Impaired macroautophagy confers substantial risk for intellectual disability in children with autism spectrum disorders.

Autism spectrum disorder (ASD) represents a complex of neurological and developmental disabilities characterized by clinical and genetic heterogeneity. While the causes of ASD are still unknown, many ASD risk factors are found to converge on intracellular quality control mechanisms that are essential for cellular homeostasis, including the autophagy-lysosomal degradation pathway. Studies have reported impaired autophagy in ASD human brain and ASD-like synapse pathology and behaviors in mouse models of brain autophagy deficiency, highlighting an essential role for defective autophagy in ASD pathogenesis. To determine whether altered autophagy in the brain may also occur in peripheral cells that might provide useful biomarkers, we assessed activities of autophagy in lympoblasts from ASD and control subjects. We find that lymphoblast autophagy is compromised in a subset of ASD participants due to impaired autophagy induction. Similar changes in autophagy are detected in postmortem human brains from ASD individuals and in brain and peripheral blood mononuclear cells from syndromic ASD mouse models. Remarkably, we find a strong correlation between impaired autophagy and intellectual disability in ASD participants. By depleting the key autophagy gene Atg7 from different brain cells, we provide further evidence that autophagy deficiency causes cognitive impairment in mice. Together, our findings suggest autophagy dysfunction as a convergent mechanism that can be detected in peripheral blood cells from a subset of autistic individuals, and that lymphoblast autophagy may serve as a biomarker to stratify ASD patients for the development of targeted interventions.

Cytoplasmic vacuolization and ectopic formation of perineuronal nets are characteristic pathologies of cytomegalic neurons in tuberous sclerosis.

Cytomegalic neurons, characterized by increased size and a hyperactive mechanistic target of rapamycin complex 1 (mTORC1), are pathognomonic for tuberous sclerosis complex (TSC). To model these neurons, we recently generated a murine Tsc1 conditional knockout model in which Tsc1 deletion in late embryonic radial glia results in neuronal hypertrophy of a subset of isocortical pyramidal neurons. In the current study, we compared the cellular pathology of these cytomegalic neurons to those of the enlarged neurons in human cortical tubers. Neurons from the mice showed unique features, such as cytoplasmic vacuoles associated with Golgi complexes and the ectopic formation of perineuronal nets (PNNs), a feature of inhibitory neurons, rarely present in excitatory cortical neurons. The membranes of these vacuoles were enriched for the plasma membrane proteins CD44, KCC2, and Na+/K+ ATPase, suggesting deficits in Golgi membrane trafficking. These aberrant features in the mouse appeared only after the onset of seizures, probably due to the prolonged seizure activity in the context of constitutive mTORC1 activation. Similar PNNs and cytoplasmic vacuoles were present in the cytomegalic neurons of human cortical tubers. Our findings reveal novel pathological features of Golgi complexes and PNNs in the cytomegalic neurons in TSC.

Huntington disease oligodendrocyte maturation deficits revealed by single-nucleus RNAseq are rescued by thiamine-biotin supplementation.

The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.

Synaptic hyperexcitability of cytomegalic pyramidal neurons contributes to epileptogenesis in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a developmental disorder associated with epilepsy, autism, and cognitive impairment. Despite inactivating mutations in the TSC1 or TSC2 genes and hyperactive mechanistic target of rapamycin (mTOR) signaling, the mechanisms underlying TSC-associated neurological symptoms remain incompletely understood. Here we generate a Tsc1 conditional knockout (CKO) mouse model in which Tsc1 inactivation in late embryonic radial glia causes social and cognitive impairment and spontaneous seizures. Tsc1 depletion occurs in a subset of layer 2/3 cortical pyramidal neurons, leading to development of cytomegalic pyramidal neurons (CPNs) that mimic dysplastic neurons in human TSC, featuring abnormal dendritic and axonal overgrowth, enhanced glutamatergic synaptic transmission, and increased susceptibility to seizure-like activities. We provide evidence that enhanced synaptic excitation in CPNs contributes to cortical hyperexcitability and epileptogenesis. In contrast, astrocytic regulation of synapse formation and synaptic transmission remains unchanged after late embryonic radial glial Tsc1 inactivation, and astrogliosis evolves secondary to seizures.

Mutant glucocerebrosidase impairs α-synuclein degradation by blockade of chaperone-mediated autophagy.

The most common genetic risk factors for Parkinson's disease (PD) are a set of heterozygous mutant (MT) alleles of the GBA1 gene that encodes ß-glucocerebrosidase (GCase), an enzyme normally trafficked through the ER/Golgi apparatus to the lysosomal lumen. We found that half of the GCase in lysosomes from postmortem human GBA-PD brains was present on the lysosomal surface and that this mislocalization depends on a pentapeptide motif in GCase used to target cytosolic protein for degradation by chaperone-mediated autophagy (CMA). MT GCase at the lysosomal surface inhibits CMA, causing accumulation of CMA substrates including α-synuclein. Single-cell transcriptional analysis and proteomics of brains from GBA-PD patients confirmed reduced CMA activity and proteome changes comparable to those in CMA-deficient mouse brain. Loss of the MT GCase CMA motif rescued primary substantia nigra dopaminergic neurons from MT GCase-induced neuronal death. We conclude that MT GBA1 alleles block CMA function and produce α-synuclein accumulation.

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knockout Mice.

FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome.

Fragility and robustness of self-sustained oscillations in an epidemiological model on small-world networks.

We investigate the susceptible-infected-recovered-susceptible epidemic model, typical of mathematical epidemiology, with the diversity of the durations of infection and recovery of the individuals on small-world networks. Infection spreads from infected to healthy nodes, whose infection and recovery periods denoted by τI and τR, respectively, are either fixed or uniformly distributed around a specified mean. Whenever τI and τR are narrowly distributed around their mean values, the epidemic prevalence in the stationary state is found to reach its maximal level in the typical small-world region. This non-monotonic behavior of the final epidemic prevalence is thought to be similar to the efficient navigation in small worlds with cost minimization. Besides, pronounced oscillatory behavior of the fraction of infected nodes emerges when the number of shortcuts on the underlying network become sufficiently large. Remarkably, we find that the synchronized oscillation of infection incidences is quite fragile to the variability of the two characteristic time scales τI and τR. Specifically, even in the limit of a random network (where the amplest oscillations are expected to arise for fixed τI and τR), increasing the variability of the duration of the infectious period and/or that of the refractory period will push the system to change from a self-sustained oscillation to a fixed point with negligible fluctuations in the steady state. Interestingly, negative correlation between τI and τR can give rise to the robustness of the self-sustained oscillatory phenomenon. Our findings thus highlight the pivotal role of, apart from the external seasonal driving force and demographic stochasticity, the intrinsic characteristic of the system itself in understanding the cycle of outbreaks of recurrent epidemics.

Roles for neuronal and glial autophagy in synaptic pruning during development.

The dendritic protrusions known as spines represent the primary postsynaptic location for excitatory synapses. Dendritic spines are critical for many synaptic functions, and their formation, modification, and turnover are thought to be important for mechanisms of learning and memory. At many excitatory synapses, dendritic spines form during the early postnatal period, and while many spines are likely being formed and removed throughout life, the net number are often gradually "pruned" during adolescence to reach a stable level in the adult. In neurodevelopmental disorders, spine pruning is disrupted, emphasizing the importance of understanding its governing processes. Autophagy, a process through which cytosolic components and organelles are degraded, has recently been shown to control spine pruning in the mouse cortex, but the mechanisms through which autophagy acts remain obscure. Here, we draw on three widely studied prototypical synaptic pruning events to focus on two governing principles of spine pruning: 1) activity-dependent synaptic competition and 2) non-neuronal contributions. We briefly review what is known about autophagy in the central nervous system and its regulation by metabolic kinases. We propose a model in which autophagy in both neurons and non-neuronal cells contributes to spine pruning, and how other processes that regulate spine pruning could intersect with autophagy. We further outline future research directions to address outstanding questions on the role of autophagy in synaptic pruning.

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations.

Heterozygous mutations in GBA, the gene encoding the lysosomal enzyme glucosylceramidase beta/ß-glucocerebrosidase, comprise the most common genetic risk factor for Parkinson disease (PD), but the mechanisms underlying this association remain unclear. Here, we show that in GbaL444P/WT knockin mice, the L444P heterozygous Gba mutation triggers mitochondrial dysfunction by inhibiting autophagy and mitochondrial priming, two steps critical for the selective removal of dysfunctional mitochondria by autophagy, a process known as mitophagy. In SHSY-5Y neuroblastoma cells, the overexpression of L444P GBA impeded mitochondrial priming and autophagy induction when endogenous lysosomal GBA activity remained intact. By contrast, genetic depletion of GBA inhibited lysosomal clearance of autophagic cargo. The link between heterozygous GBA mutations and impaired mitophagy was corroborated in postmortem brain tissue from PD patients carrying heterozygous GBA mutations, where we found increased mitochondrial content, mitochondria oxidative stress and impaired autophagy. Our findings thus suggest a mechanistic basis for mitochondrial dysfunction associated with GBA heterozygous mutations. Abbreviations: AMBRA1: autophagy/beclin 1 regulator 1; BECN1: beclin 1, autophagy related; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chloroyphenylhydrazone; CYCS: cytochrome c, somatic; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; GBA: glucosylceramidase beta; GBA-PD: Parkinson disease with heterozygous GBA mutations; GD: Gaucher disease; GFP: green fluorescent protein; LC3B: microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated form of microtubule-associated protein 1 light chain 3 beta; MitoGreen: MitoTracker Green; MitoRed: MitoTracker Red; MMP: mitochondrial membrane potential; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH transcription factor; NBR1: NBR1, autophagy cargo receptor; Non-GBA-PD: Parkinson disease without GBA mutations; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; ROS: reactive oxygen species; SNCA: synuclein alpha; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1/Porin: voltage dependent anion channel 1; WT: wild type.

Ultraflexible Transparent Film Heater Made of Ag Nanowire/PVA Composite for Rapid-Response Thermotherapy Pads.

Ultraflexible transparent film heaters have been fabricated by embedding conductive silver (Ag) nanowires into a thin poly(vinyl alcohol) film (AgNW/PVA). A cold-pressing method was used to rationally adjust the sheet resistance of the composite films and thus the heating powers of the AgNW/PVA film heaters at certain biases. The film heaters have a favorable optical transmittance (93.1% at 26 Ω/sq) and an outstanding mechanical flexibility (no visible change in sheet resistance after 10 000 bending cycles and at a radius of curvature ≤1 mm). The film heaters have an environmental endurance, and there is no significant performance degradation after being kept at high temperature (80 °C) and high humidity (45 °C, 80% humidity) for half a year. The efficient Joule heating can increase the temperature of the film heaters (20 Ω/sq) to 74 °C in ∼20 s at a bias of 5 V. The fast-heating characteristics at low voltages (a few volts) associated with its transparent and flexibility properties make the poly(dimethylsiloxane)/AgNW/PVA composite film a potential candidate in medical thermotherapy pads.

Ligation-free ribosome profiling of cell type-specific translation in the brain.

Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.

Decreased Coenzyme Q10 Levels in Multiple System Atrophy Cerebellum.

In familial and sporadic multiple system atrophy (MSA) patients, deficiency of coenzyme Q10 (CoQ10) has been associated with mutations in COQ2, which encodes the second enzyme in the CoQ10 biosynthetic pathway. Cerebellar ataxia is the most common presentation of CoQ10 deficiency, suggesting that the cerebellum might be selectively vulnerable to low levels of CoQ10 To investigate whether CoQ10 deficiency represents a common feature in the brains of MSA patients independent of the presence of COQ2 mutations, we studied CoQ10 levels in postmortem brains of 12 MSA, 9 Parkinson disease (PD), 9 essential tremor (ET) patients, and 12 controls. We also assessed mitochondrial respiratory chain enzyme activities, oxidative stress, mitochondrial mass, and levels of enzymes involved in CoQ biosynthesis. Our studies revealed CoQ10 deficiency in MSA cerebellum, which was associated with impaired CoQ biosynthesis and increased oxidative stress in the absence of COQ2 mutations. The levels of CoQ10 in the cerebella of ET and PD patients were comparable or higher than in controls. These findings suggest that CoQ10 deficiency may contribute to the pathogenesis of MSA. Because no disease modifying therapies are currently available, increasing CoQ10 levels by supplementation or upregulation of its biosynthesis may represent a novel treatment strategy for MSA patients.

Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits.

Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

Lingo-1 expression is increased in essential tremor cerebellum and is present in the basket cell pinceau.

The Lingo-1 sequence variant has been associated with essential tremor (ET) in several genome-wide association studies. However, the role that Lingo-1 might play in pathogenesis of ET is not understood. Since Lingo-1 protein is a negative regulator of axonal regeneration and neurite outgrowth, it could contribute to Purkinje cell (PC) or basket cell axonal pathology observed in postmortem studies of ET brains. In this study, we used Western blotting and immunohistochemistry to examine Lingo-1 protein in ET vs. control brains. In Western blots, Lingo-1 protein expression level was significantly increased in cerebellar cortex (1.56 ± 0.46 in ET cases vs. 0.99 ± 0.20 in controls, p = 0.002), but was similar in the occipital cortex (p = 1.00) of ET cases vs. controls. Lingo-1 immunohistochemistry in cerebellum revealed that Lingo-1 was enriched in the distal axonal processes of basket cells, which formed a "pinceau" structure around the PC axon initial segment (AIS). We found that some Lingo-1-positive pinceau had abnormally elongated processes, targeting PC axon segments distal to the AIS. In ET cases, the percentage of Lingo-1-positive pinceau that were ≥30 or ≥40 µm in length was increased 2.4- to 4.1-fold, respectively, vs. pinceau seen in control brains (p < 0.0001). Elongated Lingo-1-positive pinceau strongly correlated with number of PC axonal torpedoes and a rating of basket cell axonal pathology. The increased cerebellar Lingo-1 expression and elongated Lingo-1-positive pinceau processes could contribute to the abnormal PC and basket cell axonal pathology and cerebellar dysfunction observed in ET.

Mitochondrial abnormalities in temporal lobe of autistic brain.

Autism spectrum disorder (ASD) consists of a group of complex developmental disabilities characterized by impaired social interactions, deficits in communication and repetitive behavior. Multiple lines of evidence implicate mitochondrial dysfunction in ASD. In postmortem BA21 temporal cortex, a region that exhibits synaptic pathology in ASD, we found that compared to controls, ASD patients exhibited altered protein levels of mitochondria respiratory chain protein complexes, decreased Complex I and IV activities, decreased mitochondrial antioxidant enzyme SOD2, and greater oxidative DNA damage. Mitochondrial membrane mass was higher in ASD brain, as indicated by higher protein levels of mitochondrial membrane proteins Tom20, Tim23 and porin. No differences were observed in either mitochondrial DNA or levels of the mitochondrial gene transcription factor TFAM or cofactor PGC1α, indicating that a mechanism other than alterations in mitochondrial genome or mitochondrial biogenesis underlies these mitochondrial abnormalities. We further identified higher levels of the mitochondrial fission proteins (Fis1 and Drp1) and decreased levels of the fusion proteins (Mfn1, Mfn2 and Opa1) in ASD patients, indicating altered mitochondrial dynamics in ASD brain. Many of these changes were evident in cortical pyramidal neurons, and were observed in ASD children but were less pronounced or absent in adult patients. Together, these findings provide evidence that mitochondrial function and intracellular redox status are compromised in pyramidal neurons in ASD brain and that mitochondrial dysfunction occurs during early childhood when ASD symptoms appear.

Harmaline-induced tremor in mice: videotape documentation and open questions about the model.

BACKGROUND: Harmaline-induced tremor in rodents has been extensively used as an animal model for essential tremor (ET). However, there is no visual documentation in the published literature. METHODS: We injected mice subcutaneously with either 20 mg/kg of harmaline hydrochloride or saline and then videotaped the responses. RESULTS: Action and postural tremor in the mouse began 5 minutes after subcutaneous harmaline injection and peaked at approximately 30 minutes. The tremor involved the head, trunk, tail, and four limbs and lasted for approximately 2 hours. The forelimb tremor was postural or action tremor, similar to that observed in ET. DISCUSSION: This video segment provides the first visual documentation of the phenomenology of harmaline-induced tremor in a mouse. We also raise several unanswered questions regarding the use of harmaline-induced tremor to model ET.

Regulation of presynaptic neurotransmission by macroautophagy.

mTOR is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and autophagic degradation of cellular components. When triggered by mTOR inactivation, macroautophagy degrades long-lived proteins and organelles via sequestration into autophagic vacuoles. mTOR further regulates synaptic plasticity, and neurodegeneration occurs when macroautophagy is deficient. It is nevertheless unknown whether macroautophagy modulates presynaptic function. We find that the mTOR inhibitor rapamycin induces formation of autophagic vacuoles in prejunctional dopaminergic axons with associated decreased axonal profile volumes, synaptic vesicle numbers, and evoked dopamine release. Evoked dopamine secretion was enhanced and recovery was accelerated in transgenic mice in which macroautophagy deficiency was restricted to dopaminergic neurons; rapamycin failed to decrease evoked dopamine release in the striatum of these mice. Macroautophagy that follows mTOR inhibition in presynaptic terminals, therefore, rapidly alters presynaptic structure and neurotransmission.

Macroautophagy abnormality in essential tremor.

Macroautophagy is a cellular mechanism for the clearance of protein aggregates and damaged organelles. Impaired macroautophagy has been observed in neurodegenerative disorders. We investigated the macroautophagy pathway in essential tremor (ET) cases compared to age-matched controls. We analyzed microtubule-associated protein light chain 3-II (LC3-II), S6K, phosphorylated S6K, beclin-1, and mitochondrial membrane proteins levels by Western blot in the post-mortem cerebellum of 10 ET cases and 11 controls. We also performed immunohistochemistry in 12 ET cases and 13 controls to quantify LC3 clustering in Purkinje cells (PCs). LC3-II protein levels were significantly lower in ET cases vs. controls on Western blot (0.84 ± 0.14 vs. 1.00 ± 0.14, p = 0.02), and LC3-II clustering in PCs by immunohistochemistry was significantly lower in ET cases vs. controls (2.03 ± 3.45 vs. 8.80 ± 9.81, p = 0.03). In ET cases, disease duration was inversely correlated with LC3-II protein level (r = -0.64, p = 0.046). We found that mitochondrial membrane proteins were accumulated in ET (TIM23: 1.36 ± 0.11 in ET cases vs. 1.00 ± 0.08 in controls, p = 0.02; TOMM20: 1.63 ± 0.87 in ET cases vs. 1.00 ± 0.14 in controls, p = 0.03). Beclin-1, which is involved in macroautophagy, was strikingly deficient in ET (0.42 ± 0.13 vs. 1.00 ± 0.35, p<0.001). Decreased macroautophagy was observed in the ET cerebellum, and this could be due to a decrease in beclin-1 levels, which subsequently lead to mitochondrial accumulation as a result of autophagic failure. This provides a possible means by which perturbed macroautophagy could contribute to PC pathology in ET.

Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease.

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington's disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Oligomers of mutant glial fibrillary acidic protein (GFAP) Inhibit the proteasome system in alexander disease astrocytes, and the small heat shock protein alphaB-crystallin reverses the inhibition.

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, alphaB-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.