How neurons maintain their axons long-term: an integrated view of axon biology and pathology.

Axons are processes of neurons, up to a metre long, that form the essential biological cables wiring nervous systems. They must survive, often far away from their cell bodies and up to a century in humans. This requires self-sufficient cell biology including structural proteins, organelles, and membrane trafficking, metabolic, signalling, translational, chaperone, and degradation machinery-all maintaining the homeostasis of energy, lipids, proteins, and signalling networks including reactive oxygen species and calcium. Axon maintenance also involves specialised cytoskeleton including the cortical actin-spectrin corset, and bundles of microtubules that provide the highways for motor-driven transport of components and organelles for virtually all the above-mentioned processes. Here, we aim to provide a conceptual overview of key aspects of axon biology and physiology, and the homeostatic networks they form. This homeostasis can be derailed, causing axonopathies through processes of ageing, trauma, poisoning, inflammation or genetic mutations. To illustrate which malfunctions of organelles or cell biological processes can lead to axonopathies, we focus on axonopathy-linked subcellular defects caused by genetic mutations. Based on these descriptions and backed up by our comprehensive data mining of genes linked to neural disorders, we describe the 'dependency cycle of local axon homeostasis' as an integrative model to explain why very different causes can trigger very similar axonopathies, providing new ideas that can drive the quest for strategies able to battle these devastating diseases.

COPI-regulated mitochondria-ER contact site formation maintains axonal integrity.

Coat protein complex I (COPI) is best known for its role in Golgi-endoplasmic reticulum (ER) trafficking, responsible for the retrograde transport of ER-resident proteins. The ER is crucial to neuronal function, regulating Ca2+ homeostasis and the distribution and function of other organelles such as endosomes, peroxisomes, and mitochondria via functional contact sites. Here we demonstrate that disruption of COPI results in mitochondrial dysfunction in Drosophila axons and human cells. The ER network is also disrupted, and the neurons undergo rapid degeneration. We demonstrate that mitochondria-ER contact sites (MERCS) are decreased in COPI-deficient axons, leading to Ca2+ dysregulation, heightened mitophagy, and a decrease in respiratory capacity. Reintroducing MERCS is sufficient to rescue not only mitochondrial distribution and Ca2+ uptake but also ER morphology, dramatically delaying neurodegeneration. This work demonstrates an important role for COPI-mediated trafficking in MERC formation, which is an essential process for maintaining axonal integrity.

Acyl-Ghrelin Attenuates Neurochemical and Motor Deficits in the 6-OHDA Model of Parkinson's Disease.

The feeding-related hormone, acyl-ghrelin, protects dopamine neurones in murine 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-based models of experimental Parkinson's disease (PD). However, the potential protective effect of acyl-ghrelin on substantia nigra pars compacta (SNpc) dopaminergic neurones and consequent behavioural correlates in the more widely used 6-hydroxydopamine (6-OHDA) rat medial forebrain bundle (MFB) lesion model of PD are unknown. To address this question, acyl-ghrelin levels were raised directly by mini-pump infusion for 7 days prior to unilateral injection of 6-OHDA into the MFB with assessment of amphetamine-induced rotations on days 27 and 35, and immunohistochemical analysis of dopaminergic neurone survival. Whilst acyl-ghrelin treatment was insufficient to elevate food intake or body weight, it attenuated amphetamine-induced circling behaviour and SNpc dopamine neurone loss induced by 6-OHDA. These data support the notion that elevating circulating acyl-ghrelin may be a valuable approach to slow or impair progression of neurone loss in PD.

Live Imaging of Mitochondria in the Intact Fly Wing.

Detailed mechanisms governing the transport of mitochondria in neurons have recently emerged, although it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. Here, we provide a protocol to study the intracellular dynamics of mitochondria in the wing neurons of adult Drosophila in situ. The mounting and imaging procedures that we describe are suitable for use on most microscopes, and they can be easily implemented in any laboratory. Our noninvasive mounting procedures, combined with the translucency of the wing cuticle in adult animals, makes the wing nervous system accessible to advanced microscopy studies in a physiological environment. Combining the powerful genetics of Drosophila with time-lapse live imaging, users of this protocol will be able to analyze mitochondrial dynamics over time in a subset of sensory neurons in the wing. These cells extend long axons with a stereotypical plus-end-out microtubule orientation that represents a unique model to understand the logic of neuronal cargo transport, including the mitochondria. Finally, the neurons in this tissue respond to mechanical and chemical stimulation of the sensory organs of the wing, opening up the possibility of coupling the study of mitochondrial dynamics with the modulation of neuronal activity in aging Drosophila We anticipate that the unique characteristics of this in vivo system will contribute to the discovery of novel mechanisms that regulate mitochondrial dynamics within an organismal context with relevant implications for the pathogenesis of age-dependent neurological disorders.

Clonal Imaging of Mitochondria in the Dissected Fly Wing.

Mitochondria are essential for long-term neuronal function and survival. They are maintained in neurons, including long axonal stretches, through dynamic processes such as fission, fusion, biogenesis, and mitophagy. Here, we describe a protocol for the in-depth morphological analysis of individual mitochondria in axons in vivo. Most mitochondrial analysis of axons is currently performed in vitro with neurons in a developmental state. Therefore, an understanding of the axonal mitochondrial network during aging in fully differentiated neurons and the long-term consequence of gene knockout is often not developed. By using a clonal system paired with fluorescent genetically encoded markers in the Drosophila wing, we can visualize individual neurons (out of the whole bundle), including their long axons and the mitochondria that they contain, using confocal imaging. The clonal system also allows visualization of neurons with genetic perturbations that would otherwise be lethal if present in the whole organism, allowing investigators to bypass lethality. This protocol can further be adapted to measure the physiological and biochemical state of the mitochondria. Mitochondrial morphology and health in axons are tightly linked to aging, axon injury, and neurodegeneration; therefore, this method can be used to investigate mitochondrial dysfunction associated with novel genes or those linked to neurodegenerative disease and axonopathy.

Analysis of Mitochondrial Dynamics in Adult Drosophila Axons.

Neuronal survival depends on the generation of ATP from an ever-changing mitochondrial network. This requires a fine balance between the constant degradation of damaged mitochondria, biogenesis of new mitochondria, movement along microtubules, dynamic processes, and adequate functional capacity to meet firing demands. The distribution of mitochondria needs to be tightly controlled throughout the entire neuron, including its projections. Axons in particular can be enormous structures compared to the size of the cell soma, and how mitochondria are maintained in these compartments is poorly defined. Mitochondrial dysfunction in neurons is associated with aging and neurodegenerative diseases, with the axon being preferentially vulnerable to destruction. Drosophila offer a unique way to study these organelles in fully differentiated adult neurons in vivo. Here, we briefly review the regulation of neuronal mitochondria in health, aging, and disease and introduce two methodological approaches to study mitochondrial dynamics and transport in axons using the Drosophila wing system.

A nod and a Wnk to axon branching and destruction.

In this issue of Neuron, Izadifar et al. (2021) have identified a conserved molecule Wnk as a key regulator in both developmental axon branching and long-term survival of neurons, characterizing its interplay with axon destruction genes including Sarm. The discovery of Wnk will be important to our understanding of neurodevelopmental and neurodegenerative diseases.

TSG101 negatively regulates mitochondrial biogenesis in axons.

There is a tight association between mitochondrial dysfunction and neurodegenerative diseases and axons that are particularly vulnerable to degeneration, but how mitochondria are maintained in axons to support their physiology remains poorly defined. In an in vivo forward genetic screen for mutants altering axonal mitochondria, we identified tsg101 Neurons mutant for tsg101 exhibited an increase in mitochondrial number and decrease in mitochondrial size. TSG101 is best known as a component of the endosomal sorting complexes required for transport (ESCRT) complexes; however, loss of most other ESCRT components did not affect mitochondrial numbers or size, suggesting TSG101 regulates mitochondrial biology in a noncanonical, ESCRT-independent manner. The TSG101-mutant phenotype was not caused by lack of mitophagy, and we found that autophagy blockade was detrimental only to the mitochondria in the cell bodies, arguing mitophagy and autophagy are dispensable for the regulation of mitochondria number in axons. Interestingly, TSG101 mitochondrial phenotypes were instead caused by activation of PGC-1ɑ/Nrf2-dependent mitochondrial biogenesis, which was mTOR independent and TFEB dependent and required the mitochondrial fission-fusion machinery. Our work identifies a role for TSG101 in inhibiting mitochondrial biogenesis, which is essential for the maintenance of mitochondrial numbers and sizes, in the axonal compartment.

Genetic diversity of axon degenerative mechanisms in models of Parkinson's disease.

Parkinson's disease (PD) is the most common form of neurodegenerative movement disorder, associated with profound loss of dopaminergic neurons from the basal ganglia. Though loss of dopaminergic neuron cell bodies from the substantia nigra pars compacta is a well-studied feature, atrophy and loss of their axons within the nigrostriatal tract is also emerging as an early event in disease progression. Genes that drive the Wallerian degeneration, like Sterile alpha and toll/interleukin-1 receptor motif containing (Sarm1), are excellent candidates for driving this axon degeneration, given similarities in the morphology of axon degeneration after axotomy and in PD. In the present study we assessed whether Sarm1 contributes to loss of dopaminergic projections in mouse models of PD. In Sarm1 deficient mice, we observed a significant delay in the degeneration of severed dopaminergic axons distal to a 6-OHDA lesion of the medial forebrain bundle (MFB) in the nigrostriatal tract, and an accompanying rescue of morphological, biochemical and behavioural phenotypes. However, we observed no difference compared to controls when striatal terminals were lesioned with 6-OHDA to induce a dying back form of neurodegeneration. Likewise, when PD phenotypes were induced using AAV-induced alpha-synuclein overexpression, we observed similar modest loss of dopaminergic terminals in Sarm1 knockouts and controls. Our data argues that axon degeneration after MFB lesion is Sarm1-dependent, but that other models for PD do not require Sarm1, or that Sarm1 acts with other redundant genetic pathways. This work adds to a growing body of evidence indicating Sarm1 contributes to some, but not all types of neurodegeneration, and supports the notion that while axon degeneration in many context appears morphologically similar, a diversity of axon degeneration programs exist.

Dopaminergic Progenitors Derived From Epiblast Stem Cells Function Similarly to Primary VM-Derived Progenitors When Transplanted Into a Parkinson's Disease Model.

Neural transplantation in neurodegenerative diseases such as Parkinson's disease (PD) offers to replace cells lost during the progression of the disease process. Primary fetal ventral mesencephalon (VM), the origin of bona fide midbrain dopaminergic (DAergic) precursors, is currently the gold standard source of cells for transplantation in PD. However, the use of tissue from this source raises ethical and logistical constraints necessitating the need for alternative supplies of donor cells. The requirement of any alternative donor cell source is to have the capability to generate authentic mature DAergic neurons, which could be utilized in cell-replacement strategies. Mouse pluripotent stem cells can efficiently generate electrochemically mature midbrain DAergic precursors in vitro using a stepwise control of FGF signaling. Here, we have compared DAergic transplants derived from two progenitor cell sources in an allograft system: mouse epiblast stem cells (EpiSC) and primary fetal mouse VM tissue. Cells were transplanted into the striatum of 6-OHDA lesioned mice pre-treated with L-DOPA. Drug-induced rotations, a number of motor tests and drug-induced abnormal involuntary movements (AIMs) were assessed. Functional improvements were demonstrated post-transplantation in some behavioral tests, with no difference in graft volume or the number of TH immuno-positive cells in the grafts of the two transplant groups. L-DOPA-induced AIMs and amphetamine-induced AIMs were observed in both transplant groups, with no differences in rate or severity between the two groups. Collectively, in this mouse-to-mouse allograft system, we report no significant differences in the functional ability between the gold standard primary VM derived and pluripotent stem cell-derived DAergic transplants.

Autophagic and endo-lysosomal dysfunction in neurodegenerative disease.

Due to their post-mitotic state, metabolic demands and often large polarised morphology, the function and survival of neurons is dependent on an efficient cellular waste clearance system both for generation of materials for metabolic processes and removal of toxic components. It is not surprising therefore that deficits in protein clearance can tip the balance between neuronal health and death. Here we discuss how autophagy and lysosome-mediated degradation pathways are disrupted in several neurological disorders. Both genetic and cell biological evidence show the diversity and complexity of vesicular clearance dysregulation in cells, and together may ultimately suggest a unified mechanism for neuronal demise in degenerative conditions. Causative and risk-associated mutations in Alzheimer's disease, Frontotemporal Dementia, Amyotrophic Lateral Sclerosis, Parkinson's disease, Huntington's disease and others have given the field a unique mechanistic insight into protein clearance processes in neurons. Through their broad implication in neurodegenerative diseases, molecules involved in these genetic pathways, in particular those involved in autophagy, are emerging as appealing therapeutic targets for intervention in neurodegeneration.

Glutathione S-Transferase Regulates Mitochondrial Populations in Axons through Increased Glutathione Oxidation.

Mitochondria are essential in long axons to provide metabolic support and sustain neuron integrity. A healthy mitochondrial pool is maintained by biogenesis, transport, mitophagy, fission, and fusion, but how these events are regulated in axons is not well defined. Here, we show that the Drosophila glutathione S-transferase (GST) Gfzf prevents mitochondrial hyperfusion in axons. Gfzf loss altered redox balance between glutathione (GSH) and oxidized glutathione (GSSG) and initiated mitochondrial fusion through the coordinated action of Mfn and Opa1. Gfzf functioned epistatically with the thioredoxin peroxidase Jafrac1 and the thioredoxin reductase 1 TrxR-1 to regulate mitochondrial dynamics. Altering GSH:GSSG ratios in mouse primary neurons in vitro also induced hyperfusion. Mitochondrial changes caused deficits in trafficking, the metabolome, and neuronal physiology. Changes in GSH and oxidative state are associated with neurodegenerative diseases like Alzheimer's. Our demonstration that GSTs are key in vivo regulators of axonal mitochondrial length and number provides a potential mechanistic link.

Influence of chronic L-DOPA treatment on immune response following allogeneic and xenogeneic graft in a rat model of Parkinson's disease.

Although intrastriatal transplantation of fetal cells for the treatment of Parkinson's disease had shown encouraging results in initial open-label clinical trials, subsequent double-blind studies reported more debatable outcomes. These studies highlighted the need for greater preclinical analysis of the parameters that may influence the success of cell therapy. While much of this has focused on the cells and location of the transplants, few have attempted to replicate potentially critical patient centered factors. Of particular relevance is that patients will be under continued L-DOPA treatment prior to and following transplantation, and that typically the grafts will not be immunologically compatible with the host. The aim of this study was therefore to determine the effect of chronic L-DOPA administered during different phases of the transplantation process on the survival and function of grafts with differing degrees of immunological compatibility. To that end, unilaterally 6-OHDA lesioned rats received sham surgery, allogeneic or xenogeneic transplants, while being treated with L-DOPA before and/or after transplantation. Irrespective of the L-DOPA treatment, dopaminergic grafts improved function and reduced the onset of L-DOPA induced dyskinesia. Importantly, although L-DOPA administered post transplantation was found to have no detrimental effect on graft survival, it did significantly promote the immune response around xenogeneic transplants, despite the administration of immunosuppressive treatment (cyclosporine). This study is the first to systematically examine the effect of L-DOPA on graft tolerance, which is dependent on the donor-host compatibility. These findings emphasize the importance of using animal models that adequately represent the patient paradigm.

Using Drosophila models of Huntington's disease as a translatable tool.

The Huntingtin (Htt) protein is essential for a wealth of intracellular signaling cascades and when mutated, causes multifactorial dysregulation of basic cellular processes. Understanding the contribution to each of these intracellular pathways is essential for the elucidation of mechanisms that drive pathophysiology. Using appropriate models of Huntington's disease (HD) is key to finding the molecular mechanisms that contribute to neurodegeneration. While mouse models and cell lines expressing mutant Htt have been instrumental to HD research, there has been a significant contribution to our understating of the disease from studies utilizing Drosophila melanogaster. Flies have an Htt protein, so the endogenous pathways with which it interacts are likely conserved. Transgenic flies engineered to overexpress the human mutant HTT gene display protein aggregation, neurodegeneration, behavioral deficits and a reduced lifespan. The short life span of flies, low cost of maintaining stocks and genetic tools available for in vivo manipulation make them ideal for the discovery of new genes that are involved in HD pathology. It is possible to do rapid genome wide screens for enhancers or suppressors of the mutant Htt-mediated phenotype, expressed in specific tissues or neuronal subtypes. However, there likely remain many yet unknown genes that modify disease progression, which could be found through additional screening approaches using the fly. Importantly, there have been instances where genes discovered in Drosophila have been translated to HD mouse models.

Glucocerebrosidase gene therapy prevents α-synucleinopathy of midbrain dopamine neurons.

Diminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinson's disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6 months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein.

Sustained Systemic Glucocerebrosidase Inhibition Induces Brain α-Synuclein Aggregation, Microglia and Complement C1q Activation in Mice.

AIMS: Loss-of-function mutations in GBA1, which cause the autosomal recessive lysosomal storage disease, Gaucher disease (GD), are also a key genetic risk factor for the α-synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase and reductions in this enzyme result in the accumulation of the glycolipid substrates glucosylceramide and glucosylsphingosine. Deficits in autophagy and lysosomal degradation pathways likely contribute to the pathological accumulation of α-synuclein in PD. In this report we used conduritol-ß-epoxide (CBE), a potent selective irreversible competitive inhibitor of glucocerebrosidase, to model reduced glucocerebrosidase activity in vivo, and tested whether sustained glucocerebrosidase inhibition in mice could induce neuropathological abnormalities including α-synucleinopathy, and neurodegeneration. RESULTS: Our data demonstrate that daily systemic CBE treatment over 28 days caused accumulation of insoluble α-synuclein aggregates in the substantia nigra, and altered levels of proteins involved in the autophagy lysosomal system. These neuropathological changes were paralleled by widespread neuroinflammation, upregulation of complement C1q, abnormalities in synaptic, axonal transport and cytoskeletal proteins, and neurodegeneration. INNOVATION: A reduction in brain GCase activity has been linked to sporadic PD and normal aging, and may contribute to the susceptibility of vulnerable neurons to degeneration. This report demonstrates that systemic reduction of GCase activity using chemical inhibition, leads to neuropathological changes in the brain reminiscent of α-synucleinopathy. CONCLUSIONS: These data reveal a link between reduced glucocerebrosidase and the development of α-synucleinopathy and pathophysiological abnormalities in mice, and support the development of GCase therapeutics to reduce α-synucleinopathy in PD and related disorders.

Progressive decline of glucocerebrosidase in aging and Parkinson's disease.

The principal risk factor for developing most adult onset neurodegenerative diseases is aging, with incidence rising significantly after age 50. Despite research efforts, the causes of Parkinson's disease (PD) remain unknown. As neurons age, they show signs of diminished lysosomal and mitochondrial function, including increased oxidative stress and accumulation of misfolded proteins, and these changes become exacerbated PD. We show that activity of the lysosomal hydrolase glucocerebrosidase gradually diminishes with age in the substantia nigra and putamen of healthy controls. This reduction is comparable to glucocerebrosidase activity in GBA1-mutation carrier PD patients. These data, demonstrate for the first time that an age-dependent reduction in glucocerebrosidase activity may lower the threshold for developing PD.

A Nurr1 agonist causes neuroprotection in a Parkinson's disease lesion model primed with the toll-like receptor 3 dsRNA inflammatory stimulant poly(I:C).

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) are characterized by the expression of genes required for dopamine synthesis, handling and reuptake and the expression of these genes is largely controlled by nuclear receptor related 1 (Nurr1). Nurr1 is also expressed in astrocytes and microglia where it functions to mitigate the release of proinflammatory cytokines and neurotoxic factors. Given that Parkinson's disease (PD) pathogenesis has been linked to both loss of Nurr1 expression in the SNpc and inflammation, increasing levels of Nurr1 maybe a promising therapeutic strategy. In this study a novel Nurr1 agonist, SA00025, was tested for both its efficiency to induce the transcription of dopaminergic target genes in vivo and prevent dopaminergic neuron degeneration in an inflammation exacerbated 6-OHDA-lesion model of PD. SA00025 (30mg/kg p.o.) entered the brain and modulated the expression of the dopaminergic phenotype genes TH, VMAT, DAT, AADC and the GDNF receptor gene c-Ret in the SN of naive rats. Daily gavage treatment with SA00025 (30mg/kg) for 32 days also induced partial neuroprotection of dopaminergic neurons and fibers in rats administered a priming injection of polyinosinic-polycytidylic acid (poly(I:C) and subsequent injection of 6-OHDA. The neuroprotective effects of SA00025 in this dopamine neuron degeneration model were associated with changes in microglial morphology indicative of a resting state and a decrease in microglial specific IBA-1 staining intensity in the SNpc. Astrocyte specific GFAP staining intensity and IL-6 levels were also reduced. We conclude that Nurr1 agonist treatment causes neuroprotective and anti-inflammatory effects in an inflammation exacerbated 6-OHDA lesion model of PD.

Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease.

Autologous transplantation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons is a potential clinical approach for treatment of neurological disease. Preclinical demonstration of long-term efficacy, feasibility, and safety of iPSC-derived dopamine neurons in non-human primate models will be an important step in clinical development of cell therapy. Here, we analyzed cynomolgus monkey (CM) iPSC-derived midbrain dopamine neurons for up to 2 years following autologous transplantation in a Parkinson's disease (PD) model. In one animal, with the most successful protocol, we found that unilateral engraftment of CM-iPSCs could provide a gradual onset of functional motor improvement contralateral to the side of dopamine neuron transplantation, and increased motor activity, without a need for immunosuppression. Postmortem analyses demonstrated robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the transplanted putamen. Our proof of concept findings support further development of autologous iPSC-derived cell transplantation for treatment of PD.

ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress.

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10µM), or large inclusions (≥10µM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS.