We determined the effects of intravenous infusion of amino acids (AA) at serum insulin of 5, 30, 72, and 167 mU/l on anabolic signaling, expression of ubiquitin-proteasome components, and protein turnover in muscles of healthy young men. Tripling AA availability at 5 mU/l insulin doubled incorporation of [1-¹³C]leucine [i.e., muscle protein synthesis (MPS), P < 0.01] without affecting the rate of leg protein breakdown (LPB; appearance of d₅-phenylalanine). While keeping AA availability constant, increasing insulin to 30 mU/l halved LPB ( P < 0.05) without further inhibition at higher doses, whereas rates of MPS were identical to that at 5 mU/l insulin. The phosphorylation of PKB Ser⁴⁷³ and p70^S6k Thr³⁸⁹ increased concomitantly with insulin, but whereas raising insulin to 30 mU/l increased the phosphorylation of mTOR Ser²⁴⁴⁸, 4E-BP1 Thr^37/46, or GSK3β Ser⁹ and decreased that of eEF2 Thr⁵⁶, higher insulin doses to 72 and 167 mU/l did not augment these latter responses. MAFbx and proteasome C2 subunit proteins declined as insulin increased, with MuRF-1 expression largely unchanged. Thus increasing AA and insulin availability causes changes in anabolic signaling and amounts of enzymes of the ubiquitin-proteasome pathway, which cannot be easily reconciled with observed effects on MPS or LPB.

Alzheimer's disease is the most common cause of dementia in the elderly. Although several genetic defects have been identified in patients with a family history of this disease, the majority of cases involve individuals with no known genetic predisposition. A mutant form of ubiquitin, termed Ub ⁺¹ , has been selectively observed in the brains of Alzheimer's patients, including those with nonfamilial Alzheimer's disease, but it has been unclear why Ub ⁺¹ expression should be deleterious. Here we show that Ub ⁺¹ is an efficient substrate for polyubiquitination in vitro and in transfected human cells. The resulting polyubiquitin chains are refractory to disassembly by deubiquitinating enzymes and potently inhibit the degradation of a polyubiquitinated substrate by purified 26S proteasomes. Thus, expression of Ub ⁺¹ in aging brain could result in dominant inhibition of the Ub-proteasome system, leading to neuropathologic consequences.

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation ofPsmc1(Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome andPsmc1conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and α-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Interactions of CNS cells lead to the establishment of complex neural systems. Specifically, oligodendrocytes form myelin sheaths around axons that enable rapid electrical conduction of impulses. Recent evidence has emerged that oligodendrocytes may also release trophic factors promoting neuronal survival. We therefore studied the effects of factors released from cells of the oligodendrocyte lineage on neuronal survival and also on the morphology of neurons. Neurons derived from rat embryonic cortices were cultured and exposed to media conditioned by oligodendrocyte precursor cells (OPCs) or differentiated oligodendrocytes. In line with previous studies, exposure of OPC and oligodendrocyte-conditioned media (OCM) increased survival, a phosphatidylinositol 3′-kinase (PI3kinase)/Akt-dependent phenomenon. In addition, exposure of neurons to OCM but not OPC conditioned media resulted in increased axonal length per neuron, as detected by antibodies to phosphorylated neurofilaments. OCM exposure resulted in activation of the MAPkinase/extracellular signal-regulated kinase pathway, inhibition of which significantly reduced oligodendrocyte-mediated enhancement of axonal length but, unlike PI3kinase inhibition, had no effect on neuronal survival. Furthermore, we identify glial cell line-derived neurotrophic factor (GDNF) production by differentiated oligodendrocytes and provide evidence that implicates GDNF in OCM-mediated axonal effects, independent of its effect on neuronal survival. Therefore, we have shown that factors released by OPCs and oligodendrocytes induce the activation of distinct intracellular pathways within neurons, which have different functional effects on the cell.

Filamentous inclusions composed of the microtubule‐associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non‐transfected SH‐SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi‐directional, proceeding from both N‐ and C‐termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C‐terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin‐independent pathways for the proteasomal degradation of unfolded proteins.