Huntington’s disease Also called Huntington Chorea, affects men and women. 4 to 10 cases in 100,000 people (frequency between 0.004% and 0.01%). Chorea is defined as brief, irregular, unpredictable, purposeless movements that flow from one body part to another without a rhythmic pattern. HD is a Hyperkinetic Disorder. The symptoms in HD are characterized by Excessive motor activity Involuntary movements (dyskinesias) Decreased muscle tone Behavioral and emotional alteration and cognitive decline, which may precede motor abnormalities. Importance of cognitive and neuropsychological test to identify the pre- symptomatic carriers of the disease. HD is a dominant autosomal inherited disease characterized by the expansion of a triplet repeat CAG, that encodes for Glutamine, in the N-terminal domain of the protein.

George Huntington ( )

Specific localization of Basal Ganglia in a coronal section

HD is caused by selective degeneration of the GABAergic neurons in the striatum, in particular in the caudate

HD Normal Volumetric analysis in Huntington disease (HD)

Age of onset: HD is thought to be a true dominant disorder, since homozygous carriers of the disease are no more severely affected than heterozygous carriers. In a normal individual, the number of CAG repeats are <36. In HD patients, age of onset when the repeats are 46 ranges between 25 and 52 years old. However, many factors influence the onset of the disease, such as: Mitochondrial dysfunction Cell death by apoptosis Loss of neurotrophic factors Neuron excitotoxicity Length of the CAG repeat and onset of the disease: Inverse correlation, but not when onset is >50 years of age.

Deposition of fibrillar proteinacious material in Huntington’s disease Huntington’s disease: a progressive neurodegenerative disease characterized by CAG repeats causing glutamine expansion motifs (polyQ) in the N-terminal region of the protein huntingtin. Onset of the disease inversely correlates with the number of CAG repeates. Huntingtin can aggregate forming intracellular inclusion bodies that contain also proteasome proteins and ubiquitin. Huntingtin aggregates contain  -sheet structures similar to amyloid.

In homo sapiens, the gene for huntingtin encodes a large, cytosolic protein, comprised of 3144 aa and with a molecular weight of about 348 kDa. NH 2 COOH aa3144 PolyQP In normal individuals, PolyQ 36 aa1 Huntingtin

NH 2 aa3144 PolyQP aa1 Cleavage by caspase 2, 3, 6 calpain N-terminal fragment C-terminal fragment COOH NH 2 Processing of PolyQ, mutant huntingtin Cdk5, Akt N-term htt forms aggregates

In HD, the aggregates are present in the affected brain regions, although not necessarily in the degenerating neurons In HD, are the aggregates protecting from neurodegeneration? Are htt oligomers the most toxic species?

Huntington’s disease: loss of function of the wild type huntingtin and gain of toxic function of the mutant huntingtin -Heterozygous patients are NOT different in any way from the homozygous patients: in heterozygous patients, the amount of wild-type “good” huntingtin does not make it to “buffer’ the effects of the mutant, PolyQ expanded huntingtin. -Mutant, PolyQ expanded huntingtin can recruit wild type huntingtin into insoluble aggregates to form the inclusions. -Neurons with larger inclusions are less compromised than those ones with diffused mutant htt (which might contain oligomers not aggregated into inclusions).

Potential mechanism of cell death in Huntington’s disease

Which is the physiological role of wild type huntingtin? -Huntingtin is a molecule required during development, as hungtintin KO mice die at 7.5 postnatal day. Heterozygosity is enough to reach adulthood. -Huntingtin interacts with several proteins, i.e. cytoskeletal proteins and others that are associated with membrane vesicles, suggesting a role in intracellular trafficking, membrane recycling and retrograde fast axonal transport. -The presence of Q repeats (CAG) followed by PolyP domain associates with transcriptional regulatory proteins. Inverse correlation between number of CAG repeats and transcriptional activity. After interacting, huntingtin transports transcriptional regulatory proteins in the cytosol and within the nucleus. (BDNF).

Physiological roles of wild type huntingtin

Physiologic function of wild-type huntingtin: Cell survival, anti-apoptotic function

Immortalized embrional striatal cells Normal Serum withdrawal N548Mu N548WT Rigamonti et al., 2000 Cell death is prevented by huntingtin wt, but is promoted by huntingtin PolyQ mutant

Rigamonti et al., 2000 Temperature-dependent apoptotic DNA fragmentation is sustained by huntingtin PolyQ mutant, but is prevented by huntingtin wt

Physiologic function of wild-type huntingtin: modulating the expression and the transport of BDNF (Brain Derived Growth Factor) to striatal neurons

-Peculiarity of HD is that lethality occurs specifically in striatal neurons, although huntingtin is ubiquitously expressed. -It is known that huntingtin promotes neuronal survival (and is required during development) -BDNF has certainly a role in maintaining the “health” of striatal neurons. In facts, BDNF: a)promotes growth and differentiation of striatal neurons b)protects striatal neurons from excitotoxicity

Wild-type huntingtin, but not mutant PolyQ huntingtin, specifically increase the levels of secreted and cellular BDNF in cell culture Zuccato et al., 2002

BDNF seems likely to be produced in cortex and hippocampus, and to be subsequently transported by afferents to the striatal neurons What happens in the cortex of HD patients?

Levels of BDNF mRNA are reduced in cortex of HD patient Zuccato et al., 2002

Pathogenic mechanisms of mutant PolyQ huntingtin: Htt processing and formation of aggregates

NH 2 aa3144 PolyQP aa1 Cleavage by caspase 2, 3, 6 calpain N-terminal fragment C-terminal fragment COOH NH 2 Processing of PolyQ, mutant huntingtin Cdk5, Akt N-term fragment forms aggregates

N-terminal fragment NH 2 Capabilities to form aggregates nucleus cytosol Soluble form Inhibits Glu Uptake Glu vesicle Different translocation of PolyQ, mutant huntingtin N-terminal fragment activates caspase-1 positive feedback in regulating apoptosis

How does the CAG, PolyQ expanded motif cause HD? -It induces mitochondrial dysfunction, that leads to mitochondrial disruption of Ca ++ homeostasis: trigger of cytochrome c and caspase- dependent apoptosis. -It avoids certain post-translation modifications (like SUMOylation and palmitoylation) to occur on the protein, with consequences on the protein’s functionality. -The N-terminal fragment translocates to the nucleus where it causes: a) reduction of the synthesis of neurotrophic factors (like BDNF Brain- Derived Neurotrophic Factor), that are essential for the neuronal growth and life (REST/NRSF). b) formation of intranuclear aggregates that recruit also transcription factors (CREB binding protein CBP), inducing transcriptional deregulation.

Pathogenic mechanisms of mutant PolyQ huntingtin: Mitochondrial dysfunction

Membrane Potential: Difference in the voltage between the two sides of a membrane, caused by an influx and efflux of ions to and from a cell or neuron and an organelle. In the mitochondrion, the membrane potential is caused in particular by exchange of Ca ++ ions between the mitochondrion and the cytosol. Ca ++ PTPs: Permeability Transition Pores PTP

Panov et al., 2002 Membrane potential and calcium levels are reduced in HD mitochondria Lymphoblasts

Juvenile HD patient: 65 repeats Adult HD patient: 46 repeats Panov et al., 2002 In HD patients, mitochondrial depolarization occurs at lower Ca++ levels PTP compromised? Physiologically, the potential drops when the PTP open

The tendency of the PTP to be open directly correlates with the number of PolyQ repeats in huntingtin mutant molecule

Huntingtin aggregates are visible on the mitochondrial membrane of PolyQ mutant huntingtin transgenic mice, but not of wild-type huntingtin transgenic mice Huntingtin PolyQ mutant transgenic mice Huntingtin wild-type transgenic mice Panov et al., 2002

Evidences that Calcium homeostasis is altered 1-in mitochondria of HD patients 2- in mitochondria of transgenic mice over-expressing mutant htt, but not in mice over-expressing wild type htt 3-administration of synthetic PolyQ repeats to human wild-type mitochondria in vitro causes alteration of calcium homeostasis Altered PTP and mitochondrial calcium homeostasis EARLY EVENT in HD

Mutant PolyQ huntingtin disrupts Calcium homeostasis, probably forming aggregates on the mitochondrial membrane that cause abnormal opening of the Permeability Transition Pores. Can mutant htt cause mitochondrial dysfunction also using different mechanisms?

Serum deprivation increases mitochondrial fragmentation in cells over-expressing mutant htt HeLa cells Serum deprivation to induce oxidative stress

Mutant htt sensitizes cells to mitochondrial abnormalities following oxidative stress

The only over-expression of toxic mutant htt causes mitochondrial fragmentation and dysfunction

Mutant htt affecting mitochondrial dynamics?

Mitochondrial fusion seems to occur only in cells over-expressing wt htt, but not mutant htt

Abolishing fission (Drp1 mutant) or inducing fusion (Mfn2 expression) RESCUES the mitochondrial fragmentation phenotype Cellular death by apoptosis caused by mutant htt is rescued too

Mutant htt PREDISPOSES cells to toxic stimuli leading to increased mitochondrial fission and therefore dysfunction A role for unbalanced fusion/fission in mitochondrial dysfunction in HD?

Mutant htt causes mitochondrial fragmentation in fibroblasts of HD patients

PolyQ expansion associated with HD impairs mitochondrial movement

WT YAC128 PolyQ expansion associated with HD affects mitochondrial size …

…and structure: loss of cristae’s density and presence of constrictions Side view of outer membrane “sagittal” “coronal” normal YAC128

Human BrainsMitochondrial lysates from mouse brain Mutant HTT associates with DRP1 in HD Colocalization is also found in neurons

DRP1 No nucleotides DRP1 + nucleotides DRP1 + nucleotides +httQ ext1-53 Mutant HTT has greater affinity than wt HTT for DRP1: effects on DRP1 GTPase acrivity DRP1 forms oligomers in presence of GTP (is a GTPase), even thicker in presence of mutant htt.

Inactive Drp1 mutant rescues the effects of polyQ htt on mitochondrial fragmentation and cell death (and also transport) Same effects achieved also by over-expression on Mfn2

Re-established mitochondrial fission and fusion balance improves the toxic effects of polyQ htt on mitochondrial motility, fragmentation and on cell death.

PolyQ htt binds to Drp1 and disrupts the equilibrium between fission and fusion: a model for htt toxicity in HD

Autophagy in HD: Loss of physiologic function of mutant htt

LC3II accumulates in HD: autophagy is induced or is defective?

Rescuing autophagy with Rapamycin reverts the eye degenerating phenotype in htt mutant flies Autophagy as a protective mechanism against HD

Autophagic activity is reduced in HD cells Protein degradation in MEF from wt or mutant htt knock in mice

What happens at an autophagosome level?

Autophagosomes are correctly formed and are abundant in HD cells H O W E V E R......

The cytosolic content in HD autophagosomes seems to differ from normal autophagosomes

Inefficient engulfment of cytosolic components by autophagosomes in HD cells

Macroautophagy is inefficient in HD cells Does the cell “give up” on autophagy in HD, or does it try to compensate with other mechanisms?

Protein degradation that does not depend on macroautophagy is increased in HD cells

CMA reporter is more evident in HD cells in typical autophagic punctae…

…and co-localizes with LC3, an autophagy related protein

In HD cells, lysosomal CMA receptor LAMP-2 is upregulated and functional…

HD mouse brain Up-regulation of CMA receptor Lamp-2 also in vivo, in the brain of HD mice

…and in dopaminergic neurons too

LAMP-2 transcription and stability is increased in HD cells

Therapy for HD -Possible presymptomatic treatment, after genetic test. -Symptomatic treatments aimed at treating: Depression Psychosis Chorea Tetrabenazine, depletes DA from central neurons. -Mutant Htt siRNA -Caspase inhibitors (risks of higher susceptibility to develop cancer after a long- term treatment). Minocycline (antibiotic already in use in humans) used now in clinical trails for HD. In HD prevents apoptosis by inhibiting caspases and mitochondrial permeability. Possible other unknown mechanisms of action. -Enhancing the clearance of mutant Htt, for example by autophagy. -NMDA receptors antagonists to block glutamate-induced excitotoxicity. -To induce wt Htt physiological function

Potential therapeutic strategies in HD