientific rationale and basic biological concepts that underpin this hypothesis. Then, we provide epidemiological, cellular, and molecular data to support this idea, and we describe new and recently published observations that suggest that the former high incidence of neurodegenerative disease in Guam may have been driven by DNA damage-induced transcription errors. Finally, we explore the long-term implications of these findings on our understanding of the impact of genotoxic stress on human aging and disease. [ABSTRACT FROM AUTHOR]

The development of immune cell engagers (ICEs) can be limited by logistical and functional restrictions associated with fusion protein designs, thus limiting immune cell recruitment to solid tumors. Herein, a high affinity superantigen‐based multivalent ICE is developed for simultaneous activation and recruitment of NK and T cells for tumor treatment. Yeast library‐based directed evolution is adopted to identify superantigen variants possessing enhanced binding affinity to immunoreceptors expressed on human T cells and NK cells. High‐affinity superantigens exhibiting improved immune‐stimulatory activities are then incorporated into a superantigen‐based tri‐functional yeast‐display‐enhanced multivalent immune cell engager (STYMIE), which is functionalized with a nanobody, a Neo‐2/15 cytokine, and an Fc domain for tumor targeting, immune stimulation, and prolonged circulation, respectively. Intravenous administration of STYMIE enhances NK and T cell recruitment into solid tumors, leading to enhanced inhibition in multiple tumor models. The study offers design principles for multifunctional ICEs. [ABSTRACT FROM AUTHOR]

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction., Competing Interests: The authors have declared that no competing interests exist.

The mutant ubiquitin UBB(+1) is a substrate as well as an inhibitor of the ubiquitin-proteasome system (UPS) and accumulates in the neuropathological hallmarks of Alzheimer's disease (AD). A role for the UPS has been suggested in the generation of amyloid β (Aβ) plaques in AD. To investigate the effect of UBB(+1) expression on amyloid pathology in vivo, we crossed UBB(+1) transgenic mice with a transgenic line expressing AD-associated mutant amyloid precursor protein (APPSwe) and mutant presenilin 1 (PS1dE9), resulting in APPPS1/UBB(+1) triple transgenic mice. In these mice, we determined the Aβ levels at 3, 6, 9 and 11 months of age. Surprisingly, we found a significant decrease in Aβ deposition in amyloid plaques and levels of soluble Aβ(42) in APPPS1/UBB(+1) transgenic mice compared to APPPS1 mice at 6 months of age, without alterations in UBB(+1) protein levels or proteasomal chymotrypsin activity. These lowering effects of UBB(+1) on Aβ deposition were transient, as this relative decrease in plaque load was not significant in APPPS1/UBB(+1) mice at 9 and 11 months of age. We also show that APPPS1/UBB(+1) mice exhibit astrogliosis, indicating that they may not be improved functionally compared to APPPS1 mice despite the Aβ reduction. The molecular mechanism underlying this decrease in Aβ deposition in APPPS1/UBB(+1) mice is more complex than previously assumed because UBB(+1) is also ubiquitinated at K63 opening the possibility of additional effects of UBB(+1) (e.g. kinase activation)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

UBB(+1) is a mutant ubiquitin which accumulates in the hallmarks of tauopathies, including Alzheimer's disease. Transgenic mice expressing high levels of neuronal UBB(+1) exhibit moderately decreased proteasome activity and spatial reference memory deficits at 9months of age. In the present study, we characterized the behavioral phenotype of male UBB(+1) transgenic mice at different ages. We show that UBB(+1) transgenic mice displayed an age-related functional decline similar to wild-type littermates, without gross neurological abnormalities or alterations in procedural motor-learning and motor coordination. At 15months of age, a transgene-specific spatial learning deficit was dependent on the period of training in the Morris watermaze. This deficit could be eliminated after additional training. We conclude that the previously reported spatial reference memory deficits of UBB(+1) transgenic mice persist during aging. In addition, our results demonstrate that the subtle defect in spatial reference memory formation, caused by a decrease in forebrain proteasome activity, is a persistent defect and not a structural defect., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The ubiquitin-proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin- containing proteinaceous deposits. UBB(+1), a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB(+1) properties shift from a proteasomal ubiquitin-fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB(+1). In these mice, UBB(+1) protein is scarcely detectable in the neuronal cell population. Accumulation of UBB(+1) commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB(+1) protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB(+1) is continuously degraded and disease-related UBB(+1) accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB(+1) and its role in neurodegenerative disease., ((c) 2010 Wiley-Liss, Inc.)

UBB(+1), a mutant form of ubiquitin, is both a substrate and an inhibitor of the proteasome which accumulates in the neuropathological hallmarks of Huntington disease (HD). In vitro, expression of UBB(+1) and mutant huntingtin synergistically increase aggregate formation and polyglutamine induced cell death. We generated a UBB(+1) transgenic mouse line expressing UBB(+1) within the neurons of the striatum. In these mice lentiviral driven expression of expanded huntingtin constructs in the striatum results in a significant increase in neuronal inclusion formation. Although UBB(+1) transgenic mice show neither a decreased lifespan nor apparent neuronal loss, they appear to be more vulnerable to toxic insults like expanded polyglutamine proteins due to a modest proteasome inhibition. These findings underscore the relevance of an efficient ubiquitin-proteasome system in HD., (Copyright 2009 Elsevier Inc. All rights reserved.)

Many neurodegenerative diseases are characterized by deposits of ubiquitinated and aberrant proteins, suggesting a failure of the ubiquitin-proteasome system (UPS). The aberrant ubiquitin UBB(+1) is one of the ubiquitinated proteins accumulating in tauopathies such as Alzheimer's disease (AD) and polyglutamine diseases such as Huntington's disease. We have generated UBB(+1) transgenic mouse lines with post-natal neuronal expression of UBB(+1), resulting in increased levels of ubiquitinated proteins in the cortex. Moreover, by proteomic analysis, we identified expression changes in proteins involved in energy metabolism or organization of the cytoskeleton. These changes show a striking resemblance to the proteomic profiles of both AD brain and several AD mouse models. Moreover, UBB(+1) transgenic mice show a deficit in contextual memory in both water maze and fear conditioning paradigms. Although UBB(+1) partially inhibits the UPS in the cortex, these mice do not have an overt neurological phenotype. These mouse models do not replicate the full spectrum of AD-related changes, yet provide a tool to understand how the UPS is involved in AD pathological changes and in memory formation.

Accumulation of proteins in inclusions in neurological disorders is partly due to dysfunction of the ubiquitin-proteasome system. Proteasomal dysfunction may be caused by misexpression of one or more of its subunits. A large number of antibodies reactive with proteasome subunits were screened on material from patients exhibiting tau- and synucleinopathies. Many antisera against proteasomal subunits (11S activator, 19S regulator ATPase/non-ATPase, and 20S alpha and beta resulted in a distinct nuclear and/or cytoplasmic staining of the entorhinal-hippocampal area and the temporal cortex of Alzheimer's disease (AD) patients. In particular an antibody directed against 19S regulator ATPase subunit 6b (S6b) specifically stained the neurofibrillary tangles and dystrophic neurites in AD, Down syndrome and aged nondemented controls. In other tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy and argyrophilic grain disease), neuronal and/or glial inclusions were also S6b immunoreactive. In contrast, in synucleinopathies (Lewy body disease (LBD) and multiple system atrophy) no S6b staining was seen. Real time quantitative PCR on the temporal cortex of AD patients revealed a significant increase in S6b subunit mRNA. This increase was not found in the gyrus cinguli anterior of patients with LBD. This differential expression of S6b most likely will result in different proteomic patterns. Here we present evidence to show that S6b coexists with a reporter for proteasomal dysfunction (ubiquitin(+1)), and we conclude that S6b transcript up-regulation and the dysfunction in tauopathies may be functionally related.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites. [ABSTRACT FROM AUTHOR]

Biomarkers present in the cerebrospinal fluid (CSF) of Alzheimer Disease patients could be instrumental in guiding diagnosis and monitoring of progression of the disease. We have previously reported on the secretion of a frameshifted form of amyloid-beta precursor protein, APP+1, into the CSF of Alzheimer patients and controls. APP+1 is secreted efficiently in controls, but during the progression of Alzheimer Disease, its secretion is reduced and APP+1 accumulates in tangle-bearing neurons. Here we describe the generation of a transgenic mouse line expressing APP+1 in the brain. These mice do not suffer from overt pathology or neurodegeneration, suggesting that APP+1 is not neurotoxic. To measure APP+1 levels in the CSF, we serially sampled CSF from the cisterna magna in the same mouse over a period of months. Indeed, APP+1 is secreted into the CSF of the transgenic mice, and APP+1 levels are stable over 1 year. This mouse model may guide the study of secretion deficits as found in Alzheimer Disease.

Frameshift (+1) proteins such as APP(+1) and UBB(+1) accumulate in sporadic cases of Alzheimer disease (AD) and in older subjects with Down syndrome (DS). We investigated whether these proteins also accumulate at an early stage of neuropathogenesis in young DS individuals without neuropathology and in early-onset familial forms of AD (FAD), as well as in other tauopathies, such as Pick disease (PiD) or progressive supranuclear palsy (PSP). APP(+1) is present in many neurons and beaded neurites in very young cases of DS, which suggests that it is axonally transported. In older DS patients (>37 years), a mixed pattern of APP(+1) immunoreactivity was observed in healthy looking neurons and neurites, dystrophic neurites, in association with neuritic plaques, as well as neurofibrillary tangles. UBB(+1) immunoreactivity was exclusively present in AD type of neuropathology. A similar pattern of APP(+1) and UBB(+1) immunoreactivity was also observed for FAD and much less explicit in nondemented controls after the age of 51 years. Furthermore, we observed accumulation of +1 proteins in other types of tauopathies, such as PiD, frontotemporal dementia, PSP and argyrophylic grain disease. These data suggest that accumulation of +1 proteins contributes to the early stages of dementia and plays a pathogenic role in a number of diseases that involve the accumulation of tau.

Polyglutamine diseases are characterized by neuronal intranuclear inclusions (NIIs) of expanded polyglutamine proteins, indicating the failure of protein degradation. UBB(+1), an aberrant form of ubiquitin, is a substrate and inhibitor of the proteasome, and was previously reported to accumulate in Alzheimer disease and other tauopathies. Here, we show accumulation of UBB(+1) in the NIIs and the cytoplasm of neurons in Huntington disease and spinocerebellar ataxia type-3, indicating inhibition of the proteasome by polyglutamine proteins in human brain. We found that UBB(+1) not only increased aggregate formation of expanded polyglutamines in neuronally differentiated cell lines, but also had a synergistic effect on apoptotic cell death due to expanded polyglutamine proteins. These findings implicate UBB(+1) as an aggravating factor in polyglutamine-induced neurodegeneration, and clearly identify an important role for the ubiquitin-proteasome system in polyglutamine diseases.

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.

Molecular misreading of the ubiquitin-B (UBB) gene results in a dinucleotide deletion in UBB mRNA. The resulting mutant protein, UBB+1, accumulates in the neuropathological hallmarks of Alzheimer disease. In vitro, UBB+1 inhibits proteasomal proteolysis, although it is also an ubiquitin fusion degradation substrate for the proteasome. Using the ligase chain reaction to detect dinucleotide deletions, we report here that UBB+1 transcripts are present in each neurodegenerative disease studied (tauo- and synucleinopathies) and even in control brain samples. In contrast to UBB+1 transcripts, UBB+1 protein accumulation in the ubiquitin-containing neuropathological hallmarks is restricted to the tauopathies such as Pick disease, frontotemporal dementia, progressive supranuclear palsy, and argyrophilic grain disease. Remarkably, UBB+1 protein is not detected in the major forms of synucleinopathies (Lewy body disease and multiple system atrophy). The neurologically intact brain can cope with UBB+1 as lentivirally delivered UBB+1 protein is rapidly degraded in rat hippocampus, whereas the K29,48R mutant of UBB+1, which is not ubiquitinated, is abundantly expressed. The finding that UBB+1 protein only accumulates in tauopathies thus implies that the ubiquitin-proteasome system is impaired specifically in this group of neurodegenerative diseases and not in synucleinopathies and that the presence of UBB+1 protein reports proteasomal dysfunction in the brain.

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein (APP+1) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP+1 proficiently secreted APP+1. Because various secretory APP isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether APP+1 is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained at autopsy from 50 non-demented controls and 122 Alzheimer patients; all subjects were staged for neuropathology (Braak score). Unexpectedly, we found that the APP+1 level in the CSF of non-demented controls was much higher (1.75 ng/ml) than in the CSF of Alzheimer patients (0.51 ng/ml) (p < 0.001), and the level of APP+1 in CSF was inversely correlated with the severity of the neuropathology. Moreover the earliest neuropathological changes are already reflected in a significant decrease of the APP+1 level in CSF. These data show that APP+1 is normally secreted by neurons, preventing intra-neuronal accumulation of APP+1 in brains of non-demented controls without neurofibrillary pathology.

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood–brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients. [ABSTRACT FROM AUTHOR]

Despite decades of rigorous research and numerous clinical trials, Alzheimer's disease (AD) stands as a notable healthcare challenge of this century, with effective therapeutic solutions remaining elusive. Recently, the endocannabinoid system (ECS) has emerged as an essential therapeutic target due to its regulatory role in different physiological processes, such as neuroprotection, modulation of inflammation, and synaptic plasticity. This aligns with previous research showing that cannabinoid receptor ligands have the potential to trigger the functional structure of neuronal and brain networks, potentially impacting memory processing. Therefore, our study aims to assess the effects of prolonged, intermittent exposure (over 90 days) to JWH-133 (0.2 mg/kg) and an EU-GMP certified Cannabis sativa L. (Cannabixir® Medium Flos, 2.5 mg/kg) on recognition memory, as well as their influence on brain metabolism and modulation of the expanded endocannabinoid system in APP/PS1 mice. Chronic therapy with cannabinoid receptor ligands resulted in reduced anxiety-like behavior and partially reversed the cognitive deficits. Additionally, a reduction was observed in both the number and size of Aβ plaque deposits, along with decreased cerebral glucose metabolism, as well as a decline in the expression of mTOR and CB2 receptors. Furthermore, the study revealed enlarged astrocytes and enhanced expression of M1 mAChR in mice subjected to cannabinoid treatment. Our findings highlight the pivotal involvement of the extended endocannabinoid system in cognitive decline and pathological aspects associated with AD, presenting essential preclinical evidence to support the continued exploration and assessment of cannabinoid receptor ligands for AD treatment. [ABSTRACT FROM AUTHOR]

Semaphorin 3A (SEMA3A) plays a crucial role in the development, differentiation, and plasticity of specific types of neurons that secrete Gonadotropin-Releasing Hormone (GnRH) and regulates the acquisition and maintenance of reproductive competence in humans and mice. Its insufficient expression has been linked to reproductive disorders in humans, which are characterized by reduced or failed sexual competence. Various mutations, polymorphisms, and alternatively spliced variants of SEMA3A have been associated with infertility. One of the common causes of infertility in women of reproductive age is diminished ovarian reserve (DOR), characterized by a reduced ovarian follicular pool. Despite its clinical significance, there are no universally accepted diagnostic criteria or therapeutic interventions for DOR. In this study, we analyzed the SEMA3A plasma levels in 77 women and investigated their potential role in influencing fertility in patients with DOR. The results revealed that the SEMA3A levels were significantly higher in patients with DOR than in healthy volunteers. Furthermore, the SEMA3A levels were increased in patients who underwent fertility treatment and had positive Beta-Human Chorionic Gonadotropin (βHCG) values (β+) after controlled ovarian stimulation (COS) compared to those who had negative βHCG values (β−). These findings may serve as the basis for future investigations into the diagnosis of infertility and emphasize new possibilities for the SEMA3A-related treatment of sexual hormonal dysfunction that leads to infertility. [ABSTRACT FROM AUTHOR]

Ceramide C16 is a sphingolipid detected at high levels in several neurodegenerative disorders, including multiple sclerosis (MS). It can be generated de novo or from the hydrolysis of other sphingolipids, such as sphingomyelin or through the recycling of sphingosine, in what is known as the salvage pathway. While the myelin damage occurring in MS suggests the importance of the hydrolytic and salvage pathways, the growing interest on the importance of diet in demyelinating disorders, prompted us to investigate the involvement of de novo ceramide C16 synthesis on disease severity. A diet rich in saturated fats such as palmitic acid, as found in many highly processed foods, provides substrates for the ceramide C16 synthetic enzymes ceramide synthase 6 (CERS6) and 5 (CERS5), which are expressed in the central nervous system. Using the experimental autoimmune encephalomyelitis (EAE) model of inflammatory demyelination, we show here that mice with CamK2a+ neuronal specific deletion of both CerS6 and CerS5 show a milder course of EAE than wild type mice, even when fed a diet enriched in palmitic acid. At a cellular level, neurons lacking both CerS6 and CerS5 are protected from the mitochondrial dysfunction arising from exposure to oxidative stress and palmitic acid in the medium. These data underscore the importance of a healthy diet avoiding processed foods for demyelinating disorders and identifies endogenous neuronal synthesis of ceramide C16 as an important determinant of disease severity., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives. [ABSTRACT FROM AUTHOR]

Sporadic forms of Alzheimer’s and Parkinson’s diseases are the most frequent forms of their kind. Together with Huntington’s disease, they belong to the so called ‘conformational diseases’ as they share a common feature in the accumulation of insoluble protein deposits. In this review, we focus on the significance of the ubiquitin-proteasome system in conformational diseases and the possible consequences due to the accumulation of aberrant proteins. In all forms of Alzheimer’s and Huntington’s diseases, but not in Parkinson’s disease, we have shown the presence of misframed proteins such as misframed ubiquitin (UBB+1) of which we have determined the functional relevance in vitro and in vivo.Misframed proteins are the result of the inaccurate transcription of monotonic sequences in the genome and their subsequent translation. This process has been called ‘molecular misreading’. In the present review, we will discuss the present state of the art with regard to UBB+1 and amyloid precursor protein APP+1. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Epidemiological studies have shown that traumatic brain injury (TBI) increases the risk for developing neurodegenerative diseases (NDs). However, molecular mechanisms that underlie this risk are largely unidentified. TBI triggers widespread epigenetic modifications. Similarly, NDs such as Alzheimer’s or Parkinson’s are associated with numerous epigenetic changes. Although epigenetic changes can persist after TBI, it is unresolved if these modifications increase the risk of later ND development and/or dementia. We briefly review TBI-related epigenetic changes, and point out putative feedback loops that might contribute to longterm persistence of some modifications. We then focus on evidence suggesting persistent TBI-associated epigenetic changes may contribute to pathological processes (e.g., neuroinflammation) which may facilitate the development of specific NDs – Alzheimer’s disease, Parkinson’s disease, or chronic traumatic encephalopathy. Finally, we discuss possible directions for TBI therapies that may help prevent or delay development of NDs. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) vectors are gene therapy delivery tools that offer a promising platform for the treatment of neurodegenerative diseases. Keeping up with developments in this fast-moving area of research is a challenge. This review was thus written with the intention to introduce this field of study to those who are new to it and direct others who are struggling to stay abreast of the literature towards notable recent studies. In ten sections, we briefly highlight early milestones within this field and its first clinical success stories. We showcase current clinical trials, which focus on gene replacement, gene augmentation, or gene suppression strategies. Next, we discuss ongoing efforts to improve the tropism of rAAV vectors for brain applications and introduce pre-clinical research directed toward harnessing rAAV vectors for gene editing applications. Subsequently, we present common genetic elements coded by the single-stranded DNA of rAAV vectors, their so-called payloads. Our focus is on recent advances that are bound to increase treatment efficacies. As needed, we included studies outside the neurodegenerative disease field that showcased improved pre-clinical designs of all-in-one rAAV vectors for gene editing applications. Finally, we discuss risks associated with off-target effects and inadvertent immunogenicity that these technologies harbor as well as the mitigation strategies available to date to make their application safer. [ABSTRACT FROM AUTHOR]

Rhythmic inspiratory activity is generated in the preBötzinger complex (preBötC), a neuronal network located bilaterally in the ventrolateral medulla. Cholinergic neurotransmission affects respiratory rhythmogenic neurons and inhibitory glycinergic neurons in the preBötC. Acetylcholine has been extensively investigated given that cholinergic fibers and receptors are present and functional in the preBötC, are important in sleep/wake cycling, and modulate inspiratory frequency through its action on preBötC neurons. Despite its role in modulating inspiratory rhythm, the source of acetylcholine input to the preBötC is not known. In the present study, we used retrograde and anterograde viral tracing approaches in transgenic mice expressing Cre‐recombinase driven by the choline acetyltransferase promoter to identify the source of cholinergic inputs to the preBötC. Surprisingly, we observed very few, if any, cholinergic projections originating from the laterodorsal and pedunculopontine tegmental nuclei (LDT/PPT), two main cholinergic, state‐dependent systems long hypothesized as the main source of cholinergic inputs to the preBötC. On the contrary, we identified glutamatergic and GABAergic/glycinergic neurons in the PPT/LDT that send projections to the preBötC. Although these neurons contribute minimally to the direct cholinergic modulation of preBötC neurons, they could be involved in state‐dependent regulation of breathing. Our data also suggest that the source of cholinergic inputs to the preBötC appears to originate from cholinergic neurons in neighboring regions of the medulla, the intermediate reticular formation, the lateral paragigantocellularis, and the nucleus of the solitary tract. [ABSTRACT FROM AUTHOR]

A biomarker for declined methylation capacity is elevation of homocysteine levels. They increase the risk for onset of vascular disease and contribute to progression of chronic neurodegeneration and aging. This narrative review discusses associations between homocysteine, consumption of methyl group-donating vitamins and impact on disease-generating mechanisms in levodopa-treated patients with Parkinson's disease. We conclude to recommend levodopa-treated patients to substitute themselves with methyl group-donating vitamins. This is harmless in terms of application of folic acid, methylcobalamin or hydroxocobalamin. Moreover, we suggest a crucial discussion on the value of the various popular hypotheses on Parkinson's disease-generating mechanisms. Findings from studies with acute levodopa exposure describe oxidative stress generation and impaired methylation capacity, which causes gene dysfunction. Their repeated occurrences contribute to onset of mitochondrial dysfunction, iron enrichment and pathologic protein accumulation in the long term. Current research underestimates these epigenetic, metabolic consequences of chronic levodopa application. Supplementary treatment strategies are recommended to avoid levodopa-related side effects., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Living systems use proximity to regulate biochemical processes. Inspired by this phenomenon, bifunctional modalities that induce proximity have been developed to redirect cellular processes. An emerging example of this class is molecules that induce ubiquitin-dependent proteasomal degradation of a protein of interest, and their initial development sparked a flurry of discovery for other bifunctional modalities. Recent advances in this area include modalities that can change protein phosphorylation, glycosylation, and acetylation states, modulate gene expression, and recruit components of the immune system. In this review, we highlight bifunctional modalities that perform functions other than degradation and have great potential to revolutionize disease treatment, while also serving as important tools in basic research to explore new aspects of biology. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine kinase (NAGK) has been identified as an anchor protein that facilitates neurodevelopment with its non-canonical structural role. Similarly, small nuclear ribonucleoprotein polypeptide N (SNRPN) regulates neurodevelopment and cognitive ability. In our previous study, we revealed the interaction between NAGK and SNRPN in the neuron. However, the precise role in neurodevelopment is elusive. In this study, we investigate the role of NAGK and SNRPN in the axodendritic development of neurons. NAGK and SNRPN interaction is significantly increased in neurons at the crucial stages of neurodevelopment. Furthermore, overexpression of the NAGK and SNRPN proteins increases axodendritic branching and neuronal complexity, whereas the knockdown inhibits neurodevelopment. We also observe the interaction of NAGK and SNRPN with the dynein light-chain roadblock type 1 (DYNLRB1) protein variably during neurodevelopment, revealing the microtubule-associated delivery of the complex. Interestingly, NAGK and SNRPN proteins rescued impaired axodendritic development in an SNRPN depletion model of Prader–Willi syndrome (PWS) patient-derived induced pluripotent stem cell neurons. Taken together, these findings are crucial in developing therapeutic approaches for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Niemann–Pick disease type C1 (NP-C) is a prematurely lethal genetic lysosomal storage disorder with neurological and visceral pathology resulting from mutations in the NPC1 gene encoding the lysosomal transmembrane protein NPC1. There is currently no cure for NP-C, and the only disease modifying treatment, miglustat, slows disease progression but does not significantly attenuate neurological symptoms. AAV-mediated gene therapy is an attractive option for NP-C, but due to the large size of the human NPC1 gene, there may be packaging and truncation issues during vector manufacturing. One option is to reduce the size of DNA regulatory elements that are essential for gene expression, such as the promoter sequence. Here, we describe a novel small truncated endogenous NPC1 promoter that leads to high gene expression both in vitro and in vivo and compare its efficacy to other commonly used promoters. Following neonatal intracerebroventricular (ICV) injection into the CNS, this novel promoter provided optimal therapeutic efficacy compared to all other promoters including increased survival, improved behavioural phenotypes, and attenuated neuropathology in mouse models of NP-C. Taken together, we propose that this novel promoter can be extremely efficient in designing an optimised AAV9 vector for gene therapy for NP-C. [ABSTRACT FROM AUTHOR]

Krabbe disease is a rare neurodegenerative fatal disease. It is caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC), which results in progressive accumulation of galactolipid substrates in myelin-forming cells. However, there is still a lack of appropriate neural models and effective approaches for Krabbe disease. We generated induced pluripotent stem cells (iPSCs) from a Krabbe patient previously. Here, Krabbe patient-derived neural stem cells (K-NSCs) were induced from these iPSCs. By using nine kinds of recombinant adeno-associated virus (rAAV) vectors to infect K-NSCs, we found that the rAAV2 vector has high transduction efficiency for K-NSCs. Most importantly, rAAV2-GALC rescued GALC enzymatic activity in K-NSCs. Our findings not only establish a novel patient NSC model for Krabbe disease, but also firstly indicate the potential of rAAV2-mediated gene therapy for this devastating disease. [ABSTRACT FROM AUTHOR]

The brain relies on many forms of dynamic activities in individual neurons, from synaptic transmission to electrical activity and intracellular signaling events. Monitoring these neuronal activities with high spatiotemporal resolution in the context of animal behavior is a necessary step to achieve a mechanistic understanding of brain function. With the rapid development and dissemination of highly optimized genetically encoded fluorescent sensors, a growing number of brain activities can now be visualized in vivo. To date, cellular calcium imaging, which has been largely used as a proxy for electrical activity, has become a mainstay in systems neuroscience. While challenges remain, voltage imaging of neural populations is now possible. In addition, it is becoming increasingly practical to image over half a dozen neurotransmitters, as well as certain intracellular signaling and metabolic activities. These new capabilities enable neuroscientists to test previously unattainable hypotheses and questions. This review summarizes recent progress in the development and delivery of genetically encoded fluorescent sensors, and highlights example applications in the context of in vivo imaging. [ABSTRACT FROM AUTHOR]

Tools developed in the fields of genome engineering, precise gene regulation, and synthetic gene networks have an increasing number of applications. When shared with the scientific community, these tools can be used to further unlock the potential of precision medicine and tissue engineering. A large number of different genetic elements, as well as modifications, have been used to create many different systems and to validate some technical concepts. New studies have tended to optimize or improve existing elements or approaches to create complex synthetic systems, especially those based on the relatively new CRISPR technology. In order to maximize the output of newly developed approaches and to move from proof-of-principle experiments to applications in regenerative medicine, it is important to navigate efficiently through the vast number of genetic elements to choose those most suitable for specific needs. In this review, we have collected information regarding the main genetic elements and their modifications, which can be useful in different synthetic systems with an emphasis of those based on CRISPR technology. We have indicated the most suitable elements and approaches to choose or combine in planning experiments, while providing their deeper understanding, and have also stated some pitfalls that should be avoided. [ABSTRACT FROM AUTHOR]

Astrocytes are important for maintaining cholesterol metabolism, glutamate uptake, and neurotransmission. Indeed, inflammatory processes and neurodegeneration contribute to the altered morphology, gene expression, and function of astrocytes. Astrocytes, in collaboration with numerous microRNAs, regulate brain cholesterol levels as well as glutamatergic and inflammatory signaling, all of which contribute to general brain homeostasis. Neural electrical activity, synaptic plasticity processes, learning, and memory are dependent on the astrocyte–neuron crosstalk. Here, we review the involvement of astrocytic microRNAs that potentially regulate cholesterol metabolism, glutamate uptake, and inflammation in Alzheimer's disease (AD). The interaction between astrocytic microRNAs and long non-coding RNA and transcription factors specific to astrocytes also contributes to the pathogenesis of AD. Thus, astrocytic microRNAs arise as a promising target, as AD conditions are a worldwide public health problem. This review examines novel therapeutic strategies to target astrocyte dysfunction in AD, such as lipid nanodiscs, engineered G protein-coupled receptors, extracellular vesicles, and nanoparticles. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. The miRNA miR-106b-5p has been linked to epilepsy, but its specific role and mechanism of action remain unclear. This was investigated in the present study using a mouse model of pilocarpine-induced status epilepticus and an in vitro system of HT22 hippocampal cells treated with Mg2+-free solution and cocultured with BV2 microglia cells. We found that inhibiting miR-106b-5p expression promoted microglia M2 polarization, reduced the inflammatory response, and alleviated neuronal injury. These effects involved modulation of the repulsive guidance molecule A (RGMa)–Rac1–c-Jun N-terminal kinase (JNK)/p38–mitogen-activated protein kinase (MAPK) signaling axis. Our results suggest that therapeutic strategies targeting miR-106b-5p or downstream factors can be effective in preventing epileptogenesis or treating epilepsy. [ABSTRACT FROM AUTHOR]

Protein aggregation is one of the major pathological events in age-related Parkinson's disease (PD) pathology, predominantly regulated by the ubiquitin–proteasome system (UPS). UPS essentially requires core component ubiquitin; however, its role in PD pathology is obscure. This study aimed to investigate the role of ubiquitin-encoding genes in sporadic PD pathology. Both cellular and rat models of PD as well as SNCA C57BL/6J-Tg (Th-SNCA*A30P*A53T)39 Eric/J transgenic mice showed a decreased abundance of UBA52 in conjunction with significant downregulation of tyrosine hydroxylase (TH) and neuronal death. In silico predictions, mass spectrometric analysis, and co-immunoprecipitation findings suggested the protein–protein interaction of UBA52 with α-synuclein, HSP90 and E3-ubiquitin ligase CHIP, and its co-localization with α-synuclein in the mitochondrion. Next, in vitro ubiquitylation assay indicated an imperative requirement of the lysine-63 residue of UBA52 in CHIP-mediated HSP90 ubiquitylation. Myc-UBA52 expressed neurons inhibited alteration in PD-specific markers such as α-synuclein and TH protein along with increased proteasome activity in diseased conditions. Furthermore, Myc-UBA52 expression inhibited the altered protein abundance of HSP90 and its various client proteins, HSP75 (homolog of HSP90 in mitochondrion) and ER stress-related markers during early PD. Taken together, the data highlights the critical role of UBA52 in HSP90 ubiquitylation in parallel to its potential contribution to the modulation of various disease-related neurodegenerative signaling targets during the early phase of PD pathology. [ABSTRACT FROM AUTHOR]